U.S. patent application number 09/342653 was filed with the patent office on 2001-10-18 for chromatin associated proteins.
Invention is credited to CAHOON, REBECCA, RAFALSKI, J. ANTONI.
Application Number | 20010031465 09/342653 |
Document ID | / |
Family ID | 22235422 |
Filed Date | 2001-10-18 |
United States Patent
Application |
20010031465 |
Kind Code |
A1 |
CAHOON, REBECCA ; et
al. |
October 18, 2001 |
CHROMATIN ASSOCIATED PROTEINS
Abstract
This invention relates to an isolated nucleic acid fragment
encoding a chromatin associated protein. The invention also relates
to the construction of a chimeric gene encoding all or a portion of
the chromatin associated protein, in sense or antisense
orientation, wherein expression of the chimeric gene results in
production of altered levels of the chromatin associated protein in
a transformed host cell.
Inventors: |
CAHOON, REBECCA;
(WILMINGTON, DE) ; RAFALSKI, J. ANTONI;
(WILMINGTON, DE) |
Correspondence
Address: |
WILLIAM R MAJARIAN
E I DU PONT DE NEMOURS & COMPANY
LEGAL PATENTS
WILMINGTON
DE
19898
|
Family ID: |
22235422 |
Appl. No.: |
09/342653 |
Filed: |
June 29, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60092841 |
Jul 14, 1998 |
|
|
|
Current U.S.
Class: |
435/6.13 ;
435/189; 435/6.1; 435/91.2; 536/23.2 |
Current CPC
Class: |
Y02A 40/146 20180101;
C12N 15/8201 20130101; C12N 9/16 20130101; C12N 9/001 20130101;
C12N 15/8216 20130101; C12N 15/822 20130101; C07K 14/415 20130101;
C12N 15/8261 20130101 |
Class at
Publication: |
435/6 ; 435/91.2;
435/189; 536/23.2 |
International
Class: |
C12Q 001/68; C12P
019/34; C07H 021/04; C12N 009/02 |
Claims
What is claimed is:
1. An isolated nucleic acid fragment encoding a lamin B
receptor/sterol reductase comprising a member selected from the
group consisting of: (a) an isolated nucleic acid fragment encoding
an amino acid sequence that is at least 65% similar to the amino
acid sequence set forth in a member selected from the group
consisting of SEQ ID NO:2, 4 and 6; (b) an isolated nucleic acid
fragment that is complementary to (a).
2. The isolated nucleic acid fragment of claim 1 wherein nucleic
acid fragment is a functional RNA.
3. The isolated nucleic acid fragment of claim 1 wherein the
nucleotide sequence of the fragment comprises the sequence set
forth in a member selected from the group consisting of SEQ ID
NO:1, 3 and 5.
4. A chimeric gene comprising the nucleic acid fragment of claim 1
operably linked to suitable regulatory sequences.
5. A transformed host cell comprising the chimeric gene of claim
4.
6. A lamin B receptor/sterol reductase polypeptide comprising all
or a substantial portion of the amino acid sequence set forth in a
member selected from the group consisting of SEQ ID NO:2, 4 and
6.
7. A method of altering the level of expression of a chromatin
associated protein in a host cell comprising: (a) transforming a
host cell with the chimeric gene of claim 4; and (b) growing the
transformed host cell produced in step (a) under conditions that
are suitable for expression of the chimeric gene wherein expression
of the chimeric gene results in production of altered levels of a
chromatin associated protein in the transformed host cell.
8. A method of obtaining a nucleic acid fragment encoding all or a
substantial portion of the amino acid sequence encoding a chromatin
associated protein comprising: (a) probing a cDNA or genomic
library with the nucleic acid fragment of claim 1; (b) identifying
a DNA clone that hybridizes with the nucleic acid fragment of claim
1; (c) isolating the DNA clone identified in step (b); and (d)
sequencing the cDNA or genomic fragment that comprises the clone
isolated in step (c) wherein the sequenced nucleic acid fragment
encodes all or a substantial portion of the amino acid sequence
encoding a chromatin associated protein.
9. A method of obtaining a nucleic acid fragment encoding a
substantial portion of an amino acid sequence encoding a chromatin
associated protein comprising: (a) synthesizing an oligonucleotide
primer corresponding to a portion of the sequence set forth in any
of SEQ ID NOs:1, 3 and 5; and (b) amplifying a cDNA insert present
in a cloning vector using the oligonucleotide primer of step (a)
and a primer representing sequences of the cloning vector wherein
the amplified nucleic acid fragment encodes a substantial portion
of an amino acid sequence encoding a chromatin associated
protein.
10. The product of the method of claim 8.
11. The product of the method of claim 9.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/092,841 filed Jul. 14, 1998.
FIELD OF THE INVENTION
[0002] This invention is in the field of plant molecular biology.
More specifically, this invention pertains to nucleic acid
fragments encoding chromatin associated proteins in plants and
seeds.
BACKGROUND OF THE INVENTION
[0003] The human Lamin B receptor (LBR) belongs to the ERG4/ERG24
family of nuclear envelope inner membrane proteins. It anchors the
lamina and the heterochromatin to the inner nuclear membrane. LBR
can interact with chromodomain proteins. LBR has an amino-terminal
domain of approximately 200 amino acids followed by a
carboxyl-terminal domain that is similar in sequence to yeast and
plant sterol reductases. Two LBR-like genes have recently been
identified in humans which have strong carboxyl-terminal domains of
LBR and sterol reductases (Pezhman et al. (1998) Genomics
54(3):469-476). The human LBR/sterol reductase like proteins are
localized to the endoplasmic reticulum. These LBR/sterol reductase
proteins may define a human gene family encoding proteins of the
inner nuclear membrane and endoplasmic reticulum that function in
nuclear organization and/or sterol metabolism.
[0004] In the nucleus LBR undergoes phosphorylation by CDC2 protein
kinase in mitosis when the inner nuclear membrane breaks down into
vesicles that dissociate from the lamina and the chromatin. It is
phosphorylated by different protein kinases in interphase when the
membrane is associated with these structures. Phosphorylation of
LBR proteins may be responsible for some of the alternations in
chromatin organization and nuclear structure which occur at various
times during the cell cycle. To date, a Lamin B receptor has not
been identified in plants. A plant Lamin B receptor could be used
to manipulate cell cycle regulation and plant transformability.
[0005] Accordingly, the availability of nucleic acid sequences
encoding all or a portion of these proteins would facilitate
studies to better understand transcritional regulation, cell cycle
progression, and developmental events in eucaryotic cells. It would
also provide genetic tools for the manipulation of cell cycle
regulation and increase the efficiency of transformation.
SUMMARY OF THE INVENTION
[0006] The instant invention relates to isolated nucleic acid
fragments encoding chromatin associated proteins. Specifically,
this invention concerns an isolated nucleic acid fragment encoding
a lamin B receptor/sterol reductase and an isolated nucleic acid
fragment that is substantially similar to an isolated nucleic acid
fragment encoding a lamin B receptor/sterol reductase. In addition,
this invention relates to a nucleic acid fragment that is
complementary to the nucleic acid fragment encoding lamin B
receptor/sterol reductase.
[0007] An additional embodiment of the instant invention pertains
to a polypeptide encoding all or a substantial portion of a lamin B
receptor/sterol reductase.
[0008] In another embodiment, the instant invention relates to a
chimeric gene encoding a lamin B receptor/sterol reductase, or to a
chimeric gene that comprises a nucleic acid fragment that is
complementary to a nucleic acid fragment encoding a lamin B
receptor/sterol reductase, operably linked to suitable regulatory
sequences, wherein expression of the chimeric gene results in
production of levels of the encoded protein in a transformed host
cell that is altered (i.e., increased or decreased) from the level
produced in an untransformed host cell.
[0009] In a further embodiment, the instant invention concerns a
transformed host cell comprising in its genome a chimeric gene
encoding a lamin B receptor/sterol reductase, operably linked to
suitable regulatory sequences. Expression of the chimeric gene
results in production of altered levels of the encoded protein in
the transformed host cell. The transformed host cell can be of
eukaryotic or prokaryotic origin, and include cells derived from
higher plants and microorganisms. The invention also includes
transformed plants that arise from transformed host cells of higher
plants, and seeds derived from such transformed plants.
