U.S. patent application number 09/347331 was filed with the patent office on 2001-08-02 for chromatin associated proteins.
Invention is credited to CAHOON, REBECCA E., MIAO, GUO-HUA, ODELL, JOAN T., SAKAI, HAJIME.
Application Number | 20010010909 09/347331 |
Document ID | / |
Family ID | 22235422 |
Filed Date | 2001-08-02 |
United States Patent
Application |
20010010909 |
Kind Code |
A1 |
CAHOON, REBECCA E. ; et
al. |
August 2, 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 E.;
(WILMINGTON, DE) ; ODELL, JOAN T.; (UNIONVILLE,
PA) ; MIAO, GUO-HUA; (HOCKESSIN, DE) ; SAKAI,
HAJIME; (WILMINGTON, DE) |
Correspondence
Address: |
WILLIAM R MAJARIAN
E I DUPONT DE NEMOURS AND COMPANY
LEGAL PATENTS
WILMINGTON
DE
19898
|
Family ID: |
22235422 |
Appl. No.: |
09/347331 |
Filed: |
July 2, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60092841 |
Jul 14, 1998 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/183; 435/6.13; 435/91.2; 536/23.2 |
Current CPC
Class: |
C12N 15/8216 20130101;
C12N 15/8201 20130101; C12N 15/822 20130101; Y02A 40/146 20180101;
C12N 9/16 20130101; C12N 15/8261 20130101; C07K 14/415 20130101;
C12N 9/001 20130101 |
Class at
Publication: |
435/6 ; 435/91.2;
435/183; 536/23.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/00; C12P 019/34 |
Claims
What is claimed is:
1. An isolated nucleic acid fragment encoding a histone deacetylase
3 protein comprising a member selected from the group consisting
of: (a) an isolated nucleic acid fragment encoding the amino acid
sequence set forth in a member selected from the group consisting
of SEQ ID NO: 2, 4, 6 and 8; (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, 5 and 7.
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 histone deacetylase 3 polypeptide comprising the amino acid
sequence set forth in a member selected from the group consisting
of SEQ ID NO: 2, 4, 6 and 8.
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 comprising a nucleic acid fragment
that encodes an amino acid sequence that is at least 90% identical
to the amino acid sequence set forth in a member selected from the
group consisting of SEQ ID NO: 2, 4, 6 and 8; 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, 5 and 7; 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] Reversible acetylation of core histones may play an
important role in global transcriptional regulation in eucaryotic
cells. Increased histone acetylation has been correlated with
increased transcription (Roth et al. (1996) Cell 87:5-8) and
conversely, studies suggest that deacteylation is correlated with
transcriptional repression (Pazin et al. (1997) Cell 89:325-328). A
proposed mechanism of transcriptional regulation by histone
deacetylation may involve a histone deacetylase that is linked (via
protein-protein interactions) to a sequence specific DNA-bound
repressor protein. Transcription repression occurs upon
deacetylation of core histone proteins (Pazin et al. (1997) Cell
89:325-328). Precisely how reversible acetylation of core proteins
in turn controls gene expression is unknown however, several
mechanisms for the regulation of transcription via core acetylation
have been proposed by Pazine et al. One model suggests acetylation
of histone lysine residues increases the access of transcription
factors to the DNA. Another, suggests that acetylation of a lysine
residue in a chromatin associated protein (histone or nonhistone)
of a provides a signal that is recognized by another factor.
Accordingly, the availability of nucleic acid sequences encoding
all or a portion of histone deacetylase proteins would facilitate
studies to better understand global transcriptional regulation in
eucaryotic cells. It would also provide genetic tools for the
manipulation of histone deacetylase activity and provide mechanisms
to control transcriptional gene regulation in plants.
[0004] Several histone deacetylase proteins from corn, rice,
soybean and wheat have been discovered. In the process of
characterizing these proteins it was discovered that the histone
deacetylase proteins had significantly different amino acid
sequences, which suggested that these proteins constute a large
family of chromatin associated deacetylase proteins. Several
classes of histone deacetylase proteins were characterized (genes
1-4) by sets of conserved amino acid motifs and overall sequence
homology. Specific conserved sequence motifs were consistent for
each of the protein classes across species.
SUMMARY OF THE INVENTION
[0005] The instant invention relates to isolated nucleic acid
fragments encoding chromatin associated proteins. Specifically,
this invention concerns an isolated nucleic acid fragment encoding
a histone deacetylase gene 3 (HD3) and an isolated nucleic acid
fragment that is substantially similar to an isolated nucleic acid
fragment encoding a HD3. In addition, this invention relates to a
nucleic acid fragment that is complementary to the nucleic acid
fragment encoding HD3.
