U.S. patent application number 10/793639 was filed with the patent office on 2004-10-07 for nucleic acid molecules and other molecules associated with sterol synthesis and metabolism.
Invention is credited to Karunanandaa, Balasulojini, Kishore, Ganesh, Yu, Jaehyuk.
Application Number | 20040199940 10/793639 |
Document ID | / |
Family ID | 22502055 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040199940 |
Kind Code |
A1 |
Karunanandaa, Balasulojini ;
et al. |
October 7, 2004 |
Nucleic acid molecules and other molecules associated with sterol
synthesis and metabolism
Abstract
This invention relates to the field of biotechnology,
particularly as it pertains to the production of sterols in a
variety of host systems particularly plants. More specifically, the
invention relates to nucleic acid molecules encoding proteins and
fragments of proteins associated with sterol and phytosterol
synthesis and metabolism as well as the encoded proteins and
fragments of proteins and antibodies capable of binding to them.
The invention also relates to methods of using the nucleic acid
molecules, fragments of the nucleic acid molecules, proteins, and
fragments of proteins. The invention also relates to cells,
organisms, plants, or seeds, or progeny of any, that have been
manipulated to contain increased levels or overexpress at least one
sterol or phytosterol compound.
Inventors: |
Karunanandaa, Balasulojini;
(Creve Coeur, MO) ; Yu, Jaehyuk; (Madison, WI)
; Kishore, Ganesh; (Creve Coeur, MO) |
Correspondence
Address: |
ARNOLD & PORTER LLP
ATTN: IP DOCKETING DEPT.
555 TWELFTH STREET, N.W.
WASHINGTON
DC
20004-1206
US
|
Family ID: |
22502055 |
Appl. No.: |
10/793639 |
Filed: |
March 5, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10793639 |
Mar 5, 2004 |
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09614221 |
Jul 11, 2000 |
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6723837 |
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60142981 |
Jul 12, 1999 |
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Current U.S.
Class: |
800/281 ;
435/193; 435/419; 435/468; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 15/8243 20130101;
C07K 14/415 20130101 |
Class at
Publication: |
800/281 ;
536/023.2; 435/069.1; 435/193; 435/419; 435/468 |
International
Class: |
A01H 001/00; C12N
015/82; C07H 021/04; C12N 005/04; C12N 005/10; C12N 009/10 |
Claims
1-27. (Cancelled)
28. A substantially purified nucleic acid molecule that encodes a
protein comprising the amino acid sequence of SEQ ID NO: 625.
29. The substantially purified nucleic acid molecule of claim 28,
wherein the nucleic acid molecule comprises the nucleic acid
sequence of SEQ ID NO: 4.
30. A substantially purified nucleic acid molecule comprising a
nucleic acid sequence of SEQ ID NO: 4 or complement thereof.
31. A substantially purified nucleic acid molecule consisting of a
nucleic acid sequence of SEQ ID NO: 4 or complement thereof.
32. A substantially purified nucleic acid molecule comprising a
nucleic acid sequence having between 100% and 90% sequence identity
with a nucleic acid sequence of SEQ ID NO: 4 or complement
thereof.
33. The substantially purified nucleic acid molecule of claim 32,
wherein said nucleic acid molecule comprises a nucleic acid
sequence having between 100% and 95% sequence identity with a
nucleic acid sequence of SEQ ID NO: 4 or complement thereof.
34. The substantially purified nucleic acid molecule of claim 33,
wherein said nucleic acid molecule comprises a nucleic acid
sequence having between 100% and 98% sequence identity with a
nucleic acid sequence of SEQ ID NO: 4 or complement thereof.
35. The substantially purified nucleic acid molecule of claim 34,
wherein said nucleic acid molecule comprises a nucleic acid
sequence having between 100% and 99% sequence identity with a
nucleic acid sequence of SEQ ID NO: 4 or complement thereof.
36. A substantially purified nucleic acid molecule comprising a
nucleic acid sequence which encodes a maize HES1 protein.
37. The substantially purified nucleic acid molecule of claim 36,
wherein said nucleic acid molecule comprises a nucleic acid
sequence of SEQ ID NO: 4, or complement thereof.
38. The substantially purified nucleic acid molecule of claim 36,
wherein said nucleic acid sequence encodes a protein comprising the
amino acid sequence of SEQ ID NO: 625.
39. A transformed plant having a nucleic acid comprising, (a) an
exogenous promoter region which functions in a plant cell to cause
the production of a mRNA molecule, operably linked to, (b) a
structural nucleic acid molecule, wherein said structural nucleic
acid molecule comprises a nucleic acid sequence encoding a protein
having an amino acid sequence of SEQ ID NO: 625 or fragment
thereof, which is operably linked to, (c) a 3' non-translated
sequence that functions in a plant cells to cause termination of
transcription and addition of polyadenylated ribonucleotides to a
3' end of the mRNA molecule.
40. The transformed plant according to claim 39, wherein said
structural nucleic acid molecule is in the antisense
orientation.
41. The transformed plant according to claim 39, wherein said plant
is selected from the group consisting of rapeseed, maize, soybean,
safflower, sunflower, cotton, peanut, flax, oil palm and Cuphea.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the field of biotechnology,
particularly as it pertains to the production of sterols in a
variety of host systems particularly plants. More specifically, the
invention relates to nucleic acid molecules encoding proteins and
fragments of proteins associated with sterol and phytosterol
metabolism as well as the encoded proteins and fragments of
proteins and antibodies capable of binding to them. The invention
also relates to methods of using the nucleic acid molecules,
fragments of the nucleic acid molecules, proteins, and fragments of
proteins. The invention also relates to cells, organisms,
particularly plants, or seeds, or progeny of plants, that have been
manipulated to contain increased levels or overexpress at least one
sterol or phytosterol compound.
BACKGROUND OF THE INVENTION
[0002] Sterols are a class of essential, natural compounds required
by all eukaryotes to complete their life cycle. The types of
sterols produced and predominantly present within each of the
phylogenetic kingdoms varies. Plants produce a class of sterols
called phytosterols. A phytosterol called sitosterol predominates.
In animals, cholesterol is typically the major sterol while in
fungi it is ergosterol.
[0003] Phytosterols from plants possess a wide spectrum of
biological activities in animals and humans. Phytosterols are
considered efficacious cholesterol-lowering agents (Pelletier et
al., Annals Nutrit. Metab. 39:291-295 (1995), the entirety of which
is herein incorporated by reference). Lower cholesterol levels are
linked to a reduction in the risk to cardiovascular disease.
Phytosterols can also block cholesterol absorption in the
intestine, which would also lead to lower cholesterol levels. Thus,
enhancing the levels of phytosterols in edible plants and seeds, or
products derived from these plants and seeds, may lead to food
products with increased nutritive or therapeutic value.
[0004] In one aspect, this invention provides these desirable
plants and seeds as well as methods to produce them. Since, as will
be discussed below, the genetic manipulation made possible by this
invention involves families of related genes that cross
phylogenetic boundaries, the effects are not limited to plants
alone.
Biochemistry of Sterol Synthesis
[0005] A number of the important sterol biosynthetic enzymes,
reactions, and intermediates have been described. Sterol synthesis
uses acetyl CoA as the basic carbon building block. Multiple acetyl
CoA molecules form the five-carbon isoprene units, hence the name
isoprenoid pathway. Enzymatic combination of isoprene units leads
to the thirty-carbon squalene molecule, which is the penultimate
precursor to sterols.
[0006] Throughout plants, animals, and fungus, the reactions
proceed as: acetyl CoA_HMGCoA, mevalonate, mevalonate 5 phosphate,
mevalonate 5-pyrophosphate, isopentyl diphosphate,
5-pyrophosphatemevalonate, isopentyl pyrophosphate (PIP),
dimethylallyl pyrophosphate (DMAPP), PIP+DMAPP, geranyl
pyrophosphate+IPP, farnesyl pyrophosphate, 2 farnesyl
pyrophosphate, squalene and squalene epoxide
[0007] From squalene epoxide, the sterol biosynthesis pathway of
plants diverges from that of animals and fungi. In plants,
cycloartenol is produced next by cyclization of squalene epoxide.
The plant pathway eventually leads to the synthesis of the
predominant phytosterol, sitosterol.
[0008] Animals go on to produce lanosterol from squalene epoxide,
eventually leading to cholesterol, which is the precursor to
steroid hormones and bile acids, among other compounds. In fungi,
lanosterol leads to the production of the predominant sterol,
ergosterol.
[0009] An important regulatory control step within the pathway
consists of the HMGCoA_Mevalonate step, catalyzed by HMGCoA
reductase, and the condensation of 2 farnesyl
pyrophosphates_squalene, catalyzed by squalene synthase. An early,
reported rate-limiting step, in the pathway is the HMGCoA
reductase-catalyzed reaction.
[0010] A number of studies have focused on the regulation of HMGCoA
reductase. HMGCoA reductase (EC 1.1.1.34) catalyzes the reductive
conversion of HMGCoA to mevalonic acid (MVA). This reaction is a
reported controlling step in isoprenoid biosynthesis. The enzyme is
regulated by feedback mechanisms and by a system of activation
kinases and phosphatases (Gray, Adv. Bot. Res., 14: 25 (1987); Bach
et al., Lipids, 26: 637 (1991); Stermer et al., J. Lipid Res., 35:
1133 (1994), all of which are herein incorporated by reference in
their entirety).
[0011] Another important regulation occurs at the squalene synthase
step. Squalene synthase (EC 2.5.1.21) reductively condenses two
molecules of FPP in the presence of Mg.sup.2+ and NADPH to form
squalene. The reaction involves a head-to-head condensation and
forms a stable intermediate, presqualene diphosphate. The enzyme is
subject to regulation similar to that of HMGCoA reductase and acts
by balancing the incorporation of FPP into sterols and other
compounds.
[0012] The sterol pathway of plants diverges from that in animals
and fungi after squalene epoxide. In plants, the cyclization of
squalene epoxide occurs next, under the regulated control of
cycloartenol synthase (EC 5.4.99.8). The cyclization mechanism
proceeds from the epoxy end into a chair-boat-chair-boat sequence
that is mediated by a transient C-20 carbocationic intermediate.
The reported rate-limiting step in plant sterol synthesis occurs in
the next step, S-adenosyl-L-methionine:sterol C-24 methyl
transferase (EC 2.1.1.41) (SMT.sub.I) catalyzing the transfer of a
methyl group from a cofactor, S-adenosyl-L-methionine, to the C-24
center of the sterol side chain. This is the first of two methyl
transfer reactions. The second methyl transfer reaction occurs
further down in the pathway and has been reported to be catalyzed
by SMT.sub.II. An isoform enzyme, SMT.sub.II, catalyzes the
conversion of 24-methylene lophenol to 24-ethylidene lophenol
(Fonteneau et al., Plant Sci Lett 10:147-155(1977), the entirety of
which is herein incorporated by reference). The presence of two
distinct SMTs in plants were further confirmed by cloning cDNAs
code the enzymes from Arabidopsis (Husselstein et al., FEBS Lett
381:87-92(1996), the entirety of which is herein incorporated by
reference), soybean (Shi et al., J. Biol Chem 271: 9384-9389(1996),
the entirety of which is herein incorporated by reference), maize
(Grebenok et al., Plant Mol Biol 34: 891-896(1997), the entirety of
which is herein incorporated by reference) and tobacco
(Bouvier-Nave et al., Eur J Biochem 246: 518-529 (1997);
Bouvier-Nave et al., Eur J Biochem 256: 88-96(1998), both of which
are herein incorporated by reference in their entirety).
[0013] Later in the pathway, a sterol C-14 demethylase catalyzes
the demethylation at C-14, removing the methyl group and creating a
double bond. Interestingly, this enzyme also occurs in plants and
fungi, but at a different point in the pathway. Sterol
C14-demethylation is mediated by a cytochrome P-450 complex. A
large family of enzymes utilize the cytochrome P-450 complex. There
is, in addition, a family of cytochrome P450 complexes. For
example, sterol C-22 desaturase (EC 2.7.3.9) catalyzes the
formation of the double bond at C-22 on the side chain. The C-22
desaturase in yeast, which is the final step in the biosynthesis of
ergosterol, contains a cytochrome P450 that is distinct from the
cytochrome P450 participating in the demethylation reaction.
Additional cytochrome P450 enzymes participate in brassinosteroid
synthesis (Bishop, Plant Cell 8:959-969 (1996), the entirety of
which is herein incorporated by reference). Brassinosteroids are
steroidal compounds with plant growth regulatory properties,
including modulation of cell expansion and photomorphogenesis
(Artecal, Plant Hormones, Physiology, Biochemistry and Molecular
Biology ed. Davies, Kluwer Academic Publishers, Dordrecht, 66
(1995), Yakota, Trends in Plant Science 2:137-143 (1997), both of
which are herein incorporated by reference in their entirety).
[0014] One class of proteins, oxysterol-binding proteins, have been
reported in humans and yeast (Jiang et al., Yeast 10: 341-353
(1994), the entirety of which is herein incorporated by reference).
These proteins have been reported to modulate ergosterol levels in
yeast (Jiang et al., Yeast 10: 341-353 (1994)). In particular,
Jiang et al., reported three genes KES1, HES1 and OSH1, which
encode proteins containing an oxysterol-binding region.
Enzyme Inhibitors and Modulators
[0015] Self-regulatory and feedback regulatory mechanisms of some
of the sterol synthesis enzymes provide opportunities to effect
sterol metabolism. For example, the introduction of the feedback
inhibitor molecule inhibits enzyme action while the removal of that
molecule up-regulates the enzyme. In certain circumstances,
non-wild type enzymes can effect normal regulation. These organisms
can be generated, for example, by traditional genetic crosses,
mutation treatments and through molecular genetics. One example is
the overexpression of plant HMGCoA reductase in transgenic plants
resulting in a 6-10 fold increase in the total sterol levels (for
example, transgenic tobacco plants overproducing phytosterols in
Schaller et al., Plant Physiol. 109: 761 (1995), the entirety of
which is herein incorporated by reference).
[0016] A number of compounds have been identified that, at least
partially, exert their effects on sterol synthesis. For example,
mevinolinic acid and lovastatin are competitive inhibitors of
HMGCoA reductase and zaragonic acid is a competitive inhibitor of
squalene synthase (Alberts et al., Proc. Natl. Acad. Sci. (U.S.A.)
77:3957-61 (1993); Bergstrom et al., Proc. Natl. Acad. Sci.
(U.S.A.) 90:80-84 (1980), both of which are herein incorporated by
reference). Many fungicides and insecticides act by inhibiting
enzymes, such as those noted above or the C-14 demethylase enzyme
(Sterol Biosynthesis Inhibitors and Anti-feeding Compounds, Kato et
al., Springer-Verlag, New York (1986); Sterol biosynthesis
inhibitors: pharmaceutical and agrochemical aspects, eds. Berg and
Plempel, Ellis Horwood, Chichester, England (1988), both of which
are herein incorporated by reference in this entirety).
[0017] However, the use of these compounds can have toxic effects
that preclude their use in products destined for animal or human
consumption. Furthermore, the increase or decrease in sterol levels
possible using these compounds is limited. Typically, the changes
in levels occur over a wide spectrum of the pathway. New and more
effective methods for manipulating sterol synthesis are
desired.
[0018] The present invention provides a gene, Hes1, involved in
plant phytosterol production Expression of HES1 (protein) in
organisms such as plants can increase phytosterol biosynthesis. The
present invention also provides transgenic organisms expressing a
HES1 protein, which can enhance food and feed sources.
SUMMARY OF THE INVENTION
[0019] The present invention includes a substantially purified
nucleic acid molecule that encodes a protein comprising the amino
acid sequence of SEQ ID NO: 622.
[0020] The present invention includes a substantially purified
nucleic acid molecule that specifically hybridizes to a nucleic
acid sequence of SEQ ID NO: 1 or its complement, wherein the
nucleic acid molecule encodes a protein comprising the amino acid
sequence of SEQ ID NO: 622.
[0021] The present invention includes a substantially purified
nucleic acid molecule that encodes a protein comprising the amino
acid sequence of SEQ ID NO: 623.
[0022] The present invention includes a substantially purified
nucleic acid molecule that specifically hybridizes to a nucleic
acid sequence of SEQ ID NO: 2 or its complement, wherein the
nucleic acid molecule encodes a protein comprising the amino acid
sequence of SEQ ID NO: 623.
[0023] The present invention includes a substantially purified
nucleic acid molecule that encodes a protein comprising the amino
acid sequence of SEQ ID NO: 624.
[0024] The present invention includes a substantially purified
nucleic acid molecule that specifically hybridizes to a nucleic
acid sequence of SEQ ID NO: 3 or its complement, wherein the
nucleic acid molecule encodes a protein comprising the amino acid
sequence of SEQ ID NO: 624.
[0025] The present invention includes a substantially purified
nucleic acid molecule that encodes a protein comprising the amino
acid sequence of SEQ ID NO: 625.
[0026] The present invention includes a substantially purified
nucleic acid molecule that specifically hybridizes to a nucleic
acid sequence of SEQ ID NO: 4 or its complement, wherein the
nucleic acid molecule encodes a protein comprising the amino acid
sequence of SEQ ID NO: 625.
[0027] The present invention includes a substantially purified
nucleic acid molecule comprising a nucleic acid sequence which
encodes a plant HES1 protein.
[0028] The present invention includes an antibody capable of
specifically binding a protein comprising the amino acid sequence
of SEQ ID NO: 622.
[0029] The present invention includes an antibody capable of
specifically binding a protein comprising the amino acid sequence
of SEQ ID NO: 623.
[0030] The present invention includes an antibody capable of
specifically binding a protein comprising the amino acid sequence
of SEQ ID NO: 624.
[0031] The present invention includes an antibody capable of
specifically binding a protein comprising the amino acid sequence
of SEQ ID NO: 625.
[0032] The present invention also provides a transformed plant
having a nucleic acid molecule which comprises: (A) an exogenous
promoter region which functions in a plant cell to cause the
production of a mRNA molecule; which is linked to (B) a structural
nucleic acid molecule, wherein the structural nucleic acid molecule
comprises a nucleic acid sequence encoding a protein having an
amino acid sequence selected from the group consisting of SEQ ID
NO: 622 through SEQ ID NO: 626 or fragment thereof; which is linked
to (C) a 3' non-translated sequence that functions in the plant
cell to cause termination of transcription and addition of
polyadenylated ribonucleotides to a 3' end of the mRNA
molecule.
[0033] The present invention also provides a transformed plant
having a nucleic acid molecule which comprises: (A) an exogenous
promoter region which functions in a plant cell to cause the
production of a mRNA molecule; which is linked to (B) a transcribed
nucleic acid molecule with a transcribed strand and a
non-transcribed strand, wherein the transcribed strand is
complementary to a nucleic acid molecule comprising a nucleic acid
sequence selected from the group consisting of SEQ ID NO: 1 through
SEQ ID NO: 621 or fragment thereof; which is linked to (C) a 3'
non-translated sequence that functions in plant cells to cause
termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of the mRNA molecule.
[0034] The present invention also provides a method for determining
a level or pattern in a plant of a protein in a plant comprising:
(A) incubating, under conditions permitting nucleic acid
hybridization, a marker nucleic acid molecule, the marker nucleic
acid molecule selected from the group of marker nucleic acid
molecules which specifically hybridize to a nucleic acid molecule
having the nucleic acid sequence of SEQ ID NO: 1 through SEQ ID NO:
621 or complements thereof, with a complementary nucleic acid
molecule obtained from the plant cell or plant tissue, wherein
nucleic acid hybridization between the marker nucleic acid molecule
and the complementary nucleic acid molecule obtained from the plant
permits the detection of an mRNA for the enzyme; (B) permitting
hybridization between the marker nucleic acid molecule and the
complementary nucleic acid molecule obtained from the plant cell or
plant tissue; and (C) detecting the level or pattern of the
complementary nucleic acid, wherein the detection of the
complementary nucleic acid is predictive of the level or pattern of
the protein in the plant.
[0035] The present invention also provides a method for determining
a level or pattern of a protein in a plant under evaluation which
comprises assaying the concentration of a molecule, whose
concentration is dependent upon the expression of a gene, the gene
specifically hybridizes to a nucleic acid molecule having a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 1
through SEQ ID NO: 621 or complements thereof, in comparison to the
concentration of that molecule present in a reference plant with a
known level or pattern of the protein, wherein the assayed
concentration of the molecule is compared to the assayed
concentration of the molecule in the reference plant with the known
level or pattern of the protein.
[0036] The present invention also provides a method for determining
a mutation in a plant whose presence is predictive of a mutation
affecting a level or pattern of a protein comprising the steps: (A)
incubating, under conditions permitting nucleic acid hybridization,
a marker nucleic acid, the marker nucleic acid selected from the
group of marker nucleic acid molecules which specifically hybridize
to a nucleic acid molecule having a nucleic acid sequence selected
from the group of SEQ ID NO: 1 through SEQ ID NO: 621 or
complements thereof and a complementary nucleic acid molecule
obtained from the plant, wherein nucleic acid hybridization between
the marker nucleic acid molecule and the complementary nucleic acid
molecule obtained from the plant permits the detection of a
polymorphism whose presence is predictive of a mutation affecting
the level or pattern of the protein in the plant; (B) permitting
hybridization between the marker nucleic acid molecule and the
complementary nucleic acid molecule obtained from the plant; and
(C) detecting the presence of the polymorphism, wherein the
detection of the polymorphism is predictive of the mutation.
[0037] The present invention also provides a method of producing a
plant containing an overexpressed protein comprising: (A)
transforming the plant with a functional nucleic acid molecule,
wherein the functional nucleic acid molecule comprises a promoter
region, wherein the promoter region is linked to a structural
region, wherein the structural region has a nucleic acid sequence
selected from group consisting of SEQ ID NO: 1 through SEQ ID NO:
621, wherein the structural region is linked to a 3' non-translated
sequence that functions in the plant to cause termination of
transcription and addition of polyadenylated ribonucleotides to a
3' end of a mRNA molecule; and wherein the functional nucleic acid
molecule results in overexpression of the protein; and (B) growing
the transformed plant.
[0038] The present invention also provides a method of producing a
plant containing an overexpressed protein comprising: (A)
transforming the plant with a functional nucleic acid molecule,
wherein the functional nucleic acid molecule comprises a promoter
region, wherein the promoter region is linked to a structural
region, wherein the structural region encodes a protein comprising
an amino acid sequence selected from group consisting of SEQ ID NO:
622 through SEQ ID NO: 626, wherein the structural region is linked
to a 3' non-translated sequence that functions in the plant to
cause termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of a mRNA molecule; and wherein the
functional nucleic acid molecule results in overexpression of the
protein; and (B) growing the transformed plant.
[0039] The present invention also provides a method of producing a
plant containing reduced levels of a protein comprising: (A)
transforming the plant with a functional nucleic acid molecule,
wherein the functional nucleic acid molecule comprises a promoter
region, wherein the promoter region is linked to a structural
region, wherein the structural region comprises a nucleic acid
molecule having a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 1 through SEQ ID NO: 621; wherein the
structural region is linked to a 3' non-translated sequence that
functions in the plant to cause termination of transcription and
addition of polyadenylated ribonucleotides to a 3' end of a mRNA
molecule; and wherein the functional nucleic acid molecule results
in co-suppression of the protein; and (B) growing the transformed
plant.
[0040] The present invention also provides a method of producing a
plant containing reduced levels of a protein comprising: (A)
transforming the plant with a functional nucleic acid molecule,
wherein the functional nucleic acid molecule comprises a promoter
region, wherein the promoter region is linked to a structural
region, wherein the structural region encodes a protein comprising
an amino acid sequence selected from group consisting of SEQ ID NO:
622 through SEQ ID NO: 626; wherein the structural region is linked
to a 3' non-translated sequence that functions in the plant to
cause termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of a mRNA molecule; and wherein the
functional nucleic acid molecule results in co-suppression of the
protein; and (B) growing the transformed plant.
[0041] The present invention also provides a method for reducing
expression of a protein in a plant comprising: (A) transforming the
plant with a nucleic acid molecule, the nucleic acid molecule
having an exogenous promoter region which functions in a plant cell
to cause the production of a mRNA molecule, wherein the exogenous
promoter region is linked to a transcribed nucleic acid molecule
having a transcribed strand and a non-transcribed strand, wherein
the transcribed strand is complementary to a nucleic acid molecule
having a nucleic acid sequence selected from the group consisting
of SEQ ID NO: 1 through SEQ ID NO: 621 or fragments thereof and the
transcribed strand is complementary to an endogenous mRNA molecule;
and wherein the transcribed nucleic acid molecule is linked to a 3'
non-translated sequence that functions in the plant cell to cause
termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of a mRNA molecule; and (B) growing the
transformed plant.
[0042] The present invention also provides a method for reducing
expression of a protein in a plant comprising: (A) transforming the
plant with a nucleic acid molecule, the nucleic acid molecule
having an exogenous promoter region which functions in a plant cell
to cause the production of a mRNA molecule, wherein the exogenous
promoter region is linked to a transcribed nucleic acid molecule
having a transcribed strand and a non-transcribed strand, wherein
the transcribed strand is complementary to a nucleic acid molecule
having a nucleic acid encodes a protein comprising an amino acid
sequence selected from group consisting of SEQ ID NO: 622 through
SEQ ID NO: 626 or frgaments thereof and the transcribed strand is
complementary to an endogenous mRNA molecule; and wherein the
transcribed nucleic acid molecule is linked to a 3' non-translated
sequence that functions in the plant cell to cause termination of
transcription and addition of polyadenylated ribonucleotides to a
3' end of a mRNA molecule; and (B) growing the transformed
plant.
[0043] The present invention also provides a method of determining
an association between a polymorphism and a plant trait comprising:
(A) hybridizing a nucleic acid molecule specific for the
polymorphism to genetic material of a plant, wherein the nucleic
acid molecule has a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 1 through SEQ ID NO: 621 or complements
thereof or fragment of either; and (B) calculating the degree of
association between the polymorphism and the plant trait.
[0044] The present invention also provides a method of isolating a
nucleic acid that encodes a protein or fragment thereof comprising:
(A) incubating under conditions permitting nucleic acid
hybridization, a first nucleic acid molecule comprising a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 1
through SEQ ID NO: 621 or complements thereof or fragment of either
with a complementary second nucleic acid molecule obtained from a
plant; (B) permitting hybridization between the first nucleic acid
molecule and the second nucleic acid molecule obtained from the
plant; and (C) isolating the second nucleic acid molecule.
[0045] The present invention also provides a method for producing a
protein or fragment thereof in an organism comprising introducing a
vector comprising a nucleic acid of the present invention and
expressing the protein or fragment.
DETAILED DESCRIPTION OF THE INVENTION
[0046] One skilled in the art can refer to general reference texts
for detailed descriptions of known techniques discussed herein or
equivalent techniques. These texts include Current Protocols in
Molecular Biology Ausubel, et al., eds., John Wiley & Sons,
N.Y. (1989), and supplements through September (1998), Molecular
Cloning, A Laboratory Manual (Sambrook et al., 2nd Ed., Cold Spring
Harbor Press, Cold Spring Harbor, N.Y. (1989), for example, each of
which are specifically incorporated by reference in their
entirety). These texts can also be referred to in making or using
an aspect of the invention.
[0047] The agents of the invention will preferably be "biologically
active" with respect to either a structural attribute, such as the
capacity of a nucleic acid to hybridize to another nucleic acid
molecule, or the ability of a protein to be bound by an antibody
(or to compete with another molecule for such binding).
Alternatively, such an attribute may be catalytic and thus involve
the capacity of the agent to mediate a chemical reaction or
response.
[0048] The term "substantially purified", as used herein, refers to
a molecule separated from substantially all other molecules
normally associated with it in its native state. More preferably a
substantially purified molecule is the predominant species present
in a preparation. A substantially purified molecule may be greater
than 60% free, preferably 75% free, more preferably 90% free, and
most preferably 95% free from the other molecules (exclusive of
solvent) present in the natural mixture. The term "substantially
purified" is not intended to encompass molecules present in their
native state.
[0049] The agents of the invention may also be recombinant. As used
herein, the term recombinant means any agent (e.g., DNA, peptide
etc.), that is, or results, however indirect, from human
manipulation of a nucleic acid molecule.
[0050] It is understood that the agents of the invention may be
labeled with reagents that facilitate detection of the agent (e.g.,
fluorescent labels, Prober et al., Science 238:336-340 (1987);
Albarella et al., EP 144914; chemical labels, Sheldon et al., U.S.
Pat. No. 4,582,789; Albarella et al., U.S. Pat. No. 4,563,417;
modified bases, Miyoshi et al., EP 119448, all of which are hereby
incorporated by reference in their entirety). It is further
understood that the invention provides recombinant bacterial,
mammalian, microbial, archaebacterial, insect, fungal, and plant
cells as well as viral constructs comprising the agents of the
invention.
(a) Nucleic Acid Molecules
[0051] Agents of the invention include nucleic acid molecules and,
more preferably, nucleic acid molecules of maize, soybean, canola,
yeast, or Arabidopsis. In addition, a number of different plants
can be the ultimate source of the nucleic acid molecules of the
invention. An exemplary group of genotypes includes: B73 (Illinois
Foundation Seeds, Champaign, Ill. U.S.A.); B73 x Mo17 (Illinois
Foundation Seeds, Champaign, Ill. U.S.A.); DK604 (Dekalb Genetics,
Dekalb, Ill. U.S.A.); H99 (Illinois Foundation Seeds, Champaign,
Ill. U.S.A.); RX601 (Asgrow Seed Company, Des Moines, Iowa); and
Mo17 (Illinois Foundation Seeds, Champaign, Ill. U.S.A.). And an
exemplary group of soybean types includes: Asgrow 3244 (Asgrow Seed
Company, Des Moines, Iowa); C1944 (United States Department of
Agriculture (USDA) Soybean Germplasm Collection, Urbana, Ill.
U.S.A.); Cristalina (USDA Soybean Germplasm Collection, Urbana,
Ill. U.S.A.); FT108 (Monsoy, Brazil); Hartwig (USDA Soybean
Germplasm Collection, Urbana, Ill. U.S.A.); BW211S Null (Tohoku
University, Morioka, Japan), PI507354 (USDA Soybean Germplasm
Collection, Urbana, Ill. U.S.A.); Asgrow A4922 (Asgrow Seed
Company, Des Moines, Iowa U.S.A.); PI227687 (USDA Soybean Germplasm
Collection, Urbana, Ill. U.S.A.); PI229358 (USDA Soybean Germplasm
Collection, Urbana, Ill. U.S.A.); and Asgrow A3237 (Asgrow Seed
Company, Des Moines, Iowa U.S.A.).
[0052] A particularly preferred embodiment of the nucleic acid
molecules of the present invention are plant nucleic molecules that
comprise a nucleic acid sequence which encodes an oxysterol-binding
protein consensus sequence, for example, soybean HES1 (SEQ ID NOS:
622, 623 and 624), and maize HES1 (SEQ ID NO: 625).
[0053] Another particularly preferred embodiment of the nucleic
acid molecules of the present invention are yeast nucleic acid
molecules that comprise a nucleic acid sequence which encodes an
oxysterol-binding protein consensus sequence, for example yeast
HES1 (SEQ ID NO: 626).
[0054] A particularly preferred embodiment of the nucleic acid
molecules of the invention are nucleic acid molecules that encode a
protein or fragment thereof where the protein or fragment thereof
is selected from the group consisting of a HES1, HMGCoA reductase,
squalene synthase, cycloartenol synthase, SMTI, SMTII and UPC2. In
a more particularly preferred embodiment of the nucleic acid
molecules of the present invention are nucleic acid molecules that
encode a protein or fragment thereof where the protein or fragment
thereof is selected from the group consisting of a fungal, more
preferably a yeast HES1, a plant, more preferably a maize, soybean
or Arabidopsis HES1, a plant, more preferably a rubber or an
Arabidopsis HMGCoA reductase, a plant, more preferably an
Arabidopsis squalene synthase, a plant, more preferably an
Arabidopsis cycloartenol synthase, a plant, more preferably an
Arabidopsis SMTI or SMTII and a fungus, more preferably a yeast
UPC2.
[0055] In a preferred embodiment, the nucleic molecule encodes a
HES1 protein, preferably a plant HES1 protein comprising an
oxysterol-binding protein consensus sequence--E(K, Q) xSH (H, R)
PPx (S, T, A, C, F)A. In a further preferred embodiment, the
nucleic acid molecule encodes a HES1 protein comprising an amino
acid sequence selected from the group consisting of SEQ ID NO: 622,
SEQ ID NO: 623, SEQ ID NO: 624 and SEQ ID NO: 625. In a further
preferred embodiment, the nucleic acid molecule molecules encodes a
HES1 protein with a conservative amino acid substitution in an
amino acid sequence selected from the group consisting of SEQ ID
NO: 622, SEQ ID NO: 623, SEQ ID NO: 624 and SEQ ID NO: 625. In a
further preferred embodiment, the nucleic acid molecule molecules
encodes a HES1 protein with between 2 and 5 conservative amino acid
substitutions in an amino acid sequence selected from the group
consisting of SEQ ID NO: 622, SEQ ID NO: 623, SEQ ID NO: 624 and
SEQ ID NO: 625. In a further preferred embodiment, the nucleic acid
molecule molecules encodes a HES1 protein with between 5 and 10
conservative amino acid substitutions in an amino acid sequence
selected from the group consisting of SEQ ID NO: 622, SEQ ID NO:
623, SEQ ID NO: 624 and SEQ ID NO: 625. In a further preferred
embodiment, the nucleic acid molecule encodes a HES1 protein with
more than 10 conservative amino acid substitutions in an amino acid
sequence selected from the group consisting of SEQ ID NO: 622, SEQ
ID NO: 623, SEQ ID NO: 624 and SEQ ID NO: 625.
[0056] In another preferred embodiment, the nucleic molecule
encodes a HES1 protein, preferably a yeast HES1 protein comprising
an oxysterol-binding protein consensus sequence--E(K, Q) xSH (H, R)
PPx (S, T, A, C, F)A. In a further preferred embodiment, the
nucleic acid molecule encodes a HES1 protein comprising an amino
acid sequence SEQ ID NO: 626. In a further preferred embodiment,
the nucleic acid molecule molecules encodes a HES1 protein with a
conservative amino acid substitution in amino acid sequence SEQ ID
NO: 626. In a further preferred embodiment, the nucleic acid
molecule molecules encodes a HES1 protein with between 2 and 5
conservative amino acid substitutions in an amino acid sequence SEQ
ID NO: 626. In a further preferred embodiment, the nucleic acid
molecule molecules encodes a HES1 protein with between 5 and 10
conservative amino acid substitutions in an amino acid sequence SEQ
ID NO: 626. In a further preferred embodiment, the nucleic acid
molecule encodes a HES1 protein with more than 10 conservative
amino acid substitutions in an amino acid sequence SEQ ID NO:
626.
[0057] In an aspect of the present invention, one or more of the
nucleic acid molecules of the present invention differ in nucleic
acid sequence from those encoding a protein or fragment thereof in
SEQ ID NO: 1 through SEQ ID NO: 621 due to the degeneracy in the
genetic code in that they encode the same protein but differ in
nucleic acid sequence. In another further aspect of the present
invention, one or more of the nucleic acid molecules of the present
invention differ in nucleic acid sequence from those encoding a
protein or fragment thereof in SEQ ID NO: 1 through SEQ ID NO: 621
due to fact that the different nucleic acid sequence encodes a
protein having one or more conservative amino acid residue.
Examples of conservative substitutions are set forth in Table 1. It
is understood that codons capable of coding for such conservative
substitutions are known in the art.
1 TABLE 1 Original Residue Conservative Substitutions Ala Ser Arg
Lys Asn Gln; His Asp Glu Cys Ser; Ala Gln Asn Glu Asp Gly Pro His
Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile
Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile;
Leu
[0058] In a further aspect of the present invention, one or more of
the nucleic acid molecules of the present invention differ in
nucleic acid sequence from those encoding a protein or fragment
thereof set forth in SEQ ID NO: 1 through SEQ ID NO: 621 or
fragment thereof due to the fact that one or more codons encoding
an amino acid has been substituted for a codon that encodes a
nonessential substitution of the amino acid originally encoded.
[0059] One subset of the nucleic acid molecules of the invention is
fragment nucleic acids molecules. Fragment nucleic acid molecules
may consist of significant portion(s) of, or indeed most of, the
nucleic acid molecules of the invention, such as those specifically
disclosed. Alternatively, the fragments may comprise smaller
oligonucleotides (having from about 15 to about 400 nucleotide
residues and more preferably, about 15 to about 30 nucleotide
residues, or about 50 to about 100 nucleotide residues, or about
100 to about 200 nucleotide residues, or about 200 to about 400
nucleotide residues, or about 275 to about 350 nucleotide
residues).
[0060] A fragment of one or more of the nucleic acid molecules of
the invention may be a probe and specifically a PCR probe. A PCR
probe is a nucleic acid molecule capable of initiating a polymerase
activity while in a double-stranded structure with another nucleic
acid. Various methods for determining the structure of PCR probes
and PCR techniques exist in the art. Computer generated searches
using programs such as Primer3
(www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi), STSPipeline
(www-genome.wi.mit.edu/cgi-bin/www-STS_Pipeline), or GeneUp (Pesole
et al., BioTechniques 25:112-123 (1998)), for example, can be used
to identify potential PCR primers.
[0061] As used herein, two nucleic acid molecules are said to be
capable of specifically hybridizing to one another if the two
molecules are capable of forming an anti-parallel, double-stranded
nucleic acid structure.
[0062] A nucleic acid molecule is said to be the "complement" of
another nucleic acid molecule if they exhibit complete
complementarity. As used herein, molecules are said to exhibit
"complete complementarity" when every nucleotide of one of the
molecules is complementary to a nucleotide of the other. Two
molecules are said to be "minimally complementary" if they can
hybridize to one another with sufficient stability to permit them
to remain annealed to one another under at least conventional
"low-stringency" conditions. Similarly, the molecules are said to
be "complementary" if they can hybridize to one another with
sufficient stability to permit them to remain annealed to one
another under conventional "high-stringency" conditions.
Conventional stringency conditions are described by Sambrook et
al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring
Harbor Press, Cold Spring Harbor, N.Y. (1989) and by Haymes et al.,
Nucleic Acid Hybridization, A Practical Approach, IRL Press,
Washington, D.C. (1985). Departures from complete complementarity
are therefore permissible, as long as such departures do not
completely preclude the capacity of the molecules to form a
double-stranded structure. Thus, in order for a nucleic acid
molecule to serve as a primer or probe it need only be sufficiently
complementary in sequence to be able to form a stable
double-stranded structure under the particular solvent and salt
concentrations employed.
[0063] Appropriate stringency conditions which promote DNA
hybridization are, for example, 6.0.times.sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by a wash of
2.0.times.SSC at 20-25.degree. C., are known to those skilled in
the art or can be found in Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the
salt concentration in the wash step can be selected from a low
stringency of about 2.0.times.SSC at 50.degree. C. to a high
stringency of about 0.2.times.SSC at 65.degree. C. In addition, the
temperature in the wash step can be increased from low stringency
conditions at room temperature, about 22.degree. C., to high
stringency conditions at about 65.degree. C. Both temperature and
salt may be varied, or either the temperature or the salt
concentration may be held constant while the other variable is
changed.
[0064] In a preferred embodiment, a nucleic acid of the invention
will specifically hybridize to one or more of the nucleic acid
molecules set forth in SEQ ID NO: 1 through SEQ ID NO: 621 or
complements thereof or more preferably to a nucleic acid molecule
having a nucleic acid sequence selected from the group consisting
of SEQ ID NO: 1 through SEQ ID NO: 4, SEQ ID NO: 6 through SEQ ID
NO: 29 or complements thereof under moderately stringent
conditions, for example at about 2.0.times.SSC and about 65.degree.
C.
[0065] In a particularly preferred embodiment, a nucleic acid of
the invention will include those nucleic acid molecules that
specifically hybridize to one or more of the nucleic acid molecules
set forth in SEQ ID NO: 1 through SEQ ID NO: 621 or complements
thereof or more preferably to a nucleic acid molecule having a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 1 through SEQ ID NO: 4, SEQ ID NO: 6 through SEQ ID NO: 29 or
complements thereof under high stringency conditions such as
0.2.times.SSC and about 65.degree. C.
[0066] In one aspect of the invention, the nucleic acid molecules
of the invention have one or more of the nucleic acid sequences set
forth in SEQ ID NO: 1 through SEQ ID NO: 621 or complements thereof
or fragment thereof or more preferably to a nucleic acid molecule
having SEQ ID NO: 1 through SEQ ID NO: 4, SEQ ID NO: 6 through SEQ
ID NO: 29 or complements thereof. In another aspect of the
invention, one or more of the nucleic acid molecules of the
invention share between about 100% and 70% sequence identity with
one or more of the nucleic acid sequences set forth in SEQ ID NO: 1
through SEQ ID NO: 621 or complements thereof or more preferably to
a nucleic acid molecule having a nucleic acid sequence selected
from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 4, SEQ
ID NO: 6 through SEQ ID NO: 29 or complements thereof. In a further
aspect of the invention, one or more of the nucleic acid molecules
of the invention share between about 100% and 90% sequence identity
with one or more of the nucleic acid sequences set forth in SEQ ID
NO: 1 through SEQ ID NO: 621 or complements thereof or more
preferably to a nucleic acid molecule having a nucleic acid
sequence selected from the group consisting of SEQ ID NO: 1 through
SEQ ID NO: 4, SEQ ID NO: 6 through SEQ ID NO: 29 or complements
thereof. In a more preferred aspect of the invention, one or more
of the nucleic acid molecules of the invention share between about
100% and 95% sequence identity with one or more of the nucleic acid
sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 621 or
complements thereof or more preferably to a nucleic acid molecule
having a nucleic acid sequence selected from the group consisting
of SEQ ID NO: 1 through SEQ ID NO: 4, SEQ ID NO: 6 through SEQ ID
NO: 29 or complements thereof. In an even more preferred aspect of
the invention, one or more of the nucleic acid molecules of the
invention share between about 100% and 99% sequence identity with
one or more of the sequences set forth in SEQ ID NO: 1 through SEQ
ID NO: 621 or complements thereof or more preferably to a nucleic
acid molecule having a nucleic acid sequence selected from the
group consisting of SEQ ID NO: 1 through SEQ ID NO: 4, SEQ ID NO: 6
through SEQ ID NO: 29, or complements thereof.