[0010] An additional embodiment of the instant invention concerns a
method of altering the level of expression of a lamin B
receptor/sterol reductase in a transformed host cell comprising: a)
transforming a host cell with a chimeric gene comprising a nucleic
acid fragment encoding a lamin B receptor/sterol reductase; and b)
growing the transformed host cell under conditions that are
suitable for expression of the chimeric gene wherein expression of
the chimeric gene results in production of altered levels of lamin
B receptor/sterol reductase in the transformed host cell.
[0011] An addition embodiment of the instant invention concerns a
method for obtaining a nucleic acid fragment encoding all or a
substantial portion of an amino acid sequence encoding a lamin B
receptor/sterol reductase.
BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS
[0012] The invention can be more fully understood from the
following detailed description and the accompanying Sequence
Listing which form a part of this application.
[0013] Table 1 lists the polypeptides that are described herein,
the designation of the cDNA clones that comprise the nucleic acid
fragments encoding polypeptides representing all or a substantial
portion of these polypeptides, and the corresponding identifier
(SEQ ID NO:) as used in the attached Sequence Listing. The sequence
descriptions and Sequence Listing attached hereto comply with the
rules governing nucleotide and/or amino acid sequence disclosures
in patent applications as set forth in 37 C.F.R.
.sctn.1.821-1.825.
1TABLE 1 Chromatin Associated Proteins SEQ ID NO: Clone Protein
Designation (Nucleotide) (Amino Acid) Lamin B/Sterol bms1.pk0009.e4
1 2 Reductase (corn) Lamin B/Sterol r1r2.pk0031.g9 3 4 Reductase
(rice) Lamin B/Sterol wr1.pk0039.d9 5 6 Reductase (wheat)
[0014] The Sequence Listing contains the one letter code for
nucleotide sequence characters and the three letter codes for amino
acids as defined in conformity with the IUPAC-IUBMB standards
described in Nucleic Acids Research 13:3021-3030 (1985) and in the
Biochemical Journal 219 (No. 2):345-373 (1984) which are herein
incorporated by reference. The symbols and format used for
nucleotide and amino acid sequence data comply with the rules set
forth in 37 C.F.R. .sctn. 1.822.
DETAILED DESCRIPTION OF THE INVENTION
[0015] In the context of this disclosure, a number of terms shall
be utilized. As used herein, a "nucleic acid fragment" is a polymer
of RNA or DNA that is single- or double-stranded, optionally
containing synthetic, non-natural or altered nucleotide bases. A
nucleic acid fragment in the form of a polymer of DNA may be
comprised of one or more segments of cDNA, genomic DNA or synthetic
DNA.
[0016] As used herein, "substantially similar" refers to nucleic
acid fragments wherein changes in one or more nucleotide bases
results in substitution of one or more amino acids, but do not
affect the functional properties of the polypeptide encoded by the
nucleotide sequence. "Substantially similar" also refers to nucleic
acid fragments wherein changes in one or more nucleotide bases does
not affect the ability of the nucleic acid fragment to mediate
alteration of gene expression by gene silencing through for example
antisense or co-suppression technology. "Substantially similar"
also refers to modifications of the nucleic acid fragments of the
instant invention such as deletion or insertion of one or more
nucleotides that do not substantially affect the functional
properties of the resulting transcript vis-a-vis the ability to
mediate gene silencing or alteration of the functional properties
of the resulting protein molecule. It is therefore understood that
the invention encompasses more than the specific exemplary
nucleotide or amino acid sequences and includes functional
equivalents thereof.
[0017] For example, it is well known in the art that antisense
suppression and co-suppression of gene expression may be
accomplished using nucleic acid fragments representing less than
the entire coding region of a gene, and by nucleic acid fragments
that do not share 100% sequence identity with the gene to be
suppressed. Moreover, alterations in a nucleic acid fragment which
result in the production of a chemically equivalent amino acid at a
given site, but do not effect the functional properties of the
encoded polypeptide, are well known in the art. Thus, a codon for
the amino acid alanine, a hydrophobic amino acid, may be
substituted by a codon encoding another less hydrophobic residue,
such as glycine, or a more hydrophobic residue, such as valine,
leucine, or isoleucine. Similarly, changes which result in
substitution of one negatively charged residue for another, such as
aspartic acid for glutamic acid, or one positively charged residue
for another, such as lysine for arginine, can also be expected to
produce a functionally equivalent product. Nucleotide changes which
result in alteration of the N-terminal and C-terminal portions of
the polypeptide molecule would also not be expected to alter the
activity of the polypeptide. Each of the proposed modifications is
well within the routine skill in the art, as is determination of
retention of biological activity of the encoded products.
[0018] Moreover, substantially similar nucleic acid fragments may
also be characterized by their ability to hybridize, under
stringent conditions (0.1.times.SSC, 0.1% SDS, 65.degree. C.), with
the nucleic acid fragments disclosed herein.
[0019] Substantially similar nucleic acid fragments of the instant
invention may also be characterized by the percent similarity of
the amino acid sequences that they encode to the amino acid
sequences disclosed herein, as determined by algorithms commonly
employed by those skilled in this art. Preferred are those nucleic
acid fragments whose nucleotide sequences encode amino acid
sequences that are 80% similar to the amino acid sequences reported
herein. More preferred nucleic acid fragments encode amino acid
sequences that are 90% similar to the amino acid sequences reported
herein. Most preferred are nucleic acid fragments that encode amino
acid sequences that are 95% similar to the amino acid sequences
reported herein. Sequence alignments and percent similarity
calculations were performed using the Megalign program of the
LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison,
Wis.). Multiple alignment of the sequences was performed using the
Clustal method of alignment (Higgins and Sharp (1989) CABIOS.
5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5.
[0020] A "substantial portion" of an amino acid or nucleotide
sequence comprises an amino acid or a nucleotide sequence that is
sufficient to afford putative identification of the protein or gene
that the amino acid or nucleotide sequence comprises. Amino acid
and nucleotide sequences can be evaluated either manually by one
skilled in the art, or by using computer-based sequence comparison
and identification tools that employ algorithms such as BLAST
(Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol.
Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST- /). In
general, a sequence of ten or more contiguous amino acids or thirty
or more contiguous nucleotides is necessary in order to putatively
identify a polypeptide or nucleic acid sequence as homologous to a
known protein or gene. Moreover, with respect to nucleotide
sequences, gene-specific oligonucleotide probes comprising 30 or
more contiguous nucleotides may be used in sequence-dependent
methods of gene identification (e.g., Southern hybridization) and
isolation (e.g., in situ hybridization of bacterial colonies or
bacteriophage plaques). In addition, short oligonucleotides of 12
or more nucleotides may be used as amplification primers in PCR in
order to obtain a particular nucleic acid fragment comprising the
primers. Accordingly, a "substantial portion" of a nucleotide
sequence comprises a nucleotide sequence that will afford specific
identification and/or isolation of a nucleic acid fragment
comprising the sequence. The instant specification teaches amino
acid and nucleotide sequences encoding polypeptides that comprise
one or more particular plant proteins. The skilled artisan, having
the benefit of the sequences as reported herein, may now use all or
a substantial portion of the disclosed sequences for purposes known
to those skilled in this art. Accordingly, the instant invention
comprises the complete sequences as reported in the accompanying
Sequence Listing, as well as substantial portions of those
sequences as defined above.
[0021] "Codon degeneracy" refers to divergence in the genetic code
permitting variation of the nucleotide sequence without effecting
the amino acid sequence of an encoded polypeptide. Accordingly, the
instant invention relates to any nucleic acid fragment comprising a
nucleotide sequence that encodes all or a substantial portion of
the amino acid sequences set forth herein. The skilled artisan is
well aware of the "codon-bias" exhibited by a specific host cell in
usage of nucleotide codons to specify a given amino acid.
Therefore, when synthesizing a nucleic acid fragment for improved
expression in a host cell, it is desirable to design the nucleic
acid fragment such that its frequency of codon usage approaches the
frequency of preferred codon usage of the host cell.
