U.S. patent application number 09/756998 was filed with the patent office on 2001-08-02 for plant glycolysis and respiration enzymes.
Invention is credited to Allen, Stephen M., Lee, Jian-Ming, Lightner, Jonathan E., Odell, Joan T..
Application Number | 20010010931 09/756998 |
Document ID | / |
Family ID | 26761950 |
Filed Date | 2001-08-02 |
United States Patent
Application |
20010010931 |
Kind Code |
A1 |
Allen, Stephen M. ; et
al. |
August 2, 2001 |
Plant glycolysis and respiration enzymes
Abstract
This invention relates to an isolated nucleic acid fragment
encoding a glycolysis or respiration protein. The invention also
relates to the construction of a chimeric gene encoding all or a
portion of the glycolysis or respiration protein, in sense or
antisense orientation, wherein expression of the chimeric gene
results in production of altered levels of the glycolysis or
respiration protein in a transformed host cell.
Inventors: |
Allen, Stephen M.;
(Wilmington, DE) ; Lee, Jian-Ming; (West Caldwell,
NJ) ; Lightner, Jonathan E.; (Mulino, OR) ;
Odell, Joan T.; (Unionville, PA) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY
LEGAL DEPARTMENT - PATENTS
1007 MARKET STREET
WILMINGTON
DE
19898
US
|
Family ID: |
26761950 |
Appl. No.: |
09/756998 |
Filed: |
January 9, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09756998 |
Jan 9, 2001 |
|
|
|
09268364 |
Mar 15, 1999 |
|
|
|
6204063 |
|
|
|
|
60079387 |
Mar 26, 1998 |
|
|
|
Current U.S.
Class: |
435/194 ;
435/410; 536/23.2 |
Current CPC
Class: |
C12N 9/0055 20130101;
C12N 15/8261 20130101; C12N 9/1205 20130101; C12N 15/8243 20130101;
C12N 9/16 20130101; Y02A 40/146 20180101; C07K 14/415 20130101;
A61K 48/00 20130101 |
Class at
Publication: |
435/194 ;
435/410; 536/23.2 |
International
Class: |
C12N 005/00; C12N
005/02; C07H 021/04 |
Claims
What is claimed is:
1. An isolated nucleic acid fragment encoding all or a substantial
portion of a BCS1 protein comprising a member selected from the
group consisting of: (a) an isolated nucleic acid fragment encoding
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,
6, 8 and 10; (b) an isolated nucleic acid fragment that is
substantially similar to an isolated nucleic acid fragment encoding
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,
6, 8 and 10; and (c) an isolated nucleic acid fragment that is
complementary to (a) or (b).
2. The isolated nucleic acid fragment of claim 1 wherein the
nucleotide sequence of the fragment comprises all or a portion of
the sequence set forth in a member selected from the group
consisting of SEQ ID NO:1, 3, 5, 7 and 9.
3. A chimeric gene comprising the nucleic acid fragment of claim 1
operably linked to suitable regulatory sequences.
4. A transformed host cell comprising the chimeric gene of claim
3.
5. A BCS1 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, 6, 8 and 10.
6. An isolated nucleic acid fragment encoding all or a substantial
portion of a 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase
protein comprising a member selected from the group consisting of:
(a) an isolated nucleic acid fragment encoding all or a substantial
portion of the amino acid sequence set forth in a member selected
from the group consisting of SEQ ID NO:12, 14, 16, 18 and 20; (b)
an isolated nucleic acid fragment that is substantially similar to
an isolated nucleic acid fragment encoding all or a substantial
portion of the amino acid sequence set forth in a member selected
from the group consisting of SEQ ID NO:12, 14, 16, 18 and 20; and
(c) an isolated nucleic acid fragment that is complementary to (a)
or (b).
7. The isolated nucleic acid fragment of claim 6 wherein the
nucleotide sequence of the fragment comprises all or a portion of
the sequence set forth in a member selected from the group
consisting of SEQ ID NO:11, 13, 15, 17 and 19.
8. A chimeric gene comprising the nucleic acid fragment of claim 6
operably linked to suitable regulatory sequences.
9. A transformed host cell comprising the chimeric gene of claim
8.
10. A 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase
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:12, 14, 16, 18 and 20.