[0067] In a preferred embodiment the percent identity calculations
are performed using the Megalign program of the LASERGENE
bioinformatics computing suite (default parameters, DNASTAR Inc.,
Madison, Wis.).
[0068] In a preferred embodiment of the present invention, the
nucleic acid molecule of the present invention encodes a protein or
fragment thereof, where a protein exhibits a BLAST probability
score of greater than 1E-12, preferably a BLAST probability score
of between about 1E-30 and about 1E-12, even more preferably a
BLAST probability score of greater than 1E-30 with its
homologue.
[0069] In a preferred embodiment of the present invention, the
nucleic molecule of the present invention encodes a protein or
fragment thereof where a protein exhibits a BLAST score of greater
than 120, preferably a BLAST score of between about 1450 and about
120, even more preferably a BLAST score of greater than 1450 with
its homologue.
[0070] Nucleic acid molecules of the present invention can comprise
sequences that encode a protein or fragment thereof. Such proteins
or fragments thereof include homologues of known proteins in other
organisms.
[0071] A nucleic acid molecule of the invention can also encode a
homolog protein. As used herein, a homolog protein molecule or
fragment thereof is a counterpart protein molecule or fragment
thereof in a second species (e.g., maize HES1 is a homolog of
Arabidopsis HES1). A homolog can also be generated by molecular
evolution or DNA shuffling techniques, so that the molecule retains
at least one functional or structure characteristic of the original
protein (see, for example, U.S. Pat. No. 5,811,238).
[0072] Particularly preferred homologues are selected from the
group consisting of alfalfa, Arabidopsis, barley, Brassica,
broccoli, cabbage, citrus, cotton, garlic, oat, oilseed rape,
onion, canola, flax, an ornamental plant, maize, peanut, pepper,
potato, rice, rye, sorghum, soybean, strawberry, sugarcane,
sugarbeet, tomato, wheat, poplar, pine, fir, eucalyptus, apple,
lettuce, lentils, grape, banana, tea, turf grasses, sunflower,
soybean, and Phaseolus. A particularly preferred group of
homologues are crops harvested for seed oils, including but not
limited to rapeseed (high erucic acid rape and canola), maize,
soybean, safflower, sunflower, cotton, peanut, flax, oil palm and
Cuphea.
[0073] In a preferred embodiment, nucleic acid molecules having SEQ
ID NO: 1 through SEQ ID NO: 621 or complements and fragments of
either can be utilized to obtain such homologues.
[0074] The degeneracy of the genetic code, which allows different
nucleic acid sequences to code for the same protein or peptide, is
known in the literature. (U.S. Pat. No. 4,757,006, the entirety of
which is herein incorporated by reference).
[0075] Agents of the invention include nucleic acid molecules that
encode a substantially purified nucleic acid molecules encoding at
least about a 10 amino acid region, more preferably a 20, 30, 40,
or 50 amino acid region, of a protein selected from the group
consisting of a fungal, more preferably a yeast HES1, a plant, more
preferably a maize, soybean or Arabidopsis HES1, a plant, more
preferably a rubber or an Arabidopsis HMGCoA reductase, a plant,
more preferably an Arabidopsis squalene synthase, a plant, more
preferably an Arabidopsis cycloartenol synthase, a plant, more
preferably an Arabidopsis SMTI or SMTII and a fungus, more
preferably a yeast UPC2.
(b) Protein and Peptide Molecules
[0076] A class of agents comprises one or more of the protein or
fragments thereof or peptide molecules having a nucleic acid
sequence selected from the group consisting of SEQ ID NO: 1 through
SEQ ID NO: 621 or one or more of the protein or fragment thereof
and peptide molecules encoded by other nucleic acid agents of the
invention. A particular preferred class of proteins are those
having an amino acid sequence selected from the group consisting of
SEQ ID NO: 622 through SEQ ID NO: 625 or fragments thereof.
[0077] As used herein, the term "protein molecule" or "peptide
molecule" includes any molecule that comprises five or more amino
acids. It is well known in the art that proteins may undergo
modification, including post-translational modifications, such as,
but not limited to, disulfide bond formation, glycosylation,
phosphorylation, or oligomerization. Thus, as used herein, the term
"protein molecule" or "peptide molecule" includes any protein
molecule that is modified by any biological or non-biological
process. The terms "amino acid" and "amino acids" refer to all
naturally occurring L-amino acids. This definition is meant to
include norleucine, norvaline, ornithine, homocysteine, and
homoserine.
[0078] One or more of the protein or fragment of peptide molecules
may be produced via chemical synthesis, or more preferably, by
expressing in a suitable bacterial or eukaryotic host. Suitable
methods for expression are described by Sambrook et al., In:
Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring
Harbor Press, Cold Spring Harbor, N.Y. (1989), or similar
texts.
[0079] A "protein fragment" is a peptide or polypeptide molecule
whose amino acid sequence comprises a subset of the amino acid
sequence of that protein. A protein or fragment thereof that
comprises one or more additional peptide regions not derived from
that protein is a "fusion" protein. Such molecules may be
derivatized to contain carbohydrate or other moieties (such as
keyhole limpet hemocyanin, etc.). Fusion protein or peptide
molecules of the invention are preferably produced via recombinant
means.
[0080] Another class of agents comprise protein or peptide
molecules or fragments or fusions thereof comprising SEQ ID NO: 622
through SEQ NO: 625 or fragment thereof or encoded by SEQ ID NO: 1
through SEQ ID NO: 621 in which conservative, non-essential or
non-relevant amino acid residues have been added, replaced or
deleted. Computerized means for designing modifications in protein
structure are known in the art (Dahiyat and Mayo, Science 278:82-87
(1997), the entirety of which is herein incorporated by
reference).
[0081] A particularly preferred embodiment of the nucleic acid
molecules of the present invention are proteins comprising an amino
acid sequence which corresponds to an oxysterol-protein binding
consensus sequence.
[0082] In a preferred embodiment of the present invention, the
nucleic molecule of the present invention encodes a protein or
fragment thereof, where a protein exhibits a BLAST probability
score of greater than 1E-2, preferably a BLAST probability score of
between about 1E-30 and about 1E-12, even more preferably a BLAST
probability score of greater than 1E-30 with its homologue.
[0083] In a preferred embodiment of the present invention, the
nucleic molecule of the present invention encodes a protein or
fragment thereof where a protein exhibits a BLAST score of greater
than 120, preferably a BLAST score of between about 1450 and about
120, even more preferably a BLAST score of greater than 1450 with
its homologue.
[0084] In another preferred embodiment of the present invention,
the nucleic acid molecule encoding a protein or fragment thereof
exhibits a % identity with its homologue of between about 25% and
about 40%, more preferably of between about 40 and about 70%, even
more preferably of between about 70% and about 90% and even more
preferably between about 90% and 99%. In another preferred
embodiment of the present invention, a protein or fragments thereof
exhibits a % identity with its homologue of 100%.
[0085] In a preferred embodiment the percent identity calculations
are performed using the Megalign program of the LASERGENE
bioinformatics computing suite (default parameters, DNASTAR Inc.,
Madison, Wis.).
[0086] A protein of the invention can also be a homologue protein.
As used herein, a homologue protein molecule or fragment thereof is
a counterpart protein molecule or fragment thereof in a second
species (e.g., maize HMGCoA reductase is a homologue of Arabidopsis
HMGCoA reductase). A homologue can also be generated by molecular
evolution or DNA shuffling techniques, so that the molecule retains
at least one functional or structure characteristic of the original
(see, for example, U.S. Pat. No. 5,811,238, the entirety of which
is herein incorporated by reference).
[0087] Particularly preferred homologues are selected from the
group consisting of alfalfa, Arabidopsis, barley, Brassica,
broccoli, cabbage, citrus, cotton, garlic, oat, oilseed rape,
onion, canola, flax, an ornamental plant, maize, peanut, pepper,
potato, rice, rye, sorghum, soybean, strawberry, sugarcane,
sugarbeet, tomato, wheat, poplar, pine, fir, eucalyptus, apple,
lettuce, lentils, grape, banana, tea, turf grasses, sunflower,
soybean, and Phaseolus. A particularly preferred group of
homologues are those from oil plants such as cotton, canola and
sunflower.
[0088] In a preferred embodiment, nucleic acid molecules having SEQ
ID NO: 1 through SEQ ID NO: 621 or complements and fragments of
either can be utilized to obtain such homologues.
[0089] The degeneracy of the genetic code, which allows different
nucleic acid sequences to code for the same protein or peptide, is
known in the literature. (U.S. Pat. No. 4,757,006, the entirety of
which is herein incorporated by reference).
[0090] Agents of the invention include proteins comprising at least
about a 10 amino acid region, more preferably a 20, 30, 40 or 50
amino acid region, of a protein selected from the group consisting
of a fungal, more preferably a yeast HES1, a plant, more preferably
a maize, soybean or Arabidopsis HES1, a plant, more preferably a
rubber or an Arabidopsis HMGCoA reductase, a plant, more preferably
an Arabidopsis squalene synthase, a plant, more preferably an
Arabidopsis cycloartenol synthase, a plant, more preferably an
Arabidopsis SMTI or SMTII and a fungus, more preferably a yeast
UPC2.
(c) Plant Constructs and Plant Transformants
[0091] One or more of the nucleic acid molecules of the invention
may be used in plant transformation or transfection. Exogenous
genetic material may be transferred into a plant cell and the plant
cell regenerated into a whole, fertile or sterile plant. Exogenous
genetic material is any genetic material, whether naturally
occurring or otherwise, from any source that is capable of being
inserted into any organism. In a preferred embodiment the exogenous
genetic material includes a nucleic acid molecule of the present
invention, preferably a nucleic acid molecule having a sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 621 or complements thereof or fragments of either. Another
preferred class of exogenous genetic material are nucleic acid
molecules that encode a protein having an amino acid selected from
the group consisting of SEQ ID NO: 622 through SEQ ID NO: 626 or
fragments thereof.
[0092] Genetic material may be transferred into either
monocotyledons and dicotyledons including, but not limited to
maize, soybean, Arabidopsis, phaseolus, peanut, alfalfa, wheat,
rice, oat, sorghum, rye, tritordeum, millet, fescue, perennial
ryegrass, sugarcane, cranberry, papaya, banana, banana, muskmelon,
apple, cucumber, dendrobium, gladiolus, chrysanthemum, liliacea,
cotton, eucalyptus, sunflower, canola, turfgrass, sugarbeet, coffee
and dioscorea (Christou, In: Particle Bombardment for Genetic
Engineering of Plants, Biotechnology Intelligence Unit, Academic
Press, San Diego, Calif. (1996), the entirety of which is herein
incorporated by reference). In a particular preferred embodiment,
any seed-bearing plant may be employed as the target plant species
for modification in accordance with this invention, including
angiosperms, gymnosperms, monocotyledons, and dicotyledons. Plants
of special interest are crops harvested for seed oils, including
but not limited to rapeseed (high erucic acid rape and canola),
maize, soybean, safflower, sunflower, cotton, peanut, flax, oil
palm and Cuphea.
[0093] Transfer of a nucleic acid that encodes for a protein can
result in overexpression of that protein in a transformed cell or
transgenic plant. One or more of the proteins or fragments thereof
encoded by nucleic acid molecules of the invention may be
overexpressed in a transformed cell or transformed plant. Such
overexpression may be the result of transient or stable transfer of
the exogenous genetic material.
[0094] In another preferred aspect of the present invention,
exogenous genetic material is a nucleic acid molecule that
comprises a nucleic acid sequence which encodes a HES1 protein or
fragment thereof, more preferably a yeast HES1 protein or fragment
thereof, even more preferably a plant HES1 protein or fragment
thereof.
[0095] In a preferred embodiment, expression or overexpression of a
HES1 protein in a plant provides in that plant, relative to an
untransformed plant with a similar genetic background, an increased
level of phytosterols.
[0096] In a preferred embodiment, expression or overexpression of a
HES1 protein in a plant provides in that plant, relative to an
untransformed plant with a similar genetic background, an altered
composition of phytosterols.
[0097] In another embodiment, overexpression of a HES1 protein in a
plant provides in that plant, relative to an untransformed plant
with a similar genetic background, an increased level of a HES1
protein in a plastid.
[0098] In another preferred embodiment, overexpression of the HES1
protein in a transformed plant will result in a plant which as a
food or feed constituent exhibits an increased ability to act as a
cholesterol lowering agent relative to an untransformed plant with
a similar genetic background.
[0099] In a preferred embodiment of the present invention, the
protein or fragment thereof overexpressed in the transgenic plant
is selected from the group consisting of a HES1, HMGCoA reductase,
squalene synthase, cycloartenol synthase, SMTI, SMTII and UPC2. In
a more particularly preferred embodiment of the present invention
is a protein or fragment thereof, where the protein or fragment
thereof is selected from the group consisting of a fungal, more
preferably a yeast HES1, a plant, more preferably a maize, soybean
or Arabidopsis HES1, a plant, more preferably a rubber or an
Arabidopsis HMGCoA reductase, a plant, more preferably an
Arabidopsis squalene synthase, a plant, more preferably an
Arabidopsis cycloartenol synthase, a plant, more preferably an
Arabidopsis SMTI or SMTII and a plant, more preferably a yeast
UPC2.
[0100] In another preferred embodiment of the present invention,
the protein or fragment thereof overexpressed in the transgenic
plant is selected from the group consisting a plant HES1 HMGCoA
reductase, squalene synthase, cycloartenol synthase, SMTI, SMTII
and yeast UPC2. In a further even more particularly preferred
embodiment of the present invention the protein or fragment thereof
is a plant HES1. In an additional even more particularly preferred
embodiment of the present invention the protein or fragment thereof
is a maize, soybean or Arabidopsis HES1.
[0101] In another preferred embodiment of the present invention,
the protein or fragment thereof overexpressed in the transgenic
plant is a HES1 protein, preferably a plant HES1 protein comprising
an oxysterol-binding protein consensus sequence--E(K, Q) xSH (H, R)
PPx (S, T, A, C, F)A. In another preferred embodiment of the
present invention, the protein or fragment thereof overexpressed in
the transgenic plant is a HES1 protein that comprises an amino acid
sequence selected from the group consisting of SEQ ID NO: 622, SEQ
ID NO: 623, SEQ ID NO: 624 and SEQ ID NO: 625. In another preferred
embodiment of the present invention, the protein or fragment
thereof overexpressed in the transgenic plant is a HES1 protein
with a conservative amino acid substitution in an amino acid
sequence selected from the group consisting of SEQ ID NO: 622, SEQ
ID NO: 623, SEQ ID NO: 624 and SEQ ID NO: 625. In another preferred
embodiment of the present invention, the protein or fragment
thereof overexpressed in the transgenic plant is a HES1 protein
with between 2 and 5 conservative amino acid substitutions in an
amino acid sequence selected from the group consisting of SEQ ID
NO: 622, SEQ ID NO: 623, SEQ ID NO: 624 and SEQ ID NO: 625. In
another preferred embodiment of the present invention, the protein
or fragment thereof overexpressed in the transgenic plant is a HES1
protein with between 5 and 10 conservative amino acid substitutions
in an amino acid sequence selected from the group consisting of SEQ
ID NO: 622, SEQ ID NO: 623, SEQ ID NO: 624 and SEQ ID NO: 625. In
another preferred embodiment of the present invention, the protein
or fragment thereof overexpressed in the transgenic plant is a HES1
protein with more than 10 conservative amino acid substitutions in
an amino acid sequence selected from the group consisting of SEQ ID
NO: 622, SEQ ID NO: 623, SEQ ID NO: 624 and SEQ ID NO: 625.
[0102] In another preferred embodiment of the present invention,
the protein or fragment thereof overexpressed in the transgenic
plant is a HES1 protein that comprises an amino acid sequence SEQ
ID NO: 626. In another preferred embodiment of the present
invention, the protein or fragment thereof overexpressed in the
transgenic plant is a HES1 protein with a conservative amino acid
substitution in an amino acid sequence SEQ ID NO: 626. In another
preferred embodiment of the present invention, the protein or
fragment thereof overexpressed in the transgenic plant is a HES1
protein with between 2 and 5 conservative amino acid substitutions
in an amino acid sequence SEQ ID NO: 626. In another preferred
embodiment of the present invention, the protein or fragment
thereof overexpressed in the transgenic plant is a HES1 protein
with between 5 and 10 conservative amino acid substitutions in an
amino acid sequence SEQ ID NO: 625. In another preferred embodiment
of the present invention, the protein or fragment thereof
overexpressed in the transgenic plant is a HES1 protein with more
than 10 conservative amino acid substitutions in an amino acid
sequence SEQ ID NO: 626.
[0103] Exogenous genetic material may be transferred into a host
cell by the use of a DNA vector or construct designed for such a
purpose. Design of such a vector is generally within the skill of
the art (See, Plant Molecular Biology: A Laboratory Manual, Clark
(ed.), Springier, New York (1997), the entirety of which is herein
incorporated by reference).
[0104] A construct or vector may include a plant promoter to
express the protein or protein fragment of choice. A number of
promoters, which are active in plant cells, have been described in
the literature. These include the nopaline synthase (NOS) promoter
(Ebert et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:5745-5749 (1987),
the entirety of which is herein incorporated by reference), the
octopine synthase (OCS) promoter (which are carried on
tumor-inducing plasmids of Agrobacterium tumefaciens), the
caulimovirus promoters such as the cauliflower mosaic virus (CaMV)
19S promoter (Lawton et al., Plant Mol. Biol. 9:315-324 (1987), the
entirety of which is herein incorporated by reference) and the CaMV
35S promoter (Odell et al., Nature 313:810-812 (1985), the entirety
of which is herein incorporated by reference), the figwort mosaic
virus 35S-promoter, the light-inducible promoter from the small
subunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), the
Adh promoter (Walker et al., Proc. Natl. Acad. Sci. (U.S.A.)
84:6624-6628 (1987), the entirety of which is herein incorporated
by reference), the sucrose synthase promoter (Yang et al., Proc.
Natl. Acad. Sci. (U.S.A.) 87:4144-4148 (1990), the entirety of
which is herein incorporated by reference), the R gene complex
promoter (Chandler et al., The Plant Cell 1:1 175-1183 (1989), the
entirety of which is herein incorporated by reference) and the
chlorophyll a/b binding protein gene promoter, etc. These promoters
have been used to create DNA constructs that have been expressed in
plants; see, e.g., PCT publication WO 84/02913, herein incorporated
by reference in its entirety. The CaMV 35S promoters are preferred
for use in plants. Promoters known or found to cause transcription
of DNA in plant cells can be used in the invention.
[0105] For the purpose of expression in source tissues of the
plant, such as the leaf, seed, root or stem, it is preferred that
the promoters utilized have relatively high expression in these
specific tissues. Tissue-specific expression of a protein of the
present invention is a particularly preferred embodiment. For this
purpose, one may choose from a number of promoters for genes with
tissue- or cell-specific or -enhanced expression. Examples of such
promoters reported in the literature include the chloroplast
glutamine synthetase GS2 promoter from pea (Edwards et al., Proc.
Natl. Acad. Sci. (U.S.A.) 87:3459-3463 (1990), herein incorporated
by reference in its entirety), the chloroplast
fructose-1,6-biphosphatase (FBPase) promoter from wheat (Lloyd et
al., Mol. Gen. Genet. 225:209-216 (1991), herein incorporated by
reference in its entirety), the nuclear photosynthetic ST-LS1
promoter from potato (Stockhaus et al., EMBO J. 8:2445-2451 (1989),
herein incorporated by reference in its entirety), the
serine/threonine kinase (PAL) promoter and the glucoamylase (CHS)
promoter from Arabidopsis thaliana. Also reported to be active in
photosynthetically active tissues are the ribulose-1,5-bisphosphate
carboxylase (RbcS) promoter from eastern larch (Larix laricina),
the promoter for the cab gene, cab6, from pine (Yamamoto et al.,
Plant Cell Physiol. 35:773-778 (1994), herein incorporated by
reference in its entirety), the promoter for the Cab-1 gene from
wheat (Fejes et al., Plant Mol. Biol. 15:921-932 (1990), herein
incorporated by reference in its entirety), the promoter for the
CAB-1 gene from spinach (Lubberstedt et al., Plant Physiol.
104:997-1006 (1994), herein incorporated by reference in its
entirety), the promoter for the cab1R gene from rice (Luan et al.,
Plant Cell. 4:971-981 (1992), the entirety of which is herein
incorporated by reference), the pyruvate, orthophosphate dikinase
(PPDK) promoter from maize (Matsuoka et al., Proc. Natl. Acad. Sci.
(U.S.A.) 90: 9586-9590 (1993), herein incorporated by reference in
its entirety), the promoter for the tobacco Lhcb1*2 gene (Cerdan et
al., Plant Mol. Biol. 33:245-255 (1997), herein incorporated by
reference in its entirety), the Arabidopsis thaliana SUC2
sucrose-H+ symporter promoter (Truernit et al., Planta. 196:564-570
(1995), herein incorporated by reference in its entirety) and the
promoter for the thylakoid membrane proteins from spinach (psaD,
psaF, psae, PC, FNR, atpC, atpD, cab, rbcS). Other promoters for
the chlorophyll a/b-binding proteins may also be utilized in the
invention, such as the promoters for LhcB gene and PsbP gene from
white mustard (Sinapis alba; Kretsch et al., Plant Mol. Biol.
28:219-229 (1995), the entirety of which is herein incorporated by
reference).
[0106] For the purpose of expression in sink tissues of the plant,
such as the tuber of the potato plant, the fruit of tomato, or the
seed of maize, wheat, rice and barley, it is preferred that the
promoters utilized in the invention have relatively high expression
in these specific tissues. A number of promoters for genes with
tuber-specific or -enhanced expression are known, including the
class I patatin promoter (Bevan et al., EMBO J. 8:1899-1906 (1986);
Jefferson et al., Plant Mol. Biol. 14:995-1006 (1990), both of
which are herein incorporated by reference in their entirety), the
promoter for the potato tuber ADPGPP genes, both the large and
small subunits, the sucrose synthase promoter (Salanoubat and
Belliard, Gene 60:47-56 (1987), Salanoubat and Belliard, Gene
84:181-185 (1989), both of which are incorporated by reference in
their entirety), the promoter for the major tuber proteins
including the 22 kd protein complexes and proteinase inhibitors
(Hannapel, Plant Physiol. 101:703-704 (1993), herein incorporated
by reference in its entirety), the promoter for the granule bound
starch synthase gene (GBSS) (Visser et al., Plant Mol. Biol.
17:691-699 (1991), herein incorporated by reference in its
entirety) and other class I and II patatins promoters
(Koster-Topfer et al., Mol Gen Genet. 219:390-396 (1989); Mignery
et al., Gene. 62:27-44 (1988), both of which are herein
incorporated by reference in their entirety).
[0107] Other promoters can also be used to express a protein or
fragment thereof in specific tissues, such as seeds or fruits. The
promoter for .beta.-conglycinin (Chen et al., Dev. Genet. 10:
112-122 (1989), herein incorporated by reference in its entirety)
or other seed-specific promoters such as the napin and phaseolin
promoters, can be used. The zeins are a group of storage proteins
found in maize endosperm. Genomic clones for zein genes have been
isolated (Pedersen et al., Cell 29:1015-1026 (1982), herein
incorporated by reference in its entirety) and the promoters from
these clones, including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD and
.gamma. genes, could also be used. Other promoters known to
function, for example, in maize include the promoters for the
following genes: waxy, Brittle, Shrunken 2, Branching enzymes I and
II, starch synthases, debranching enzymes, oleosins, glutelins and
sucrose synthases. A particularly preferred promoter for maize
endosperm expression is the promoter for the glutelin gene from
rice, more particularly the Osgt-1 promoter (Zheng et al., Mol.
Cell Biol. 13:5829-5842 (1993), herein incorporated by reference in
its entirety). Examples of promoters suitable for expression in
wheat include those promoters for the ADPglucose pyrosynthase
(ADPGPP) subunits, the granule bound and other starch synthase, the
branching and debranching enzymes, the embryogenesis-abundant
proteins, the gliadins and the glutenins. Examples of such
promoters in rice include those promoters for the ADPGPP subunits,
the granule bound and other starch synthase, the branching enzymes,
the debranching enzymes, sucrose synthases and the glutelins. A
particularly preferred promoter is the promoter for rice glutelin,
Osgt-1. Examples of such promoters for barley include those for the
ADPGPP subunits, the granule bound and other starch synthase, the
branching enzymes, the debranching enzymes, sucrose synthases, the
hordeins, the embryo globulins and the aleurone specific
proteins.
[0108] Root specific promoters may also be used. An example of such
a promoter is the promoter for the acid chitinase gene (Samac et
al., Plant Mol. Biol. 25:587-596 (1994), the entirety of which is
herein incorporated by reference). Expression in root tissue could
also be accomplished by utilizing the root specific subdomains of
the CaMV35S promoter that have been identified (Lam et al., Proc.
Natl. Acad. Sci. (U.S.A.) 86:7890-7894 (1989), herein incorporated
by reference in its entirety). Other root cell specific promoters
include those reported by Conkling et al. (Conkling et al., Plant
Physiol. 93:1203-1211 (1990), the entirety of which is herein
incorporated by reference).
[0109] Additional promoters that may be utilized are described, for
example, in U.S. Pat. Nos. 5,378,619; 5,391,725; 5,428,147;
5,447,858; 5,608,144; 5,608,144; 5,614,399; 5,633,441; 5,633,435;
and 4,633,436, all of which are herein incorporated in their
entirety. In addition, a tissue specific enhancer may be used
(Fromm et al., The Plant Cell 1:977-984 (1989), the entirety of
which is herein incorporated by reference).
[0110] Constructs or vectors may also include, with the coding
region of interest, a nucleic acid sequence that acts, in whole or
in part, to terminate transcription of that region. A number of
such sequences have been isolated, including the Tr7 3' sequence
and the NOS 3' sequence (Ingelbrecht et al., The Plant Cell
1:671-680 (1989), the entirety of which is herein incorporated by
reference; Bevan et al., Nucleic Acids Res. 11:369-385 (1983), the
entirety of which is herein incorporated by reference).
[0111] A vector or construct may also include regulatory elements.
Examples of such include the Adh intron 1 (Callis et al., Genes and
Develop. 1:1 183-1200 (1987), the entirety of which is herein
incorporated by reference), the sucrose synthase intron (Vasil et
al., Plant Physiol. 91:1575-1579 (1989), the entirety of which is
herein incorporated by reference) and the TMV omega element (Gallie
et al., The Plant Cell 1:301-311 (1989), the entirety of which is
herein incorporated by reference). These and other regulatory
elements may be included when appropriate.
[0112] A vector or construct may also include a selectable marker.
Selectable markers may also be used to select for plants or plant
cells that contain the exogenous genetic material. Examples of such
include, but are not limited to: a neo gene (Potrykus et al., Mol.
Gen. Genet. 199:183-188 (1985), the entirety of which is herein
incorporated by reference), which codes for kanamycin resistance
and can be selected for using kanamycin, G418, etc.; a bar gene
which codes for bialaphos resistance; a mutant EPSP synthase gene
(Hinchee et al., Bio/Technology 6:915-922 (1988), the entirety of
which is herein incorporated by reference) which encodes glyphosate
resistance; a nitrilase gene which confers resistance to bromoxynil
(Stalker et al., J. Biol. Chem. 263:6310-6314 (1988), the entirety
of which is herein incorporated by reference); a mutant
acetolactate synthase gene (ALS) which confers imidazolinone or
sulphonylurea resistance (European Patent Application 154,204 (Sep.
11, 1985), the entirety of which is herein incorporated by
reference); and a methotrexate resistant DHFR gene (Thillet et al.,
J. Biol. Chem. 263:12500-12508 (1988), the entirety of which is
herein incorporated by reference).
[0113] A vector or construct may also include a transit peptide.
Incorporation of a suitable chloroplast transit peptide may also be
employed (European Patent Application Publication Number 0218571,
the entirety of which is herein incorporated by reference).
Translational enhancers may also be incorporated as part of the
vector DNA. DNA constructs could contain one or more 5'
non-translated leader sequences which may serve to enhance
expression of the gene products from the resulting mRNA
transcripts. Such sequences may be derived from the promoter
selected to express the gene or can be specifically modified to
increase translation of the mRNA. Such regions may also be obtained
from viral RNAs, from suitable eukaryotic genes, or from a
synthetic gene sequence. For a review of optimizing expression of
transgenes, see Koziel et al., Plant Mol. Biol. 32:393-405 (1996),
the entirety of which is herein incorporated by reference.
[0114] A vector or construct may also include a screenable marker.
Screenable markers may be used to monitor expression. Exemplary
screenable markers include: a .beta.-glucuronidase or uidA gene
(GUS) which encodes an enzyme for which various chromogenic
substrates are known (Jefferson, Plant Mol. Biol, Rep. 5:387-405
(1987), the entirety of which is herein incorporated by reference;
Jefferson et al., EMBO J. 6:3901-3907 (1987), the entirety of which
is herein incorporated by reference); an R-locus gene, which
encodes a product that regulates the production of anthocyanin
pigments (red color) in plant tissues (Dellaporta et al., Stadler
Symposium 11:263-282 (1988), the entirety of which is herein
incorporated by reference); a .beta.-lactamase gene (Sutcliffe et
al., Proc. Natl. Acad. Sci. (U.S.A.) 75:3737-3741 (1978), the
entirety of which is herein incorporated by reference), a gene
which encodes an enzyme for which various chromogenic substrates
are known (e.g., PADAC, a chromogenic cephalosporin); a luciferase
gene (Ow et al., Science 234:856-859 (1986), the entirety of which
is herein incorporated by reference); a xylE gene (Zukowsky et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 80:1101-1105 (1983), the entirety
of which is herein incorporated by reference) which encodes a
catechol dioxygenase that can convert chromogenic catechols; an
.alpha.-amylase gene (Ikatu et al., Bio/Technol. 8:241-242 (1990),
the entirety of which is herein incorporated by reference); a
tyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703-2714
(1983), the entirety of which is herein incorporated by reference)
which encodes an enzyme capable of oxidizing tyrosine to DOPA and
dopaquinone which in turn condenses to melanin; an
.alpha.-galactosidase, which will turn a chromogenic
.alpha.-galactose substrate.
[0115] Included within the terms "selectable or screenable marker
genes" are also genes which encode a secretable marker whose
secretion can be detected as a means of identifying or selecting
for transformed cells. Examples include markers which encode a
secretable antigen that can be identified by antibody interaction,
or even secretable enzymes which can be detected catalytically.
Secretable proteins fall into a number of classes, including small,
diffusible proteins which are detectable, (e.g., by ELISA), small
active enzymes which are detectable in extracellular solution
(e.g., .alpha.-amylase, .beta.-lactamase, phosphinothricin
transferase), or proteins which are inserted or trapped in the cell
wall (such as proteins which include a leader sequence such as that
found in the expression unit of extension or tobacco PR-S). Other
possible selectable and/or screenable marker genes will be apparent
to those of skill in the art.
[0116] There are many methods for introducing transforming nucleic
acid molecules into plant cells. Suitable methods are believed to
include virtually any method by which nucleic acid molecules may be
introduced into a cell, such as by Agrobacterium infection or
direct delivery of nucleic acid molecules such as, for example, by
PEG-mediated transformation, by electroporation or by acceleration
of DNA coated particles, etc (Potrykus, Ann. Rev. Plant Physiol.
Plant Mol. Biol. 42:205-225 (1991), the entirety of which is herein
incorporated by reference; Vasil, Plant Mol. Biol. 25:925-937
(1994), the entirety of which is herein incorporated by reference).
For example, electroporation has been used to transform maize
protoplasts (Fromm et al., Nature 312:791-793 (1986), the entirety
of which is herein incorporated by reference).
[0117] Other vector systems suitable for introducing transforming
DNA into a host plant cell include but are not limited to binary
artificial chromosome (BIBAC) vectors (Hamilton et al., Gene
200:107-116 (1997), the entirety of which is herein incorporated by
reference); and transfection with RNA viral vectors (Della-Cioppa
et al., Ann. N.Y. Acad. Sci. (1996), 792 (Engineering Plants for
Commercial Products and Applications), 57-61, the entirety of which
is herein incorporated by reference). Additional vector systems
also include plant selectable YAC vectors such as those described
in Mullen et al., Molecular Breeding 4:449-457 (1988), the entirety
of which is herein incorporated by reference).
[0118] Technology for introduction of DNA into cells is well known
to those of skill in the art. Four general methods for delivering a
gene into cells have been described: (1) chemical methods (Graham
and van der Eb, Virology 54:536-539 (1973), the entirety of which
is herein incorporated by reference); (2) physical methods such as
microinjection (Capecchi, Cell 22:479-488 (1980), the entirety of
which is herein incorporated by reference), electroporation (Wong
and Neumann, Biochem. Biophys. Res. Commun. 107:584-587 (1982);
Fromm et al., Proc. Natl. Acad. Sci. (U.S.A.) 82:5824-5828 (1985);
U.S. Pat. No. 5,384,253, all of which are herein incorporated in
their entirety); and the gene gun (Johnston and Tang, Methods Cell
Biol. 43:353-365 (1994), the entirety of which is herein
incorporated by reference); (3) viral vectors (Clapp, Clin.
Perinatol. 20:155-168 (1993); Lu et al., J. Exp. Med. 178:2089-2096
(1993); Eglitis and Anderson, Biotechniques 6:608-614 (1988), all
of which are herein incorporated in their entirety); and (4)
receptor-mediated mechanisms (Curiel et al., Hum. Gen. Ther.
3:147-154 (1992), Wagner et al., Proc. Natl. Acad. Sci. (USA)
89:6099-6103 (1992), both of which are incorporated by reference in
their entirety).
[0119] Acceleration methods that may be used include, for example,
microprojectile bombardment and the like. One example of a method
for delivering transforming nucleic acid molecules to plant cells
is microprojectile bombardment. This method has been reviewed by
Yang and Christou (eds.), Particle Bombardment Technology for Gene
Transfer, Oxford Press, Oxford, England (1994), the entirety of
which is herein incorporated by reference). Non-biological
particles (microprojectiles) that may be coated with nucleic acids
and delivered into cells by a propelling force. Exemplary particles
include those comprised of tungsten, gold, platinum and the
like.
[0120] A particular advantage of microprojectile bombardment, in
addition to it being an effective means of reproducibly
transforming monocots, is that neither the isolation of protoplasts
(Cristou et al., Plant Physiol. 87:671-674 (1988), the entirety of
which is herein incorporated by reference) nor the susceptibility
of Agrobacterium infection are required. An illustrative embodiment
of a method for delivering DNA into maize cells by acceleration is
a biolistics .alpha.-particle delivery system, which can be used to
propel particles coated with DNA through a screen, such as a
stainless steel or Nytex screen, onto a filter surface covered with
maize cells cultured in suspension. Gordon-Kamm et al., describes
the basic procedure for coating tungsten particles with DNA
(Gordon-Kamm et al., Plant Cell 2:603-618 (1990), the entirety of
which is herein incorporated by reference). The screen disperses
the tungsten nucleic acid particles so that they are not delivered
to the recipient cells in large aggregates. A particle delivery
system suitable for use with the invention is the helium
acceleration PDS-1000/He gun is available from Bio-Rad Laboratories
(Bio-Rad, Hercules, Calif.)(Sanford et al., Technique 3:3-16
(1991), the entirety of which is herein incorporated by
reference).
[0121] For the bombardment, cells in suspension may be concentrated
on filters. Filters containing the cells to be bombarded are
positioned at an appropriate distance below the microprojectile
stopping plate. If desired, one or more screens are also positioned
between the gun and the cells to be bombarded.
[0122] Alternatively, immature embryos or other target cells may be
arranged on solid culture medium. The cells to be bombarded are
positioned at an appropriate distance below the microprojectile
stopping plate. If desired, one or more screens are also positioned
between the acceleration device and the cells to be bombarded.
Through the use of techniques set forth herein one may obtain up to
1000 or more foci of cells transiently expressing a marker gene.
The number of cells in a focus which express the exogenous gene
product 48 hours post-bombardment often range from one to ten and
average one to three.
[0123] In bombardment transformation, one may optimize the
pre-bombardment culturing conditions and the bombardment parameters
to yield the maximum numbers of stable transformants. Both the
physical and biological parameters for bombardment are important in
this technology. Physical factors are those that involve
manipulating the DNA/microprojectile precipitate or those that
affect the flight and velocity of either the macro- or
microprojectiles. Biological factors include all steps involved in
manipulation of cells before and immediately after bombardment, the
osmotic adjustment of target cells to help alleviate the trauma
associated with bombardment and also the nature of the transforming
DNA, such as linearized DNA or intact supercoiled plasmids. It is
believed that pre-bombardment manipulations are especially
important for successful transformation of immature embryos.
[0124] In another alternative embodiment, plastids can be stably
transformed. Methods disclosed for plastid transformation in higher
plants include the particle gun delivery of DNA containing a
selectable marker and targeting of the DNA to the plastid genome
through homologous recombination (Svab et al., Proc. Natl. Acad.
Sci. (U.S.A.) 87:8526-8530 (1990); Svab and Maliga, Proc. Natl.
Acad. Sci. (U.S.A.) 90:913-917 (1993); Staub and Maliga, EMBO J.
12:601-606 (1993); U.S. Pat. Nos. 5,451,513 and 5,545,818, all of
which are herein incorporated by reference in their entirety).
[0125] Accordingly, it is contemplated that one may wish to adjust
various aspects of the bombardment parameters in small scale
studies to fully optimize the conditions. One may particularly wish
to adjust physical parameters such as gap distance, flight
distance, tissue distance and helium pressure. One may also
minimize the trauma reduction factors by modifying conditions which
influence the physiological state of the recipient cells and which
may therefore influence transformation and integration
efficiencies. For example, the osmotic state, tissue hydration and
the subculture stage or cell cycle of the recipient cells may be
adjusted for optimum transformation. The execution of other routine
adjustments will be known to those of skill in the art in light of
the present disclosure.
[0126] Agrobacterium-mediated transfer is a widely applicable
system for introducing genes into plant cells because the DNA can
be introduced into whole plant tissues, thereby bypassing the need
for regeneration of an intact plant from a protoplast. The use of
Agrobacterium-mediated plant integrating vectors to introduce DNA
into plant cells is well known in the art. See, for example the
methods described by Fraley et al., Bio/Technology 3:629-635 (1985)
and Rogers et al., Methods Enzymol. 153:253-277 (1987), both of
which are herein incorporated by reference in their entirety.
Further, the integration of the Ti-DNA is a relatively precise
process resulting in few rearrangements. The region of DNA to be
transferred is defined by the border sequences and intervening DNA
is usually inserted into the plant genome as described (Spielmann
et al., Mol. Gen. Genet. 205:34 (1986), the entirety of which is
herein incorporated by reference).
[0127] Modem Agrobacterium transformation vectors are capable of
replication in E. coli as well as Agrobacterium, allowing for
convenient manipulations as described (Klee et al., In: Plant DNA
Infectious Agents, Hohn and Schell (eds.), Springer-Verlag, New
York, pp. 179-203 (1985), the entirety of which is herein
incorporated by reference). Moreover, technological advances in
vectors for Agrobacterium-mediated gene transfer have improved the
arrangement of genes and restriction sites in the vectors to
facilitate construction of vectors capable of expressing various
polypeptide coding genes. The vectors described have convenient
multi-linker regions flanked by a promoter and a polyadenylation
site for direct expression of inserted polypeptide coding genes and
are suitable for present purposes (Rogers et al., Methods Enzymol.
153:253-277 (1987)). In addition, Agrobacterium containing both
armed and disarmed Ti genes can be used for the transformations. In
those plant strains where Agrobacterium-mediated transformation is
efficient, it is the method of choice because of the facile and
defined nature of the gene transfer.
[0128] A transgenic plant formed using Agrobacterium transformation
methods typically contains a single gene on one chromosome. Such
transgenic plants can be referred to as being heterozygous for the
added gene. More preferred is a transgenic plant that is homozygous
for the added structural gene; i.e., a transgenic plant that
contains two added genes, one gene at the same locus on each
chromosome of a chromosome pair. A homozygous transgenic plant can
be obtained by sexually mating (selfing) an independent segregant
transgenic plant that contains a single added gene, germinating
some of the seed produced and analyzing the resulting plants
produced for the gene of interest.
[0129] It is also to be understood that two different transgenic
plants can also be mated to produce offspring that contain two
independently segregating, exogenous genes. Selfing of appropriate
progeny can produce plants that are homozygous for both added,
exogenous genes that encode a polypeptide of interest.
Back-crossing to a parental plant and out-crossing with a
non-transgenic plant are also contemplated, as is vegetative
propagation.
[0130] Transformation of plant protoplasts can be achieved using
methods based on calcium phosphate precipitation, polyethylene
glycol treatment, electroporation and combinations of these
treatments (See, for example, Potrykus et al., Mol. Gen. Genet.