[0022] "Synthetic nucleic acid fragments" can be assembled from
oligonucleotide building blocks that are chemically synthesized
using procedures known to those skilled in the art. These building
blocks are ligated and annealed to form larger nucleic acid
fragments which may then be enzymatically assembled to construct
the entire desired nucleic acid fragment. "Chemically synthesized",
as related to nucleic acid fragment, means that the component
nucleotides were assembled in vitro. Manual chemical synthesis of
nucleic acid fragments may be accomplished using well established
procedures, or automated chemical synthesis can be performed using
one of a number of commercially available machines. Accordingly,
the nucleic acid fragments can be tailored for optimal gene
expression based on optimization of nucleotide sequence to reflect
the codon bias of the host cell. The skilled artisan appreciates
the likelihood of successful gene expression if codon usage is
biased towards those codons favored by the host. Determination of
preferred codons can be based on a survey of genes derived from the
host cell where sequence information is available.
[0023] "Gene" refers to a nucleic acid fragment that expresses a
specific protein, including regulatory sequences preceding (5'
non-coding sequences) and following (3' non-coding sequences) the
coding sequence. "Native gene" refers to a gene as found in nature
with its own regulatory sequences. "Chimeric gene" refers any gene
that is not a native gene, comprising regulatory and coding
sequences that are not found together in nature. Accordingly, a
chimeric gene may comprise regulatory sequences and coding
sequences that are derived from different sources, or regulatory
sequences and coding sequences derived from the same source, but
arranged in a manner different than that found in nature.
"Endogenous gene" refers to a native gene in its natural location
in the genome of an organism. A "foreign" gene refers to a gene not
normally found in the host organism, but that is introduced into
the host organism by gene transfer. Foreign genes can comprise
native genes inserted into a non-native organism, or chimeric
genes. A "transgene" is a gene that has been introduced into the
genome by a transformation procedure.
[0024] "Coding sequence" refers to a nucleotide sequence that codes
for a specific amino acid sequence. "Regulatory sequences" refer to
nucleotide sequences located upstream (5' non-coding sequences),
within, or downstream (3' non-coding sequences) of a coding
sequence, and which influence the transcription, RNA processing or
stability, or translation of the associated coding sequence.
Regulatory sequences may include promoters, translation leader
sequences, introns, and polyadenylation recognition sequences.
[0025] "Promoter" refers to a nucleotide sequence capable of
controlling the expression of a coding sequence or functional RNA.
In general, a coding sequence is located 3' to a promoter sequence.
The promoter sequence consists of proximal and more distal upstream
elements, the latter elements often referred to as enhancers.
Accordingly, an "enhancer" is a nucleotide sequence which can
stimulate promoter activity and may be an innate element of the
promoter or a heterologous element inserted to enhance the level or
tissue-specificity of a promoter. Promoters may be derived in their
entirety from a native gene, or be composed of different elements
derived from different promoters found in nature, or even comprise
synthetic nucleotide segments. It is understood by those skilled in
the art that different promoters may direct the expression of a
gene in different tissues or cell types, or at different stages of
development, or in response to different environmental conditions.
Promoters which cause a nucleic acid fragment to be expressed in
most cell types at most times are commonly referred to as
"constitutive promoters". New promoters of various types useful in
plant cells are constantly being discovered; numerous examples may
be found in the compilation by Okamuro and Goldberg (1989)
Biochemistry of Plants 15:1-82. It is further recognized that since
in most cases the exact boundaries of regulatory sequences have not
been completely defined, nucleic acid fragments of different
lengths may have identical promoter activity.
[0026] The "translation leader sequence" refers to a nucleotide
sequence located between the promoter sequence of a gene and the
coding sequence. The translation leader sequence is present in the
fully processed mRNA upstream of the translation start sequence.
The translation leader sequence may affect processing of the
primary transcript to mRNA, mRNA stability or translation
efficiency. Examples of translation leader sequences have been
described (Turner and Foster (1995) Molecular Biotechnology
3:225).
[0027] The "3' non-coding sequences" refer to nucleotide sequences
located downstream of a coding sequence and include polyadenylation
recognition sequences and other sequences encoding regulatory
signals capable of affecting mRNA processing or gene expression.
The polyadenylation signal is usually characterized by affecting
the addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor. The use of different 3' non-coding sequences is
exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.
[0028] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from posttranscriptional processing of the primary
transcript and is referred to as the mature RNA. "Messenger RNA
(mRNA)" refers to the RNA that is without introns and that can be
translated into polypeptide by the cell. "cDNA" refers to a
double-stranded DNA that is complementary to and derived from mRNA.
"Sense" RNA refers to an RNA transcript that includes the mRNA and
so can be translated into a polypeptide by the cell. "Antisense
RNA" refers to an RNA transcript that is complementary to all or
part of a target primary transcript or mRNA and that blocks the
expression of a target gene (see U.S. Pat. No. 5,107,065,
incorporated herein by reference). The complementarity of an
antisense RNA may be with any part of the specific nucleotide
sequence, i.e., at the 5' non-coding sequence, 3' non-coding
sequence, introns, or the coding sequence. "Functional RNA" refers
to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may
not be translated but yet has an effect on cellular processes.
[0029] The term "operably linked" refers to the association of two
or more nucleic acid fragments on a single nucleic acid fragment so
that the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of
the promoter). Coding sequences can be operably linked to
regulatory sequences in sense or antisense orientation.
[0030] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from the nucleic acid fragment of the invention.
Expression may also refer to translation of mRNA into a
polypeptide. "Antisense inhibition" refers to the production of
antisense RNA transcripts capable of suppressing the expression of
the target protein. "Overexpression" refers to the production of a
gene product in transgenic organisms that exceeds levels of
production in normal or non-transformed organisms. "Co-suppression"
refers to the production of sense RNA transcripts capable of
suppressing the expression of identical or substantially similar
foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated
herein by reference).
[0031] "Altered levels" refers to the production of gene product(s)
in transgenic organisms in amounts or proportions that differ from
that of normal or non-transformed organisms.
[0032] "Mature" protein refers to a post-translationally processed
polypeptide; i.e., one from which any pre- or propeptides present
in the primary translation product have been removed. "Precursor"
protein refers to the primary product of translation of mRNA; i.e.,
with pre- and propeptides still present. Pre- and propeptides may
be but are not limited to intracellular localization signals.
[0033] A "chloroplast transit peptide" is an amino acid sequence
which is translated in conjunction with a protein and directs the
protein to the chloroplast or other plastid types present in the
cell in which the protein is made. "Chloroplast transit sequence"
refers to a nucleotide sequence that encodes a chloroplast transit
peptide. A "signal peptide" is an amino acid sequence which is
translated in conjunction with a protein and directs the protein to
the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant
Mol. Biol. 42:21-53). If the protein is to be directed to a
vacuole, a vacuolar targeting signal (supra) can further be added,
or if to the endoplasmic reticulum, an endoplasmic reticulum
retention signal (supra) may be added. If the protein is to be
directed to the nucleus, any signal peptide present should be
removed and instead a nuclear localization signal included (Raikhel
(1992) Plant Phys. 100:1627-1632).
[0034] "Transformation" refers to the transfer of a nucleic acid
fragment into the genome of a host organism, resulting in
genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
organisms. Examples of methods of plant transformation include
Agrobacterium-mediated transformation (De Blaere et al. (1987)
Meth. Enzymol. 143:277) and particle-accelerated or "gene gun"
transformation technology (Klein et al. (1987) Nature (London)
327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by
reference).
[0035] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described more fully
in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold
Spring Harbor Laboratory Press: Cold Spring Harbor, 1989
(hereinafter "Maniatis").
[0036] Nucleic acid fragments encoding at least a portion of
several chromatin associated proteins have been isolated and
identified by comparison of random plant cDNA sequences to public
databases containing nucleotide and protein sequences using the
BLAST algorithms well known to those skilled in the art. The
nucleic acid fragments of the instant invention may be used to
isolate cDNAs and genes encoding homologous proteins from the same
or other plant species. Isolation of homologous genes using
sequence-dependent protocols is well known in the art. Examples of
sequence-dependent protocols include, but are not limited to,
methods of nucleic acid hybridization, and methods of DNA and RNA
amplification as exemplified by various uses of nucleic acid
amplification technologies (e.g., polymerase chain reaction, ligase
chain reaction).
