U.S. patent application number 10/081301 was filed with the patent office on 2002-09-05 for vitamin b metabolism proteins.
Invention is credited to Cahoon, Rebecca E., Falco, Saverio Carl, Rafalski, J. Antoni.
Application Number | 20020124276 10/081301 |
Document ID | / |
Family ID | 26791600 |
Filed Date | 2002-09-05 |
United States Patent
Application |
20020124276 |
Kind Code |
A1 |
Cahoon, Rebecca E. ; et
al. |
September 5, 2002 |
Vitamin B metabolism proteins
Abstract
This invention relates to an isolated nucleic acid fragment
encoding a vitamin B6 metabolic enzyme. The invention also relates
to the construction of a chimeric gene encoding all or a portion of
the vitamin B6 metabolic enzyme, in sense or antisense orientation,
wherein expression of the chimeric gene results in production of
altered levels of the vitamin B6 metabolic enzyme in a transformed
host cell.
Inventors: |
Cahoon, Rebecca E.;
(Wilmington, DE) ; Falco, Saverio Carl;
(Wilmington, DE) ; Rafalski, J. Antoni;
(Wilmington, DE) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY
LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1128
4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
26791600 |
Appl. No.: |
10/081301 |
Filed: |
February 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10081301 |
Feb 20, 2002 |
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09371056 |
Aug 9, 1999 |
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60096342 |
Aug 12, 1998 |
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Current U.S.
Class: |
800/8 ; 435/194;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 15/8243 20130101;
C12N 9/1205 20130101; C12Y 104/03005 20130101; C12Y 207/01035
20130101; C12N 9/0022 20130101 |
Class at
Publication: |
800/8 ; 435/194;
435/325; 435/320.1; 435/69.1; 536/23.2 |
International
Class: |
A01K 067/00; C12N
009/12; C07H 021/04; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated nucleic acid fragment encoding a pyridoxal kinase
comprising a member selected from the group consisting of: (a) an
isolated nucleic acid fragment comprising at least 400 contiguous
nucleotides wherein the nucleic acid fragment encodes an amino acid
sequence that is at least 80% identical to the amino acid sequence
set forth in a member selected from the group consisting of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO: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, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO: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. An isolated nucleic acid fragment encoding a
pyridoxamine-phosphate oxidase comprising a member selected from
the group consisting of: (a) an isolated nucleic acid fragment
encoding an amino acid sequence that is at least 80% identical to
the amino acid sequence set forth in a member selected from the
group consisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and
SEQ ID NO:16; (b) an isolated nucleic acid fragment that is
complementary to (a).
7. The isolated nucleic acid fragment of claim 6 wherein nucleic
acid fragment is a functional RNA.
8. The isolated nucleic acid fragment of claim 6 wherein the
nucleotide sequence of the fragment comprises the sequence set
forth in a member selected from the group consisting of SEQ ID
NO:9, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:15.
9. A chimeric gene comprising the nucleic acid fragment of claim 6
operably linked to suitable regulatory sequences.
10. A transformed host cell comprising the chimeric gene of claim
9.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/096,342, filed Aug. 12, 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 vitamin B6 metabolism in
plants and seeds.
BACKGROUND OF THE INVENTION
[0003] Vitamins are organic nutrients required in small quantities
for a variety of biochemical functions. Vitamins, generally, can
not be synthesized by the body and must therefore be supplied by
the diet. Besides being water soluble, vitamins in the B complex
have little in common from the chemical point of view. Excess B
vitamins are rarely accumulated or stored and thus must be provided
regularly.
[0004] Vitamin B 1 (thiamin) and vitamin B6 (pyridoxine, pyridoxal,
and pyridoxamine) are essential for animal nutrition. Vitamin B6
consists of three closely related pyridine derivatives: pyridoxine,
pyridoxal, and pyridoxamine. All forms of vitamin B6 are absorbed
from the intestine, but some hydrolysis of the phosphate esters
occurs during digestion. In the presence of magnesium ions and
using ATP as a cofactor, pyridoxal kinase (EC 2.7.1.35) converts
pyridoxine to pyridoxine 5'-phosphate. At least two pyridoxal
kinases involved in the salvage pathway of vitamin B6 biosynthesis
have been identified in Escherichia coli (Yang et al. (1998) J.