205:193-200 (1986); Lorz et al., Mol. Gen. Genet. 199:178 (1985);
Fromm et al., Nature 319:791 (1986); Uchimiya et al., Mol. Gen.
Genet. 204:204 (1986); Marcotte et al., Nature 335:454-457 (1988),
all of which are herein incorporated by reference in their
entirety).
[0131] Application of these systems to different plant strains
depends upon the ability to regenerate that particular plant strain
from protoplasts. Illustrative methods for the regeneration of
cereals from protoplasts are described (Fujimura et al, Plant
Tissue Culture Letters 2:74 (1985); Toriyama et al, Theor Appl.
Genet. 205:34 (1986); Yamada et al., Plant Cell Rep. 4:85 (1986);
Abdullah et al., Biotechnolog. 4:1087 (1986), all of which are
herein incorporated by reference in their entirety).
[0132] To transform plant strains that cannot be successfully
regenerated from protoplasts, other ways to introduce DNA into
intact cells or tissues can be utilized. For example, regeneration
of cereals from immature embryos or explants can be effected as
described (Vasil, Biotechnology 6:397 (1988), the entirety of which
is herein incorporated by reference). In addition, "particle gun"
or high-velocity microprojectile technology can be utilized (Vasil
et al., Bio/Technology 10:667 (1992), the entirety of which is
herein incorporated by reference).
[0133] Using the latter technology, DNA is carried through the cell
wall and into the cytoplasm on the surface of small metal particles
as described (Klein et al., Nature 328:70 (1987); Klein et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 85:8502-8505 (1988); McCabe et al.,
Bio/Technology 6:923 (1988), all of which are herein incorporated
by reference in their entirety). The metal particles penetrate
through several layers of cells and thus allow the transformation
of cells within tissue explants.
[0134] Other methods of cell transformation can also be used and
include but are not limited to introduction of DNA into plants by
direct DNA transfer into pollen (Hess et al., Intern Rev. Cytol.
107:367 (1987); Luo et al., Plant Mol Biol. Reporter 6:165 (1988),
all of which are herein incorporated by reference in their
entirety), by direct injection of DNA into reproductive organs of a
plant (Pena et al., Nature 325:274 (1987), the entirety of which is
herein incorporated by reference), or by direct injection of DNA
into the cells of immature embryos followed by the rehydration of
desiccated embryos (Neuhaus et al., Theor. Appl. Genet. 75:30
(1987), the entirety of which is herein incorporated by
reference).
[0135] The regeneration, development and cultivation of plants from
single plant protoplast transformants or from various transformed
explants is well known in the art (Weissbach and Weissbach, In:
Methods for Plant Molecular Biology, Academic Press, San Diego,
Calif., (1988), the entirety of which is herein incorporated by
reference). This regeneration and growth process typically includes
the steps of selection of transformed cells, culturing those
individualized cells through the usual stages of embryonic
development through the rooted plantlet stage. Transgenic embryos
and seeds are similarly regenerated. The resulting transgenic
rooted shoots are thereafter planted in an appropriate plant growth
medium such as soil.
[0136] The development or regeneration of plants containing the
foreign, exogenous gene that encodes a protein of interest is well
known in the art. Preferably, the regenerated plants are
self-pollinated to provide homozygous transgenic plants. Otherwise,
pollen obtained from the regenerated plants is crossed to
seed-grown plants of agronomically important lines. Conversely,
pollen from plants of these important lines is used to pollinate
regenerated plants. A transgenic plant of the invention containing
a desired polypeptide is cultivated using methods well known to one
skilled in the art.
[0137] There are a variety of methods for the regeneration of
plants from plant tissue. The particular method of regeneration
will depend on the starting plant tissue and the particular plant
species to be regenerated.
[0138] Methods for transforming dicots, primarily by use of
Agrobacterium tumefaciens and obtaining transgenic plants have been
published for cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No.
5,159,135; U.S. Pat. No. 5,518,908, all of which are herein
incorporated by reference in their entirety); soybean (U.S. Pat.
No. 5,569,834; U.S. Pat. No. 5,416,011; McCabe et al.,
Biotechnology 6:923 (1988); Christou et al., Plant Physiol.
87:671-674 (1988); all of which are herein incorporated by
reference in their entirety); Brassica (U.S. Pat. No. 5,463,174,
the entirety of which is herein incorporated by reference); peanut
(Cheng et al., Plant Cell Rep. 15:653-657 (1996), McKently et al.,
Plant Cell Rep. 14:699-703 (1995), all of which are herein
incorporated by reference in their entirety); papaya; and pea
(Grant et al., Plant Cell Rep. 15:254-258 (1995), the entirety of
which is herein incorporated by reference).
[0139] Transformation of monocotyledons using electroporation,
particle bombardment and Agrobacterium have also been reported.
Transformation and plant regeneration have been achieved in
asparagus (Bytebier et al., Proc. Natl. Acad. Sci. (USA) 84:5354
(1987), the entirety of which is herein incorporated by reference);
barley (Wan and Lemaux, Plant Physiol 104:37 (1994), the entirety
of which is herein incorporated by reference); maize (Rhodes et
al., Science 240:204 (1988); Gordon-Kamm et al., Plant Cell
2:603-618 (1990); Fromm et al., Bio/Technology 8:833 (1990); Koziel
et al., Bio/Technology 11:194 (1993); Armstrong et al., Crop
Science 35.550-557 (1995); all of which are herein incorporated by
reference in their entirety); oat (Somers et al., Bio/Technology
10:1589 (1992), the entirety of which is herein incorporated by
reference); orchard grass (Horn et al., Plant Cell Rep. 7:469
(1988), the entirety of which is herein incorporated by reference);
rice (Toriyama et al., Theor Appl. Genet. 205:34 (1986); Part et
al., Plant Mol. Biol. 32:1135-1148 (1996); Abedinia et al., Aust.
J. Plant Physiol. 24:133-141 (1997); Zhang and Wu, Theor. Appl.
Genet. 76:835 (1988); Zhang et al., Plant Cell Rep. 7:379 (1988);
Battraw and Hall, Plant Sci. 86:191-202 (1992); Christou et al.,
Bio/Technology 9:957 (1991), all of which are herein incorporated
by reference in their entirety); rye (De la Pena et al., Nature
325:274 (1987), the entirety of which is herein incorporated by
reference); sugarcane (Bower and Birch, Plant J. 2:409 (1992), the
entirety of which is herein incorporated by reference); tall fescue
(Wang et al., Bio/Technology 10:691 (1992), the entirety of which
is herein incorporated by reference) and wheat (Vasil et al.,
Bio/Technology 10:667 (1992), the entirety of which is herein
incorporated by reference; U.S. Pat. No. 5,631,152, the entirety of
which is herein incorporated by reference.) Assays for gene
expression based on the transient expression of cloned nucleic acid
constructs have been developed by introducing the nucleic acid
molecules into plant cells by polyethylene glycol treatment,
electroporation, or particle bombardment (Marcotte et al., Nature
335:454-457 (1988), the entirety of which is herein incorporated by
reference; Marcotte et al., Plant Cell 1:523-532 (1989), the
entirety of which is herein incorporated by reference; McCarty et
al., Cell 66:895-905 (1991), the entirety of which is herein
incorporated by reference; Hattori et al., Genes Dev. 6:609-618
(1992), the entirety of which is herein incorporated by reference;
Goff et al., EMBO J. 9:2517-2522 (1990), the entirety of which is
herein incorporated by reference). Transient expression systems may
be used to functionally dissect gene constructs (see generally,
Mailga et al., Methods in Plant Molecular Biology, Cold Spring
Harbor Press (1995)).
[0140] Any of the nucleic acid molecules of the invention may be
introduced into a plant cell in a permanent or transient manner in
combination with other genetic elements such as vectors, promoters,
enhancers, etc. Further, any of the nucleic acid molecules of the
invention may be introduced into a plant cell in a manner that
allows for overexpression of the protein or fragment thereof
encoded by the nucleic acid molecule.
[0141] Cosuppression is the reduction in expression levels, usually
at the level of RNA, of a particular endogenous gene or gene family
by the expression of a homologous sense construct that is capable
of transcribing mRNA of the same strandedness as the transcript of
the endogenous gene (Napoli et al., Plant Cell 2:279-289 (1990),
the entirety of which is herein incorporated by reference; van der
Krol et al., Plant Cell 2:291-299 (1990), the entirety of which is
herein incorporated by reference). Cosuppression may result from
stable transformation with a single copy nucleic acid molecule that
is homologous to a nucleic acid sequence found with the cell
(Prolls and Meyer, Plant J. 2:465-475 (1992), the entirety of which
is herein incorporated by reference) or with multiple copies of a
nucleic acid molecule that is homologous to a nucleic acid sequence
found with the cell (Mittlesten et al., Mol. Gen. Genet.
244:325-330 (1994), the entirety of which is herein incorporated by
reference). Genes, even though different, linked to homologous
promoters may result in the cosuppression of the linked genes
(Vaucheret, C.R. Acad. Sci. III 316:1471-1483 (1993), the entirety
of which is herein incorporated by reference).
[0142] This technique has, for example, been applied to generate
white flowers from red petunia and tomatoes that do not ripen on
the vine. Up to 50% of petunia transformants that contained a sense
copy of the glucoamylase (CHS) gene produced white flowers or
floral sectors; this was as a result of the post-transcriptional
loss of mRNA encoding CHS (Flavell, Proc. Natl. Acad. Sci. (U.S.A.)
91:3490-3496 (1994), the entirety of which is herein incorporated
by reference); van Blokland et al., Plant J. 6:861-877 (1994), the
entirety of which is herein incorporated by reference).
Cosuppression may require the coordinate transcription of the
transgene and the endogenous gene and can be reset by a
developmental control mechanism (Jorgensen, Trends Biotechnol.
8:340-344 (1990), the entirety of which is herein incorporated by
reference; Meins and Kunz, In: Gene Inactivation and Homologous
Recombination in Plants, Paszkowski (ed.), pp. 335-348, Kluwer
Academic, Netherlands (1994), the entirety of which is herein
incorporated by reference).
[0143] It is understood that one or more of the nucleic acids of
the invention may be introduced into a plant cell and transcribed
using an appropriate promoter with such transcription resulting in
the cosuppression of an endogenous protein.
[0144] Antisense approaches are a way of preventing or reducing
gene function by targeting the genetic material (Mol et al., FEBS
Lett. 268:427-430 (1990), the entirety of which is herein
incorporated by reference). The objective of the antisense approach
is to use a sequence complementary to the target gene to block its
expression and create a mutant cell line or organism in which the
level of a single chosen protein is selectively reduced or
abolished. Antisense techniques have several advantages over other
`reverse genetic` approaches. The site of inactivation and its
developmental effect can be manipulated by the choice of promoter
for antisense genes or by the timing of external application or
microinjection. Antisense can manipulate its specificity by
selecting either unique regions of the target gene or regions where
it shares homology to other related genes (Hiatt et al., In:
Genetic Engineering, Setlow (ed.), Vol. 11, New York: Plenum 49-63
(1989), the entirety of which is herein incorporated by
reference).
[0145] The principle of regulation by antisense RNA is that RNA
that is complementary to the target mRNA is introduced into cells,
resulting in specific RNA:RNA duplexes being formed by base pairing
between the antisense substrate and the target mRNA (Green et al.,
Annu. Rev. Biochem. 55:569-597 (1986), the entirety of which is
herein incorporated by reference). Under one embodiment, the
process involves the introduction and expression of an antisense
gene sequence. Such a sequence is one in which part or all of the
normal gene sequences are placed under a promoter in inverted
orientation so that the `wrong` or complementary strand is
transcribed into a noncoding antisense RNA that hybridizes with the
target mRNA and interferes with its expression (Takayama and
Inouye, Crit. Rev. Biochem. Mol. Biol. 25:155-184 (1990), the
entirety of which is herein incorporated by reference). An
antisense vector is constructed by standard procedures and
introduced into cells by transformation, transfection,
electroporation, microinjection, infection, etc. The type of
transformation and choice of vector will determine whether
expression is transient or stable. The promoter used for the
antisense gene may influence the level, timing, tissue,
specificity, or inducibility of the antisense inhibition.
[0146] It is understood that the activity of a protein in a plant
cell may be reduced or depressed by growing a transformed plant
cell containing a nucleic acid molecule of the present invention
whose non-transcribed strand encodes a protein or fragment
thereof.
[0147] Antibodies have been expressed in plants (Hiatt et al.,
Nature 342:76-78 (1989), the entirety of which is herein
incorporated by reference; Conrad and Fielder, Plant Mol. Biol.
26:1023-1030 (1994), the entirety of which is herein incorporated
by reference). Cytoplasmic expression of a scFv (single-chain Fv
antibodies) has been reported to delay infection by artichoke
mottled crinkle virus. Transgenic plants that express antibodies
directed against endogenous proteins may exhibit a physiological
effect (Philips et al., EMBO J. 16:4489-4496 (1997), the entirety
of which is herein incorporated by reference; Marion-Poll, Trends
in Plant Science 2:447-448 (1997), the entirety of which is herein
incorporated by reference). For example, expressed anti-abscissic
antibodies have been reported to result in a general perturbation
of seed development (Philips et al., EMBO J. 16: 4489-4496
(1997)).
[0148] Antibodies that are catalytic may also be expressed in
plants (abzymes). The principle behind abzymes is that since
antibodies may be raised against many molecules, this recognition
ability can be directed toward generating antibodies that bind
transition states to force a chemical reaction forward (Persidas,
Nature Biotechnology 15:1313-1315 (1997), the entirety of which is
herein incorporated by reference; Baca et al., Ann. Rev. Biophys.
Biomol. Struct. 26:461-493 (1997), the entirety of which is herein
incorporated by reference). The catalytic abilities of abzymes may
be enhanced by site directed mutagenesis. Examples of abzymes are,
for example, set forth in U.S. Pat. No: 5,658,753; U.S. Pat. No.
5,632,990; U.S. Pat. No. 5,631,137; U.S. Pat. No. 5,602,015; U.S.
Pat. No. 5,559,538; U.S. Pat. No. 5,576,174; U.S. Pat. No.
5,500,358; U.S. Pat. No. 5,318,897; U.S. Pat. No. 5,298,409; U.S.
Pat. No. 5,258,289 and U.S. Pat. No. 5,194,585, all of which are
herein incorporated in their entirety.
[0149] It is understood that any of the antibodies of the invention
may be expressed in plants and that such expression can result in a
physiological effect. It is also understood that any of the
expressed antibodies may be catalytic.
(d) Antibodies
[0150] One aspect of the invention concerns antibodies,
single-chain antigen binding molecules, or other proteins that
specifically bind to one or more of the protein or peptide
molecules of the invention and their homologues, fusions or
fragments. In a preferred embodiment, an antibody of the present
invention binds to an amino acid selected from the group consisting
of SEQ ID NO: 622 through 625. Such antibodies may be used to
quantitatively or qualitatively detect the protein or peptide
molecules of the invention. As used herein, an antibody or peptide
is said to "specifically bind" to a protein or peptide molecule of
the invention if such binding is not competitively inhibited by the
presence of non-related molecules.
[0151] Nucleic acid molecules that encode all or part of the
protein of the invention can be expressed, via recombinant means,
to yield protein or peptides that can in turn be used to elicit
antibodies that are capable of binding the expressed protein or
peptide. Such antibodies may be used in immunoassays for that
protein. Such protein-encoding molecules, or their fragments may be
a "fusion" molecule (i.e., a part of a larger nucleic acid
molecule) such that, upon expression, a fusion protein is produced.
It is understood that any of the nucleic acid molecules of the
invention may be expressed, via recombinant means, to yield
proteins or peptides encoded by these nucleic acid molecules.
[0152] The antibodies that specifically bind proteins and protein
fragments of the invention may be polyclonal or monoclonal and may
comprise intact immunoglobulins, or antigen binding portions of
immunoglobulins fragments (such as (F(ab'), F(ab').sub.2), or
single-chain immunoglobulins producible, for example, via
recombinant means. It is understood that practitioners are familiar
with the standard resource materials which describe specific
conditions and procedures for the construction, manipulation and
isolation of antibodies (see, for example, Harlow and Lane, In:
Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold
Spring Harbor, N.Y. (1988), the entirety of which is herein
incorporated by reference).
[0153] Murine monoclonal antibodies are particularly preferred.
BALB/c mice are preferred for this purpose, however, equivalent
strains may also be used. The animals are preferably immunized with
approximately 25 .mu.g of purified protein (or fragment thereof)
that has been emulsified in a suitable adjuvant (such as TiterMax
adjuvant (Vaxcel, Norcross, Ga.)). Immunization is preferably
conducted at two intramuscular sites, one intraperitoneal site and
one subcutaneous site at the base of the tail. An additional i.v.
injection of approximately 25 .mu.g of antigen is preferably given
in normal saline three weeks later. After approximately 11 days
following the second injection, the mice may be bled and the blood
screened for the presence of anti-protein or peptide antibodies.
Preferably, a direct binding Enzyme-Linked Immunoassay (ELISA) is
employed for this purpose.
[0154] More preferably, the mouse having the highest antibody titer
is given a third i.v. injection of approximately 25 .mu.g of the
same protein or fragment. The splenic leukocytes from this animal
may be recovered 3 days later and then permitted to fuse, most
preferably, using polyethylene glycol, with cells of a suitable
myeloma cell line (such as, for example, the P3X63Ag8.653 myeloma
cell line). Hybridoma cells are selected by culturing the cells
under "HAT" (hypoxanthine-aminopterin-thy- mine) selection for
about one week. The resulting clones may then be screened for their
capacity to produce monoclonal antibodies ("mAbs"), preferably by
direct ELISA.
[0155] In one embodiment, anti-protein or peptide monoclonal
antibodies are isolated using a fusion of a protein or peptide of
the invention, or conjugate of a protein or peptide of the
invention, as immunogens. Thus, for example, a group of mice can be
immunized using a fusion protein emulsified in Freund's complete
adjuvant (e.g., approximately 50 .mu.g of antigen per
immunization). At three week intervals, an identical amount of
antigen is emulsified in Freund's incomplete adjuvant and used to
immunize the animals. Ten days following the third immunization,
serum samples are taken and evaluated for the presence of antibody.
If antibody titers are too low, a fourth booster can be employed.
Polysera capable of binding the protein or peptide can also be
obtained using this method.
[0156] In a preferred procedure for obtaining monoclonal
antibodies, the spleens of the above-described immunized mice are
removed, disrupted and immune splenocytes are isolated over a
ficoll gradient. The isolated splenocytes are fused, using
polyethylene glycol with BALB/c-derived HGPRT (hypoxanthine guanine
phosphoribosyl transferase) deficient P3x63xAg8.653 plasmacytoma
cells. The fused cells are plated into 96 well microtiter plates
and screened for hybridoma fusion cells by their capacity to grow
in culture medium supplemented with hypothanthine, aminopterin and
thymidine for approximately 2-3 weeks.
[0157] Hybridoma cells that arise from such incubation are
preferably screened for their capacity to produce an immunoglobulin
that binds to a protein of interest. An indirect ELISA may be used
for this purpose. In brief, the supernatants of hybridomas are
incubated in microtiter wells that contain immobilized protein.
After washing, the titer of bound immunoglobulin can be determined
using, for example, a goat anti-mouse antibody conjugated to
horseradish peroxidase. After additional washing, the amount of
immobilized enzyme is determined (for example through the use of a
chromogenic substrate). Such screening is performed as quickly as
possible after the identification of the hybridoma in order to
ensure that a desired clone is not overgrown by non-secreting
neighbor cells. Desirably, the fusion plates are screened several
times since the rates of hybridoma growth vary. In a preferred
sub-embodiment, a different antigenic form may be used to screen
the hybridoma. Thus, for example, the splenocytes may be immunized
with one immunogen, but the resulting hybridomas can be screened
using a different immunogen. It is understood that any of the
protein or peptide molecules of the invention may be used to raise
antibodies.
[0158] As discussed below, such antibody molecules or their
fragments may be used for diagnostic purposes. Where the antibodies
are intended for diagnostic purposes, it may be desirable to
derivatize them, for example with a ligand group (such as biotin)
or a detectable marker group (such as a fluorescent group, a
radioisotope or an enzyme).
[0159] The ability to produce antibodies that bind the protein or
peptide molecules of the invention permits the identification of
mimetic compounds derived from those molecules. These mimetic
compounds may contain a fragment of the protein or peptide or
merely a structurally similar region and nonetheless exhibits an
ability to specifically bind to antibodies directed against that
compound.
[0160] It is understood that any of the agents of the invention can
be substantially purified and/or be biologically active and/or
recombinant.
(e) Exemplary Uses
[0161] Nucleic acid molecules and fragments thereof of the
invention may be employed to obtain other nucleic acid molecules
from the same species (nucleic acid molecules from maize may be
utilized to obtain other nucleic acid molecules from maize). Such
nucleic acid molecules include the nucleic acid molecules that
encode the complete coding sequence of a protein and promoters and
flanking sequences of such molecules. In addition, such nucleic
acid molecules include nucleic acid molecules that encode for other
isozymes or gene family members. Such molecules can be readily
obtained by using the above-described nucleic acid molecules or
fragments thereof to screen cDNA or genomic libraries. Methods for
forming such libraries are well known in the art.
[0162] Nucleic acid molecules and fragments thereof of the
invention may also be employed to obtain nucleic acid homologs.
Such homologs include the nucleic acid molecule of other plants or
other organisms (e.g., alfalfa, Arabidopsis, barley, Brassica,
broccoli, cabbage, citrus, cotton, garlic, oat, oilseed rape,
onion, canola, flax, an ornamental plant, pea, peanut, pepper,
potato, rice, rye, sorghum, strawberry, sugarcane, sugarbeet,
tomato, wheat, poplar, pine, fir, eucalyptus, apple, lettuce,
lentils, grape, banana, tea, turf grasses, sunflower, oil palm,
Phaseolus, etc.) including the nucleic acid molecules that encode,
in whole or in part, protein homologs of other plant species or
other organisms, sequences of genetic elements, such as promoters
and transcriptional regulatory elements. Particularly preferred
plants are selected from the group consisting of maize, canola,
soybean, crambe, mustard, castor bean, peanut, sesame, cottonseed,
linseed, safflower, oil palm, flax and sunflower.
[0163] Such molecules can be readily obtained by using the
above-described nucleic acid molecules or fragments thereof to
screen cDNA or genomic libraries obtained from such plant species.
Methods for forming such libraries are well known in the art. Such
homolog molecules may differ in their nucleotide sequences from
those found in one or more of SEQ ID NOs: 1-4, 6-29 or complements
thereof because complete complementarity is not needed for stable
hybridization. The nucleic acid molecules of the invention
therefore also include molecules that, although capable of
specifically hybridizing with the nucleic acid molecules may lack
"complete complementarity."
[0164] Any of a variety of methods may be used to obtain one or
more of the above-described nucleic acid molecules (Zamechik et
al., Proc. Natl. Acad. Sci. (U.S.A.) 83:4143-4146 (1986); Goodchild
et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:5507-5511 (1988);
Wickstrom et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:1028-1032
(1988); Holt et al., Molec. Cell. Biol. 8:963-973 (1988); Gerwirtz
et al., Science 242:1303-1306 (1988); Anfossi et al., Proc. Natl.
Acad. Sci. (U.S.A.) 86:3379-3383 (1989); Becker et al., EMBO J.
8:3685-3691 (1989)). Automated nucleic acid synthesizers may be
employed for this purpose. In lieu of such synthesis, the disclosed
nucleic acid molecules may be used to define a pair of primers that
can be used with the polymerase chain reaction (Mullis et al., Cold
Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich et al.,
European Patent 50,424; European Patent 84,796; European Patent
258,017; European Patent 237,362; Mullis, European Patent 201,184;
Mullis et al., U.S. Pat. No. 4,683,202; Erlich, U.S. Pat. No.
4,582,788; and Saiki et al., U.S. Pat. No. 4,683,194) to amplify
and obtain any desired nucleic acid molecule or fragment.
[0165] Promoter sequences and other genetic elements, including but
not limited to transcriptional regulatory flanking sequences,
associated with one or more of the disclosed nucleic acid sequences
can also be obtained using the disclosed nucleic acid sequence
provided herein. In one embodiment, such sequences are obtained by
incubating nucleic acid molecules of the present invention with
members of genomic libraries and recovering clones that hybridize
to such nucleic acid molecules thereof. In a second embodiment,
methods of "chromosome walking," or inverse PCR may be used to
obtain such sequences (Frohman et al., Proc. Natl. Acad. Sci.
(U.S.A.) 85:8998-9002 (1988); Ohara et al., Proc. Natl. Acad.
Sci.(U.S.A.) 86:5673-5677 (1989); Pang et al., Biotechniques
22:1046-1048 (1977); Huang et al., Methods Mol. Biol. 69:89-96
(1997); Huang et al., Method Mol. Biol. 67:287-294 (1997); Benkel
et al., Genet. Anal. 13:123-127 (1996); Hartl et al., Methods Mol.
Biol. 58:293-301 (1996)). The term "chromosome walking" means a
process of extending a genetic map by successive hybridization
steps.
[0166] The nucleic acid molecules of the invention may be used to
isolate promoters of cell enhanced, cell specific, tissue enhanced,
tissue specific, developmentally or environmentally regulated
expression profiles. Isolation and functional analysis of the 5'
flanking promoter sequences of these genes from genomic libraries,
for example, using genomic screening methods and PCR techniques
would result in the isolation of useful promoters and
transcriptional regulatory elements. These methods are known to
those of skill in the art and have been described (See, for
example, Birren et al., Genome Analysis: Analyzing DNA, 1, (1997),
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Promoters obtained utilizing the nucleic acid molecules of the
invention could also be modified to affect their control
characteristics. Examples of such modifications would include but
are not limited to enhancer sequences. Such genetic elements could
be used to enhance gene expression of new and existing traits for
crop improvement.
[0167] Another subset of the nucleic acid molecules of the
invention includes nucleic acid molecules that are markers. The
markers can be used in a number of conventional ways in the field
of molecular genetics. Such markers include nucleic acid molecules
SEQ ID NOs: 1-4, 6-29 or complements thereof or fragments of either
that can act as markers and other nucleic acid molecules of the
present invention that can act as markers.
[0168] Genetic markers of the invention include "dominant" or
"codominant" markers. "Codominant markers" reveal the presence of
two or more alleles (two per diploid individual) at a locus.
"Dominant markers" reveal the presence of only a single allele per
locus. The presence of the dominant marker phenotype (e.g., a band
of DNA) is an indication that one allele is in either the
homozygous or heterozygous condition. The absence of the dominant
marker phenotype (e.g., absence of a DNA band) is merely evidence
that "some other" undefined allele is present. In the case of
populations where individuals are predominantly homozygous and loci
are predominately dimorphic, dominant and codominant markers can be
equally valuable. As populations become more heterozygous and
multi-allelic, codominant markers often become more informative of
the genotype than dominant markers. Marker molecules can be, for
example, capable of detecting polymorphisms such as single
nucleotide polymorphisms (SNPs).
[0169] The genomes of animals and plants naturally undergo
spontaneous mutation in the course of their continuing evolution
(Gusella, Ann. Rev. Biochem. 55:831-854 (1986)). A "polymorphism"
is a variation or difference in the sequence of the gene or its
flanking regions that arises in some of the members of a species.
The variant sequence and the "original" sequence co-exist in the
species' population. In some instances, such co-existence is in
stable or quasi-stable equilibrium.
[0170] A polymorphism is thus said to be "allelic," in that, due to
the existence of the polymorphism, some members of a species may
have the original sequence (i.e., the original "allele") whereas
other members may have the variant sequence (i.e., the variant
"allele"). In the simplest case, only one variant sequence may
exist and the polymorphism is thus said to be di-allelic. In other
cases, the species' population may contain multiple alleles and the
polymorphism is termed tri-allelic, etc. A single gene may have
multiple different unrelated polymorphisms. For example, it may
have a di-allelic polymorphism at one site and a multi-allelic
polymorphism at another site.
[0171] The variation that defines the polymorphism may range from a
single nucleotide variation to the insertion or deletion of
extended regions within a gene. In some cases, the DNA sequence
variations are in regions of the genome that are characterized by
short tandem repeats (STRs) that include tandem di- or
tri-nucleotide repeated motifs of nucleotides. Polymorphisms
characterized by such tandem repeats are referred to as "variable
number tandem repeat" ("VNTR") polymorphisms. VNTRs have been used
in identity analysis (Weber, U.S. Pat. No. 5,075,217; Armour et
al., FEBS Lett. 307:113-115 (1992); Jones et al., Eur. J. Haematol.
39:144-147 (1987); Horn et al., PCT Patent Application WO91/14003;
Jeffreys, European Patent Application 370,719; Jeffreys, U.S. Pat.
No. 5,175,082; Jeffreys et al., Amer. J. Hum. Genet. 39:11-24
(1986); Jeffreys et al., Nature 316:76-79 (1985); Gray et al.,
Proc. R. Acad. Soc. Lond. 243:241-253 (1991); Moore et al.,
Genomics 10:654-660 (1991); Jeffreys et al., Anim. Genet. 18:1-15
(1987); Hillel et al., Anim. Genet. 20:145-155 (1989); Hillel et
al., Genet. 124:783-789 (1990)).
[0172] The detection of polymorphic sites in a sample of DNA may be
facilitated through the use of nucleic acid amplification methods.
Such methods specifically increase the concentration of
polynucleotides that span the polymorphic site, or include that
site and sequences located either distal or proximal to it. Such
amplified molecules can be readily detected by gel electrophoresis
or other means.
[0173] In an alternative embodiment, such polymorphisms can be
detected through the use of a marker nucleic acid molecule that is
physically linked to such polymorphism(s). For this purpose, marker
nucleic acid molecules comprising a nucleotide sequence of a
polynucleotide located within 1 mb of the polymorphism(s) and more
preferably within 100 kb of the polymorphism(s) and most preferably
within 10 kb of the polymorphism(s) can be employed.
[0174] The identification of a polymorphism can be determined in a
variety of ways. By correlating the presence or absence of it in a
plant with the presence or absence of a phenotype, it is possible
to predict the phenotype of that plant. If a polymorphism creates
or destroys a restriction endonuclease cleavage site, or if it
results in the loss or insertion of DNA (e.g., a VNTR
polymorphism), it will alter the size or profile of the DNA
fragments that are generated by digestion with that restriction
endonuclease. As such, organisms that possess a variant sequence
can be distinguished from those having the original sequence by
restriction fragment analysis. Polymorphisms that can be identified
in this manner are termed "restriction fragment length
polymorphisms" ("RFLPs") (Glassberg, UK Patent Application 2135774;
Skolnick et al., Cytogen. Cell Genet. 32:58-67 (1982); Botstein et
al., Ann. J. Hum. Genet. 32:314-331 (1980); Fischer et al., (PCT
Application WO90/13668; Uhlen, PCT Application WO90/11369).
[0175] Polymorphisms can also be identified by Single Strand
Conformation Polymorphism (SSCP) analysis (Elles, Methods in
Molecular Medicine: Molecular Diagnosis of Genetic Diseases, Humana
Press (1996)); Orita et al., Genomics 5:874-879 (1989)). A number
of protocols have been described for SSCP including, but not
limited to, Lee et al., Anal. Biochem. 205:289-293 (1992); Suzuki
et al., Anal. Biochem. 192:82-84 (1991); Lo et al., Nucleic Acids
Research 20:1005-1009 (1992); Sarkar et al., Genomics 13:441-443
(1992). It is understood that one or more of the nucleic acids of
the invention, may be utilized as markers or probes to detect
polymorphisms by SSCP analysis.
[0176] Polymorphisms may also be found using a DNA fingerprinting
technique called amplified fragment length polymorphism (AFLP),
which is based on the selective PCR amplification of restriction
fragments from a total digest of genomic DNA to profile that DNA
(Vos et al., Nucleic Acids Res. 23:4407-4414 (1995)). This method
allows for the specific co-amplification of high numbers of
restriction fragments, which can be visualized by PCR without
knowledge of the nucleic acid sequence. It is understood that one
or more of the nucleic acids of the invention may be utilized as
markers or probes to detect polymorphisms by AFLP analysis or for
fingerprinting RNA.
[0177] Polymorphisms may also be found using random amplified
polymorphic DNA (RAPD) (Williams et al., Nucl. Acids Res.
18:6531-6535 (1990)) and cleaveable amplified polymorphic sequences
(CAPS) (Lyamichev et al., Science 260:778-783 (1993)). It is
understood that one or more of the nucleic acid molecules of the
invention, may be utilized as markers or probes to detect
polymorphisms by RAPD or CAPS analysis.
[0178] Single Nucleotide Polymorphisms (SNPs) generally occur at
greater frequency than other polymorphic markers and are spaced
with a greater uniformity throughout a genome than other reported
forms of polymorphism. The greater frequency and uniformity of SNPs
means that there is greater probability that such a polymorphism
will be found near or in a genetic locus of interest than would be
the case for other polymorphisms. SNPs are located in
protein-coding regions and noncoding regions of a genome. Some of
these SNPs may result in defective or variant protein expression
(e.g., as a result of mutations or defective splicing). Analysis
(genotyping) of characterized SNPs can require only a plus/minus
assay rather than a lengthy measurement, permitting easier
automation.
[0179] SNPs can be characterized using any of a variety of methods.
Such methods include the direct or indirect sequencing of the site,
the use of restriction enzymes (Botstein et al., Am. J. Hum. Genet.
32:314-331 (1980), the entirety of which is herein incorporated
reference; Konieczny and Ausubel, Plant J. 4:403-410 (1993), the
entirety of which is herein incorporated by reference), enzymatic
and chemical mismatch assays (Myers et al., Nature 313:495-498
(1985), the entirety of which is herein incorporated by reference),
allele-specific PCR (Newton et al., Nucl. Acids Res. 17:2503-2516
(1989), the entirety of which is herein incorporated by reference;
Wu et al., Proc. Natl. Acad. Sci. USA 86:2757-2760 (1989), the
entirety of which is herein incorporated by reference), ligase
chain reaction (Barany, Proc. Natl. Acad. Sci. USA 88:189-193
(1991), the entirety of which is herein incorporated by reference),
single-strand conformation polymorphism analysis (Labrune et al.,
Am. J. Hum. Genet. 48: 1115-1120 (1991), the entirety of which is
herein incorporated by reference), single base primer extension
(Kuppuswamy et al., Proc. Natl. Acad. Sci. USA 88:1143-1147 (1991),
Goelet U.S. Pat. No. 6,004,744; Goelet U.S. Pat. No. 5,888,819; all
of which are herein incorporated by reference in their entirety ),
solid-phase ELISA-based oligonucleotide ligation assays (Nikiforov
et al., Nucl. Acids Res. 22:4167-4175 (1994), dideoxy
fingerprinting (Sarkar et al., Genomics 13:441-443 (1992), the
entirety of which is herein incorporated by reference),
oligonucleotide fluorescence-quenching assays (Livak et al., PCR
Methods Appl. 4:357-362 (1995a), the entirety of which is herein
incorporated by reference), 5'-nuclease allele-specific
hybridization TaqMan.TM. assay (Livak et al., Nature Genet.
9:341-342 (1995), the entirety of which is herein incorporated by
reference), template-directed dye-terminator incorporation (TDI)
assay (Chen and Kwok, Nucl. Acids Res. 25:347-353 (1997), the
entirety of which is herein incorporated by reference),
allele-specific molecular beacon assay (Tyagi et al., Nature
Biotech. 16: 49-53 (1998), the entirety of which is herein
incorporated by reference), PinPoint assay (Haff and Smirnov,
Genome Res. 7: 378-388 (1997), the entirety of which is herein
incorporated by reference), dCAPS analysis (Neff et al., Plant J.
14:387-392 (1998), the entirety of which is herein incorporated by
reference), pyrosequencing (Ronaghi et al, Analytical Biochemistry
267:65-71 (1999); Ronaghi et al PCT application WO 98/13523; Nyren
et al PCT application WO 98/28440, all of which are herein
incorporated by reference in their entirety;
http//www.pyrosequencing.com), using mass spectrometry, e.g. the
Masscode.TM. system (Howbert et al WO 99/05319; Howber et al WO
97/27331, all of which are herein incorporated by reference in
their entirety; http//www.rapigene.com; Becker et al PCT
application WO 98/26095; Becker et al PCT application; WO 98/12355;
Becker et al PCT application WO 97/33000; Monforte et al U.S. Pat.
No. 5,965,363, all of which are herein incorporated by reference in
their entirety), invasive cleavage of oligonucleotide probes
(Lyamichev et al Nature Biotechnology 17:292-296, herein
incorporated by reference in its entirety; http//www.twt.com), and
using high density oligonucleotide arrays (Hacia et al Nature
Genetics 22:164-167; herein incorporated by reference in its
entirety; http//www.affymetrix.com).
[0180] Polymorphisms may also be detected using allele-specific
oligonucleotides (ASO), which, can be for example, used in
combination with hybridization based technology including southern,
northern, and dot blot hybridizations, reverse dot blot
hybridizations and hybridizations performed on microarray and
related technology.
[0181] The stringency of hybridization for polymorphism detection
is highly dependent upon a variety of factors, including length of
the allele-specific oligonucleotide, sequence composition, degree
of complementarity (i.e. presence or absence of base mismatches),
concentration of salts and other factors such as formamide, and
temperature. These factors are important both during the
hybridization itself and during subsequent washes performed to
remove target polynucleotide that is not specifically hybridized.
-In practice, the conditions of the final, most stringent wash are
most critical. In addition, the amount of target polynucleotide
that is able to hybridize to the allele-specific oligonucleotide is
also governed by such factors as the concentration of both the ASO
and the target polynucleotide, the presence and concentration of
factors that act to "tie up" water molecules, so as to effectively
concentrate the reagents (e.g., PEG, dextran, dextran sulfate,
etc.), whether the nucleic acids are immobilized or in solution,
and the duration of hybridization and washing steps.
[0182] Hybridizations are preferably performed below the melting
temperature (T.sub.m) of the ASO. The closer the hybridization
and/or washing step is to the T.sub.m, the higher the stringency.
T.sub.m for an oligonucleotide may be approximated, for example,
according to the following formula:
T.sub.m=81.5+16.6.times.(log 10[Na+])+0.41.times.(% G+C)-675/n;
where [Na+] is the molar salt
[0183] concentration of Na+ or any other suitable cation and
n=number of bases in the oligonucleotide. Other formulas for
approximating T.sub.m are available and are known to those of
ordinary skill in the art.
[0184] Stringency is preferably adjusted so as to allow a given ASO
to differentially hybridize to a target polynucleotide of the
correct allele and a target polynucleotide of the incorrect allele.
Preferably, there will be at least a two-fold differential between
the signal produced by the ASO hybridizing to a target
polynucleotide of the correct allele and the level of the signal
produced by the ASO cross-hybridizing to a target polynucleotide of
the incorrect allele (e.g., an ASO specific for a mutant allele
cross-hybridizing to a wild-type allele). In more preferred
embodiments of the present invention, there is at least a five-fold
signal differential. In highly preferred embodiments of the present
invention, there is at least an order of magnitude signal
differential between the ASO hybridizing to a target polynucleotide
of the correct allele and the level of the signal produced by the
ASO cross-hybridizing to a target polynucleotide of the incorrect
allele.
[0185] While certain methods for detecting polymorphisms are
described herein, other detection methodologies may be utilized.
For example, additional methodologies are known and set forth, in
Birren et al., Genome Analysis, 4:135-186, A Laboratory Manual.
Mapping Genomes, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1999); Maliga et al., Methods in Plant Molecular
Biology. A Laboratory Course Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1 995); Paterson, Biotechnology
Intelligence Unit: Genome Mapping in Plants, R.G. Landes Co.,
Georgetown, Tex., and Academic Press, San Diego, Calif. (1996); The
Maize Handbook, Freeling and Walbot, eds., Springer-Verlag, New
York, N.Y. (1994); Methods in Molecular Medicine: Molecular
Diagnosis of Genetic Diseases, Elles, ed., Humana Press, Totowa,
N.J. (1996); Clark, ed., Plant Molecular Biology: A Laboratory
Manual, Clark, ed., Springer-Verlag, Berlin, Germany (1997), all of
which are herein incorporated by reference in their entirety.
[0186] Requirements for marker-assisted selection in a plant
breeding program are: (1) the marker(s) should co-segregate or be
closely linked with the desired trait; (2) an efficient means of
screening large populations for the molecular marker(s) should be
available; and (3) the screening technique should have high
reproducibility across laboratories and preferably be economical to
use and be user-friendly.
[0187] The genetic linkage of marker molecules can be established
by a gene mapping model such as, without limitation, the flanking
marker model reported by Lander and Botstein, Genetics 121:185-199
(1989) and the interval mapping, based on maximum likelihood
methods described by Lander and Botstein, Genetics 121:185-199
(1989) and implemented in the software package MAPMAKER/QTL
(Lincoln and Lander, Mapping Genes Controlling Quantitative Traits
Using MAPMAKER/QTL, Whitehead Institute for Biomedical Research,
Massachusetts, (1990). Additional software includes Qgene, Version
2.23 (1996), Department of Plant Breeding and Biometry, 266 Emerson
Hall, Cornell University, Ithaca, N.Y.). Use of Qgene software is a
particularly preferred approach.
[0188] A maximum likelihood estimate (MLE) for the presence of a
marker is calculated, together with an MLE assuming no QTL effect,
to avoid false positives. A log.sub.10 of an odds ratio (LOD) is
then calculated as: LOD=log.sub.10 (MLE for the presence of a
QTL/MLE given no linked QTL).