[0037] For example, genes encoding other lamin B receptor/sterol
reductase, either as cDNAs or genomic DNAs, could be isolated
directly by using all or a portion of the instant nucleic acid
fragments as DNA hybridization probes to screen libraries from any
desired plant employing methodology well known to those skilled in
the art. Specific oligonucleotide probes based upon the instant
nucleic acid sequences can be designed and synthesized by methods
known in the art (Maniatis). Moreover, the entire sequences can be
used directly to synthesize DNA probes by methods known to the
skilled artisan such as random primer DNA labeling, nick
translation, or end-labeling techniques, or RNA probes using
available in vitro transcription systems. In addition, specific
primers can be designed and used to amplify a part or all of the
instant sequences. The resulting amplification products can be
labeled directly during amplification reactions or labeled after
amplification reactions, and used as probes to isolate full length
cDNA or genomic fragments under conditions of appropriate
stringency.
[0038] In addition, two short segments of the instant nucleic acid
fragments may be used in polymerase chain reaction protocols to
amplify longer nucleic acid fragments encoding homologous genes
from DNA or RNA. The polymerase chain reaction may also be
performed on a library of cloned nucleic acid fragments wherein the
sequence of one primer is derived from the instant nucleic acid
fragments, and the sequence of the other primer takes advantage of
the presence of the polyadenylic acid tracts to the 3' end of the
mRNA precursor encoding plant genes. Alternatively, the second
primer sequence may be based upon sequences derived from the
cloning vector. For example, the skilled artisan can follow the
RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA
85:8998) to generate cDNAs by using PCR to amplify copies of the
region between a single point in the transcript and the 3' or 5'
end. Primers oriented in the 3' and 5' directions can be designed
from the instant sequences. Using commercially available 3' RACE or
5' RACE systems (BRL), specific 3' or 5' cDNA fragments can be
isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 86:5673;
Loh et al. (1989) Science 243:217). Products generated by the 3'
and 5' RACE procedures can be combined to generate full-length
cDNAs (Frohman and Martin (1989) Techniques 1:165).
[0039] Availability of the instant nucleotide and deduced amino
acid sequences facilitates immunological screening of cDNA
expression libraries. Synthetic peptides representing portions of
the instant amino acid sequences may be synthesized. These peptides
can be used to immunize animals to produce polyclonal or monoclonal
antibodies with specificity for peptides or proteins comprising the
amino acid sequences. These antibodies can be then be used to
screen cDNA expression libraries to isolate full-length cDNA clones
of interest (Lerner (1984) Adv. Immunol. 36:1; Maniatis).
[0040] The nucleic acid fragments of the instant invention may be
used to create transgenic plants in which the disclosed
polypeptides are present at higher or lower levels than normal or
in cell types or developmental stages in which they are not
normally found. This would have the effect of altering chromatin
organization and nuclear structure in those cells.
[0041] Overexpression of the proteins of the instant invention may
be accomplished by first constructing a chimeric gene in which the
coding region is operably linked to a promoter capable of directing
expression of a gene in the desired tissues at the desired stage of
development. For reasons of convenience, the chimeric gene may
comprise promoter sequences and translation leader sequences
derived from the same genes. 3' Non-coding sequences encoding
transcription termination signals may also be provided. The instant
chimeric gene may also comprise one or more introns in order to
facilitate gene expression.
[0042] Plasmid vectors comprising the instant chimeric gene can
then constructed. The choice of plasmid vector is dependent upon
the method that will be used to transform host plants. The skilled
artisan is well aware of the genetic elements that must be present
on the plasmid vector in order to successfully transform, select
and propagate host cells containing the chimeric gene. The skilled
artisan will also recognize that different independent
transformation events will result in different levels and patterns
of expression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida
et al. (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple
events must be screened in order to obtain lines displaying the
desired expression level and pattern. Such screening may be
accomplished by Southern analysis of DNA, Northern analysis of mRNA
expression, Western analysis of protein expression, or phenotypic
analysis.
[0043] For some applications it may be useful to direct the instant
polypeptides to different cellular compartments, or to facilitate
its secretion from the cell. It is thus envisioned that the
chimeric gene described above may be further supplemented by
altering the coding sequence to encode the instant polypeptides
with appropriate intracellular targeting sequences such as transit
sequences (Keegstra (1989) Cell 56:247-253), signal sequences or
sequences encoding endoplasmic reticulum localization (Chrispeels
(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear
localization signals (Raikhel (1992) Plant Phys.100:1627-1632)
added and/or with targeting sequences that are already present
removed. While the references cited give examples of each of these,
the list is not exhaustive and more targeting signals of utility
may be discovered in the future.
[0044] It may also be desirable to reduce or eliminate expression
of genes encoding the instant polypeptides in plants for some
applications. In order to accomplish this, a chimeric gene designed
for co-suppression of the instant polypeptide can be constructed by
linking a gene or gene fragment encoding that polypeptide to plant
promoter sequences. Alternatively, a chimeric gene designed to
express antisense RNA for all or part of the instant nucleic acid
fragment can be constructed by linking the gene or gene fragment in
reverse orientation to plant promoter sequences. Either the
co-suppression or antisense chimeric genes could be introduced into
plants via transformation wherein expression of the corresponding
endogenous genes are reduced or eliminated.
[0045] Molecular genetic solutions to the generation of plants with
altered gene expression have a decided advantage over more
traditional plant breeding approaches. Changes in plant phenotypes
can be produced by specifically inhibiting expression of one or
more genes by antisense inhibition or cosuppression (U.S. Pat. Nos.
5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression
construct would act as a dominant negative regulator of gene
activity. While conventional mutations can yield negative
regulation of gene activity these effects are most likely
recessive. The dominant negative regulation available with a
transgenic approach may be advantageous from a breeding
perspective. In addition, the ability to restrict the expression of
specific phenotype to the reproductive tissues of the plant by the
use of tissue specific promoters may confer agronomic advantages
relative to conventional mutations which may have an effect in all
tissues in which a mutant gene is ordinarily expressed.
[0046] The person skilled in the art will know that special
considerations are associated with the use of antisense or
cosuppresion technologies in order to reduce expression of
particular genes. For example, the proper level of expression of
sense or antisense synthase enzyme genes may require the use of
different chimeric genes utilizing different regulatory elements
known to the skilled artisan. Once transgenic plants are obtained
by one of the methods described above, it will be necessary to
screen individual transgenics for those that most effectively
display the desired phenotype. It is well known to those skilled in
the art that individual transgenic plants carrying the same
construct may differ in expression levels; this phenomenon is
commonly referred to as "position effect". For example, when the
construct in question is designed to express higher levels of the
gene of interest, individual plants will vary in the amount of the
protein produced and thus in enzyme activity; this in turn will
effect the phenotype. Thus, in the use of these techniques their
efficiency in an individual transgenic plant is unpredictable, but
given a large transgenic population individuals with suppressed
gene expression will be obtained. In either case, in order to save
time, the person skilled in the art will make multiple genetic
constructs containing one or more different parts of the gene to be
suppressed, since the art does not teach a method to predict which
will be most effective for a particular gene. Furthermore, even the
most effective constructs will give an effective suppression
phenotype only in a fraction of the individual transgenic lines
isolated. For example, WO 93/11245 and WO 94/11516 disclose that
when attempting to suppress the expression of fatty acid desaturase
genes in canola, actual suppression was obtained in less than 1% of
the lines tested. In other species the percentage is somewhat
higher, but in no case does the percentage reach 100. This should
not be seen as a limitation on the present invention, but instead
as practical matter that is appreciated and anticipated by the
person skilled in this art. Accordingly, the skilled artisan will
develop methods for screening large numbers of transformants. The
nature of these screens will generally be chosen on practical
grounds, and is not an inherent part of the invention. For example,
one can screen by looking for changes in gene expression by using
antibodies specific for the protein encoded by the gene being
suppressed, or one could establish assays that specifically measure
enzyme activity. A preferred method will be one which allows large
numbers of samples to be processed rapidly, since it will be
expected that the majority of samples will be negative.