[0006] An additional embodiment of the instant invention pertains
to a polypeptide encoding all or a substantial portion of a
chromatin associated protein selected from the group consisting of
HD3.
[0007] In another embodiment, the instant invention relates to a
chimeric gene encoding a HD3 or to a chimeric gene that comprises a
nucleic acid fragment that is complementary to a nucleic acid
fragment encoding a HD3, 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.
[0008] In a further embodiment, the instant invention concerns a
transformed host cell comprising in its genome a chimeric gene
encoding a HD3 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.
[0009] An additional embodiment of the instant invention concerns a
method of altering the level of expression of a HD3 in a
transformed host cell comprising: a) transforming a host cell with
a chimeric gene comprising a nucleic acid fragment encoding a HD3;
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 HD3 in
the transformed host cell.
[0010] 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 HD3.
BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS
[0011] The invention can be more fully understood from the
following detailed description and the accompanying Sequence
Listing which form a part of this application.
[0012] 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: (Amino Protein
Clone Designation (Nucleotide) Acid) Histone p0016.ctscg42rb 1 2
Deacetylase Gene 3 rds2c.pk005.d13 3 4 Contig composed of: 5 6
r10n.pk117.n14 rsl1n.pk013.p12 Contig composed of: 7 8
sfl1.pk0071.h1 src3c.pk017.p14
[0013] 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
[0014] 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.
[0015] As used herein, "contig" refers to a nucleotide sequence
that is assembled from two or more constituent nucleotide sequences
that share common or overlapping regions of sequence homology. For
example, the nucleotide sequences of two or more nucleic acid
fragments can be compared and aligned in order to identify common
or overlapping sequences. Where common or overlapping sequences
exist between two or more nucleic acid fragments, the sequences
(and thus their corresponding nucleic acid fragments) can be
assembled into a single contiguous nucleotide sequence.
[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. Estimates of
such homology are provided by either DNA-DNA or DNA-RNA
hybridization under conditions of stringency as is well understood
by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic
Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions
can be adjusted to screen for moderately similar fragments, such as
homologous sequences from distantly related organisms, to highly
similar fragments, such as genes that duplicate functional enzymes
from closely related organisms. Post-hybridization washes determine
stringency conditions. One set of preferred conditions uses a
series of washes starting with 6.times. SSC, 0.5% SDS at room
temperature for 15 min, then repeated with 2.times. SSC, 0.5% SDS
at 45.degree. C. for 30 min, and then repeated twice with
0.2.times. SSC, 0.5% SDS at 50.degree. C. for 30 min. A more
preferred set of stringent conditions uses higher temperatures in
which the washes are identical to those above except for the
temperature of the final two 30 min washes in 0.2.times. SSC, 0.5%
SDS was increased to 60.degree. C. Another preferred set of highly
stringent conditions uses two final washes in 0.1.times. SSC, 0.1%
SDS at 65.degree. C.
[0019] Substantially similar nucleic acid fragments of the instant
invention may also be characterized by the percent identity 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 HD3 proteins, 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 the level of
histone acetylation in those cells which in turn could alter global
gene expression.
[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 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. 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 a large
number of transformants will be negative for the desired
phenotype.
[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,
soybean and wheat tissues were prepared. The characteristics of the
libraries are described below.
2TABLE 2 cDNA Libraries from Corn, Rice, Soybean and Wheat Library
Tissue Clone p0016 Corn tassel shoots, 0.1-1.4 cm, pooled
p0016.ctscg42rb rds2c Rice developing seeds in the middle of the
rds2c.pk005.d13 plant r10n Rice 15 day old leaf* r10n.pk0014.d8
rsl1n Rice 15 day old seedling* rsl1n.pk013.p12 sfl1 Soybean
immature flower sfl1.pk0071.h1 src3c Soybean 8 day old root
infected with cyst src3c.pk017.p14 nematode, Heterodera glycines
*These libraries were normalized essentially as described in U.S.
Pat. No. 5,482,845, incorporated herein by reference.