11. A method of altering the level of expression of a glycolysis or
respiration protein in a host cell comprising: (a) transforming a
host cell with the chimeric gene of any of claims 3 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 glycolysis or respiration protein in the
transformed host cell.
12. A method of obtaining a nucleic acid fragment encoding all or a
substantial portion of the amino acid sequence encoding a
glycolysis or respiration protein comprising: (a) probing a cDNA or
genomic library with the nucleic acid fragment of any of claims 1
and 6; (b) identifying a DNA clone that hybridizes with the nucleic
acid fragment of any of claims 1 and 6; (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 glycolysis or
respiration protein.
13. A method of obtaining a nucleic acid fragment encoding a
substantial portion of an amino acid sequence encoding a glycolysis
or respiration 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, 7, 9, 11, 13, 15, 17 and
19; 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 glycolysis or respiration protein.
14. The product of the method of claim 12.
15. The product of the method of claim 13.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/079,387, filed Mar. 26, 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 proteins involved in glycolysis and respiration
in plants and seeds.
BACKGROUND OF THE INVENTION
[0003] Glycolysis is the main pathway for carbohydrate catabolism.
It is a process in which monosaccharides are broken down to pyruvic
acid, two molecules of which are formed per monosaccharide residue.
In plants D-glucose and D-fructose are the main monosaccharides
catabolized by glycolysis although other monosaccharides that can
be converted to glucose or fructose can be handled by this
catabolic pathway. In cells where photosynthesis is not taking
place glycolysis is a key metabolic component of the respiratory
process which generates energy in the form of ATP. Typically the
cells of germinating seedlings and non-photosynthetic cells of
mature plants utilize this metabolic pathway. The glycolytic
pathway is controlled in part by the potent allosteric regulator
fructose-2,6-bisphosphate (F2,6P). This regulatory molecule
activates the enzymatic activity of phosphofructosekinase (PFK)
which stimulates the flow of carbon through the glycolytic pathway
to pyruvate. PFK plays a central role in the control of glycolysis
because it catalyzes one of the pathway's rate-determining
reactions. F2,6P also inhibits the activity of fructose
bisphosphatase (FBPase) which stimulates the flow of carbon through
gluconeogenesis, to form glucose. The concentration of F2,6P in the
cell depends on the action of 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase (PFK-2/FBPase). The formation and degradation of
F2,6P is catalyzed by PFK-2 and FBPase-2, two enzyme activities
that occur on different domains of the same protein molecule
(Algaier, J. et al. (1988) Biochem Biophys Res Commun
153(1):328-333). Thus, 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase is a key regulatory enzyme that controls carbon
flux through glycolysis vs. gluconeogenesis. Because PFK-2/FBPase
regulates the abundance of a key allosteric regulator, manipulating
the activity of this enzyme either by controlling expression or by
directed mutagenesis could be used to control carbon flux through
the glycolytic of gluconeogenic pathways. This could be very
important in bioprocessing in plants.
[0004] Respiration (aerobic metabolism) takes place in the
mitochondria in most eukaryotes. The ubiquinol-cytochrome C
reductase (bc1) complex is an important component of the
mitochondrial electron transport system. The BCS1 gene encodes a
product that has been shown to be necessary for the expression of
the Rieske iron-sulfur protein a component of the bc1complex
(Nobrega, F. G. et al. (1992) EMBO 11:3821-3829). By controlling
the expression of BCS1 it may be possible to modulate the level of
the Rieske iron-sulfur protein in plant cells which it turn would
regulate the amount of functional ubiquinol-cytochrome C reductase
complexes in mitochondria.
[0005] Few of the genes encoding the 6-phosphofructo
2-kinase/fructose 2,6-bisphosphatase and BCS1 proteins in corn,
Momordica, rice and wheat, have been isolated and sequenced. For
example, no corn, Momordica, rice or wheat genes have been reported
for 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase and no
plant genes have been reported for BCS1. Accordingly, the
availability of nucleic acid sequences encoding all or a portion of
these proteins would facilitate studies to better understand carbon
flux and respiration, provide genetic tools for the manipulation of
these metabolic pathways, and provide a means to control glycolysis
and respiration in plant cells.
SUMMARY OF THE INVENTION
[0006] The instant invention relates to isolated nucleic acid
fragments encoding proteins involved in glycolysis and respiration.