[0047] The instant polypeptides (or portions thereof) may be
produced in heterologous host cells, particularly in the cells of
microbial hosts, and can be used to prepare antibodies to the these
proteins by methods well known to those skilled in the art. The
antibodies are useful for detecting the polypeptides of the instant
invention in situ in cells or in vitro in cell extracts. Preferred
heterologous host cells for production of the instant polypeptides
are microbial hosts. Microbial expression systems and expression
vectors containing regulatory sequences that direct high level
expression of foreign proteins are well known to those skilled in
the art. Any of these could be used to construct a chimeric gene
for production of the instant polypeptides. This chimeric gene
could then be introduced into appropriate microorganisms via
transformation to provide high level expression of the encoded
chromatin associated protein. An example of a vector for high level
expression of the instant polypeptides in a bacterial host is
provided (Example 6).
[0048] All or a substantial portion of the nucleic acid fragments
of the instant invention may also be used as probes for genetically
and physically mapping the genes that they are a part of, and as
markers for traits linked to those genes. Such information may be
useful in plant breeding in order to develop lines with desired
phenotypes. For example, the instant nucleic acid fragments may be
used as restriction fragment length polymorphism (RFLP) markers.
Southern blots (Maniatis) of restriction-digested plant genomic DNA
may be probed with the nucleic acid fragments of the instant
invention. The resulting banding patterns may then be subjected to
genetic analyses using computer programs such as MapMaker (Lander
et al. (1987) Genomics 1:174-181) in order to construct a genetic
map. In addition, the nucleic acid fragments of the instant
invention may be used to probe Southern blots containing
restriction endonuclease-treated genomic DNAs of a set of
individuals representing parent and progeny of a defined genetic
cross. Segregation of the DNA polymorphisms is noted and used to
calculate the position of the instant nucleic acid sequence in the
genetic map previously obtained using this population (Botstein et
al. (1980) Am. J. Hum. Genet. 32:314-331).
[0049] The production and use of plant gene-derived probes for use
in genetic mapping is described in Bernatzky and Tanksley (1986)
Plant Mol. Biol. Reporter 4(1):37-41. Numerous publications
describe genetic mapping of specific cDNA clones using the
methodology outlined above or variations thereof. For example, F2
intercross populations, backcross populations, randomly mated
populations, near isogenic lines, and other sets of individuals may
be used for mapping. Such methodologies are well known to those
skilled in the art.
[0050] Nucleic acid probes derived from the instant nucleic acid
sequences may also be used for physical mapping (i.e., placement of
sequences on physical maps; see Hoheisel et al. In: Nonmammalian
Genomic Analysis: A Practical Guide, Academic press 1996, pp.
319-346, and references cited therein).
[0051] In another embodiment, nucleic acid probes derived from the
instant nucleic acid sequences may be used in direct fluorescence
in situ hybridization (FISH) mapping (Trask (1991) Trends Genet.
7:149-154). Although current methods of FISH mapping favor use of
large clones (several to several hundred KB; see Laan et al. (1995)
Genome Research 5:13-20), improvements in sensitivity may allow
performance of FISH mapping using shorter probes.
[0052] A variety of nucleic acid amplification-based methods of
genetic and physical mapping may be carried out using the instant
nucleic acid sequences. Examples include allele-specific
amplification (Kazazian (1989) J. Lab. Clin. Med. 114(2):95-96),
polymorphism of PCR-amplified fragments (CAPS; Sheffield et al.
(1993) Genomics 16:325-332), allele-specific ligation (Landegren et
al. (1988) Science 241:1077-1080), nucleotide extension reactions
(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid
Mapping (Walter et al. (1997) Nature Genetics 7:22-28) and Happy
Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For
these methods, the sequence of a nucleic acid fragment is used to
design and produce primer pairs for use in the amplification
reaction or in primer extension reactions. The design of such
primers is well known to those skilled in the art. In methods
employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the
mapping cross in the region corresponding to the instant nucleic
acid sequence. This, however, is generally not necessary for
mapping methods.
[0053] Loss of function mutant phenotypes may be identified for the
instant cDNA clones either by targeted gene disruption protocols or
by identifying specific mutants for these genes contained in a
maize population carrying mutations in all possible genes
(Ballinger and Benzer (1989) Proc. Natl. Acad. Sci USA 86:9402;
Koes et al. (1995) Proc. Natl. Acad. Sci USA 92:8149; Bensen et al.
(1995) Plant Cell 7:75). The latter approach may be accomplished in
two ways. First, short segments of the instant nucleic acid
fragments may be used in polymerase chain reaction protocols in
conjunction with a mutation tag sequence primer on DNAs prepared
from a population of plants in which Mutator transposons or some
other mutation-causing DNA element has been introduced (see Bensen,
supra). The amplification of a specific DNA fragment with these
primers indicates the insertion of the mutation tag element in or
near the plant gene encoding the instant polypeptides.
Alternatively, the instant nucleic acid fragment may be used as a
hybridization probe against PCR amplification products generated
from the mutation population using the mutation tag sequence primer
in conjunction with an arbitrary genomic site primer, such as that
for a restriction enzyme site-anchored synthetic adaptor. With
either method, a plant containing a mutation in the endogenous gene
encoding the instant polypeptides can be identified and obtained.
This mutant plant can then be used to determine or confirm the
natural function of the instant polypeptides disclosed herein.
EXAMPLES
[0054] The present invention is further defined in the following
Examples, in which all parts and percentages are by weight and
degrees are Celsius, unless otherwise stated. It should be
understood that these Examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only. From the above discussion and these Examples, one skilled in
the art can ascertain the essential characteristics of this
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions.
Example 1
Composition of cDNA Libraries; Isolation and Sequencing of cDNA
Clones
[0055] cDNA libraries representing mRNAs from various corn, rice
and wheat tissues were prepared. The characteristics of the
libraries are described below.
2TABLE 2 cDNA Libraries from Corn, Rice and Wheat Library Tissue
Clone bms1 Corn (Zea mays L., BMS) cell culture 1 day
bms1.pk0009.e4 after subculture r1r2 Rice leaf, 15 days after
germination, 12 r1r2.pk0031.g9 hours after infection of strain
Magaporthe grisea 4360-R-62 (AVR2-YAMO) wr1 Wheat (Triticum
aestivum L.) root; 7 day old wr1.pk0039.d9 seedling, light
grown
[0056] cDNA libraries may be prepared by any one of many methods
available. For example, the cDNAs may be introduced into plasmid
vectors by first preparing the cDNA libraries in Uni-ZAP.TM. XR
vectors according to the manufacturer's protocol (Stratagene
Cloning Systems, La Jolla, Calif.). The Uni-ZAP.TM. XR libraries
are converted into plasmid libraries according to the protocol
provided by Stratagene. Upon conversion, cDNA inserts will be
contained in the plasmid vector pBluescript. In addition, the cDNAs
may be introduced directly into precut Bluescript II SK(+) vectors
(Stratagene) using T4 DNA ligase (New England Biolabs), followed by
transfection into DH10B cells according to the manufacturer's
protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid
vectors, plasmid DNAs are prepared from randomly picked bacterial
colonies containing recombinant pBluescript plasmids, or the insert
cDNA sequences are amplified via polymerase chain reaction using
primers specific for vector sequences flanking the inserted cDNA
sequences. Amplified insert DNAs or plasmid DNAs are sequenced in
dye-primer sequencing reactions to generate partial cDNA sequences
(expressed sequence tags or "ESTs"; see Adams et al., (1991)
Science 252:1651). The resulting ESTs are analyzed using a Perkin
Elmer Model 377 fluorescent sequencer.
Example 2
Identification of cDNA Clones
[0057] cDNA clones encoding chromatin associated proteins were
identified by conducting BLAST (Basic Local Alignment Search Tool;
Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also
www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences
contained in the BLAST "nr" database (comprising all non-redundant
GenBank CDS translations, sequences derived from the 3-dimensional
structure Brookhaven Protein Data Bank, the last major release of
the SWISS-PROT protein sequence database, EMBL, and DDBJ
databases). The cDNA sequences obtained in Example 1 were analyzed
for similarity to all publicly available DNA sequences contained in
the "nr" database using the BLASTN algorithm provided by the
National Center for Biotechnology Information (NCBI). The DNA
sequences were translated in all reading frames and compared for
similarity to all publicly available protein sequences contained in
the "nr" database using the BLASTX algorithm (Gish and States
(1993) Nature Genetics 3:266-272) provided by the NCBI. For
convenience, the P-value (probability) of observing a match of a
cDNA sequence to a sequence contained in the searched databases
merely by chance as calculated by BLAST are reported herein as
"pLog" values, which represent the negative of the logarithm of the
reported P-value. Accordingly, the greater the pLog value, the
greater the likelihood that the cDNA sequence and the BLAST "hit"
represent homologous proteins.