[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* XR
vectors according to the manufacturer's protocol (Stratagene
Cloning Systems, La Jolla, Calif.). The Uni-ZAP* 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 Histone Deacetylase Gene
3
[0058] The BLASTX search using the EST sequences from clones listed
in Table 3 revealed similarity of the polypeptides encoded by the
cDNAs to histone deacetylase from Gallus gallus (NCBI Identifier
Nos: gi 3023929 and 3023932) and Strongylocentrotus purpuratus
(NCBI Identifier No: gi 3023930). Shown in Table 3 are the BLAST
results for individual ESTs ("EST") and contigs assembled from two
or more ESTs ("Contig"):
3TABLE 3 BLAST Results for Sequences Encoding Polypeptides
Homologous to Histone Deacetylase Gene 3 Clone Status BLAST pLog
Score p0016.ctscg42rb EST 93.00 (gi 3023929) rds2c.pk005.d13 EST
69.40 (gi 3023932) Contig composed of: Contig 14.00 (gi 3023930)
r10n.pk117.n14 rsl1n.pk013.p12 Contig composed of: Contig 94.15 (gi
3023930) sfl1.pk0071 h1 src3c.pk017.p14
[0059] The data in Table 4 represents a calculation of the percent
identity of the amino acid sequences set forth in SEQ ID NOs: 2, 4,
6 and 8 and the Gallus gallus and Strongylocentrotus purpuratus
sequences (SEQ ID NOs: 9, 10 and 11).
4TABLE 4 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Histone Deacetylase Gene 3 SEQ ID NO. Percent
Identity to 2 54% (gi 3023929) 4 62% (gi 3023932) 6 31% (gi
3023930) 8 66% (gi 3023930)
[0060] Sequence alignments and percent identity 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 histone deacetylase
gene 3. These sequences represent the first corn, rice and soybean
sequences encoding histone deacetylase gene 3.
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 T 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 p35 S/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. PDS 1000/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.. 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
11 1 1056 DNA Zea mays 1 ccacgcgtcc gctcaagtac catgccaggg
ttctttatat tgacattgat gtccatcatg 60 gagatggagt tgaagaagcc
ttttatttca ctgacagggt aatgactgtg agtttccaca 120 agtatggtga
cctgttcttt cctggaacag gtgatattaa ggatatagga gaaagggaag 180
gaaaatatta tgccatcaac attccactta aagatgggat agatgacact agctttactc
240 ggctttttaa aacaattatt gccaaagttg ttgagacata tctgcctggt
gctattgttc 300 ttcaatgtgg ggctgattca ttggcgaggg atcgtttagg
ctgcttcaat ctctctattg 360 aaggccatgc tgaatgtgta aagtttgtca
agaaattcaa tattcccctt ctggtaactg 420 gaggtggtgg atacaccaag
gagaatgtag cacggtgttg ggctgttgaa actggggtcc 480 ttttagacac
agaactccca aatgagattc caaaaaatga atatattgag tactttgctc 540
caggattata cattgaaagt tccaaatttg agacatggag caatttgaac agtaagacct