Specifically, this invention concerns an isolated nucleic acid
fragment encoding a BCS1 or 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase protein. In addition, this invention relates to
a nucleic acid fragment that is complementary to the nucleic acid
fragment encoding a BCS1 or 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase protein.
[0007] An additional embodiment of the instant invention pertains
to a polypeptide encoding all or a substantial portion of a protein
involved in glycolysis or respiration selected from the group
consisting of BCS1 and 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase.
[0008] In another embodiment, the instant invention relates to a
chimeric gene encoding a BCS1 or 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase protein, or to a chimeric gene that comprises a
nucleic acid fragment that is complementary to a nucleic acid
fragment encoding a BCS1 or 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase protein, 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 BCS1 or 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase protein, 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 BCS1 or
6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein in a
transformed host cell comprising: a) transforming a host cell with
a chimeric gene comprising a nucleic acid fragment encoding a BCS1
or 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein;
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 BCS1
or 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein 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 BCS1 or
6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS
[0012] The invention can be more fully understood from the
following detailed description and the accompanying drawings and
Sequence Listing which form a part of this application.
[0013] FIG. 1 presents an alignment of the amino acid sequence set
forth in SEQ ID NO:4 and the Saccharomyces cerevisiae BCS1 protein
(NCBI Identifier No. gi 2506091; SEQ ID NO:21).
[0014] The following 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.
[0015] SEQ ID NO:1 is the nucleotide sequence comprising a contig
assembled from the cDNA inserts in clones cr1n.pk0185.g6,
p0010.cbpca28r, p0126.cn1cr73r, p0126.cn1dc60r and cpf1c.pk009.116
encoding a portion of a corn BCS1 protein.
[0016] SEQ ID NO:2 is the deduced amino acid sequence of a portion
of a BCS1 protein derived from the nucleotide sequence of SEQ ID
NO:1.
[0017] SEQ ID NO:3 is the nucleotide sequence comprising a portion
of the cDNA insert in clone rr1.pk0025.d4 encoding a portion of a
rice BCS1 protein.
[0018] SEQ ID NO:4 is the deduced amino acid sequence of a portion
of a BCS1 protein derived from the nucleotide sequence of SEQ ID
NO:3.
[0019] SEQ ID NO:5 is the nucleotide sequence comprising a portion
of the cDNA insert in clone rr1.pk0026.e10 encoding a portion of a
rice BCS1 protein.
[0020] SEQ ID NO:6 is the deduced amino acid sequence of a portion
of a BCS1 protein derived from the nucleotide sequence of SEQ ID
NO:5.
[0021] SEQ ID NO:7 is the nucleotide sequence comprising a portion
of the cDNA insert in clone s12.pk127.m2 encoding a portion of a
soybean BCS1 protein.
[0022] SEQ ID NO:8 is the deduced amino acid sequence of a portion
of a BCS1 protein derived from the nucleotide sequence of SEQ ID
NO:7.
[0023] SEQ ID NO:9 is the nucleotide sequence comprising a portion
of the cDNA insert in clone wre1n.pk0059.e1 encoding a portion of a
wheat BCS1 protein.
[0024] SEQ ID NO:10 is the deduced amino acid sequence of a portion
of a BCS1 protein derived from the nucleotide sequence of SEQ ID
NO:9.
[0025] SEQ ID NO:11 is the nucleotide sequence comprising a portion
of the cDNA insert in clone cs1.pk0039.d2 encoding a portion of a
corn 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase
protein.
[0026] SEQ ID NO:12 is the deduced amino acid sequence of a portion
of a 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein
derived from the nucleotide sequence of SEQ ID NO:11.
[0027] SEQ ID NO:13 is the nucleotide sequence comprising a portion
of the cDNA insert in clone fds.pk0026.a2 encoding a portion of a
Momordica 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase
protein.
[0028] SEQ ID NO:14 is the deduced amino acid sequence of a portion
of a 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein
derived from the nucleotide sequence of SEQ ID NO:13.
[0029] SEQ ID NO:15 is the nucleotide sequence comprising a contig
assembled from the cDNA inserts in clones r1s6.pk0007.b6,
rds2c.pk005.d2, r1r6.pk0085.b4 and r1s48.pk0013.b4 encoding a
portion of a rice 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase protein.
[0030] SEQ ID NO:16 is the deduced amino acid sequence of a portion
of a 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein
derived from the nucleotide sequence of SEQ ID NO:15.
