U.S. patent application number 11/507094 was filed with the patent office on 2007-05-17 for corynebacterium glutamicum genes encoding proteins involved in membrane synthesis and membrane transport.
This patent application is currently assigned to BASF Aktiengesellschaft. Invention is credited to Gregor Haberhauer, Burkhard Kroger, Markus Pompejus, Hartwig Schroder, Oskar Zelder.
Application Number | 20070111230 11/507094 |
Document ID | / |
Family ID | 31499772 |
Filed Date | 2007-05-17 |
United States Patent
Application |
20070111230 |
Kind Code |
A1 |
Pompejus; Markus ; et
al. |
May 17, 2007 |
Corynebacterium glutamicum genes encoding proteins involved in
membrane synthesis and membrane transport
Abstract
Isolated nucleic acid molecules, designated MCT nucleic acid
molecules, which encode novel MCT proteins from Corynebacterium
glutamicum are described. The invention also provides antisense
nucleic acid molecules, recombinant expression vectors containing
MCT nucleic acid molecules, and host cells into which the
expression vectors have been introduced. The invention still
further provides isolated MCT proteins, mutated MCT proteins,
fusion proteins, antigenic peptides and methods for the improvement
of production of a desired compound from C. glutamicum based on
genetic engineering of MCT genes in this organism.
Inventors: |
Pompejus; Markus;
(Freinsheim, DE) ; Kroger; Burkhard;
(Limburgerhof, DE) ; Schroder; Hartwig; (Nussloch,
DE) ; Zelder; Oskar; (Speyer, DE) ;
Haberhauer; Gregor; (Limburgerhof, DE) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP
ONE POST OFFICE SQUARE
BOSTON
MA
02109-2127
US
|
Assignee: |
BASF Aktiengesellschaft
Ludwigshafen
DE
|
Family ID: |
31499772 |
Appl. No.: |
11/507094 |
Filed: |
August 18, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10627476 |
Jul 25, 2003 |
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11507094 |
Aug 18, 2006 |
|
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09602787 |
Jun 23, 2000 |
6696561 |
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10627476 |
Jul 25, 2003 |
|
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60141031 |
Jun 25, 1999 |
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Current U.S.
Class: |
435/134 ;
435/252.3; 435/471; 435/69.3; 435/7.32; 530/350; 536/23.7 |
Current CPC
Class: |
C07K 14/34 20130101;
C12N 9/18 20130101; G01N 33/56911 20130101; C12Q 1/689 20130101;
C12N 9/00 20130101 |
Class at
Publication: |
435/006 ;
536/023.7; 435/007.32; 435/069.3; 435/252.3; 435/471; 530/350 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/554 20060101 G01N033/554; C07H 21/04 20060101
C07H021/04; C12N 15/74 20060101 C12N015/74; G01N 33/569 20060101
G01N033/569; C07K 14/195 20060101 C07K014/195 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 8, 1999 |
DE |
19931454.3 |
Jul 8, 1999 |
DE |
19931478.0 |
Jul 8, 1999 |
DE |
19931563.9 |
Jul 9, 1999 |
DE |
19932122.1 |
Jul 9, 1999 |
DE |
19932124.8 |
Jul 9, 1999 |
DE |
19932125.6 |
Jul 9, 1999 |
DE |
19932128.0 |
Jul 9, 1999 |
DE |
19932180.9 |
Jul 9, 1999 |
DE |
19932182.5 |
Jul 9, 1999 |
DE |
19932190.6 |
Jul 9, 1999 |
DE |
19932191.4 |
Jul 9, 1999 |
DE |
19932209.0 |
Jul 9, 1999 |
DE |
19932212.0 |
Jul 9, 1999 |
DE |
19932227.9 |
Jul 9, 1999 |
DE |
19932228.7 |
Jul 9, 1999 |
DE |
19932229.5 |
Jul 9, 1999 |
DE |
19932230.9 |
Jul 14, 1999 |
DE |
19932927.3 |
Jul 14, 1999 |
DE |
19933005.0 |
Jul 14, 1999 |
DE |
19933006.9 |
Aug 27, 1999 |
DE |
19940764.9 |
Aug 27, 1999 |
DE |
19940765.7 |
Aug 27, 1999 |
DE |
19940766.5 |
Aug 27, 1999 |
DE |
19940830.0 |
Aug 27, 1999 |
DE |
19940831.9 |
Aug 27, 1999 |
DE |
19940832.7 |
Aug 27, 1999 |
DE |
19940833.5 |
Aug 31, 1999 |
DE |
19941378.9 |
Aug 31, 1999 |
DE |
19941379.7 |
Aug 31, 1999 |
DE |
19941395.9 |
Sep 3, 1999 |
DE |
19942077.7 |
Sep 3, 1999 |
DE |
19942078.5 |
Sep 3, 1999 |
DE |
19942079.3 |
Sep 3, 1999 |
DE |
19942088.2 |
Claims
1. An isolated nucleic acid molecule selected from the group
consisting of a) an isolated nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:77, or a complement thereof; b) an
isolated nucleic acid molecule which encodes a polypeptide
comprising the amino acid sequence of SEQ ID NO:78, or a complement
thereof; c) an isolated nucleic acid molecule which encodes a
naturally occurring allelic variant of a polypeptide comprising the
amino acid sequence of SEQ ID NO:78, or a complement thereof; d) an
isolated nucleic acid molecule comprising a nucleotide sequence
which is at least 50% identical to the entire nucleotide sequence
of SEQ ID NO:77, or a complement thereof; and e) an isolated
nucleic acid molecule comprising a fragment of at least 15
contiguous nucleotides of the nucleotide sequence of SEQ ID NO:77,
or a complement thereof.
2. An isolated nucleic acid molecule comprising the nucleic acid
molecule of claim 1 and a nucleotide sequence encoding a
heterologous polypeptide.
3. A vector comprising the nucleic acid molecule of claim 1.
4. The vector of claim 3, which is an expression vector.
5. A host cell transfected with the expression vector of claim
4.
6. The host cell of claim 5, wherein said cell is a
microorganism.
7. The host cell of claim 6, wherein said cell-belongs to the genus
Corynebacterium or Brevibacterium.
8. A method of producing a polypeptide comprising culturing the
host cell of claim 5 in an appropriate culture medium to, thereby,
produce the polypeptide.
9. A method for producing a fine chemical, comprising culturing the
cell of claim 5 such that the fine chemical is produced.
10. The method of claim 9, wherein said method further comprises
the step of recovering the fine chemical from said culture.
11. The method of claim 9, wherein said cell belongs to the genus
Corynebacterium or Brevibacterium.
12. The method of claim 9, wherein said cell is selected from the
group consisting of Corynebacterium glutamicum, Corynebacterium
herculis, Corynebacterium, lilium, Corynebacterium
acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium
acetophilum, Corynebacterium ammoniagenes, Corynebacterium
fujiokense, Corynebacterium nitrilophilus, Brevibacterium
ammoniagenes, Brevibacterium butanicum, Brevibacterium divaricatum,
Brevibacterium flavum, Brevibacterium healii, Brevibacterium
ketoglutamicum, Brevibacterium ketosoreductum, Brevibacterium
lactofermentum, Brevibacterium linens, Brevibacterium
paraffinolyticum, and those strains set forth in Table 3.
13. The method of claim 9, wherein expression of the nucleic acid
molecule from said vector results in modulation of production of
said fine chemical.
14. The method of claim 9, wherein said fine chemical is selected
from the group consisting of organic acids, proteinogenic and
nonproteinogenic amino acids, purine and pyrimidine bases,
nucleosides, nucleotides, lipids, saturated and unsaturated fatty
acids, diols, carbohydrates, aromatic compounds, vitamins,
cofactors, polyketides, and enzymes.
15. The method of claim 9, wherein said fine chemical is an amino
acid selected from the group consisting of lysine, glutamate,
glutamine, alanine, aspartate, glycine, serine, threonine,
methionine, cysteine, valine, leucine, isoleucine, arginine,
proline, histidine, tyrosine, phenylalanine, and tryptophan.
16. An isolated polypeptide selected from the group consisting of
a) an isolated polypeptide comprising the amino acid sequence of
SEQ ID NO:78; b) an isolated polypeptide comprising a naturally
occurring allelic variant of a polypeptide comprising the amino
acid sequence of SEQ ID NO:78; c) an isolated polypeptide which is
encoded by a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:77; d) an isolated polypeptide which is
encoded by a nucleic acid molecule comprising a nucleotide sequence
which is at least 50% identical to the entire nucleotide sequence
of SEQ ID NO:77; e) an isolated polypeptide comprising an amino
acid sequence which is at least 50% identical to the entire amino
acid sequence of SEQ ID NO:78; and f) an isolated polypeptide
comprising a fragment of a polypeptide comprising the amino acid
sequence of SEQ ID NO:78, wherein said polypeptide fragment
maintains a biological activity of the polypeptide comprising the
amino sequence.
17. The isolated polypeptide of claim 16, further comprising
heterologous amino acid sequences.
18. A method for diagnosing the presence or activity of
Corynebacterium diphtheriae in a subject, comprising detecting the
presence of at least one of the nucleic acid molecules of claim 1,
thereby diagnosing the presence or activity of Corynebacterium
diphtheriae in the subject.
19. A method for diagnosing the presence or activity of
Corynebacterium diphtheriae in a subject, comprising detecting the
presence of at least one of the polypeptide molecules of claim 16,
thereby diagnosing the presence or activity of Corynebacterium
diphtheriae in the subject.
20. A host cell comprising a nucleic acid molecule selected from
the group consisting of a) the nucleic acid molecule of SEQ ID
NO:77, wherein the nucleic acid molecule is disrupted by at least
one technique selected from the group consisting of a point
mutation, a truncation, an inversion, a deletion, an addition, a
substitution and homologous recombination; b) the nucleic acid
molecule of SEQ ID NO:77, wherein the nucleic acid molecule
comprises one or more nucleic acid modifications as compared to the
sequence of SEQ ID NO:77, wherein the modification is selected from
the group consisting of a point mutation, a truncation, an
inversion, a deletion, an addition and a substitution; and c) the
nucleic acid molecule of SEQ ID NO:77, wherein the regulatory
region of the nucleic acid molecule is modified relative to the
wild-type regulatory region of the molecule by at least one
technique selected from the group consisting of a point mutation, a
truncation, an inversion, a deletion, an addition, a substitution
and homologous recombination.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/627,476, filed Jul. 25, 2003, which is a continuation of
U.S. application Ser. No. 09/602,787, filed Jun. 23, 2000, which,
in turn, claims priority to prior filed U.S. Provisional Patent
Application Ser. No. 60/141031, filed Jun. 25, 1999. This
application also claims priority to German Patent Application No.
19931454.3, filed Jul. 8, 1999, German Patent Application No.
19931478.0, filed Jul. 8, 1999, German Patent Application No.
19931563.9, filed Jul. 8, 1999, German Patent Application No.
19932122.1, filed Jul. 9, 1999, German Patent Application No.
19932124.8, filed Jul. 9, 1999, German Patent Application No.
19932125.6, filed Jul. 9, 1999, German Patent Application No.
19932128.0, filed Jul. 9, 1999, German Patent Application No.
19932180.9, filed Jul. 9, 1999, German Patent Application No.
19932182.5, filed Jul. 9, 1999, German Patent Application No.
19932190.6, filed Jul. 9, 1999, German Patent Application No.
19932191.4, filed Jul. 9, 1999, German Patent Application No.
19932209.0, filed Jul. 9, 1999, German Patent Application No.
19932212.0, filed Jul. 9, 1999, German Patent Application No.
19932227.9, filed Jul. 9, 1999, German Patent Application No.
19932228.7, filed Jul. 9, 1999, German Patent Application No.
19932229.5, filed Jul. 9, 1999, German Patent Application No.
19932230.9, filed Jul. 9, 1999, German Patent Application No.
19932927.3, filed Jul. 14, 1999, German Patent Application No.
19933005.0, filed Jul. 14, 1999, German Patent Application No.
19933006.9, filed Jul. 14, 1999, German Patent Application No.
19940764.9, filed Aug. 27, 1999, German Patent Application No.
19940765.7, filed Aug. 27, 1999, German Patent Application No.
19940766.5, filed Aug. 27, 1999, German Patent Application No.
19940830.0, filed Aug. 27, 1999, German Patent Application No.
19940831.9, filed Aug. 27, 1999, German Patent Application No.
19940832.7, filed Aug. 27, 1999, German Patent Application No.
19940833.5, filed Aug. 27, 1999, German Patent Application No.
19941378.9 filed Aug. 31, 1999, German Patent Application No.
19941379.7, filed Aug. 31, 1999, German Patent Application No.
19941395.9, filed Aug. 31, 1999, German Patent Application No.
19942077.7, filed Sep. 3, 1999, German Patent Application No.
19942078.5, filed September 3, 1999, German Patent Application No.
19942079.3, filed Sep. 3, 1999, and German Patent Application No.
19942088.2, filed Sep. 3, 1999. The entire contents of all of the
above referenced applications are hereby expressly incorporated
herein by this reference.
INCORPORATION OF MATERIAL SUBMITTED ON COMPACT DISCS
[0002] This application incorporates herein by reference the
material contained on the compact discs submitted herewith as part
of this application. Specifically, the file "seqlistcorr2" (2.43
MB) contained on each of Copy 1, Copy 2 and the CRF copy of the
Sequence Listing is hereby incorporated herein by reference. This
file was created on Jul. 31, 2006. In addition, the files "Appendix
A" (399 KB) and "Appendix B" (140 KB) contained on each of the
compact disks entitled "Appendices Copy 1" and "Appendices Copy 2"
are hereby incorporated herein by reference. Each of these files
were created on Jul. 31, 2006.
BACKGROUND OF THE INVENTION
[0003] Certain products and by-products of naturally-occurring
metabolic processes in cells have utility in a wide array of
industries, including the food, feed, cosmetics, and pharmaceutical
industries. These molecules, collectively termed `fine chemicals`,
include organic acids, both proteinogenic and non-proteinogenic
amino acids, nucleotides and nucleosides, lipids and fatty acids,
diols, carbohydrates, aromatic compounds, vitamins and cofactors,
and enzymes. Their production is most conveniently performed
through the large-scale culture of bacteria developed to produce
and secrete large quantities of one or more desired molecules. One
particularly useful organism for this purpose is Corynebacterium
glutamicum, a gram positive, nonpathogenic bacterium. Through
strain selection, a number of mutant strains have been developed
which produce an array of desirable compounds. However, selection
of strains improved for the production of a particular molecule is
a time-consuming and difficult process.
SUMMARY OF THE INVENTION
[0004] The invention provides novel bacterial nucleic acid
molecules which have a variety of uses. These uses include the
identification of microorganisms which can be used to produce fine
chemicals, the modulation of fine chemical production in C.
glutamicum or related bacteria, the typing or identification of C.
glutamicum or related bacteria, as reference points for mapping the
C. glutamicum genome, and as markers for transformation. These
novel nucleic acid molecules encode proteins, referred to herein as
membrane construction and membrane transport (MCT) proteins.
[0005] C. glutamicum is a gram positive, aerobic bacterium which is
commonly used in industry for the large-scale production of a
variety of fine chemicals, and also for the degradation of
hydrocarbons (such as in petroleum spills) and for the oxidation of
terpenoids. The MCT nucleic acid molecules of the invention,
therefore, can be used to identify microorganisms which can be used
to produce fine chemicals, e.g., by fermentation processes.
Modulation of the expression of the MCT nucleic acids of the
invention, or modification of the sequence of the MCT nucleic acid
molecules of the invention, can be used to modulate the production
of one or more fine chemicals from a microorganism (e.g., to
improve the yield or production of one or more fine chemicals from
a Corynebacterium or Brevibacterium species).
[0006] The MCT nucleic acids of the invention may also be used to
identify an organism as being Corynebacterium glutamicum or a close
relative thereof, or to identify the presence of C. glutamicum or a
relative thereof in a mixed population of microorganisms. The
invention provides the nucleic acid sequences of a number of C.
glutamicum genes; by probing the extracted genomic DNA of a culture
of a unique or mixed population of microorganisms under stringent
conditions with a probe spanning a region of a C. glutamicum gene
which is unique to this organism, one can ascertain whether this
organism is present. Although Corynebacterium glutamicum itself is
nonpathogenic, it is related to species pathogenic in humans, such
as Corynebacterium diphtheriae (the causative agent of diphtheria);
the detection of such organisms is of significant clinical
relevance.
[0007] The MCT nucleic acid molecules of the invention may also
serve as reference points for mapping of the C. glutamicum genome,
or of genomes of related organisms. Similarly, these molecules, or
variants or portions thereof, may serve as markers for genetically
engineered Corynebacterium or Brevibacterium species. e.g.e.g. The
MCT proteins encoded by the novel nucleic acid molecules of the
invention are capable of, for example, performing a function
involved in the metabolism (e.g., the biosynthesis or degradation)
of compounds necessary for membrane biosynthesis, or of assisting
in the transmembrane transport of one or more compounds either into
or out of the cell. Given the availability of cloning vectors for
use in Corynebacterium glutamicum, such as those disclosed in
Sinskey et al., U.S. Pat. No. 4,649,119, and techniques for genetic
manipulation of C. glutamicum and the related Brevibacterium
species (e.g., lactofermentum) (Yoshihama et al, J. Bacteriol. 162:
591-597 (1985); Katsumata et al., J. Bacteriol. 159: 306-311
(1984); and Santamaria et al., J. Gen. Microbiol. 130: 2237-2246
(1984)), the nucleic acid molecules of the invention may be
utilized in the genetic engineering of this organism to make it a
better or more efficient producer of one or more fine chemicals.
This improved production or efficiency of production of a fine
chemical may be due to a direct effect of manipulation of a gene of
the invention, or it may be due to an indirect effect of such
manipulation.
[0008] There are a number of mechanisms by which the alteration of
an MCT protein of the invention may directly affect the yield,
production, and/or efficiency of production of a fine chemical from
a C. glutamicum strain incorporating such an altered protein. Those
MCT proteins involved in the export of fine chemical molecules from
the cell may be increased in number or activity such that greater
quantities of these compounds are secreted to the extracellular
medium, from which they are more readily recovered. Similarly,
those MCT proteins involved in the import of nutrients necessary
for the biosynthesis of one or more fine chemicals (e.g.,
phosphate, sulfate, nitrogen compounds, etc.) may be increased in
number or activity such that these precursors, cofactors, or
intermediate compounds are increased in concentration within the
cell. Further, fatty acids and lipids themselves are desirable fine
chemicals; by optimizing the activity or increasing the number of
one or more MCT proteins of the invention which participate in the
biosynthesis of these compounds, or by impairing the activity of
one or more MCT proteins which are involved in the degradation of
these compounds, it may be possible to increase the yield,
production, and/or efficiency of production of fatty acid and lipid
molecules from C. glutamicum.
[0009] The mutagenesis of one or more MCT genes of the invention
may also result in MCT proteins having altered activities which
indirectly impact the production of one or more desired fine
chemicals from C. glutamicum. For example, MCT proteins of the
invention involved in the export of waste products may be increased
in number or activity such that the normal metabolic wastes of the
cell (possibly increased in quantity due to the overproduction of
the desired fine chemical) are efficiently exported before they are
able to damage nucleotides and proteins within the cell (which
would decrease the viability of the cell) or to interfere with fine
chemical biosynthetic pathways (which would decrease the yield,
production, or efficiency of production of the desired fine
chemical). Further, the relatively large intracellular quantities
of the desired fine chemical may in itself be toxic to the cell, so
by increasing the activity or number of transporters able to export
this compound from the cell, one may increase the viability of the
cell in culture, in turn leading to a greater number of cells in
the culture producing the desired fine chemical. The MCT proteins
of the invention may also be manipulated such that the relative
amounts of different lipid and fatty acid molecules are produced.
This may have a profound effect on the lipid composition of the
membrane of the cell. Since each type of lipid has different
physical properties, an alteration in the lipid composition of a
membrane may significantly alter membrane fluidity. Changes in
membrane fluidity can impact the transport of molecules across the
membrane, as well as the integrity of the cell, both of which have
a profound effect on the production of fine chemicals from C.
glutamicum in large-scale fermentative culture.
[0010] The invention provides novel nucleic acid molecules which
encode proteins, referred to herein as MCT proteins, which are
capable of, for example, participating in the metabolism of
compounds necessary for the construction of cellular membranes in
C. glutamicum, or in the transport of molecules across these
membranes. Nucleic acid molecules encoding an MCT protein are
referred to herein as MCT nucleic acid molecules. In a preferred
embodiment, the MCT protein participates in the metabolism of
compounds necessary for the construction of cellular membranes in
C. glutamicum, or in the transport of molecules across these
membranes. Examples of such proteins include those encoded by the
genes set forth in Table 1.
[0011] Accordingly, one aspect of the invention pertains to
isolated nucleic acid molecules (e.g., cDNAs, DNAs, or RNAs)
comprising a nucleotide sequence encoding an MCT protein or
biologically active portions thereof, as well as nucleic acid
fragments suitable as primers or hybridization probes for the
detection or amplification of MCT-encoding nucleic acid (e.g., DNA
or mRNA). In particularly preferred embodiments, the isolated
nucleic acid molecule comprises one of the nucleotide sequences set
forth in Appendix A or the coding region or a complement thereof of
one of these nucleotide sequences. In other particularly preferred
embodiments, the isolated nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes to or is at least
about 50%, preferably at least about 60%, more preferably at least
about 70%, 80% or 90%, and even more preferably at least about 95%,
96%, 97%, 98%, 99% or more homologous to a nucleotide sequence set
forth in Appendix A, or a portion thereof. In other preferred
embodiments, the isolated nucleic acid molecule encodes one of the
amino acid sequences set forth in Appendix B. The preferred MCT
proteins of the present invention also preferably possess at least
one of the MCT activities described herein.
[0012] In another embodiment, the isolated nucleic acid molecule
encodes a protein or portion thereof wherein the protein or portion
thereof includes an amino acid sequence which is sufficiently
homologous to an amino acid sequence of Appendix B, e.g.,
sufficiently homologous to an amino acid sequence of Appendix B
such that the protein or portion thereof maintains an MCT activity.
Preferably, the protein or portion thereof encoded by the nucleic
acid molecule maintains the ability to participate in the
metabolism of compounds necessary for the construction of cellular
membranes in C. glutamicum, or in the transport of molecules across
these membranes. In one embodiment, the protein encoded by the
nucleic acid molecule is at least about 50%, preferably at least
about 60%, and more preferably at least about 70%, 80%, or 90% and
most preferably at least about 95%, 96%, 97%, 98%, or 99% or more
homologous to an amino acid sequence of Appendix B (e.g., an entire
amino acid sequence selected from those sequences set forth in
Appendix B). In another preferred embodiment, the protein is a full
length C. glutamicum protein which is substantially homologous to
an entire amino acid sequence of Appendix B (encoded by an open
reading frame shown in Appendix A).
[0013] In another preferred embodiment, the isolated nucleic acid
molecule is derived from C. glutamicum and encodes a protein (e.g.,
an MCT fusion protein) which includes a biologically active domain
which is at least about 50% or more homologous to one of the amino
acid sequences of Appendix B and is able to participate in the
metabolism of compounds necessary for the construction of cellular
membranes in C. glutamicum, or in the transport of molecules across
these membranes, or has one or more of the activities set forth in
Table 1, and which also includes heterologous nucleic acid
sequences encoding a heterologous polypeptide or regulatory
regions.