600 atctcagttc aatcaaagtg caagtgatgg agagtttgcg gtacatacag
catgctcctg 660 gtgttcaaat gcaagaggtt cctcccgatt tttatatccc
ggactttgat gaagatgaat 720 tggatcctga tgaacgtgtt gaccagcaca
ctcaagacaa gcagattcac cgtgatgatg 780 agtactatga aggtgacaat
gacaacgatc acgacgacgg cacacgctaa tctgcttctt 840 ctgaggccct
ggtgtaaatg gaaacctgag attcttcact gttgtgtgta gttcagcagc 900
cttgagatgt aatagtttgt cagttgacag cagggatcta atttagcagg tcaaagtggt
960 ttagactttt ataagggatt ctgacccctc ctgaattaca cattgtagta
caagtctgca 1020 tatttttaag catgcaaaaa ttcaaatttc tcaaaa 1056 2 275
PRT Zea mays 2 Thr Arg Pro Leu Lys Tyr His Ala Arg Val Leu Tyr Ile
Asp Ile Asp 1 5 10 15 Val His His Gly Asp Gly Val Glu Glu Ala Phe
Tyr Phe Thr Asp Arg 20 25 30 Val Met Thr Val Ser Phe His Lys Tyr
Gly Asp Leu Phe Phe Pro Gly 35 40 45 Thr Gly Asp Ile Lys Asp Ile
Gly Glu Arg Glu Gly Lys Tyr Tyr Ala 50 55 60 Ile Asn Ile Pro Leu
Lys Asp Gly Ile Asp Asp Thr Ser Phe Thr Arg 65 70 75 80 Leu Phe Lys
Thr Ile Ile Ala Lys Val Val Glu Thr Tyr Leu Pro Gly 85 90 95 Ala
Ile Val Leu Gln Cys Gly Ala Asp Ser Leu Ala Arg Asp Arg Leu 100 105
110 Gly Cys Phe Asn Leu Ser Ile Glu Gly His Ala Glu Cys Val Lys Phe
115 120 125 Val Lys Lys Phe Asn Ile Pro Leu Leu Val Thr Gly Gly Gly
Gly Tyr 130 135 140 Thr Lys Glu Asn Val Ala Arg Cys Trp Ala Val Glu
Thr Gly Val Leu 145 150 155 160 Leu Asp Thr Glu Leu Pro Asn Glu Ile
Pro Lys Asn Glu Tyr Ile Glu 165 170 175 Tyr Phe Ala Pro Gly Leu Tyr
Ile Glu Ser Ser Lys Phe Glu Thr Trp 180 185 190 Ser Asn Leu Asn Ser
Lys Thr Tyr Leu Ser Ser Ile Lys Val Gln Val 195 200 205 Met Glu Ser
Leu Arg Tyr Ile Gln His Ala Pro Gly Val Gln Met Gln 210 215 220 Glu
Val Pro Pro Asp Phe Tyr Ile Pro Asp Phe Asp Glu Asp Glu Leu 225 230
235 240 Asp Pro Asp Glu Arg Val Asp Gln His Thr Gln Asp Lys Gln Ile
His 245 250 255 Arg Asp Asp Glu Tyr Tyr Glu Gly Asp Asn Asp Asn Asp
His Asp Asp 260 265 270 Gly Thr Arg 275 3 533 DNA Oryza sativa
unsure (13) unsure (22) unsure (26) unsure (54) unsure (56)..(57)
unsure (71) unsure (74) unsure (89) unsure (92) unsure (99) 3
cgaacattgc tancaattgg gntggnggga ttgcatcacc gcaaaagaag tgcnanngca
60 tcaaagggtc ngcntacatt aatgatccng gnttttggng aattctggag
cttctcaagt 120 accatgccag ggttctctat attgatatcg atgttcatca
cggggatgga gttgaagaag 180 ccttttattt cactgacagg gtaatgactg
taagtttcca caagtatggt gattttttct 240 tccctggcac aggtgatatt
aaggatatag gagaaagaga aggaaaatat tatgccatta 300 acattccact
taaagatggg atagatgact ccggctttac tcgccttttt aaaacagtta 360
ttgccaaagt tgttgagaca tatctgccag gtgctattgt tcttcaatgc ggggctgatt
420 ccttggcacg ggaccgtctg gggtgcttca atctgtccat tgaaggccat
gctgaatgtg 480 tgaagtttgt caagaaattc aatatccctc tactgggtga
ctggggggtg gtg 533 4 171 PRT Oryza sativa UNSURE (4) UNSURE (7)