[0031] SEQ ID NO:17 is the nucleotide sequence comprising a portion
of the cDNA insert in clone src2c.pk003.p13 encoding a portion of a
soybean 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase
protein.
[0032] SEQ ID NO:18 is the deduced amino acid sequence of a portion
of a 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein
derived from the nucleotide sequence of SEQ ID NO:17.
[0033] SEQ ID NO:19 is the nucleotide sequence comprising a contig
assembled from the cDNA inserts in clones w1su2. pk029.111 and
wkm2c.pk006.h13 encoding a portion of a wheat 6-phosphofructo
2-kinase/fructose 2,6-bisphosphatase protein.
[0034] SEQ ID NO:20 is the deduced amino acid sequence of a portion
of a 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein
derived from the nucleotide sequence of SEQ ID NO:19.
[0035] SEQ ID NO:21 is the amino acid sequence of the Saccharomyces
cerevisiae BCS1 sequence set forth in NCBI Identifier No. gi
2506091.
[0036] 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
[0037] In the context of this disclosure, a number of terms shall
be utilized. As used herein, an "isolated 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. An isolated 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. As used herein, "contig" refers to an
assemblage of overlapping nucleic acid sequences to form one
contiguous nucleotide sequence. For example, several DNA sequences
can be compared and aligned to identify common or overlapping
regions. The individual sequences can then be assembled into a
single contiguous nucleotide sequence.
[0038] 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 protein encoded by the DNA
sequence.
[0039] "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 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 alteration of gene
expression by antisense or co-suppression technology or alteration
of the functional properties of the resulting protein molecule. It
is therefore understood that the invention encompasses more than
the specific exemplary sequences.
[0040] 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 gene 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 protein,
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 protein molecule
would also not be expected to alter the activity of the protein.
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.
[0041] Moreover, substantially similar nucleic acid fragments may
also be characterized by their ability to hybridize, under
stringent conditions (0.1X SSC, 0.1% SDS, 65.degree. C.), with the
nucleic acid fragments disclosed herein.
[0042] 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, D. G. and Sharp, P. M. (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.
[0043] A "substantial portion" of an amino acid or nucleotide
sequence comprises enough of the amino acid sequence of a
polypeptide or the nucleotide sequence of a gene to afford putative
identification of that polypeptide or gene, either by manual
evaluation of the sequence by one skilled in the art, or by
computer-automated sequence comparison and identification using
algorithms such as BLAST (Basic Local Alignment Search Tool;
Altschul, S. F., 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 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 20-30 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 12-15 bases 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 enough of the sequence
to afford specific identification and/or isolation of a nucleic
acid fragment comprising the sequence. The instant specification
teaches partial or complete amino acid and nucleotide sequences
encoding 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.
[0044] "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 that encodes
all or a substantial portion of the amino acid sequence encoding
the BCS1 and 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase
proteins as set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18
and 20. 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 gene for
improved expression in a host cell, it is desirable to design the
gene such that its frequency of codon usage approaches the
frequency of preferred codon usage of the host cell.
[0045] "Synthetic genes" 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 gene segments which are then
enzymatically assembled to construct the entire gene. "Chemically
synthesized", as related to a sequence of DNA, means that the
component nucleotides were assembled in vitro. Manual chemical
synthesis of DNA 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 genes 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.
[0046] "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.
[0047] "Coding sequence" refers to a DNA 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.
[0048] "Promoter" refers to a DNA 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 DNA 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 DNA 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 gene 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, DNA
fragments of different lengths may have identical promoter
activity.
[0049] The "translation leader sequence" refers to a DNA 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, R. and Foster, G. D. (1995) Molecular Biotechnology
3:225).
[0050] The "3' non-coding sequences" refer to DNA 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.
[0051] "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 protein by the cell. "cDNA" refers to a
double-stranded DNA that is complementary to and derived from mRNA.
"Sense" RNA refers to RNA transcript that includes the mRNA and so
can be translated into protein by the cell. "Antisense RNA" refers
to a 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 (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 gene transcript, 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.
[0052] The term "operably linked" refers to the association of
nucleic acid sequences 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.
[0053] 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).
[0054] "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.
[0055] "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.
[0056] 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, J. J., (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).
[0057] "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).