Bacteriol. 180:1814-1821). Pyridoxine 5'-phosphate is converted to
pyridoxal 5'-phosphate by the action of pyridoxamine 5'-phosphate
oxidase (EC 1.4.3.5). Pyridoxal 5'-phosphate is the major form of
B6 transported in plasma. Pyridoxal phosphate is used in
transamination and is an integral part in the enzymes which
breakdown glycogen. Studies in yeast and bacteria show that loss of
function perturbs amino acid, fatty acid, and sterol metabolism
(Lam, H. M. and Winkler, M. E. (1992) Bacteriol. 174:6033-6045;
Zhao and Winkler (1995) J. Bacteriol. 177:883-891; Loubbardi et al.
(1995) J. Bacteriol. 1 77:1817-1823).
[0005] Thiamin and Vitamin B6 are present in almost all plant and
animal tissues commonly used as foods, but the content is usually
small. Accordingly, enzymes responsible for their biosynthesis are
potential targets for future antibiotics, fungicides, and
herbicides. Thus, a detailed understanding of the activation,
structure, mechanism, kinetics, and substrate-binding properties of
the vitamin B1 and vitamin B6 biosynthetic enzymes from plants (and
other organisms) would aid in the rational design of chemical or
other kinds of herbicides. Isolation and purification of the
enzymes from plants would provide a valuable tool for the in vitro
screening of inhibitors of vitamin B1 and vitamin B6
biosynthesis.
SUMMARY OF THE INVENTION
[0006] The instant invention relates to isolated nucleic acid
fragments encoding enzymes involved in vitamin B6 metabolism.
Specifically, this invention concerns an isolated nucleic acid
fragment encoding a pyridoxal kinase or a pyridoxamine-phosphate
oxidase and an isolated nucleic acid fragment that is substantially
similar to an isolated nucleic acid fragment encoding a pyridoxal
kinase or a pyridoxamine-phosphate oxidase. In addition, this
invention relates to a nucleic acid fragment that is complementary
to the nucleic acid fragment encoding pyridoxal kinase or
pyridoxamine-phosphate oxidase.
[0007] An additional embodiment of the instant invention pertains
to a polypeptide encoding all or a substantial portion of a vitamin
B6 metabolic enzyme selected from the group consisting of pyridoxal
kinase and pyridoxamine-phosphate oxidase.
[0008] In another embodiment, the instant invention relates to a
chimeric gene encoding a pyridoxal kinase or a
pyridoxamine-phosphate oxidase, or to a chimeric gene that
comprises a nucleic acid fragment that is complementary to a
nucleic acid fragment encoding a pyridoxal kinase or a
pyridoxamine-phosphate oxidase, 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 pyridoxal kinase or a pyridoxamine-phosphate oxidase,
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 pyridoxal kinase or
a pyridoxamine-phosphate oxidase in a transformed host cell
comprising: a) transforming a host cell with a chimeric gene
comprising a nucleic acid fragment encoding a pyridoxal kinase or a
pyridoxamine-phosphate oxidase; 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 pyridoxal kinase or
pyridoxamine-phosphate oxidase 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 pyridoxal
kinase or a pyridoxamine-phosphate oxidase.
[0012] A further embodiment of the instant invention is a method
for evaluating at least one compound for its ability to inhibit the
activity of a pyridoxal kinase or a pyridoxamine-phosphate oxidase,
the method comprising the steps of: (a) transforming a host cell
with a chimeric gene comprising a nucleic acid fragment encoding a
pyridoxal kinase or a pyridoxamine-phosphate oxidase, operably
linked to suitable regulatory sequences; (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 pyridoxal kinase or
pyridoxamine-phosphate oxidase in the transformed host cell; (c)
optionally purifying the pyridoxal kinase or the
pyridoxamine-phosphate oxidase expressed by the transformed host
cell; (d) treating the pyridoxal kinase or the
pyridoxamine-phosphate oxidase with a compound to be tested; and
(e) comparing the activity of the pyridoxal kinase or the
pyridoxamine-phosphate oxidase that has been treated with a test
compound to the activity of an untreated pyridoxal kinase or
pyridoxamine-phosphate oxidase, thereby selecting compounds with
potential for inhibitory activity.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0013] The invention can be more fully understood from the
following detailed description and the accompanying Sequence
Listing which form a part of this application.