Example 3
Characterization of cDNA Clones Encoding Lamin B Receptor/Sterol
Reductase
[0058] The BLASTX search using the EST sequences from clones listed
in Table 3 revealed similarity of the polypeptides encoded by the
cDNAs to lamin B receptor/sterol reductase from Homo sapiens (NCBI
Identifier No. gi 4191396). Shown in Table 3 are the BLAST results
for the sequences of the entire cDNA inserts comprising the
indicated cDNA clones ("FIS"):
3TABLE 3 BLAST Results for Sequences Encoding Polypeptides
Homologous to Lamin B Receptor/Sterol Reductase BLAST pLog Score
Clone Status gi 4191396 bms1.pk0009.e4 FIS 18.70 r1r2.pk0031.g9 FIS
34.52 wr1.pk0039.d9 FIS 25.22
[0059] The data in Table 4 represents a calculation of the percent
similarity of the amino acid sequences set forth in SEQ ID NOs:2, 4
and 6 and the Homo sapiens sequence (SEQ ID NO:7).
4TABLE 4 Percent Similarity of Amino Acid Sequences Deduced From
the Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Lamin B Receptor/Sterol Reductase SEQ ID NO. Percent
Similarity to gi 4191396 2 61% 4 46% 6 58%
[0060] Sequence alignments and percent similarity calculations were
performed using the Megalign program of the LASARGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the Clustal
method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)
with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5. Sequence alignments and BLAST scores and probabilities
indicate that the nucleic acid fragments comprising the instant
cDNA clones encode a substantial portion of a lamin B
receptor/sterol reductase. These sequences represent the first
corn, rice and wheat sequences encoding lamin B receptor/sterol
reductase.
Example 4
Expression of Chimeric Genes in Monocot Cells
[0061] A chimeric gene comprising a cDNA encoding the instant
polypeptides in sense orientation with respect to the maize 27 kD
zein promoter that is located 5' to the cDNA fragment, and the 10
kD zein 3' end that is located 3' to the cDNA fragment, can be
constructed. The cDNA fragment of this gene may be generated by
polymerase chain reaction (PCR) of the cDNA clone using appropriate
oligonucleotide primers. Cloning sites (NcoI or SmaI) can be
incorporated into the oligonucleotides to provide proper
orientation of the DNA fragment when inserted into the digested
vector pML103 as described below. Amplification is then performed
in a standard PCR. The amplified DNA is then digested with
restriction enzymes NcoI and SmaI and fractionated on an agarose
gel. The appropriate band can be isolated from the gel and combined
with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. Plasmid
pML103 has been deposited under the terms of the Budapest Treaty at
ATCC (American Type Culture Collection, 10801 University Blvd.,
Manassas, Va. 20110-2209), and bears accession number ATCC 97366.
The DNA segment from pML103 contains a 1.05 kb SalI-NcoI promoter
fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI
fragment from the 3' end of the maize 10 kD zein gene in the vector
pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at
15.degree. C. overnight, essentially as described (Maniatis). The
ligated DNA may then be used to transform E. coli XL1-Blue
(Epicurian Coli XL-1 Blue.TM.; Stratagene). Bacterial transformants
can be screened by restriction enzyme digestion of plasmid DNA and
limited nucleotide sequence analysis using the dideoxy chain
termination method (Sequenase.TM. DNA Sequencing Kit; U.S.
Biochemical). The resulting plasmid construct would comprise a
chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD
zein promoter, a cDNA fragment encoding the instant polypeptides,
and the 10 kD zein 3' region.
[0062] The chimeric gene described above can then be introduced
into corn cells by the following procedure. Immature corn embryos
can be dissected from developing caryopses derived from crosses of
the inbred corn lines H99 and LH132. The embryos are isolated 10 to
11 days after pollination when they are 1.0 to 1.5 mm long. The
embryos are then placed with the axis-side facing down and in
contact with agarose-solidified N6 medium (Chu et al. (1975) Sci.
Sin. Peking 18:659-668). The embryos are kept in the dark at
27.degree. C. Friable embryogenic callus consisting of
undifferentiated masses of cells with somatic proembryoids and
embryoids borne on suspensor structures proliferates from the
scutellum of these immature embryos. The embryogenic callus
isolated from the primary explant can be cultured on N6 medium and
sub-cultured on this medium every 2 to 3 weeks.
[0063] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst
Ag, Frankfurt, Germany) may be used in transformation experiments
in order to provide for a selectable marker. This plasmid contains
the Pat gene (see European Patent Publication 0 242 236) which
encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT
confers resistance to herbicidal glutamine synthetase inhibitors
such as phosphinothricin. The pat gene in p35S/Ac is under the
control of the 35S promoter from Cauliflower Mosaic Virus (Odell et
al. (1985) Nature 313:810-812) and the 3' region of the nopaline
synthase gene from the T-DNA of the Ti plasmid of Agrobacterium
tumefaciens.
[0064] The particle bombardment method (Klein et al. (1987) Nature
327:70-73) may be used to transfer genes to the callus culture
cells. According to this method, gold particles (1 .mu.m in
diameter) are coated with DNA using the following technique. Ten
.mu.g of plasmid DNAs are added to 50 .mu.L of a suspension of gold
particles (60 mg per mL). Calcium chloride (50 .mu.L of a 2.5 M
solution) and spermidine free base (20 .mu.L of a 1.0 M solution)
are added to the particles. The suspension is vortexed during the
addition of these solutions. After 10 minutes, the tubes are
briefly centrifuged (5 sec at 15,000 rpm) and the supernatant
removed. The particles are resuspended in 200 .mu.L of absolute
ethanol, centrifuged again and the supernatant removed. The ethanol
rinse is performed again and the particles resuspended in a final
volume of 30 .mu.L of ethanol. An aliquot (5 .mu.L) of the
DNA-coated gold particles can be placed in the center of a
Kapton.TM. flying disc (Bio-Rad Labs). The particles are then
accelerated into the corn tissue with a Biolistic.TM. PDS-1000/He
(Bio-Rad Instruments, Hercules Calif.), using a helium pressure of
1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0
cm.
[0065] For bombardment, the embryogenic tissue is placed on filter
paper over agarose-solidified N6 medium. The tissue is arranged as
a thin lawn and covered a circular area of about 5 cm in diameter.
The petri dish containing the tissue can be placed in the chamber
of the PDS-1000/He approximately 8 cm from the stopping screen. The
air in the chamber is then evacuated to a vacuum of 28 inches of
Hg. The macrocarrier is accelerated with a helium shock wave using
a rupture membrane that bursts when the He pressure in the shock
tube reaches 1000 psi.
[0066] Seven days after bombardment the tissue can be transferred
to N6 medium that contains gluphosinate (2 mg per liter) and lacks
casein or proline. The tissue continues to grow slowly on this
medium. After an additional 2 weeks the tissue can be transferred
to fresh N6 medium containing gluphosinate. After 6 weeks, areas of
about 1 cm in diameter of actively growing callus can be identified
on some of the plates containing the glufosinate-supplemented
medium. These calli may continue to grow when sub-cultured on the
selective medium.
[0067] Plants can be regenerated from the transgenic callus by
first transferring clusters of tissue to N6 medium supplemented
with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be
transferred to regeneration medium (Fromm et al. (1990)
Bio/Technology 8:833-839).
Example 5
Expression of Chimeric Genes in Dicot Cells
[0068] A seed-specific expression cassette composed of the promoter
and transcription terminator from the gene encoding the .beta.
subunit of the seed storage protein phaseolin from the bean
Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem.
261:9228-9238) can be used for expression of the instant
polypeptides in transformed soybean. The phaseolin cassette
includes about 500 nucleotides upstream (5') from the translation
initiation codon and about 1650 nucleotides downstream (3') from
the translation stop codon of phaseolin. Between the 5' and 3'
regions are the unique restriction endonuclease sites Nco I (which
includes the ATG translation initiation codon), Sma I, Kpn I and
Xba I. The entire cassette is flanked by Hind III sites.