[0058] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described more fully
in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning:
A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold
Spring Harbor, 1989 (hereinafter "Maniatis").
[0059] Nucleic acid fragments encoding at least a portion of
several proteins involved in glycolysis and respiration 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. Table 1 lists the proteins that are described herein, and
the designation of the cDNA clones that comprise the nucleic acid
fragments encoding these proteins.
1TABLE 1 Glycolysis and Respiration Proteins Enzyme Clone Plant
BCS1 cr1n.pk0185.g6 Corn p0010.cbpca28r Corn p0126.cnlcr73r Corn
p01 26.cnldc60r Corn cpf1c.pk009.116 Corn rr1.pk0025.d4 Rice
rr1.pk0026.e10 Rice sl2.pk127.m2 Soybean wreln.pk0059.el Wheat
6-phosphofructo cs1.pk0039.d2 Corn 2-kinase/fructose fds.pk0026.a2
Momordica 2,6-bisphosphatase rls6.pk0007.b6 Rice rds2c.pk005.d2
Rice rlr6.pk0085.b4 Rice r1s48.pk0013.b4 Rice src2c.pk003.pl3
Soybean wlsu2.pk029.l11 Wheat wkm2c.pk006.h13 Wheat
[0060] 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).
[0061] For example, genes encoding other BCS1 or 6-phosphofructo
2-kinase/fructose 2,6-bisphosphatase 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.
[0062] 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) PNAS 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) PNAS 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, M. A. and Martin, G. R.,
(1989) Techniques 1:165).
[0063] 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, R. A. (1984) Adv. Immunol. 36:1;
Maniatis).
[0064] The nucleic acid fragments of the instant invention may be
used to create transgenic plants in which the disclosed BCS1 or
6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase proteins 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 modulating respiration or altering the
level of carbon flux in glycolysis in those cells.
[0065] Overexpression of the BCS1 or 6-phosphofructo
2-kinase/fructose 2,6-bisphosphatase 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.
[0066] 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.
[0067] For some applications it may be useful to direct the instant
glycolysis and respiration proteins 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 a
BCS1 or 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase
protein with appropriate intracellular targeting sequences such as
transit sequences (Keegstra, K. (1989) Cell 56:247-253), signal
sequences or sequences encoding endoplasmic reticulum localization
(Chrispeels, J. J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol.
42:21-53), or nuclear localization signals (Raikhel, N. (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.
[0068] It may also be desirable to reduce or eliminate expression
of genes encoding BCS1 or 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase proteins in plants for some applications. In
order to accomplish this, a chimeric gene designed for
co-suppression of the instant glycolysis and respiration proteins
can be constructed by linking a gene or gene fragment encoding a
BCS1 or 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase
protein 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.
[0069] The instant BCS1 or 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase proteins (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 BCS1 or 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase proteins in situ in cells or in vitro in cell
extracts. Preferred heterologous host cells for production of the
instant BCS1 or 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase proteins 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 BCS1 or
6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase proteins. This
chimeric gene could then be introduced into appropriate
microorganisms via transformation to provide high level expression
of the encoded glycolysis or respiration protein. An example of a
vector for high level expression of the instant BCS1 or
6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase proteins in a
bacterial host is provided (Example 7).
[0070] 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 at., (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, D.
et al., (1980) Am. J. Hum. Genet. 32:314-331).
[0071] The production and use of plant gene-derived probes for use
in genetic mapping is described in R. Bernatzky, R. and Tanksley,
S. D. (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.
[0072] 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, J. D., et al., In:
Nonmammalian Genomic Analysis: A Practical Guide, Academic press
1996, pp. 319-346, and references cited therein).
[0073] 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, B. J. (1991) Trends
Genet. 7:149-154). Although current methods of FISH mapping favor
use of large clones (several to several hundred KB; see Laan, M. et
al. (1995) Genome Research 5:13-20), improvements in sensitivity
may allow performance of FISH mapping using shorter probes.
[0074] 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, H. H. (1989) J. Lab. Clin. Med.
114(2):95-96), polymorphism of PCR-amplified fragments (CAPS;
Sheffield, V. C. et al. (1993) Genomics 16:325-332),
allele-specific ligation (Landegren, U. et al. (1988) Science
241:1077-1080), nucleotide extension reactions (Sokolov, B. P.