[0014] 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 Vitamin B6 Metabolic Enzymes SEQ ID NO: Protein Clone
Designation (Nucleotide) (Amino Acid) Corn Pyridoxal Contig of: 1 2
Kinase cc1.mn0001.h9 cr1n.pk0161.c12 p0004.cb1hc14r p0038.crvae17r
Rice Pyridoxal rlr6.pk0096.a8 3 4 Kinase Soybean Pyridoxal Contig
of: 5 6 Kinase sgc6c.pk001.o1 srm.pk0038.g4 Wheat Pyridoxal Contig
of: 7 8 Kinase wdk4c.pk006.119 wl1n.pk0106.e8 Corn Pyridoxamine-
Contig of: 9 10 Phosphate Oxidase cbn10.pk0048.g12 cpd1c.pk007.15
cr1n.pk0063.f3 csi1n.pk0050.f8 p0010.cbpcs54r p0072.comfr39r
p0072.comfu19r Rice Pyridoxamine- Contig of: 11 12 Phosphate
Oxidase rlr48.pk0022.b10 rr1.pk097.c18 Soybean Contig of: 13 14
Pyridoxamine- sfl1.pk0095.g3 Phosphate Oxidase sr1.pk0006.b5 Wheat
wr1.pk0018.f9 15 16 Pyridoxamine- Phosphate Oxidase
[0015] 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
[0016] 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.
[0017] 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.
[0018] 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--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.
[0019] 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.
[0020] 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.
[0021] 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% identical to the amino acid sequences reported herein.
More preferred nucleic acid fragments encode amino acid sequences
that are 90% identical to the amino acid sequences reported herein.
Most preferred are nucleic acid fragments that encode amino acid
sequences that are 95% identical to the amino acid sequences
reported herein. Sequence alignments and percent identity
calculations were performed using the Megalign program of the
LASERGENE 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.
[0022] 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.
[0023] "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.
[0024] "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.
[0025] "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.
[0026] "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.
[0027] "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.
[0028] 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).
[0029] 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.
[0030] "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.
[0031] 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.
[0032] 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).
[0033] "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.
[0034] "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.
[0035] "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).
[0036] 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").
[0037] Nucleic acid fragments encoding at least a portion of
several vitamin B6 metabolic enzymes 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).
[0038] For example, genes encoding other pyridoxal kinases or
pyridoxamine-phosphate oxidases, 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.
[0039] 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
85:8998-9002) 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
86:5673-5677; Loh et al. (1989) Science 243:217-220). Products
generated by the 3' and 5' RACE procedures can be combined to
generate full-length cDNAs (Frohman and Martin (1989) Techniques
1:165).
[0040] 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).
[0041] 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
active vitamin B1 or vitamin B6 in those cells.
[0042] 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.
[0043] Plasmid vectors comprising the instant chimeric gene can
then be 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] The person skilled in the art will know that special
considerations are associated with the use of antisense or
cosuppression 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.
[0048] 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
vitamin B6 metabolic enzyme. An example of a vector for high level
expression of the instant polypeptides in a bacterial host is
provided (Example 7).
[0049] Additionally, the instant polypeptides can be used as a
targets to facilitate design and/or identification of inhibitors of
those enzymes that may be useful as herbicides. This is desirable
because the polypeptides described herein catalyze various steps in
vitamin B6 metabolism. Accordingly, inhibition of the activity of
one or more of the enzymes described herein could lead to
inhibition of plant growth. Thus, the instant polypeptides could be
appropriate for new herbicide discovery and design.
[0050] 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).
[0051] The production and use of plant gene-derived probes for use
in genetic mapping is described in Bematzky 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.
[0052] 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).
[0053] 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.
[0054] 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 11:95-96),
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. 1 7:6795-6807). 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.