[0014] In another embodiment, the isolated nucleic acid molecule is
at least 15 nucleotides in length and hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of Appendix A. Preferably, the isolated nucleic acid
molecule corresponds to a naturally-occurring nucleic acid
molecule. More preferably, the isolated nucleic acid encodes a
naturally-occurring C. glutamicum MCT protein, or a biologically
active portion thereof.
[0015] Another aspect of the invention pertains to vectors, e.g.,
recombinant expression vectors, containing the nucleic acid
molecules of the invention, and host cells into which such vectors
have been introduced. In one embodiment, such a host cell is used
to produce an MCT protein by culturing the host cell in a suitable
medium. The MCT protein can be then isolated from the medium or the
host cell.
[0016] Yet another aspect of the invention pertains to a
genetically altered microorganism in which an MCT gene has been
introduced or altered. In one embodiment, the genome of the
microorganism has been altered by introduction of a nucleic acid
molecule of the invention encoding wild-type or mutated MCT
sequence as a transgene. In another embodiment, an endogenous MCT
gene within the genome of the microorganism has been altered, e.g.,
functionally disrupted, by homologous recombination with an altered
MCT gene. In another embodiment, an endogenous or introduced MCT
gene in a microorganism has been altered by one or more point
mutations, deletions, or inversions, but still encodes a functional
MCT protein. In still another embodiment, one or more of the
regulatory regions (e.g., a promoter, repressor, or inducer) of an
MCT gene in a microorganism has been altered (e.g., by deletion,
truncation, inversion, or point mutation) such that the expression
of the MCT gene is modulated. In a preferred embodiment, the
microorganism belongs to the genus Corynebacterium or
Brevibacterium, with Corynebacterium glutamicum being particularly
preferred. In a preferred embodiment, the microorganism is also
utilized for the production of a desired compound, such as an amino
acid, with lysine being particularly preferred.
[0017] In another aspect, the invention provides a method of
identifying the presence or activity of Cornyebacterium diphtheriae
in a subject. This method includes detection of one or more of the
nucleic acid or amino acid sequences of the invention (e.g., the
sequences set forth in Appendix A or Appendix B) in a subject,
thereby detecting the presence or activity of Corynebacterium
diphtheriae in the subject.
[0018] Still another aspect of the invention pertains to an
isolated MCT protein or a portion, e.g., a biologically active
portion, thereof. In a preferred embodiment, the isolated MCT
protein or portion thereof can participate in the metabolism of
compounds necessary for the construction of cellular membranes in
C. glutamicum, or in the transport of molecules across these
membranes. In another preferred embodiment, the isolated MCT
protein or portion thereof is sufficiently homologous to an amino
acid sequence of Appendix B such that the protein or portion
thereof maintains the ability to participate in the metabolism of
compounds necessary for the construction of cellular membranes in
C. glutamicum, or in the transport of molecules across these
membranes.
[0019] The invention also provides an isolated preparation of an
MCT protein. In preferred embodiments, the MCT protein comprises an
amino acid sequence of Appendix B. In another preferred embodiment,
the invention pertains to an isolated full length protein which is
substantially homologous to an entire amino acid sequence of
Appendix B (encoded by an open reading frame set forth in Appendix
A). In yet another embodiment, the protein is at least about 50%,
preferably at least about 60%, and more preferably at least about
70%, 80%, or 90%, and most preferably at least about 95%, 96%, 97%,
98%, or 99% or more homologous to an entire amino acid sequence of
Appendix B. In other embodiments, the isolated MCT protein
comprises an amino acid sequence which is at least about 50% or
more homologous to one of the amino acid sequences of Appendix B
and is able to participate in the metabolism of compounds necessary
for the construction of cellular membranes in C. glutamicum, or in
the transport of molecules across these membranes, or has one or
more of the activities set forth in Table 1.
[0020] Alternatively, the isolated MCT protein can comprise an
amino acid sequence which is encoded by a nucleotide sequence which
hybridizes, e.g., hybridizes under stringent conditions, or is at
least about 50%, preferably at least about 60%, more preferably at
least about 70%, 80%, or 90%, and even more preferably at least
about 95%, 96%, 97%, 98,%, or 99% or more homologous, to a
nucleotide sequence of Appendix B. It is also preferred that the
preferred forms of MCT proteins also have one or more of the MCT
bioactivities described herein.
[0021] The MCT polypeptide, or a biologically active portion
thereof, can be operatively linked to a non-MCT polypeptide to form
a fusion protein. In preferred embodiments, this fusion protein has
an activity which differs from that of the MCT protein alone. In
other preferred embodiments, this fusion protein participate in the
metabolism of compounds necessary for the construction of cellular
membranes in C. glutamicum, or in the transport of molecules across
these membranes. In particularly preferred embodiments, integration
of this fusion protein into a host cell modulates production of a
desired compound from the cell.
[0022] In another aspect, the invention provides methods for
screening molecules which modulate the activity of an MCT protein,
either by interacting with the protein itself or a substrate or
binding partner of the MCT protein, or by modulating the
transcription or translation of an MCT nucleic acid molecule of the
invention.
[0023] Another aspect of the invention pertains to a method for
producing a fine chemical. This method involves the culturing of a
cell containing a vector directing the expression of an MCT nucleic
acid molecule of the invention, such that a fine chemical is
produced. In a preferred embodiment, this method further includes
the step of obtaining a cell containing such a vector, in which a
cell is transfected with a vector directing the expression of an
MCT nucleic acid. In another preferred embodiment, this method
further includes the step of recovering the fine chemical from the
culture. In a particularly preferred embodiment, the cell is from
the genus Corynebacterium or Brevibacterium, or is selected from
those strains set forth in Table 3.
[0024] Another aspect of the invention pertains to methods for
modulating production of a molecule from a microorganism. Such
methods include contacting the cell with an agent which modulates
MCT protein activity or MCT nucleic acid expression such that a
cell associated activity is altered relative to this same activity
in the absence of the agent. In a preferred embodiment, the cell is
modulated for one or more C. glutamicum metabolic pathways for cell
membrane components or is modulated for the transport of compounds
across such membranes, such that the yields or rate of production
of a desired fine chemical by this microorganism is improved. The
agent which modulates MCT protein activity can be an agent which
stimulates MCT protein activity or MCT nucleic acid expression.
Examples of agents which stimulate MCT protein activity or MCT
nucleic acid expression include small molecules, active MCT
proteins, and nucleic acids encoding MCT proteins that have been
introduced into the cell. Examples of agents which inhibit MCT
activity or expression include small molecules and antisense MCT
nucleic acid molecules.
[0025] Another aspect of the invention pertains to methods for
modulating yields of a desired compound from a cell, involving the
introduction of a wild-type or mutant MCT gene into a cell, either
maintained on a separate plasmid or integrated into the genome of
the host cell. If integrated into the genome, such integration can
be random, or it can take place by homologous recombination such
that the native gene is replaced by the introduced copy, causing
the production of the desired compound from the cell to be
modulated. In a preferred embodiment, said yields are increased. In
another preferred embodiment, said chemical is a fine chemical. In
a particularly preferred embodiment, said fine chemical is an amino
acid. In especially preferred embodiments, said amino acid is
L-lysine.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention provides MCT nucleic acid and protein
molecules which are involved in the metabolism of cellular membrane
components in C. glutamicum or in the transport of compounds across
such membranes. The molecules of the invention may be utilized in
the modulation of production of fine chemicals from microorganisms,
such as C. glutamicum, either directly (e.g., where overexpression
or optimization of a fatty acid biosynthesis protein has a direct
impact on the yield, production, and/or efficiency of production of
the fatty acid from modified C. glutamicum), or may have an
indirect impact which nonetheless results in an increase of yield,
production, and/or efficiency of production of the desired compound
(e.g., where modulation of the metabolism of cell membrane
components results in alterations in the yield, production, and/or
efficiency of production or the composition of the cell membrane,
which in turn may impact the production of one or more fine
chemicals). Aspects of the invention are further explicated
below.
I. Fine Chemicals
[0027] The term `fine chemical` is art-recognized and includes
molecules produced by an organism which have applications in
various industries, such as, but not limited to, the
pharmaceutical, agriculture, and cosmetics industries. Such
compounds include organic acids, such as tartaric acid, itaconic
acid, and diaminopimelic acid, both proteinogenic and
non-proteinogenic amino acids, purine and pyrimidine bases,
nucleosides, and nucleotides (as described e.g. in Kuninaka, A.
(1996) Nucleotides and related compounds, p. 561-612, in
Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, and
references contained therein), lipids, both saturated and
unsaturated fatty acids (e.g., arachidonic acid), diols (e.g.,
propane diol, and butane diol), carbohydrates (e.g, hyaluronic acid
and trehalose), aromatic compounds (e.g, aromatic amines, vanillin,
and indigo), vitamins and cofactors (as described in Ullmann's
Encyclopedia of Industrial Chemistry, vol. A27, "Vitamins", p.
443-613 (1996) VCH: Weinheim and references therein; and Ong, A.S.,
Niki, E. & Packer, L. (1995) "Nutrition, Lipids, Health, and
Disease" Proceedings of the UNESCO/Confederation of Scientific and
Technological Associations in Malaysia, and the Society for Free
Radical Research--Asia, held Sept. 1-3, 1994 at Penang, Malaysia,
AOCS Press, (1995)), enzymes, polyketides (Cane et al. (1998)
Science 282: 63-68), and all other chemicals described in Gutcho
(1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN:
0818805086 and references therein. The metabolism and uses of
certain of these fine chemicals are further explicated below.
A. Amino Acid Metabolism and Uses
[0028] Amino acids comprise the basic structural units of all
proteins, and as such are essential for normal cellular functioning
in all organisms. The term "amino acid" is art-recognized. The
proteinogenic amino acids, of which there are 20 species, serve as
structural units for proteins, in which they are linked by peptide
bonds, while the nonproteinogenic amino acids (hundreds of which
are known) are not normally found in proteins (see Ulmann's
Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH:
Weinheim (1985)). Amino acids may be in the D- or L-optical
configuration, though L-amino acids are generally the only type
found in naturally-occurring proteins. Biosynthetic and degradative
pathways of each of the 20 proteinogenic amino acids have been well
characterized in both prokaryotic and eukaryotic cells (see, for
example, Stryer, L. Biochemistry, 3.sup.rd edition, pages 578-590
(1988)). The `essential` amino acids (histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, threonine, tryptophan,
and valine), so named because they are generally a nutritional
requirement due to the complexity of their biosyntheses, are
readily converted by simple biosynthetic pathways to the remaining
11 `nonessential` amino acids (alanine, arginine, asparagine,
aspartate, cysteine, glutamate, glutamine, glycine, proline,
serine, and tyrosine). Higher animals do retain the ability to
synthesize some of these amino acids, but the essential amino acids
must be supplied from the diet in order for normal protein
synthesis to occur.
[0029] Aside from their function in protein biosynthesis, these
amino acids are interesting chemicals in their own right, and many
have been found to have various applications in the food, feed,
chemical, cosmetics, agriculture, and pharmaceutical industries.
Lysine is an important amino acid in the nutrition not only of
humans, but also of monogastric animals such as poultry and swine.
Glutamate is most commonly used as a flavor additive (mono-sodium
glutamate, MSG) and is widely used throughout the food industry, as
are aspartate, phenylalanine, glycine, and cysteine. Glycine,
L-methionine and tryptophan are all utilized in the pharmaceutical
industry. Glutamine, valine, leucine, isoleucine, histidine,
arginine, proline, serine and alanine are of use in both the
pharmaceutical and cosmetics industries. Threonine, tryptophan, and
D/ L-methionine are common feed additives. (Leuchtenberger, W.
(1996) Amino aids--technical production and use, p. 466-502 in Rehm
et al. (eds.) Biotechnology vol. 6, chapter 14a, VCH: Weinheim).
Additionally, these amino acids have been found to be useful as
precursors for the synthesis of synthetic amino acids and proteins,
such as N-acetylcysteine, S-carboxymethyl-L-cysteine,
(S)-5-hydroxytryptophan, and others described in Ulmann's
Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97, VCH:
Weinheim, 1985.
[0030] The biosynthesis of these natural amino acids in organisms
capable of producing them, such as bacteria, has been well
characterized (for review of bacterial amino acid biosynthesis and
regulation thereof, see Umbarger, H. E.(1978) Ann. Rev. Biochem.
47: 533-606). Glutamate is synthesized by the reductive amination
of .alpha.-ketoglutarate, an intermediate in the citric acid cycle.
Glutamine, proline, and arginine are each subsequently produced
from glutamate. The biosynthesis of serine is a three-step process
beginning with 3-phosphoglycerate (an intermediate in glycolysis),
and resulting in this amino acid after oxidation, transamination,
and hydrolysis steps. Both cysteine and glycine are produced from
serine; the former by the condensation of homocysteine with serine,
and the latter by the transferal of the side-chain .beta.-carbon
atom to tetrahydrofolate, in a reaction catalyzed by serine
transhydroxymethylase. Phenylalanine, and tyrosine are synthesized
from the glycolytic and pentose phosphate pathway precursors
erythrose 4-phosphate and phosphoenolpyruvate in a 9-step
biosynthetic pathway that differ only at the final two steps after
synthesis of prephenate. Tryptophan is also produced from these two
initial molecules, but its synthesis is an 11-step pathway.
Tyrosine may also be synthesized from phenylalanine, in a reaction
catalyzed by phenylalanine hydroxylase. Alanine, valine, and
leucine are all biosynthetic products of pyruvate, the final
product of glycolysis. Aspartate is formed from oxaloacetate, an
intermediate of the citric acid cycle. Asparagine, methionine,
threonine, and lysine are each produced by the conversion of
aspartate. Isoleucine is formed from threonine. A complex 9-step
pathway results in the production of histidine from
5-phosphoribosyl-1-pyrophosphate, an activated sugar.
[0031] Amino acids in excess of the protein synthesis needs of the
cell cannot be stored, and are instead degraded to provide
intermediates for the major metabolic pathways of the cell (for
review see Stryer, L. Biochemistry .sub.3.sup.rd ed. Ch. 21 "Amino
Acid Degradation and the Urea Cycle" p. 495-516 (1988)). Although
the cell is able to convert unwanted amino acids into useful
metabolic intermediates, amino acid production is costly in terms
of energy, precursor molecules, and the enzymes necessary to
synthesize them. Thus it is not surprising that amino acid
biosynthesis is regulated by feedback inhibition, in which the
presence of a particular amino acid serves to slow or entirely stop
its own production (for overview of feedback mechanisms in amino
acid biosynthetic pathways, see Stryer, L. Biochemistry, 3.sup.rd
ed. Ch. 24: "Biosynthesis of Amino Acids and Heme" p. 575-600
(1988)). Thus, the output of any particular amino acid is limited
by the amount of that amino acid present in the cell.
B. Vitamin, Cofactor, and Nutraceutical Metabolism and Uses
[0032] Vitamins, cofactors, and nutraceuticals comprise another
group of molecules which the higher animals have lost the ability
to synthesize and so must ingest, although they are readily
synthesized by other organisms such as bacteria. These molecules
are either bioactive substances themselves, or are precursors of
biologically active substances which may serve as electron carriers
or intermediates in a variety of metabolic pathways. Aside from
their nutritive value, these compounds also have significant
industrial value as coloring agents, antioxidants, and catalysts or
other processing aids. (For an overview of the structure, activity,
and industrial applications of these compounds, see, for example,
Ullman's Encyclopedia of Industrial Chemistry, "Vitamins" vol. A27,
p. 443-613, VCH: Weinheim, 1996.) The term "vitamin" is
art-recognized, and includes nutrients which are required by an
organism for normal functioning, but which that organism cannot
synthesize by itself. The group of vitamins may encompass cofactors
and nutraceutical compounds. The language "cofactor" includes
nonproteinaceous compounds required for a normal enzymatic activity
to occur. Such compounds may be organic or inorganic; the cofactor
molecules of the invention are preferably organic. The term
"nutraceutical" includes dietary supplements having health benefits
in plants and animals, particularly humans. Examples of such
molecules are vitamins, antioxidants, and also certain lipids
(e.g., polyunsaturated fatty acids).
[0033] The biosynthesis of these molecules in organisms capable of
producing them, such as bacteria, has been largely characterized
(Ullman's Encyclopedia of Industrial Chemistry, "Vitamins" vol.
A27, p. 443-613, VCH: Weinheim, 1996; Michal, G. (1999) Biochemical
Pathways: An Atlas of Biochemistry and Molecular Biology, John
Wiley & Sons; Ong, A.S., Niki, E. & Packer, L. (1995)
"Nutrition, Lipids, Health, and Disease" Proceedings of the
UNESCO/Confederation of Scientific and Technological Associations
in Malaysia, and the Society for Free Radical Research--Asia, held
Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press: Champaign, IL X,
374 S).
[0034] Thiamin (vitamin B.sub.1) is produced by the chemical
coupling of pyrimidine and thiazole moieties. Riboflavin (vitamin
B.sub.2) is synthesized from guanosine-5'-triphosphate (GTP) and
ribose-5'-phosphate. Riboflavin, in turn, is utilized for the
synthesis of flavin mononucleotide (FMN) and flavin adenine
dinucleotide (FAD). The family of compounds collectively termed
`vitamin B.sub.6` (e.g., pyridoxine, pyridoxamine,
pyridoxa-5'-phosphate, and the commercially used pyridoxin
hydrochloride) are all derivatives of the common structural unit,
5-hydroxy-6-methylpyridine. Pantothenate (pantothenic acid,
(R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-.beta.-alanine)
can be produced either by chemical synthesis or by fermentation.
The final steps in pantothenate biosynthesis consist of the
ATP-driven condensation of .beta.-alanine and pantoic acid. The
enzymes responsible for the biosynthesis steps for the conversion
to pantoic acid, to .beta.-alanine and for the condensation to
panthotenic acid are known. The metabolically active form of
pantothenate is Coenzyme A, for which the biosynthesis proceeds in
5 enzymatic steps. Pantothenate, pyridoxal-5'-phosphate, cysteine
and ATP are the precursors of Coenzyme A. These enzymes not only
catalyze the formation of panthothante, but also the production of
(R)-pantoic acid, (R)-pantolacton, (R)-panthenol (provitamin
B.sub.5), pantetheine (and its derivatives) and coenzyme A.
[0035] Biotin biosynthesis from the precursor molecule pimeloyl-CoA
in microorganisms has been studied in detail and several of the
genes involved have been identified. Many of the corresponding
proteins have been found to also be involved in Fe-cluster
synthesis and are members of the nifS class of proteins. Lipoic
acid is derived from octanoic acid, and serves as a coenzyme in
energy metabolism, where it becomes part of the pyruvate
dehydrogenase complex and the .alpha.-ketoglutarate dehydrogenase
complex. The folates are a group of substances which are all
derivatives of folic acid, which is turn is derived from L-glutamic
acid, p-amino-benzoic acid and 6-methylpterin. The biosynthesis of
folic acid and its derivatives, starting from the metabolism
intermediates guanosine-5'-triphosphate (GTP), L-glutamic acid and
p-amino-benzoic acid has been studied in detail in certain
microorganisms.
[0036] Corrinoids (such as the cobalamines and particularly vitamin
B.sub.12) and porphyrines belong to a group of chemicals
characterized by a tetrapyrole ring system. The biosynthesis of
vitamin B.sub.12 is sufficiently complex that it has not yet been
completely characterized, but many of the enzymes and substrates
involved are now known. Nicotinic acid (nicotinate), and
nicotinamide are pyridine derivatives which are also termed
`niacin`. Niacin is the precursor of the important coenzymes NAD
(nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine
dinucleotide phosphate) and their reduced forms.
[0037] The large-scale production of these compounds has largely
relied on cell-free chemical syntheses, though some of these
chemicals have also been produced by large-scale culture of
microorganisms, such as riboflavin, Vitamin B.sub.6, pantothenate,
and biotin. Only Vitamin B.sub.12 is produced solely by
fermentation, due to the complexity of its synthesis. In vitro
methodologies require significant inputs of materials and time,
often at great cost.
C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and
Uses
[0038] Purine and pyrimidine metabolism genes and their
corresponding proteins are important targets for the therapy of
tumor diseases and viral infections. The language "purine" or
"pyrimidine" includes the nitrogenous bases which are constituents
of nucleic acids, co-enzymes, and nucleotides. The term
"nucleotide" includes the basic structural units of nucleic acid
molecules, which are comprised of a nitrogenous base, a pentose
sugar (in the case of RNA, the sugar is ribose; in the case of DNA,
the sugar is D-deoxyribose), and phosphoric acid. The language
"nucleoside" includes molecules which serve as precursors to
nucleotides, but which are lacking the phosphoric acid moiety that
nucleotides possess. By inhibiting the biosynthesis of these
molecules, or their mobilization to form nucleic acid molecules, it
is possible to inhibit RNA and DNA synthesis; by inhibiting this
activity in a fashion targeted to cancerous cells, the ability of
tumor cells to divide and replicate may be inhibited. Additionally,
there are nucleotides which do not form nucleic acid molecules, but
rather serve as energy stores (i.e., AMP) or as coenzymes (i.e.,
FAD and NAD).
[0039] Several publications have described the use of these
chemicals for these medical indications, by influencing purine
and/or pyrimidine metabolism (e.g Christopherson, R. I. and Lyons,
S. D. (1990) "Potent inhibitors of de novo pyrimidine and purine
biosynthesis as chemotherapeutic agents." Med. Res. Reviews 10:
505-548). Studies of enzymes involved in purine and pyrimidine
metabolism have been focused on the development of new drugs which
can be used, for example, as immunosuppressants or
anti-proliferants (Smith, J. L., (1995) "Enzymes in nucleotide
synthesis." Curr. Opin. Struct. Biol. 5: 752-757; (1995) Biochem
Soc. Transact. 23: 877-902). However, purine and pyrimidine bases,
nucleosides and nucleotides have other utilities: as intermediates
in the biosynthesis of several fine chemicals (e.g., thiamine,
S-adenosyl-methionine, folates, or riboflavin), as energy carriers
for the cell (e.g., ATP or GTP), and for chemicals themselves,
commonly used as flavor enhancers (e.g., IMP or GMP) or for several
medicinal applications (see, for example, Kuninaka, A. (1996)
Nucleotides and Related Compounds in Biotechnology vol. 6, Rehm et
al., eds. VCH: Weinheim, p. 561-612). Also, enzymes involved in
purine, pyrimidine, nucleoside, or nucleotide metabolism are
increasingly serving as targets against which chemicals for crop
protection, including fungicides, herbicides and insecticides, are
developed.
[0040] The metabolism of these compounds in bacteria has been
characterized (for reviews see, for example, Zalkin, H. and Dixon,
J. E. (1992) "de novo purine nucleotide biosynthesis", in: Progress
in Nucleic Acid Research and Molecular Biology, vol. 42, Academic
Press:, p. 259-287; and Michal, G. (I 999) "Nucleotides and
Nucleosides", Chapter 8 in: Biochemical Pathways: An Atlas of
Biochemistry and Molecular Biology, Wiley: New York). Purine
metabolism has been the subject of intensive research, and is
essential to the normal functioning of the cell. Impaired purine
metabolism in higher animals can cause severe disease, such as
gout. Purine nucleotides are synthesized from ribose-5-phosphate,
in a series of steps through the intermediate compound
inosine-5'-phosphate (IMP), resulting in the production of
guanosine-5'-monophosphate (GMP) or adenosine-5'-monophosphate
(AMP), from which the triphosphate forms utilized as nucleotides
are readily formed. These compounds are also utilized as energy
stores, so their degradation provides energy for many different
biochemical processes in the cell. Pyrimidine biosynthesis proceeds
by the formation of uridine-5'-monophosphate (UMP) from
ribose-5-phosphate. UMP, in turn, is converted to
cytidine-5'-triphosphate (CTP). The deoxy- forms of all of these
nucleotides are produced in a one step reduction reaction from the
diphosphate ribose form of the nucleotide to the diphosphate
deoxyribose form of the nucleotide. Upon phosphorylation, these
molecules are able to participate in DNA synthesis.