(1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping
(Walter, M. A. et al. (1997) Nature Genetics 7:22-28) and Happy
Mapping (Dear, P. H. and Cook, P. R. (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.
[0075] 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 BCS1 or
6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase protein.
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 a BCS1 or 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase protein can be identified and obtained. This
mutant plant can then be used to determine or confirm the natural
function of the BCS1 or 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase protein gene product.
EXAMPLES
[0076] 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
[0077] cDNA libraries representing mRNAs from various corn,
Momordica, rice, soybean and wheat tissues were prepared. The
characteristics of the libraries are described below.
2TABLE 2 eDNA Libraries from Corn, Momordica, Rice, Soybean and
Wheat Library Tissue Clone cpflc Corn pooled BMS treated with
chemicals related to protein cpflc.pk009.116 synthesis** cr1n Corn
root from 7 day seedlings grown in light* crln.pk0185.g6 cs1 Corn
leaf, sheath 5 wk old plant cs1.pk0039.d2 fds Momordica charantia
developing seed fds.pk0026.a2 p0010 Corn log phase suspension cells
(BMS) treated with A23187 p0010.cbpca28r to induce mass
apoptosis**** p0126 Corn, night harvested leaf tissue; V8-V10***
p0126.cnlcr73r p0126.cnldc60r rds2c Rice developing seeds in the
middle of the plant. rds2c.pk005.d2 rls6 Rice leaf, 15 day after
germination, 6 hrs after infection of rls6.pk0007.b6 Magaporthe
grisea strain 4360-R-67 (avr2-yamo); rlr6.pk0085.b4 Susceptible
rls48 Rice leaf. 15 days after germination, 48 hours after
infection rls48.pk0013.b4 of strain Magaporthe grisea 4360-R-67
(avr2-yamo); Susceptible rr1 Rice root of two week old developing
seedling rr1.pk0025.d4 rr1.pk0026.e10 sl2 Soybean two week old
developing seedlings treated with 2.5 sl2.pk127.m2 ppm chlorimuron
src2c Soybean 8 day old root inoculated with eggs of cyst
src2c.pk003.p13 nematode Heterodera glycines (Race 1) for 4 days
wkm2c Wheat kernel malted 55 hours at 22.degree. C. wkm2c.pk006.h13
wlsu2 Wheat seedlings 8 hr after fungicide***** treatment,
wlsu2.pk029.l11 subtracted with cDNAs from wheat seedlings 0 hr
after inoculation with Erysiphe graminis f. sp tritici wre1n Wheat
root; 7 day old etiolated seedling* wreln.pk0059.el *These
libraries were normalized essentially as described in U.S. Pat. No.
5,482,845 ***V8-V10 refer to stages of corn growth. The
descriptions can be found in "How a Corn Plant Develops" Special
Report No.48, Iowa State University of Science and Technology
Cooperative Extension Service Ames, Iowa, Reprinted February 1996.
****A23187 is commercially available from Calbiochem-Noavbiochem
Corp. *****Application of
6-iodo-2-propoxy-3-propyl-4(3H)-quinazolinone synthesis and methods
of using this compound are described in USSN 08/545,827,
incorporated herein by reference.
[0078] cDNA libraries were prepared in Uni-ZAP.TM. XR vectors
according to the manufacturer's protocol (Stratagene Cloning
Systems, La Jolla, Calif.). Conversion of the Uni-ZAP.TM. XR
libraries into plasmid libraries was accomplished according to the
protocol provided by Stratagene. Upon conversion, cDNA inserts were
contained in the plasmid vector pBluescript. cDNA inserts from
randomly picked bacterial colonies containing recombinant
pBluescript plasmids were amplified via polymerase chain reaction
using primers specific for vector sequences flanking the inserted
cDNA sequences or plasmid DNA was prepared from cultured bacterial
cells. Amplified insert DNAs or plasmid DNAs were sequenced in
dye-primer sequencing reactions to generate partial cDNA sequences
(expressed sequence tags or "ESTs"; see Adams, M. D. et al., (1991)
Science 252:1651). The resulting ESTs were analyzed using a Perkin
Elmer Model 377 fluorescent sequencer.