UNSURE (18)..(19) UNSURE (33) 4 Asn Ile Ala Xaa Asn Trp Xaa Gly Gly
Ile Ala Ser Pro Gln Lys Lys 1 5 10 15 Cys Xaa Xaa Ile Lys Gly Ser
Ala Tyr Ile Asn Asp Pro Gly Phe Trp 20 25 30 Xaa Ile Leu Glu Leu
Leu Lys Tyr His Ala Arg Val Leu Tyr Ile Asp 35 40 45 Ile Asp Val
His His Gly Asp Gly Val Glu Glu Ala Phe Tyr Phe Thr 50 55 60 Asp
Arg Val Met Thr Val Ser Phe His Lys Tyr Gly Asp Phe Phe Phe 65 70
75 80 Pro Gly Thr Gly Asp Ile Lys Asp Ile Gly Glu Arg Glu Gly Lys
Tyr 85 90 95 Tyr Ala Ile Asn Ile Pro Leu Lys Asp Gly Ile Asp Asp
Ser Gly Phe 100 105 110 Thr Arg Leu Phe Lys Thr Val Ile Ala Lys Val
Val Glu Thr Tyr Leu 115 120 125 Pro Gly Ala Ile Val Leu Gln Cys Gly
Ala Asp Ser Leu Ala Arg Asp 130 135 140 Arg Leu Gly Cys Phe Asn Leu
Ser Ile Glu Gly His Ala Glu Cys Val 145 150 155 160 Lys Phe Val Lys
Lys Phe Asn Ile Pro Leu Leu 165 170 5 388 DNA Oryza sativa unsure
(69) unsure (138) unsure (244) unsure (299) unsure (313) unsure
(353) unsure (362) 5 acgggcgccg ccgccgccgc gctcggtcta atcggaatct
atttctcgac gcgactccgc 60 ttccccggnt cccctctcac ccttccgcgc
cgccgccgcc gccgcttgta gtggttgggg 120 ggcgagcggc cgctcganag
cgaagcgatg ctggagaaaa gacaggatag cctacttcta 180 cgatggcgat
gtgggcaatg tctactttgg gccaaatcac ccgatgaaac cacatcgact 240
ttgnatgaca catcatcttg tgctttcata tgatcttcac aagaacgatg ggggatatnt
300 aggccccaca aangcatatc caacagagct cgcacagttc cattctgctg
gantaatgtg 360 gnattcttgc atcggctaaa ctcctgac 388 6 49 PRT Oryza
sativa UNSURE (7) UNSURE (26) UNSURE (30) UNSURE (44) UNSURE (47) 6
Met Lys Pro His Arg Leu Xaa Met Thr His His Leu Val Leu Ser Tyr 1 5
10 15 Asp Leu His Lys Asn Asp Gly Gly Tyr Xaa Gly Pro Thr Xaa Ala
Tyr 20 25 30 Pro Thr Glu Leu Ala Gln Phe His Ser Ala Gly Xaa Met
Trp Xaa Ser 35 40 45 Cys 7 741 DNA Glycine max unsure (9) unsure
(214) unsure (219) unsure (222) unsure (224) 7 agcacatana
gtgattaggc tggtatcaag cgcgacatgg cactgcagtg caggttaagc 60
tcacaccatt gatatcacta gccatattct cctttcgcta attcccgcaa gctactacct
120 tcttcaacgc tctctctgca aaagatgcgc tccaaggaca gaatcgctta
cttctacgac 180 ggtgatgtcg gtagtgttta ctttggggcg aagnatccna
tnangcccca ccggctttgc 240 atgactcatc atcttgttct ctcatacgat
cttcataaga agatggagat ttaccgtcca 300 cacaaggctt atcctgttga
gcttgcccag tttcattcag ctgattatgt tgagtttttg 360 aacaggatta
cacctgacac tcagcacttg ttcttgaagg aactgacaaa atataatctt 420
ggagaagact gccctgtatt tgacaactta tttgaatttt gtcagattta tgctggtgga
480 actatagatg ctgcacgtcg attaaacaat caattgtgtg atattgctat
aaactgggcc 540 ggtggactac atcatgctaa gaaatgcgag gcatctggat
tttgttacat caatgacttg 600 gttttaggaa tcttggagct tcttaaatat
catgctcgtg ttttgtatat tgatatagat 660 gtgcaccatg gtgatggtgt
agaagaagcc ttctacttca ctgacagggt gatgactgtc 720 agttttcaca
agtacggaga g 741 8 199 PRT Glycine max UNSURE (24) UNSURE