D. Trehalose Metabolism and Uses
[0041] Trehalose consists of two glucose molecules, bound in
.alpha., .alpha.-1,1 linkage. It is commonly used in the food
industry as a sweetener, an additive for dried or frozen foods, and
in beverages. However, it also has applications in the
pharmaceutical, cosmetics and biotechnology industries (see, for
example, Nishimoto et al., (1998) U.S. Pat. No. 5,759,610; Singer,
M. A. and Lindquist, S. (1998) Trends Biotech. 16: 460-467; Paiva,
C. L. A. and Panek, A. D. (1996) Biotech. Ann. Rev. 2: 293-314; and
Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose is produced by
enzymes from many microorganisms and is naturally released into the
surrounding medium, from which it can be collected using methods
known in the art.
II. Membrane Biosynthesis and Transmembrane Transport
[0042] Cellular membranes serve a variety of functions in a cell.
First and foremost, a membrane differentiates the contents of a
cell from the surrounding environment, thus giving integrity to the
cell. Membranes may also serve as barriers to the influx of
hazardous or unwanted compounds, and also to the efflux of desired
compounds. Cellular membranes are by nature impervious to the
unfacilitated diffusion of hydrophilic compounds such as proteins,
water molecules and ions due to their structure: a bilayer of lipid
molecules in which the polar head groups face outwards (towards the
exterior and interior of the cell, respectively) and the nonpolar
tails face inwards at the center of the bilayer, forming a
hydrophobic core (for a general review of membrane structure and
function, see Gennis, R. B. (1989) Biomembranes, Molecular
Structure and Function, Springer: Heidelberg). This barrier enables
cells to maintain a relatively higher concentration of desired
compounds and a relatively lower concentration of undesired
compounds than are contained within the surrounding medium, since
the diffusion of these compounds is effectively blocked by the
membrane. However, the membrane also presents an effective barrier
to the import of desired compounds and the export of waste
molecules. To overcome this difficulty, cellular membranes
incorporate many kinds of transporter proteins which are able to
facilitate the transmembrane transport of different kinds of
compounds. There are two general classes of these transport
proteins: pores or channels and transporters. The former are
integral membrane proteins, sometimes complexes of proteins, which
form a regulated hole through the membrane. This regulation, or
`gating` is generally specific to the molecules to be transported
by the pore or channel, rendering these transmembrane constructs
selectively permeable to a specific class of substrates; for
example, a potassium channel is constructed such that only ions
having a like charge and size to that of potassium may pass
through. Channel and pore proteins tend to have discrete
hydrophobic and hydrophilic domains, such that the hydrophobic face
of the protein may associate with the interior of the membrane
while the hydrophilic face lines the interior of the channel, thus
providing a sheltered hydrophilic environment through which the
selected hydrophilic molecule may pass. Many such pores/channels
are known in the art, including those for potassium, calcium,
sodium, and chloride ions.
[0043] This pore and channel-mediated system of facilitated
diffusion is limited to very small molecules, such as ions, because
pores or channels large enough to permit the passage of whole
proteins by facilitated diffusion would be unable to prevent the
passage of smaller hydrophilic molecules as well. Transport of
molecules by this process is sometimes termed `facilitated
diffusion` since the driving force of a concentration gradient is
required for the transport to occur. Permeases also permit
facilitated diffusion of larger molecules, such as glucose or other
sugars, into the cell when the concentration of these molecules on
one side of the membrane is greater than that on the other (also
called `uniport`). In contrast to pores or channels, these integral
membrane proteins (often having between 6-14 membrane-spanning
.beta.-helices) do not form open channels through the membrane, but
rather bind to the target molecule at the surface of the membrane
and then undergo a conformational shift such that the target
molecule is released on the opposite side of the membrane.
[0044] However, cells frequently require the import or export of
molecules against the existing concentration gradient (`active
transport`), a situation in which facilitated diffusion cannot
occur. There are two general mechanisms used by cells for such
membrane transport: symport or antiport, and energy-coupled
transport such as that mediated by the ABC transporters. Symport
and antiport systems couple the movement of two different molecules
across the membrane (via permeases having two separate binding
sites for the two different molecules); in symport, both molecules
are transported in the same direction, while in antiport, one
molecule is imported while the other is exported. This is possible
energetically because one of the two molecules moves in accordance
with a concentration gradient, and this energetically favorable
event is permitted only upon concomitant movement of a desired
compound against the prevailing concentration gradient. Single
molecules may be transported across the membrane against the
concentration gradient in an energy-driven process, such as that
utilized by the ABC transporters. In this system, the transport
protein located in the membrane has an ATP-binding cassette; upon
binding of the target molecule, the ATP is converted to ADP+Pi, and
the resulting release of energy is used to drive the movement of
the target molecule to the opposite face of the membrane,
facilitated by the transporter. For more detailed descriptions of
all of these transport systems, see: Bamberg, E. et al., (1993)
"Charge transport of ion pumps on lipid bilayer membranes", Q. Rev.
Biophys. 26: 1-25; Findlay, J. B. C. (1991) "Structure and function
in membrane transport systems", Curr. Opin. Struct. Biol.
1:804-810; Higgins, C. F. (1992) "ABC transporters from
microorganisms to man", Ann. Rev. Cell Biol. 8: 67-113; Gennis, R.
B. (1989) "Pores, Channels and Transporters", in: Biomembranes,
Molecular Structure and Function, Springer: Heidelberg, p. 270-322;
and Nikaido, H. and Saier, H. (1992) "Transport proteins in
bacteria: common themes in their design", Science 258: 936-942, and
references contained within each of these references.
[0045] The synthesis of membranes is a well-characterized process
involving a number of components, the most important of which are
lipid molecules. Lipid synthesis may be divided into two parts: the
synthesis of fatty acids and their attachment to
sn-glycerol-3-phosphate, and the addition or modification of a
polar head group. Typical lipids utilized in bacterial membranes
include phospholipids, glycolipids, sphingolipids, and
phosphoglycerides. Fatty acid synthesis begins with the conversion
of acetyl CoA either to malonyl CoA by acetyl CoA carboxylase, or
to acetyl-ACP by acetyltransacylase. Following a condensation
reaction, these two product molecules together form
acetoacetyl-ACP, which is converted by a series of condensation,
reduction and dehydration reactions to yield a saturated fatty acid
molecule having a desired chain length. The production of
unsaturated fatty acids from such molecules is catalyzed by
specific desaturases either aerobically, with the help of molecular
oxygen, or anaerobically (for reference on fatty acid synthesis,
see F. C. Neidhardt et al. (1996) E. coli and Salmonella. ASM
Press: Washington, D.C., p. 612-636 and references contained
therein; Lengeler et al. (eds) (1999) Biology of Procaryotes.
Thieme: Stuttgart, New York, and references contained therein; and
Magnuson, K. et al., (1993) Microbiological Reviews 57: 522-542,
and references contained therein). The cyclopropane fatty acids
(CFA) are synthesized by a specific CFA-synthase using SAM as a
cosubstrate. Branched chain fatty acids are synthesized from
branched chain amino acids that are deaminated to yield branched
chain 2-oxo-acids (see Lengeler et al., eds. (1999) Biology of
Procaryotes. Thieme: Stuttgart, New York, and references contained
therein). Another essential step in lipid synthesis is the transfer
of fatty acids onto the polar head groups by, for example,
glycerol-phosphate-acyltransferases. The combination of various
precursor molecules and biosynthetic enzymes results in the
production of different fatty acid molecules, which has a profound
effect on the composition of the membrane.
III. Elements and Methods of the Invention
[0046] The present invention is based, at least in part, on the
discovery of novel molecules, referred to herein as MCT nucleic
acid and protein molecules, which control the production of
cellular membranes in C. glutamicum and govern the movement of
molecules across such membranes. In one embodiment, the MCT
molecules participate in the metabolism of compounds necessary for
the construction of cellular membranes in C. glutamicum, or in the
transport of molecules across these membranes. In a preferred
embodiment, the activity of the MCT molecules of the present
invention to regulate membrane component production and membrane
transport has an impact on the production of a desired fine
chemical by this organism. In a particularly preferred embodiment,
the MCT molecules of the invention are modulated in activity, such
that the C. glutamicum metabolic pathways which the MCT proteins of
the invention regulate are modulated in yield, production, and/or
efficiency of production and the transport of compounds through the
membranes is altered in efficiency, which either directly or
indirectly modulates the yield, production, and/or efficiency of
production of a desired fine chemical by C. glutamicum.
[0047] The language, "MCT protein" or "MCT polypeptide" includes
proteins which participate in the metabolism of compounds necessary
for the construction of cellular membranes in C. glutamicum, or in
the transport of molecules across these membranes. Examples of MCT
proteins include those encoded by the MCT genes set forth in Table
1 and Appendix A. The terms "MCT gene" or "MCT nucleic acid
sequence" include nucleic acid sequences encoding an MCT protein,
which consist of a coding region and also corresponding
untranslated 5' and 3' sequence regions. Examples of MCT genes
include those set forth in Table 1. The terms "production" or
"productivity" are art-recognized and include the concentration of
the fermentation product (for example, the desired fine chemical)
formed within a given time and a given fermentation volume (e.g.,
kg product per hour per liter). The term "efficiency of production"
includes the time required for a particular level of production to
be achieved (for example, how long it takes for the cell to attain
a particular rate of output of a fine chemical). The term "yield"
or "product/carbon yield" is art-recognized and includes the
efficiency of the conversion of the carbon source into the product
(i.e., fine chemical). This is generally written as, for example,
kg product per kg carbon source. By increasing the yield or
production of the compound, the quantity of recovered molecules, or
of useful recovered molecules of that compound in a given amount of
culture over a given amount of time is increased. The terms
"biosynthesis" or a "biosynthetic pathway" are art-recognized and
include the synthesis of a compound, preferably an organic
compound, by a cell from intermediate compounds in what may be a
multistep and highly regulated process. The terms "degradation" or
a "degradation pathway" are art-recognized and include the
breakdown of a compound, preferably an organic compound, by a cell
to degradation products (generally speaking, smaller or less
complex molecules) in what may be a multistep and highly regulated
process. The language "metabolism" is art-recognized and includes
the totality of the biochemical reactions that take place in an
organism. The metabolism of a particular compound, then, (e.g., the
metabolism of an amino acid such as glycine) comprises the overall
biosynthetic, modification, and degradation pathways in the cell
related to this compound.
[0048] In another embodiment, the MCT molecules of the invention
are capable of modulating the production of a desired molecule,
such as a fine chemical, in a microorganism such as C. glutamicum.
There are a number of mechanisms by which the alteration of an MCT
protein of the invention may directly affect the yield, production,
and/or efficiency of production of a fine chemical from a C.
glutamicum strain incorporating such an altered protein. Those MCT
proteins involved in the export of fine chemical molecules from the
cell may be increased in number or activity such that greater
quantities of these compounds are secreted to the extracellular
medium, from which they are more readily recovered. Similarly,
those MCT proteins involved in the import of nutrients necessary
for the biosynthesis of one or more fine chemicals (e.g.,
phosphate, sulfate, nitrogen compounds, etc.) may be increased in
number or activity such that these precursor, cofactor, or
intermediate compounds are increased in concentration within the
cell. Further, fatty acids and lipids themselves are desirable fine
chemicals; by optimizing the activity or increasing the number of
one or more MCT proteins of the invention which participate in the
biosynthesis of these compounds, or by impairing the activity of
one or more MCT proteins which are involved in the degradation of
these compounds, it may be possible to increase the yield,
production, and/or efficiency of production of fatty acid and lipid
molecules from C. glutamicum.
[0049] The mutagenesis of one or more MCT genes of the invention
may also result in MCT proteins having altered activities which
indirectly impact the production of one or more desired fine
chemicals from C. glutamicum. For example, MCT proteins of the
invention involved in the export of waste products may be increased
in number or activity such that the normal metabolic wastes of the
cell (possibly increased in quantity due to the overproduction of
the desired fine chemical) are efficiently exported before they are
able to damage nucleotides and proteins within the cell (which
would decrease the viability of the cell) or to interfere with fine
chemical biosynthetic pathways (which would decrease the yield,
production, or efficiency of production of the desired fine
chemical). Further, the relatively large intracellular quantities
of the desired fine chemical may in itself be toxic to the cell, so
by increasing the activity or number of transporters able to export
this compound from the cell, one may increase the viability of the
cell in culture, in turn leading to a greater number of cells in
the culture producing the desired fine chemical. The MCT proteins
of the invention may also be manipulated such that the relative
amounts of different lipid and fatty acid molecules are produced.
This may have a profound effect on the lipid composition of the
membrane of the cell. Since each type of lipid has different
physical properties, an alteration in the lipid composition of a
membrane may significantly alter membrane fluidity. Changes in
membrane fluidity can impact the transport of molecules across the
membrane, as well as the integrity of the cell, both of which have
a profound effect on the production of fine chemicals from C.
glutamicum in large-scale fermentative culture.
[0050] The isolated nucleic acid sequences of the invention are
contained within the genome of a Corynebacterium glutamicum strain
available through the American Type Culture Collection, given
designation ATCC 13032. The nucleotide sequence of the isolated C.
glutamicum MCT DNAs and the predicted amino acid sequences of the
C. glutamicum MCT proteins are shown in Appendices A and B,
respectively. Computational analyses were performed which
classified and/or identified these nucleotide sequences as
sequences which encode proteins involved in the metabolism of
cellular membrane components or proteins involved in the transport
of compounds across such membranes.
[0051] The present invention also pertains to proteins which have
an amino acid sequence which is substantially homologous to an
amino acid sequence of Appendix B. As used herein, a protein which
has an amino acid sequence which is substantially homologous to a
selected amino acid sequence is least about 50% homologous to the
selected amino acid sequence, e.g., the entire selected amino acid
sequence. A protein which has an amino acid sequence which is
substantially homologous to a selected amino acid sequence can also
be least about 50-60%, preferably at least about 60-70%, and more
preferably at least about 70-80%, 80-90%, or 90-95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
the selected amino acid sequence.
[0052] The MCT protein or a biologically active portion or fragment
thereof of the invention can participate in the metabolism of
compounds necessary for the construction of cellular membranes in
C. glutamicum, or in the transport of molecules across these
membranes, or have one or more of the activities set forth in Table
1.
[0053] Various aspects of the invention are described in further
detail in the following subsections:
A. Isolated Nucleic Acid Molecules
[0054] One aspect of the invention pertains to isolated nucleic
acid molecules that encode MCT polypeptides or biologically active
portions thereof, as well as nucleic acid fragments sufficient for
use as hybridization probes or primers for the identification or
amplification of MCT-encoding nucleic acid (e.g., MCT DNA). As used
herein, the term "nucleic acid molecule" is intended to include DNA
molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g.,
mRNA) and analogs of the DNA or RNA generated using nucleotide
analogs. This term also encompasses untranslated sequence located
at both the 3' and 5' ends of the coding region of the gene: at
least about 100 nucleotides of sequence upstream from the 5' end of
the coding region and at least about 20 nucleotides of sequence
downstream from the 3' end of the coding region of the gene. The
nucleic acid molecule can be single-stranded or double-stranded,
but preferably is double-stranded DNA. An "isolated" nucleic acid
molecule is one which is separated from other nucleic acid
molecules which are present in the natural source of the nucleic
acid. Preferably, an "isolated" nucleic acid is free of sequences
which naturally flank the nucleic acid (i.e., sequences located at
the 5' and 3' ends of the nucleic acid) in the genomic DNA of the
organism from which the nucleic acid is derived. For example, in
various embodiments, the isolated MCT nucleic acid molecule can
contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1
kb of nucleotide sequences which naturally flank the nucleic acid
molecule in genomic DNA of the cell from which the nucleic acid is
derived (e.g, a C. glutamicum cell). Moreover, an "isolated"
nucleic acid molecule, such as a DNA molecule, can be substantially
free of other cellular material, or culture medium when produced by
recombinant techniques, or chemical precursors or other chemicals
when chemically synthesized.
[0055] A nucleic acid molecule of the present invention, e.g., a
nucleic acid molecule having a nucleotide sequence of Appendix A,
or a portion thereof, can be isolated using standard molecular
biology techniques and the sequence information provided herein.
For example, a C. glutamicum MCT DNA can be isolated from a C.
glutamicum library using all or portion of one of the sequences of
Appendix A as a hybridization probe and standard hybridization
techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and
Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989). Moreover, a nucleic acid molecule
encompassing all or a portion of one of the sequences of Appendix A
can be isolated by the polymerase chain reaction using
oligonucleotide primers designed based upon this sequence (e.g., a
nucleic acid molecule encompassing all or a portion of one of the
sequences of Appendix A can be isolated by the polymerase chain
reaction using oligonucleotide primers designed based upon this
same sequence of Appendix A). For example, mRNA can be isolated
from normal endothelial cells (e.g., by the guanidinium-thiocyanate
extraction procedure of Chirgwin et al. (1979) Biochemistry 18:
5294-5299) and DNA can be prepared using reverse transcriptase
(e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL,
Bethesda, Md.; or AMV reverse transcriptase, available from
Seikagaku America, Inc., St. Petersburg, Fla.). Synthetic
oligonucleotide primers for polymerase chain reaction amplification
can be designed based upon one of the nucleotide sequences shown in
Appendix A. A nucleic acid of the invention can be amplified using
cDNA or, alternatively, genomic DNA, as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to an MCT nucleotide
sequence can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[0056] In a preferred embodiment, an isolated nucleic acid molecule
of the invention comprises one of the nucleotide sequences shown in
Appendix A. The sequences of Appendix A correspond to the
Corynebacterium glutamicum MCT DNAs of the invention. This DNA
comprises sequences encoding MCT proteins (i.e., the "coding
region", indicated in each sequence in Appendix A), as well as 5'
untranslated sequences and 3' untranslated sequences, also
indicated in Appendix A. Alternatively, the nucleic acid molecule
can comprise only the coding region of any of the sequences in
Appendix A.
[0057] For the purposes of this application, it will be understood
that each of the sequences set forth in Appendix A has an
identifying RXA, RXN, RXS, or RXC number having the designation
"RXA", "RXN", "RXS" or "RXC" followed by 5 digits (i.e., RXA02099,
RXN03097, RXS00148, or RXC01748). Each of these sequences comprises
up to three parts: a 5' upstream region, a coding region, and a
downstream region. Each of these three regions is identified by the
same RXA, RXN, RXS, or RXC designation to eliminate confusion. The
recitation "one of the sequences in Appendix A", then, refers to
any of the sequences in Appendix A, which may be distinguished by
their differing RXA, RXN, RXS, or RXC designations. The coding
region of each of these sequences is translated into a
corresponding amino acid sequence, which is set forth in Appendix
B. The sequences of Appendix B are identified by the same RXA, RXN,
RXS, or RXC designations as Appendix A, such that they can be
readily correlated. For example, the amino acid sequences in
Appendix B designated RXA02099, RXN03097, RXS00148, and RXC01748
are translations of the coding region of the nucleotide sequences
of nucleic acid molecules RXA02099, RXN03097, RXS00148, and
RXC01748, respectively, in Appendix A. Each of the RXA, RXN, RXS,
and RXC nucleotide and amino acid sequences of the invention has
also been assigned a SEQ ID NO, as indicated in Table 1. For
example, as set forth in Table 1, the nucleotide sequence of
RXA00104 is SEQ ID NO:5, and the amino acid sequence of RXA00104 is
SEQ ID NO:6.
[0058] Several of the genes of the invention are "F-designated
genes". An F-designated gene includes those genes set forth in
Table 1 which have an `F` in front of the RXA, RXN, RXS, or RXC
designation. For example, SEQ ID NO:11, designated, as indicated on
Table 1, as "F RXA02581", is an F-designated gene, as are SEQ ID
NOs: 31, 33, and 43 (designated on Table 1 as "F RXA02487", "F
RXA02490", and "F RXA02809", respectively).
[0059] In one embodiment, the nucleic acid molecules of the present
invention are not intended to include those compiled in Table 2. In
the case of the dapD gene, a sequence for this gene was published
in Wehrmann, A., et al. (1998) J. Bacteriol. 180(12): 3159-3165.
However, the sequence obtained by the inventors of the present
application is significantly longer than the published version. It
is believed that the published version relied on an incorrect start
codon, and thus represents only a fragment of the actual coding
region.
[0060] In another preferred embodiment, an isolated nucleic acid
molecule of the invention comprises a nucleic acid molecule which
is a complement of one of the nucleotide sequences shown in
Appendix A, or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences shown in Appendix
A is one which is sufficiently complementary to one of the
nucleotide sequences shown in Appendix A such that it can hybridize
to one of the nucleotide sequences shown in Appendix A, thereby
forming a stable duplex.
[0061] In still another preferred embodiment, an isolated nucleic
acid molecule of the invention comprises a nucleotide sequence
which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, or 70%%, more preferably at least about
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and
even more preferably at least about 95%, 96%, 97%, 98%, 99% or more
homologous to a nucleotide sequence shown in Appendix A, or a
portion thereof. Ranges and identity values intermediate to the
above-recited ranges, (e.g., 70-90% identical or 80-95% identical)
are also intended to be encompassed by the present invention. For
example, ranges of identity values using a combination of any of
the above values recited as upper and/or lower limits are intended
to be included. In an additional preferred embodiment, an isolated
nucleic acid molecule of the invention comprises a nucleotide
sequence which hybridizes, e.g., hybridizes under stringent
conditions, to one of the nucleotide sequences shown in Appendix A,
or a portion thereof.
[0062] Moreover, the nucleic acid molecule of the invention can
comprise only a portion of the coding region of one of the
sequences in Appendix A, for example a fragment which can be used
as a probe or primer or a fragment encoding a biologically active
portion of an MCT protein. The nucleotide sequences determined from
the cloning of the MCT genes from C. glutamicum allows for the
generation of probes and primers designed for use in identifying
and/or cloning MCT homologues in other cell types and organisms, as
well as MCT homologues from other Corynebacteria or related
species. The probe/primer typically comprises substantially
purified oligonucleotide. The oligonucleotide typically comprises a
region of nucleotide sequence that hybridizes under stringent
conditions to at least about 12, preferably about 25, more
preferably about 40, 50 or 75 consecutive nucleotides of a sense
strand of one of the sequences set forth in Appendix A, an
anti-sense sequence of one of the sequences set forth in Appendix
A, or naturally occurring mutants thereof. Primers based on a
nucleotide sequence of Appendix A can be used in PCR reactions to
clone MCT homologues. Probes based on the MCT nucleotide sequences
can be used to detect transcripts or genomic sequences encoding the
same or homologous proteins. In preferred embodiments, the probe
further comprises a label group attached thereto, e.g. the label
group can be a radioisotope, a fluorescent compound, an enzyme, or
an enzyme co-factor. Such probes can be used as a part of a
diagnostic test kit for identifying cells which misexpress an MCT
protein, such as by measuring a level of an MCT-encoding nucleic
acid in a sample of cells, e.g., detecting MCT mRNA levels or
determining whether a genomic MCT gene has been mutated or
deleted.