Example 2
Identification of cDNA Clones
[0079] ESTs encoding glycolysis and respiration proteins were
identified by conducting BLAST (Basic Local Alignment Search Tool;
Altschul, S. F., 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, W. and States,
D. J. (1993) Nature Genetics 3:266-272 and Altschul, Stephen F., et
al. (1997) Nucleic Acids Res. 25:3389-3402) 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 BCS1 Proteins
[0080] The BLASTX search using the EST sequences from clones
cr1n.pk0185.g6, p0010.cbpca28r, p0126.cn1cr73r, p0126.cn1dc60r,
cpf1c.pk009.116, rr1.pk0026.e10, s12.pk127.m2 and wre1n.pk0059.e1
revealed similarity of the proteins encoded by the cDNAs to BCS1
protein from Saccharomyces cerevisiae (NCBI Identifier No. gi
2506091).
[0081] A BLASTP search using the amino acid sequence encoded by the
cDNA from clone rr1.pk0025.d4 revealed similarity of the encoded
protein to BCS1 protein from Saccharomyces cerevisiae (NCBI
Identifier No. gi 2506091).
[0082] In the process of comparing the ESTs it was found that corn
clones cr1n.pk0185.g6, p0010.cbpca28r, p0126.cn1cr73r,
p0126.cn1dc60r and cpf1c.pk009.116 had overlapping regions of
homology. Using this homology it was possible to align the ESTs and
assemble a contig encoding a unique corn BCS1 protein. The BLAST
results for the corn contig and each of the ESTs are shown in Table
3:
3TABLE 3 BLAST Results for Clones Encoding Polypeptides Homologous
to Saccharomyces cerevisiae BCS 1 Protein Clone BLAST pLog Score
Contig composed of: 23.00 cr1n.pk0185.g6 p0010.cbpca28r
p0126.cn1cr73r p0126.cn1dc60r cpflc.pk009.116 rr1.pk0025.d4 10.30
rr1.pk0026.e10 13.00 sl2.pk127.m2 9.10 wreln.pk0059.el 10.50
[0083] The sequence of the corn contig composed of clones
cr1n.pk0185.g6, p0010.cbpca28r, p0126.cn1cr73r, p0126.cn1dc60r and
cpf1c.pk009.116 is shown in SEQ ID NO:1; the deduced amino acid
sequence of this contig, which represents 49% of the protein
(middle region), is shown in SEQ ID NO:2.
[0084] The sequence of the entire cDNA insert in clone
rr1.pk0025.d4 was determined and is shown in SEQ ID NO:3; the
deduced amino acid sequence of this cDNA, which represents 50% of
the protein (C-terminal region), is shown in SEQ ID NO:4. FIG. 1
presents an alignment of the amino acid sequence set forth in SEQ
ID NO:4 and the Saccharomyces cerevisiae sequence. A calculation of
the percent similarity of the amino acid sequence set forth in SEQ
ID NO:4 and the Saccharomyces cerevisiae sequence (using the
Clustal Algorithm) revealed that the protein encoded by the cDNA
insert in clone rr1.pk0025.d4 is 20% similar to the Saccharomyces
cerevisiae BCS1 protein.
[0085] The sequence of a portion of the cDNA insert from clone
rr1.pk0026.e10 is shown in SEQ ID NO:5; the deduced amino acid
sequence of this cDNA, which represents 20% of the protein (middle
region), is shown in SEQ ID NO:6. The sequence of a portion of the
cDNA insert from clone s12.pk127.m2 is shown in SEQ ID NO:7; the
deduced amino acid sequence of this cDNA, which represents 26% of
the protein (middle region) is shown in SEQ ID NO:8. The sequence
of a portion of the cDNA insert from clone wre1n.pk0059.e1 is shown
in SEQ ID NO:9; the deduced amino acid sequence of this cDNA, which
represents 12% of the protein (middle region) is shown in SEQ ID
NO:10.
[0086] BLAST scores and probabilities indicate that the instant
nucleic acid fragments encode portions of BCS1 proteins. These
sequences represent the first corn, rice, soybean and wheat
sequences encoding BCS1 proteins.