[0189] The LOD score essentially indicates how much more likely the
data are to have arisen assuming the presence of a QTL than in its
absence. The LOD threshold value for avoiding a false positive with
a given confidence, say 95%, depends on the number of markers and
the length of the genome. Graphs indicating LOD thresholds are set
forth in Lander and Botstein, Genetics 121:185-199 (1989) and
further described by Ars and Moreno-Gonzlez, Plant Breeding,
Hayward et al., (eds.) Chapman & Hall, London, pp. 314-331
(1993).
[0190] In a preferred embodiment of the present invention the
nucleic acid marker exhibits a LOD score of greater than 2.0, more
preferably 2.5, even more preferably greater than 3.0 or 4.0 with
the trait or phenotype of interest. In a preferred embodiment, the
trait of interest is altered, preferably increased phytosterol
levels or compositions.
[0191] Additional models can be used. Many modifications and
alternative approaches to interval mapping have been reported,
including the use non-parametric methods (Kruglyak and Lander,
Genetics 139:1421-1428 (1995)). Multiple regression methods or
models can be also be used, in which the trait is regressed on a
large number of markers (Jansen, Biometrics in Plant Breeding, van
Oijen and Jansen (eds.), Proceedings of the Ninth Meeting of the
Eucarpia Section Biometrics in Plant Breeding, The Netherlands, pp.
116-124 (1994); Weber and Wricke, Advances in Plant Breeding,
Blackwell, Berlin, 16 (1994)). Procedures combining interval
mapping with regression analysis, whereby the phenotype is
regressed onto a single putative QTL at a given marker interval and
at the same time onto a number of markers that serve as
`cofactors,` have been reported by Jansen and Stam, Genetics
136:1447-1455 (1994), and Zeng, Genetics 136:1457-1468 (1994).
Generally, the use of cofactors reduces the bias and sampling error
of the estimated QTL positions (Utz and Melchinger, Biometrics in
Plant Breeding, van Oijen and Jansen (eds.) Proceedings of the
Ninth Meeting of the Eucarpia Section Biometrics in Plant Breeding,
The Netherlands, pp. 195-204 (1994), thereby improving the
precision and efficiency of QTL mapping (Zeng, Genetics
136:1457-1468 (1994), herein incorporated by reference in its
entirety). These models can be extended to multi-environment
experiments to analyze genotype-environment interactions (Jansen et
al., Theo. Appl. Genet. 91:33-37 (1995), herein incorporated by
reference in its entirety).
[0192] It is understood that one or more of the nucleic acid
molecules of the invention may be used as molecular markers. It is
also understood that one or more of the protein molecules of the
invention may be used as molecular markers.
[0193] In a preferred embodiment, the polymorphism is present and
screened for in a mapping population, e.g. a collection of plants
capable of being used with markers such as polymorphic markers to
map genetic position of traits. The choice of appropriate mapping
population often depends on the type of marker systems employed
(Tanksley et al., J.P. Gustafson and R. Appels (eds.). Plenum
Press, New York, pp. 157-173 (1988), the entirety of which is
herein incorporated by reference). Consideration must be given to
the source of parents (adapted vs. exotic) used in the mapping
population. Chromosome pairing and recombination rates can be
severely disturbed (suppressed) in wide crosses (adapted x exotic)
and generally yield greatly reduced linkage distances. Wide crosses
will usually provide segregating populations with a relatively
large number of polymorphisms when compared to progeny in a narrow
cross (adapted x adapted).
[0194] An F.sub.2 population is the first generation of selfing
(self-pollinating) after the hybrid seed is produced. Usually a
single F.sub.1 plant is selfed to generate a population segregating
for all the genes in Mendelian (1:2:1) pattern. Maximum genetic
information is obtained from a completely classified F.sub.2
population using a codominant marker system (Mather, Measurement of
Linkage in Heredity: Methuen and Co., (1938), the entirety of which
is herein incorporated by reference). In the case of dominant
markers, progeny tests (e.g., F.sub.3, BCF.sub.2) are required to
identify the heterozygotes, in order to classify the population.
However, this procedure is often prohibitive because of the cost
and time involved in progeny testing. Progeny testing of F.sub.2
individuals is often used in map construction where phenotypes do
not consistently reflect genotype (e.g. disease resistance) or
where trait expression is controlled by a QTL. Segregation data
from progeny test populations e.g. F.sub.3 or BCF.sub.2) can be
used in map construction. Marker-assisted selection can then be
applied to cross progeny based on marker-trait map associations
(F.sub.2, F.sub.3), where linkage groups have not been completely
disassociated by recombination events (i.e., maximum
disequilibrium).
[0195] Recombinant inbred lines (RIL) (genetically related lines;
usually >F.sub.5, developed from continuously selfing F.sub.2
lines towards homozygosity) can be used as a mapping population.
Information obtained from dominant markers can be maximized by
using RIL because all loci are homozygous or nearly so. Under
conditions of tight linkage (i.e., about <10% recombination),
dominant and co-dominant markers evaluated in RIL populations
provide more information per individual than either marker type in
backcross populations (Reiter. Proc. Natl. Acad. Sci. (U.S.A.)
89:1477-1481 (1992), the entirety of which is herein incorporated
by reference). However, as the distance between markers becomes
larger (i.e., loci become more independent), the information in RIL
populations decreases dramatically when compared to codominant
markers.
[0196] Backcross populations (e.g., generated from a cross between
a successful variety (recurrent parent) and another variety (donor
parent) carrying a trait not present in the former) can be utilized
as a mapping population. A series of backcrosses to the recurrent
parent can be made to recover most of its desirable traits. Thus a
population is created consisting of individuals nearly like the
recurrent parent but each individual carries varying amounts or
mosaic of genomic regions from the donor parent. Backcross
populations can be useful for mapping dominant markers if all loci
in the recurrent parent are homozygous and the donor and recurrent
parent have contrasting polymorphic marker alleles (Reiter et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 89:1477-1481 (1992), the entirety
of which is herein incorporated by reference). Information obtained
from backcross populations using either codominant or dominant
markers is less than that obtained from F.sub.2 populations because
one, rather than two, recombinant gamete is sampled per plant.
Backcross populations, however, are more informative (at low marker
saturation) when compared to RILs as the distance between linked
loci increases in RIL populations (i.e. about 0.15% recombination).
Increased recombination can be beneficial for resolution of tight
linkages, but may be undesirable in the construction of maps with
low marker saturation.
[0197] Near-isogenic lines (NIL) (created by many backcrosses to
produce a collection of individuals that is nearly identical in
genetic composition except for the trait or genomic region under
interrogation) can be used as a mapping population. In mapping with
NILs, only a portion of the polymorphic loci is expected to map to
a selected region.
[0198] Bulk segregant analysis (BSA) is a method developed for the
rapid identification of linkage between markers and traits of
interest (Michelmore et al., Proc. Natl. Acad. Sci. U.S.A.
88:9828-9832 (1991), the entirety of which is herein incorporated
by reference). In BSA, two bulked DNA samples are drawn from a
segregating population originating from a single cross. These bulks
contain individuals that are identical for a particular trait
(resistant or susceptible to particular disease) or genomic region
but arbitrary at unlinked regions (i.e. heterozygous). Regions
unlinked to the target region will not differ between the bulked
samples of many individuals in BSA.
[0199] In an aspect of the present invention, one or more of the
nucleic molecules of the present invention are used to determine
the level (i.e., the concentration of mRNA in a sample, etc.) in a
plant (preferably maize, canola, soybean, crambe, mustard, castor
bean, peanut, sesame, cottonseed, linseed, safflower, oil palm,
flax or sunflower) or pattern (i.e., the kinetics of expression,
rate of decomposition, stability profile, etc.) of the expression
of a protein encoded in part or whole by one or more of the nucleic
acid molecule of the present invention (collectively, the
"Expression Response" of a cell or tissue).
[0200] As used herein, the Expression Response manifested by a cell
or tissue is said to be "altered" if it differs from the Expression
Response of cells or tissues of plants not exhibiting the
phenotype. To determine whether a Expression Response is altered,
the Expression Response manifested by the cell or tissue of the
plant exhibiting the phenotype is compared with that of a similar
cell or tissue sample of a plant not exhibiting the phenotype. As
will be appreciated, it is not necessary to re-determine the
Expression Response of the cell or tissue sample of plants not
exhibiting the phenotype each time such a comparison is made;
rather, the Expression Response of a particular plant may be
compared with previously obtained values of normal plants. As used
herein, the phenotype of the organism is any of one or more
characteristics of an organism (e.g. disease resistance, pest
tolerance, environmental tolerance such as tolerance to abiotic
stress, male sterility, quality improvement or yield etc.). A
change in genotype or phenotype may be transient or permanent. Also
as used herein, a tissue sample is any sample that comprises more
than one cell. In a preferred aspect, a tissue sample comprises
cells that share a common characteristic (e.g. derived from root,
seed, flower, leaf, stem or pollen etc.).
[0201] In one aspect of the present invention, an evaluation can be
conducted to determine whether a particular mRNA molecule is
present. One or more of the nucleic acid molecules of the present
invention are utilized to detect the presence or quantity of the
mRNA species. Such molecules are then incubated with cell or tissue
extracts of a plant under conditions sufficient to permit nucleic
acid hybridization. The detection of double-stranded probe-mRNA
hybrid molecules is indicative of the presence of the mRNA; the
amount of such hybrid formed is proportional to the amount of mRNA.
Thus, such probes may be used to ascertain the level and extent of
the mRNA production in a plant's cells or tissues. Such nucleic
acid hybridization may be conducted under quantitative conditions
(thereby providing a numerical value of the amount of the mRNA
present). Alternatively, the assay may be conducted as a
qualitative assay that indicates either that the mRNA is present,
or that its level exceeds a user set, predefined value.
[0202] A number of methods can be used to compare the expression
response between two or more samples of cells or tissue. These
methods include hybridization assays, such as northerns, RNAse
protection assays, and in situ hybridization. Alternatively, the
methods include PCR-type assays. In a preferred method, the
expression response is compared by hybridizing nucleic acids from
the two or more samples to an array of nucleic acids. The array
contains a plurality of suspected sequences known or suspected of
being present in the cells or tissue of the samples.
[0203] An advantage of in situ hybridization over more conventional
techniques for the detection of nucleic acids is that it allows an
investigator to determine the precise spatial population (Angerer
et al., Dev. Biol. 101:477-484 (1984); Angerer et al., Dev. Biol.
112:157-166 (1985); Dixon et al., EMBO J. 10:1317-1324 (1991)). In
situ hybridization may be used to measure the steady-state level of
RNA accumulation (Hardin et al., J. Mol. Biol. 202:417-431 (1989)).
A number of protocols have been devised for in situ hybridization,
each with tissue preparation, hybridization and washing conditions
(Meyerowitz, Plant Mol. Biol. Rep. 5:242-250 (1987); Cox and
Goldberg, In: Plant Molecular Biology: A Practical Approach, Shaw
(ed.), pp. 1-35, IRL Press, Oxford (1988); Raikhel et al., In situ
RNA hybridization in plant tissues, In: Plant Molecular Biology
Manual, vol. B9:1-32, Kluwer Academic Publisher, Dordrecht, Belgium
(1989)).
[0204] In situ hybridization also allows for the localization of
proteins within a tissue or cell (Wilkinson, In Situ Hybridization,
Oxford University Press, Oxford (1992); Langdale, In Situ
Hybridization In: The Maize Handbook, Freeling and Walbot (eds.),
pp. 165-179, Springer-Verlag, New York (1994)). It is understood
that one or more of the molecules of the invention, preferably one
or more of the nucleic acid molecules or fragments thereof of the
invention or one or more of the antibodies of the invention may be
utilized to detect the level or pattern of a protein or mRNA
thereof by in situ hybridization.
[0205] Fluorescent in situ hybridization allows the localization of
a particular DNA sequence along a chromosome, which is useful,
among other uses, for gene mapping, following chromosomes in hybrid
lines, or detecting chromosomes with translocations, transversions
or deletions. In situ hybridization has been used to identify
chromosomes in several plant species (Griffor et al., Plant Mol.
Biol. 17:101-109 (1991); Gustafson et al., Proc. Natl. Acad. Sci.
(U.S.A.) 87:1899-1902 (1990); Mukai and Gill, Genome 34:448-452
(1991); Schwarzacher and Heslop-Harrison, Genome 34:317-323 (1991);
Wang et al., Jpn. J. Genet. 66:313-316 (1991); Parra and Windle,
Nature Genetics 5:17-21 (1993)). It is understood that the nucleic
acid molecules of the invention may be used as probes or markers to
localize sequences along a chromosome.
[0206] Another method to localize the expression of a molecule is
tissue printing. Tissue printing provides a way to screen, at the
same time on the same membrane many tissue sections from different
plants or different developmental stages (Yomo and Taylor, Planta
112:35-43 (1973); Harris and Chrispeels, Plant Physiol. 56:292-299
(1975); Cassab and Varner, J. Cell. Biol. 105:2581-2588 (1987);
Spruce et al., Phytochemistry 26:2901-2903 (1987); Barres et al.,
Neuron 5:527-544 (1990); Reid and Pont-Lezica, Tissue Printing:
Tools for the Study of Anatomy, Histochemistry and Gene Expression,
Academic Press, New York, N.Y. (1992); Reid et al., Plant Physiol.
93:160-165 (1990); Ye et al., Plant J. 1:175-183 (1991)).
[0207] A microarray-based method for high-throughput monitoring of
gene expression may be utilized to measure expression response.
This `chip`-based approach involves microarrays of nucleic acid
molecules as gene-specific hybridization targets to quantitatively
measure expression of the corresponding mRNA (Schena et al.,
Science 270:467-470 (1995), the entirety of which is herein
incorporated by reference;
http://cmgm.stanford.edu/pbrown/array.html; Shalon, Ph.D. Thesis,
Stanford University (1996), the entirety of which is herein
incorporated by reference). Hybridization to a microarray can be
used to efficiently analyze the presence and/or amount of a number
of nucleotide sequences simultaneously.
[0208] Several microarray methods have been described. One method
compares the sequences to be analyzed by hybridization to a set of
oligonucleotides representing all possible subsequences (Bains and
Smith, J. Theor. Biol. 135:303-307 (1989), the entirety of which is
herein incorporated by reference). A second method hybridizes the
sample to an array of oligonucleotide or cDNA molecules. An array
consisting of oligonucleoties complementary to subsequences of a
target sequence can be used to determine the identity of a target
sequence, measure its amount, and detect single nucleotide
differences between the target and a reference sequence. Nucleic
acid molecule microarrays may also be screened with protein
molecules or fragments thereof to determine nucleic acid molecules
that specifically bind protein molecules or fragments thereof.
[0209] The microarray approach may be used with polypeptide targets
(U.S. Pat. No. 5,445,934; U.S. Pat. No: 5,143,854; U.S. Pat. No.
5,079,600; U.S. Pat. No. 4,923,901, all of which are herein
incorporated by reference in their entirety). Essentially,
polypeptides are synthesized on a substrate (microarray) and these
polypeptides can be screened with either protein molecules or
fragments thereof or nucleic acid molecules in order to screen for
either protein molecules or fragments thereof or nucleic acid
molecules that specifically bind the target polypeptides. (Fodor et
al., Science 251:767-773 (1991), the entirety of which is herein
incorporated by reference). It is understood that one or more of
the nucleic acid molecules or protein or fragments thereof of the
invention may be utilized in a microarray-based method.
[0210] In a preferred embodiment of the present invention
microarrays may be prepared that comprise nucleic acid molecules
where preferably at least 10%, preferably at least 25%, more
preferably at least 50% and even more preferably at least 75%, 80%,
85%, 90% or 95% of the nucleic acid molecules located on that array
are selected from the group of nucleic acid molecules that
specifically hybridize to one or more nucleic acid molecule having
a nucleic acid sequence selected from the group of SEQ ID NO: 1
through SEQ ID NO: 621 or complements thereof or fragments of
either.
[0211] In another preferred embodiment of the present invention
microarrays may be prepared that comprise nucleic acid molecules
where preferably at least 10%, preferably at least 25%, more
preferably at least 50% and even more preferably at least 75%, 80%,
85%, 90% or 95% of the nucleic acid molecules located on that array
are selected from the group of nucleic acid molecules having a
nucleic acid sequence selected from the group of SEQ ID NO: 1
through SEQ ID NO: 621 or complements thereof or fragments of
either.
[0212] In a preferred embodiment of the present invention
microarrays may be prepared that comprise nucleic acid molecules
where such nucleic acid molecules encode at least one, preferably
at least two, more preferably at least three, even more preferably
at least four, five or six proteins or fragments thereof selected
from the group consisting of HES1, HMGCoA reductase, squalene
synthase, cycloartenol synthase, SMTII and UPC2. In even more
preferred embodiment of the present invention microarrays may be
prepared that comprise nucleic acid molecules where such nucleic
acid molecules encode at least one, preferably at least two, more
preferably at least three, even more preferably at least four, five
or six proteins or fragments thereof selected from the group
consisting of a fungal, more preferably a yeast HES1, a plant, more
preferably a maize, soybean or Arabidopsis HES1, a plant, more
preferably a rubber or an Arabidopsis HMGCoA reductase, a plant,
more preferably an Arabidopsis squalene synthase, a plant, more
preferably an Arabidopsis cycloartenol synthase, a plant, more
preferably an Arabidopsis SMTII and a fungus , more preferably an
yeast UPC2.
[0213] Site directed mutagenesis may be utilized to modify nucleic
acid sequences, particularly as it is a technique that allows one
or more of the amino acids encoded by a nucleic acid molecule to be
altered (e.g., a threonine to be replaced by a methionine). At
least three basic methods for site directed mutagenesis can be
employed. These are cassette mutagenesis (Wells et al., Gene
34:315-323 (1985), the entirety of which is herein incorporated by
reference), primer extension (Gilliam et al., Gene 12:129-137
(1980), the entirety of which is herein incorporated by reference;
Zoller and Smith, Methods Enzymol. 100:468-500 (1983), the entirety
of which is herein incorporated by reference; Dalbadie-McFarland et
al., Proc. Natl. Acad. Sci. (U.S.A.) 79:6409-6413 (1982), the
entirety of which is herein incorporated by reference) and methods
based upon PCR (Scharf et al., Science 233:1076-1078 (1986), the
entirety of which is herein incorporated by reference; Higuchi et
al., Nucleic Acids Res. 16:7351-7367 (1988), the entirety of which
is herein incorporated by reference). Site directed mutagenesis
approaches are also described in U.S. Pat. No. 5,811,238, European
Patent 0 385 962, the entirety of which is herein incorporated by
reference; European Patent 0 359 472, the entirety of which is
herein incorporated by reference; and PCT Patent Application WO
93/07278, the entirety of which is herein incorporated by
reference.
[0214] Site directed mutagenesis strategies have been applied to
plants for both in vitro as well as in vivo site directed
mutagenesis (Lanz et al., J. Biol. Chem. 266:9971-9976 (1991), the
entirety of which is herein incorporated by reference; Kovgan and
Zhdanov, Biotekhnologiya 5:148-154, No. 207160n, Chemical Abstracts
110:225 (1989), the entirety of which is herein incorporated by
reference; Ge et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:4037-4041
(1989), the entirety of which is herein incorporated by reference;
Zhu et al., J. Biol. Chem. 271:18494-18498 (1996), the entirety of
which is herein incorporated by reference; Chu et al., Biochemistry
33:6150-6157 (1994), the entirety of which is herein incorporated
by reference; Small et al., EMBO J. 11:1291-1296 (1992), the
entirety of which is herein incorporated by reference; Cho et al.,
Mol. Biotechnol. 8:13-16 (1997), the entirety of which is herein
incorporated by reference; Kita et al., J. Biol. Chem.
271:26529-26535 (1996), the entirety of which is herein
incorporated by reference, Jin et al., Mol. Microbiol. 7:555-562
(1993), the entirety of which is herein incorporated by reference;
Hatfield and Vierstra, J. Biol. Chem. 267:14799-14803 (1992), the
entirety of which is herein incorporated by reference; Zhao et al.,
Biochemistry 31:5093-5099 (1992), the entirety of which is herein
incorporated by reference).
[0215] Any of the nucleic acid molecules of the invention may
either be modified by site directed mutagenesis or used as, for
example, nucleic acid molecules that are used to target other
nucleic acid molecules for modification. It is understood that
mutants with more than one altered nucleotide can be constructed
using techniques that practitioners are familiar with, such as
isolating restriction fragments and ligating such fragments into an
expression vector (see, for example, Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989)).
[0216] Sequence-specific DNA-binding proteins play a role in the
regulation of transcription. The isolation of recombinant cDNAs
encoding these proteins facilitates the biochemical analysis of
their structural and functional properties. Genes encoding such
DNA-binding proteins have been isolated using classical genetics
(Vollbrecht et al., Nature 350: 241-243 (1991), the entirety of
which is herein incorporated by reference) and molecular
biochemical approaches, including the screening of recombinant cDNA
libraries with antibodies (Landschulz et al., Genes Dev. 2:786-800
(1988), the entirety of which is herein incorporated by reference)
or DNA probes (Bodner et al., Cell 55:505-518 (1988), the entirety
of which is herein incorporated by reference). In addition, an in
situ screening procedure has been used and has facilitated the
isolation of sequence-specific DNA-binding proteins from various
plant species (Gilmartin et al., Plant Cell 4:839-849 (1992), the
entirety of which is herein incorporated by reference; Schindler et
al., EMBO J. 11:1261-1273 (1992), the entirety of which is herein
incorporated by reference). An in situ screening protocol does not
require the purification of the protein of interest (Vinson et al.,
Genes Dev. 2:801-806 (1988), the entirety of which is herein
incorporated by reference; Singh et al., Cell 52:415-423 (1988),
the entirety of which is herein incorporated by reference).
[0217] Two steps may be employed to characterize DNA-protein
interactions. The first is to identify sequence fragments that
interact with DNA-binding proteins, to titrate binding activity, to
determine the specificity of binding and to determine whether a
given DNA-binding activity can interact with related DNA sequences
(Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd
edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989)). Electrophoretic mobility-shift assay is a widely used
assay. The assay provides a rapid and sensitive method for
detecting DNA-binding proteins based on the observation that the
mobility of a DNA fragment through a nondenaturing, low-ionic
strength polyacrylamide gel is retarded upon association with a
DNA-binding protein (Fried and Crother, Nucleic Acids Res.
9:6505-6525 (1981), the entirety of which is herein incorporated by
reference). When one or more specific binding activities have been
identified, the exact sequence of the DNA bound by the protein may
be determined.
[0218] Several procedures for characterizing protein/DNA-binding
sites are used, including methylation and ethylation interference
assays (Maxam and Gilbert, Methods Enzymol. 65:499-560 (1980), the
entirety of which is herein incorporated by reference; Wissman and
Hillen, Methods Enzymol. 208:365-379 (1991), the entirety of which
is herein incorporated by reference), footprinting techniques
employing DNase I (Galas and Schmitz, Nucleic Acids Res.
5:3157-3170 (1978), the entirety of which is herein incorporated by
reference), 1,10-phenanthroline-copper ion methods (Sigman et al.,
Methods Enzymol. 208:414-433 (1991), the entirety of which is
herein incorporated by reference) and hydroxyl radicals methods
(Dixon et al., Methods Enzymol. 208:414-433 (1991), the entirety of
which is herein incorporated by reference). It is understood that
one or more of the nucleic acid molecules of the invention may be
utilized to identify a protein or fragment thereof that
specifically binds to a nucleic acid molecule of the invention. It
is also understood that one or more of the protein molecules or
fragments thereof of the invention may be utilized to identify a
nucleic acid molecule that specifically binds to it.
[0219] A two-hybrid system is based on the fact that proteins, such
as transcription factors that interact (physically) with one
another carry out many cellular functions. Two-hybrid systems have
been used to probe the function of new proteins (Chien et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 88:9578-9582 (1991) the entirety of
which is herein incorporated by reference; Durfee et al., Genes
Dev. 7:555-569 (1993) the entirety of which is herein incorporated
by reference; Choi et al., Cell 78:499-512 (1994), the entirety of
which is herein incorporated by reference; Kranz et al., Genes Dev.
8:313-327 (1994), the entirety of which is herein incorporated by
reference).
[0220] Interaction mating techniques have facilitated a number of
two-hybrid studies of protein-protein interaction. Interaction
mating has been used to examine interactions between small sets of
tens of proteins (Finley and Brent, Proc. Natl. Acad. Sci. (U.S.A.)
91:12098-12984 (1994), the entirety of which is herein incorporated
by reference), larger sets of hundreds of proteins (Bendixen et
al., Nucl. Acids Res. 22:1778-1779 (1994), the entirety of which is
herein incorporated by reference) and to comprehensively map
proteins encoded by a small genome (Bartel et al., Nature Genetics
12:72-77 (1996), the entirety of which is herein incorporated by
reference). This technique utilizes proteins fused to the
DNA-binding domain and proteins fused to the activation domain.
They are expressed in two different haploid yeast strains of
opposite mating type and the strains are mated to determine if the
two proteins interact. Mating occurs when haploid yeast strains
come into contact and result in the fusion of the two haploids into
a diploid yeast strain. An interaction can be determined by the
activation of a two-hybrid reporter gene in the diploid strain.
[0221] The CLONTECH laboratories, Inc. provides the MATCHMAKER
two-hybrid System kit (Cat. No. K1605-1) in which the sequences
encoding the two functional domains of the GAL4 transcriptional
activator, DNA binding domain and activation domain, are cloned
into two different shuttle/expression vectors (pGBT9 and pGAD424)
(Bartel et al. In Cellular Interactions in Development: A Practical
Approach, D. A. Hartley, ed., Oxford University Press, Oxford
153-179 (1993), the entirety of which is herein incorporated by
reference). The gene code for the target protein is cloned into the
pGBT9 to generate a hybrid of GAL4-DNA binding domain with a target
protein and the gene(s) encode for potentially interacting
protein(s) are cloned into the pGAD424 to create hybrid protein(s)
of GAL4-activation domain with potentially interacting protein or
with a collection of random proteins in a fusion library. The both
plasmids carrying hybrid proteins are cotransformed into one yeast
strain. Both hybrid proteins are targeted to the yeast nucleus by
nuclear localization signal. If the target protein and the
potentially interacting protein interact with each other, the GAL4
DNA binding domain and the GAL4 activation domain are brought to
proximity and proper function of the transcriptional activator unit
will be reconstituted resulting in transcription of reporter gene
(lacZ or HIS3). An advantage of this technique is that it reduces
the number of yeast transformations needed to test individual
interactions. It is understood that the protein-protein
interactions of protein or fragments thereof of the invention may
be investigated using the two-hybrid system and that any of the
nucleic acid molecules of the invention that encode such proteins
or fragments thereof may be used to transform yeast in the
two-hybrid system.
(f) Fungal Constructs and Fungal Transformants
[0222] The invention also relates to a fungal recombinant vector
comprising exogenous genetic material. The invention also relates
to a fungal cell comprising a fungal recombinant vector. The
invention also relates to methods for obtaining a recombinant
fungal host cell comprising introducing into a fungal host cell
exogenous genetic material.
[0223] Exogenous genetic material may be transferred into a fungal
cell. In a preferred embodiment the exogenous genetic material
includes a nucleic acid molecule of the present invention,
preferably a nucleic acid molecule having a sequence selected from
the group consisting of SEQ ID NO: 1 through SEQ ID NO: 621 or
complements thereof or fragments of either. Another preferred class
of exogenous genetic material are nucleic acid molecules that
encode a protein having an amino acid selected from the group
consisting of SEQ ID NO: 622 through SEQ ID NO: 626 or fragments
thereof.
[0224] The fungal recombinant vector may be any vector which can be
conveniently subjected to recombinant DNA procedures. The choice of
a vector will typically depend on the compatibility of the vector
with the fungal host cell into which the vector is to be
introduced. The vector may be a linear or a closed circular
plasmid. The vector system may be a single vector or plasmid or two
or more vectors or plasmids which together contain the total DNA to
be introduced into the genome of the fungal host.
[0225] The fungal vector may be an autonomously replicating vector,
i.e., a vector which exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into the fungal cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. This integration may be the result of
homologous or non-homologous recombination.
[0226] Integration of a vector or nucleic acid into the genome by
homologous recombination, regardless of the host being considered,
relies on the nucleic acid sequence of the vector. Typically, the
vector contains nucleic acid sequences for directing integration by
homologous recombination into the genome of the host. These nucleic
acid sequences enable the vector to be integrated into the host
cell genome at a precise location or locations in one or more
chromosomes. To increase the likelihood of integration at a precise
location, there should be preferably two nucleic acid sequences
that individually contain a sufficient number of nucleic acids,
preferably 400 bp to 1500 bp, more preferably 800 bp to 1000 bp,
which are highly homologous with the corresponding host cell target
sequence. This enhances the probability of homologous
recombination. These nucleic acid sequences may be any sequence
that is homologous with a host cell target sequence and,
furthermore, may or may not encode proteins.
[0227] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. Examples of origin of
replications for use in a yeast host cell are the 2 micron origin
of replication and the combination of CEN3 and ARS 1. Any origin of
replication may be used which is compatible with the fungal host
cell of choice.
[0228] The fungal vectors of the invention preferably contain one
or more selectable markers which permit easy selection of
transformed cells. A selectable marker is a gene the product of
which provides, for example biocide or viral resistance, resistance
to heavy metals, prototrophy to auxotrophs and the like. The
selectable marker may be selected from the group including, but not
limited to, amdS (acetamidase), argB (ornithine
carbamoyltransferase), bar (phosphinothricin acetyltransferase),
hygB (hygromycin phosphotransferase), niaD (nitrate reductase),
pyrG (orotidine-5'-phosphate decarboxylase) and sC (sulfate
adenyltransferase) and trpC (anthranilate synthase). Preferred for
use in an Aspergillus cell are the amdS and pyrG markers of
Aspergillus nidulans or Aspergillus oryzae and the bar marker of
Streptomyces hygroscopicus. Furthermore, selection may be
accomplished by co-transformation, e.g., as described in WO
91/17243, the entirety of which is herein incorporated by
reference. A nucleic acid sequence of the invention may be operably
linked to a suitable promoter sequence. The promoter sequence is a
nucleic acid sequence which is recognized by the fungal host cell
for expression of the nucleic acid sequence. The promoter sequence
contains transcription and translation control sequences which
mediate the expression of the protein or fragment thereof.
[0229] A promoter may be any nucleic acid sequence which shows
transcriptional activity in the fungal host cell of choice and may
be obtained from genes encoding polypeptides either homologous or
heterologous to the host cell. Examples of suitable promoters for
directing the transcription of a nucleic acid construct of the
invention in a filamentous fungal host are promoters obtained from
the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor
miehei aspartic proteinase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase and hybrids thereof. In a yeast host, a useful promoter
is the Saccharomyces cerevisiae enolase (eno-1) promoter.
Particularly preferred promoters are the TAKA amylase, NA2-tpi (a
hybrid of the promoters from the genes encoding Aspergillus niger
neutral alpha -amylase and Aspergillus oryzae triose phosphate
isomerase), glaA, Saccharomyces cerevisiae GAL 1 (galactokinase)
and Saccharomyces cerevisiae GPD (glyceraldehyde-3-phosphate
dehydrogenase) promoters.
[0230] A protein or fragment thereof encoding nucleic acid molecule
of the invention may also be operably linked to a terminator
sequence at its 3' terminus. The terminator sequence may be native
to the nucleic acid sequence encoding the protein or fragment
thereof or may be obtained from foreign sources. Any terminator
which is functional in the fungal host cell of choice may be used
in the invention, but particularly preferred terminators are
obtained from the genes encoding Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, Saccharomyces
cerevisiae cytochrome-c oxidase (CYC1) and Saccharomyces cerevisiae
enolase.
[0231] A protein or fragment thereof encoding nucleic acid molecule
of the invention may also be operably linked to a suitable leader
sequence. A leader sequence is a nontranslated region of a mRNA
which is important for translation by the fungal host. The leader
sequence is operably linked to the 5' terminus of the nucleic acid
sequence encoding the protein or fragment thereof. The leader
sequence may be native to the nucleic acid sequence encoding the
protein or fragment thereof or may be obtained from foreign
sources. Any leader sequence which is functional in the fungal host
cell of choice may be used in the invention, but particularly
preferred leaders are obtained from the genes encoding Aspergillus
oryzae TAKA amylase and Aspergillus oryzae triose phosphate
isomerase.
[0232] A polyadenylation sequence may also be operably linked to
the 3' terminus of the nucleic acid sequence of the invention. The
polyadenylation sequence is a sequence which when transcribed is
recognized by the fungal host to add polyadenosine residues to
transcribed mRNA. The polyadenylation sequence may be native to the
nucleic acid sequence encoding the protein or fragment thereof or
may be obtained from foreign sources. Any polyadenylation sequence
which is functional in the fungal host of choice may be used in the
invention, but particularly preferred polyadenylation sequences are
obtained from the genes encoding Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase and Saccharomyces
cerevisiae cytochrome-c oxidase (CYC1).
[0233] To avoid the necessity of disrupting the cell to obtain the
protein or fragment thereof and to minimize the amount of possible
degradation of the expressed protein or fragment thereof within the
cell, it is preferred that expression of the protein or fragment
thereof gives rise to a product secreted outside the cell. To this
end, a protein or fragment thereof of the invention may be linked
to a signal peptide linked to the amino terminus of the protein or
fragment thereof. A signal peptide is an amino acid sequence which
permits the secretion of the protein or fragment thereof from the
fungal host into the culture medium. The signal peptide may be
native to the protein or fragment thereof of the invention or may
be obtained from foreign sources. The 5' end of the coding sequence
of the nucleic acid sequence of the invention may inherently
contain a signal peptide coding region naturally linked in
translation reading frame with the segment of the coding region
which encodes the secreted protein or fragment thereof.
Alternatively, the 5' end of the coding sequence may contain a
signal peptide coding region which is foreign to that portion of
the coding sequence which encodes the secreted protein or fragment
thereof. The foreign signal peptide may be required where the
coding sequence does not normally contain a signal peptide coding
region. Alternatively, the foreign signal peptide may simply
replace the natural signal peptide to obtain enhanced secretion of
the desired protein or fragment thereof. The foreign signal peptide
coding region may be obtained from a glucoamylase or an amylase
gene from an Aspergillus species, a lipase or proteinase gene from
Rhizomucor miehei, the gene for the alpha-factor from Saccharomyces
cerevisiae, or the calf preprochymosin gene. An effective signal
peptide for fungal host cells is the Aspergillus oryzae TAKA
amylase signal, Aspergillus niger neutral amylase signal, the
Rhizomucor miehei aspartic proteinase signal, the Humicola
lanuginosus cellulase signal, or the Rhizomucor miehei lipase
signal. However, any signal peptide capable of permitting secretion
of the protein or fragment thereof in a fungal host of choice may
be used in the invention.
[0234] A protein or fragment thereof encoding nucleic acid molecule
of the invention may also be linked to a propeptide coding region.
A propeptide is an amino acid sequence found at the amino terminus
of aproprotein or proenzyme. Cleavage of the propeptide from the
proprotein yields a mature biochemically active protein. The
resulting polypeptide is known as a propolypeptide or proenzyme (or
a zymogen in some cases). Propolypeptides are generally inactive
and can be converted to mature active polypeptides by catalytic or
autocatalytic cleavage of the propeptide from the propolypeptide or
proenzyme. The propeptide coding region may be native to the
protein or fragment thereof or may be obtained from foreign
sources. The foreign propeptide coding region may be obtained from
the Saccharomyces cerevisiae alpha-factor gene or Myceliophthora
thermophila laccase gene (WO 95/33836, the entirety of which is
herein incorporated by reference).
[0235] The procedures used to ligate the elements described above
to construct the recombinant expression vector of the invention are
well known to one skilled in the art (see, for example, Sambrook et
al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring
Harbor, N.Y., (1989)).
[0236] The invention also relates to recombinant fungal host cells
produced by the methods of the invention which are advantageously
used with the recombinant vector of the invention. The cell is
preferably transformed with a vector comprising a nucleic acid
sequence of the invention followed by integration of the vector
into the host chromosome. The choice of fungal host cells will to a
large extent depend upon the gene encoding the protein or fragment
thereof and its source. The fungal host cell may, for example, be a
yeast cell or a filamentous fungal cell.
[0237] "Yeast" as used herein includes Ascosporogenous yeast
(Endomycetales), Basidiosporogenous yeast and yeast belonging to
the Fungi Imperfecti (Blastomycetes). The Ascosporogenous yeasts
are divided into the families Spermophthoraceae and
Saccharomycetaceae. The latter is comprised of four subfamilies,
Schizosaccharomycoideae (for example, genus Schizosaccharomyces),
Nadsonioideae, Lipomycoideae and Saccharomycoideae (for example,
genera Pichia, Kluyveromyces and Saccharomyces). The
Basidiosporogenous yeasts include the genera Leucosporidim,
Rhodosporidium, Sporidiobolus, Filobasidium and Filobasidiella.
Yeast belonging to the Fungi Imperfecti are divided into two
families, Sporobolomycetaceae (for example, genera Sorobolomyces
and Bullera) and Cryptococcaceae (for example, genus Candida).
Since the classification of yeast may change in the future, for the
purposes of this invention, yeast shall be defined as described in
Biology and Activities of Yeast (Skinner et al., Soc. App.
Bacteriol. Symposium Series No. 9, (1980), the entirety of which is
herein incorporated by reference). The biology of yeast and
manipulation of yeast genetics are well known in the art (see, for
example, Biochemistry and Genetics of Yeast, Bacil et al. (ed.),
2nd edition, 1987; The Yeasts, Rose and Harrison (eds.), 2nd ed.,
(1987); and The Molecular Biology of the Yeast Saccharomyces,
Strathern et al. (eds.), (1981), all of which are herein
incorporated by reference in their entirety).
[0238] "Fungi" as used herein includes the phyla Ascomycota,
Basidiomycota, Chytridiomycota and Zygomycota (as defined by
Hawksworth et al., In: Ainsworth and Bisby's Dictionary of The
Fungi, 8.sup.th edition, 1995, CAB International, University Press,
Cambridge, UK; the entirety of which is herein incorporated by
reference) as well as the Oomycota (as cited in Hawksworth et al.,
In: Ainsworth and Bisby's Dictionary of The Fungi, 8.sup.th
edition, 1995, CAB International, University Press, Cambridge, UK)
and all mitosporic fungi (Hawksworth et al., In: Ainsworth and
Bisby's Dictionary of The Fungi, 8.sup.th edition, 1995, CAB
International, University Press, Cambridge, UK). Representative
groups of Ascomycota include, for example, Neurospora,
Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotiun
(=Aspergillus) and the true yeasts listed above. Examples of
Basidiomycota include mushrooms, rusts and smuts. Representative
groups of Chytridiomycota include, for example, Allomyces,
Blastocladiella, Coelomomyces and aquatic fungi. Representative
groups of Oomycota include, for example, Saprolegniomycetous
aquatic fungi (water molds) such as Achlya. Examples of mitosporic
fungi include Aspergillus, Penicilliun, Candida and Alternaria.
Representative groups of Zygomycota include, for example, Rhizopus
and Mucor.
[0239] "Filamentous fungi" include all filamentous forms of the
subdivision Eumycota and Oomycota (as defined by Hawksworth et al.,
In: Ainsworth and Bisby's Dictionary of The Fungi, 8.sup.th
edition, 1995, CAB International, University Press, Cambridge, UK).
The filamentous fungi are characterized by a vegetative mycelium
composed of chitin, cellulose, glucan, chitosan, mannan and other
complex polysaccharides. Vegetative growth is by hyphal elongation
and carbon catabolism is obligately aerobic. In contrast,
vegetative growth by yeasts such as Saccharomyces cerevisiae is by
budding of a unicellular thallus and carbon catabolism may be
fermentative.
[0240] In one embodiment, the fungal host cell is a yeast cell. In
a preferred embodiment, the yeast host cell is a cell of the
species of Candida, Kluyveromyces, Saccharomyces,
Schizosaccharomyces, Pichia and Yarrowia. In a preferred
embodiment, the yeast host cell is a Saccharomyces cerevisiae cell,
a Saccharomyces carlsbergensis, Saccharomyces diastaticus cell, a
Saccharomyces douglasii cell, a Saccharomyces kluyveri cell, a
Saccharomyces norbensis cell, or a Saccharomyces oviformis cell. In
another preferred embodiment, the yeast host cell is a
Kluyveromyces lactis cell. In another preferred embodiment, the
yeast host cell is a Yarrowia lipolytica cell.
[0241] In another embodiment, the fungal host cell is a filamentous
fungal cell. In a preferred embodiment, the filamentous fungal host
cell is a cell of the species of, but not limited to, Acremonium,
Aspergillus, Fusarium, Humicola, Myceliophthora, Mucor, Neurospora,
Penicillium, Thielavia, Tolypocladium and Trichoderma.
[0242] The recombinant fungal host cells of the invention may
further comprise one or more sequences which encode one or more
factors that are advantageous in the expression of the protein or
fragment thereof, for example, an activator (e.g., a trans-acting
factor), a chaperone and a processing protease. The nucleic acids
encoding one or more of these factors are preferably not operably
linked to the nucleic acid encoding the protein or fragment
thereof. An activator is a protein which activates transcription of
a nucleic acid sequence encoding a polypeptide (Kudla et al., EMBO
9:1355-1364(1990); Jarai and Buxton, Current Genetics
26:2238-244(1994); Verdier, Yeast 6:271-297(1990), all of which are
herein incorporated by reference in their entirety). The nucleic
acid sequence encoding an activator may be obtained from the genes
encoding Saccharomyces cerevisiae heme activator protein 1 (hap1),
Saccharomyces cerevisiae galactose metabolizing protein 4 (gal4)
and Aspergillus nidulans ammonia regulation protein (areA). For
further examples, see Verdier, Yeast 6:271-297 (1990); MacKenzie et
al., Journal of Gen. Microbiol. 139:2295-2307 (1993), both of which
are herein incorporated by reference in their entirety). A
chaperone is a protein which assists another protein in folding
properly (Hartl et al., TIBS 19:20-25 (1994); Bergeron et al., TIBS
19:124-128 (1994); Demolder et al., J. Biotechnology 32:179-189
(1994); Craig, Science 260:1902-1903(1993); Gething and Sambrook,
Nature 355:33-45 (1992); Puig and Gilbert, J. Biol. Chem.