[0055] 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
86:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci USA
92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). 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
[0056] 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
[0057] 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 cbn10 Corn Developing Kernel (Embryo and
cbn10.pk0048.g12 Endosperm); 10 Days After Pollination cc1 Corn
Undifferentiated Callus cc1.mn0001.h9 cpd1c Corn Treated with
Chemicals Related to cpd1c.pk007.15 Protein Kinases* cr1n Corn Root
From 7 Day Old Seedlings** cr1n.pk0063.f3 cr1n.pk0161.c12 csi1n
Corn Silk** csi1n.pk0050.f8 p0004 Corn Immature Ear p0004.cb1hc14r
p0010 Corn Log Phase Suspension Cells Treated p0010.cbpcs54r With
A23187*** to Induce Mass Apoptosis p0038 Corn V5-Stage**** Roots
p0038.crvae17r p0072 Corn Mesocotyl 14 Days After Planting
p0072.comfr39r Etiolated Seedling p0072.comfu19r rlr48 Rice Leaf 15
Days After Germination, 48 rlr48.pk0022.b10 Hours After Infection
of Strain Magaporthe grisea 4360-R-62 (AVR2-YAMO); Resistant rlr6
Rice Leaf 15 Days After Germination, 6 rlr6.pk0096.a8 Hours After
Infection of Strain Magaporthe grisea 4360-R-62 (AVR2-YAMO);
Resistant rr1 Rice Root of Two Week Old Developing rr1.pk097.c18
Seedling sfl1 Soybean Immature Flower sfl1.pk0095.g3 sgc6c Soybean
Cotyledon 16-26 Days After sgc6c.pk001.o1 Germination (all yellow)
sr1 Soybean Root sr1.pk0006.b5 srm Soybean Root Meristem
srm.pk0038.g4 wdk4c Wheat Developing Kernel, 21 Days After
wdk4c.pk006.119 Anthesis wl1n Wheat Leaf From 7 Day Old Seedling**
wl1.pk0106.e8 wr1 Wheat Root From 7 Day Old Seedling wr1.pk0018.f9
*Chemicals used included 1,2-didecanoyl rac glycerol,
straurosporine, K-252, A3, H-7, olomoucine, rapamycin; which are
commercially available from Calbiochem-Novabiochem Corp. **These
libraries were normalized essentially as described in U.S. Pat. No.
5,482,845, incorporated herein by reference. ***A23187 is
commercially available from several vendors including Calbiochem.
****Corn developmental stages are explained in the publication "How
a corn plant develops" from the Iowa State University Coop. Ext.
Service Special Report No. 48 reprinted June 1993.
[0058] cDNA libraries may be prepared by any one of many methods
available. For example, the cDNAs may be introduced into plasmid
vectors by first preparing the cDNA libraries in Uni-ZAP.TM. XR
vectors according to the manufacturer's protocol (Stratagene
Cloning Systems, La Jolla, Calif.). The Uni-ZAP.TM. XR libraries
are converted into plasmid libraries according to the protocol
provided by Stratagene. Upon conversion, cDNA inserts will be
contained in the plasmid vector pBluescript. In addition, the cDNAs
may be introduced directly into precut Bluescript II SK(+) vectors
(Stratagene) using T4 DNA ligase (New England Biolabs), followed by
transfection into DH10B cells according to the manufacturer's
protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid
vectors, plasmid DNAs are prepared from randomly picked bacterial
colonies containing recombinant pBluescript plasmids, or the insert
cDNA sequences are amplified via polymerase chain reaction using
primers specific for vector sequences flanking the inserted cDNA
sequences. Amplified insert DNAs or plasmid DNAs are sequenced in
dye-primer sequencing reactions to generate partial cDNA sequences
(expressed sequence tags or "ESTs"; see Adams et al., (1991)
Science 252:1651-1656). The resulting ESTs are analyzed using a
Perkin Elmer Model 377 fluorescent sequencer.
Example 2
Identification of cDNA Clones
[0059] cDNA clones encoding vitamin B6 metabolic enzymes were
identified by conducting BLAST (Basic Local Alignment Search Tool;
Altschul et al. (1993) J. Mol. Biol. 2:5:403-410; see also
www.nebi.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 Pyridoxal Kinase
[0060] The BLASTX search using the EST sequences from clones listed
in Table 3 revealed similarity of the polypeptides encoded by the
cDNAs to pyridoxal kinase from Sus scrofa and Homo sapiens (NCBI
General Identifier Nos. 2773404 and 4505701, respectively). Shown
in Table 3 are the BLAST results for individual ESTs ("EST"), or
contigs assembled from two or more ESTs ("Contig"):
3TABLE 3 BLAST Results for Sequences Encoding Polypeptides
Homologous to Pyridoxal Kinase BLAST pLog Score Clone Status
2773404 4505701 Contig of: Contig 76.15 75.22 cc1.mn0001.h9
cr1n.pk0161.c12 p0004.cb1hc14r p0038.crvae17r rlr6.pk0096.a8 EST
11.10 12.00 Contig of: Contig 63.10 60.70 sgc6c.pk001.o1
srm.pk0038.g4 Contig of: Contig 65.52 65.40 wdk4c.pk006.119
wl1n.pk0106.e8
[0061] 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 Sus scrofa and Homo sapiens (NCBI General
Identifier Nos. 2773404 and 4505701, respectively).