Example 4
Characterization of cDNA Clones Encoding 6-Phosphofructo
2-Kinase/Fructose 2,6-Bisphosphatase
[0087] The BLASTX search using the EST sequence from clone
cs1.pk0039.d2 revealed similarity of the protein encoded by the
cDNA to 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase from
Spinacia oleracea (NCBI Identifier No. gi 3170230). The BLASTX
search using the EST sequences from clones fds.pk0026.a2, r1s6.
pk0007.b6, rds2c.pk005.d2, r1r6.pk0085.b4, r1s48. pk0013.b4,
src2c.pk003.p13, w1su2.pk029.111 and wkm2c.pk006.h13 revealed
similarity of the proteins encoded by the cDNAs to 6-phosphofructo
2-kinase/fructose 2,6-bisphosphatase from Solanum tuberosum (NCBI
Identifier No. gi 3309583).
[0088] In the process of comparing the ESTs it was found that rice
clones r1s6.pk0007.b6, rds2c.pk005.d2, r1r6.pk0085.b4 and
r1s48.pk0013.b4 had overlapping regions of homology. Wheat clones
w1su2.pk029.111 and wkm2c.pk006.h13 were also found to have
overlapping regions of homology. Using this homology it was
possible to align the ESTs and assemble two contigs encoding a
unique rice and wheat 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase proteins.
[0089] The BLAST results for each of these ESTs and the rice and
wheat contigs are shown in Table 4:
4TABLE 4 BLAST Results for Clones Encoding Polypeptides Homologous
to Spinacia oleracea and Solanum tuberosum 6-Phosphofructo
2-Kinase/Fructose 2,6-Bisphosjphatase Proteins Clone BLAST pLog
Score cs1.pk0039.d2 82.00 fds.pk0026.a2 58.10 Contig composed of:
151.00 rls6.pk0007.b6 rds2c.pk005.d2 rlr6.pk0085.b4 rls48.pk0013.b4
src2c.pk003.p13 101.00 Contig composed of: 84.70 wlsu2.pk029.l11
wkm2c.pk006.h13
[0090] The sequence of a portion of the cDNA insert from clone
cs1.pk0039.d2 is shown in SEQ ID NO:11; the deduced amino acid
sequence of this cDNA, which represents 20% of the protein (middle
region), is shown in SEQ ID NO:12. The sequence of a portion of the
cDNA insert from clone fds.pk0026.a2 is shown in SEQ ID NO:13; the
deduced amino acid sequence of this cDNA, which represents 32% of
the protein (middle region), is shown in SEQ ID NO:14. The sequence
of the rice contig composed of clones r1s6.pk0007.b6,
rds2c.pk005.d2, r1r6.pk0085.b4 and r1s48.pk0013.b4 is shown in SEQ
ID NO:15; the deduced amino acid sequence of this contig, which
represents 50% of the protein (C-terminal region), is shown in SEQ
ID NO:16. The sequence of a portion of the cDNA insert from clone
src2c.pk003.p13 is shown in SEQ ID NO:17; the deduced amino acid
sequence of this cDNA, which represents 36% of the protein (middle
region), is shown in SEQ ID NO:18. The sequence of the wheat contig
composed of clones w1su2.pk029.111 and wkm2c.pk006.h13 is shown in
SEQ ID NO:19; the deduced amino acid sequence of this contig, which
represents 28% of the protein (C-terminal region), is shown in SEQ
ID NO:20.
[0091] BLAST scores and probabilities indicate that the instant
nucleic acid fragments encode portions of 6-phosphofructo
2-kinase/fructose 2,6-bisphosphatase proteins. These sequences
represent the first corn, Momordica, rice, soybean and wheat
sequences encoding 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase.
Example 5
Expression of Chimeric Genes in Monocot Cells
[0092] A chimeric gene comprising a cDNA encoding a glycolysis or
respiration protein 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 a glycolysis or respiration
protein, and the 10 kD zein 3' region.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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 6
Expression of Chimeric Genes in Dicot Cells
[0099] 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 glycolysis
and respiration proteins 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.
[0100] 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.
[0101] Soybean embroys may then be transformed with the expression
vector comprising a sequences encoding a glycolysis or respiration
protein. 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.
[0102] 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.
[0103] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Kline 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.
[0104] 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
glycolysis or respiration protein 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.
[0105] 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.
[0106] 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.
[0107] 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 7
Expression of Chimeric Genes in Microbial Cells
[0108] The cDNAs encoding the instant glycolysis and respiration
proteins 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.
[0109] 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 decribed 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 glycolysis or respiration protein are then screened
for the correct orientation with respect to the T7 promoter by
restriction enzyme analysis.
[0110] 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 0
0
* * * * *
References