[0063] In one embodiment, the nucleic acid molecule of the
invention encodes a protein or portion thereof which includes an
amino acid sequence which is sufficiently homologous to an amino
acid sequence of Appendix B such that the protein or portion
thereof maintains the ability to participate in the metabolism of
compounds necessary for the construction of cellular membranes in
C. glutamicum, or in the transport of molecules across these
membranes. As used herein, the language "sufficiently homologous"
refers to proteins or portions thereof which have amino acid
sequences which include a minimum number of identical or equivalent
(e.g., an amino acid residue which has a similar side chain as an
amino acid residue in one of the sequences of Appendix B) amino
acid residues to an amino acid sequence of Appendix B such that the
protein or portion thereof is able to participate in the metabolism
of compounds necessary for the construction of cellular membranes
in C. glutamicum, or in the transport of molecules across these
membranes. Protein members of such membrane component metabolic
pathways or membrane transport systems, as described herein, may
play a role in the production and secretion of one or more fine
chemicals. Examples of such activities are also described herein.
Thus, "the function of an MCT protein" contributes either directly
or indirectly to the yield, production, and/or efficiency of
production of one or more fine chemicals. Examples of MCT protein
activities are set forth in Table 1.
[0064] In another embodiment, the protein is at least about 50-60%,
preferably at least about 60-70%, and more preferably at least
about 70-80%, 80-90%, 90-95%, and most preferably at least about
96%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence of Appendix B.
[0065] Portions of proteins encoded by the MCT nucleic acid
molecules of the invention are preferably biologically active
portions of one of the MCT proteins. As used herein, the term
"biologically active portion of an MCT protein" is intended to
include a portion, e.g., a domain/motif, of an MCT protein that
participates in the metabolism of compounds necessary for the
construction of cellular membranes in C. glutamicum, or in the
transport of molecules across these membranes, or has an activity
as set forth in Table 1. To determine whether an MCT protein or a
biologically active portion thereof can participate in the
metabolism of compounds necessary for the construction of cellular
membranes in C. glutamicum, or in the transport of molecules across
these membranes, an assay of enzymatic activity may be performed.
Such assay methods are well known to those of ordinary skill in the
art, as detailed in Example 8 of the Exemplification.
[0066] Additional nucleic acid fragments encoding biologically
active portions of an MCT protein can be prepared by isolating a
portion of one of the sequences in Appendix B, expressing the
encoded portion of the MCT protein or peptide (e.g., by recombinant
expression in vitro) and assessing the activity of the encoded
portion of the MCT protein or peptide.
[0067] The invention further encompasses nucleic acid molecules
that differ from one of the nucleotide sequences shown in Appendix
A (and portions thereof) due to degeneracy of the genetic code and
thus encode the same MCT protein as that encoded by the nucleotide
sequences shown in Appendix A. In another embodiment, an isolated
nucleic acid molecule of the invention has a nucleotide sequence
encoding a protein having an amino acid sequence shown in Appendix
B. In a still further embodiment, the nucleic acid molecule of the
invention encodes a full length C. glutamicum protein which is
substantially homologous to an amino acid sequence of Appendix B
(encoded by an open reading frame shown in Appendix A).
[0068] It will be understood by one of ordinary skill in the art
that in one embodiment the sequences of the invention are not meant
to include the sequences of the prior art, such as those Genbank
sequences set forth in Tables 2 or 4 which were available prior to
the present invention. In one embodiment, the invention includes
nucleotide and amino acid sequences having a percent identity to a
nucleotide or amino acid sequence of the invention which is greater
than that of a sequence of the prior art (e.g., a Genbank sequence
(or the protein encoded by such a sequence) set forth in Tables 2
or 4). For example, the invention includes a nucleotide sequence
which is greater than and/or at least 38% identical to the
nucleotide sequence designated RXA01420 (SEQ ID NO:7), a nucleotide
sequence which is greater than and/or at least 43% identical to the
nucleotide sequence designated RXA00104 (SEQ ID NO:5), and a
nucleotide sequence which is greater than and/or at least 45%
identical to the nucleotide sequence designated RXA02173 (SEQ ID
NO:25). One of ordinary skill in the art would be able to calculate
the lower threshold of percent identity for any given sequence of
the invention by examining the GAP-calculated percent identity
scores set forth in Table 4 for each of the three top hits for the
given sequence, and by subtracting the highest GAP-calculated
percent identity from 100 percent. One of ordinary skill in the art
will also appreciate that nucleic acid and amino acid sequences
having percent identities greater than the lower threshold so
calculated (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and
even more preferably at least about 95%, 96%, 97%, 98%, 99% or more
identical) are also encompassed by the invention.
[0069] In addition to the C. glutamicum MCT nucleotide sequences
shown in Appendix A, it will be appreciated by one of ordinary
skill in the art that DNA sequence polymorphisms that lead to
changes in the amino acid sequences of MCT proteins may exist
within a population (e.g., the C. glutamicum population). Such
genetic polymorphism in the MCT gene may exist among individuals
within a population due to natural variation. As used herein, the
terms "gene" and "recombinant gene" refer to nucleic acid molecules
comprising an open reading frame encoding an MCT protein,
preferably a C. glutamicum MCT protein. Such natural variations can
typically result in 1-5% variance in the nucleotide sequence of the
MCT gene. Any and all such nucleotide variations and resulting
amino acid polymorphisms in MCT that are the result of natural
variation and that do not alter the functional activity of MCT
proteins are intended to be within the scope of the invention.
[0070] Nucleic acid molecules corresponding to natural variants and
non-C. glutamicum homologues of the C. glutamicum MCT DNA of the
invention can be isolated based on their homology to the C.
glutamicum MCT nucleic acid disclosed herein using the C.
glutamicum DNA, or a portion thereof, as a hybridization probe
according to standard hybridization techniques under stringent
hybridization conditions. Accordingly, in another embodiment, an
isolated nucleic acid molecule of the invention is at least 15
nucleotides in length and hybridizes under stringent conditions to
the nucleic acid molecule comprising a nucleotide sequence of
Appendix A. In other embodiments, the nucleic acid is at least 30,
50, 100, 250 or more nucleotides in length. As used herein, the
term "hybridizes under stringent conditions" is intended to
describe conditions for hybridization and washing under which
nucleotide sequences at least 60% homologous to each other
typically remain hybridized to each other. Preferably, the
conditions are such that sequences at least about 65%, more
preferably at least about 70%, and even more preferably at least
about 75% or more homologous to each other typically remain
hybridized to each other. Such stringent conditions are known to
those of ordinary skill in the art and can be found in Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),
6.3.1-6.3.6. A preferred, non-limiting example of stringent
hybridization conditions are hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more washes in 0.2.times.SSC, 0.1% SDS at 50-65.degree. C.
Preferably, an isolated nucleic acid molecule of the invention that
hybridizes under stringent conditions to a sequence of Appendix A
corresponds to a naturally-occurring nucleic acid molecule. As used
herein, a "naturally-occurring" nucleic acid molecule refers to an
RNA or DNA molecule having a nucleotide sequence that occurs in
nature (e.g., encodes a natural protein). In one embodiment, the
nucleic acid encodes a natural C. glutamicum MCT protein.
[0071] In addition to naturally-occurring variants of the MCT
sequence that may exist in the population, one of ordinary skill in
the art will further appreciate that changes can be introduced by
mutation into a nucleotide sequence of Appendix A, thereby leading
to changes in the amino acid sequence of the encoded MCT protein,
without altering the functional ability of the MCT protein. For
example, nucleotide substitutions leading to amino acid
substitutions at "non-essential" amino acid residues can be made in
a sequence of Appendix A. A "non-essential" amino acid residue is a
residue that can be altered from the wild-type sequence of one of
the MCT proteins (Appendix B) without altering the activity of said
MCT protein, whereas an "essential" amino acid residue is required
for MCT protein activity. Other amino acid residues, however,
(e.g., those that are not conserved or only semi-conserved in the
domain having MCT activity) may not be essential for activity and
thus are likely to be amenable to alteration without altering MCT
activity.
[0072] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding MCT proteins that contain changes
in amino acid residues that are not essential for MCT activity.
Such MCT proteins differ in amino acid sequence from a sequence
contained in Appendix B yet retain at least one of the MCT
activities described herein. In one embodiment, the isolated
nucleic acid molecule comprises a nucleotide sequence encoding a
protein, wherein the protein comprises an amino acid sequence at
least about 50% homologous to an amino acid sequence of Appendix B
and is capable of participate in the metabolism of compounds
necessary for the construction of cellular membranes in C.
glutamicum, or in the transport of molecules across these
membranes, or has one or more activities set forth in Table 1.
Preferably, the protein encoded by the nucleic acid molecule is at
least about 50-60% homologous to one of the sequences in Appendix
B, more preferably at least about 60-70% homologous to one of the
sequences in Appendix B, even more preferably at least about
70-80%, 80-90%, 90-95% homologous to one of the sequences in
Appendix B, and most preferably at least about 96%, 97%, 98%, or
99% homologous to one of the sequences in Appendix B.
[0073] To determine the percent homology of two amino acid
sequences (e.g., one of the sequences of Appendix B and a mutant
form thereof) or of two nucleic acids, the sequences are aligned
for optimal comparison purposes (e.g., gaps can be introduced in
the sequence of one protein or nucleic acid for optimal alignment
with the other protein or nucleic acid). The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in one sequence (e.g.,
one of the sequences of Appendix B) is occupied by the same amino
acid residue or nucleotide as the corresponding position in the
other sequence (e.g., a mutant form of the sequence selected from
Appendix B), then the molecules are homologous at that position
(i.e., as used herein amino acid or nucleic acid "homology" is
equivalent to amino acid or nucleic acid "identity"). The percent
homology between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % homology=# of
identical positions/total# of positions.times.100).
[0074] An isolated nucleic acid molecule encoding an MCT protein
homologous to a protein sequence of Appendix B can be created by
introducing one or more nucleotide substitutions, additions or
deletions into a nucleotide sequence of Appendix A such that one or
more amino acid substitutions, additions or deletions are
introduced into the encoded protein. Mutations can be introduced
into one of the sequences of Appendix A by standard techniques,
such as site-directed mutagenesis and PCR-mediated mutagenesis.
Preferably, conservative amino acid substitutions are made at one
or more predicted non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in an MCT protein is
preferably replaced with another amino acid residue from the same
side chain family. Alternatively, in another embodiment, mutations
can be introduced randomly along all or part of an MCT coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for an MCT activity described herein to
identify mutants that retain MCT activity. Following mutagenesis of
one of the sequences of Appendix A, the encoded protein can be
expressed recombinantly and the activity of the protein can be
determined using, for example, assays described herein (see Example
8 of the Exemplification).
[0075] In addition to the nucleic acid molecules encoding MCT
proteins described above, another aspect of the invention pertains
to isolated nucleic acid molecules which are antisense thereto. An
"antisense" nucleic acid comprises a nucleotide sequence which is
complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. Accordingly, an
antisense nucleic acid can hydrogen bond to a sense nucleic acid.
The antisense nucleic acid can be complementary to an entire MCT
coding strand, or to only a portion thereof. In one embodiment, an
antisense nucleic acid molecule is antisense to a "coding region"
of the coding strand of a nucleotide sequence encoding an MCT
protein. The term "coding region" refers to the region of the
nucleotide sequence comprising codons which are translated into
amino acid residues (e.g., the entire coding region of SEQ ID NO:5
(RXA00104 in Appendix A) comprises nucleotides 1 to 756). In
another embodiment, the antisense nucleic acid molecule is
antisense to a "noncoding region" of the coding strand of a
nucleotide sequence encoding MCT. The term "noncoding region"
refers to 5' and 3' sequences which flank the coding region that
are not translated into amino acids (i.e., also referred to as 5'
and 3' untranslated regions).
[0076] Given the coding strand sequences encoding MCT disclosed
herein (e.g., the sequences set forth in Appendix A), antisense
nucleic acids of the invention can be designed according to the
rules of Watson and Crick base pairing. The antisense nucleic acid
molecule can be complementary to the entire coding region of MCT
mRNA, but more preferably is an oligonucleotide which is antisense
to only a portion of the coding or noncoding region of MCT mRNA.
For example, the antisense oligonucleotide can be complementary to
the region surrounding the translation start site of MCT mRNA. An
antisense oligonucleotide can be, for example, about 5, 10, 15, 20,
25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense
nucleic acid of the invention can be constructed using chemical
synthesis and enzymatic ligation reactions using procedures known
in the art. For example, an antisense nucleic acid (e.g., an
antisense oligonucleotide) can be chemically synthesized using
naturally occurring nucleotides or variously modified nucleotides
designed to increase the biological stability of the molecules or
to increase the physical stability of the duplex formed between the
antisense and sense nucleic acids, e.g., phosphorothioate
derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides which can be used to generate the
antisense nucleic acid include 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0077] The antisense nucleic acid molecules of the invention are
typically administered to a cell or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding an MCT protein to thereby inhibit expression of the
protein, e.g., by inhibiting transcription and/or translation. The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule which binds to DNA duplexes, through specific
interactions in the major groove of the double helix. The antisense
molecule can be modified such that it specifically binds to a
receptor or an antigen expressed on a selected cell surface, e.g.,
by linking the antisense nucleic acid molecule to a peptide or an
antibody which binds to a cell surface receptor or antigen. The
antisense nucleic acid molecule can also be delivered to cells
using the vectors described herein. To achieve sufficient
intracellular concentrations of the antisense molecules, vector
constructs in which the antisense nucleic acid molecule is placed
under the control of a strong prokaryotic, viral, or eukaryotic
promoter are preferred.
[0078] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0079] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. Ribozymes are catalytic RNA molecules
with ribonuclease activity which are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can
be used to catalytically cleave MCT mRNA transcripts to thereby
inhibit translation of MCT mRNA. A ribozyme having specificity for
an MCT-encoding nucleic acid can be designed based upon the
nucleotide sequence of an MCT DNA disclosed herein (i.e., SEQ ID
NO. 5 (RXA00104) in Appendix A)). For example, a derivative of a
Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide
sequence of the active site is complementary to the nucleotide
sequence to be cleaved in an MCT-encoding mRNA. See, e.g., Cech et
al. U.S. Pat. No. 4,987,071 and Cech et al. U.S. Pat. No.
5,116,742. Alternatively, MCT mRNA can be used to select a
catalytic RNA having a specific ribonuclease activity from a pool
of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993)
Science 261:1411-1418.
[0080] Alternatively, MCT gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of an MCT nucleotide sequence (e.g., an MCT promoter and/or
enhancers) to form triple helical structures that prevent
transcription of an MCT gene in target cells. See generally,
Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et
al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992)
Bioassays 14(12):807-15.
B. Recombinant Expression Vectors and Host Cells
[0081] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding
an MCT protein (or a portion thereof). As used herein, the term
"vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. One type of
vector is a "plasmid", which refers to a circular double stranded
DNA loop into which additional DNA segments can be ligated. Another
type of vector is a viral vector, wherein additional DNA segments
can be ligated into the viral genome. Certain vectors are capable
of autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively linked. Such vectors are referred to herein as
"expression vectors". In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids. In
the present specification, "plasmid" and "vector" can be used
interchangeably as the plasmid is the most commonly used form of
vector. However, the invention is intended to include such other
forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0082] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, which is operatively linked to the nucleic acid
sequence to be expressed. Within a recombinant expression vector,
"operably linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
which allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many
types of host cell and those which direct expression of the
nucleotide sequence only in certain host cells. Preferred
regulatory sequences are, for example, promoters such as cos-,
tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacI.sup.q-, T7-,
T5-, T3-, gal-, trc-, ara-, SP6-, amy, SPO2, .lamda.-P.sub.R-or
.lamda. P.sub.L, which are used preferably in bacteria. Additional
regulatory sequences are, for example, promoters from yeasts and
fungi, such as ADC1, MF.alpha., AC, P-60, CYC1, GAPDH, TEF, rp28,
ADH, promoters from plants such as CaMV/35S, SSU, OCS, lib4, usp,
STLS1, B33, nos or ubiquitin- or phaseolin-promoters. It is also
possible to use artificial promoters. It will be appreciated by one
of ordinary skill in the art that the design of the expression
vector can depend on such factors as the choice of the host cell to
be transformed, the level of expression of protein desired, etc.
The expression vectors of the invention can be introduced into host
cells to thereby produce proteins or peptides, including fusion
proteins or peptides, encoded by nucleic acids as described herein
(e.g., MCT proteins, mutant forms of MCT proteins, fusion proteins,
etc.).
[0083] The recombinant expression vectors of the invention can be
designed for expression of MCT proteins in prokaryotic or
eukaryotic cells. For example, MCT genes can be expressed in
bacterial cells such as C. glutamicum, insect cells (using
baculovirus expression vectors), yeast and other fungal cells (see
Romanos, M. A. et al. (1992) "Foreign gene expression in yeast: a
review", Yeast 8: 423-488; van den Hondel, C.A.M.J.J. et al. (1991)
"Heterologous gene expression in filamentous fungi" in: More Gene
Manipulations in Fungi, J. W. Bennet & L. L. Lasure, eds., p.
396-428: Academic Press: San Diego; and van den Hondel, C.A.M.J.J.
& Punt, P. J. (1991) "Gene transfer systems and vector
development for filamentous fungi, in: Applied Molecular Genetics
of Fungi, Peberdy, J. F. et al., eds., p. 1-28, Cambridge
University Press: Cambridge), algae and multicellular plant cells
(see Schmidt, R. and Willmitzer, L. (1988) High efficiency
Agrobacterium tumefaciens--mediated transformation of Arabidopsis
thaliana leaf and cotyledon explants" Plant Cell Rep.: 583-586), or
mammalian cells. Suitable host cells are discussed further in
Goeddel, Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. (1990). Alternatively, the
recombinant expression vector can be transcribed and translated in
vitro, for example using T7 promoter regulatory sequences and T7
polymerase.
[0084] Expression of proteins in prokaryotes is most often carried
out with vectors containing constitutive or inducible promoters
directing the expression of either fusion or non-fusion proteins.
Fusion vectors add a number of amino acids to a protein encoded
therein, usually to the amino terminus of the recombinant protein
but also to the C-terminus or fused within suitable regions in the
proteins. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase.
[0085] Typical fusion expression vectors include pGEX (Pharmacia
Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) which fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein. In one embodiment, the coding sequence
of the MCT protein is cloned into a pGEX expression vector to
create a vector encoding a fusion protein comprising, from the
N-terminus to the C-terminus, GST-thrombin cleavage site-X protein.
The fusion protein can be purified by affinity chromatography using
glutathione-agarose resin. Recombinant MCT protein unfused to GST
can be recovered by cleavage of the fusion protein with
thrombin.
[0086] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) pLG338,
pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236,
pMBL24, pLG200, pUR290, pIN-III113-B1, .lamda.gt11, pBdC1, and pET
11d (Studier et al, Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89 ;
and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York
IBSN 0 444 904018). Target gene expression from the pTrc vector
relies on host RNA polymerase transcription from a hybrid trp-lac
fusion promoter. Target gene expression from the pET 11d vector
relies on transcription from a T7 gn10-lac fusion promoter mediated
by a coexpressed viral RNA polymerase (T7 gnl). This viral
polymerase is supplied by host strains BL21(DE3) or HMS174(DE3)
from a resident .lamda. prophage harboring a T7 gn1 gene under the
transcriptional control of the lacUV 5 promoter. For transformation
of other varieties of bacteria, appropriate vectors may be
selected. For example, the plasmids pIJ101, pIJ364, pIJ702 and
pIJ361 are known to be useful in transforming Streptomyces, while
plasmids pUB110, pC194, or pBD214 are suited for transformation of
Bacillus species. Several plasmids of use in the transfer of
genetic information into Corynebacterium include pHM1519, pBL1,
pSA77, or pAJ667 (Pouwels et al., eds. (1985) Cloning Vectors.
Elsevier: N.Y. IBSN 0 444 904018).
[0087] One strategy to maximize recombinant protein expression is
to express the protein in a host bacteria with an impaired capacity
to proteolytically cleave the recombinant protein (Gottesman, S.,
Gene Expression Technology: Methods in Enzymology 185, Academic
Press, San Diego, Calif. (1990) 119-128). Another strategy is to
alter the nucleic acid sequence of the nucleic acid to be inserted
into an expression vector so that the individual codons for each
amino acid are those preferentially utilized in the bacterium
chosen for expression, such as C. glutamicum (Wada et al. (1992)
Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid
sequences of the invention can be carried out by standard DNA
synthesis techniques.
[0088] In another embodiment, the MCT protein expression vector is
a yeast expression vector. Examples of vectors for expression in
yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) Embo
J. 6:229-234), 2.mu., pAG-1, Yep6, Yep13, pEMBLYe23, pMFa (Kurjan
and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al.,
(1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San
Diego, Calif.). Vectors and methods for the construction of vectors
appropriate for use in other fungi, such as the filamentous fungi,
include those detailed in: van den Hondel, C.A.M.J.J. & Punt,
P. J. (1991) "Gene transfer systems and vector development for
filamentous fungi, in: Applied Molecular Genetics of Fungi, J. F.
Peberdy, et al., eds., p. 1-28, Cambridge University Press:
Cambridge, and Pouwels et al., eds. (1985) Cloning Vectors.
Elsevier: N.Y. (IBSN 0 444 904018).
[0089] Alternatively, the MCT proteins of the invention can be
expressed in insect cells using baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf9 cells) include the pAc series
(Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL
series (Lucklow and Summers (1989) Virology 170:31-39).
[0090] In another embodiment, the MCT proteins of the invention may
be expressed in unicellular plant cells (such as algae) or in plant
cells from higher plants (e.g., the spermatophytes, such as crop
plants). Examples of plant expression vectors include those
detailed in: Becker, D., Kemper, E., Schell, J. and Masterson, R.
(1992) "New plant binary vectors with selectable markers located
proximal to the left border", Plant Mol. Biol. 20: 1195-1197; and
Bevan, M. W. (1984) "Binary Agrobacterium vectors for plant
transformation", Nuc. Acid Res. 12: 8711-8721, and include pLGV23,
pGHlac+, pBIN19, pAK2004, and pDH51 (Pouwels et al., eds. (1985)
Cloning Vectors. Elsevier: N.Y. IBSN 0 444 904018).
[0091] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)
EMBO J. 6:187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E.
F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd,
ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989.
[0092] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) PNAS
86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)
Science 230:912-916), and mammary gland-specific promoters (e.g.,
milk whey promoter; U.S. Pat. No. 4,873,316 and European
Application Publication No. 264,166). Developmentally-regulated
promoters are also encompassed, for example the murine hox
promoters (Kessel and Gruss (1990) Science 249:374-379) and the
.alpha.-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.
3:537-546).
[0093] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively linked to a regulatory sequence in a manner
which allows for expression (by transcription of the DNA molecule)
of an RNA molecule which is antisense to MCT mRNA. Regulatory
sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen which direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen which direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see Weintraub, H. et al.,
Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[0094] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0095] A host cell can be any prokaryotic or eukaryotic cell. For
example, an MCT protein can be expressed in bacterial cells such as
C. glutamicum, insect cells, yeast or mammalian cells (such as
Chinese hamster ovary cells (CHO) or COS cells). Other suitable
host cells are known to one of ordinary skill in the art.