(26)..(27) 8 Met Arg Ser Lys Asp Arg Ile Ala Tyr Phe Tyr Asp Gly
Asp Val Gly 1 5 10 15 Ser Val Tyr Phe Gly Ala Lys Xaa Pro Xaa Xaa
Pro His Arg Leu Cys 20 25 30 Met Thr His His Leu Val Leu Ser Tyr
Asp Leu His Lys Lys Met Glu 35 40 45 Ile Tyr Arg Pro His Lys Ala
Tyr Pro Val Glu Leu Ala Gln Phe His 50 55 60 Ser Ala Asp Tyr Val
Glu Phe Leu Asn Arg Ile Thr Pro Asp Thr Gln 65 70 75 80 His Leu Phe
Leu Lys Glu Leu Thr Lys Tyr Asn Leu Gly Glu Asp Cys 85 90 95 Pro
Val Phe Asp Asn Leu Phe Glu Phe Cys Gln Ile Tyr Ala Gly Gly 100 105
110 Thr Ile Asp Ala Ala Arg Arg Leu Asn Asn Gln Leu Cys Asp Ile Ala
115 120 125 Ile Asn Trp Ala Gly Gly Leu His His Ala Lys Lys Cys Glu
Ala Ser 130 135 140 Gly Phe Cys Tyr Ile Asn Asp Leu Val Leu Gly Ile
Leu Glu Leu Leu 145 150 155 160 Lys Tyr His Ala Arg Val Leu Tyr Ile
Asp Ile Asp Val His His Gly 165 170 175 Asp Gly Val Glu Glu Ala Phe
Tyr Phe Thr Asp Arg Val Met Thr Val 180 185 190 Ser Phe His Lys Tyr
Gly Glu 195 9 480 PRT Gallus gallus 9 Met Ala Leu Thr Gln Gly Thr
Lys Arg Lys Val Cys Tyr Tyr Tyr Asp 1 5 10 15 Gly Asp Val Gly Asn
Tyr Tyr Tyr Gly Gln Gly His Pro Met Lys Pro 20 25 30 His Arg Ile
Arg Met Thr His Asn Leu Leu Leu Asn Tyr Gly Leu Tyr 35 40 45 Arg
Lys Met Glu Ile Tyr Arg Pro His Lys Ala Asn Ala Glu Glu Met 50 55
60 Thr Lys Tyr His Ser Asp Asp Tyr Ile Lys Phe Leu Arg Ser Ile Arg
65 70 75 80 Pro Asp Asn Met Ser Glu Tyr Ser Lys Gln Met Gln Arg Phe
Asn Val 85 90 95 Gly Glu Asp Cys Pro Val Phe Asp Gly Leu Phe Glu
Phe Cys Gln Leu 100 105 110 Ser Ala Gly Gly Ser Val Ala Ser Ala Val
Lys Leu Asn Lys Gln Gln 115 120 125 Thr Asp Ile Ala Val Asn Trp Ala
Gly Gly Leu His His Ala Lys Lys 130 135 140 Ser Glu Ala Ser Gly Phe
Cys Tyr Val Asn Asp Ile Val Leu Ala Ile 145 150 155 160 Leu Glu Leu
Leu Lys Tyr His Gln Arg Val Leu Tyr Ile Asp Ile Asp 165 170 175 Ile
His His Gly Asp Gly Val Glu Glu Ala Phe Tyr Thr Thr Asp Arg 180 185
190 Val Met Thr Val Ser Phe His Lys Tyr Gly Glu Tyr Phe Pro Gly Thr
195 200 205 Gly Asp Leu Arg Asp Ile Gly Ala Gly Lys Gly Lys Tyr Tyr
Ala Val 210 215 220 Asn Tyr Pro Leu Arg Asp Gly Ile Asp Asp Glu Ser
Tyr Glu Ala Ile 225 230 235 240 Phe Lys Pro Val Ile Ser Lys Val Met
Glu Thr Phe Gln Pro Ser Ala 245 250 255 Val Val Leu Gln Cys Gly Ser
Asp Ser Leu Ser Gly Asp Arg Leu Gly 260 265 270 Cys Phe Asn Leu Thr
Ile Lys Gly His Ala Lys Cys Val Glu Phe Val 275 280 285 Lys Ser Phe
Asn Leu Pro Met Leu Met Leu Gly Gly Gly Gly Tyr Thr 290 295 300 Ile
Arg Asn Val Ala Arg Cys Trp Thr Tyr Glu Thr Ala Val Ala Leu 305 310
315 320 Asp Thr Glu Ile Pro Asn Glu Leu Pro Tyr Asn Asp Tyr Phe Glu
Tyr 325 330 335 Phe Gly Pro Asp Phe Lys Leu His Ile Ser Pro Ser Asn
Met Thr Asn 340 345 350 Gln Asn Thr Asn Glu Tyr Leu Glu Lys Ile Lys
Gln Arg Leu Phe Glu 355 360 365 Asn Leu Arg Met Leu Pro His Ala Pro