4TABLE 4 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Pyridoxal Kinase Percent Identity to SEQ ID NO.
2773404 4505701 2 44.5 42.9 4 24.3 28.7 6 54.6 49.5 8 47.3 45.7
[0062] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE
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 an entire corn, and a substantial portion of a
rice, a soybean and a wheat pyridoxal kinase. These sequences
represent the first corn, rice, soybean and wheat sequences
encoding pyridoxal kinase.
Example 4
Characterization of cDNA Clones Encoding Pyridoxamine-Phosphate
Oxidase
[0063] The BLASTX search using the EST sequences from clones listed
in Table 5 revealed similarity of the polypeptides encoded by the
cDNAs to pyridoxamine-phosphate oxidase from Synechocystis sp.,
Caenorhabditis elegans and Rattus norvegicus (NCBI General
Identifier Nos. 3122599, 3979940 and 3237304, respectively). Shown
in Table 5 are the BLAST results for individual ESTs ("EST"),
contigs assembled from the sequences of the entire cDNA inserts
comprising the indicated cDNA clones and an EST ("Contig*"), or
contigs assembled from two or more ESTs ("Contig"):
5TABLE 5 BLAST Results for Sequences Encoding Polypeptides
Homologous to Pyridoxamine-Phosphate Oxidase NCBI General BLAST
Clone Status Identifier No. pLog Score Contig of: Contig 3122599
55.10 cbn10.pk0048.g12 cpd1c.pk007.15 cr1n.pk0063.f3
csi1n.pk0050.f8 p0010.cbpcs54r p0072.comfr39r p0072.comfu19r Contig
of: Contig 3979940 8.30 rlr48.pk0022.b10 rr1.pk097.c18 Contig of:
Contig 3237304 57.10 sfl1.pk0095.g3:fis sr1.pk0006.b5 wr1.pk0018.f9
EST 3237304 37.40
[0064] The data in Table 6 represents a calculation of the percent
identity of the amino acid sequences set forth in SEQ ID NOs:10,
12, 14 and 16 and the Rattus norvegicus sequence (NCBI General
Identifier No. 3237304).
6TABLE 6 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Pyridoxamine-Phosphate Oxidase Percent Identity SEQ
ID NO. to 3237304 10 38.3 12 14.5 14 40.2 16 45.3
[0065] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE
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
pyridoxamine-phosphate oxidase. These sequences represent the first
corn, rice, soybean and wheat sequences encoding
pyridoxamine-phosphate oxidase.
Example 5
Expression of Chimeric Genes in Monocot Cells
[0066] A chimeric gene comprising a cDNA encoding the instant
polypeptides in sense orientation with respect to the maize 27 kD
zein promoter that is located 5' to the cDNA fragment, and the 10
kD zein 3' end that is located 3' to the cDNA fragment, can be
constructed. The cDNA fragment of this gene may be generated by
polymerase chain reaction (PCR) of the cDNA clone using appropriate
oligonucleotide primers. Cloning sites (NcoI or SmaI) can be
incorporated into the oligonucleotides to provide proper
orientation of the DNA fragment when inserted into the digested
vector pML103 as described below. Amplification is then performed
in a standard PCR. The amplified DNA is then digested with
restriction enzymes NcoI and SmaI and fractionated on an agarose
gel. The appropriate band can be isolated from the gel and combined
with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. Plasmid
pML103 has been deposited under the terms of the Budapest Treaty at
ATCC (American Type Culture Collection, 10801 University Blvd.,
Manassas, Va. 20110-2209), and bears accession number ATCC 97366.