Microorganisms related to Corynebacterium glutamicum which may be
conveniently used as host cells for the nucleic acid and protein
molecules of the invention are set forth in Table 3.
[0096] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection",
"conjugation" and "transduction" are intended to refer to a variety
of art-recognized techniques for introducing foreign nucleic acid
(e.g., linear DNA or RNA (e.g., a linearized vector or a gene
construct alone without a vector) or nucleic acid in the form of a
vector (e.g., a plasmid, phage, phasmid, phagemid, transposon or
other DNA) into a host cell, including calcium phosphate or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection,
lipofection, natural competence, chemical-mediated transfer, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0097] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding an MCT protein or can be introduced on a separate
vector. Cells stably transfected with the introduced nucleic acid
can be identified by, for example, drug selection (e.g., cells that
have incorporated the selectable marker gene will survive, while
the other cells die).
[0098] To create a homologous recombinant microorganism, a vector
is prepared which contains at least a portion of an MCT gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the MCT gene.
Preferably, this MCT gene is a Corynebacterium glutamicum MCT gene,
but it can be a homologue from a related bacterium or even from a
mammalian, yeast, or insect source. In a preferred embodiment, the
vector is designed such that, upon homologous recombination, the
endogenous MCT gene is functionally disrupted (i.e., no longer
encodes a functional protein; also referred to as a "knock out"
vector). Alternatively, the vector can be designed such that, upon
homologous recombination, the endogenous MCT gene is mutated or
otherwise altered but still encodes functional protein (e.g., the
upstream regulatory region can be altered to thereby alter the
expression of the endogenous MCT protein). In the homologous
recombination vector, the altered portion of the MCT gene is
flanked at its 5' and 3' ends by additional nucleic acid of the MCT
gene to allow for homologous recombination to occur between the
exogenous MCT gene carried by the vector and an endogenous MCT gene
in a microorganism. The additional flanking MCT nucleic acid is of
sufficient length for successful homologous recombination with the
endogenous gene. Typically, several kilobases of flanking DNA (both
at the 5' and 3' ends) are included in the vector (see e.g.,
Thomas, K. R., and Capecchi, M. R. (1987) Cell 51: 503 for a
description of homologous recombination vectors). The vector is
introduced into a microorganism (e.g., by electroporation) and
cells in which the introduced MCT gene has homologously recombined
with the endogenous MCT gene are selected, using art-known
techniques.
[0099] In another embodiment, recombinant microorganisms can be
produced which contain selected systems which allow for regulated
expression of the introduced gene. For example, inclusion of an MCT
gene on a vector placing it under control of the lac operon permits
expression of the MCT gene only in the presence of IPTG. Such
regulatory systems are well known in the art.
[0100] In another embodiment, an endogenous MCT gene in a host cell
is disrupted (e.g., by homologous recombination or other genetic
means known in the art) such that expression of its protein product
does not occur. In another embodiment, an endogenous or introduced
MCT gene in a host cell has been altered by one or more point
mutations, deletions, or inversions, but still encodes a functional
MCT protein. In still another embodiment, one or more of the
regulatory regions (e.g., a promoter, repressor, or inducer) of an
MCT gene in a microorganism has been altered (e.g., by deletion,
truncation, inversion, or point mutation) such that the expression
of the MCT gene is modulated. One of ordinary skill in the art will
appreciate that host cells containing more than one of the
described MCT gene and protein modifications may be readily
produced using the methods of the invention, and are meant to be
included in the present invention.
[0101] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) an MCT protein. Accordingly, the invention further
provides methods for producing MCT proteins using the host cells of
the invention. In one embodiment, the method comprises culturing
the host cell of invention (into which a recombinant expression
vector encoding an MCT protein has been introduced, or into which
genome has been introduced a gene encoding a wild-type or altered
MCT protein) in a suitable medium until MCT protein is produced. In
another embodiment, the method further comprises isolating MCT
proteins from the medium or the host cell.
C. Isolated MCT Proteins
[0102] Another aspect of the invention pertains to isolated MCT
proteins, and biologically active portions thereof. An "isolated"
or "purified" protein or biologically active portion thereof is
substantially free of cellular material when produced by
recombinant DNA techniques, or chemical precursors or other
chemicals when chemically synthesized. The language "substantially
free of cellular material" includes preparations of MCT protein in
which the protein is separated from cellular components of the
cells in which it is naturally or recombinantly produced. In one
embodiment, the language "substantially free of cellular material"
includes preparations of MCT protein having less than about 30% (by
dry weight) of non-MCT protein (also referred to herein as a
"contaminating protein"), more preferably less than about 20% of
non-MCT protein, still more preferably less than about 10% of
non-MCT protein, and most preferably less than about 5% non-MCT
protein. When the MCT protein or biologically active portion
thereof is recombinantly produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, more preferably less than about
10%, and most preferably less than about 5% of the volume of the
protein preparation. The language "substantially free of chemical
precursors or other chemicals" includes preparations of MCT protein
in which the protein is separated from chemical precursors or other
chemicals which are involved in the synthesis of the protein. In
one embodiment, the language "substantially free of chemical
precursors or other chemicals" includes preparations of MCT protein
having less than about 30% (by dry weight) of chemical precursors
or non-MCT chemicals, more preferably less than about 20% chemical
precursors or non-MCT chemicals, still more preferably less than
about 10% chemical precursors or non-MCT chemicals, and most
preferably less than about 5% chemical precursors or non-MCT
chemicals. In preferred embodiments, isolated proteins or
biologically active portions thereof lack contaminating proteins
from the same organism from which the MCT protein is derived.
Typically, such proteins are produced by recombinant expression of,
for example, a C. glutamicum MCT protein in a microorganism such as
C. glutamicum.
[0103] An isolated MCT protein or a portion thereof of the
invention can participate in the metabolism of compounds necessary
for the construction of cellular membranes in C. glutamicum, or in
the transport of molecules across these membranes, or has one or
more of the activities set forth in Table 1. In preferred
embodiments, the protein or portion thereof comprises an amino acid
sequence which is sufficiently homologous to an amino acid sequence
of Appendix B such that the protein or portion thereof maintains
the ability participate in the metabolism of compounds necessary
for the construction of cellular membranes in C. glutamicum, or in
the transport of molecules across these membranes. The portion of
the protein is preferably a biologically active portion as
described herein. In another preferred embodiment, an MCT protein
of the invention has an amino acid sequence shown in Appendix B. In
yet another preferred embodiment, the MCT protein has an amino acid
sequence which is encoded by a nucleotide sequence which
hybridizes, e.g., hybridizes under stringent conditions, to a
nucleotide sequence of Appendix A. In still another preferred
embodiment, the MCT protein has an amino acid sequence which is
encoded by a nucleotide sequence that is at least about 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least
about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more
preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%,
or 91%, 92%, 93%, 94%, and even more preferably at least about 95%,
96%, 97%, 98%, 99% or more homologous to one of the nucleic acid
sequences of Appendix A, or a portion thereof. Ranges and identity
values intermediate to the above-recited values, (e.g., 70-90%
identical or 80-95% identical) are also intended to be encompassed
by the present invention. For example, ranges of identity values
using a combination of any of the above values recited as upper
and/or lower limits are intended to be included. The preferred MCT
proteins of the present invention also preferably possess at least
one of the MCT activities described herein. For example, a
preferred MCT protein of the present invention includes an amino
acid sequence encoded by a nucleotide sequence which hybridizes,
e.g., hybridizes under stringent conditions, to a nucleotide
sequence of Appendix A, and which can participate in the metabolism
of compounds necessary for the construction of cellular membranes
in C. glutamicum, or in the transport of molecules across these
membranes, or which has one or more of the activities set forth in
Table 1.
[0104] In other embodiments, the MCT protein is substantially
homologous to an amino acid sequence of Appendix B and retains the
functional activity of the protein of one of the sequences of
Appendix B yet differs in amino acid sequence due to natural
variation or mutagenesis, as described in detail in subsection I
above. Accordingly, in another embodiment, the MCT protein is a
protein which comprises an amino acid sequence which is at least
about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%,
preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,
69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably
at least about 95%, 96%, 97%, 98%, 99% or more homologous to an
entire amino acid sequence of Appendix B and which has at least one
of the MCT activities described herein. Ranges and identity values
intermediate to the above-recited values, (e.g., 70-90% identical
or 80-95% identical) are also intended to be encompassed by the
present invention. For example, ranges of identity values using a
combination of any of the above values recited as upper and/or
lower limits are intended to be included. In another embodiment,
the invention pertains to a full length C. glutamicum protein which
is substantially homologous to an entire amino acid sequence of
Appendix B.
[0105] Biologically active portions of an MCT protein include
peptides comprising amino acid sequences derived from the amino
acid sequence of an MCT protein, e.g., the an amino acid sequence
shown in Appendix B or the amino acid sequence of a protein
homologous to an MCT protein, which include fewer amino acids than
a full length MCT protein or the full length protein which is
homologous to an MCT protein, and exhibit at least one activity of
an MCT protein. Typically, biologically active portions (peptides,
e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36,
37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a
domain or motif with at least one activity of an MCT protein.
Moreover, other biologically active portions, in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the activities
described herein. Preferably, the biologically active portions of
an MCT protein include one or more selected domains/motifs or
portions thereof having biological activity.
[0106] MCT proteins are preferably produced by recombinant DNA
techniques. For example, a nucleic acid molecule encoding the
protein is cloned into an expression vector (as described above),
the expression vector is introduced into a host cell (as described
above) and the MCT protein is expressed in the host cell. The MCT
protein can then be isolated from the cells by an appropriate
purification scheme using standard protein purification techniques.
Alternative to recombinant expression, an MCT protein, polypeptide,
or peptide can be synthesized chemically using standard peptide
synthesis techniques. Moreover, native MCT protein can be isolated
from cells (e.g., endothelial cells), for example using an anti-MCT
antibody, which can be produced by standard techniques utilizing an
MCT protein or fragment thereof of this invention.
[0107] The invention also provides MCT chimeric or fusion proteins.
As used herein, an MCT "chimeric protein" or "fusion protein"
comprises an MCT polypeptide operatively linked to a non-MCT
polypeptide. An "MCT polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to an MCT protein, whereas a
"non-MCT polypeptide" refers to a polypeptide having an amino acid
sequence corresponding to a protein which is not substantially
homologous to the MCT protein, e.g., a protein which is different
from the MCT protein and which is derived from the same or a
different organism. Within the fusion protein, the term
"operatively linked" is intended to indicate that the MCT
polypeptide and the non-MCT polypeptide are fused in-frame to each
other. The non-MCT polypeptide can be fused to the N-terminus or
C-terminus of the MCT polypeptide. For example, in one embodiment
the fusion protein is a GST-MCT fusion protein in which the MCT
sequences are fused to the C-terminus of the GST sequences. Such
fusion proteins can facilitate the purification of recombinant MCT
proteins. In another embodiment, the fusion protein is an MCT
protein containing a heterologous signal sequence at its
N-terminus. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of an MCT protein can be increased
through use of a heterologous signal sequence.
[0108] Preferably, an MCT chimeric or fusion protein of the
invention is produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, for example by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al. John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). An MCT-encoding nucleic acid can be cloned into
such an expression vector such that the fusion moiety is linked
in-frame to the MCT protein.
[0109] Homologues of the MCT protein can be generated by
mutagenesis, e.g., discrete point mutation or truncation of the MCT
protein. As used herein, the term "homologue" refers to a variant
form of the MCT protein which acts as an agonist or antagonist of
the activity of the MCT protein. An agonist of the MCT protein can
retain substantially the same, or a subset, of the biological
activities of the MCT protein. An antagonist of the MCT protein can
inhibit one or more of the activities of the naturally occurring
form of the MCT protein, by, for example, competitively binding to
a downstream or upstream member of the cell membrane component
metabolic cascade which includes the MCT protein, or by binding to
an MCT protein which mediates transport of compounds across such
membranes, thereby preventing translocation from taking place.
[0110] In an alternative embodiment, homologues of the MCT protein
can be identified by screening combinatorial libraries of mutants,
e.g., truncation mutants, of the MCT protein for MCT protein
agonist or antagonist activity. In one embodiment, a variegated
library of MCT variants is generated by combinatorial mutagenesis
at the nucleic acid level and is encoded by a variegated gene
library. A variegated library of MCT variants can be produced by,
for example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of
potential MCT sequences is expressible as individual polypeptides,
or alternatively, as a set of larger fusion proteins (e.g., for
phage display) containing the set of MCT sequences therein. There
are a variety of methods which can be used to produce libraries of
potential MCT homologues from a degenerate oligonucleotide
sequence. Chemical synthesis of a degenerate gene sequence can be
performed in an automatic DNA synthesizer, and the synthetic gene
then ligated into an appropriate expression vector. Use of a
degenerate set of genes allows for the provision, in one mixture,
of all of the sequences encoding the desired set of potential MCT
sequences. Methods for synthesizing degenerate oligonucleotides are
known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3;
Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al.
(1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res.
11:477.
[0111] In addition, libraries of fragments of the MCT protein
coding can be used to generate a variegated population of MCT
fragments for screening and subsequent selection of homologues of
an MCT protein. In one embodiment, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of an MCT coding sequence with a nuclease under conditions
wherein nicking occurs only about once per molecule, denaturing the
double stranded DNA, renaturing the DNA to form double stranded DNA
which can include sense/antisense pairs from different nicked
products, removing single stranded portions from reformed duplexes
by treatment with S1 nuclease, and ligating the resulting fragment
library into an expression vector. By this method, an expression
library can be derived which encodes N-terminal, C-terminal and
internal fragments of various sizes of the MCT protein.
[0112] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of MCT homologues. The most widely used techniques,
which are amenable to high through-put analysis, for screening
large gene libraries typically include cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates isolation of the vector encoding the
gene whose product was detected. Recursive ensemble mutagenesis
(REM), a new technique which enhances the frequency of functional
mutants in the libraries, can be used in combination with the
screening assays to identify MCT homologues (Arkin and Yourvan
(1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein
Engineering 6(3):327-331).
[0113] In another embodiment, cell based assays can be exploited to
analyze a variegated MCT library, using methods well known in the
art.
D. Uses and Methods of the Invention
[0114] The nucleic acid molecules, proteins, protein homologues,
fusion proteins, primers, vectors, and host cells described herein
can be used in one or more of the following methods: identification
of C. glutamicum and related organisms; mapping of genomes of
organisms related to C. glutamicum ; identification and
localization of C. glutamicum sequences of interest; evolutionary
studies; determination of MCT protein regions required for
function; modulation of an MCT protein activity; modulation of the
metabolism of one or more cell membrane components; modulation of
the transmembrane transport of one or more compounds; and
modulation of cellular production of a desired compound, such as a
fine chemical.
[0115] The MCT nucleic acid molecules of the invention have a
variety of uses. First, they may be used to identify an organism as
being Corynebacterium glutamicum or a close relative thereof. Also,
they may be used to identify the presence of C. glutamicum or a
relative thereof in a mixed population of microorganisms. The
invention provides the nucleic acid sequences of a number of C.
glutamicum genes; by probing the extracted genomic DNA of a culture
of a unique or mixed population of microorganisms under stringent
conditions with a probe spanning a region of a C. glutamicum gene
which is unique to this organism, one can ascertain whether this
organism is present.
[0116] Although Corynebacterium glutamicum itself is nonpathogenic,
it is related to pathogenic species, such as Corynebacterium
diphtheriae. Corynebacterium diphtheriae is the causative agent of
diphtheria, a rapidly developing, acute, febrile infection which
involves both local and systemic pathology. In this disease, a
local lesion develops in the upper respiratory tract and involves
necrotic injury to epithelial cells; the bacilli secrete toxin
which is disseminated through this lesion to distal susceptible
tissues of the body. Degenerative changes brought about by the
inhibition of protein synthesis in these tissues, which include
heart, muscle, peripheral nerves, adrenals, kidneys, liver and
spleen, result in the systemic pathology of the disease. Diphtheria
continues to have high incidence in many parts of the world,
including Africa, Asia, Eastern Europe and the independent states
of the former Soviet Union. An ongoing epidemic of diphtheria in
the latter two regions has resulted in at least 5,000 deaths since
1990. In one embodiment, the invention provides a method of
identifying the presence or activity of Cornyebacterium diphtheriae
in a subject. This method includes detection of one or more of the
nucleic acid or amino acid sequences of the invention (e.g., the
sequences set forth in Appendix A or Appendix B) in a subject,
thereby detecting the presence or activity of Corynebacterium
diphtheriae in the subject. C. glutamicum and C. diphtheriae are
related bacteria, and many of the nucleic acid and protein
molecules in C. glutamicum are homologous to C. diphtheriae nucleic
acid and protein molecules, and can therefore be used to detect C.
diphtheriae in a subject.
[0117] The nucleic acid and protein molecules of the invention may
also serve as markers for specific regions of the genome. This has
utility not only in the mapping of the genome, but also for
functional studies of C. glutamicum proteins. For example, to
identify the region of the genome to which a particular C.
glutamicum DNA-binding protein binds, the C. glutamicum genome
could be digested, and the fragments incubated with the DNA-binding
protein. Those which bind the protein may be additionally probed
with the nucleic acid molecules of the invention, preferably with
readily detectable labels; binding of such a nucleic acid molecule
to the genome fragment enables the localization of the fragment to
the genome map of C. glutamicum, and, when performed multiple times
with different enzymes, facilitates a rapid determination of the
nucleic acid sequence to which the protein binds. Further, the
nucleic acid molecules of the invention may be sufficiently
homologous to the sequences of related species such that these
nucleic acid molecules may serve as markers for the construction of
a genomic map in related bacteria, such as Brevibacterium
lactofermentum.
[0118] The MCT nucleic acid molecules of the invention are also
useful for evolutionary and protein structural studies. The
metabolic and transport processes in which the molecules of the
invention participate are utilized by a wide variety of prokaryotic
and eukaryotic cells; by comparing the sequences of the nucleic
acid molecules of the present invention to those encoding similar
enzymes from other organisms, the evolutionary relatedness of the
organisms can be assessed. Similarly, such a comparison permits an
assessment of which regions of the sequence are conserved and which
are not, which may aid in determining those regions of the protein
which are essential for the functioning of the enzyme. This type of
determination is of value for protein engineering studies and may
give an indication of what the protein can tolerate in terms of
mutagenesis without losing function.
[0119] Manipulation of the MCT nucleic acid molecules of the
invention may result in the production of MCT proteins having
functional differences from the wild-type MCT proteins. These
proteins may be improved in efficiency or activity, may be present
in greater numbers in the cell than is usual, or may be decreased
in efficiency or activity.
[0120] The invention provides methods for screening molecules which
modulate the activity of an MCT protein, either by interacting with
the protein itself or a substrate or binding partner of the MCT
protein, or by modulating the transcription or translation of an
MCT nucleic acid molecule of the invention. In such methods, a
microorganism expressing one or more MCT proteins of the invention
is contacted with one or more test compounds, and the effect of
each test compound on the activity or level of expression of the
MCT protein is assessed.
[0121] There are a number of mechanisms by which the alteration of
an MCT protein of the invention may directly affect the yield,
production, and/or efficiency of production of a fine chemical from
a C. glutamicum strain incorporating such an altered protein.
Recovery of fine chemical compounds from large-scale cultures of C.
glutamicum is significantly improved if C. glutamicum secretes the
desired compounds, since such compounds may be readily purified
from the culture medium (as opposed to extracted from the mass of
C. glutamicum cells). By either increasing the number or the
activity of transporter molecules which export fine chemicals from
the cell, it may be possible to increase the amount of the produced
fine chemical which is present in the extracellular medium, thus
permitting greater ease of harvesting and purification. Conversely,
in order to efficiently overproduce one or more fine chemicals,
increased amounts of the cofactors, precursor molecules, and
intermediate compounds for the appropriate biosynthetic pathways
are required. Therefore, by increasing the number and/or activity
of transporter proteins involved in the import of nutrients, such
as carbon sources (i.e., sugars), nitrogen sources (i.e., amino
acids, ammonium salts), phosphate, and sulfur, it may be possible
to improve the production of a fine chemical, due to the removal of
any nutrient supply limitations on the biosynthetic process.
Further, fatty acids and lipids are themselves desirable fine
chemicals, so by optimizing the activity or increasing the number
of one or more MCT proteins of the invention which participate in
the biosynthesis of these compounds, or by impairing the activity
of one or more MCT proteins which are involved in the degradation
of these compounds, it may be possible to increase the yield,
production, and/or efficiency of production of fatty acid and lipid
molecules from C. glutamicum.
[0122] The engineering of one or more MCT genes of the invention
may also result in MCT proteins having altered activities which
indirectly impact the production of one or more desired fine
chemicals from C. glutamicum. For example, the normal biochemical
processes of metabolism result in the production of a variety of
waste products (e.g., hydrogen peroxide and other reactive oxygen
species) which may actively interfere with these same metabolic
processes (for example, peroxynitrite is known to nitrate tyrosine
side chains, thereby inactivating some enzymes having tyrosine in
the active site (Groves, J. T. (1999) Curr. Opin. Chem. Biol. 3(2):
226-235). While these waste products are typically excreted, the C.
glutamicum strains utilized for large-scale fermentative production
are optimized for the overproduction of one or more fine chemicals,
and thus may produce more waste products than is typical for a
wild-type C. glutamicum. By optimizing the activity of one or more
MCT proteins of the invention which are involved in the export of
waste molecules, it may be possible to improve the viability of the
cell and to maintain efficient metabolic activity. Also, the
presence of high intracellular levels of the desired fine chemical
may actually be toxic to the cell, so by increasing the ability of
the cell to secrete these compounds, one may improve the viability
of the cell.
[0123] Further, the MCT proteins of the invention may be
manipulated such that the relative amounts of various lipid and
fatty acid molecules produced are altered. This may have a profound
effect on the lipid composition of the membrane of the cell. Since
each type of lipid has different physical properties, an alteration
in the lipid composition of a membrane may significantly alter
membrane fluidity. Changes in membrane fluidity can impact the
transport of molecules across the membrane, which, as previously
explicated, may modify the export of waste products or the produced
fine chemical or the import of necessary nutrients. Such membrane
fluidity changes may also profoundly affect the integrity of the
cell; cells with relatively weaker membranes are more vulnerable in
the large-scale fermentor environment to mechanical stresses which
may damage or kill the cell. By manipulating MCT proteins involved
in the production of fatty acids and lipids for membrane
construction such that the resulting membrane has a membrane
composition more amenable to the environmental conditions extant in
the cultures utilized to produce fine chemicals, a greater
proportion of the C. glutamicum cells should survive and multiply.
Greater numbers of C. glutamicum cells in a culture should
translate into greater yields, production, or efficiency of
production of the fine chemical from the culture.
[0124] The aforementioned mutagenesis strategies for MCT proteins
to result in increased yields of a fine chemical from C. glutamicum
are not meant to be limiting; variations on these strategies will
be readily apparent to one of ordinary skill in the art. Using such
strategies, and incorporating the mechanisms disclosed herein, the
nucleic acid and protein molecules of the invention may be utilized
to generate C. glutamicum or related strains of bacteria expressing
mutated MCT nucleic acid and protein molecules such that the yield,
production, and/or efficiency of production of a desired compound
is improved. This desired compound may be any natural product of C.
glutamicum, which includes the final products of biosynthesis
pathways and intermediates of naturally-occurring metabolic
pathways, as well as molecules which do not naturally occur in the
metabolism of C. glutamicum, but which are produced by a C.
glutamicum strain of the invention.