[0069] The cDNA fragment of this gene may be generated by
polymerase chain reaction (PCR) of the cDNA clone using appropriate
oligonucleotide primers. Cloning sites can be incorporated into the
oligonucleotides to provide proper orientation of the DNA fragment
when inserted into the expression vector. Amplification is then
performed as described above, and the isolated fragment is inserted
into a pUC18 vector carrying the seed expression cassette.
[0070] Soybean embroys may then be transformed with the expression
vector comprising sequences encoding the instant polypeptides. To
induce somatic embryos, cotyledons, 3-5 mm in length dissected from
surface sterilized, immature seeds of the soybean cultivar A2872,
can be cultured in the light or dark at 26.degree. C. on an
appropriate agar medium for 6-10 weeks. Somatic embryos which
produce secondary embryos are then excised and placed into a
suitable liquid medium. After repeated selection for clusters of
somatic embryos which multiplied as early, globular staged embryos,
the suspensions are maintained as described below.
[0071] Soybean embryogenic suspension cultures can maintained in 35
mL liquid media on a rotary shaker, 150 rpm, at 26.degree. C. with
florescent lights on a 16:8 hour day/night schedule. Cultures are
subcultured every two weeks by inoculating approximately 35 mg of
tissue into 35 mL of liquid medium.
[0072] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein et al.
(1987) Nature (London) 327:70, U.S. Pat. No. 4,945,050). A DuPont
Biolistic.TM. PDS1000/HE instrument (helium retrofit) can be used
for these transformations.
[0073] A selectable marker gene which can be used to facilitate
soybean transformation is a chimeric gene composed of the 35S
promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature
313:810-812), the hygromycin phosphotransferase gene from plasmid
pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188) and the
3' region of the nopaline synthase gene from the T-DNA of the Ti
plasmid of Agrobacterium tumefaciens. The seed expression cassette
comprising the phaseolin 5' region, the fragment encoding the
instant polypeptides and the phaseolin 3' region can be isolated as
a restriction fragment. This fragment can then be inserted into a
unique restriction site of the vector carrying the marker gene.
[0074] To 50 .mu.L of a 60 mg/mL 1 .mu.m gold particle suspension
is added (in order): 5 .mu.L DNA (1 .mu.g/.mu.L), 20 .mu.l
spermidine (0.1 M), and 50 .mu.L CaCl.sub.2 (2.5 M). The particle
preparation is then agitated for three minutes, spun in a microfuge
for 10 seconds and the supernatant removed. The DNA-coated
particles are then washed once in 400 .mu.L 70% ethanol and
resuspended in 40 .mu.L of anhydrous ethanol. The DNA/particle
suspension can be sonicated three times for one second each. Five
.mu.L of the DNA-coated gold particles are then loaded on each
macro carrier disk.
[0075] Approximately 300-400 mg of a two-week-old suspension
culture is placed in an empty 60.times.15 mm petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately 5-10 plates of tissue are
normally bombarded. Membrane rupture pressure is set at 1100 psi
and the chamber is evacuated to a vacuum of 28 inches mercury. The
tissue is placed approximately 3.5 inches away from the retaining
screen and bombarded three times. Following bombardment, the tissue
can be divided in half and placed back into liquid and cultured as
described above.
[0076] Five to seven days post bombardment, the liquid media may be
exchanged with fresh media, and eleven to twelve days post
bombardment with fresh media containing 50 mg/mL hygromycin. This
selective media can be refreshed weekly. Seven to eight weeks post
bombardment, green, transformed tissue may be observed growing from
untransformed, necrotic embryogenic clusters. Isolated green tissue
is removed and inoculated into individual flasks to generate new,
clonally propagated, transformed embryogenic suspension cultures.
Each new line may be treated as an independent transformation
event. These suspensions can then be subcultured and maintained as
clusters of immature embryos or regenerated into whole plants by
maturation and germination of individual somatic embryos.
Example 6
Expression of Chimeric Genes in Microbial Cells
[0077] The cDNAs encoding the instant polypeptides can be inserted
into the T7 E. coli expression vector pBT430. This vector is a
derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135)
which employs the bacteriophage T7 RNA polymerase/T7 promoter
system. Plasmid pBT430 was constructed by first destroying the EcoR
I and Hind III sites in pET-3a at their original positions. An
oligonucleotide adaptor containing EcoR I and Hind III sites was
inserted at the BamH I site of pET-3a. This created pET-3aM with
additional unique cloning sites for insertion of genes into the
expression vector. Then, the Nde I site at the position of
translation initiation was converted to an Nco I site using
oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM
in this region, 5'-CATATGG, was converted to 5'-CCCATGG in
pBT430.
[0078] Plasmid DNA containing a cDNA may be appropriately digested
to release a nucleic acid fragment encoding the protein. This
fragment may then be purified on a 1% NuSieve GTG.TM. low melting
agarose gel (FMC). Buffer and agarose contain 10 .mu.g/ml ethidium
bromide for visualization of the DNA fragment. The fragment can
then be purified from the agarose gel by digestion with GELase.TM.
(Epicentre Technologies) according to the manufacturer's
instructions, ethanol precipitated, dried and resuspended in 20
.mu.L of water. Appropriate oligonucleotide adapters may be ligated
to the fragment using T4 DNA ligase (New England Biolabs, Beverly,
Mass.). The fragment containing the ligated adapters can be
purified from the excess adapters using low melting agarose as
described above. The vector pBT430 is digested, dephosphorylated
with alkaline phosphatase (NEB) and deproteinized with
phenol/chloroform as described above. The prepared vector pBT430
and fragment can then be ligated at 16.degree. C. for 15 hours
followed by transformation into DH5 electrocompetent cells (GIBCO
BRL). Transformants can be selected on agar plates containing LB
media and 100 .mu.g/mL ampicillin. Transformants containing the
gene encoding the instant polypeptides are then screened for the
correct orientation with respect to the T7 promoter by restriction
enzyme analysis.
[0079] For high level expression, a plasmid clone with the cDNA
insert in the correct orientation relative to the T7 promoter can
be transformed into E. coli strain BL21(DE3) (Studier et al. (1986)
J. Mol. Biol. 189:113-130). Cultures are grown in LB medium
containing ampicillin (100 mg/L) at 25.degree. C. At an optical
density at 600 nm of approximately 1, IPTG
(isopropylthio-.beta.-galactoside, the inducer) can be added to a
final concentration of 0.4 mM and incubation can be continued for 3
h at 25.degree. C. Cells are then harvested by centrifugation and
re-suspended in 50 .mu.L of 50 mM Tris-HCl at pH 8.0 containing 0.1
mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of
1 mm glass beads can be added and the mixture sonicated 3 times for
about 5 seconds each time with a microprobe sonicator. The mixture
is centrifuged and the protein concentration of the supernatant
determined. One .mu.g of protein from the soluble fraction of the
culture can be separated by SDS-polyacrylamide gel electrophoresis.
Gels can be observed for protein bands migrating at the expected
molecular weight.