269:7764-7771 (1994); Wang and Tsou, FASEB Journal 7:1515-11157
(1993); Robinson et al., Bio/Technology 1:381-384 (1994), all of
which are herein incorporated by reference in their entirety). The
nucleic acid sequence encoding a chaperone may be obtained from the
genes encoding Aspergillus oryzae protein disulphide isomerase,
Saccharomyces cerevisiae calnexin, Saccharomyces cerevisiae
BiP/GRP78 and Saccharomyces cerevisiae Hsp70. For further examples,
see Gething and Sambrook, Nature 355:33-45 (1992); Hartl et al.,
TIBS 19:20-25 (1994). A processing protease is a protease that
cleaves a propeptide to generate a mature biochemically active
polypeptide (Enderlin and Ogrydziak, Yeast 10:67-79 (1994); Fuller
et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:1434-1438 (1989); Julius
et al., Cell 37:1075-1089 (1984); Julius et al., Cell 32:839-852
(1983), all of which are incorporated by reference in their
entirety). The nucleic acid sequence encoding a processing protease
may be obtained from the genes encoding Aspergillus niger Kex2,
Saccharomyces cerevisiae dipeptidylaminopeptidase, Saccharomyces
cerevisiae Kex2 and Yarrowia lipolytica dibasic processing
endoprotease (xpr6). Any factor that is functional in the fungal
host cell of choice may be used in the invention.
[0243] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus host cells are
described in EP 238 023 and Yelton et al., Proc. Natl. Acad. Sci.
(U.S.A.) 81:1470-1474 (1984), both of which are herein incorporated
by reference in their entirety. A suitable method of transforming
Fusarium species is described by Malardier et al., Gene 78:147-156
(1989), the entirety of which is herein incorporated by reference.
Yeast may be transformed using the procedures described by Becker
and Guarente, In: Abelson and Simon, (eds.), Guide to Yeast
Genetics and Molecular Biology, Methods Enzymol. Volume 194, pp.
182-187, Academic Press, Inc., New York; Ito et al., J.
Bacteriology 153:163 (1983); Hinnen et al., Proc. Natl. Acad. Sci.
(U.S.A.) 75:1920 (1978), all of which are herein incorporated by
reference in their entirety.
[0244] The invention also relates to methods of producing the
protein or fragment thereof comprising culturing the recombinant
fungal host cells under conditions conducive for expression of the
protein or fragment thereof. The fungal cells of the invention are
cultivated in a nutrient medium suitable for production of the
protein or fragment thereof using methods known in the art. For
example, the cell may be cultivated by shake flask cultivation,
small-scale or large-scale fermentation (including continuous,
batch, fed-batch, or solid state fermentations) in laboratory or
industrial fermentors performed in a suitable medium and under
conditions allowing the protein or fragment thereof to be expressed
and/or isolated. The cultivation takes place in a suitable nutrient
medium comprising carbon and nitrogen sources and inorganic salts,
using procedures known in the art (see, e.g., Bennett and LaSure
(eds.), More Gene Manipulations in Fungi, Academic Press, CA,
(1991), the entirety of which is herein incorporated by reference).
Suitable media are available from commercial suppliers or may be
prepared according to published compositions (e.g., in catalogues
of the American Type Culture Collection, Manassas, Va.). If the
protein or fragment thereof is secreted into the nutrient medium, a
protein or fragment thereof can be recovered directly from the
medium. If the protein or fragment thereof is not secreted, it is
recovered from cell lysates.
[0245] The expressed protein or fragment thereof may be detected
using methods known in the art that are specific for the particular
protein or fragment. These detection methods may include the use of
specific antibodies, formation of an enzyme product, or
disappearance of an enzyme substrate. For example, if the protein
or fragment thereof has enzymatic activity, an enzyme assay may be
used. Alternatively, if polyclonal or monoclonal antibodies
specific to the protein or fragment thereof are available,
immunoassays may be employed using the antibodies to the protein or
fragment thereof. The techniques of enzyme assay and immunoassay
are well known to those skilled in the art.
[0246] The resulting protein or fragment thereof may be recovered
by methods known in the arts. For example, the protein or fragment
thereof may be recovered from the nutrient medium by conventional
procedures including, but not limited to, centrifugation,
filtration, extraction, spray-drying, evaporation, or
precipitation. The recovered protein or fragment thereof may then
be further purified by a variety of chromatographic procedures,
e.g., ion exchange chromatography, gel filtration chromatography,
affinity chromatography, or the like.
(g) Mammalian Constructs and Transformed Mammalian Cells
[0247] The invention also relates to methods for obtaining a
recombinant mammalian host cell, comprising introducing into a
mammalian host cell exogenous genetic material. The invention also
relates to a mammalian cell comprising a mammalian recombinant
vector. The invention also relates to methods for obtaining a
recombinant mammalian host cell, comprising introducing into a
mammalian cell exogenous genetic material. In a preferred
embodiment the exogenous genetic material includes a nucleic acid
molecule of the present invention, preferably a nucleic acid
molecule having a sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 621 or complements thereof or
fragments of either. Another preferred class of exogenous genetic
material are nucleic acid molecules that encode a protein having an
amino acid selected from the group consisting of SEQ ID NO: 622
through SEQ ID NO: 626 or fragments thereof.
[0248] Mammalian cell lines available as hosts for expression are
known in the art and include many immortalized cell lines available
from the American Type Culture Collection (ATCC, Manassas, Va.),
such as HeLa cells, Chinese hamster ovary (CHO) cells, baby hamster
kidney (BHK) cells and a number of other cell lines.
[0249] Suitable promoters for mammalian cells are also known in the
art and include viral promoters, such as those from Simian Virus 40
(SV40) (Fiers et al., Nature 273: 113 (1978)), Rous sarcoma virus
(RSV), adenovirus (ADV), cytomegalovirus (CMV), and bovine
papilloma virus (BPV), as well as mammalian cell-derived promoters.
An exemplary, non-limiting, list includes: a hematopoietic stem
cell-specific promoter, such as the CD34 promoter (Bum et al., U.S.
Pat. No. 5,556,954); the glucose-6-phosphotase promoter (Yoshiuchi
et al., J, Clin. Endocrin. Metab. 83:1016-1019 (1998));
interleukin-1 alpha promoter (Mori and Prager, Leuk. Lymphoma
26:421-433 (1997)); CMV promoter (Tong et al., Anticancer Res.
18:719-725 (1998), Norman et al., Vaccine 15:801-803 (1997)); RSV
promoter (Elshami et al., Cancer Gene Ther. 4:213-221 (1997);
Baldwin et al., Gene Ther. 4:1142-1149 (1997)); SV40 promoter
(Harms and Splitter, Hum. Gene Ther. 6:1291-1297 (1995)); CD11c
integrin gene promoter (Corbi and Lopez-Rodriguez, Leuk. Lymphoma
25:415-425 (1997)), GM-CSF promoter (Shannon et al., Crit. Rev.
Immunol. 17:301-323 (1997)); interleukin-5R alpha promoter (Sun et
al., Curr. Top. Microbiol. Immunol 211:173-187 (1996));
interleukin-2 promoter (Serfing et al., Biochim. Biophys. Acta
1263:181-200 (1995); O'Neill et al., Transplant Proc. 23:2862-2866
(1991)); c-fos promoter (Janknecht, Immunobiology 193:137-142
(1995), Janknecht et al., Carcinogenesis 16:443-450 (1995), Takai
et al., Princess Takamatsu Symp. 22:197-204 (1991)); h-ras promoter
(Rachal et al., EXS 64:330-342 (1993)); and DMD gene promoter (Ray
et al., Adv. Exp. Med. Biol. 280:107-111 (1990). All of the above
documents are incorporated by reference in their entirety and can
be relied on to make or use aspects of this invention, especially
in designing and constructing appropriate vector and host
expression systems.
[0250] Vectors used in mammalian cell expression systems may also
include additional functional sequences. For example, terminator
sequences, poly-A addition sequences, and internal ribosome entry
site (IRES) sequences. Enhancer sequences, which increase
expression, may also be included and sequences that promote
amplification of the gene may also be desirable (for example,
methotrexate resistance genes). One of skill in the art is familiar
with numerous examples of these additional functional sequences, as
well as other functional sequences, that may optionally be included
in an expression vector.
[0251] Vectors suitable for replication in mammalian cells may
include viral replicons, or sequences which insure integration of
the appropriate sequences encoding HCV epitopes into the host
genome. For example, another vector used to express foreign DNA is
vaccinia virus. In this case, for example, a nucleic acid molecule
encoding a protein or fragment thereof is inserted into the
vaccinia genome. Techniques for the insertion of foreign DNA into
the vaccinia virus genome are known in the art and may utilize, for
example, homologous recombination. Such heterologous DNA is
generally inserted into a gene which is non-essential to the virus,
for example, the thymidine kinase gene (tk), which also provides a
selectable marker. Plasmid vectors that greatly facilitate the
construction of recombinant viruses have been described (see, for
example, Mackett et al, J. Virol. 49:857 (1984); Chakrabarti et
al., Mol. Cell. Biol. 5:3403 (1985); Moss, In: Gene Transfer
Vectors For Mammalian Cells (Miller and Calos, eds., Cold Spring
Harbor Laboratory, N.Y., p. 10, (1987); all of which are herein
incorporated by reference in their entirety). Expression of the HCV
polypeptide then occurs in cells or animals which are infected with
the live recombinant vaccinia virus.
[0252] The sequence to be integrated into the mammalian sequence
may be introduced into the primary host by any convenient means,
which includes calcium precipitated DNA, spheroplast fusion,
transformation, electroporation, biolistics, lipofection,
microinjection, or other convenient means. Where an amplifiable
gene is being employed, the amplifiable gene may serve as the
selection marker for selecting hosts into which the amplifiable
gene has been introduced. Alternatively, one may include with the
amplifiable gene another marker, such as a drug resistance marker,
e.g., neomycin resistance (G418 in mammalian cells), hygromycin in
resistance etc., or an auxotrophy marker (HIS3, TRP1, LEU2, URA3,
ADE2, LYS2, etc.) for use in yeast cells.
[0253] Depending upon the nature of the modification and associated
targeting construct, various techniques may be employed for
identifying targeted integration. Conveniently, the DNA may be
digested with one or more restriction enzymes and the fragments
probed with an appropriate DNA fragment which will identify the
properly sized restriction fragment associated with
integration.
[0254] One may use different promoter sequences, enhancer
sequences, or other sequence which will allow for enhanced levels
of expression in the expression host. Thus, one may combine an
enhancer from one source, a promoter region from another source, a
5'-noncoding region upstream from the initiation methionine from
the same or different source as the other sequences and the like.
One may provide for an intron in the non-coding region with
appropriate splice sites or for an alternative 3'-untranslated
sequence or polyadenylation site. Depending upon the particular
purpose of the modification, any of these sequences may be
introduced, as desired.
[0255] Where selection is intended, the sequence to be integrated
will have with it a marker gene, which allows for selection. The
marker gene may conveniently be downstream from the target gene and
may include resistance to a cytotoxic agent, e.g., antibiotics,
heavy metals, or the like, resistance or susceptibility to HAT,
gancyclovir, etc., complementation to an auxotrophic host,
particularly by using an auxotrophic yeast as the host for the
subject manipulations, or the like. The marker gene may also be on
a separate DNA molecule, particularly with primary mammalian cells.
Alternatively, one may screen the various transformants, due to the
high efficiency of recombination in yeast, by using hybridization
analysis, PCR, sequencing, or the like.
[0256] For homologous recombination, constructs can be prepared
where the amplifiable gene will be flanked, normally on both sides
with DNA homologous with the DNA of the target region. Depending
upon the nature of the integrating DNA and the purpose of the
integration, the homologous DNA will generally be within 100 kb,
usually 50 kb, preferably about 25 kb, of the transcribed region of
the target gene, more preferably within 2 kb of the target gene.
Where modeling of the gene is intended, homology will usually be
present proximal to the site of the mutation. The homologous DNA
may include the 5'-upstream region outside of the transcriptional
regulatory region or comprising any enhancer sequences,
transcriptional initiation sequences, adjacent sequences, or the
like. The homologous region may include a portion of the coding
region, where the coding region may be comprised only of an open
reading frame or combination of exons and introns. The homologous
region may comprise all or a portion of an intron, where all or a
portion of one or more exons may also be present. Alternatively,
the homologous region may comprise the 3'-region, so as to comprise
all or a portion of the transcriptional termination region, or the
region 3' of this region. The homologous regions may extend over
all or a portion of the target gene or be outside the target gene
comprising all or a portion of the transcriptional regulatory
regions and/or the structural gene.
[0257] The integrating constructs may be prepared in accordance
with conventional ways, where sequences may be synthesized,
isolated from natural sources, manipulated, cloned, ligated,
subjected to in vitro mutagenesis, primer repair, or the like. At
various stages, the joined sequences may be cloned and analyzed by
restriction analysis, sequencing, or the like. Usually during the
preparation of a construct where various fragments are joined, the
fragments, intermediate constructs and constructs will be carried
on a cloning vector comprising a replication system functional in a
prokaryotic host, e.g., E. coli and a marker for selection, e.g.,
biocide resistance, complementation to an auxotrophic host, etc.
Other functional sequences may also be present, such as
polylinkers, for ease of introduction and excision of the construct
or portions thereof, or the like. A large number of cloning vectors
are available such as pBR322, the pUC series, etc. These constructs
may then be used for integration into the primary mammalian
host.
[0258] In the case of the primary mammalian host, a replicating
vector may be used. Usually, such vector will have a viral
replication system, such as SV40, bovine papilloma virus,
adenovirus, or the like. The linear DNA sequence vector may also
have a selectable marker for identifying transfected cells.
Selectable markers include the neo gene, allowing for selection
with G418, the herpes tk gene for selection with HAT medium, the
gpt gene with mycophenolic acid, complementation of an auxotrophic
host, etc.
[0259] The vector may or may not be capable of stable maintenance
in the host. Where the vector is capable of stable maintenance, the
cells will be screened for homologous integration of the vector
into the genome of the host, where various techniques for curing
the cells may be employed. Where the vector is not capable of
stable maintenance, for example, where a temperature sensitive
replication system is employed, one may change the temperature from
the permissive temperature to the non-permissive temperature, so
that the cells may be cured of the vector. In this case, only those
cells having integration of the construct comprising the
amplifiable gene and, when present, the selectable marker, will be
able to survive selection.
[0260] Where a selectable marker is present, one may select for the
presence of the targeting construct by means of the selectable
marker. Where the selectable marker is not present, one. may select
for the presence of the construct by the amplifiable gene. For the
neo gene or the herpes tk gene, one could employ a medium for
growth of the transformants of about 0.1-1 mg/ml of G418 or may use
HAT medium, respectively. Where DHFR is the amplifiable gene, the
selective medium may include from about 0.01-0.5 .mu.M of
methotrexate or be deficient in glycine-hypoxanthine-thymidine and
have dialysed serum (GHT media).
[0261] The DNA can be introduced into the expression host by a
variety of techniques that include calcium phosphate/DNA
co-precipitates, microinjection of DNA into the nucleus,
electroporation, yeast protoplast fusion with intact cells,
transfection, polycations, e.g., polybrene, polyomithine, etc., or
the like. The DNA may be single or double stranded DNA, linear or
circular. The various techniques for transforming mammalian cells
are well known (see Keown et al., Methods Enzymol. (1989); Keown et
al., Methods Enzymol. 185:527-537 (1990); Mansour et al., Nature
336:348-352, (1988); all of which are herein incorporated by
reference in their entirety).
(h) Insect Constructs and Transformed Insect Cells
[0262] The invention also relates to an insect recombinant vectors
comprising exogenous genetic material. The invention also relates
to an insect cell comprising an insect recombinant vector. The
invention also relates to methods for obtaining a recombinant
insect host cell, comprising introducing into an insect cell
exogenous genetic material. In a preferred embodiment the exogenous
genetic material includes a nucleic acid molecule of the present
invention, preferably a nucleic acid molecule having a sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 621 or complements thereof or fragments of either. Another
preferred class of exogenous genetic material are nucleic acid
molecules that encode a protein having an amino acid selected from
the group consisting of SEQ ID NO: 622 through SEQ ID NO: 626 or
fragments thereof.
[0263] The insect recombinant vector may be any vector which can be
conveniently subjected to recombinant DNA procedures and can bring
about the expression of the nucleic acid sequence. The choice of a
vector will typically depend on the compatibility of the vector
with the insect host cell into which the vector is to be
introduced. The vector may be a linear or a closed circular
plasmid. The vector system may be a single vector or plasmid or two
or more vectors or plasmids which together contain the total DNA to
be introduced into the genome of the insect host. In addition, the
insect vector may be an expression vector. Nucleic acid molecules
can be suitably inserted into a replication vector for expression
in the insect cell under a suitable promoter for insect cells. Many
vectors are available for this purpose and selection of the
appropriate vector will depend mainly on the size of the nucleic
acid molecule to be inserted into the vector and the particular
host cell to be transformed with the vector. Each vector contains
various components depending on its function (amplification of DNA
or expression of DNA) and the particular host cell with which it is
compatible. The vector components for insect cell transformation
generally include, but are not limited to, one or more of the
following: a signal sequence, origin of replication, one or more
marker genes and an inducible promoter.
[0264] The insect vector may be an autonomously replicating vector,
i.e., a vector which exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into the insect cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. For integration, the vector may rely on the
nucleic acid sequence of the vector for stable integration of the
vector into the genome by homologous or nonhomologous
recombination. Alternatively, the vector may contain additional
nucleic acid sequences for directing integration by homologous
recombination into the genome of the insect host. The additional
nucleic acid sequences enable the vector to be integrated into the
host cell genome at a precise location(s) in the chromosome(s). To
increase the likelihood of integration at a precise location, there
should be preferably two nucleic acid sequences which individually
contain a sufficient number of nucleic acids, preferably 400 bp to
1500 bp, more preferably 800 bp to 1000 bp, which are highly
homologous with the corresponding target sequence to enhance the
probability of homologous recombination. These nucleic acid
sequences may be any sequence that is homologous with a target
sequence in the genome of the insect host cell and, furthermore,
may be non-encoding or encoding sequences.
[0265] Baculovirus expression vectors (BEVs) have become important
tools for the expression of foreign genes, both for basic research
and for the production of proteins with direct clinical
applications in human and veterinary medicine (Doerfler, Curr. Top.
Microbiol. Immunol. 131:51-68 (1968); Luckow and Summers,
Bio/Technology 6:47-55 (1988a); Miller, Annual Review of Microbiol.
42:177-199 (1988); Summers, Curr. Comm. Molecular Biology, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1988); all of which
are herein incorporated by reference in their entirety). BEVs are
recombinant insect viruses in which the coding sequence for a
chosen foreign gene has been inserted behind a baculovirus promoter
in place of the viral gene, e.g., polyhedrin (Smith and Summers,
U.S. Pat. No., 4,745,051, the entirety of which is incorporated
herein by reference).
[0266] The use of baculovirus vectors relies upon the host cells
being derived from Lepidopteran insects such as Spodoptera
frugiperda or Trichoplusia ni. The preferred Spodoptera frugiperda
cell line is the cell line Sf9. The Spodoptera frugiperda Sf9 cell
line was obtained from American Type Culture Collection (Manassas,
Va.) and is assigned accession number ATCC CRL 1711 (Summers and
Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell
Culture Procedures, Texas Ag. Exper. Station Bulletin No. 1555
(1988), the entirety of which is herein incorporated by reference).
Other insect cell systems, such as the silkworm B. mori may also be
used.
[0267] The proteins expressed by the BEVs are, therefore,
synthesized, modified and transported in host cells derived from
Lepidopteran insects. Most of the genes that have been inserted and
produced in the baculovirus expression vector system have been
derived from vertebrate species. Other baculovirus genes in
addition to the polyhedrin promoter may be employed to advantage in
a baculovirus expression system. These include immediate-early
(alpha), delayed-early (.beta.), late (.gamma.), or very late
(delta), according to the phase of the viral infection during which
they are expressed. The expression of these genes occurs
sequentially, probably as the result of a "cascade" mechanism of
transcriptional regulation. (Guarino and Summers, J. Virol.
57:563-571 (1986); Guarino and Summers, J. Virol. 61:2091-2099
(1987); Guarino and Summers, Virol. 162:444-451 (1988); all of
which are herein incorporated by reference in their entirety).
[0268] Insect recombinant vectors are useful as intermediates for
the infection or transformation of insect cell systems. For
example, an insect recombinant vector containing a nucleic acid
molecule encoding a baculovirus transcriptional promoter followed
downstream by an insect signal DNA sequence is capable of directing
the secretion of the desired biologically active protein from the
insect cell. The vector may utilize a baculovirus transcriptional
promoter region derived from any of the over 500 baculoviruses
generally infecting insects, such as for example the Orders
Lepidoptera, Diptera, Orthoptera, Coleoptera and Hymenoptera,
including for example but not limited to the viral DNAs of
Autographa californica MNPV, Bombyx mori NPV, Trichoplusia ni MNPV,
Rachiplusia ou MNPV or Galleria mellonella MNPV, wherein said
baculovirus transcriptional promoter is a baculovirus
immediate-early gene IE1 or IEN promoter; an immediate-early gene
in combination with a baculovirus delayed-early gene promoter
region selected from the group consisting of 39K and a HindIII-k
fragment delayed-early gene; or a baculovirus late gene promoter.
The immediate-early or delayed-early promoters can be enhanced with
transcriptional enhancer elements. The insect signal DNA sequence
may code for a signal peptide of a Lepidopteran adipokinetic
hormone precursor or a signal peptide of the Manduca sexta
adipokinetic hormone precursor (Summers, U.S. Pat. No. 5,155,037;
the entirety of which is herein incorporated by reference). Other
insect signal DNA sequences include a signal peptide of the
Orthoptera Schistocerca gregaria locust adipokinetic hormone
precursor and the Drosophila melanogaster cuticle genes CP1, CP2,
CP3 or CP4 or for an insect signal peptide having substantially a
similar chemical composition and function (Summers, U.S. Pat. No.
5,155,037).
[0269] Insect cells are distinctly different from animal cells.
Insects have a unique life cycle and have distinct cellular
properties such as the lack of intracellular plasminogen activators
in which are present in vertebrate cells. Another difference is the
high expression levels of protein products ranging from 1 to
greater than 500 mg/liter and the ease at which cDNA can be cloned
into cells (Frasier, In Vitro Cell. Dev. Biol. 25:225 (1989);
Summers and Smith, In: A Manual of Methods for Baculovirus Vectors
and Insect Cell Culture Procedures, Texas Ag. Exper. Station
Bulletin No. 1555 (1988), both of which are incorporated by
reference in their entirety).
[0270] Recombinant protein expression in insect cells is achieved
by viral infection or stable transformation. For viral infection,
the desired gene is cloned into baculovirus at the site of the
wild-type polyhedron gene (Webb and Summers, Technique
2:173-(1990); Bishop and Posse, Adv. Gene Technol. 1:55 (1990);
both of which are incorporated by reference in their entirety). The
polyhedron gene is a component of a protein coat in occlusions
which encapsulate virus particles. Deletion or insertion in the
polyhedron gene results the failure to form occlusion bodies.
Occlusion negative viruses are morphologically different from
occlusion positive viruses and enable one skilled in the art to
identify and purify recombinant viruses.
[0271] The vectors of invention preferably contain one or more
selectable markers, which permit easy selection of transformed
cells. A selectable marker is a gene the product of which provides,
for example biocide or viral resistance, resistance to heavy
metals, prototrophy to auxotrophs and the like. Selection may be
accomplished by co-transformation, e.g., as described in WO
91/17243, a nucleic acid sequence of the invention may be operably
linked to a suitable promoter sequence. The promoter sequence is a
nucleic acid sequence, which is recognized by the insect host cell
for expression of the nucleic acid sequence. The promoter sequence
contains transcription and translation control sequences, which
mediate the expression of the protein or fragment thereof. The
promoter may be any nucleic acid sequence, which shows
transcriptional activity in the insect host cell of choice and may
be obtained from genes encoding polypeptides either homologous or
heterologous to the host cell.
[0272] For example, a nucleic acid molecule encoding a protein or
fragment thereof may also be operably linked to a suitable leader
sequence. A leader sequence is a nontranslated region of a mRNA,
which is important for translation by the fungal host. The leader
sequence is operably linked to the 5' terminus of the nucleic acid
sequence encoding the protein or fragment thereof. The leader
sequence may be native to the nucleic acid sequence encoding the
protein or fragment thereof or may be obtained from foreign
sources. Any leader sequence, which is functional in the insect
host cell of choice may be used in the invention.
[0273] A polyadenylation sequence may also be operably linked to
the 3' terminus of the nucleic acid sequence of the invention. The
polyadenylation sequence is a sequence which when transcribed is
recognized by the insect host to add polyadenosine residues to
transcribed mRNA. The polyadenylation sequence may be native to the
nucleic acid sequence encoding the protein or fragment thereof or
may be obtained from foreign sources. Any polyadenylation sequence,
which is functional in the fungal host of choice, may be used in
the invention.
[0274] To avoid the necessity of disrupting the cell to obtain the
protein or fragment thereof and to minimize the amount of possible
degradation of the expressed polypeptide within the cell, it is
preferred that expression of the polypeptide gene gives rise to a
product secreted outside the cell. To this end, the protein or
fragment thereof of the invention may be linked to a signal peptide
linked to the amino terminus of the protein or fragment thereof. A
signal peptide is an amino acid sequence which permits the
secretion of the protein or fragment thereof from the insect host
into the culture medium. The signal peptide may be native to the
protein or fragment thereof of the invention or may be obtained
from foreign sources. The 5' end of the coding sequence of the
nucleic acid sequence of the invention may inherently contain a
signal peptide coding region naturally linked in translation
reading frame with the segment of the coding region which encodes
the secreted protein or fragment thereof.
[0275] At present, a mode of achieving secretion of a foreign gene
product in insect cells is by way of the foreign gene's native
signal peptide. Because the foreign genes are usually from
non-insect organisms, their signal sequences may be poorly
recognized by insect cells and, hence, levels of expression may be
suboptimal. However, the efficiency of expression of foreign gene
products seems to depend primarily on the characteristics of the
foreign protein. On average, nuclear localized or non-structural
proteins are most highly expressed, secreted proteins are
intermediate and integral membrane proteins are the least
expressed. One factor generally affecting the efficiency of the
production of foreign gene products in a heterologous host system
is the presence of native signal sequences (also termed
presequences, targeting signals, or leader sequences) associated
with the foreign gene. The signal sequence is generally coded by a
DNA sequence immediately following (5' to 3') the translation start
site of the desired foreign gene.
[0276] The expression dependence on the type of signal sequence
associated with a gene product can be represented by the following
example. If a foreign gene is inserted at a site downstream from
the translational start site of the baculovirus polyhedrin gene so
as to produce a fusion protein (containing the N-terminus of the
polyhedrin structural gene), the fused gene is highly expressed.
But less expression is achieved when a foreign gene is inserted in
a baculovirus expression vector immediately following the
transcriptional start site and totally replacing the polyhedrin
structural gene.
[0277] Insertions into the region -50 to -1 significantly alter
(reduce) steady state transcription which, in turn, reduces
translation of the foreign gene product. Use of the pVL941 vector
optimizes transcription of foreign genes to the level of the
polyhedrin gene transcription. Even though the transcription of a
foreign gene may be optimal, optimal translation may vary because
of several factors involving processing: signal peptide
recognition, mRNA and ribosome binding, glycosylation, disulfide
bond formation, sugar processing, oligomerization, for example.
[0278] The properties of the insect signal peptide are expected to
be more optimal for the efficiency of the translation process in
insect cells than those from vertebrate proteins. This phenomenon
can generally be explained by the fact that proteins secreted from
cells are synthesized as precursor molecules containing hydrophobic
N-terminal signal peptides. The signal peptides direct transport of
the select protein to its target membrane and are then cleaved by a
peptidase on the membrane, such as the endoplasmic reticulum, when
the protein passes through it.
[0279] Another exemplary insect signal sequence is the sequence
encoding for Drosophila cuticle proteins such as CP1, CP2, CP3 or
CP4 (Summers, U.S. Pat. No. 5,278,050; the entirety of which is
herein incorporated by reference). Most of a 9 kb region of the
Drosophila genome containing genes for the cuticle proteins has
been sequenced. Four of the five cuticle genes contains a signal
peptide coding sequence interrupted by a short intervening sequence
(about 60 base pairs) at a conserved site. Conserved sequences
occur in the 5' mRNA untranslated region, in the adjacent 35 base
pairs of upstream flanking sequence and at -200 base pairs from the
mRNA start position in each of the cuticle genes.
[0280] Standard methods of insect cell culture, cotransfection and
preparation of plasmids are set forth in Summers and Smith (Summers
and Smith, A Manual of Methods for Baculovirus Vectors and Insect
Cell Culture Procedures, Texas Agricultural Experiment Station
Bulletin No. 1555, Texas A&M University (1987)). Procedures for
the cultivation of viruses and cells are described in Volkman and
Summers, J. Virol 19:820-832 (1975) and Volkman et al., J. Virol
19:820-832 (1976); both of which are herein incorporated by
reference in their entirety.
(i) Bacterial Constructs and Transformed Bacterial Cells
[0281] The invention also relates to a bacterial recombinant vector
comprising exogenous genetic material. The invention also relates
to a bacteria cell comprising a bacterial recombinant vector. The
invention also relates to methods for obtaining a recombinant
bacteria host cell, comprising introducing into a bacterial host
cell exogenous genetic material. In a preferred embodiment the
exogenous genetic material includes a nucleic acid molecule of the
present invention, preferably a nucleic acid molecule having a
sequence selected from the group consisting of SEQ ID NO: 1 through
SEQ ID NO: 621 or complements thereof or fragments of either.
Another preferred class of exogenous genetic material are nucleic
acid molecules that encode a protein having an amino acid selected
from the group consisting of SEQ ID NO: 622 through SEQ ID NO: 626
or fragments thereof.
[0282] The bacterial recombinant vector may be any vector that can
be conveniently subjected to recombinant DNA procedures. The choice
of a vector will typically depend on the compatibility of the
vector with the bacterial host cell into which the vector is to be
introduced. The vector may be a linear or a closed circular
plasmid. The vector system may be a single vector or plasmid or two
or more vectors or plasmids, which together contain the total DNA
to be introduced into the genome of the bacterial host. In
addition, the bacterial vector may be an expression vector. Nucleic
acid molecules encoding protein homologues or fragments thereof
can, for example, be suitably inserted into a replicable vector for
expression in the bacterium under the control of a suitable
promoter for bacteria. Many vectors are available for this purpose
and selection of the appropriate vector will depend mainly on the
size of the nucleic acid to be inserted into the vector and the
particular host cell to be transformed with the vector. Each vector
contains various components depending on its function
(amplification of DNA or expression of DNA) and the particular host
cell with which it is compatible. The vector components for
bacterial transformation generally include, but are not limited to,
one or more of the following: a signal sequence, an origin of
replication, one or more marker genes and an inducible
promoter.
[0283] In general, plasmid vectors containing replicon and control
sequences that are derived from species compatible with the host
cell are used in connection with bacterial hosts. The vector
ordinarily carries a replication site, as well as marking sequences
that are capable of providing phenotypic selection in transformed
cells. For example, E. coli is typically transformed using pBR322,
a plasmid derived from an E. coli species (see, e.g., Bolivar et
al., Gene 2:95 (1977); the entirety of which is herein incorporated
by reference). The plasmid pBR322 contains genes for ampicillin and
tetracycline resistance and thus provides easy means for
identifying transformed cells. The pBR322 plasmid, or other
microbial plasmid or phage, also generally contains, or is modified
to contain, promoters that can be used by the microbial organism
for expression of the selectable marker genes.
[0284] Nucleic acid molecules encoding protein or fragments thereof
may be expressed not only directly, but also as a fusion with
another polypeptide, preferably a signal sequence or other
polypeptide having a specific cleavage site at the N-terminus of
the mature polypeptide. In general, the signal sequence may be a
component of the vector, or it may be a part of the polypeptide DNA
that is inserted into the vector. The heterologous signal sequence
selected should be one that is recognized and processed (i.e.,
cleaved by a signal peptidase) by the host cell. For bacterial host
cells that do not recognize and process the native polypeptide
signal sequence, the signal sequence is substituted by a bacterial
signal sequence selected, for example, from the group consisting of
the alkaline phosphatase, penicillinase, lpp, or heat-stable
enterotoxin II leaders.
[0285] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA and includes origins of replication or autonomously
replicating sequences. Such sequences are well known for a variety
of bacteria. The origin of replication from the plasmid pBR322 is
suitable for most Gram-negative bacteria.
[0286] Expression and cloning vectors also generally contain a
selection gene, also termed a selectable marker. This gene encodes
a protein necessary for the survival or growth of transformed host
cells grown in a selective culture medium. Host cells not
transformed with the vector containing the selection gene will not
survive in the culture medium. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli. One example of a selection scheme
utilizes a drug to arrest growth of a host cell. Those cells that
are successfully transformed with a heterologous protein homologue
or fragment thereof produce a protein conferring drug resistance
and thus survive the selection regimen.
[0287] The expression vector for producing a protein or fragment
thereof can also contains an inducible promoter that is recognized
by the host bacterial organism and is operably linked to the
nucleic acid encoding, for example, the nucleic acid molecule
encoding the protein homologue or fragment thereof of interest.
Inducible promoters suitable for use with bacterial hosts include
the .beta.-lactamase and lactose promoter systems (Chang et al.,
Nature 275:615 (1978); Goeddel et al., Nature 281:544 (1979); both
of which are herein incorporated by reference in their entirety),
the arabinose promoter system (Guzman et al., J. Bacteriol.
174:7716-7728 (1992); the entirety of which is herein incorporated
by reference), alkaline phosphatase, a tryptophan (trp) promoter
system (Goeddel, Nucleic Acids Res. 8:4057 (1980); EP 36,776; both
of which are herein incorporated by reference in their entirety)
and hybrid promoters such as the tac promoter (deBoer et al., Proc.
Natl. Acad. Sci. (USA) 80:21-25 (1983); the entirety of which is
herein incorporated by reference). However, other known bacterial
inducible promoters are suitable (Siebenlist et al., Cell 20:269
(1980); the entirety of which is herein incorporated by
reference).
[0288] Promoters for use in bacterial systems also generally
contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA
encoding the polypeptide of interest. The promoter can be removed
from the bacterial source DNA by restriction enzyme digestion and
inserted into the vector containing the desired DNA.
[0289] Construction of suitable vectors containing one or more of
the above-listed components employs standard ligation techniques.
Isolated plasmids or DNA fragments are cleaved, tailored and
re-ligated in the form desired to generate the plasmids required.
Examples of available bacterial expression vectors include, but are
not limited to: the multifunctional E. coli cloning and expression
vectors such as Bluescript.TM. (Stratagene, La Jolla, Calif.), in
which, for example, encoding an A. nidulans protein homologue or
fragment thereof homologue, may be ligated into the vector in frame
with sequences for the amino-terminal Met and the subsequent 7
residues of .beta.-galactosidase so that a hybrid protein is
produced; pIN vectors (Van Heeke and Schuster, J. Biol. Chem.
264:5503-5509 (1989), the entirety of which is herein incorporated
by reference); and the like. pGEX vectors (Promega, Madison Wis.
U.S.A.) may also be used to express foreign polypeptides as fusion
proteins with glutathione S-transferase (GST). In general, such
fusion proteins are soluble and can easily be purified from lysed
cells by adsorption to glutathione-agarose beads followed by
elution in the presence of free glutathione. Proteins made in such
systems are designed to optionally include a heparin, thrombin, or
factor XA protease cleavage sites so that the cloned polypeptide of
interest can be released from the GST moiety at will. Proteins or
polypeptides of the invention can be expressed as variants that
facilitate purification. For example, a fusion protein to such
proteins as maltose binding protein (MBP),
glutathione-S-transferase (GST) or thioredoxin (TRX) are known in
the art [New England BioLab, Beverly, Mass., Pharmacia, Piscataway,
N.J., and InVitrogen, San Diego, Calif.]. The polypeptide or
protein can also be a tagged variant to facilitate purification,
such as with histidine or methionine rich regions (His-Tag;
available from LifeTechnologies Inc, Gaithersburg, Md.) that bind
to metal ion affinity chromatography columns, or with an epitope
that binds to a specific antibody (Flag, available from Kodak, New
Haven, Conn.). An exemplary, non-limiting list of commercially
available vectors suitable for fusion protein expression includes:
pBR322 (Promega); pGEX (Amersham); pT7 (USB); pET (Novagen); pIBI
(IBI); pProEX-1 (Gibco/BRL); pBluescript II (Stratagene); pTZ18R
and pTZ19R (USB); pSE420 (Invitrogen); pVL1392 (Invitrogen);
pBlueBac (Invitrogen); pBAcPAK (Clontech); pHIL (Invitrogen); pYES2
(Invitrogen); pCDNA (Invitrogen); and pREP (Invitrogen). A number
of other purification methods or means are also known and can be
used. Reverse-phase high performance liquid chromatography
(RP-HPLC), optionally employing hydrophobic RP-HPLC media, e.g.,
silica gel, further purify the protein. Combinations of methods and
means can also be employed to provide a substantially purified
recombinant polypeptide or protein. Isolated plasmids or DNA
fragments are cleaved, tailored, and re-ligated in the form desired
to generate the plasmids required. Examples of available bacterial
expression vectors include, but are not limited to, the
multifunctional E. coli cloning and expression vectors such as
Bluescript Registered.TM. (Stratagene, La Jolla, Calif.), in which,
for example, encoding an gene homologue or fragment thereof
homologue, may be ligated into the vector in frame with sequences
for the amino-terminal Met and the subsequent 7 residues of beta
-galactosidase so that a hybrid protein is produced; pIN vectors
(Van Heeke and Schuster J. Biol. Chem. 264: 5503-5509 (1989). The
entirety of which is herein incorporated by reference); and the
like. pGEX vectors (Promega, Madison Wis.) may also be used to
express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. Proteins made in such systems are designed to
include heparin, thrombin or factor XA protease cleavage sites so
that the cloned polypeptide of interest can be released from the
GST moiety at will.
[0290] Suitable host bacteria for a bacterial vector include
archaebacteria and eubacteria, especially eubacteria and most
preferably Enterobacteriaceae. Examples of useful bacteria include
Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus,
Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella,
Rhizobia, Vitreoscilla and Paracoccus. Suitable E. coli hosts
include E. coli W3 110 (American Type Culture Collection (ATCC)
27,325, Manassas, Va. U.S.A.), E. coli 294 (ATCC 31,446), E. coli B
and E. coli X1776 (ATCC 31,537). These examples are illustrative
rather than limiting. Mutant cells of any of the above-mentioned
bacteria may also be employed. It is, of course, necessary to
select the appropriate bacteria taking into consideration
replicability of the replicon in the cells of a bacterium. For
example, E. coli, Serratia, or Salmonella species can be suitably
used as the host when well known plasmids such as pBR322, pBR325,
pACYC177, or pKN410 are used to supply the replicon. E. coli strain
W3110 is a preferred host or parent host because it is a common
host strain for recombinant DNA product fermentations. Preferably,
the host cell should secrete minimal amounts of proteolytic
enzymes.
[0291] Host cells are transfected and preferably transformed with
the above-described vectors and cultured in conventional nutrient
media modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
[0292] Numerous methods of transfection are known to the ordinarily
skilled artisan, for example, calcium phosphate and
electroporation. Depending on the host cell used, transformation is
done using standard techniques appropriate to such cells. The
calcium treatment employing calcium chloride, as described in
section 1.82 of Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Laboratory Press, (1989), is
generally used for bacterial cells that contain substantial
cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO, as described in Chung and Miller (Chung
and Miller, Nucleic Acids Res. 16:3580 (1988); the entirety of
which is herein incorporated by reference). Yet another method is
the use of the technique termed electroporation.
[0293] Bacterial cells used to produce the polypeptide of interest
for purposes of this invention are cultured in suitable media in
which the promoters for the nucleic acid encoding the heterologous
polypeptide can be artificially induced as described generally,
e.g., in Sambrook et al., Molecular Cloning: A Laboratory Manual,
New York: Cold Spring Harbor Laboratory Press, (1989). Examples of
suitable media are given in U.S. Pat. Nos. 5,304,472 and 5,342,763;
both of which are incorporated by reference in their entirety.
[0294] In addition to the above discussed procedures, practitioners
are familiar with the standard resource materials which describe
specific conditions and procedures for the construction,
manipulation and isolation of macromolecules (e.g., DNA molecules,
plasmids, etc.), generation of recombinant organisms and the
screening and isolating of clones, (see for example, Sambrook et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Press (1989); Mailga et al., Methods in Plant Molecular Biology,
Cold Spring Harbor Press (1995), the entirety of which is herein
incorporated by reference; Birren et al., Genome Analysis:
Analyzing DNA, 1, Cold Spring Harbor, N.Y., the entirety of which
is herein incorporated by reference).