[0125] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patent applications, patents, published patent
applications, Tables, Appendices, and the sequence listing cited
throughout this application are hereby incorporated by
reference.
Exemplification
EXAMPLE 1
Preparation of Total Genomic DNA of Corynebacterium glutamicum ATCC
13032
[0126] A culture of Corynebacterium glutamicum (ATCC 13032) was
grown overnight at 30.degree. C. with vigorous shaking in BHI
medium (Difco). The cells were harvested by centrifugation, the
supernatant was discarded and the cells were resuspended in 5 ml
buffer-I (5% of the original volume of the culture--all indicated
volumes have been calculated for 100 ml of culture volume).
Composition of buffer-I: 140.34 g/l sucrose, 2.46 g/l
MgSO.sub.4.times.7H.sub.2O, 10 ml/l KH.sub.2PO.sub.4 solution (100
g/l, adjusted to pH 6.7 with KOH), 50 ml/l M12 concentrate (10 g/l
(NH.sub.4).sub.2SO.sub.4, 1 g/l NaCl, 2 g/l MgSO.sub.4.times.7
H.sub.2O, 0.2 g/l CaCl.sub.2, 0.5 g/l yeast extract (Difco), 10
ml/l trace-elements-mix (200 mg/l FeSO.sub.4.times.H.sub.2O, 10
mg/l ZnSO.sub.4.times.7 H.sub.2O, 3 mg/l MnCl.sub.2.times.4
H.sub.2O, 30 mg/l H.sub.3BO.sub.3 20 mg/l CoCl.sub.2.times.6
H.sub.2O, 1 mg/l NiCl.sub.2.times.6 H.sub.2O, 3 mg/l
Na.sub.2MoO.sub.4.times.2 H.sub.2O, 500 mg/l complexing agent (EDTA
or critic acid), 100 ml/l vitamins-mix (0.2 mg/l biotin, 0.2 mg/l
folic acid, 20 mg/l p-amino benzoic acid, 20 mg/l riboflavin, 40
mg/l ca-panthothenate, 140 mg/l nicotinic acid, 40 mg/l pyridoxole
hydrochloride, 200 mg/l myo-inositol). Lysozyme was added to the
suspension to a final concentration of 2.5 mg/ml. After an
approximately 4 h incubation at 37.degree. C., the cell wall was
degraded and the resulting protoplasts are harvested by
centrifugation. The pellet was washed once with 5 ml buffer-1 and
once with 5 ml TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The
pellet was resuspended in 4 ml TE-buffer and 0.5 ml SDS solution
(10%) and 0.5 ml NaCl solution (5 M) are added. After adding of
proteinase K to a final concentration of 200 .mu.g/ml, the
suspension is incubated for ca. 18 h at 37.degree. C. The DNA was
purified by extraction with phenol,
phenol-chloroform-isoamylalcohol and chloroform-isoamylalcohol
using standard procedures. Then, the DNA was precipitated by adding
1/50 volume of 3 M sodium acetate and 2 volumes of ethanol,
followed by a 30 min incubation at -20.degree. C. and a 30 min
centrifugation at 12,000 rpm in a high speed centrifuge using a
SS34 rotor (Sorvall). The DNA was dissolved in 1 ml TE-buffer
containing 20 .mu.g/ml RNaseA and dialysed at 4.degree. C. against
1000 ml TE-buffer for at least 3 hours. During this time, the
buffer was exchanged 3 times. To aliquots of 0.4 ml of the dialysed
DNA solution, 0.4 ml of 2 M LiCl and 0.8 ml of ethanol are added.
After a 30 min incubation at -20.degree. C., the DNA was collected
by centrifugation (13,000 rpm, Biofuge Fresco, Heraeus, Hanau,
Germany). The DNA pellet was dissolved in TE-buffer. DNA prepared
by this procedure could be used for all purposes, including
southern blotting or construction of genomic libraries.
EXAMPLE 2
Construction of Genomic Libraries in Escherichia coli of
Corynebacterium glutamicum ATCC13032.
[0127] Using DNA prepared as described in Example 1, cosmid and
plasmid libraries were constructed according to known and well
established methods (see e.g., Sambrook, J. et al. (1989)
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press, or Ausubel, F. M. et al. (1994) "Current
Protocols in Molecular Biology", John Wiley & Sons.)
[0128] Any plasmid or cosmid could be used. Of particular use were
the plasmids pBR322 (Sutcliffe, J. G. (1979) Proc. Natl. Acad. Sci.
USA, 75:3737-3741); pACYC177 (Change & Cohen (1978) J.
Bacteriol 134:1141-1156), plasmids of the pBS series (pBSSK+,
pBSSK- and others; Stratagene, LaJolla, USA), or cosmids as
SuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T. J.,
Rosenthal A. and Waterson, R. H. (1987) Gene 53:283-286. Gene
libraries specifically for use in C. glutamicum may be constructed
using plasmid pSL109 (Lee, H.-S. and A. J. Sinskey (1994) J.
Microbiol. Biotechnol. 4: 256-263).
EXAMPLE 3
DNA Sequencing and Computational Functional Analysis
[0129] Genomic libraries as described in Example 2 were used for
DNA sequencing according to standard methods, in particular by the
chain termination method using ABI377 sequencing machines (see
e.g., Fleischman, R. D. et al. (1995) "Whole-genome Random
Sequencing and Assembly of Haemophilus Influenzae Rd., Science,
269:496-512). Sequencing primers with the following nucleotide
sequences were used: 5'-GGAAACAGTATGACCATG-3' or
5'-GTAAAACGACGGCCAGT-3'.
EXAMPLE 4
In vivo Mutagenesis
[0130] In vivo mutagenesis of Corynebacterium glutamicum can be
performed by passage of plasmid (or other vector) DNA through E.
coli or other microorganisms (e.g. Bacillus spp. or yeasts such as
Saccharomyces cerevisiae) which are impaired in their capabilities
to maintain the integrity of their genetic information. Typical
mutator strains have mutations in the genes for the DNA repair
system (e.g., mutHLS, mutD, mutT, etc.; for reference, see Rupp, W.
D. (1996) DNA repair mechanisms, in: Escherichia coli and
Salmonella, p. 2277-2294, ASM: Washington.) Such strains are well
known to those of ordinary skill in the art. The use of such
strains is illustrated, for example, in Greener, A. and Callahan,
M. (1994) Strategies 7: 32-34.
EXAMPLE 5
DNA Transfer Between Escherichia coli and Corynebacterium
glutamicum
[0131] Several Corynebacterium and Brevibacterium species contain
endogenous plasmids (as e.g., pHM1519 or pBL1) which replicate
autonomously (for review see, e.g., Martin, J. F. et al. (1987)
Biotechnology, 5:137-146). Shuttle vectors for Escherichia coli and
Corynebacterium glutamicum can be readily constructed by using
standard vectors for E. coli (Sambrook, J. et al. (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) to which a origin or
replication for and a suitable marker from Corynebacterium
glutamicum is added. Such origins of replication are preferably
taken from endogenous plasmids isolated from Corynebacterium and
Brevibacterium species. Of particular use as transformation markers
for these species are genes for kanamycin resistance (such as those
derived from the Tn5 or Tn903 transposons) or chloramphenicol
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology, VCH, Weinheim). There are numerous examples in the
literature of the construction of a wide variety of shuttle vectors
which replicate in both E. coli and C. glutamicum, and which can be
used for several purposes, including gene over-expression (for
reference, see e.g., Yoshihama, M. et al. (1985) J. Bacteriol.
162:591-597, Martin J. F. et al. (1987) Biotechnology, 5:137-146
and Eikmanns, B. J. et al. (1991) Gene, 102:93-98).
[0132] Using standard methods, it is possible to clone a gene of
interest into one of the shuttle vectors described above and to
introduce such a hybrid vectors into strains of Corynebacterium
glutamicum. Transformation of C. glutamicum can be achieved by
protoplast transformation (Kastsumata, R. et al. (1984) J.
Bacteriol. 159306-311), electroporation (Liebl, E. et al. (1989)
FEMS Microbiol. Letters, 53:399-303) and in cases where special
vectors are used, also by conjugation (as described e.g. in
Schafer, A et al. (1990) J. Bacteriol. 172:1663-1666). It is also
possible to transfer the shuttle vectors for C. glutamicum to E.
coli by preparing plasmid DNA from C. glutamicum (using standard
methods well-known in the art) and transforming it into E. coli.
This transformation step can be performed using standard methods,
but it is advantageous to use an Mcr-deficient E. coli strain, such
as NM522 (Gough & Murray (1983) J. Mol. Biol. 166:1-19).
[0133] Genes may be overexpressed in C. glutamicum strains using
plasmids which comprise pCG1 (U.S. Pat. No. 4,617,267) or fragments
thereof, and optionally the gene for kanamycin resistance from
TN903 (Grindley, N. D. and Joyce, C. M. (1980) Proc. Natl. Acad.
Sci. USA 77(12): 7176-7180). In addition, genes may be
overexpressed in C. glutamicum strains using plasmid pSL109 (Lee,
H.-S. and A. J. Sinskey (1994) J. Microbiol. Biotechnol. 4:
256-263).
[0134] Aside from the use of replicative plasmids, gene
overexpression can also be achieved by integration into the genome.
Genomic integration in C. glutamicum or other Corynebacterium or
Brevibacterium species may be accomplished by well-known methods,
such as homologous recombination with genomic region(s),
restriction endonuclease mediated integration (REMI) (see, e.g., DE
Patent 19823834), or through the use of transposons. It is also
possible to modulate the activity of a gene of interest by
modifying the regulatory regions (e.g., a promoter, a repressor,
and/or an enhancer) by sequence modification, insertion, or
deletion using site-directed methods (such as homologous
recombination) or methods based on random events (such as
transposon mutagenesis or REMI). Nucleic acid sequences which
function as transcriptional terminators may also be inserted 3' to
the coding region of one or more genes of the invention; such
terminators are well-known in the art and are described, for
example, in Winnacker, E. L. (1987) From Genes to
Clones--Introduction to Gene Technology. VCH: Weinheim.
EXAMPLE 6
Assessment of the Expression of the Mutant Protein
[0135] Observations of the activity of a mutated protein in a
transformed host cell rely on the fact that the mutant protein is
expressed in a similar fashion and in a similar quantity to that of
the wild-type protein. A useful method to ascertain the level of
transcription of the mutant gene (an indicator of the amount of
mRNA available for translation to the gene product) is to perform a
Northern blot (for reference see, for example, Ausubel et al.
(1988) Current Protocols in Molecular Biology, Wiley: N.Y.), in
which a primer designed to bind to the gene of interest is labeled
with a detectable tag (usually radioactive or chemiluminescent),
such that when the total RNA of a culture of the organism is
extracted, run on gel, transferred to a stable matrix and incubated
with this probe, the binding and quantity of binding of the probe
indicates the presence and also the quantity of mRNA for this gene.
This information is evidence of the degree of transcription of the
mutant gene. Total cellular RNA can be prepared from
Corynebacterium glutamicum by several methods, all well-known in
the art, such as that described in Bormann, E. R. et al. (1992)
Mol. Microbiol. 6: 317-326.
[0136] To assess the presence or relative quantity of protein
translated from this mRNA, standard techniques, such as a Western
blot, may be employed (see, for example, Ausubel et al. (1988)
Current Protocols in Molecular Biology, Wiley: N.Y.). In this
process, total cellular proteins are extracted, separated by gel
electrophoresis, transferred to a matrix such as nitrocellulose,
and incubated with a probe, such as an antibody, which specifically
binds to the desired protein. This probe is generally tagged with a
chemiluminescent or colorimetric label which may be readily
detected. The presence and quantity of label observed indicates the
presence and quantity of the desired mutant protein present in the
cell.
EXAMPLE 7
Growth of Genetically Modified Corynebacterium glutamicum--Media
and Culture Conditions
[0137] Genetically modified Corynebacteria are cultured in
synthetic or natural growth media. A number of different growth
media for Corynebacteria are both well-known and readily available
(Lieb et al. (1989) Appl. Microbiol. Biotechnol., 32:205-210; von
der Osten et al. (1998) Biotechnology Letters, 11:11-16; Patent DE
4,120,867; Liebl (1992) "The Genus Corynebacterium, in: The
Procaryotes, Volume II, Balows, A. et al., eds. Springer-Verlag).
These media consist of one or more carbon sources, nitrogen
sources, inorganic salts, vitamins and trace elements. Preferred
carbon sources are sugars, such as mono-, di-, or polysaccharides.
For example, glucose, fructose, mannose, galactose, ribose,
sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or
cellulose serve as very good carbon sources. It is also possible to
supply sugar to the media via complex compounds such as molasses or
other by-products from sugar refinement. It can also be
advantageous to supply mixtures of different carbon sources. Other
possible carbon sources are alcohols and organic acids, such as
methanol, ethanol, acetic acid or lactic acid. Nitrogen sources are
usually organic or inorganic nitrogen compounds, or materials which
contain these compounds. Exemplary nitrogen sources include ammonia
gas or ammonia salts, such as NH.sub.4Cl or
(NH.sub.4).sub.2SO.sub.4, NH.sub.4OH, nitrates, urea, amino acids
or complex nitrogen sources like corn steep liquor, soy bean flour,
soy bean protein, yeast extract, meat extract and others.
[0138] Inorganic salt compounds which may be included in the media
include the chloride-, phosphorous- or sulfate- salts of calcium,
magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc,
copper and iron. Chelating compounds can be added to the medium to
keep the metal ions in solution. Particularly useful chelating
compounds include dihydroxyphenols, like catechol or
protocatechuate, or organic acids, such as citric acid. It is
typical for the media to also contain other growth factors, such as
vitamins or growth promoters, examples of which include biotin,
riboflavin, thiamin, folic acid, nicotinic acid, pantothenate and
pyridoxin. Growth factors and salts frequently originate from
complex media components such as yeast extract, molasses, corn
steep liquor and others. The exact composition of the media
compounds depends strongly on the immediate experiment and is
individually decided for each specific case. Information about
media optimization is available in the textbook "Applied Microbiol.
Physiology, A Practical Approach (eds. P. M. Rhodes, P. F.
Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). It is
also possible to select growth media from commercial suppliers,
like standard 1 (Merck) or BHI (grain heart infusion, DIFCO) or
others.
[0139] All medium components are sterilized, either by heat (20
minutes at 1.5 bar and 121.degree. C.) or by sterile filtration.
The components can either be sterilized together or, if necessary,
separately. All media components can be present at the beginning of
growth, or they can optionally be added continuously or
batchwise.
[0140] Culture conditions are defined separately for each
experiment. The temperature should be in a range between 15.degree.
C. and 45.degree. C. The temperature can be kept constant or can be
altered during the experiment. The pH of the medium should be in
the range of 5 to 8.5, preferably around 7.0, and can be maintained
by the addition of buffers to the media. An exemplary buffer for
this purpose is a potassium phosphate buffer. Synthetic buffers
such as MOPS, HEPES, ACES and others can alternatively or
simultaneously be used. It is also possible to maintain a constant
culture pH through the addition of NaOH or NH.sub.4OH during
growth. If complex medium components such as yeast extract are
utilized, the necessity for additional buffers may be reduced, due
to the fact that many complex compounds have high buffer
capacities. If a fermentor is utilized for culturing the
micro-organisms, the pH can also be controlled using gaseous
ammonia.
[0141] The incubation time is usually in a range from several hours
to several days. This time is selected in order to permit the
maximal amount of product to accumulate in the broth. The disclosed
growth experiments can be carried out in a variety of vessels, such
as microtiter plates, glass tubes, glass flasks or glass or metal
fermentors of different sizes. For screening a large number of
clones, the microorganisms should be cultured in microtiter plates,
glass tubes or shake flasks, either with or without baffles.
Preferably 100 ml shake flasks are used, filled with 10% (by
volume) of the required growth medium. The flasks should be shaken
on a rotary shaker (amplitude 25 mm) using a speed-range of 100-300
rpm. Evaporation losses can be diminished by the maintenance of a
humid atmosphere; alternatively, a mathematical correction for
evaporation losses should be performed.
[0142] If genetically modified clones are tested, an unmodified
control clone or a control clone containing the basic plasmid
without any insert should also be tested. The medium is inoculated
to an OD.sub.600 of 0.5-1.5 using cells grown on agar plates, such
as CM plates (10 g/l glucose, 2,5 g/l NaCl, 2 g/l urea, 10 g/l
polypeptone, 5 g/l yeast extract, 5 g/l meat extract, 22 g/l NaCl,
2 g/l urea, 10 g/l polypeptone, 5 g/l yeast extract, 5 g/l meat
extract, 22 g/l agar, pH 6.8 with 2M NaOH) that had been incubated
at 30.degree. C. Inoculation of the media is accomplished by either
introduction of a saline suspension of C. glutamicum cells from CM
plates or addition of a liquid preculture of this bacterium.
EXAMPLE 8
In vitro Analysis of the Function of Mutant Proteins
[0143] The determination of activities and kinetic parameters of
enzymes is well established in the art. Experiments to determine
the activity of any given altered enzyme must be tailored to the
specific activity of the wild-type enzyme, which is well within the
ability of one of ordinary skill in the art. Overviews about
enzymes in general, as well as specific details concerning
structure, kinetics, principles, methods, applications and examples
for the determination of many enzyme activities may be found, for
example, in the following references: Dixon, M., and Webb, E. C.,
(1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure
and Mechanism. Freeman: New York; Walsh, (1979) Enzymatic Reaction
Mechanisms. Freeman: San Francisco; Price, N. C., Stevens, L.
(1982) Fundamentals of Enzymology. Oxford Univ. Press: Oxford;
Boyer, P. D., ed. (1983) The Enzymes, 3.sup.rd ed. Academic Press:
New York; Bisswanger, H., (1994) Enzymkinetik, 2.sup.nd ed. VCH:
Weinheim (ISBN 3527300325); Bergmeyer, H. U., Bergmeyer, J.,
Gral.beta.l, M., eds. (1983-1986) Methods of Enzymatic Analysis,
3.sup.rd ed., vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's
Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes".
VCH: Weinheim, p. 352-363.
[0144] The activity of proteins which bind to DNA can be measured
by several well-established methods, such as DNA band-shift assays
(also called gel retardation assays). The effect of such proteins
on the expression of other molecules can be measured using reporter
gene assays (such as that described in Kolmar, H. et al. (1995)
EMBO J. 14: 3895-3904 and references cited therein). Reporter gene
test systems are well known and established for applications in
both pro- and eukaryotic cells, using enzymes such as
beta-galactosidase, green fluorescent protein, and several
others.
[0145] The determination of activity of membrane-transport proteins
can be performed according to techniques such as those described in
Gennis, R. B. (1989) "Pores, Channels and Transporters", in
Biomembranes, Molecular Structure and Function, Springer:
Heidelberg, p. 85-137; 199-234; and 270-322.
EXAMPLE 9
Analysis of Impact of Mutant Protein on the Production of the
Desired Product
[0146] The effect of the genetic modification in C. glutamicum on
production of a desired compound (such as an amino acid) can be
assessed by growing the modified microorganism under suitable
conditions (such as those described above) and analyzing the medium
and/or the cellular component for increased production of the
desired product (i.e., an amino acid). Such analysis techniques are
well known to one of ordinary skill in the art, and include
spectroscopy, thin layer chromatography, staining methods of
various kinds, enzymatic and microbiological methods, and
analytical chromatography such as high performance liquid
chromatography (see, for example, Ullman, Encyclopedia of
Industrial Chemistry, vol. A2, p. 89-90 and p. 443-613, VCH:
Weinheim (1985); Fallon, A. et al., (1987) "Applications of HPLC in
Biochemistry" in: Laboratory Techniques in Biochemistry and
Molecular Biology, vol. 17; Rehm et al. (1993) Biotechnology, vol.
3, Chapter III: "Product recovery and purification", page 469-714,
VCH: Weinheim; Belter, P. A. et al. (1988) Bioseparations:
downstream processing for biotechnology, John Wiley and Sons;
Kennedy, J. F. and Cabral, J. M. S. (1992) Recovery processes for
biological materials, John Wiley and Sons; Shaeiwitz, J. A. and
Henry, J. D. (1988) Biochemical separations, in: Ulmann's
Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page
1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation and
purification techniques in biotechnology, Noyes Publications.)
[0147] In addition to the measurement of the final product of
fermentation, it is also possible to analyze other components of
the metabolic pathways utilized for the production of the desired
compound, such as intermediates and side-products, to determine the
overall efficiency of production of the compound. Analysis methods
include measurements of nutrient levels in the medium (e.g.,
sugars, hydrocarbons, nitrogen sources, phosphate, and other ions),
measurements of biomass composition and growth, analysis of the
production of common metabolites of biosynthetic pathways, and
measurement of gasses produced during fermentation. Standard
methods for these measurements are outlined in Applied Microbial
Physiology, A Practical Approach, P. M. Rhodes and P. F. Stanbury,
eds., IRL Press, p. 103-129; 131-163; and 165-192 (ISBN:
0199635773) and references cited therein.
EXAMPLE 10
Purification of the Desired Product from C. glutamicum Culture
[0148] Recovery of the desired product from the C. glutamicum cells
or supernatant of the above-described culture can be performed by
various methods well known in the art. If the desired product is
not secreted from the cells, the cells can be harvested from the
culture by low-speed centrifugation, the cells can be lysed by
standard techniques, such as mechanical force or sonication. The
cellular debris is removed by centrifligation, and the supernatant
fraction containing the soluble proteins is retained for further
purification of the desired compound. If the product is secreted
from the C. glutamicum cells, then the cells are removed from the
culture by low-speed centrifugation, and the supernate fraction is
retained for further purification.
[0149] The supernatant fraction from either purification method is
subjected to chromatography with a suitable resin, in which the
desired molecule is either retained on a chromatography resin while
many of the impurities in the sample are not, or where the
impurities are retained by the resin while the sample is not. Such
chromatography steps may be repeated as necessary, using the same
or different chromatography resins. One of ordinary skill in the
art would be well-versed in the selection of appropriate
chromatography resins and in their most efficacious application for
a particular molecule to be purified. The purified product may be
concentrated by filtration or ultrafiltration, and stored at a
temperature at which the stability of the product is maximized.
[0150] There are a wide array of purification methods known to the
art and the preceding method of purification is not meant to be
limiting. Such purification techniques are described, for example,
in Bailey, J. E. & Ollis, D. F. Biochemical Engineering
Fundamentals, McGraw-Hill: N.Y. (1986).
[0151] The identity and purity of the isolated compounds may be
assessed by techniques standard in the art. These include
high-performance liquid chromatography (HPLC), spectroscopic
methods, staining methods, thin layer chromatography, NIRS,
enzymatic assay, or microbiologically. Such analysis methods are
reviewed in: Patek et al. (1994) Appl. Environ. Microbiol. 60:
133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27-32; and
Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's
Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH:
Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, 575-581 and
p. 581-587; Michal, G. (1999) Biochemical Pathways: An Atlas of
Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A.
et al. (1987) Applications of HPLC in Biochemistry in: Laboratory
Techniques in Biochemistry and Molecular Biology, vol. 17.
EXAMPLE 11
Analysis of the Gene Sequences of the Invention
[0152] The comparison of sequences and determination of percent
homology between two sequences are art-known techniques, and can be
accomplished using a mathematical algorithm, such as the algorithm
of Karlin and Altschul (1990) Proc. Natl. Acad Sci. USA 87:2264-68,
modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci.