Sequence CWU 1
1
7 1 413 DNA Zea mays 1 gcacgagcgg agacctgctg ctagcacttt cgttcagctt
gccctgtgga gtgagttccg 60 tggtcccata cttctacccc acgtacctgc
tcattctact ggtcttgagg gaaaggcgcg 120 atgaggcgag gtgctcgcag
aagtacaggg agatctgggc agagtactgc aagctcgtgc 180 cgtggaggat
cctgccttat gtgtactgaa gagacggtag aaaccaaggc agctcatggc 240
cctgggccag ctgtaaacct tattttgttt gcccttaacc agttggtgaa tgttgatgta
300 gcactcggta aactgtgacc gtgcaaactt ttgttattgt tggtccatac
atgtttggaa 360 tcgtgaatca gaccgcctca cttggtggca aaaaaaaaaa
aaaaaaaaaa aaa 413 2 68 PRT Zea mays 2 Thr Ser Gly Asp Leu Leu Leu
Ala Leu Ser Phe Ser Leu Pro Cys Gly 1 5 10 15 Val Ser Ser Val Val
Pro Tyr Phe Tyr Pro Thr Tyr Leu Leu Ile Leu 20 25 30 Leu Val Leu
Arg Glu Arg Arg Asp Glu Ala Arg Cys Ser Gln Lys Tyr 35 40 45 Arg
Glu Ile Trp Ala Glu Tyr Cys Lys Leu Val Pro Trp Arg Ile Leu 50 55
60 Pro Tyr Val Tyr 65 3 604 DNA Oryza sativa 3 gcacgagatc
actgggatgg tggcttttga gaaacaaagt ggagctgtcc cttttggctg 60
ctgtagttaa ctgcttcatt ttcgttattg gctatcttgt gttcagagga gccaacaaac
120 aaaaacatat cttcaagaag aaccctaaag ctcttatttg gggtaaacct
cccaaacttg 180 tcggggggaa gctacttgta tctggctact ggggaattgc
aaagcactgc aattatcttg 240 gggatatact gctagctctt tcatttagct
taccctgtgg aaccagttcg gtgatcccat 300 acttctaccc aacatacctg
ttcattttgc tgatatggag ggaacgaagg gacgaagcaa 360 ggtgctcaga
gaagtacaag gagatctggg tagaatattg caagcttgtg ccttggagga 420
tctttcctta cgtgtattaa atccaaatat tttgcctagc aggtgcatcg ttgtagaacc
480 aagagcgttg ttgtgctatt tgaacatgta aaattcacca agattcctgt
tgtttatttg 540 tagctgacat ccgtgttgaa tatcaattaa catagatttt
gttgaaaaaa aaaaaaaaaa 600 aaaa 604 4 145 PRT Oryza sativa 4 Thr Arg
Ser Leu Gly Trp Trp Leu Leu Arg Asn Lys Val Glu Leu Ser 1 5 10 15
Leu Leu Ala Ala Val Val Asn Cys Phe Ile Phe Val Ile Gly Tyr Leu 20
25 30 Val Phe Arg Gly Ala Asn Lys Gln Lys His Ile Phe Lys Lys Asn
Pro 35 40 45 Lys Ala Leu Ile Trp Gly Lys Pro Pro Lys Leu Val Gly
Gly Lys Leu 50 55 60 Leu Val Ser Gly Tyr Trp Gly Ile Ala Lys His
Cys Asn Tyr Leu Gly 65 70 75 80 Asp Ile Leu Leu Ala Leu Ser Phe Ser
Leu Pro Cys Gly Thr Ser Ser 85 90 95 Val Ile Pro Tyr Phe Tyr Pro
Thr Tyr Leu Phe Ile Leu Leu Ile Trp 100 105 110 Arg Glu Arg Arg Asp
Glu Ala Arg Cys Ser Glu Lys Tyr Lys Glu Ile 115 120 125 Trp Val Glu
Tyr Cys Lys Leu Val Pro Trp Arg Ile Phe Pro Tyr Val 130 135 140 Tyr
145 5 572 DNA Triticum aestivum 5 tacttgtatc tggctactgg ggcattgcaa
ggcactgcaa ttaccttgga gatctgcttc 60 tggcactctc attcagcttg
ccttgtggag ccagctccgt gatcccgtac ttctacccga 120 cctacctgct
gatcctgctg atatggagag aacgaagaga cgaggcgagg tgctcagaga 180
agtacaagga catctgggca gagtactgca agcttgtgcc ctggaggatt ctaccttacg
240 tgtactgatt agttaaagaa ccagaaggcc atgttgtatt gttgtttttg
gccctgatga 300 tcctgcataa ctaaatggta aggtcttttg tacgtttttc
ttggatatcc agttttaaat 360 tgaagctgca tcgatctttt agctttgttg
gggaagtgct gctaattttc atttgagctg 420 tccctttttt tcttcatccc
cttctattgc tgaaagaaga gaataccgtt gggaaaaaaa 480 aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaat 540
aaaaaaaaat ctcgaggggg gcgccgtacc ca 572 6 81 PRT Triticum aestivum
6 Leu Val Ser Gly Tyr Trp Gly Ile Ala Arg His Cys Asn Tyr Leu Gly 1
5 10 15 Asp Leu Leu Leu Ala Leu Ser Phe Ser Leu Pro Cys Gly Ala Ser
Ser 20 25 30 Val Ile Pro Tyr Phe Tyr Pro Thr Tyr Leu Leu Ile Leu
Leu Ile Trp 35 40 45 Arg Glu Arg Arg Asp Glu Ala Arg Cys Ser Glu
Lys Tyr Lys Asp Ile 50 55 60 Trp Ala Glu Tyr Cys Lys Leu Val Pro
Trp Arg Ile Leu Pro Tyr Val 65 70 75 80 Tyr 7 418 PRT Homo sapiens
7 Met Ala Pro Thr Gln Gly Pro Arg Ala Pro Leu Glu Phe Gly Gly Pro 1
5 10 15 Leu Gly Ala Ala Ala Leu Leu Leu Leu Leu Pro Ala Thr Met Phe
His 20 25 30 Leu Leu Leu Ala Ala Arg Ser Gly Pro Ala Arg Leu Leu
Gly Pro Pro 35 40 45 Ala Ser Leu Pro Gly Leu Glu Val Leu Trp Ser
Pro Arg Ala Leu Leu 50 55 60 Leu Trp Leu Ala Trp Leu Gly Leu Gln
Ala Ala Leu Tyr Leu Leu Pro 65 70 75 80 Ala Arg Lys Val Ala Glu Gly
Gln Glu Leu Lys Asp Lys Ser Arg Leu 85 90 95 Arg Tyr Pro Ile Asn
Gly Phe Gln Ala Leu Val Leu Thr Ala Leu Leu 100 105 110 Val Gly Leu
Gly Met Ser Ala Gly Leu Pro Leu Gly Ala Leu Pro Glu 115 120 125 Met
Leu Leu Pro Leu Ala Phe Val Ala Thr Leu Thr Ala Phe Ile Phe 130 135
140 Ser Leu Phe Leu Tyr Met Lys Ala Gln Val Ala Pro Val Ser Ala Leu
145 150 155 160 Ala Pro Gly Gly Asn Ser Gly Asn Pro Ile Tyr Asp Phe
Phe Leu Gly 165 170 175 Arg Glu Leu Asn Pro Arg Ile Cys Phe Phe Asp
Phe Lys Tyr Phe Cys 180 185 190 Glu Leu Arg Pro Gly Leu Ile Gly Trp
Val Leu Ile Asn Leu Ala Leu 195 200 205 Leu Met Lys Glu Ala Glu Leu
Arg Gly Ser Pro Ser Leu Ala Met Trp 210 215 220 Leu Val Asn Gly Phe
Gln Leu Leu Tyr Val Gly Asp Ala Leu Trp His 225 230 235 240 Glu Glu
Ala Val Leu Thr Thr Met Asp Ile Thr His Asp Gly Phe Gly 245 250 255
Phe Met Leu Ala Phe Gly Asp Met Ala Trp Val Pro Phe Thr Tyr Ser 260
265 270 Leu Gln Ala Gln Phe Leu Leu His His Pro Gln Pro Leu Gly Leu
Pro 275 280 285 Met Ala Ser Val Ile Cys Leu Ile Asn Ala Ile Gly Tyr
Tyr Ile Phe 290 295 300 Arg Gly Ala Asn Ser Gln Lys Asn Thr Phe Arg
Lys Asn Pro Ser Asp 305 310 315 320 Pro Arg Val Ala Gly Leu Glu Thr
Ile Ser Thr Ala Thr Gly Arg Lys 325 330 335 Leu Leu Val Ser Gly Trp
Trp Gly Met Val Arg His Pro Asn Tyr Leu 340 345 350 Gly Asp Leu Ile
Met Ala Leu Ala Trp Ser Leu Pro Cys Gly Val Ser 355 360 365 His Leu
Leu Pro Tyr Phe Tyr Leu Leu Tyr Phe Thr Ala Leu Leu Val 370 375 380
His Arg Glu Ala Arg Asp Glu Arg Gln Cys Leu Gln Lys Tyr Gly Leu 385
390 395 400 Ala Trp Gln Glu Tyr Cys Arg Arg Val Pro Tyr Arg Ile Met
Pro Tyr 405 410 415 Ile Tyr
* * * * *
References