(j) Algal Constructs and Algal Transformants
[0295] The present invention also relates to an algal recombinant
vector comprising exogenous genetic material. The present invention
also relates to an algal cell comprising an algal recombinant
vector. The present invention also relates to methods for obtaining
a recombinant algal host cell comprising introducing into an algal
host cell exogenous genetic material.
[0296] Exogenous genetic material is any genetic material, whether
naturally occurring or otherwise, from any source that is capable
of being inserted into any organism. Exogenous genetic material may
be transferred into an algal cell. In a preferred embodiment the
exogenous genetic material includes a nucleic acid molecule having
a sequence selected from the group consisting of SEQ ID NO: 1
through SEQ ID NO: 621 or complements thereof. Another preferred
class of exogenous genetic material are nucleic acid molecules that
encode a protein having an amino acid selected from the group
consisting of SEQ ID NO: 622 through SEQ ID NO: 626 or fragments
thereof.
[0297] The algal recombinant vector may be any vector which can be
conveniently subjected to recombinant DNA procedures. The choice of
a vector will typically depend on the compatibility of the vector
with the algal host cell into which the vector is to be introduced.
The vector may be a linear or a closed circular plasmid. The vector
system may be a single vector or plasmid or two or more vectors or
plasmids which together contain the total DNA to be introduced into
the genome of the algal host.
[0298] The algal vector may be an autonomously replicating vector,
i.e., a vector which exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into the algal cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. For integration, the vector may rely on the
nucleic acid sequence of the vector for stable integration of the
vector into the genome by homologous or nonhomologous
recombination. Alternatively, the vector may contain additional
nucleic acid sequences for directing integration by homologous
recombination into the genome of the algal host. The additional
nucleic acid sequences enable the vector to be integrated into the
host cell genome at a precise location(s) in the chromosome(s). To
increase the likelihood of integration at a precise location, there
should be preferably two nucleic acid sequences which individually
contain a sufficient number of nucleic acids, preferably 400 bp to
1500 bp, more preferably 800 bp to 1000 bp, which are highly
homologous with the corresponding target sequence to enhance the
probability of homologous recombination. These nucleic acid
sequences may be any sequence that is homologous with a target
sequence in the genome of the algal host cell, and, furthermore,
may be non-encoding or encoding sequences.
[0299] The vectors of the present invention preferably contain one
or more selectable markers which permit easy selection of
transformed cells. A selectable marker is a gene, the product of
which confers upon an algal cell resistance to a compound to which
the algal would otherwise be sensitive. The compound can be
selected from the group consisting of antibiotics, fungicides,
herbicides, and heavy metals. The selectable marker may be selected
from any known or subsequently identified selectable markers,
including markers derived from algal, fungal, and bacterial
sources. Preferred selectable markers can be selected from the
group including, but not limited to, amdS (acetamidase), argB
(ornithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), ble (bleomycin binding protein), cat
(chloramphenicol acetyltransferase), hygB (hygromycin B
phosphotransferase), nat (nourseothricin acetyltransferase), niaD
(nitrate reductase), neo (neomycin phosphotransferase), pac
(puromycin acetyltransferase), pyrG (orotidine-5'-phosphate
decarboxylase), sat (streptothricin acetyltransferase), sC (sulfate
adenyltransferase), trpC (anthranilate synthase), and glyphosate
resistant EPSPS genes. Furthermore, selection may be accomplished
by co-transformation, e.g., as described in WO 91/17243, herein
incorporated by reference in its entirety.
[0300] A nucleic acid sequence of the present invention may be
operably linked to a suitable promoter sequence. The promoter
sequence is a nucleic acid sequence which is recognized by the
algal host cell for expression of the nucleic acid sequence. The
promoter sequence contains transcription and translation control
sequences which mediate the expression of the protein or fragment
thereof.
[0301] A promoter may be any nucleic acid sequence which shows
transcriptional activity in the algal host cell of choice and may
be obtained from genes encoding polypeptides either homologous or
heterologous to the host cell. Examples of suitable promoters for
directing the transcription of a nucleic acid construct of the
invention in an algal host are light harvesting protein promoters
obtained from photosynthetic organisms, Chlorella virus
methyltransferase promoters, CaMV 35 S promoter, PL promoter from
bacteriophage .lambda., nopaline synthase promoter from the Ti
plasmid of Agrobacterium tumefaciens, and bacterial trp
promoter.
[0302] A protein or fragment thereof encoding nucleic acid molecule
of the present invention may also be operably linked to a
terminator sequence at its 3' terminus. The terminator sequence may
be native to the nucleic acid sequence encoding the protein or
fragment thereof or may be obtained from foreign sources. Any
terminator which is functional in the algal host cell of choice may
be used in the present invention.
[0303] A protein or fragment thereof encoding nucleic acid molecule
of the present invention may also be operably linked to a suitable
leader sequence. A leader sequence is a nontranslated region of a
mRNA which is important for translation by the algal host. The
leader sequence is operably linked to the 5' terminus of the
nucleic acid sequence encoding the protein or fragment thereof. The
leader sequence may be native to the nucleic acid sequence encoding
the protein or fragment thereof or may be obtained from foreign
sources. Any leader sequence which is functional in the algal host
cell of choice may be used in the present invention.
[0304] A polyadenylation sequence may also be operably linked to
the 3' terminus of the nucleic acid sequence of the present
invention. The polyadenylation sequence is a sequence which when
transcribed is recognized by the algal host to add polyadenosine
residues to transcribed mRNA. The polyadenylation sequence may be
native to the nucleic acid sequence encoding the protein or
fragment thereof or may be obtained from foreign sources. Any
polyadenylation sequence which is functional in the algal host of
choice may be used in the present invention.
[0305] The procedures used to ligate the elements described above
to construct the recombinant expression vector of the present
invention are well known to one skilled in the art (see, for
example, Sambrook, 2nd ed., et al., Molecular Cloning, A Laboratory
Manual Cold Spring Harbor, N.Y., (1 989), herein incorporated by
reference in its entirety).
[0306] The present invention also relates to recombinant algal host
cells produced by the methods of the present invention which are
advantageously used with the recombinant vector of the present
invention. The cell is preferably transformed with a vector
comprising a nucleic acid sequence of the invention followed by
integration of the vector into the host chromosome. The choice of
algal host cells will to a large extent depend upon the gene
encoding the protein or fragment thereof and its source.
[0307] Algal cells may be transformed by a variety of known
techniques, including but not limit to, microprojectile
bombardment, protoplast fusion, electroporation, microinjection,
and vigorous agitation in the presence of glass beads. Suitable
procedures for transformation of green algal host cells are
described in EP 108 580, herein incorporated by reference in its
entirety. A suitable method of transforming Chlorella species is
described by Jarvis and Brown, Curr. Genet. 19: 317-321 (1991),
herein incorporated by reference in its entirety. A suitable method
of transforming cells of diatom Phaeodactylum tricornutum species
is described in WO 97/39106, herein incorporated by reference in
its entirety. Chlorophyll C-containing algae may be transformed
using the procedures described in U.S. Pat. No. 5,661,017, herein
incorporated by reference in its entirety.
[0308] The expressed protein or fragment thereof may be detected
using methods known in the art that are specific for the particular
protein or fragment. These detection methods may include the use of
specific antibodies, formation of an enzyme product, or
disappearance of an enzyme substrate. For example, if the protein
or fragment thereof has enzymatic activity, an enzyme assay may be
used. Alternatively, if polyclonal or monoclonal antibodies
specific to the protein or fragment thereof are available,
immunoassays may be employed using the antibodies to the protein or
fragment thereof. The techniques of enzyme assay and immunoassay
are well known to those skilled in the art.
[0309] The resulting protein or fragment thereof may be recovered
by methods known in the arts. For example, the protein or fragment
thereof may be recovered from the nutrient medium by conventional
procedures including, but not limited to, centrifugation,
filtration, extraction, spray-drying, evaporation, or
precipitation. The recovered protein or fragment thereof may then
be further purified by a variety of chromatographic procedures,
e.g., ion exchange chromatography, gel filtration chromatography,
affinity chromatography, or the like.
[0310] Computer Readable Media
[0311] The nucleotide or amino acid sequence provided in SEQ ID NO:
1 through SEQ ID NO: 626, or fragment thereof, or complement
thereof, or a nucleotide or an amino acid sequence at least 70%
identical, preferably 90% identical even more preferably 99% or
about 100% identical to the sequence provided in SEQ ID NO: 1
through SEQ ID NO: 626, or where appropriate complement thereof or
fragments of either, can be "provided" in a variety of mediums to
facilitate use. Such a medium can also provide a subset thereof in
a form that allows a skilled artisan to examine the sequences.
[0312] A further preferred subset of nucleic acid sequences is
where the subset of sequences is two proteins or fragments thereof,
more preferably three proteins or fragments thereof and even more
preferable four proteins or fragments thereof.
[0313] In one application of this embodiment, a nucleotide sequence
of the invention can be recorded on computer readable media so that
a computer-readable medium comprises one or more of the nucleotide
sequences of the invention. As used herein, "computer readable
media" refers to any medium that can be read and accessed directly
by a computer. Such media include, but are not limited to: magnetic
storage media, such as floppy discs, hard disc, storage medium and
magnetic tape; optical storage media such as CD-ROM; electrical
storage media such as RAM and ROM; and hybrids of these categories
such as magnetic/optical storage media.
[0314] Any number of the sequences, or sequence fragments, of the
nucleic acid molecules or proteins of the invention, or fragments
of either, can be included, in any number of combinations, on a
computer-readable medium. Specifically, any one or more of SEQ ID
NO: 1-626, or where appropriate, complements thereof, can be
included.
[0315] A skilled artisan can readily appreciate how any computer
readable medium can be used to create a machine or method
comprising a computer readable medium having recorded thereon a
nucleotide sequence of the invention. As used herein, "recorded"
refers to a process for storing information on computer readable
medium. A skilled artisan can readily adopt any method for
recording information on computer readable medium to generate media
comprising the nucleotide sequence information of the invention. A
variety of data storage structures are available to a skilled
artisan for creating a computer readable medium having recorded
thereon a nucleotide sequence of the invention. The choice of the
data storage structure will generally be based on the means chosen
to access the stored information. In addition, a variety of data
processor programs and formats can be used to store the nucleotide
sequence information of the invention on computer readable medium.
The sequence information can be represented in a word processing
text file, formatted in commercially-available software such as
WordPerfect or Microsoft Word, or represented in the form of an
ASCII file, stored in a database application, such as DB2, Sybase,
Oracle, or the like. A skilled artisan can readily adapt any number
of data processor structuring formats (e.g., text file or database)
in order to obtain computer readable medium having recorded thereon
the nucleotide sequence information of the invention.
[0316] By providing one or more of nucleotide sequences of the
invention, a skilled artisan can routinely access the sequence
information for a variety of purposes. Computer software is
publicly available that allows a skilled artisan to access sequence
information provided in a computer readable medium. The examples
which follow demonstrate how software which implements the BLAST
(Altschul et al., J. Mol. Biol. 215:403-410 (1990), the entirety of
which is herein incorporated by reference) and BLAZE (Brutlag et
al., Comp. Chem. 17:203-207 (1993), the entirety of which is herein
incorporated by reference) search algorithms on a Sybase system can
be used to identify open reading frames (ORFs) within the genome
that contain homology to ORFs or proteins from other organisms.
Such ORFs are protein-encoding fragments within the sequences of
the invention and are useful in producing commercially important
proteins such as enzymes used in amino acid biosynthesis,
metabolism, transcription, translation, RNA processing, nucleic
acid and a protein degradation, protein modification and DNA
replication, restriction, modification, recombination and
repair.
[0317] The invention further provides systems, particularly
computer-based systems, which contain the sequence information
described herein. Such systems are designed to identify
commercially important fragments of the nucleic acid molecule of
the invention. As used herein, "a computer-based system" refers to
the hardware means, software means and data storage means used to
analyze the nucleotide sequence information of the invention. The
minimum hardware means of the computer-based systems of the
invention comprises a central processing unit (CPU), input means,
output means and data storage means. A skilled artisan can readily
appreciate that any one of the currently available computer-based
system are suitable for use in the invention.
[0318] As indicated above, the computer-based systems of the
invention comprise a data storage means having stored therein a
nucleotide sequence of the invention and the necessary hardware
means and software means for supporting and implementing a search
means. As used herein, "data storage means" refers to memory that
can store nucleotide sequence information of the invention, or a
memory access means which can access manufactures having recorded
thereon the nucleotide sequence information of the invention. As
used herein, "search means" refers to one or more programs which
are implemented on the computer-based system to compare a target
sequence or target structural motif with the sequence information
stored within the data storage means. Search means are used to
identify fragments or regions of the sequence of the invention that
match a particular target sequence or target motif. A variety of
known algorithms are disclosed publicly and a variety of
commercially available software for conducting search means are
available can be used in the computer-based systems of the
invention. Examples of such software include, but are not limited
to, MacPattern (EMBL), BLASTIN and BLASTIX (NCBIA). One of the
available algorithms or implementing software packages for
conducting homology searches can be adapted for use in the present
computer-based systems.
[0319] The most preferred sequence length of a target sequence is
from about 10 to 100 amino acids or from about 30 to 300 nucleotide
residues. However, it is well recognized that during searches for
commercially important fragments of the nucleic acid molecules of
the invention, such as sequence fragments involved in gene
expression and protein processing, may be of shorter length.
[0320] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequences the sequence(s) are chosen
based on a three-dimensional configuration which is formed upon the
folding of the target motif. There are a variety of target motifs
known in the art. Protein target motifs include, but are not
limited to, enzymatic active sites and signal sequences. Nucleic
acid target motifs include, but are not limited to, promoter
sequences, cis elements, hairpin structures and inducible
expression elements (protein binding sequences).
[0321] Thus, the invention further provides an input means for
receiving a target sequence, a data storage means for storing the
target sequences of the invention sequence identified using a
search means as described above and an output means for outputting
the identified homologous sequences. A variety of structural
formats for the input and output means can be used to input and
output information in the computer-based systems of the invention.
A preferred format for an output means ranks fragments of the
sequence of the invention by varying degrees of homology to the
target sequence or target motif. Such presentation provides a
skilled artisan with a ranking of sequences which contain various
amounts of the target sequence or target motif and identifies the
degree of homology contained in the identified fragment.
[0322] A variety of comparing means can be used to compare a target
sequence or target motif with the data storage means to identify
sequence fragments sequence of the invention. For example,
implementing software which implement the BLAST and BLAZE
algorithms (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) can
be used to identify open frames within the nucleic acid molecules
of the invention. A skilled artisan can readily recognize that any
one of the publicly available homology search programs can be used
as the search means for the computer-based systems of the
invention.
[0323] Having now described the invention, the following examples
are provided by way of illustration and are not intended to limit
the scope of the invention, unless specified.
EXAMPLE 1
Identification of Yeast HES1
[0324] The yeast strain LPY9 (MATa, leu2, Ura3, his3) is grown
overnight and inoculated into SD+ hul (histidine, uracil, leucine)
media. Aliquots of the culture are treated with ketoconazole (an
inhibitor of C-14.alpha. demethylase (P450.sub.14DM) enzyme) at 10
ug/ml, 50 ug/ml, and 100 ug/ml, corresponding to 10 ppm, 50 ppm,
and 100 ppm, respectively. A sample of each is collected at 2, 4,
and 6 hours after treatment. Control samples treated with DMSO
(dimethyl sulfoxide-solvent for ketoconazole) but not with
ketoconazole are also collected. Total RNA from each sample is
collected by conventional methods, such as a Zirconium/Silica bead
binding and extraction method. The sequence content of each sample
is analyzed and compared by hybridizing each of them to a number of
yeast ORF sequences immobilized on a Nylon membrane in an array
format.
[0325] A similar comparison of a wild type yeast strain and a
double mutant strain is made. The double mutant CJ517 (MATa,
erg11::URA3, erg3::LEU2, leu2, ura3, his4)[erg11, erg3 double
mutant] is compared to LPY9 after growth in both YPD and SD+hul
media. Samples are collected at approximately 0, 2, 4, and 6 hours
after inoculation.
[0326] Table 2, below, lists the RNAs in each sample whose
abundance is effected by ketoconazole treatment or whose abundance
differs between wild type and the double mutant strain. The table
also lists the corresponding gene or sequence identifier for those
RNAs. The RNAs are ranked by the ratio of either ketocanozole vs.
control or mutant vs. control, using the ratio of 50 ppm
ketocanozole/control as a basis for comparison.
2TABLE 2* Seq. CJ-4 hr/ Num. Clone ID ALIAS LP-4 hr K-50/CK
K-100/CK Gene Description 30 YOR237W (HES1) 134.648161 1417.6262
1358.1235 Protein implicated in ergosterol biosynthesis, member of
the KES1/HES1/OSH1/YKR003W family of oxysterol-binding (OSBP)
proteins 31 YKL198C (PTK1) 68.5845326 111.1984 233.11762
Serine/threonine protein kinase, activator of low-affinity, low-
capacity polyamine transport 32 YLR465C -- 97.9601498 104.52215
133.57826 Protein of unknown function, questionable ORF 33 YMR129W
(POM152) 5.10206225 82.813831 15.392788 Nuclear pore membrane
glycoprotein, type II integral membrane protein with N-terminal
region on pore side and C-terminal region in the cisternae 34
YBR284W -- 4.92774291 60.027955 8.5359554 Protein with similarity
to AMP deaminase 35 YKL158W -- 11.6717854 59.827307 75.220412
Protein of unknown function 36 YOR083W -- 31.7378598 51.606081
42.301568 Protein of unknown function 37 YOL095C -- 3.60507866
49.740211 21.834188 Protein with similarity to S. aureus DNA
helicase PCRA 38 YOR188W (MSB1) 2.19997209 42.446767 61.303817
Protein that may play a role in polarity establishment and bud
formation 39 YBL109W -- 0.08616121 38.653463 75.964757 -- 40
YLR091W -- 17.5946744 38.325073 44.556481 Protein of unknown
function 41 YNL106C (INP52) 2.52986454 35.205536 17.376557 -- 42
YDR213W -- 18.2079478 32.136065 58.358612 Protein with similarity
to transcription factors, has ZN[2]- CYS[6] fungal-type binuclear
cluster domain in the N-terminal region 43 YBL004W -- 8.49387973
28.614573 28.645633 Protein with similarity to members of the major
facilitator superfamily (MFS) 44 YIR019C (MUC1) 48.7538739
27.594853 137.77885 Glucoamylase I (alpha-1,4-glucan glucosidase),
extracellular enzyme 45 YJL182C -- 2.53469593 26.891434 29.499298
Protein of unknown function 46 YMR254C -- 0.19897977 26.633459
10.625738 Protein of unknown function, questionable ORF 47 YDL134C
(PPH21) 3.51284473 22.849241 0 -- 48 YCR098C (GIT1) 2.27672091
21.746838 24.724171 Protein involved in inositol metabolism 49
YPL150W -- 4.72964069 21.633895 34.40982 Serine/threonine protein
kinase of unknown function 50 YKL110C (KTI12) 19.7752946 21.085633
16.303432 Protein involved in resistance to kluyveromyces lactis
killer toxin 51 YER011W (TIR1) 31.4723195 20.454605 17.935906
Stress-induced cell wall structural protein of the PAU1 family 52
YDL024C -- 3.96163383 20.381493 30.488098 Protein with similarity
to acid phosphatases 53 YGR013W -- 0.10491681 20.364081 0 -- 54
YOR325W -- 47.3518002 20.211317 29.305064 Protein of unknown
function 55 YJR150C -- 159.265973 19.793221 13.560079 -- 56 YDL126C
(CDC48) 42.7590386 19.0472 15.014024 Protein of the AAA family of
ATPases, required for cell division and homotypic membrane fusion
57 YLR464W -- 12.4297115 18.580843 36.516503 Protein with
similarity to other subtelomerically-coded proteins 58 YLR124W --
0.13902212 18.351487 11.026125 Protein of unknown function 59
YLR463C -- 8.49721471 18.007814 29.811632 Protein with similarity
to other subtelomerically-coded proteins 60 YMR297W (PRC1)
6.20117404 17.995865 24.291751 Carboxypeptidase Y (CPY) (YSCY),
serine-type protease 61 YFL029C (CAK1) 17.1104765 16.96782
44.352291 CDK-activating kinase (serine/threonine protein kinase)
responsible for in vivo activation of CDC28P, also involved in
spore wall formation 62 YER054C (GIP2) 2.14214491 16.442373
15.284537 GLC7P-interacting protein, possible regulatory subunit
for the PP1 family protein phosphatase GLC7P 63 YER060W-A (FCY22)
2.61677424 15.768882 20.550953 Purine-cytosine permease with
similarity to FCY2P, member of the purine/cytosine family of the
major facilitator superfamily (MFS) 64 YEL076C -- 13.7918147
14.372278 26.325282 Protein with similarity to other
subtelomerically-encoded proteins 65 YGL176C -- 9.0823019 14.17085
16.23816 Protein with similarity to discopyge OMMATA CA++ channel
alpha1 subunit protein B47447 66 YNR005C -- 12.9230524 14.032659
13.011356 Protein of unknown function, questionable ORF 67
YML032C-A -- 6.92372404 13.847081 5.501802 -- 68 YGR190C --
22.9885796 13.701633 42.22779 Protein of unknown function 69
YHR213W -- 17.3140804 13.267403 21.010074 Protein with similarity
to the N- terminus of FLO1P and identical to YAR062P, probable
pseudogene 70 YPL272C -- 24.778114 12.93877 11.647985 Protein of
unknown function 71 YBL100C -- 4.8456884 12.432421 16.193059
Protein of unknown function 72 YLR024C -- 11.2130442 11.927798
17.73046 Protein with similarity to ubiquitin- protein ligase (E3)
UBR1P 73 YMR102C -- 4.61311719 11.865115 16.370862 -- 74 YGR177C
(ATF2) 3.7081426 11.830167 12.555269 Alcohol O-acetyltransferase 75
YFR034C (PHO4) 14.8112083 11.216073 20.844515 Basic
helix-loop-helix (BHLH) transcription factor required for
expression of phosphate pathway, hyperphosphorylation by
PHO80P-PHO85P cyclin-dependent protein kinase complex causes
inactivation 76 YNL282W -- 5.01708646 10.943286 13.050614 -- 77
YPL176C -- 7.30789994 10.664169 18.424583 Protein with similarity
to SSP134P 78 YMR015C (ERG5) 10.2651358 10.313689 9.3557963
Cytochrome P450 (C-22 sterol desaturase) 79 YCR061W -- 4.07462743
10.291287 12.602668 Protein of unknown function 80 YHL030W (ECM29)
4.85453872 10.275837 8.9818305 Protein possibly involved in cell
wall structure or biosynthesis 81 YPL036W (PMA2) 7.19300398
10.171951 12.917306 H+-transporting P-type ATPase of the plasma
membrane, expression not detected under normal growth conditions 82
YFR007W -- 2.58144987 10.102403 6.0105766 Protein of unknown
function 83 YOL067C (RTG1) 30.4142081 10.027065 27.36633 Basic
helix-loop-helix (BHLH) transcription factor involved in
inter-organelle communication between mitochondria, peroxisomes,
and nucleus 84 YGR265W -- 22.156977 9.9554618 5.672919 Protein of
unknown function 85 YGR293C -- 51.4998515 9.7686634 8.066486
Protein of unknown function 86 YMR008C (PLB1) 5.68517668 9.602215
11.309345 Phospholipase B (lysophospholipase), releases fatty acids
from lysophospholipids 87 YOR140W -- 6.33829162 9.2015298 12.881145
-- 88 YML034W -- 4.44092944 9.2011248 15.848216 Protein of unknown
function 89 YGR176W -- 4.56487981 8.8866015 12.598661 Protein of
unknown function 90 YOR014W (RTS1) 7.03478812 8.8422619 11.590438
Protein serine/threonine phosphatase 2A (PP2A), B' regulatory
subunit, involved in regulation of stress-related responses and the
cell cycle 91 YMR317W -- 25.9636363 8.6834125 11.973301 Protein of
unknown function 92 YOR301W -- 11.3702021 8.6327901 13.589223
Protein of unknown function 93 YER119C-A -- 8.9509545 8.4086333
6.8517264 -- 94 YOR385W -- 6.30021483 8.3714543 10.537348 Protein
of unknown function 95 YGL156W (AMS1) 11.9450551 8.2732125
9.9190578 Alpha-mannosidase, hydrolyzes terminal non-reducing
alpha-D- mannose residues from alpha-D- mannosides 96 YJL219W
(HXT9) 6.10093958 8.1969449 14.860533 Hexose transporter, member of
the sugar permease family 97 YFL053W -- 3.55404282 8.1217569
6.04425 -- 98 YNL279W -- 2.75618909 8.0041323 12.470971 Protein of
unknown function 99 YHR007C (ERG11) 5.511691 7.8623796 8.6320676
Cytochrome P450 (lanosterol 14alpha-demethylase), essential for
biosynthesis of ergosterol 100 YJL127C (SPT10) 4.01528284 7.8394427
10.096027 Protein that amplifies the magnitude of transcriptional
regulation at various loci 101 YPL044C -- 2.61973879 7.8291062
4.5399013 Protein of unknown function 102 YOR030W (DFG16)
4.97362211 7.8182123 10.573213 Protein involved in invasive growth
upon nitrogen starvation 103 YIL011W -- 4.59710634 7.3954743
6.7112038 Protein with similarity to YIL176P, YIR041P and other
members of the PAU1 family 104 YNR069C -- 14.3161508 7.3694614
14.104044 Protein of unknown function 105 YNL083W -- 2.06305137
7.3050052 15.674556 Protein of the mitochondrial carrier (MCF)
family 106 YJL020C -- 6.76775321 7.0352757 5.3432583 Protein of
unknown function 107 YFL065C -- 13.5712126 7.0075571 16.704839
Protein with similarity to other subtelomerically-encoded proteins
including YHL049P, YIL177P, YJL225P, YER190P, YHR218P, and YEL076P
108 YNL329C (PAS8) 3.75487269 6.7699941 25.980939 -- 109 YHR006W
(STP2) 6.44648003 6.5480808 9.270283 Protein involved in TRNA
splicing and branched-chain amino acid uptake 110 YJL221C (FSP2)
2.37104879 6.4365653 6.3055084 Protein with similarity to alpha-D-
glucosidase (maltase) (FSP2 and YIL172C code for identical
proteins) (YIL172C and YGR287C are nearly identical) 111 YMR037C
(MSN2) 6.80686734 6.4235969 7.6612989 Zinc-finger transcriptional
activator for genes involved in the multistress response and genes
regulated through SNF1P 112 YLR379W -- 6.34038543 6.4227358
6.8206953 Protein of unknown function 113 YLR056W (ERG3) 0.03858406
6.2735601 5.191422 C-5 sterol desaturase, an iron non- heme
oxygen-requiring enzyme of the ergosterol biosynthesis pathway 114
YMR319C (FET4) 3.5515443 6.2641804 8.194608 Low-affinity Fe(II)
transport protein 115 YBR045C (GIP1) 5.88011982 6.254107 3.8135044
GLC7P-interacting protein, possible regulatory subunit for the PP1
family protein phosphatases GLC7P 116 YKL147C -- 4.54862611
6.2431328 10.034699 Protein of unknown function 117 YMR135W-A --
15.3287997 6.1049555 4.611173 -- 118 YCR048W (ARE1) 9.11370518
6.1039374 10.531291 Acyl-COA: sterol acyltransferase (sterol-ester
synthetase) 119 YBR235W -- 2.65851474 6.1026186 2.9854465 Protein
with similarity to human SLC12A1 gene for which mutations are the
cause of Bartter's Syndrome 120 YJL160C -- 5.14571281 6.0795621
6.0193217 Protein with similarity to members of the
PIR1P/HSP150P/PIR3P family 121 YNL287W (SEC21) 5.55890054 6.0742978
5.8985117 Coatomer complex gamma chain (gamma-COP) of secretory
pathway vesicles, required for retrograde Golgi to endoplasmic
reticulum transport 122 YLR458W -- 28.2501296 5.9435623 4.6311951
-- 123 YLR121C -- 4.04284936 5.9154936 8.131848 -- 124 YLR347C
(KAP95) 3.84797845 5.8759152 6.4154978 Karyopherin-beta, acts to
target proteins with nuclear localization (NLS) sequences to the
nuclear pore complex 125 YDL023C -- 3.26329833 5.8589624 4.7058193
Protein of unknown function 126 YAL010C (MDM10) 5.34077952 5.807758
8.9195451 Protein involved in mitochondrial morphology and
inheritance, mutant has large spherical mitochondria that do not
move into the bud 127 YDR077W (SED1) 3.30340602 5.6959082 5.9206909
Abundant cell surface glycoprotein, overexpression suppresses
growth defect of ERD2 128 YDR247W -- 3.28497642 5.6793015 6.7651448
Serine/threonine protein kinase with similarity to S. pombe RAN1
negative regulator of sexual conjugation and meiosis (GB: Z49701)
129 YBL011W -- 3.59243122 5.650363 8.393684 -- 130 YDL025C --
2.91426204 5.5604876 3.9241843 Serine/threonine protein kinase with
similarity to members of the NPR1 subfamily 131 YAL013W (DEP1)
8.79366086 5.5463386 6.42501 Regulator of phospholipid metabolism
132 YIL084C (SDS3) 1.99582364 5.5430688 6.9074225 Suppressor of
silencing defect 133 YJL213W -- 7.09632444 5.4980741 5.5079382
Protein with weak similarity to nocardia aryldialkylphosphatase 134
YKR053C -- 5.37724431 5.4952302 6.4562635 -- 135 YNR042W --
17.7115615 5.4798109 7.5527661 Protein of unknown function 136
YCR072C -- 5.34712592 5.4565375 4.5985045 Protein with similarity
to nuclear MRNA processing protein PRP4P, member of WD (WD-40)
repeat family 137 YER086W (ILV1) 4.55278717 5.4449008 4.2437712
Serine and threonine dehydratase (anabolic), first step in
isoleucine biosynthesis pathway 138 YJL076W -- 11.4128793 5.4277219
6.6898119 -- 139 YLR072W -- 5.19287856 5.4152299 7.2827024 Protein
of unknown function 140 YDR301W (YHH1) 2.51614995 5.4121298
7.0975432 -- 141 YIL055C -- 2.0005314 5.3410327 4.6542324 Protein
of unknown function 142 YEL076W-C -- 13.2032684 5.3265661 8.0731692
-- 143 YNR047W -- 4.44731559 5.3217828 6.1790659 Serine/threonine
protein kinase of unknown function 144 YGL211W -- 4.00934024
5.2957602 5.5379668 Protein of unknown function 145 YGL012W (ERG4)
4.57738431 5.2945042 4.833773 Sterol C-24 reductase 146 YCL014W
(BUD3) 2.0970839 5.2855114 3.3963317 Protein localized at the neck
filament ring required for axial budding, may provide a memory of
the previous bud site 147 YBR106W -- 5.74228482 5.2537051 9.2061479
-- 148 YHR095W -- 5.25923706 5.2434619 2.2666062 Protein of unknown
function 149 YEL010W -- 3.39547744 5.2424909 3.9026395 Protein of
unknown function 150 YBR005W -- 5.58242328 5.2283592 7.5591013
Protein of unknown function 151 YPL183C -- 3.25331232 5.2150911
4.034456 Protein of unknown function, has WD (WD-40) repeats 152
YJL159W -- 5.95901062 5.2095163 5.0420867 -- 153 YBL065W --
5.04084137 5.1918263 10.287249 Protein of unknown function 154
YDL071C -- 7.24874297 5.1844239 7.5184825 Protein of unknown
function 155 YGR197C (SNG1) 6.43784806 5.17339 7.8870948 Probable
transport protein that confers resistance to MNNG and
nitrosoguanidine 156 YLL028W -- 9.27382002 5.0519624 5.3421753
Member of major facilitator superfamily (MFS) multidrug- resistance
(MFS-MDR) protein family 157 YKR034W (DAL80) 3.91750209 5.0436172
7.2838566 GATA-type zinc finger transcriptional repressor for
allantoin and 4-aminobutyric acid (GABA) catabolic genes 158
YDR430C -- 2.19022255 5.0401778 3.1989703 Protein with similarity
to Class I family of aminoacyl-TRNA synthetases 159 YPL274W --
5.4156341 5.0164198 6.1307085 Protein with similarity to GAP1P and
other amino acid permeases 160 YMR261C (TPS3) 3.96385669 4.94376
3.7501015 Component of the trehalose-6- phosphate
synthase/phosphatase complex, alternate third subunit with TLS1P
161 YOL118C -- 3.20265396 4.936553 5.7544219 Protein of unknown
function 162 YOR005C (DNL4) 4.47086248 4.8815521 3.6707508
ATP-dependent DNA ligase IV, involved in non-homologous DNA end
joining 163 YNL332W -- 3.33896215 4.8789948 4.7570682 -- 164
YDR069C (DOA4) 3.37810593 4.8769723 5.1947947 Ubiquitin-specific
protease (ubiquitin C-terminal hydrolase) of the 26S proteasome
complex, involved in vacuole biogenesis and osmoregulation 165
YOR009W -- 59.4543494 4.8708102 5.2948993 Protein with similarity
to members of the PAU1 family 166 YMR035W (IMP2) 9.23409301
4.8492871 5.7664813 Inner membrane protease of mitochondria, acts
in complex with IMP1P but has different substrate specificity for
removal of signal peptidase 167 YER089C (PTC2) 2.23920866 4.8455014
5.8687657 Protein serine/threonine phosphatase of the PP2C family
168 YJR018W -- 5.54754057 4.8389334 4.4934937 Protein of unknown
function 169 YLR088W (GAA1) 3.1893544 4.814116 4.0142997 Protein
required for attachment of GPI anchor onto proteins, affects
endocytosis 170 YOL163W -- 3.92239312
4.8014959 4.5124682 Protein with weak similarity to pseudomonas
putida phthalate transporter 171 YLR462W -- 3.32915042 4.7928645
7.1350658 Protein of unknown function 172 YLR098C (CHA4) 2.05280928
4.7564347 5.5866465 Zinc-finger protein required for activation of
CHA1, has A ZN[2]- CYS[6] fungal-type binuclear cluster domain 173
YNR053C -- 2.55991235 4.7234659 3.8186389 Protein with similarity
to human breast tumor-associated autoantigen 174 YDL246C --
2.43826188 4.6757263 3.5757353 Protein with similarity to SOR1P
(SOR1 and YDL246C code for nearly identical proteins) 175 YOL045W
-- 3.55662236 4.672513 2.0538279 Serine/threonine protein kinase of
unknown function 176 YKL176C -- 3.32695888 4.6429893 5.4538239
Protein of unknown function 177 YJR114W -- 3.00664482 4.6389866
4.0045917 Protein of unknown function 178 YER091C (MET6) 6.67067887
4.6224571 2.9292597 Homocysteine methyltransferase
(5-methyltetrahydropteroyl triglutamate - homocysteine
methyltransferase), methionine synthase, cobalamin-independent 179
YHL049C -- 5.15537247 4.5637645 9.5066446 Protein with similarity
to other subtelomerically-encoded proteins including YER189P,
YML133P, and YJL225P, coded from a subtelomeric Y' region 180
YDR389W (SAC7) 3.89197011 4.5609599 4.3143109 GTPase-activating
protein for RHO1P 181 YMR202W (ERG2) 9.58572292 4.5446614 5.575174
Sterol C8-C7 isomerase (C-8 sterol isomerase), enzyme of the
ergosterol biosynthesis pathway 182 YBL019W -- 3.45990928 4.4694518
4.1655454 -- 183 YGR287C -- 10.2933872 4.4595137 9.7718104 Protein
with similarity to alpha-D- glucosidase (maltase) (YGR287C IS
nearly identical to FSP2 and YILI72C) 184 YJL082W -- 7.42175571
4.4522595 5.556901 Protein of unknown function 185 YHR098C --
2.51284975 4.4353768 4.3716652 Protein of unknown function 186
YOR371C -- 2.47743776 4.4289864 5.2783501 Protein of unknown
function 187 YDR530C (APA2) 2.40849553 4.3993312 2.7389073 ATP
adenylyltransferase II (AP4A phosphorylase) 188 YKL119C (VPH2)
0.16462534 4.3613346 0 Vacuolar H-ATPase (V-ATPase) assembly
protein acting in the endoplasmic reticulum 189 YOR273C --
13.0544715 4.3469302 10.649131 Protein with similarity to members
of major facilitator superfamily (MFS) multidrug-resistance (MFS-
MDR) protein family 190 YPL042C (SSN3) 6.78272968 4.3344728
3.9578568 Cyclin-dependent serine/threonine protein kinase of the
RNA polymerase II holoenzyme complex and Kornberg's mediator (SRB)
subcomplex 191 YGR268C -- 4.77373538 4.3329069 5.2744105 Protein of
unknown function 192 YPR011C -- 2.0077462 4.3123349 4.2742986
Protein with similarity to human Grave's Disease carrier protein
(SP: P16260) and to bovine homolog of Grave's Disease carrier
protein (SP: Q01888) 193 YPL022W (RAD1) 4.48327554 4.3036056
6.5285426 Component of the nucleotide excision repairosome, homolog
of human XPF xeroderma pigmentosum gene product and the mammalian
ERCC-4 protein 194 YGL207W (SPT16) 5.34289635 4.3033021 3.5727713
General chromatin factor required for adequate expression of CLN
and other genes 195 YGL167C (PMR1) 4.12359747 4.2628564 4.8141347
CA++-transporting P-type ATPase of Golgi membrane involved in CA++
import into Golgi 196 YJR091C (JSN1) 4.56429439 4.2419881 4.7804157
Protein that when overexpressed can suppress the hyperstable
microtubule phenotype of TUB2- 150 197 YDR238C (SEC26) 4.48641405
4.2179222 3.8109695 Coatomer complex beta chain (beta-COP) of
secretory pathway vesicles, required for retrograde transport from
Golgi to endoplasmic reticulum 198 YDL012C -- 2.90930997 4.2158147
2.0519798 Protein of unknown function 199 YDR044W (HEM13)
14.9283272 4.2136787 3.4946018 Coproporphyrinogen III oxidase,
oxygen-repressed, sixth step in heme-biosynthetic pathway 200
YGL114W -- 3.22707938 4.2023503 5.0073787 Protein with similarity
to S. pombe ISP4 protein, member of the major facilitator
superfamily (MFS) 201 YGL055W (OLE1) 2.29875509 4.1923045 3.5992372
Stearoyl-COA desaturase (delta-9 fatty acid desaturase), required
for synthesis of unsaturated fatty acids 202 YDL088C (ASM4)
4.39685251 4.1757265 3.321034 Suppressor of temperature- sensitive
mutations in POL3P (DNA polymerase delta) 203 YKL171W -- 2.64137608
4.1581147 8.2933538 Serine/threonine protein kinase of unknown
function 204 YPL190C -- 5.94196213 4.1575162 3.202837 -- 205
YMR140W -- 5.24432896 4.157179 5.4545409 Protein of unknown
function 206 YBL005W (PDR3) 3.75060207 4.1449054 6.0827305
Transcription factor related to PDR1P, contains a ZN[2]-CYS[6]
fungal-type binuclear cluster domain in the N-terminal region 207
YML032C (RAD52) 3.13968668 4.1330793 3.0832115 Protein required for
recombination and repair of X-ray damage, has a late function in
meiotic recombination 208 YFR018C -- 5.28886874 4.1041589 6.4001917
Protein with similarity to human glutaminyl-peptide
cyclotransferase 209 YGL125W (MET11) 6.80542292 4.0762178 4.4382484
-- 210 YCR057C (PWP2) 3.34704165 4.0555292 3.4118145 -- 211 YBL044W
-- 4.67885642 4.0526493 9.1998322 Protein of unknown function 212
YPL268W (PLC1) 2.90633764 4.0372127 2.1993847
Phosphoinositide-specific phospholipase C (1-
phosphatidylinositol-4,5- bisphosphate phosphodiesterase 1),
produces diacylglycerol and inositol 1,4,5-trisphosphate 213
YOR204W (DED1) 2.52920945 4.0291663 3.0830731 ATP-dependent RNA
helicase of dead box family involved in protein synthesis 214
YPL171C (OYE3) 4.94122983 4.0225239 0.2747214 NAPDH dehydrogenase
(old yellow enzyme), isoform 3 215 YOR203W -- 3.473713 3.9900922
3.2232019 Protein of unknown function 216 YNL295W -- 2.855052
3.9889367 2.1389666 Protein of unknown function 217 YEL042W (GDA1)
2.00067395 3.9856733 3.8058139 Guanosine diphosphatase of Golgi
membrane 218 YLR339C -- 4.12619065 3.9844972 3.506939 Protein of
unknown function 219 YIL007C -- 5.11957134 3.9770005 2.318674
Protein of unknown function 220 YIR007W -- 3.71972069 3.9670484
5.0861072 Protein with similarity to endoglucanase 221 YER114C
(BOI2) 2.5967273 3.9643546 6.3042836 Protein with SH3 domain
involved in bud formation, binds to BEM1P 222 YLR092W (SUL2)
6.02237117 3.9547891 4.5438793 High-affinity sulfate transporter
223 YEL060C (PRB1) 5.60961951 3.939317 4.7370327 Protease B (YSCB)
(PRB) (cerevisin), serine protease of the subtilisin family with
broad proteolytic specificity 224 YAL051W -- 2.40553928 3.9334781
4.5099518 -- 225 YJR147W -- 2.05911726 3.9267937 5.1956856 -- 226
YOR386W (PHR1) 1.99823774 3.9204076 5.7096569 Deoxyribodipyrimidine
photolyase, involved in light-dependent repair of pyrimidine dimers
227 YCR037C (PHO87) 2.81200613 3.8982171 4.0311884 Member of the
phosphate permease family of the major facilitator superfamily 228
YOL100W -- 3.04632994 3.8854218 9.2592192 Serine/threonine protein