The DNA segment from pML103 contains a 1.05 kb SalI-NcoI promoter
fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI
fragment from the 3' end of the maize 10 kD zein gene in the vector
pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at
15.degree. C. overnight, essentially as described (Maniatis). The
ligated DNA may then be used to transform E. coli XL1-Blue
(Epicurian Coli XL-1 Blue.TM.; Stratagene). Bacterial transformants
can be screened by restriction enzyme digestion of plasmid DNA and
limited nucleotide sequence analysis using the dideoxy chain
termination method (Sequenase.TM. DNA Sequencing Kit; U.S.
Biochemical). The resulting plasmid construct would comprise a
chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD
zein promoter, a cDNA fragment encoding the instant polypeptides,
and the 10 kD zein 3' region.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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
[0073] 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), SmaI, KpnI and
XbaI. The entire cassette is flanked by Hind III sites.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein et al.
(1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A
DuPont Biolistic.TM. PDS1000/HE instrument (helium retrofit) can be
used for these transformations.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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
[0082] 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.
[0083] 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.
[0084] 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.
Example 8
Evaluating Compounds for Their Ability to Inhibit the Activity of
Vitamin B6 Metabolic Enzymes
[0085] The polypeptides described herein may be produced using any
number of methods known to those skilled in the art. Such methods
include, but are not limited to, expression in bacteria as
described in Example 7, or expression in eukaryotic cell culture,
in planta, and using viral expression systems in suitably infected
organisms or cell lines. The instant polypeptides may be expressed
either as mature forms of the proteins as observed in vivo or as
fusion proteins by covalent attachment to a variety of enzymes,
proteins or affinity tags. Common fusion protein partners include
glutathione S-transferase ("GST"), thioredoxin ("Trx"), maltose
binding protein, and C- and/or N-terminal hexahistidine polypeptide
("(His).sub.6"). The fusion proteins may be engineered with a
protease recognition site at the fusion point so that fusion
partners can be separated by protease digestion to yield intact
mature enzyme. Examples of such proteases include thrombin,
enterokinase and factor Xa. However, any protease can be used which
specifically cleaves the peptide connecting the fusion protein and
the enzyme.
[0086] Purification of the instant polypeptides, if desired, may
utilize any number of separation technologies familiar to those
skilled in the art of protein purification. Examples of such
methods include, but are not limited to, homogenization,
filtration, centrifugation, heat denaturation, ammonium sulfate
precipitation, desalting, pH precipitation, ion exchange
chromatography, hydrophobic interaction chromatography and affinity
chromatography, wherein the affinity ligand represents a substrate,
substrate analog or inhibitor. When the instant polypeptides are
expressed as fusion proteins, the purification protocol may include
the use of an affinity resin which is specific for the fusion
protein tag attached to the expressed enzyme or an affinity resin
containing ligands which are specific for the enzyme. For example,
the instant polypeptides may be expressed as a fusion protein
coupled to the C-terminus of thioredoxin. In addition, a
(His).sub.6 peptide may be engineered into the N-terminus of the
fused thioredoxin moiety to afford additional opportunities for
affinity purification. Other suitable affinity resins could be
synthesized by linking the appropriate ligands to any suitable
resin such as Sepharose-4B. In an alternate embodiment, a
thioredoxin fusion protein may be eluted using dithiothreitol;
however, elution may be accomplished using other reagents which
interact to displace the thioredoxin from the resin. These reagents
include .beta.-mercaptoethanol or other reduced thiol. The eluted
fusion protein may be subjected to further purification by
traditional means as stated above, if desired. Proteolytic cleavage
of the thioredoxin fusion protein and the enzyme may be
accomplished after the fusion protein is purified or while the
protein is still bound to the ThioBond.TM. affinity resin or other
resin.
[0087] Crude, partially purified or purified enzyme, either alone
or as a fusion protein, may be utilized in assays for the
evaluation of compounds for their ability to inhibit enzymatic
activation of the instant polypeptides disclosed herein. Assays may
be conducted under well known experimental conditions which permit
optimal enzymatic activity. For example, assays for pyridoxal
kinase and pyridoxamine-phosphate oxidase are presented by Merrill
and Wang (1986) Methods Enzymol. 122:110-116.
Sequence CWU 0
0
* * * * *
References