USA 90:5873-77. Such an algorithm is incorporated into the NBLAST
and XBLAST programs (version 2.0) of Altschul, et al. (1990) J.
Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed
with the NBLAST program, score=100, wordlength=12 to obtain
nucleotide sequences homologous to MCT nucleic acid molecules of
the invention. BLAST protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to MCT protein molecules of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al., (1997) Nucleic Acids
Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST
programs, one of ordinary skill in the art will know how to
optimize the parameters of the program (e.g., XBLAST and NBLAST)
for the specific sequence being analyzed.
[0153] Another example of a mathematical algorithm utilized for the
comparison of sequences is the algorithm of Meyers and Miller
((1988) Comput. Appl. Biosci. 4: 11-17). Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the GCG sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM 120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Additional algorithms for sequence analysis are known
in the art, and include ADVANCE and ADAM. described in Torelli and
Robotti (1994) Comput. Appl. Biosci. 10:3-5; and FASTA, described
in Pearson and Lipman (1988) P.N.A.S. 85:2444-8.
[0154] The percent homology between two amino acid sequences can
also be accomplished using the GAP program in the GCG software
package (available at http://www.gcg.com), using either a Blosum 62
matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4
and a length weight of 2, 3, or 4. The percent homology between two
nucleic acid sequences can be accomplished using the GAP program in
the GCG software package, using standard parameters, such as a gap
weight of 50 and a length weight of 3.
[0155] A comparative analysis of the gene sequences of the
invention with those present in Genbank has been performed using
techniques known in the art (see, e.g., Bexevanis and Ouellette,
eds. (1998) Bioinformatics: A Practical Guide to the Analysis of
Genes and Proteins. John Wiley and Sons: New York). The gene
sequences of the invention were compared to genes present in
Genbank in a three-step process. In a first step, a BLASTN analysis
(e.g., a local alignment analysis) was performed for each of the
sequences of the invention against the nucleotide sequences present
in Genbank, and the top 500 hits were retained for further
analysis. A subsequent FASTA search (e.g., a combined local and
global alignment analysis, in which limited regions of the
sequences are aligned) was performed on these 500 hits. Each gene
sequence of the invention was subsequently globally aligned to each
of the top three FASTA hits, using the GAP program in the GCG
software package (using standard parameters). In order to obtain
correct results, the length of the sequences extracted from Genbank
were adjusted to the length of the query sequences by methods
well-known in the art. The results of this analysis are set forth
in Table 4. The resulting data is identical to that which would
have been obtained had a GAP (global) analysis alone been performed
on each of the genes of the invention in comparison with each of
the references in Genbank, but required significantly reduced
computational time as compared to such a database-wide GAP (global)
analysis. Sequences of the invention for which no alignments above
the cutoff values were obtained are indicated on Table 4 by the
absence of alignment information. It will further be understood by
one of ordinary skill in the art that the GAP alignment homology
percentages set forth in Table 4 under the heading "% homology
(GAP)" are listed in the European numerical format, wherein a `,`
represents a decimal point. For example, a value of "40,345" in
this column represents "40.345%".
EXAMPLE 12
Construction and Operation of DNA Microarrays
[0156] The sequences of the invention may additionally be used in
the construction and application of DNA microarrays (the design,
methodology, and uses of DNA arrays are well known in the art, and
are described, for example, in Schena, M. et al. (1995) Science
270: 467-470; Wodicka, L. et al. (1997) Nature Biotechnology 15:
1359-1367; DeSaizieu, A. et al. (1998) Nature Biotechnology 16:
45-48; and DeRisi, J. L. et al. (1997) Science 278: 680-686).
[0157] DNA microarrays are solid or flexible supports consisting of
nitrocellulose, nylon, glass, silicone, or other materials. Nucleic
acid molecules may be attached to the surface in an ordered manner.
After appropriate labeling, other nucleic acids or nucleic acid
mixtures can be hybridized to the immobilized nucleic acid
molecules, and the label may be used to monitor and measure the
individual signal intensities of the hybridized molecules at
defined regions. This methodology allows the simultaneous
quantification of the relative or absolute amount of all or
selected nucleic acids in the applied nucleic acid sample or
mixture. DNA microarrays, therefore, permit an analysis of the
expression of multiple (as many as 6800 or more) nucleic acids in
parallel (see, e.g., Schena, M. (1996) BioEssays 18(5):
427-431).
[0158] The sequences of the invention may be used to design
oligonucleotide primers which are able to amplify defined regions
of one or more C. glutamicum genes by a nucleic acid amplification
reaction such as the polymerase chain reaction. The choice and
design of the 5' or 3' oligonucleotide primers or of appropriate
linkers allows the covalent attachment of the resulting PCR
products to the surface of a support medium described above (and
also described, for example, Schena, M. et al. (1995) Science 270:
467-470).
[0159] Nucleic acid microarrays may also be constructed by in situ
oligonucleotide synthesis as described by Wodicka, L. et al. (1997)
Nature Biotechnology 15: 1359-1367. By photolithographic methods,
precisely defined regions of the matrix are exposed to light.
Protective groups which are photolabile are thereby activated and
undergo nucleotide addition, whereas regions that are masked from
light do not undergo any modification. Subsequent cycles of
protection and light activation permit the synthesis of different
oligonucleotides at defined positions. Small, defined regions of
the genes of the invention may be synthesized on microarrays by
solid phase oligonucleotide synthesis.
[0160] The nucleic acid molecules of the invention present in a
sample or mixture of nucleotides may be hybridized to the
microarrays. These nucleic acid molecules can be labeled according
to standard methods. In brief, nucleic acid molecules (e.g., mRNA
molecules or DNA molecules) are labeled by the incorporation of
isotopically or fluorescently labeled nucleotides, e.g., during
reverse transcription or DNA synthesis. Hybridization of labeled
nucleic acids to microarrays is described (e.g., in Schena, M. et
al. (1995) supra; Wodicka, L. et al. (1997), supra; and DeSaizieu
A. et al. (1998), supra). The detection and quantification of the
hybridized molecule are tailored to the specific incorporated
label. Radioactive labels can be detected, for example, as
described in Schena, M. et al. (1995) supra) and fluorescent labels
may be detected, for example, by the method of Shalon et al. (1996)
Genome Research 6: 639-645).
[0161] The application of the sequences of the invention to DNA
microarray technology, as described above, permits comparative
analyses of different strains of C. glutamicum or other
Corynebacteria. For example, studies of inter-strain variations
based on individual transcript profiles and the identification of
genes that are important for specific and/or desired strain
properties such as pathogenicity, productivity and stress tolerance
are facilitated by nucleic acid array methodologies. Also,
comparisons of the profile of expression of genes of the invention
during the course of a fermentation reaction are possible using
nucleic acid array technology.
EXAMPLE 13
Analysis of the Dynamics of Cellular Protein Populations
(Proteomics)
[0162] The genes, compositions, and methods of the invention may be
applied to study the interactions and dynamics of populations of
proteins, termed `proteomics`. Protein populations of interest
include, but are not limited to, the total protein population of C.
glutamicum (e.g., in comparison with the protein populations of
other organisms), those proteins which are active under specific
environmental or metabolic conditions (e.g., during fermentation,
at high or low temperature, or at high or low pH), or those
proteins which are active during specific phases of growth and
development.
[0163] Protein populations can be analyzed by various well-known
techniques, such as gel electrophoresis. Cellular proteins may be
obtained, for example, by lysis or extraction, and may be separated
from one another using a variety of electrophoretic techniques.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) separates proteins largely on the basis of their
molecular weight. Isoelectric focusing polyacrylamide gel
electrophoresis (IEF-PAGE) separates proteins by their isoelectric
point (which reflects not only the amino acid sequence but also
posttranslational modifications of the protein). Another, more
preferred method of protein analysis is the consecutive combination
of both IEF-PAGE and SDS-PAGE, known as 2-D-gel electrophoresis
(described, for example, in Hermann et al. (1998) Electrophoresis
19: 3217-3221; Fountoulakis et al. (1998) Electrophoresis 19:
1193-1202; Langen et al. (1997) Electrophoresis 18: 1184-1192;
Antelmann et al. (1997) Electrophoresis 18: 1451-1463). Other
separation techniques may also be utilized for protein separation,
such as capillary gel electrophoresis; such techniques are well
known in the art.
[0164] Proteins separated by these methodologies can be visualized
by standard techniques, such as by staining or labeling. Suitable
stains are known in the art, and include Coomassie Brilliant Blue,
silver stain, or fluorescent dyes such as Sypro Ruby (Molecular
Probes). The inclusion of radioactively labeled amino acids or
other protein precursors (e.g., .sup.35S-methionine,
.sup.35S-cysteine, .sup.14C-labelled amino acids, .sup.15N-amino
acids, .sup.15NO.sub.3 or .sup.15NH.sub.4.sup.+ or
.sup.13C-labelled amino acids) in the medium of C. glutamicum
permits the labeling of proteins from these cells prior to their
separation. Similarly, fluorescent labels may be employed. These
labeled proteins can be extracted, isolated and separated according
to the previously described techniques.
[0165] Proteins visualized by these techniques can be further
analyzed by measuring the amount of dye or label used. The amount
of a given protein can be determined quantitatively using, for
example, optical methods and can be compared to the amount of other
proteins in the same gel or in other gels. Comparisons of proteins
on gels can be made, for example, by optical comparison, by
spectroscopy, by image scanning and analysis of gels, or through
the use of photographic films and screens. Such techniques are
well-known in the art.
[0166] To determine the identity of any given protein, direct
sequencing or other standard techniques may be employed. For
example, N- and/or C-terminal amino acid sequencing (such as Edman
degradation) may be used, as may mass spectrometry (in particular
MALDI or ESI techniques (see, e.g., Langen et al. (1997)
Electrophoresis 18: 1184-1192)). The protein sequences provided
herein can be used for the identification of C. glutamicum proteins
by these techniques.
[0167] The information obtained by these methods can be used to
compare patterns of protein presence, activity, or modification
between different samples from various biological conditions (e.g.,
different organisms, time points of fermentation, media conditions,
or different biotopes, among others). Data obtained from such
experiments alone, or in combination with other techniques, can be
used for various applications, such as to compare the behavior of
various organisms in a given (e.g., metabolic) situation, to
increase the productivity of strains which produce fine chemicals
or to increase the efficiency of the production of fine
chemicals.
Equivalents
[0168] Those of ordinary skill in the art will recognize, or will
be able to ascertain using no more than routine experimentation,
many equivalents to the specific embodiments of the invention
described herein. Such equivalents are intended to be encompassed
by the following claims. TABLE-US-00001 TABLE 1 GENES IN THE
APPLICATION Nucleic Amino Acid Acid SEQ SEQ ID ID Identification NT
NT NO NO Code Contig. Start Stop Function 1 2 RXN03097 VV0062 3 557
AMMONIUM TRANSPORT SYSTEM 3 4 RXA02099 GR00630 6198 6470 AMMONIUM
TRANSPORT SYSTEM 5 6 RXA00104 GR00014 15895 16650 CYSQ PROTEIN,
ammonium transport protein Polyketide Synthesis 7 8 RXA01420
GR00416 775 17 4''-MYCAROSYL ISOVALERYL-COA TRANSFERASE (EC
2.--.--.--) 9 10 RXN02581 VV0098 30482 28623 POLYKETIDE SYNTHASE 11
12 F RXA02581 GR00741 1 1527 POLYKETIDE SYNTHASE 13 14 RXA02582
GR00741 1890 6719 PROBABLE POLYKETIDE SYNTHASE CY338.20 15 16
RXA01138 GR00318 1656 2072 ACTINORHODIN POLYKETIDE DIMERASE (EC
--.--.--.--) 17 18 RXA01980 GR00573 1470 838 POLYKETIDE CYCLASE 19
20 RXN01007 VV0021 2572 866 FRNA 21 22 RXN00784 VV0103 27531 28265
FRNE Fatty acid and lipid synthesis 23 24 RXA02335 GR00672 550 2322
BIOTIN CARBOXYLASE (EC 6.3.4.14) 25 26 RXA02173 GR00641 7473 8924
ACETYL-COENZYME A CARBOXYLASE CARBOXYL TRANSFERASE SUBUNIT BETA (EC
6.4.1.2) 27 28 RXA01764 GR00500 2178 3110 3-OXOACYL-[ACYL-CARRIER
PROTEIN] REDUCTASE (EC 1.1.1.100) 29 30 RXN02487 VV0007 6367 4664
LONG-CHAIN-FATTY-ACID--COA LIGASE (EC 6.2.1.3) 31 32 F RXA02487
GR00718 4937 4650 LONG-CHAIN-FATTY-ACID--COA LIGASE (EC 6.2.1.3) 33
34 F RXA02490 GR00720 817 5 LONG-CHAIN-FATTY-ACID--COA LIGASE (EC
6.2.1.3) 35 36 RXA01467 GR00422 920 1210 ACYL CARRIER PROTEIN 37 38
RXA00796 GR00212 202 5 Acyl carrier protein phosphodiesterase 39 40
RXA01897 GR00544 617 1159 Acyl carrier protein phosphodiesterase 41
42 RXN02809 VV0342 380 6 Acyl carrier protein phosphodiesterase 43
44 F RXA02809 GR00790 277 5 Acyl carrier protein phosphodiesterase
45 46 RXN00113 VV0129 103 5724 FATTY ACID SYNTHASE (EC 2.3.1.85)
[INCLUDES: EC 2.3.1.38; EC 2.3.1.39; EC 2.3.1.41; 47 48 F RXA00113
GR00017 2 3295 FATTY-ACID SYNTHASE (EC 2.3.1.85) 49 50 RXN03111
VV0084 6040 5 FATTY ACID SYNTHASE (EC 2.3.1.85) [INCLUDES: EC
2.3.1.38; EC 2.3.1.39; EC 2.3.1.41; EC 1.1.1.100; EC 4.2.1.61; EC
1.3.1.10; EC 3.1.2.14] 51 52 F RXA00158 GR00024 2088 4 FATTY ACID
SYNTHASE (EC 2.3.1.85) 53 54 F RXA00572 GR00155 2 3832 FATTY ACID
SYNTHASE (EC 2.3.1.85) 55 56 RXA02582 GR00741 1890 6719 PROBABLE
POLYKETIDE SYNTHASE CY338.20 57 58 RXA02691 GR00754 15347 14541
FATTY ACYL RESPONSIVE REGULATOR 59 60 RXA00880 GR00242 6213 8057
LONG-CHAIN-FATTY-ACID--COA LIGASE (EC 6.2.1.3) 61 62 RXA01060
GR00296 9566 10489 OMEGA-3 FATTY ACID DESATURASE (EC 1.14.99.--) 63
64 RXN01722 VV0036 2938 1214 MEDIUM-CHAIN-FATTY-ACID--COA LIGASE
(EC 6.2.1.--) 65 66 F RXA01722 GR00488 5746 4022
MEDIUM-CHAIN-FATTY-ACID--COA LIGASE (EC 6.2.1.--) 67 68 RXA01644
GR00456 9854 8577 CYCLOPROPANE-FATTY-ACYL-PHOSPHOLIPID SYNTHASE (EC
2.1.1.79) 69 70 RXA02029 GR00618 356 1669
CYCLOPROPANE-FATTY-ACYL-PHOSPHOLIPID SYNTHASE (EC 2.1.1.79) 71 72
RXA01801 GR00509 3396 2380 ENOYL-COA HYDRATASE (EC 4.2.1.17) 73 74
RXN02512 VV0171 16147 15185 LIPID A BIOSYNTHESIS LAUROYL
ACYLTRANSFERASE (EC 2.3.1.--) 75 76 F RXA02512 GR00721 3303 4259
LIPID A BIOSYNTHESIS LAUROYL ACYLTRANSFERASE (EC 2.3.1.--) 77 78
RXA00899 GR00245 1599 2864 CARDIOLIPIN SYNTHETASE (EC 2.7.8.--) 79
80 RXN00819 VV0054 18127 19455 ACYL-COA DEHYDROGENASE (EC
1.3.99.--) 81 82 F RXA00819 GR00221 18 1007 ACYL-COA DEHYDROGENASE
(EC 1.3.99.--) 83 84 F RXA01766 GR00500 4081 4371 ACYL-COA
DEHYDROGENASE (EC 1.3.99.--) 85 86 RXN01762 VV0054 15318 13783
LONG-CHAIN-FATTY-ACID--COA LIGASE (EC 6.2.1.3) 87 88 F RXA01762
GR00500 1272 10 LONG-CHAIN-FATTY-ACID--COA LIGASE (EC 6.2.1.3) 89
90 RXA00681 GR00179 3405 2662 3-OXOACYL-[ACYL-CARRIER PROTEIN]
REDUCTASE (EC 1.1.1.100) 91 92 RXA00802 GR00214 3803 4516
3-OXOACYL-[ACYL-CARRIER PROTEIN] REDUCTASE (EC 1.1.1.100) 93 94
RXA02133 GR00639 3 308 3-OXOACYL-[ACYL-CARRIER PROTEIN] REDUCTASE
(EC 1.1.1.100) 95 96 RXN01114 VV0182 9118 10341 3-KETOACYL-COA
THIOLASE (EC 2.3.1.16) 97 98 F RXA01114 GR00308 2 793
3-KETOACYL-COA THIOLASE (EC 2.3.1.16) 99 100 RXA01894 GR00542 1622
2476 PHOSPHATIDATE CYTIDYLYLTRANSFERASE (EC 2.7.7.41) 101 102
RXA02599 GR00742 3179 3655 PHOSPHATIDYLGLYCEROPHOSPHATASE B (EC
3.1.3.27) 103 104 RXN02638 VV0098 54531 53656
1-ACYL-SN-GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE (EC 2.3.1.51) 105
106 F RXA02638 GR00749 8 511 1-ACYL-SN-GLYCEROL-3-PHOSPHATE
ACYLTRANSFERASE (EC 2.3.1.51) 107 108 RXA00856 GR00232 720 1256
CDP-DIACYLGLYCEROL--GLYCEROL-3-PHOSPHATE 3- PHOSPHATIDYLTRANSFERASE
(EC 2.7.8.5) 109 110 RXA02511 GR00721 2621 3277
CDP-DIACYLGLYCEROL--GLYCEROL-3-PHOSPHATE 3- PHOSPHATIDYLTRANSFERASE
(EC 2.7.8.5) 111 112 RXN02836 VV0102 32818 33372 KETOACYL REDUCTASE
HETN (EC 1.3.1.--) 113 114 F RXA02836 GR00827 106 411 KETOACYL
REDUCTASE HETN (EC 1.3.1.--) 115 116 RXA02578 GR00740 2438 3541
PUTATIVE ACYLTRANSFERASE 117 118 RXA02150 GR00639 18858 19658
1-ACYL-SN-GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE (EC 2.3.1.51) 119
120 RXA00607 GR00160 1869 2249 POLY(3-HYDROXYALKANOATE) POLYMERASE
(EC 2.3.1.--) 121 122 RXA02397 GR00698 1688 2683
POLY-BETA-HYDROXYBUTYRATE POLYMERASE (EC 2.3.1.--) 123 124 RXN03110
VV0083 16568 17929 HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3.1.2.6)
125 126 F RXA00660 GR00171 1027 5 HYDROXYACYLGLUTATHIONE HYDROLASE
(EC 3.1.2.6) 127 128 RXA00801 GR00214 3138 3770
HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3.1.2.6) 129 130 RXA00821
GR00221 1469 2311 HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3.1.2.6) 131