kinase of unknown function 229 YBL047C -- 3.40597241 3.88363
5.2814809 Protein with similarity to cytoskeletal protein USO1P,
PAN1P, and mouse tyrosine kinase substrate EPS15 230 YAR014C --
2.53512963 3.823947 4.4065791 Protein of unknown function 231
YKL182W (FAS1) 3.75368336 3.8068781 5.3493259 Fatty-acyl-COA
synthase, beta chain (contains acetyl transferase, enoyl reductase,
dehydratase, and malonyl/palmitoyl transferase) 232 YLR331C --
3.94099666 3.795387 3.3715843 Protein of unknown function 233
YEL031W (SPF1) 7.77512435 3.7891074 4.357615 Protein with
similarity to CA++- transporting ATPases 234 YHR078W -- 2.2941334
3.7838221 4.6151917 Protein of unknown function, has 4 potential
transmembrane domains 235 YPL155C (KIP2) 3.29502679 3.7807978
9.392792 Kinesin-related protein 236 YNR074C -- 4.3061075 3.7638306
5.7991531 Protein with similarity to Bacillus subtilis nitrite
reductase (NIRB) 237 YMR303C (ADH2) 4.56919214 3.7542967 3.1957867
Alcohol dehydrogenase II, glucose- repressed 238 YLR134W (PDC5)
4.9450653 3.7528169 3.2704832 Pyruvate decarboxylase isozyme 2 239
YKL067W (YNK1) 4.49102455 3.7325797 3.6497934 Nucleoside
diphosphate kinase, responsible for synthesis of all nucleoside
triphosphates except ATP 240 YLR136C (TIS11) 2.88004451 3.7255421
5.1711798 -- 241 YDR443C (SCA1) 2.75733315 3.7068432 6.9331513 --
242 YGL071W (RCS1) 3.39203358 3.6963077 4.5310166 Regulatory
protein involved in IRON uptake 243 YBR293W -- 2.25740646 3.6840827
3.0384171 Member of major facilitator superfamily (MFS) multidrug-
resistance (MFS-MDR) protein family 244 YMR324C -- 3.33053542
3.6802526 2.8779503 Protein with similarity to members of the
YBL108P/YCR103P/ YKL223P family 245 YFL051C -- 2.07690974 3.6611179
4.7476201 Protein with similarity to FLO1P family of proteins 246
YBR276C (PPS1) 2.35950244 3.6550406 3.4539593 Protein tyrosine
phosphatase (PTPase) with dual specificity 247 YFL042C --
3.57726533 3.6509118 4.5694594 Protein of unknown function, has
similarity to YHR080P 248 YPL263C (KEL3) 4.50871509 3.6484792
3.8498382 Protein with similarity to KEL1P and KEL2P 249 YLR188W
(MDL1) 5.00498919 3.6478982 4.3936321 ATP-binding cassette (ABC)
superfamily member, equivalent to a "half-molecule" ABC protein
plus an ATP-binding domain 250 YPR021C -- 2.21061647 3.6466639
3.2312479 Protein with similarity to proteins of the mitochondrial
carrier (MCF) family (GB: Z49274) 251 YKL138C (MRPL31) 3.22538649
3.6454084 4.0488722 Mitochondrial ribosomal protein of the large
subunit (YML31) 252 YNL148C (ALF1) 3.33997835 3.6391378 5.2515594
Alpha-tubulin foldin, cofactor B 253 YLR302C -- 10.5636377
3.6318383 0.3621924 Protein of unknown function 254 YBR298C (MAL31)
5.44502575 3.6302693 9.8311328 High affinity maltoaseH+ symporter
(maltose permease) member of the sugar permease family 255 YAR044W
(OSH1) 4.12112011 3.624939 3.8839622 Protein implicated in
ergosterol biosynthesis, member of the KES1/HES1/OSH1/YKR003W
family of oxysterol-binding (OSBP) proteins 256 YLR120C (YAP3)
6.14265883 3.6229845 4.4298562 Transcription factor of the basic
leucine zipper (BZIP) family, one of eight members of a novel
fungal-specific family of BZIP proteins 257 YGR134W -- 2.8756723
3.6189405 1.9505784 Protein of unknown function 258 YMR088C --
3.01763425 3.574571 2.5742717 Member of major facilitator
superfamily (MFS) multidrug- resistance (MFS-MDR) protein family 2
259 YDR291W -- 4.95353348 3.5637613 2.8803997 Protein with
similarity to SGS1P and other DNA helicases 260 YJR017C (ESS1)
2.98118086 3.5587415 3.2208256 Processing/termination factor,
involved in transcription termination or 3'-end processing of
pre-MRNA 261 YGL178W (MPT5) 4.28561965 3.558276 3.3338238 Protein
required for high temperature growth, recovery from alpha-factor
arrest, and normal lifespan of yeast cells 262 YHR086W (NAM8)
2.63503306 3.556686 4.1441189 U1 SNRNA-associated protein,
essential for meiotic recombination and suppressor of mitochondrial
splicing defects, has 3 RNA recognition (RRM) domains 263 YGR178C
(PBP1) 2.95926792 3.5559294 3.6095103 poly(A)-binding protein 264
YBL022C (PIM1) 4.1993836 3.5255118 3.2518435 Serine protease
required for intramitochondrial proteolysis and maintenance of
respiratory function, related to E. coli ATP- dependent protease LA
265 YJL083W -- 3.34026267 3.5131828 5.6812601 Protein with
similarity to IRS4P 266 YJR053W -- 2.12253894 3.5096202 4.5401439
Protein involved in efficiency of mating 267 YJL175W -- 6.11781731
3.5040684 3.7938536 Protein of unknown function 268 YMR016C --
3.67893179 3.4720987 3.3111279 -- 269 YLL051C (FRE6) 2.59520796
3.4643555 4.4566151 Protein with similarity to ferric reductase
FRE2P 270 YJL212C -- 4.42206996 3.458335 4.0764022 Protein with
similarity to S. pombe ISP4+ which is induced by sexual
differentiation 271 YMR019W (STB4) 3.2576922 3.4414621 3.397646
SIN3P-binding protein, has ZN[2]- CYS[6] fungal-type binuclear
cluster domain in the N-terminal region 272 YHL047C -- 3.02606918
3.4089434 3.007693 Member of major facilitator superfamily (MFS)
multidrug- resistance (MFS-MDR) protein family 273 YBR038W (CHS2)
2.03060756 3.3885338 2.8509884 Chitin synthase II, responsible for
primary septum disk 274 YLR023C -- 2.68880866 3.3876183 2.5555381
Protein of unknown function 275 YPL009C -- 5.28314415 3.3856037
0.8412905 Protein of unknown function 276 YGL008C (PMA1) 2.09210526
3.3844005 3.725269 H+-transporting P-type ATPase of the plasma
membrane, activity is rate-limiting for growth at low pH 277
YMR033W (ARP9) 3.08586194 3.3800103 2.9005564 Protein with
similarity to actin and actin-related proteins ARP1P-ARP10P 278
YLR153C (ACS2) 3.45528019 3.3785446 3.1285812 Acetyl-COA synthetase
(acetate- COA ligase) 279 YLL061W -- 10.9366799 3.369477 3.0795633
Protein with similarity to GAP1P and other amino acid permeases 280
YNL192W (CHS1) 3.72575719 3.358192 3.7457248 Chitin synthase I, has
a repair function during cell separation 281 YEL058W (PCM1)
4.56623631 3.3482618 3.4456437 Hexosephosphate mutase
(phosphoacetylglucosamine mutase) (N-acetylglucosamine- phosphate
mutase), converts N- acetyl-D-glucosamine 1-phosphate to
N-acetyl-D-glucosamine 6- phosphate 282 YLR099C -- 4.72209646
3.3290462 2.9774757 Protein of unknown function 283 YDL057W --
3.21787484 3.316811 4.4492894 Protein of unknown function 284
YLR195C (NMT1) 3.4535546 3.3142347 3.1727409
N-myristoyltransferase, adds myristoyl group to N-terminal glycine
of certain proteins 285 YAL005C (SSA1) 3.48582964 3.3068323
2.9388227 Heat shock protein of HSP70 family, cytoplasmic 286
YPL222W -- 2.79485782 3.2974442 2.9997551 Protein of unknown
function 287 YJL056C -- 2.36206747 3.2790296 3.3129634 -- 288
YKR021W -- 2.33705924 3.269552 2.936447 Protein of unknown function
289 YPL119C (DBP1) 5.87199247 3.2464223 2.197366 ATP-dependent RNA
helicase of dead box family, suppressor of SPP81/DED1 290 YGL014W
-- 3.11296478 3.2294295 3.6382821 Protein with pumilio repeats that
is involved with MPT5P in relocalization of SIR3P and SIR4P from
telomeres to the nucleolus 291 YER010C -- 11.4713039 3.2179542
2.3807886 Protein of unknown function 292 YJR151C -- 41.4229667
3.2130608 4.5216913 Protein of unknown function 293 YPL207W --
2.48831068 3.2080219 2.9864022 Protein of unknown function 294
YER130C -- 2.01652303 3.2075344 3.3332725 Protein of unknown
function 295 YNR065C -- 2.86361451 3.2060768 7.3945002 Protein with
similarity to PEP1P 296 YGL192W (IME4) 2.89030953 3.170381
6.4784105 Positive transcription factor for IME1 and IME2, mediates
control of meiosis by carrying signals regarding mating type
(A/alpha) and nutritional status 297 YMR047C (NUP116) 2.56622055
3.1702234 4.7742052 Nuclear pore protein (nucleoporin) of the GLFG
family, may be
involved in binding and translocation of nuclear proteins 298
YDR256C (CTA1) 4.54027942 3.158248 8.0093186 Catalase A
(peroxisomal) 299 YDR208W (MSS4) 2.61164524 3.154316 3.0423151
Potential PI P 5-kinase, multicopy suppressor of STT4 mutation 300
YHR214W -- 4.54013428 3.1513793 5.6325011 Protein of unknown
function (YAR066W and YHR214W code for identical proteins) 301
YLR249W (YEF3) 3.59397167 3.1445334 2.631954 Translation elongation
factor EF- 3A, member of ATP-binding cassette (ABC) superfamily 302
YNL331C -- 3.44801501 3.1185277 6.5303136 Probable aryl-alcohol
reductase 303 YPR115W -- 2.4843458 3.1174643 2.5667848 Protein of
unknown function 304 YJL178C -- 2.60257256 3.1121969 2.7705734
Protein of unknown function 305 YAR042W (SWH1) 18.1940127 3.0992362
6.2488302 Protein of unknown function 306 YDR015C -- 0.09079169
3.0861607 0.3097629 Protein of unknown function 307 YBL067C (UBP13)
3.41427731 3.0820393 2.4717411 Ubiquitin C-terminal hydrolase 308
YHR072W (ERG7) 3.5569619 3.0809956 3.4311189 Lanosterol synthase,
carries out complex cyclization step of squalene to lanosterol in
ergosterol biosynthesis pathway 309 YAL028W -- 9.17485562 3.0726043
3.8109858 Protein of unknown function 310 YIR015W -- 2.80351347
3.066482 3.4328314 Subunit of RNase P 311 YMR308C (PSE1) 2.69422447
3.0659484 2.6409014 -- 312 YOR345C -- 5.73841888 3.0523183
2.2898958 Deoxycytidyl transferase involved in mutagenic
translesion DNA synthesis 313 YPL193W -- 3.60415592 3.0500696
2.8450987 Protein of unknown function 314 YFR012W -- 3.31259823
3.0316711 0 Protein of unknown function 315 YPL205C -- 13.258257
3.0208358 0.7999155 Protein of unknown function 316 YDR476C --
8.1273943 3.0155987 3.8636781 Protein of unknown function 317
YCR052W (RSC6) 2.2744649 3.0112438 2.6017436 Component of abundant
chromatin remodeling complex (RSC) 318 YGL022W (STT3) 3.64275733
3.0050118 3.8854905 Oligosaccharyltransferase subunit, member of a
complex of eight ER proteins that transfers core oligosaccharide
from dolichol carrier to Asn-X-Ser/Thr motif 319 YMR109W --
19.0544656 3.0044499 5.6886658 -- 320 YHR032W -- 9.30722933
2.9855823 4.5560581 Protein of unknown function, member of the
major facilitator superfamily(MFS) 321 YLR236C -- 2.6190617
2.9810987 3.7681402 -- 322 YOR337W (TEA1) 2.13152473 2.9790715
4.8228581 TY1 enhancer activator of the GAL4P-type family of DNA-
binding proteins 323 YFR055W -- 2.35867872 2.9771983 3.0622139
Protein with similarity to E. coli cystathionine beta-lyase 324
YHR212C -- 4.01639255 2.9769438 4.4451423 Protein identical to
YAR060P/RAA19P 325 YLR001C -- 2.77031036 2.9663037 2.7628132
Protein of unknown function 326 YOR034C -- 3.38439363 2.9543526
2.5499862 -- 327 YPR076W -- 3.86182393 2.9410933 3.2728075 Protein
of unknown function 328 YKL183W -- 2.9718977 2.9334031 5.2561547
Protein of unknown function 329 YBR004C -- 3.05485559 2.9257736
2.8905869 Protein expressed between 3 and 6 hours after transfer to
sporulation medium 330 YJR071W -- 3.39019477 2.924417 1.768982
Protein of unknown function 331 YCR084C (TUP1) 2.40138822 2.9219843
3.2718264 General repressor of transcription (with SSN6P), member
of WD (WD-40) repeat family 332 YFR030W (MET10) 33.6060485
2.9138815 2.0879079 Assimilatory sulfite reductase subunit,
flavin-binding (alpha) subunit, part of the sulfate assimilation
pathway 333 YKL148C (SDH1) 2.72554507 2.9036242 2.5317298 Succinate
dehydrogenase (ubiquinone) flavoprotein (FP) subunit, converts
succinate + ubiquinone to fumarate + ubiquinol in the TCA cycle 334
YER044C -- 3.6669641 2.9002716 2.6807728 Protein of unknown
function 335 YLR045C (STU2) 2.16969039 2.8946579 2.9923107
Component of the spindle pole body 336 YPL226W -- 2.45263084
2.8885678 2.5557944 Protein with similarity to members of the
ATP-binding cassette (ABC) superfamily 337 YHR161C -- 2.86345744
2.8873374 2.9469349 -- 338 YJR109C (CPA2) 4.31426739 2.8803515
3.1263529 Carbamoylphosphate synthase (glutamine-hydrolyzing)
arginine- specific, large chain 339 YGR250C -- 2.20914388 2.8752914
3.8774955 Protein of unknown function, has three RNA recognition
(RRM) domains 340 YLR149C -- 3.39994503 2.8694003 4.6627573 Protein
of unknown function 341 YCL057W (PRD1) 3.49569406 2.8641379
2.7495149 Proteinase YSCD, saccharolysin, contains zinc
metalloendoprotease motif HEXXH 342 YLR114C -- 2.27233205 2.8496505
1.8650501 Protein with weak similarity in the C-terminus to
drosophila melanogaster bicaudal-D protein 343 YML075C (HMG1)
2.71708812 2.8491957 3.2059005 3-hydroxy-3-methylglutaryl- coenzyme
A reductase 1, rate limiting enzyme for sterol biosynthesis,
converts HMG-COA to mevalonate 344 YLR397C (AFG2) 2.56801854
2.8469125 2.7385515 Protein of the AAA family of ATPases, has
similarity to mammalian valosin-containing protein (VCP) 345
YJR019C (TES1) 4.07777555 2.8303235 2.0724897 Acyl-COA thioesterase
346 YBL008W (HIR1) 7.24580603 2.8284713 2.8866813 Histone
transcription inhibitor, required for periodic repression of 3 of
the 4 histone gene loci and for autogenous repression of HTA1- HTB1
locus by H2A and H2B 347 YGL062W (PYC1) 2.649771 2.8279558
3.1059191 Pyruvate carboxylase 1, converts pyruvate to oxaloacetate
for gluconeogenesis 348 YPL244C -- 3.43385233 2.8218119 3.3274479
Protein of unknown function 349 YGL001C -- 3.91981575 2.8214816
1.9852785 Protein with similarity to nocardia SP. cholesterol
dehydrogenase 350 YMR302C (PRP12) 2.92335545 2.8146501 2.7190981 --
351 YPL160W (CDC60) 2.25327101 2.8142723 1.7426948 Leucyl-TRNA
synthetase, cytoplasmic 352 YLL024C (SSA2) 4.09160949 2.8142088
2.4784071 Heat shock protein of HSP70 family, cytoplasmic 353
YEL077C -- 3.20718793 2.8098429 3.9054119 -- 354 YMR205C (PFK2)
2.27470363 2.8050429 2.2843952 Phosphofructokinase beta subunit,
part of a complex with PFK1P which carries out key regulatory step
in glycolysis 355 YPL114W -- 4.16484234 2.7962162 1.717967 Protein
of unknown function 356 YPL221W -- 4.08515832 2.7886642 3.960997
Protein of unknown function 357 YJR137C (ECM17) 26.5435466 2.787597
2.0763181 Putative sulfite reductase (ferredoxin) 358 YKL164C
(PIR1) 2.11125363 2.7864791 2.3925674 Protein required for
tolerance to heat shock, member of the PIR1P/HSP150P/PIR3P family
359 YCL037C (SRO9) 8.35007693 2.7855748 2.393588 Suppressor of YPT6
null and RHO3 mutations 360 YHR082C (KSP1) 2.14499054 2.7799591
3.4962633 Serine/threonine kinase that suppresses PRP20 mutant when
overproduced 361 YPR074C -- 3.19760669 2.7711859 2.476508 -- 362
YBR184W (MEL1) 5.06354303 2.7711448 3.5340388 Alpha-galactosidase
(melibiase), converts melibiose into galactose + glucose, converts
melibiose to galactose and glucose 363 YOL157C -- 2.70064964
2.7668777 3.6204284 Probable alpha-glucosidase 364 YFL066C --
2.94443276 2.753026 3.5848427 Protein with similarity to other
subtelomerically-encoded proteins including YIL177P, YHL050P, and
YER190P 365 YLL029W -- 2.22657399 2.7389102 3.1468025 Protein of
unknown function 366 YJL198W -- 2.98124683 2.7343513 4.6395823
Protein with strong similarity to PHO87P, member of the phosphate
permease family of the major facilitator superfamily (MFS) 367
YDR088C (SLU7) 2.07293165 2.7339627 2.6876744 Pre-MRNA splicing
factor affecting 3' splice site choice, required only for the
second catalytic step of splicing 368 YJR132W (NMD5) 3.2005363
2.7333821 3.208398 Member of the karyopherin-beta family, possibly
involved in nuclear transport 369 YIL078W (THS1) 3.31778832
2.7330794 1.6123939 Threonyl-TRNA synthetase, cytoplasmic, member
of Class II family of aminoacyl-TRNA synthetases 370 YGL113W --
2.33404789 2.7249323 3.3810122 Protein of unknown function 371
YMR086W -- 2.69384376 2.7191747 2.9840404 Protein of unknown
function 372 YGL233W (SEC15) 2.61433498 2.7141295 2.7961427
Component of exocyst complex required for exocytosis 373 YGL144C --
2.26752066 2.7069494 2.6236889 Protein of unknown function 374
YOR137C -- 3.14249753 2.7031211 4.9526236 Protein of unknown
function 375 YJR143C (PMT4) 2.80130312 2.6954799 2.4879264
Mannosyltransferase (dolichyl phosphate-D-mannose: protein O-
D-mannosyltransferase), involved in initiation of O-glycosylation
376 YBR289W (SNF5) 2.00671327 2.6881295 3.038619 Component of
SWI/SNF global transcription activator complex, acts to assist
gene-specific activators through chromatin remodeling 377 YNL240C
-- 5.13894557 2.685901 3.523963 Protein with similarity to
kluyveromyces MARX. LET1 protein 378 YML013W -- 3.62672833
2.6831604 2.9292996 Protein of unknown function 379 YKL168C --
2.43589311 2.6791837 3.1896257 -- 380 YGL151W (NUT1) 2.47823061
2.6787971 2.5683618 Protein that affects expression of HO 381
YNL197C (WHI3) 2.51493336 2.6764555 3.4233233 Protein involved in
regulation of cell size, has 1 RNA recognition (RRM) domain 382
YMR192W -- 2.18376269 2.6732126 2.7489187 Protein with similarity
to mouse TBC1 protein 383 YAL038W (CDC19) 2.63679951 2.6714535
2.7525692 -- 384 YEL075C -- 5.12225893 2.6632638 3.778537 Protein
with similarity to other subtelomerically-encoded proteins
including YHL049P, YIL177P, and YJL225P 385 YHR219W -- 3.76398139
2.6619567 4.0207342 Protein with similarity to other
subtelomerically-encoded proteins 386 YJL069C -- 2.65731007
2.6517254 2.6568606 Protein of unknown function 387 YLR125W --
6.28348756 2.642933 3.1335402 Protein of unknown function 388
YML035C (AMD1) 2.23864371 2.6401405 1.6690608 AMP deaminase,
converts AMP to IMP and ammonia 389 YMR165C (SMP2) 2.58642399
2.6310411 3.3572604 Protein whose deletion causes increased plasmid
stability 390 YDL223C -- 3.16684859 2.6240147 2.2340877 Protein of
unknown function 391 YLR138W -- 2.69090586 2.6158483 2.9821754 --
392 YAR020C -- 3.79173888 2.6111125 2.0234222 -- 393 YLR337C (VRP1)
6.57336326 2.6027011 3.7504037 Proline-rich protein verprolin,
involved in cytoskeletal organization and cellular growth 394
YLR060W (FRS1) 2.61550639 2.5992071 1.9335426 Phenylalanyl-TRNA
synthetase, alpha subunit, cytoplasmic 395 YLL013C -- 2.93447915
2.5901954 4.1297767 Protein with similarity to drosophila pumilio
protein 396 YIR003W -- 2.41363594 2.5863745 2.874494 Protein with
similarity to E. coli and Bacillus subtilis mind, has potential
coiled-coil region 397 YIL137C -- 2.27968603 2.5792356 1.9624104
Protein with similarity to aminopeptidases 398 YBL081W --
2.34404421 2.573205 3.2079939 Protein with 37% identity to
drosophila L not protein 399 YOR171C -- 3.59659097 2.5718305
2.4035974 -- 400 YPL237W (SUI3) 2.5966981 2.5628077 2.5479456
Translation initiation factor EIF2beta subunit 401 YHR142W --
3.52383057 2.5597096 2.9887896 Protein of unknown function 402
YLL012W -- 3.25020683 2.550591 2.7451737 Protein with similarity to
human triacylglycerol lipase 403 YFR025C (HIS2) 2.5112362 2.5457991
2.8789156 Histidinol phosphatase 404 YGR240C (PFK1) 2.24103063
2.5388997 2.4938739 Phosphofructokinase alpha subunit, part of a
complex with PFK2P which carries out A key regulatory step in
glycolysis 405 YPL101W -- 4.18961695 2.5351198 2.6201803 Protein of
unknown function 406 YOR127W (RGA1) 3.85804733 2.5316649 2.5697341
RHO-type GTPase-activating protein (GAP) for CDC42P 407 YBR088C
(POL30) 2.5383718 2.5276319 4.0628861 Proliferating cell nuclear
antigen (PCNA), required for DNA synthesis and DNA repair 408
YBR295W (PCA1) 4.16669535 2.525791 1.1221384 P-type
copper-transporting ATPase 409 YCL044C -- 2.35958836 2.519608
3.263571 Protein of unknown function 410 YBR110W (ALG1) 2.23384099
2.5141215 3.2250999 Beta-mannosyltransferase involved in
N-glycosylation (transfers MAN from GDP-MAN to DOL-PP- GLCNAC2) 411
YGR175C (ERG1) 6.02726287 2.5103577 1.8132661 Squalene
monooxygenase (squalene epoxidase), enzyme of the ergosterol
biosynthesis pathway 412 YLR116W -- 2.98116702 2.5079761 3.9707409
-- 413 YCR068W -- 3.32107678 2.4920381 3.6811994 Protein of unknown
function 414 YJR105W -- 2.20476096 2.4908887 1.7029385 Protein with
similarity to ribokinase 415 YKL157W (APE2) 2.18209838 2.4866194
2.093134 Aminopeptidase II (YSCII), plays a nutritional role in
releasing leucine from peptides externally cleaved at leucine 416
YFR009W (GCN20) 2.63782118 2.4859544 2.1613378 Component of a
protein complex required for activation of GCN2P protein kinase in
response to amino acid starvation, member of ATP- binding cassette
(ABC) superfamily 417 YDR211W (GCD6) 2.22567451 2.4835485 1.8240639
Translation initiation factor EIF2B (guanine nucleotide exchange
factor), 81 KDA (beta) subunit 418 YAR060C -- 4.88485967 2.482682
4.6114571 Protein identical to YHR212P, has a predicted
mitochondrial transit peptide 419 YJL187C (SWE1) 2.01161328
2.4809757 2.6294797 Serine/tyrosine dual-specificity protein kinase
able to phosphorylate CDC28P on tyrosine and inhibit its activity
420 YDR387C -- 2.3225348 2.4746572 3.0481024 Protein with
similarity to ITR1P and ITR2P 421 YDR251W (PAM1) 2.09471237
2.4744652 2.3344613 Coiled-coil protein and multicopy suppressor of
loss of PP2A (genes PPH21, PPH22, and PPH3) 422 YJL172W (CPS1)
2.4464951 2.473092 2.228723 GLY-X carboxypeptidase YSCS, involved
in nitrogen metabolism 423 YMR277W (FCP1) 2.51675116 2.466666
2.2346158 TFIIF-interacting component of the C-terminal domain
phosphatase 424 YDL047W (SIT4) 2.40214863 2.4572974 2.7529791
Protein serine/threonine phosphatase involved in cell cycle
regulation, member of the PPP family of protein phosphatases and
related to PP2A phosphatases 425 YML117W -- 2.2473701 2.4482108
2.8054783 Protein of unknown function, contains an ATP/GTP-binding
site motif A (P-loop) 426 YHR039C-A -- 2.49103418 2.4469729
1.7368373 -- 427 YLL003W (SFI1) 3.03031186 2.4467012 2.2685901
Protein of unknown function 428 YKR048C (NAP1) 3.02222721 2.4404483
3.002619 Nucleosome assembly protein that plays a role in assembly
of histones into octamer, required for full expression of CLB2P
functions 429 YOR197W -- 2.87645711 2.438206 1.9784081 Protein of
unknown function 430 YEL046C (GLY1) 2.40664526 2.4369367 2.6795853
Protein required for glycine prototrophy in SHMT1 SHMT2 double
mutant 431 YJL029C -- 2.36823878 2.43429 2.3384644 Protein of
unknown function, has similarity to C. elegans hypothetical protein
T05G5.8 432 YOR233W (KIN4) 3.52231883 2.4312627 3.0678435
Serine/threonine protein kinase related to KIN1P and KIN2P,
catalytic domain is most related to SNF1P 433 YOR299W (BUD7)
2.16058794 2.4312223 2.9585581 Protein required for bipolar budding
pattern 434 YHR218W -- 2.37694362 2.4297245 4.1990669 Protein with
similarity to other subtelomerically-encoded proteins including
YHR219P and YFL065P, probable pseudogene
435 YGL026C (TRP5) 3.92053304 2.4267316 2.4752996 Tryptophan
synthase, last (fifth) step in tryptophan biosynthesis pathway 436
YJL017W -- 2.745014 2.4179146 2.8613495 Protein of unknown function
437 YNL161W -- 4.7525671 2.4161417 2.324762 Serine/threonine
protein kinase of unknown function 438 YOR141C (ARP8) 5.68817037
2.4122798 1.7395537 Protein with similarity to actin and
actin-related proteins ARP1P- ARP10P 439 YAL042W -- 2.84377325
2.4057529 3.7961408 Protein of unknown function, has 2 potential
transmembrane domains 440 YGR270W (YTA7) 2.68803581 2.4056715
1.945755 Protein with similarity to members of the AAA family of
ATPases 441 YBR119W (MUD1) 2.83912216 2.4051525 1.3987642 U1 SNRNP
A protein (SNRNA- associated protein) with 2 RNA recognition (RRM)
domains 442 YDR052C (DBF4) 6.85835185 2.4036928 1.5834976
Regulatory subunit for CDC7P protein kinase, required for G1/S
transition 443 YEL069C (HXT13) 2.69020108 2.4013304 3.6711431
Protein with strong similarity to hexose transporters, member of
the sugar permease family 444 YDR285W (ZIP1) 8.03633767 2.3921886
0.2216256 Structural protein of the synaptonemal element central
element, has predicted coiled-coil domain 445 YJL047C -- 2.8960182
2.3885065 2.0814157 Protein with similarity to clathrin heavy chain
in one domain 446 YKL101W (HSL1) 4.2235071 2.3780286 2.4485279
Serine/threonine protein kinase that interacts genetically with
histone mutations 447 YIL143C (SSL2) 2.16202858 2.3668818 1.9944618
DNA helicase component of RNA polymerase transcription initiation
factor TFIIH (factor B) 448 YBR182C -- 3.15043584 2.3653183
2.517131 -- 449 YER189W -- 2.65287612 2.3630614 5.1724275 Protein
with similarity to subtelomerically-encoded proteins including
YIL177P, YHL049P, and YJL225P 450 YLR194C -- 3.11287981 2.3617044
2.923307 Protein of unknown function 451 YGR160W -- 2.13853989
2.3577684 1.8132562 Protein of unknown function 452 YGR258C (RAD2)
2.06944636 2.3572245 2.1751698 Structure-specific single-stranded
DNA endonuclease of the nucleotide excision repairosome 453 YGR162W
(TIF4631) 2.28099935 2.3554791 1.7039222 MRNA CAP-binding protein
(EIF4F) 150 K subunit 454 YJR036C -- 3.21027204 2.3546452 5.0712893
Possible ubiquitin-protein ligase (E3) 455 YGR124W (ASN2)
3.37829988 2.3505148 2.4742017 Asparagine synthetase (L-aspartate:
L-glutamine amidoligase [AMP- forming]), ASN1P and ASN2P are
isozymes 456 YDL180W -- 2.20643197 2.3467843 1.8047293 Protein of
unknown function 457 YDR266C -- 3.29383065 2.3411759 2.3118864
Protein of unknown function 458 YAR073W -- 7.67257484 2.3325262
1.6890618 Protein with strong similarity to PUR5P, may be an
inosine-5'- monophosphate dehydrogenase 459 YPL048W (CAM1)
2.18528771 2.3294863 3.3106924 -- 460 YEL030W (ECM10) 1.99868799
2.3236082 2.3835153 Protein possibly involved in cell wall
structure or biosynthesis 461 YLL058W -- 6.13836096 2.3223158
2.3541199 Protein with similarity to neurospora crassa O-
succinylhomoserine (thiol)-lyase 462 YJR010W (MET3) 8.36384636
2.3172147 1.5113084 ATP-sulfurylase (sulfate adenylyltransferase)
463 YER110C (KAP123) 3.02732098 2.3160572 1.8042941
Karyopherin-beta, involved in nuclear import of ribosomal proteins
464 YGL063W (PUS2) 2.17517427 2.3124794 4.1754638 Pseudouridine
synthase 465 YPL184C -- 3.3404012 2.3122475 2.2563666 Protein of
unknown function 466 YGR254W (ENO1) 2.05650599 2.3095639 1.9054756
Enolase 1 (2-phosphoglycerate dehydratase), converts 2-phospho-
D-glycerate to phosphoenolpyruvate in glycolysis 467 YIL108W --
3.15926869 2.3086561 2.5782072 Protein of unknown function 468
YDR388W (RVS167) 2.34115713 2.3058518 2.6912527 Protein with A SH3
domain that affects actin distribution and bipolar budding 469
YNL323W -- 2.29668952 2.3038327 2.0645985 Protein with similarity
to YCX1P 470 YBL076C (ILS1) 2.31635893 2.3036041 1.7634202
Isoleucyl-TRNA synthetase 471 YLR217W -- 2.57939547 2.2859565
1.6611523 Protein of unknown function 472 YGR294W -- 8.48668724
2.2857763 1.7132102 Protein of the PAU1 family 473 YDL070W --
2.16064033 2.2854538 3.7599153 -- 474 YOL044W -- 2.15373467
2.2849315 2.1736446 -- 475 YGL145W (TIP20) 4.18903489 2.2829973
1.6161221 Cytoplasmic protein that interacts physically with
SEC20P, required for ER to Golgi transport 476 YLR044C (PDC1)
2.21772333 2.2774972 1.9431592 Pyruvate decarboxylase isozyme 1 477
YNR013C -- 2.0080141 2.2770842 2.4893728 Protein with similarity to
PHO87P and YJL198P, member of the phosphate permease family of the
major facilitator superfamily (MFS) 478 YML049C -- 2.08547393
2.2761395 2.0329879 -- 479 YDR221W -- 2.53153283 2.2731861
1.8131644 Protein with similarity to the beta subunit of human
glucosidase II 480 YMR135C -- 4.75727106 2.2636411 4.3609747
Protein of unknown function 481 YKR001C (VPS1) 2.48277065 2.2630712
1.5678763 Vacuolar sorting protein, member of the dynamin family of
GTPases 482 YLR413W -- 2.80009402 2.2629262 2.3695083 Protein of
unknown function 483 YDR122W (KIN1) 2.0434064 2.2623436 2.3432635
Serine/threonine protein kinase, related to KIN2P and S. pombe KIN1
484 YIL154C (IMP2') 2.216207 2.2548739 2.2466776 -- 485 YKL068W
(NUP100) 2.2598003 2.2529093 2.7012733 Nuclear pore protein
(nucleoporin) of the GLFG family, may be involved in binding and
translation of proteins during nucleocytoplasmic transport 486
YHR190W (ERG9) 2.81318531 2.2475123 1.7238705 Squalene synthetase
(farnesyl- diphosphate farnesyltransferase), acts at a branch point
in the isoprenoid biosynthesis pathway 487 YGL179C -- 4.83814707
2.2398396 3.8786749 Serine/threonine protein kinase with similarity
to ELM1P and KIN82P 488 YOL017W -- 3.01741322 2.2303862 2.2459064
Protein of unknown function 489 YHR189W -- 2.0021212 2.22911
2.2289936 Putative peptidyl-TRNA hydrolase (PTH) 490 YNL208W --
3.64860898 2.2181817 2.5247363 Protein of unknown function 491
YHR041C (SRB2) 2.27216109 2.2178582 2.4847273 Component of the RNA
polymerase II holoenzyme and Kornberg's mediator (SRB) subcomplex
492 YPR080W (TEF1) 2.50402057 2.2115095 1.8587879 Translation
elongation factor EF- 1alpha (TEF1 and TEF2 code for identical
proteins) 493 YBR229C (ROT2) 2.45186053 2.2034499 2.1844611
Catalytic (alpha) subunit of glucosidase II 494 YGR262C --
2.83275613 2.2029336 1.9251756 Protein with similarity to apple
tree calcium/calmodulin-binding protein kinase PIR: JQ2251 495
YER144C (UBP5) 3.38126089 2.1994294 2.7106303 Ubiquitin-specific
protease (ubiquitin C-terminal hydrolase), homologous to DOA4P and
human TRE-2 496 YDR264C (AKR1) 3.13151279 2.1983967 2.7516536
Ankyrin repeat-containing protein that has an inhibitory effect on
signaling in the pheromone pathway 497 YLR427W -- 2.24985411
2.1938243 2.5059695 Protein of unknown function 498 YLR374C --
2.26923061 2.1927227 2.6395044 Protein of unknown function 499
YMR092C (AIP1) 2.2241966 2.1917074 2.1939749 Actin interacting
protein, has 4 WD (WD-40) repeats 500 YDR294C -- 2.20085342
2.1899557 2.3333139 -- 501 YMR296C (LCB1) 2.1334221 2.1891645
1.9030014 Component of serine C- palmitoyltransferase, first step
in biosynthesis of long-chain base component of sphingolipids 502
YKR039W (GAP1) 1.99105648 2.1881751 1.2556866 General amino acid
permease, proton symport transporter for all naturally-occurring
L-amino acids, 4-aminobutyric acid (GABA), ornithine, citrulline,
some D-amino acids, and some toxic analogs 503 YDR422C (SIP1)
2.62373247 2.1870761 2.0836347 Multicopy suppressor of SNF1,
related to GAL83P/SPM1P and SPM2P 504 YMR080C (NAM7) 2.82340116
2.1828046 2.1714828 Protein involved with NMD2P and UPF3P in decay
of MRNA containing nonsense codons 505 YBL106C -- 2.38138747
2.1809814 2.7798326 -- 506 YEL043W -- 3.44125375 2.1784956
2.8076042 Protein of unknown function 507 YBR222C (FAT2) 5.13679804
2.1781103 3.0936394 Peroxisomal AMP-binding protein 508 YDR004W
(RAD57) 2.26389978 2.1754266 2.076582 Component of recombinosome
complex involved in meiotic recombination and recombinational
repair, with RAD55P promotes DNA strand exchange by RAD51P
recombinase 509 YHR174W (ENO2) 2.38714668 2.1702816 1.9697417
Enolase 2 (2-phosphoglycerate dehydratase), converts 2-phospho-
D-glycerate to phosphoenolpyruvate in glycolysis 510 YER043C (SAH1)
3.73200717 2.1669937 1.6246235 Adenosylhomocysteinase (S-
adenosylhomocysteine hydrolase) 511 YKR012C -- 2.41358469 2.1555775
1.1414615 Protein of unknown function 512 YOL007C -- 3.17872347
2.1529948 1.2712945 -- 513 YMR220W (ERG8) 2.68816133 2.1489328
2.0693924 Phosphomevalonate kinase, converts mevalonate-5-phosphate
to mevalonate pyrophosphate, involved in isoprene and ergosterol
biosynthesis pathways 514 YDR062W (LCB2) 2.54448949 2.1430094
1.9627647 Subunit of serine C- palmitoyltransferase, first step in
sphingolipic biosynthesis, and suppressor of calcium-sensitivity of
CSG2 515 YAL048C -- 5.02313141 2.1384748 4.221132 Protein with weak
similarity to RAS1P, RAS2P, and other GTP- binding proteins of the
RAS superfamily 516 YBL111C -- 2.17340644 2.1313903 3.8030907 --
517 YJL108C -- 4.56646166 2.1302533 2.8609713 Protein of unknown
function, contains 8 potential transmembrane domains 518 YJL141C
(YAK1) 2.80000608 2.1277388 2.8291776 Serine/threonine protein
kinase, negative regulator of cell growth acting in opposition to
CAMP- dependent protein kinase A 519 YJL102W (MEF2) 2.08592026
2.1220696 1.6098307 Mitochondrial translation elongation factor,
promotes GTP- dependent translocation of nascent chain from A-site
to P-site of ribosome 520 YDL174C (DLD1) 2.28050309 2.1220649
2.4305801 D-lactate dehydrogenase (cytochrome), (D-lactate
ferricytochrome C oxidoreductase) (D-LCR), mitochondrial 521
YMR011W (HXT2) 7.25080973 2.1188378 1.6420019 High-affinity hexose
transporter, member of sugar permease family 522 YLR129W (DIP2)
3.36115373 2.1126408 2.0325416 DOM34P-interacting protein, has WD
(WD-40) repeats 523 YML008C (ERG6) 2.51872662 2.1091692 1.7889829
S-adenosylmethionine delta-24- sterol-C-methyltransferase, carries
out methylation of zymosterol as part of the ergosterol
biosynthesis pathway 524 YGL245W -- 2.30162026 2.1065078 1.4267053
Glutamyl-TRNA synthetase, member of the Class I aminoacyl TRNA
synthetase family 525 YGL024W -- 2.67631735 2.1046757 1.4610387
Protein of unknown function 526 YHL027W (RIM101) 2.57210755
2.1033157 2.5927892 Zinc-finger protein involved in induction of
IME1 527 YGR281W (YOR1) 4.18259907 2.0935061 2.3634092
Oligomycin-resistance factor, member of the ATP-binding cassette
(ABC) superfamily 528 YIL175W -- 2.10803474 2.0859355 2.4771166 --
529 YHL019C (APM2) 1.9986708 2.0848729 3.0618718
Clathrin-associated protein (AP) complex, medium subunit 530
YAL019W (FUN30) 5.19927199 2.0806959 1.7340212 -- 531 YGL112C
(TAF60) 2.21463331 2.0765265 2.1891308 Component of TAF(II) complex
(TBP-associated protein complex) required for activated
transcription by RNA polymerase II 532 YNL218W -- 2.28887465 2.0761
1.6749939 Protein with similarity to E. coli DNA polymerase III
gamma and TAU subunits 533 YML058C-A -- 217.969407 2.0723568
3.2869214 534 YOL156W (HXT11) 5.12784966 2.0709411 2.2192118
Low-affinity glucose permease 535 YGR218W (CRM1) 2.32581989
2.0675233 1.5505702 Exportin, beta-karyopherin 536 YGR296W --
3.15948331 2.0664535 3.7402619 Protein with similarity to other
subtelomerically-encoded proteins including YER190P (YPL283 and
YGR296W code for identical proteins) 537 YLR176C -- 2.54329087
2.0627475 1.4892288 -- 538 YDL229W (SSB1) 5.21935107 2.0615889
2.0067653 Heat shock protein of HSP70 family involved in the
translational apparatus 539 YER034W -- 2.57654853 2.0562947
1.9025056 Protein of unknown function 540 YKR050W (TRK2) 2.23638067
2.056259 4.703529 Potassium transporter of the plasma membrane,
moderate affinity, member of the potassium permease family of the
major facilitator superfamily 541 YIL113W -- 7.07756282 2.0539759
2.28618 Dual-specificity protein phosphatase 542 YCR023C --
2.01851078 2.0520751 2.2109695 Member of major facilitator
superfamily (MFS) multidrug- resistance protein family 2 543
YMR069W -- 4.45745957 2.0520592 0 Protein of unknown function 544
YAL020C (ATS1) 3.02597511 2.050802 2.0781706 Protein with
similarity to human RCC1 protein, suppressor of mutations in alpha
tubulin 545 YNL256W -- 3.16308725 2.045577 1.8697374 Protein with
similarity to bacterial dihydropteroate synthase 546 YMR124W --
2.65610298 2.0431312 2.2988806 Protein of unknown function, has
potential coiled-coil region (GB: Z49273) 547 YOR162C -- 2.4478098
2.0361958 2.1075035 -- 548 YOR353C -- 2.20965265 2.0220258
1.7471747 Protein with weak similarity to adenylate cyclases 549
YPL028W (ERG10) 2.86559138 2.0185951 1.6989337 Acetyl-COA
acetyltransferase (acetoacetyl-COA thiolase), first step in
mevalonate/sterol pathway 550 YIL114C (POR2) 2.24322702 2.0152799
2.367678 Outer mitochondrial membrane porin (voltage-dependent
anion- selective channel) 551 YDL029W (ACT2) 2.07186888 2.0140172
1.810394 -- 552 YDL143W (CCT4) 2.3041307 2.0128325 1.6478427
Component of chaperonin- containing T-complex (TCP ring complex,
TRIC), homologous to mouse CCT4 553 YPL267W -- 2.06501413 2.0119076
1.6761922 Protein of unknown function 554 YOL105C -- 2.79225712
2.0026061 2.23737 -- 555 YML004C (GLO1) 2.19630894 2.0015677
1.7985136 Glyoxalase I, converts methylglyoxal and glutathione into
S-D-lactoylglutathione 556 YMR266W -- 2.47393267 1.991188 1.727182
Protein of unknown function, probable integral membrane
glycoprotein 557 YPL194W -- 2.87006368 0.4961465 1.5346869 -- 558
YOR152C -- 2.74047761 0.4915256 0.2221023 Protein of unknown
function 559 YDR242W (AMD2) 8.28951711 0.4819032 0.9215489 Protein
with similarity to amidases 560 YFL054C -- 7.43223753 0.4793582
0.6136582 Protein with similarity to FPS1P and YPR192P, member of
MIP family of transmembrane channels 561 YAR068W -- 3.24259317
0.479021 1.2297001 Protein with similarity to ICWP protein 562
YAL001C (TFC3) 2.94740587 0.4742746 1.1915566 RNA polymerase
transcription initiation factor TFIIIC (TAU), 138 KDA subunit 563
YLR454W -- 5.72921213 0.4716283 1.641906 Protein of unknown
function 564 YDL020C (SON1) 2.27378766 0.4591519 0.8208918 -- 565
YMR225C (MRPL44) 0.19372389 0.4430311 0.4019617 Mitochondrial
ribosomal protein of the large subunit (YMR44) 566 YJR038C --
9.06373624 0.4422872 4.1801655 Protein of unknown function 567
YDR380W -- 0.1136124 0.4417559 0.8241167 Protein with similarity to
pyruvate decarboxylase, pyruvate oxidase, acetolactate synthase
(large subunit), and other enzymes
that require thiamine pyrophosphate 568 YKL170W (MRPL38) 0.20347891
0.4296401 0.4533368 Mitochondrial ribosomal protein of the large
subunit (YML38) (E. coli L14), belongs to the L14 family of
prokaryotic ribosomal proteins 569 YGR248W (SOL4) 0.17664863
0.4293198 0.4062793 Protein of unknown function 570 YER058W
(PET117) 0.18996331 0.4289442 0.4202828 Protein involved in
assembly of cytochrome oxidase 571 YBR039W (ATP3) 0.18787084
0.4197886 0.2837245 F1-gamma ATP synthase 572 YDL102W (CDC2)
17.5853214 0.4169873 0.0258767 -- 573 YJR153W -- 3.65551445
0.4116558 0.6086987 -- 574 YMR188C -- 0.20743995 0.4113817
0.3381207 Protein with similarity to 30S ribosomal proteins (S17)
575 YBR244W -- 0.16093632 0.4035137 0.3438917 Protein with
similarity to glutathione peroxidase 576 YDR523C (SPS1) 10.8815611
0.4014712 0.3371725 Serine/threonine protein kinase involved in
middle/late stage of meiosis 577 YDL031W -- 2.0561223 0.3989968
0.6791327 Protein with similarity to RNA helicases of dead/DEAH box
family 578 YER109C (FLO8B) 2.33584341 0.3826502 2.0529509 -- 579
YIR017C (MET28) 2.97658904 0.3775372 0.3008895 Transcriptional
activator of the basic leucine zipper (BZIP) family, works with
MET4P and CBF1P to regulation sulfur amino acid metabolism 580
YDL016C -- 3.9417341 0.374232 0.2672688 Protein of unknown function
581 YIR028W (DAL4) 2.5006493 0.3741716 3.0010653 Allantoin
permease, member of the uracil/allantoin permease family of the
major facilitator superfamily (MFS) 582 YOR124C (UBP2) 2.8382974
0.3622925 0.3859773 Ubiquitin-specific protease (ubiquitin
C-terminal hydrolase), cleaves at the C-terminus of ubiquitin 583
YBL108W -- 0.1900473 0.3575329 0.5467376 Protein of unknown
function 584 YDR259C -- 5.62713626 0.3429355 0.2335082 -- 585
YDR253C (MET32) 2.86314943 0.3397175 0.3279043 Zinc-finger protein
involved in transcriptional regulation of methionine metabolism 586
YJL196C (ELO1) 0.17135352 0.3378086 0.3752547 Fatty acid elongation
protein involved in elongation of tetradecanoic acid to
hexadecanoic acid 587 YDR141C -- 0.09160554 0.3290633 0.1693929
Protein of unknown function, member of the major facilitator
superfamily (MFS) 588 YBR069C (VAP1) 3.0181038 0.3157547 1.2268269
Amino acid permease for valine, leucine, isoleucine, tyrosine, and
tryptophan 589 YOR314W -- 2.65430513 0.2917342 0.3312621 Protein of
unknown function 590 YDL068W -- 0.11556176 0.2684108 0.1521109
Protein of unknown function 591 YPL136W -- 2.17418921 0.2530647
3.1708409 Protein of unknown function 592 YGL034C -- 0.1411795
0.2524039 0.3723439 Protein of unknown function 593 YLR162W --
4.13626663 0.2515583 0.6851592 Protein of unknown function 594
YMR193C-A -- 3.34099753 0.2354896 0.3596816 -- 595 YMR146C (TIF34)
5.0351989 0.2248204 0.7193538 Translation initiation factor EIF3,
P39 subunit, has 2 WD (WD-40) repeats 596 YFL012W -- 71.9436495
0.2235373 1.5215902 Protein of unknown function 597 YER096W --
7.21258235 0.1766673 0.4170679 -- 598 YNR071C -- 2.01488788
0.1446196 0.0535063 Protein with similarity to UDPglucose
4-epimerase 599 YLR419W -- 0.20769335 0.1102431 0.9141258 Protein
with similarity to several pre-MRNA splicing factors 600 YKL105C --
3.23146223 0.086572 5.0836556 Protein of unknown function 601
YLR142W (PUT1) 2.2907881 0.0854218 0.6671487 Proline oxidase, first
step in synthesis of glutamate from proline 602 YDL239C --
7.81000565 0.0417738 0.347901 Protein of unknown function 603
YHR137W (ARO9) 0.07724918 0.0347684 0.0703134 Aromatic amino acid
aminotransferase II 604 YDR374C -- 17.25276 0 4.6059679 Protein of
unknown function 605 YIL100W -- 9.97598883 0 2.8122773 Protein of
unknown function, questionable ORF 606 YPL025C -- 9.52247441 0
20.22382 Protein of unknown function 607 YOR072W -- 7.48662389 0
6.2287404 Protein of unknown function 608 YNL242W -- 6.47720448 0
2.5753253 Protein of unknown function 609 YIR027C (DAL1) 5.64113227
0 0 Allantoinase, first step in the degradation of allantoin as a
secondary nitrogen source 610 YOR139C -- 5.46648132 0 11.760995
Transcription factor with domains homologous to MYC oncoprotein and
yeast HSF1P, required for normal cell surface assembly and
flocculence 611 YEL019C (MMS21) 3.34008483 0 2.069236 Protein
involved in DNA repair 612 YDL132W (cdc53) 3.16426832 0 0.1467847
-- 613 YOR177C -- 2.97842594 0 0.435871 Protein of unknown function
614 YML042W (CAT2) 2.76437696 0 16.65885 Carnitine
O-acetyltransferase, peroxisomal and mitochondrial 615 YER044C-A
(MEI4) 2.5971776 0 0 Protein required early in meiosis for meiotic
recombination, chromosome synapsis, and viable spore formation 616
YGR083C (GCD2) 2.32134339 0 0 Translation initiation factor EIF2B
(guanine nucleotide exchange factor), 71 KDA (delta) subunit 617
YAR030C -- 2.06301879 0 0 Protein of unknown function, probable
non-coding ORF 618 YJR157W -- 0.2073771 0 0.6711879 Protein of
unknown function 619 YHR217C -- 0.2061042 0 0.549346 Protein of
unknown function 620 YKL100C -- 0.12715731 0 40.399169 Protein of
unknown function * Table Headings: Clone ID: A clone ID designation
number. Alias: Alternative gene names used in the literature. This
information is provided by YPD .TM., Hodges et al. Nucl. Acids Res.