Gly Val Gln Met Gln Pro Ile 370 375 380 Pro Glu Asp Ala Val Gln Glu
Asp Ser Gly Asp Glu Glu Glu Glu Asp 385 390 395 400 Pro Glu Lys Arg
Ile Ser Ile Arg Asn Ser Asp Lys Arg Ile Ser Cys 405 410 415 Asp Glu
Glu Phe Ser Asp Ser Glu Asp Glu Gly Glu Gly Gly Arg Lys 420 425 430
Asn Val Ala Asn Phe Lys Lys Ala Lys Arg Val Lys Thr Glu Glu Glu 435
440 445 Lys Glu Glu Glu Glu Lys Lys Asp Glu Lys Glu Glu Glu Lys Ala
Lys 450 455 460 Glu Glu Lys Ala Glu Pro Lys Gly Val Lys Glu Glu Thr
Lys Ser Thr 465 470 475 480 10 428 PRT Gallus gallus 10 Met Ala Lys
Thr Val Ala Tyr Phe Tyr Asp Pro Asp Val Gly Asn Phe 1 5 10 15 His
Tyr Gly Ala Gly His Pro Met Lys Pro His Arg Leu Ala Leu Thr 20 25
30 His Ser Leu Val Leu His Tyr Gly Leu Tyr Lys Lys Met Ile Val Phe
35 40 45 Lys Pro Tyr Gln Ala Ser Gln His Asp Met Cys Arg Phe His
Ser Glu 50 55 60 Asp Tyr Ile Asp Phe Leu Gln Arg Val Ser Pro Asn
Asn Met Gln Gly 65 70 75 80 Phe Thr Lys Ser Leu Asn Ala Phe Asn Val
Gly Asp Asp Cys Pro Val 85 90 95 Phe Pro Gly Leu Phe Glu Phe Cys
Ser Arg Tyr Thr Gly Ala Ser Leu 100 105 110 Gln Gly Ala Thr Gln Leu
Asn Asn Lys Ile Cys Asp Ile Ala Ile Asn 115 120 125 Trp Ala Gly Gly
Leu His His Ala Lys Lys Phe Glu Ala Ser Gly Phe 130 135 140 Cys Tyr
Val Asn Asp Ile Val Ile Gly Ile Leu Glu Leu Leu Lys Tyr 145 150 155
160 His Pro Arg Val Leu Tyr Ile Asp Ile Asp Ile His His Gly Asp Gly
165 170 175 Val Gln Glu Ala Phe Tyr Leu Thr Asp Arg Val Met Thr Val
Ser Phe 180 185 190 His Lys Tyr Gly Asn Tyr Phe Phe Pro Gly Thr Gly
Asp Met Tyr Glu 195 200 205 Val Gly Ala Glu Ser Gly Arg Tyr Tyr Ala
Leu Asn Val Pro Leu Arg 210 215 220 Asp Gly Ile Asp Asp Gln Ser Tyr
Lys His Leu Phe Gln Pro Val Ile 225 230 235 240 Asn Gln Val Val Asp
Tyr Tyr Gln Pro Thr Cys Ile Val Leu Gln Cys 245 250 255 Gly Ala Asp
Ser Leu Gly Arg Asp Arg Leu Gly Cys Phe Asn Leu Ser 260 265 270 Ile
Arg Gly His Gly Glu Cys Val Glu Tyr Val Lys Ser Phe Asn Ile 275 280
285 Pro Leu Leu Val Leu Gly Gly Gly Gly Tyr Thr Val Arg Asn Val Ala
290 295 300 Arg Cys Trp Thr Tyr Glu Thr Ser Leu Leu Val Asp Glu Ala
Ile Ser 305 310 315 320 Glu Glu Leu Pro Tyr Ser Glu Tyr Phe Glu Tyr
Phe Ala Pro Asp Phe 325 330 335 Thr Leu His Pro Asp Val Ser Thr Arg
Ile Glu Asn Gln Asn Ser Arg 340 345 350 Gln Tyr Leu Asp Gln Ile Arg
Gln Thr Ile Phe Glu Asn Leu Lys Met 355 360 365 Leu Asn His Ala Pro
Ser Val Gln Ile His Asp Val Pro Ser Asp Leu 370 375 380 Leu Ser Tyr
Asp Arg Thr Asp Glu Pro Asp Pro Glu Glu Arg Gly Ser 385 390 395 400
Glu Glu Asn Tyr Ser Arg Pro Glu Ala Ala Asn Glu Phe Tyr Asp Gly 405
410 415 Asp His Asp Asn Asp Lys Glu Ser Asp Val Glu Ile 420 425 11
576 PRT Strongylocentrotus purpuratus 11 Met Ala Ser Thr Gly Thr
Lys Lys Arg Val Cys Tyr Tyr Tyr Asp Gly 1 5 10 15 Asp Val Gly Asn
Tyr Tyr Tyr Gly Gln Gly His Pro Met Lys Pro His 20 25 30 Arg Ile
Arg Met Thr His Asn Leu Ile Leu Asn Tyr Gly Leu Tyr Arg 35 40 45
Lys Met Glu Ile Tyr Arg Pro His Lys Ala Val Met Glu Glu Met Thr 50
55 60 Lys Tyr His Ser Asp Asp Tyr Val Lys Phe Leu Arg Thr Ile Arg
Pro 65 70 75 80 Asp Asn Met Ser Glu Tyr Thr Lys Gln Met Gln Arg Phe
Asn Val Gly 85 90 95 Glu Asp Cys Pro Val Phe Asp Gly Leu Tyr Glu
Phe Cys Gln Leu Ser 100 105 110 Ser Gly Gly Ser Val Ala Gly Ala Val
Lys Leu Asn Lys Gln Gln Thr 115 120 125 Asp Ile Ala Ile Asn Trp Ala
Gly Gly Leu His His Ala Lys Lys Ser 130 135 140 Glu Ala Ser Gly Phe
Cys Tyr Val Asn Asp Ile Val Leu Ala Ile Leu 145 150 155 160 Glu Leu
Leu Lys Tyr His Gln Arg Val Leu Tyr Ile Asp Ile Asp Ile 165 170 175
His His Gly Asp Gly Val Glu Glu Ala Phe Tyr Thr Thr Asp Arg Val 180
185 190 Met Thr Val Ser Phe His Lys Tyr Gly Glu Tyr Phe Pro Gly Thr
Gly 195 200 205 Asp Leu Arg Asp Ile Gly Ala Gly Lys Gly Lys Tyr Tyr
Ala Val Asn 210 215 220 Phe Pro Leu Arg Asp Gly Ile Asp Asp Glu Ser
Tyr Asp Lys Ile Phe 225 230 235 240 Lys Pro Ile Met Cys Lys Val Met
Glu Met Tyr Gln Pro Ser Ala Ile 245 250 255 Cys Leu Gln Cys Gly Ala
Asp Ser Leu Ser Gly Asp Arg Leu Gly Cys 260 265 270 Phe Asn Leu Thr
Leu Lys Gly His Ala Lys Cys Val Glu Phe Met Lys 275 280 285
Gln Tyr Asn Leu Pro Leu Leu Leu Met Gly Gly Gly Gly Tyr Thr Ile 290
295 300 Arg Asn Val Ala Arg Cys Trp Thr Tyr Glu Thr Ser Thr Ala Leu
Gly 305 310 315 320 Val Glu Ile Ala Asn Glu Leu Pro Tyr Asn Asp Tyr
Phe Glu Tyr Phe 325 330 335 Gly Pro Asp Phe Lys Leu His Ile Ser Pro
Ser Asn Met Thr Asn Gln 340 345 350 Asn Thr Gly Glu Tyr Leu Asp Lys
Ile Lys Thr Arg Leu Tyr Glu Asn 355 360 365 Met Arg Met Ile Pro His
Ala Pro Gly Val Gln Met Gln Pro Ile Pro 370 375 380 Glu Asp Ala Ile
Pro Asp Asp Ser Asp Ala Glu Asp Glu Ala Glu Asn 385 390 395 400 Pro
Asp Lys Arg Ile Ser Ile Met Ala Gln Asp Lys Arg Ile Gln Arg 405 410
415 Asp Asp Glu Phe Ser Asp Ser Glu Asp Glu Gly Glu Thr Arg Leu Pro
420 425 430 Gly Glu Gly Gly Arg Arg Asp His Arg Ser His Lys Ala Lys
Arg Ser 435 440 445 Lys Ile Asp Asp Ser Pro Gly Lys Glu Ala Asp Lys
Glu Ala Lys Ser 450 455 460 Ser Asp Ala Ser Lys Glu Ala Lys Pro Ala
Ala Glu Pro Gln Ala Val 465 470 475 480 Pro Met Asp Thr Thr Pro Ala
Pro Pro Pro Lys Lys Ser Glu Asp Lys 485 490 495 Pro Glu Ala Ser Lys
Pro Thr Glu Val Lys Ala Lys Pro Ala Glu Lys 500 505 510 Glu Pro Gly
Glu Gly Glu Ala Ser Pro Ala Asp Leu Val Val Pro Val 515 520 525 Pro
Lys Val Ser Ala Pro Ser Glu Gly Ala Thr Leu Pro Ala Val Thr 530 535
540 Ile Pro Pro Ser Ser Gly Thr Ser Gln Pro Pro Ala Asp Pro Pro Val
545 550 555 560 Ser Ala Pro Thr Pro Thr Pro Ala Ser Ala Pro Ala Glu
Lys Gln Asp 565 570 575
* * * * *
References