27: 69-73 (1999), the entirety of which is herein incorporated by
reference. CJ-4 hr/LP-4 hr: Expression level in the mutant CJ517 as
compared with the respective wild type strain LPY9 at 4 hr sampling
of log phase growth of yeast (ratio of mutant expression
level/control expression level). CJ refers to the mutant CJ517 (The
mutant is defective in the gene (ERG11) codes for C14 #demethylase
enzyme in the sterol biosynthetic pathway). LP refers to the
respective wild type strain LPY9, used to compare the gene
expression profile with the mutant. K-50/CK: Expression level in
the wild type yeast LPY9, at 2 hr after treatment with 50 micro
gram/ml ketoconazole as compared to the wild type LPY9 strain
without ketoconazole treatment (ratio of treatment expression
level/control expression level). K refers to ketoconazole
treatment. The clones listed in Table #2 are either up or down
regulated in the mutant (CJ517) as well as in response to
ketoconazole treatment. K-100/CK: Expression level in the wild type
yeast LPY9, at 2 hr after treatment with 100 micro gram/ml
ketoconazole as compared to the wild type LPY9 strain without
ketoconazole treatment (ratio of treatment expression level/control
expression level). Gene Description: Description of the clone
listed in column 1.
[0327] Table 3, below, lists the RNAs from Table 2 that correspond
to genes or structural regions implicated in transcription
regulation.
3TABLE 3* Seq. CJ-4 hr/ Num. Clone ID ALIAS LP-4 hr K-50/CK
K-100/CK Gene Description 30 YOR237W (HES1) 134.648161 1417.62621
1358.12348 Protein implicated in ergosterol biosynthesis, member of
the KES1/HES1/OSH1/YKR003W family of oxysterol-binding (OSBP)
proteins 42 YDR213W -- 18.2079478 32.1360646 58.3586116 Protein
with similarity to transcription factors, has ZN[2]- CYS[6]
fungal-type binuclear cluster domain in the N-terminal region 74
YGR177C (ATF2) 3.7081426 11.830167 12.5552685 Alcohol
O-acetyltransferase 75 YFR034C (PHO4) 14.8112083 11.2160731
20.8445145 Basic helix-loop-helix (BHLH) transcription factor
required for expression of phosphate pathway, hyperphosphorylation
by PHO80P-PHO85P cyclin-dependent protein kinase complex causes
inactivation 83 YOL067C (RTG1) 30.4142081 10.0270648 27.3663295
Basic helix-loop-helix (BHLH) transcription factor involved in
inter-organelle communication between mitochondria, peroxisomes,
and nucleus 100 YJL127C (SPT10) 4.01528284 7.83944269 10.0960266
Protein that amplifies the magnitude of transcriptional regulation
at various loci 111 YMR037C (MSN2) 6.80686734 6.42359685 7.66129891
Zinc-finger transcriptional activator for genes involved in the
multistress response and genes regulated through SNF1P 118 YCR048W
(ARE1) 9.11370518 6.1039374 10.5312906 Acyl-COA: sterol
acyltransferase (sterol-ester synthetase) 131 YAL013W (DEP1)
8.79366086 5.54633863 6.42500999 Regulator of phospholipid
metabolism 132 YIL084C (SDS3) 1.99582364 5.54306878 6.90742248
Suppressor of silencing defect 157 YKR034W (DAL80) 3.91750209
5.0436172 7.28385659 GATA-type zinc finger transcriptional
repressor for allantoin and 4-aminobutyric acid (GABA) catabolic
genes 172 YLR098C (CHA4) 2.05280928 4.75643469 5.58664651
Zinc-finger protein required for activation of CHA1, has A ZN[2]-
CYS[6] fungal-type binuclear cluster domain 180 YDR389W (SAC7)
3.89197011 4.56095992 4.31431086 GTPase-activating protein for
RHO1P 202 YDL088C (ASM4) 4.39685251 4.17572645 3.32103404
Suppressor of temperature- sensitive mutations in POL3P (DNA
polymerase delta) 206 YBL005W (PDR3) 3.75060207 4.14490535
6.08273054 Transcription factor related to PDR1P, contains A
ZN[2]-CYS[6] fungal-type binuclear cluster domain in the N-terminal
region 242 YGL071W (RCS1) 3.39203358 3.69630773 4.53101664
Regulatory protein involved in iron uptake 255 YAR044W (OSH1)
4.12112011 3.624939 3.88396219 Protein implicated in ergosterol
biosynthesis, member of the KES1/HES1/OSH1/YKR003W family of
oxysterol-binding (OSBP) proteins 256 YLR120C (YAP3) 6.14265883
3.62298451 4.42985615 Transcription factor of the basic leucine
zipper (BZIP) family, one of eight members of a novel
fungal-specific family of BZIP proteins 260 YJR017C (ESS1)
2.98118086 3.55874146 3.22082555 Processing/termination factor,
involved in transcription termination or 3'-end processing of
pre-MRNA 271 YMR019W (STB4) 3.2576922 3.44146214 3.39764598
SIN3P-binding protein, has ZN[2]- CYS[6] fungal-type binuclear
cluster domain in the N-terminal region 278 YLR153C (ACS2)
3.45528019 3.37854457 3.12858117 Acetyl-COA synthetase (acetate-
COA ligase) 289 YPL119C (DBP1) 5.87199247 3.24642228 2.19736599
ATP-dependent RNA helicase of dead box family, suppressor of
SPP81/DED1 290 YGL014W -- 3.11296478 3.22942947 3.6382821 Protein
with pumilio repeats that is involved with MPT5P in relocalization
of SIR3P and SIR4P from telomeres to the nucleolus 296 YGL192W
(IME4) 2.89030953 3.17038103 6.47841053 Positive transcription
factor for IME1 and IME2, mediates control of meiosis by carrying
signals regarding mating type (A/alpha) and nutritional status 297
YMR047C (NUP116) 2.56622055 3.17022339 4.77420515 Nuclear pore
protein (nucleoporin) of the GLFG family, may be involved in
binding and translocation of nuclear proteins 301 YLR249W (YEF3)
3.59397167 3.14453335 2.63195398 Translation elongation factor EF-
3A, member of ATP-binding cassette (ABC) superfamily 322 YOR337W
(TEA1) 2.13152473 2.97907151 4.82285812 TY1 enhancer activator of
the GAL4P-type family of DNA- binding proteins 331 YCR084C (TUP1)
2.40138822 2.92198431 3.27182635 General repressor of transcription
(with SSN6P), member of WD (WD-40) repeat family 336 YPL226W --
2.45263084 2.88856775 2.55579443 Protein with similarity to members
of the ATP-binding cassette (ABC) superfamily 345 YJR019C (TES1)
4.07777555 2.83032346 2.07248965 Acyl-COA thioesterase 346 YBL008W
(HIR1) 7.24580603 2.82847131 2.88668127 Histone transcription
inhibitor, required for periodic repression of 3 of the 4 histone
gene loci and for autogenous repression of HTA1- HTB1 locus by H2A
and H2B 349 YGL001C -- 3.91981575 2.82148161 1.98527852 Protein
with similarity to nocardia SP. cholesterol dehydrogenase 359
YCL037C (SRO9) 8.35007693 2.78557477 2.39358801 Suppressor of YPT6
null and RHO3 mutations 367 YDR088C (SLU7) 2.07293165 2.73396273
2.68767436 Pre-MRNA splicing factor affecting 3' splice site
choice, required only for the second catalytic step of splicing 376
YBR289W (SNF5) 2.00671327 2.68812945 3.03861899 Component of
SWI/SNF global transcription activator complex, acts to assist
gene-specific activators through chromatin remodeling 400 YPL237W
(SUI3) 2.5966981 2.5628077 2.54794558 Translation initiation factor
EIF2 beta subunit 406 YOR127W (RGA1) 3.85804733 2.53166489
2.56973414 RHO-type GTPase-activating protein (GAP) for CDC42P 416
YFR009W (GCN20) 2.63782118 2.48595438 2.16133777 Component of a
protein complex required for activation of GCN2P protein kinase in
response to amino acid starvation, member of ATP- binding cassette
(ABC) superfamily 417 YDR211W (GCD6) 2.22567451 2.48354852
1.82406386 Translation initiation factor EIF2B (guanine nucleotide
exchange factor), 81 KDA (beta) subunit 440 YGR270W (YTA7)
2.68803581 2.4056715 1.94575504 Protein with similarity to members
of the AAA family of ATPases 441 YBR119W (MUD1) 2.83912216
2.40515252 1.39876418 U1 SNRNP A protein (SNRNA- associated
protein) with 2 RNA recognition (RRM) domains 442 YDR052C (DBF4)
6.85835185 2.40369283 1.58349756 Regulatory subunit for CDC7P
protein kinase, required for G1/S transition 492 YPR080W (TEF1)
2.50402057 2.21150946 1.85878786 Translation elongation factor EF-
1alpha (TEF1 and TEF2 code for identical proteins) 496 YDR264C
(AKR1) 3.13151279 2.19839665 2.75165355 Ankyrin repeat-containing
protein that has an inhibitory effect on signaling in the pheromone
pathway 503 YDR422C (SIP1) 2.62373247 2.18707608 2.08363472
Multicopy suppressor of SNF1, related to GAL83P/SPM1P and SPM2P 504
YMR080C (NAM7) 2.82340116 2.1828046 2.17148277 Protein involved
with NMD2P and UPF3P in decay of MRNA containing nonsense codons
515 YAL048C -- 5.02313141 2.13847476 4.22113197 Protein with weak
similarity to RAS1P, RAS2P, and other GTP- binding proteins of the
RAS superfamily 526 YHL027W (RIM101) 2.57210755 2.10331571
2.59278915 Zinc-finger protein involved in induction of IME1 531
YGL112C (TAF60) 2.21463331 2.07652653 2.18913076 Component of
TAF(II) complex (TBP-associated protein complex) required for
activated transcription by RNA polymerase II 549 YPL028W (ERG10)
2.86559138 2.01859514 1.69893374 Acetyl-COA acetyltransferase
(acetoacetyl-COA thiolase), first step in mevalonate/sterol pathway
621 YNR019W (ARE2) 2.1 1.79103463 2.85442 Acyl-COA: sterol
acyltransferase (sterol-ester synthetase) 560 YFL054C -- 7.43223753
0.47935821 0.61365816 Protein with similarity to FPS1P and YPR192P,
member of MIP family of transmembrane channels 562 YAL001C (TFC3)
2.94740587 0.47427458 1.19155655 RNA polymerase transcription
initiation factor TFIIIC (TAU), 138 KDA subunit 579 YIR017C (MET28)
2.97658904 0.3775372 0.30088953 Transcriptional activator of the
basic leucine zipper (BZIP) family, works with MET4P and CBF1P to
regulation sulfur amino acid metabolism 585 YDR253C (MET32)
2.86314943 0.33971751 0.32790428 Zinc-finger protein involved in
transcriptional regulation of methionine metabolism 595 YMR146C
(TIF34) 5.0351989 0.22482039 0.71935381 Translation initiation
factor EIF3, P39 subunit, has 2 WD (WD-40) repeats 616 YGR083C
(GCD2) 2.32134339 0 0 Translation initiation factor EIF2B (guanine
nucleotide exchange factor), 71 KDA (delta) subunit 610 YOR139C
(SFL1) 5.46648132 0 11.7609951 Transcription factor with domains
homologous to MYC oncoprotein and yeast HSF1P, required for normal
cell surface assembly and flocculence *Table Headings: Clone ID: A
clone ID designation number. CJ-4 hr/LP-4 hr: Expression level in
the mutant CJ517 as compared with the respective wild type strain
LPY9 at 4 hr sampling of log phase growth of yeast (ratio of mutant
expression level/control expression level). Genes in the Table are
either up or down regulated in the mutant (CJ517) as well as in
response to ketoconazole treatment. K-50/CK: Expression level in
the wild type yeast LPY9, at 2 hr after treatment with 50 micro
gram/ml ketoconazole as compared to the wild type LPY9 strain
without ketoconazole treatment (ratio of treatment expression
level/control expression level). K-100/CK: Expression level in the
wild type yeast LPY9, at 2 hr after treatment with 100 micro
gram/ml ketoconazole as compared to the wild type LPY9 strain
without ketoconazole treatment (ratio of treatment expression
level/control expression level). Gene Description: Description of
the clone listed in column 1.
[0328] In addition, for example, Table 2 identifies a yeast HES1
gene as a gene with an associated change in mRNA levels in the two
different comparisons. Fang et al. EMBO J 15:6447-59 (1996), the
entirety of which is herein incorporated by reference, reported a
mutation in HES1, which caused a 55% reduction in carbon flux
through the mevalonate pathway in yeast.
[0329] Each of the sequences listed in Table 2 or 3 represents a
gene that effects sterol levels, directly or indirectly, or whose
expression changes as a result of alterations in the sterol
synthesis pathway.
EXAMPLE 2
[0330] Sequences that encode for the yeast HES1 protein are used to
search databases for homologues from other species. A number of
different databases can be used for these searches, including, for
example, dbEST, GenBank, EMBL, SwissProt, PIR, and GENES. In
addition, various algorithms for searching can be selected, such
as, for example, the BLAST suite of programs at the default values.
Typically, matches found with BLAST P values equal or less than
0.001 (probability) or BLAST Score of equal or greater than 90 are
classified as hits. If the program used to determine the hit is
HMMSW then the score refers to HMMSW score. The GenBank database is
searched with BLASTN and BLASTX (default values). Sequences that
pass the hit probability threshold of 10e.sup.-8 are considered
hits.
4TABLE 4 Seq. Sequence: Num. Clone ID DNA/Protein Hit description
Library 1 701100307CPR9855 DNA Yeast HES 1 homolog SOYMON028 2
701001443CPR9857 DNA Yeast HES 1 homolog SOYMON018 3
701010572CPR9854 DNA Yeast HES 1 homolog SOYMON019 4
701176735CPR9736 DNA Yeast HES 1 homolog SATMONN05 5 Z75145 DNA
Protein implicated in ergosterol biosynthesis, -- member of the
KES1/HES1/OSH1/YKR003W family of oxysterol-binding (OSBP) proteins
622 701100307CPR9855 Protein Yeast HES 1 homolog SOYMON028 623
701001443CPR9857 Protein Yeast HES 1 homolog SOYMON018 624
701010572CPR9854 Protein Yeast HES 1 homolog SOYMON019 625
701176735CPR9736 Protein Yeast HES 1 homolog SATMONN05 626 Z75145
Protein Protein implicated in ergosterol biosynthesis, -- member of
the KES1/HES1/OSH1/YKR003W family of oxysterol-binding (OSBP)
proteins 6 701003888H1 DNA Yeast HES 1 homolog SOYMON019 7
701001351H1 DNA Yeast HES 1 homolog SOYMON018 8 700672545H1 DNA
Yeast HES 1 homolog SOYMON006 9 700664054H1 DNA Yeast HES 1 homolog
SOYMON005 10 700665644H1 DNA Yeast HES 1 homolog SOYMON005 11
700764248H1 DNA Yeast HES 1 homolog SOYMON020 12 700851444H1 DNA
Yeast HES 1 homolog SOYMON023 13 700971910H1 DNA Yeast HES 1
homolog SOYMON005 14 700652932H1 DNA Yeast HES 1 homolog SOYMON003
15 700982894H1 DNA Yeast HES 1 homolog SOYMON009 16 701120140H1 DNA
Yeast HES 1 homolog SOYMON037 17 701064234H1 DNA Yeast HES 1
homolog SOYMON034 18 700954013H1 DNA Yeast HES 1 homolog SOYMON022
19 701129375H1 DNA Yeast HES 1 homolog SOYMON037 20 701043941H1 DNA
Yeast HES 1 homolog SOYMON032 21 LIB24-114-Q1-E1-H8 DNA Arabidopsis
HES 1 homolog LIB24 22 LIB22-016-Q1-E1-F3 DNA Arabidopsis HES 1
homolog LIB22 23 LIB25-101-Q1-E1-F1 DNA Arabidopsis HES 1 homolog
LIB25 24 AA042357 DNA Arabidopsis HES 1 homolog -- 25 AA720163 DNA
Arabidopsis HES 1 homolog -- 26 Z29936 DNA Arabidopsis HES 1
homolog -- 27 T76850 DNA Arabidopsis HES 1 homolog -- 28 T76580 DNA
Arabidopsis HES 1 homolog -- 29 AA586043 DNA Arabidopsis HES 1
homolog --
[0331] Homologues to yeast HES1 are also identified in the
following libraries: SOYMON003, SOYMON005, SOYMON006, SOYMON009,
SOYMON018, SOYMON019, SOYMON020, SOYMON022, SOYMON028, SOYMON023,
SOYMON032, SOYMON034, SOYMON027, SATMONN05, LIB22, LIB24, and LIB25
These libraries are prepared as follows:
[0332] The SATMONN05 cDNA library is a normalized library generated
from maize (B73 x Mo17, Illinois Foundation Seeds, Champaign Ill.,
U.S.A.) root tissue at the V6 development stage. Seeds are planted
at a depth of approximately 3 cm into 2-3 inch peat pots containing
Metro 200 growing medium. After 2-3 weeks growth they are
transplanted into 10 inch pots containing the same growing medium.
Plants are watered daily before transplantation and three times a
week after transplantation. Peters 15-16-17 fertilizer is applied
three times per week after transplanting at a strength of 150 ppm
N. Two to three times during the lifetime of the plant, from
transplanting to flowering, a total of 900 mg Fe is added to each
pot. Maize plants are grown in a greenhouse in 15 hr day/9 hr night
cycles. The daytime temperature is approximately 80.degree. F. and
the nighttime temperature is approximately 70.degree. F.
Supplemental lighting is provided by 1000 W sodium vapor lamps.
Tissue is collected when the maize plant is at the 6-leaf
development stage. The root system is cut from the mature maize
plant and washed with water to free it from the soil. The tissue is
immediately frozen in liquid nitrogen and the harvested tissue is
then stored at -80.degree. C. until RNA preparation. The RNA is
purified from the stored tissue. The library is normalized in two
rounds using conditions adapted from Soares et al., Proc. Natl.
Acad. Sci. (U.S.A.) 91:9928 (1994), the entirety of which is herein
incorporated by reference and Bonaldo et al., Genome Res. 6: 791
(1996), the entirety of which is herein incorporated by reference
except that a significantly longer (48-hours/round) reannealing
hybridization was used. SATMON003 is a root tissue library from the
same donor.
[0333] The SOYMON003 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
hypocotyl axis tissue from seedlings 2 day after-imbibition. Seeds
are planted at a depth of approximately 2 cm into 2-3 inch peat
pots containing Metromix 350 medium. Trays are placed in an
environmental chamber and grown at 12 hr daytime/12 hr nighttime
cycles. The daytime temperature is approximately 29.degree. C, and
the nighttime temperature approximately 24.degree. C. Soil is
checked and watered daily to maintain even moisture conditions.
Tissue is collected 2 days after the start of imbibition. The 2
days after imbibition samples are separated into 3 collections
after removal of any adhering seed coat. At 2 days after imbibition
under the above conditions, the seedlings have significant
expansion of the axis and are close to emerging from the soil. A
few seedlings have cracked the soil surface and exhibited slight
greening of the exposed cotyledons. The seedlings are washed in
water to remove soil, hypocotyl axis harvested and immediately
frozen in liquid nitrogen. The harvested tissue is then stored at
-80.degree. C. until RNA preparation. The RNA is purified from the
stored tissue and the cDNA library is constructed.
[0334] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0335] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0336] The SOYMON005 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
hypocotyl axis tissue from seeds 6 hour post-imbibition. Seeds are
planted at a depth of approximately 2 cm into 2-3 inch peat pots
containing Metromix 350 medium. Trays are placed in an
environmental chamber and grown at 12 hr daytime/12 hr nighttime
cycles. The daytime temperature is approximately 29.degree. C, and
the nighttime temperature approximately 24.degree. C. Soil is
checked and watered daily to maintain even moisture conditions.
Tissue is collected 6 hours after the start of imbibition. The 6
hours after imbibition sample is collected over the course of
approximately 2 hours starting at 6 hours post imbibition. At the 6
hours after imbibition stage, not all cotyledons have become fully
hydrated and germination. Radicle protrusion has not occurred. The
seedlings are washed in water to remove soil, then the hypocotyl
axis is harvested and immediately frozen in liquid nitrogen. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation. The RNA is purified from the stored tissue and the
cDNA library is constructed.
[0337] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0338] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0339] The SOYMON006 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
cotyledons from seeds 6 hour post-imbibition. Seeds are planted at
a depth of approximately 2 cm into 2-3 inch peat pots containing
Metromix 350 medium. Trays are placed in an environmental chamber
and grown at 12 hr daytime/12 hr nighttime cycles. The daytime
temperature is approximately 29.degree. C., and the nighttime
temperature approximately 24.degree. C. Soil is checked and watered
daily to maintain even moisture conditions. Tissue is collected 6
hours after imbibition. The 6 hours after imbibition sample is
collected over the course of approximately 2 hours starting at 6
hours post-imbibition. At the 6 hours after imbibition, not all
cotyledons have become fully hydrated and germination. Radicle
protrusion has not occurred. The seedlings are washed in water to
remove soil, then the cotyledon is harvested and immediately frozen
in liquid nitrogen. The harvested tissue is then stored at
-80.degree. C. until RNA preparation. The RNA is purified from the
stored tissue and the cDNA library is constructed.
[0340] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0341] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0342] The SOYMON009 cDNA library is generated from soybean
cutlivar C1944 (USDA Soybean Germplasm Collection, Urbana, Ill.
U.S.A.) pod and seed tissue harvested 15 days post-flowering. Pods
from field grown plants are harvested 15 days post-flowering. The
pods are picked from all over the plant, placed into 14 ml
polystyrene tubes and immediately immersed in dry-ice.
Approximately 3 g of pod tissue is harvested. The harvested tissue
is then stored at -80.degree. C. until RNA preparation. The RNA is
purified from the stored tissue and the cDNA library is
constructed. The RNA is purified from the stored tissue and the
cDNA library is constructed.
[0343] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0344] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0345] The SOYMON018 cDNA is generated from soybean cultivar Asgrow
3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.) leaf tissue
harvested from plants grown in a field in Jerseyville 45 and 55
days after flowering. Leaves from field grown plants are harvested
45 and 55 days after flowering from the fourth node. Approximately
27 g and 33 g of leaves are collected from the 45 and 55 days after
flowering plants, placed into 14 ml polystyrene tubes and
immediately immersed in dry ice. The harvested tissue is then
stored at -80.degree. C. until RNA preparation. Total RNA is
prepared from the combination of equal amounts of leaf tissue from
both time points and the cDNA library is constructed.
[0346] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0347] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0348] The SOYMON019 cDNA library is generated from soybean
cultivars Cristalina (USDA Soybean Germplasm Collection, Urbana,
Ill. U.S.A.) and FT108 (Monsoy, Brazil) (tropical germ plasma) root
tissue. Roots are harvested from plants grown in an environmental
chamber under 12 hr daytime/12 hr nighttime cycles. The daytime
temperature is approximately 29.degree. C., and the nighttime
temperature approximately 24.degree. C. Soil is checked and watered
daily to maintain even moisture conditions. Approximately 50 g and
56 g of roots are harvested from each of the Cristalina and FT108
cultivars and immediately frozen in dry ice. The plants are
uprooted and the roots quickly rinsed in a pail of water. The root
tissue is then cut from the plants, placed immediately in 14 ml
polystyrene tubes and immersed in dry-ice. The harvested tissue is
then stored at -80.degree. C. until RNA preparation. Total RNA is
prepared from the combination of equal amounts of root tissue from
each cultivar and the cDNA library is constructed.
[0349] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0350] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0351] The SOYMON020 cDNA is generated from soybean cultivar Asgrow
3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.) seeds harvested
from plants grown in a field in Jerseyville 65 and 75 days
post-flowering. The seed pods are picked from all over the plant
and the seeds extracted from the pods. Approximately 14 g and 31 g
of seeds are harvested from the respective seed pods and
immediately frozen in dry ice. The harvested tissue is then stored
at -80.degree. C. until RNA preparation. Total RNA is prepared from
the combination of equal numbers of seeds from 65 and 75 days after
flowering and the cDNA library is constructed.
[0352] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT-beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0353] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0354] The SOYMON022 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
partially to fully opened flower tissue, which is harvested from
plants grown in an environmental chamber. Seeds are planted in
moist Metromix 350 medium at a depth of approximately 2 cm. Trays
are placed in an environmental chamber set to a 12 h day/12 h night
cycle, 29.degree. C. daytime temperature, 24.degree. C. night
temperature and 70% relative humidity. Daytime light levels are
measured at 450 .mu.Einsteins/m.sup.2. Soil is checked and watered
daily to maintain even moisture conditions. Flowers are removed
from the plant at the pedicel. Flower buds showing petal color to
fully open flowers are selected for collection. A total of 3 g of
flower tissue is harvested and immediately frozen in dry ice. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation. Total RNA is prepared from a mixture of opened and
partially opened flowers and the cDNA library is constructed.
[0355] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0356] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0357] The SOYMON023 cDNA library is generated from soybean
genotype BW211S Null (Tohoku University, Morioka, Japan) seed
tissue harvested from plants grown in a field in Jerseyville. After
15 and 40 days, pods are harvested from all over the plant and
seeds are dissected out from the pods. Approximately, 0.7 g and
14.2 g of seeds are harvested from the plants at the 15 and 40 days
after flowering timepoints. The seeds are placed into 14 ml
polystyrene tubes and immersed in dry-ice. The tissue is then
transferred to a -80.degree. C. freezer for storage. The harvested
tissue is then stored at -80.degree. C. until RNA preparation.
Total RNA is prepared from the combination of 0.5 g and 1.0 g of
seeds from the 15 and 40 days after flowering timepoints and the
cDNA library is constructed.
[0358] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0359] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0360] The SOYMON028 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
drought-stressed root tissue. Seeds are planted in moist Metromix
350 medium at a depth of approximately 2 cm in trays. The trays are
placed in an environmental chamber set to a 12 h day/12 h night
cycle, 26.degree. C. daytime temperature, 21.degree. C. night
temperature and 70% relative humidity. Daytime light levels are
measured at 300 .mu.Einsteins/m.sup.2. Soil is checked and watered
daily to maintain even moisture conditions. At the R3 stage of
development, water is withheld from half of the plant collection
(drought stressed population). After 3 days, half of the plants
from the drought stressed condition and half of the plants from the
control population are harvested. After another 3 days (6 days post
drought induction) the remaining plants are harvested. A total of
27 g and 40 g of root tissue is harvested from plants at two time
points and immediately frozen in dry ice. The harvested tissue is
then stored at -80.degree. C. until RNA preparation. Total RNA is
prepared from the combination of equal amounts of drought stressed
root tissue from both time points and the cDNA library is
constructed.
[0361] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0362] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0363] The SOYMON032 cDNA library is prepared from the Asgrow
cultivar A4922 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
rehydrated dry soybean seed meristem tissue. Surface sterilized
seeds are germinated in liquid media for 24 hours. The seed axis is
then excised from the barely germinating seed, placed on tissue
culture media and incubated overnight at 20.degree. C. in the dark.
The supportive tissue is removed from the explant prior to harvest.
Approximately 570 mg of tissue is harvested and frozen in liquid
nitrogen. The harvested tissue is then stored at -80.degree. C.
until RNA preparation. The RNA is purified from the stored tissue
and the cDNA library is constructed.
[0364] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0365] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0366] The SOYMON034 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
cold-shocked seedling tissue without cotyledons. Seeds are imbibed
and germinated in vermiculite for 2 days under constant
illumination (ca. 510 Lux). After 48 hours, the seedlings are
transferred to a cold room set at 5.degree. C. under constant
illumination (ca. 560 Lux). After 30, 60 and 180 minutes seedlings
are harvested and dissected. The seedlings after 2 days of
imbibition are beginning to emerge from the vermiculite surface.
The apical hooks are dark green in appearance. A portion of the
seedling consisting of the root, hypocotyl and apical hook is
frozen in liquid nitrogen and stored at -80.degree. C. Total RNA is
prepared from equal amounts of pooled tissue and the cDNA library
is constructed.
[0367] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0368] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0369] The SOYMON037 cDNA library is generated from soybean
cultivar A3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
etiolated axis and radical tissue. Seeds are planted in moist
vermiculite, wrapped and kept at room temperature in complete
darkness until harvest. Etiolated axis and hypocotyl tissue is
harvested at 2, 3 and 4 days post-planting. Samples are frozen in
liquid nitrogen upon harvesting and stored at -80.degree. C. until
RNA preparation. 1 gram of each sample (axis+hypocotyl at day 2, 3
and 4) is pooled for RNA isolation. The RNA is purified from the
pooled tissue and the cDNA library is constructed.
[0370] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0371] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0372] The cDNA library of the present invention designated LIB22,
is prepared from Arabidopsis thaliana Columbia ecotype root tissue.
Wild type Arabidopsis thaliana seeds are planted in commonly used
planting pots and grown in an environmental chamber. After 5-6
weeks the plants are in the reproductive growth phase. Stems are
bolting from the base of the plants. After 7 weeks, more stems and
floral buds appear, and a few flowers are starting to open. Roots
of 7-week old plants from pots are rinsed intensively with tap
water to wash away dirt, and briefly blotted by paper towel to take
away free water. The tissues are immediately frozen in liquid
nitrogen and stored at -80.degree. C. until total RNA
extraction.
[0373] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0374] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0375] The cDNA library of the present invention designated LIB24,
is prepared from Arabidopsis thaliana, Columbia ecotype, flower bud
tissue. Wild type Arabidopsis thaliana seeds are planted in
commonly used planting pots and grown in an environmental chamber.
Flower buds are green and unopened and are harvested about seven
weeks after planting. The tissue is immediately frozen in liquid
nitrogen and stored at -80.degree. C. until total RNA
extraction.
[0376] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0377] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0378] The cDNA library of the present invention designated LIB25,
is prepared from Arabidopsis thaliana, Columbia ecotype, open
flower tissue. Wild type Arabidopsis thaliana seeds are planted in
commonly used planting pots and grown in an environmental chamber.
Flower are completely opened with all parts of floral structure
observable, but no siliques are appearing, and are harvested about
seven weeks after planting. The tissue was immediately frozen in
liquid nitrogen and stored at -80.degree. C. until total RNA
extraction.
[0379] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, N.Y. U.S.A.).
[0380] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
EXAMPLE 3
Detection of Changes in Sterol Metabolism
[0381] A labeled acetyl-CoA molecule, squalene molecule, or acetate
are used in a variety of assays to detect changes in sterol
production, secretion, localization, protein-binding, degradation,
and trafficking known in the art. The example below
illustrates.
[0382] Cells from transformed plants are cultured in an appropriate
medium. Labeled acetate, preferably .sup.14C-labeled, is added to a
concentration of about 1 uCi/ml. After a period of growth, the
cells are collected, the lipids extracted, and resolved by
thin-layer chromatography or run over HPLC column using known
methods. The levels of each sterol resolved can be compared to
control cells fed the same labeled .sup.14C acetate and the amount
of .sup.14C-labeled sterol for each determined from the resolved
sterols.
References
[0383] In addition to those references cited and incorporated by
reference above, the below references are incorporated in their
entirety. In addition, these references, as well as each of those
cited in the Summary and Detailed Description above, can be relied
upon to make and use aspects of the invention.
[0384] Jiang, et al., A new family of yeast genes implicated in
ergosterol synthesis is related to the human oxysterol-binding
protein. Yeast 10: 341-53 (1994).
[0385] Fang, et al., Kes1p shares homology with human
oxysterol-binding protein and participates in a novel regulatory
pathway for yeast Golgi-derived transport vesicle biogenesis. EMBO
J. 15: 6447-59(1996).
[0386] Crowley, et al., A mutation in a purported regulatory gene
affects control of sterol uptake in Saccharomyces cerevisiae.
Journal of Bacteriology, 180(16): 4177-83 (1998).
[0387] Casperand Holt. Expression of the green fluorescent
protein-encoding gene from a tobacco mosaic virus-based vector.
Gene 173: 69-73 (1996).
Sequence CWU 0
0
* * * * *
References