132 RXN02966 VV0143 12056 13462 HYDROXYACYLGLUTATHIONE HYDROLASE
(EC 3.1.2.6) 133 134 F RXA01833 GR00517 1666 260
HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3.1.2.6) 135 136 RXA01853
GR00525 5561 5010 HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3.1.2.6) 137
138 RXN02424 VV0116 10570 11169 HYDROXYACYLGLUTATHIONE HYDROLASE
(EC 3.1.2.6) 139 140 F RXA02424 GR00706 808 428
HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3.1.2.6) 141 142 RXN00419
VV0112 1024 266 ACETOACETYL-COA REDUCTASE (EC 1.1.1.36) 143 144 F
RXA00419 GR00095 3 464 ACETOACETYL-COA REDUCTASE (EC 1.1.1.36) 145
146 F RXA00421 GR00096 565 723 ACETOACETYL-COA REDUCTASE (EC
1.1.1.36) 147 148 RXN02923 VV0088 3301 2564 ACETOACETYL-COA
REDUCTASE (EC 1.1.1.36) 149 150 RXN02922 VV0321 11407 10328
ACYL-COA DEHYDROGENASE, SHORT-CHAIN SPECIFIC (EC 1.3.99.2) 151 152
RXN03065 VV0038 6237 6629 HOLO-[ACYL-CARRIER PROTEIN] SYNTHASE (EC
2.7.8.7) 153 154 RXN03132 VV0127 39053 39472
POLY-BETA-HYDROXYBUTYRATE POLYMERASE (EC 2.3.1.--) 155 156 RXN03157
VV0188 1607 1170 LIPOPOLYSACCHARIDE CORE BIOSYNTHESIS PROTEIN KDTB
157 158 RXN00934 VV0171 15181 14099 (AE000805) LPS biosynthesis
RfbU related protein [Methanobacterium thermoautotrophicum] 159 160
RXN00792 VV0321 10328 9132 ACYL-COA DEHYDROGENASE, SHORT-CHAIN
SPECIFIC (EC 1.3.99.2) 161 162 RXN00931 VV0171 13011 12166 ACYL-COA
THIOESTERASE II (EC 3.1.2.--) 163 164 F RXA00931 GR00253 4959 4114
thioesterase II 165 166 RXN01421 VV0122 16024 15638 ACYLTRANSFERASE
(EC 2.3.1.--) 167 168 RXN02342 VV0078 3460 4266
BIOTIN--[ACETYL-COA-CARBOXYLASE] SYNTHETASE (EC 6.3.4.15) 169 170
RXN00563 VV0038 1 2739 FATTY ACID SYNTHASE (EC 2.3.1.85) [INCLUDES:
EC 2.3.1.38; EC 2.3.1.39; EC 2.3.1.41; EC 1.1.1.100; EC 4.2.1.61;
EC 1.3.1.10; EC 3.1.2.14] 171 172 RXN02168 VV0100 2894 81 FATTY
ACID SYNTHASE (EC 2.3.1.85) [INCLUDES: EC 2.3.1.38; EC 2.3.1.39; EC
2.3.1.41; EC 1.1.1.100; EC 4.2.1.61; EC 1.3.1.10; EC 3.1.2.14] 173
174 RXN01090 VV0155 6483 5686 KETOACYL REDUCTASE HETN (EC 1.3.1.--)
175 176 RXN02062 VV0222 3159 1990 Lipopolysaccharide
N-acetylglucosaminyltransferase 177 178 RXN02148 VV0300 16561 17703
Lipopolysaccharide N-acetylglucosaminyltransferase 179 180 RXN02595
VV0098 11098 9935 Lipopolysaccharide
N-acetylglucosaminyltransferase 181 182 RXS00148 VV0167 9849 12059
METHYLMALONYL-COA MUTASE ALPHA-SUBUNIT (EC 5.4.99.2) 183 184
RXS00149 VV0167 7995 9842 METHYLMALONYL-COA MUTASE BETA-SUBUNIT (EC
5.4.99.2) 185 186 RXS02106 VV0123 22649 21594 LIPOATE-PROTEIN
LIGASE A (EC 6.--.--.--) 187 188 RXS01746 VV0185 934 1686
LIPOATE-PROTEIN LIGASE B (EC 6.--.--.--) 189 190 RXS01747 VV0185
1826 2869 LIPOIC ACID SYNTHETASE 191 192 RXC01748 VV0185 3001 3780
protein involved in lipid metabolism 193 194 RXC00354 VV0135 33604
32792 Cytosolic Protein involved in lipid metabolism 195 196
RXC01749 VV0185 3953 5569 Membrane Spanning Protein involved in
lipid metabolism Fatty acid degradation 197 198 RXA02268 GR00655
2182 3081 LIPASE (EC 3.1.1.3) 199 200 RXA02269 GR00655 3094 4065
LIPASE (EC 3.1.1.3) 201 202 RXA01614 GR00449 8219 7197
LYSOPHOSPHOLIPASE L2 (EC 3.1.1.5) 203 204 RXA01983 GR00573 3559
3053 LIPASE (EC 3.1.1.3) 205 206 RXN02947 VV0078 1319 6
PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC 6.4.1.3) 207 208 F
RXA02320 GR00667 593 6 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC
6.4.1.3) 209 210 F RXA02851 GR00851 524 6 PROPIONYL-COA CARBOXYLASE
BETA CHAIN (EC 6.4.1.3) 211 212 RXN02321 VV0078 3291 1663
PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC 6.4.1.3) 213 214 F
RXA02321 GR00667 1380 937 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC
6.4.1.3) 215 216 F RXA02343 GR00675 1403 1816 PROPIONYL-COA
CARBOXYLASE BETA CHAIN (EC 6.4.1.3) 217 218 F RXA02850 GR00850 2
493 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC 6.4.1.3) 219 220
RXA02583 GR00741 6743 8290 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC
6.4.1.3) 221 222 RXA00870 GR00239 809 2320
METHYLMALONATE-SEMIALDEHYDE DEHYDROGENASE (ACYLATING) (EC 1.2.1.27)
2-Methyl-3-oxopropanoate: NAD+ oxidoreductase (CoA-propanoylating)
223 224 RXA01260 GR00367 2381 1200 LIPOAMIDE DEHYDROGENASE
COMPONENT (E3) OF BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE
COMPLEX (EC 1.8.1.4) 225 226 RXA01261 GR00367 2607 2437 LIPOAMIDE
DEHYDROGENASE COMPONENT (E3) OF BRANCHED-CHAIN ALPHA-KETO ACID
DEHYDROGENASE COMPLEX (EC 1.8.1.4) 227 228 RXA01136 GR00318 685
1116 ISOVALERYL-COA DEHYDROGENASE (EC 1.3.99.10) 229 230 RXN00559
VV0103 7568 6552 PROTEIN VDLD 231 232 F RXA00559 GR00149 218 6
PROTEIN VDLD 233 234 RXA01580 GR00440 707 6 Glycerophosphoryl
diester phosphodiesterase 235 236 RXA02677 GR00754 3119 3877
GLYCEROPHOSPHORYL DIESTER PHOSPHODIESTERASE (EC 3.1.4.46) 237 238
RXS01166 VV0117 18142 16838 EXTRACELLULAR LIPASE PRECURSOR (EC
3.1.1.3) Terpenoid biosynthesis 239 240 RXA00875 GR00241 2423 1857
ISOPENTENYL-DIPHOSPHATE DELTA-ISOMERASE (EC 5.3.3.2) 241 242
RXA01292 GR00373 1204 2388 PHYTOENE DEHYDROGENASE (EC 1.3.--.--)
243 244 RXA01293 GR00373 2370 2696 PHYTOENE DEHYDROGENASE (EC
1.3.--.--) 245 246 RXA02310 GR00665 1132 2394 GERANYLGERANYL
HYDROGENASE 247 248 RXA02718 GR00758 18539 19585 GERANYLGERANYL
PYROPHOSPHATE SYNTHASE (EC 2.5.1.1) 249 250 RXA01067 GR00298 1453
2181 undecaprenyl-diphosphate synthase (EC 2.5.1.31) 251 252
RXA01269 GR00367 20334 19894 UNDECAPRENYL-PHOSPHATE
GALACTOSEPHOSPHOTRANSFERASE (EC 2.7.8.6) 253 254 RXA01205 GR00346 3
533 PUTATIVE UNDECAPRENYL-PHOSPHATE ALPHA-N-
ACETYLGLUCOSAMINYLTRANSFERASE (EC 2.4.1.--) 255 256 RXA01576
GR00438 8053 8811 DOLICHYL-PHOSPHATE BETA-GLUCOSYLTRANSFERASE (EC
2.4.1.117) 257 258 RXN02309 VV0025 28493 29542
OCTAPRENYL-DIPHOSPHATE SYNTHASE (EC 2.5.1.--) 259 260 F RXA02309
GR00665 978 4 OCTAPRENYL-DIPHOSPHATE SYNTHASE (EC 2.5.1.--) 261 262
RXN00477 VV0086 38905 37262 PHYTOENE DEHYDROGENASE (EC 1.3.--.--)
263 264 F RXA00477 GR00119 13187 11544 PHYTOENE DEHYDROGENASE (EC
1.3.--.--) 265 266 RXA00478 GR00119 14020 13190 PHYTOENE SYNTHASE
(EC 2.5.1.--) 267 268 RXA01291 GR00373 345 1277 PHYTOENE SYNTHASE
(EC 2.5.1.--)
269 270 RXA00480 GR00119 17444 16329 FARNESYL DIPHOSPHATE SYNTHASE
(EC 2.5.1.1) (EC 2.5.1.10) 271 272 RXS01879 VV0105 1505 573
isopentenyl-phosphate kinase (EC 2.7.4.--) 273 274 RXS02023 VV0160
3234 4001 P450 cytochrome, isopentenyltransf, ferridox 275 276
RXS00948 VV0107 4266 5384 12-oxophytodienoate reductase (EC
1.3.1.42) 277 278 RXS02228 VV0068 1876 2778 TRNA
DELTA(2)-ISOPENTENYLPYROPHOSPHATE TRANSFERASE (EC 2.5.1.8) 279 280
RXC01971 VV0105 4545 3715 Metal-Dependent Hydrolase involved in
metabolism of terpenoids 281 282 RXC02697 VV0017 31257 32783
membrane protein involved in metabolism of terpenoids
ABC-Transporter 283 284 RXN01946 VV0228 2 1276 Hypothetical ABC
Transporter ATP-Binding Protein 285 286 F RXA01946 GR00559 1849 575
(AL021184) ABC transporter ATP binding protein [Mycobacterium
tuberculosis] 287 288 RXN00164 VV0232 1782 94 Hypothetical ABC
Transporter ATP-Binding Protein 289 290 F RXA00164 GR00025 1782 94
, P, G, R ATPase subunits of ABC transporters 291 292 RXN00243
VV0057 28915 27899 , P, G, R ATPase subunits of ABC transporters
293 294 F RXA00243 GR00037 930 4 , P, G, R ATPase subunits of ABC
transporters 295 296 RXA00259 GR00039 8469 6268 , P, G, R ATPase
subunits of ABC transporters 297 298 RXN00410 VV0086 51988 51323
GLUTAMINE TRANSPORT ATP-BINDING PROTEIN GLNQ 299 300 F RXA00410
GR00092 829 164 , P, G, R ATPase subunits of ABC transporters 301
302 RXN00456 VV0076 6780 8156 , P, G, R ATPase subunits of ABC
transporters 303 304 F RXA00456 GR00114 316 5 , P, G, R ATPase
subunits of ABC transporters 305 306 F RXA00459 GR00115 1231 245 ,
P, G, R ATPase subunits of ABC transporters 307 308 RXN01604 VV0137
8117 7470 , P, G, R ATPase subunits of ABC transporters 309 310 F
RXA01604 GR00448 2 607 , P, G, R ATPase subunits of ABC
transporters 311 312 RXN02547 VV0057 27726 25588 , P, G, R ATPase
subunits of ABC transporters 313 314 F RXA02547 GR00726 22055 19932
, P, G, R ATPase subunits of ABC transporters 315 316 RXN02571
VV0101 12331 13359 MALTOSE/MALTODEXTRIN TRANSPORT ATP-BINDING
PROTEIN MALK 317 318 F RXA02571 GR00736 1469 2497 , P, G, R ATPase
subunits of ABC transporters 319 320 RXN02074 VV0318 12775 11153
TRANSPORT ATP-BINDING PROTEIN CYDD 321 322 F RXA02074 GR00628 5798
4176 , P, G, R ATPase subunits of ABC transporters 323 324 RXA02095
GR00629 14071 15474 , P, G, R ATPase subunits of ABC transporters
325 326 RXA02225 GR00652 3156 2275 , P, G, R ATPase subunits of ABC
transporters 327 328 RXA02253 GR00654 20480 21406 , P, G, R ATPase
subunits of ABC transporters 329 330 RXN01881 VV0105 529 95
Hypothetical ABC Transporter ATP-Binding Protein 331 332 F RXA01881
GR00537 3092 3532 ATPase components of ABC transporters with
duplicated ATPase domains 333 334 RXA00526 GR00136 1353 664
Hypothetical ABC Transporter ATP-Binding Protein 335 336 RXN00733
VV0132 1647 2531 Hypothetical ABC Transporter ATP-Binding Protein
337 338 F RXA00733 GR00197 411 4 Hypothetical ABC Transporter
ATP-Binding Protein 339 340 RXA00735 GR00198 849 181 Hypothetical
ABC Transporter ATP-Binding Protein 341 342 RXA00878 GR00242 3733
1871 Hypothetical ABC Transporter ATP-Binding Protein 343 344
RXN01191 VV0169 10478 12067 Hypothetical ABC Transporter
ATP-Binding Protein 345 346 F RXA01191 GR00341 1571 165
Hypothetical ABC Transporter ATP-Binding Protein 347 348 RXN01212
VV0169 3284 4207 Hypothetical ABC Transporter ATP-Binding Protein
349 350 F RXA01212 GR00350 1 813 Hypothetical ABC Transporter
ATP-Binding Protein 351 352 RXA02749 GR00764 4153 5028 Hypothetical
ABC Transporter ATP-Binding Protein 353 354 RXA02224 GR00652 2271
475 Hypothetical ABC Transporter ATP-Binding Protein 355 356
RXN01602 VV0229 1109 2638 Hypothetical ABC Transporter ATP-Binding
Protein 357 358 RXN02515 VV0087 962 1717 Hypothetical ABC
Transporter ATP-Binding Protein 359 360 RXN00525 VV0079 26304 27566
Hypothetical ABC Transporter Permease Protein 361 362 RXN02096
VV0126 20444 22135 Hypothetical ABC Transporter Permease Protein
363 364 RXN00412 VV0086 53923 52844 Hypothetical Amino Acid ABC
Transporter ATP-Binding Protein 365 366 RXN00411 VV0086 52844 52170
Hypothetical Amino Acid ABC Transporter Permease Protein 367 368
RXN02614 VV0313 5964 5236 TAURINE TRANSPORT ATP-BINDING PROTEIN
TAUB 369 370 RXN02613 VV0313 5223 4267 TAURINE-BINDING PERIPLASMIC
PROTEIN PRECURSOR 371 372 RXN00368 VV0226 2300 726
SPERMIDINE/PUTRESCINE TRANSPORT ATP-BINDING PROTEIN POTA 373 374 F
RXA00368 GR00076 1 579 SPERMIDINE/PUTRESCINE TRANSPORT ATP-BINDING
PROTEIN POTA 375 376 F RXA00370 GR00077 6 803 SPERMIDINE/PUTRESCINE
TRANSPORT ATP-BINDING PROTEIN POTA 377 378 RXN01285 VV0215 1780
1055 FERRIC ENTEROBACTIN TRANSPORT ATP-BINDING PROTEIN FEPC 379 380
RXN00523 VV0194 1363 338 FERRIC ENTEROBACTIN TRANSPORT PROTEIN FEPG
381 382 RXN01142 VV0077 5805 6302 NITRATE TRANSPORT ATP-BINDING
PROTEIN NRTD 383 384 RXN01141 VV0077 4644 5468 NITRATE TRANSPORT
PROTEIN NRTA 385 386 RXN01002 VV0106 8858 8055 PHOSPHONATES
TRANSPORT ATP-BINDING PROTEIN PHNC 387 388 RXN01000 VV0106 7252
6407 PHOSPHONATES TRANSPORT SYSTEM PERMEASE PROTEIN PHNE 389 390
RXN01732 VV0106 9944 8895 PHOSPHONATES-BINDING PERIPLASMIC PROTEIN
PRECURSOR 391 392 RXN03080 VV0045 1670 2449 FERRIC ENTEROBACTIN
TRANSPORT ATP-BINDING PROTEIN FEPC 393 394 RXN03081 VV0045 2476
2934 FERRIENTEROBACTIN-BINDING PERIPLASMIC PROTEIN PRECURSOR 395
396 RXN03082 VV0045 3131 3451 FERRIENTEROBACTIN-BINDING PERIPLASMIC
PROTEIN PRECURSOR Other transporters 397 398 RXA02261 GR00654 30936
32291 AMMONIUM TRANSPORT SYSTEM 399 400 RXA02020 GR00613 1015 5
AROMATIC AMINO ACID TRANSPORT PROTEIN AROP 401 402 RXA00281 GR00043
4721 5404 BACITRACIN TRANSPORT ATP-BINDING PROTEIN BCRA 403 404
RXN00570 VV0147 855 4 BENZOATE MEMBRANE TRANSPORT PROTEIN 405 406 F
RXA00570 GR00153 1 498 BENZOATE MEMBRANE TRANSPORT PROTEIN 407 408
RXN00571 VV173 1298 42 BENZOATE MEMBRANE TRANSPORT PROTEIN 409 410
F RXA00571 GR00154 2 1186 BENZOATE MEMBRANE TRANSPORT PROTEIN 411
412 RXA00962 GR00268 2 667 BENZOATE MEMBRANE TRANSPORT PROTEIN 413
414 RXA02811 GR00792 177 560 BENZOATE MEMBRANE TRANSPORT PROTEIN
415 416 RXA02115 GR00635 2 1198 BENZOATE MEMBRANE TRANSPORT PROTEIN
417 418 RXN00590 VV0178 5043 6230 BRANCHED CHAIN AMINO ACID
TRANSPORT SYSTEM II CARRIER PROTEIN 419 420 F RXA00590 GR00157 178
564 BRANCHED CHAIN AMINO ACID TRANSPORT SYSTEM II CARRIER PROTEIN
421 422 F RXA01538 GR00427 5040 5429 BRANCHED CHAIN AMINO ACID
TRANSPORT SYSTEM II CARRIER PROTEIN 423 424 RXA01727 GR00489 1471
194 BRANCHED-CHAIN AMINO ACID TRANSPORT SYSTEM CARRIER PROTEIN 425
426 RXA00623 GR00163 6525 7862 C4-DICARBOXYLATE TRANSPORT PROTEIN
427 428 RXA01584 GR00441 55 597 CHROMATE TRANSPORT PROTEIN 429 430
RXA00852 GR00231 3137 2448 COBALT TRANSPORT ATP-BINDING PROTEIN
CBIO 431 432 RXA00690 GR00181 1213 68 COBALT TRANSPORT PROTEIN CBIQ
433 434 RXA00827 GR00223 1319 567 COBALT TRANSPORT PROTEIN CBIQ 435
436 RXA00851 GR00231 2448 1840 COBALT TRANSPORT PROTEIN CBIQ 437
438 RXS03220 D-XYLOSE-PROTON SYMPORT 439 440 F RXA02762 GR00768 346
630 D-XYLOSE PROTON-SYMPORTER 441 442 RXN00092 VV0129 27509 26844
GLUTAMINE TRANSPORT ATP-BINDING PROTEIN GLNQ 443 444 F RXA00092
GR00014 1 204 GLUTAMINE TRANSPORT ATP-BINDING PROTEIN GLNQ 445 446
RXN03060 VV0030 6227 5376 GLUTAMINE TRANSPORT ATP-BINDING PROTEIN
GLNQ 447 448 F RXA02618 GR00745 1914 2351 GLUTAMINE TRANSPORT
ATP-BINDING PROTEIN GLNQ 449 450 F RXA02900 GR10040 2979 2128
GLUTAMINE TRANSPORT ATP-BINDING PROTEIN GLNQ 451 452 RXS03212
GLYCINE BETAINE TRANSPORTER BETP 453 454 F RXA01591 GR00446 3 947
GLYCINE BETAINE TRANSPORTER BETP 455 456 RXN00201 VV0096 197 6 HIGH
AFFINITY RIBOSE TRANSPORT PROTEIN RBSD 457 458 F RXA00201 GR00032
191 6 HIGH AFFINITY RIBOSE TRANSPORT PROTEIN RBSD 459 460 RXA01221
GR00354 2108 2833 HIGH-AFFINITY BRANCHED-CHAIN AMINO ACID TRANSPORT
ATP-BINDING PROTEIN BRAG 461 462 RXA01222 GR00354 2844 3542
HIGH-AFFINITY BRANCHED-CHAIN AMINO ACID TRANSPORT ATP-BINDING
PROTEIN LIVF 463 464 RXA01219 GR00354 151 1032 HIGH-AFFINITY
BRANCHED-CHAIN AMINO ACID TRANSPORT PERMEASE PROTEIN LIVH 465 466
RXA01220 GR00354 1032 2108 HIGH-AFFINITY BRANCHED-CHAIN AMINO ACID
TRANSPORT PERMEASE PROTEIN LIVM 467 468 RXA00091 GR00013 7762 8514
IRON(III) DICITRATE TRANSPORT ATP-BINDING PROTEIN FECE 469 470
RXA00228 GR00032 29232 28642 IRON(III) DICITRATE TRANSPORT
ATP-BINDING PROTEIN FECE 471 472 RXA00346 GR00064 1054 1743
IRON(III) DICITRATE TRANSPORT ATP-BINDING PROTEIN FECE 473 474
RXA00524 GR00135 779 1111 IRON(III) DICITRATE TRANSPORT ATP-BINDING
PROTEIN FECE 475 476 RXA01823 GR00516 591 1367 IRON(III) DICITRATE
TRANSPORT ATP-BINDING PROTEIN FECE 477 478 RXA02767 GR00770 1032
1814 IRON(III) DICITRATE TRANSPORT ATP-BINDING PROTEIN FECE 479 480
RXA02792 GR00777 8581 7829 IRON(III) DICITRATE TRANSPORT
ATP-BINDING PROTEIN FECE 481 482 RXN02929 VV0090 36837 37874
IRON(III) DICITRATE TRANSPORT SYSTEM PERMEASE PROTEIN FECD 483 484
F RXA01235 GR00358 1165 194 IRON(III) DICITRATE TRANSPORT SYSTEM
PERMEASE PROTEIN FECD 485 486 RXN02794 VV0134 10625 9552 IRON(III)
DICITRATE TRANSPORT SYSTEM PERMEASE PROTEIN FECD 487 488 F RXA01419
GR00415 888 1151 IRON(III) DICITRATE TRANSPORT SYSTEM PERMEASE
PROTEIN FECD 489 490 F RXA02794 GR00777 10172 9552 IRON(III)
DICITRATE TRANSPORT SYSTEM PERMEASE PROTEIN FECD 491 492 RXN03079
VV0045 644 1660 IRON(III) DICITRATE TRANSPORT SYSTEM PERMEASE
PROTEIN FECD 493 494 F RXA02865 GR10007 3832 2816 IRON(III)
DICITRATE TRANSPORT SYSTEM PERMEASE PROTEIN FECD 495 496 RXA00181
GR00028 3954 2383 PROLINE TRANSPORT SYSTEM 497 498 RXA00591 GR00158
229 1581 PROLINE/BETAINE TRANSPORTER 499 500 RXA01629 GR00453 3476
1965 PROLINE/BETAINE TRANSPORTER 501 502 RXA02030 GR00618 3072 1687
PROLINE/BETAINE TRANSPORTER 503 504 RXA00186 GR00028 12242 12988
SHORT-CHAIN FATTY ACIDS TRANSPORTER 505 506 RXA00187 GR00028 13097
13447 SHORT-CHAIN FATTY ACIDS TRANSPORTER 507 508 RXA01667 GR00464
703 1908 SODIUM/GLUTAMATE SYMPORT CARRIER PROTEIN 509 510 RXA02171
GR00641 6571 4919 SODIUM/PROLINE SYMPORTER 511 512 RXA00902 GR00245
4643 5875 SODIUM-DEPENDENT PHOSPHATE TRANSPORT PROTEIN 513 514
RXA00941 GR00257 1999 683 sodium-dependent phosphate transport
protein 515 516 RXN00449 VV0112 30992 32572 Sodium-Dicarboxylate
Symport Protein 517 518 F RXA00449 GR00109 2040 1036
Sodium-Dicarboxylate Symport Protein 519 520 F RXA01755 GR00498 352
5 Sodium-Dicarboxylate Symport Protein 521 522 RXA00269 GR00041
1826 1038 SPERMIDINE/PUTRESCINE TRANSPORT ATP-BINDING PROTEIN POTA
523 524 RXA00369 GR00076 583 1299 SPERMIDINE/PUTRESCINE TRANSPORT
ATP-BINDING PROTEIN POTA 525 526 RXA02073 GR00628 4176 2647
TRANSPORT ATP-BINDING PROTEIN CYDC 527 528 RXA01399 GR00409 1 1119
TRANSPORT ATP-BINDING PROTEIN CYDD 529 530 RXA01339 GR00389 8408
7164 TYROSINE-SPECIFIC TRANSPORT PROTEIN 531 532 RXA02527 GR00725
5519 6847 2-OXOGLUTARATE/MALATE TRANSLOCATOR PRECURSOR 533 534
RXN00298 VV0176 40228 42072 HIGH-AFFINITY CHOLINE TRANSPORT PROTEIN
535 536 F RXA00298 GR00048 4459 6303 Ectoine/Proline/Glycine
betaine carrier ectP 537 538 RXA00596 GR00159 335 787 potassium
efflux system protein phaE 539 540 RXA02364 GR00686 841 215
C4-DICARBOXYLATE-BINDING PERIPLASMIC PROTEIN PRECURSOR, transport
protein 541 542 RXN01411 VV0050 26015 26779 SHIKIMATE TRANSPORTER
543 544 RXN00960 VV0075 1139 105 PROTON/SODIUM-GLUTAMATE SYMPORT
PROTEIN 545 546 RXN02447 VV0107 14297 13203 GALACTOSE-PROTON
SYMPORT
547 548 RXN02395 VV0176 16747 14858 GLYCINE BETAINE TRANSPORTER
BETP 549 550 RXN02348 VV0078 6027 7910 KUP SYSTEM POTASSIUM UPTAKE
PROTEIN 551 552 RXN00297 VV0176 38630 39541 Hypothetical Malonate
Transporter 553 554 RXN03103 VV0070 845 1087 GLUTAMATE-BINDING
PROTEIN PRECURSOR 555 556 RXN02993 VV0071 736 65 GLUTAMINE-BINDING
PROTEIN 557 558 RXN00349 VV0135 35187 36653 Hypothetical Trehalose
Transport Protein 559 560 RXN03095 VV0057 4056 4424 CADMIUM EFFLUX
SYSTEM ACCESSORY PROTEIN HOMOLOG 561 562 RXN03160 VV0189 5150 5617
CHROMATE TRANSPORT PROTEIN 563 564 RXN02955 VV0176 8666 9187
DICARBOXYLATE TRANSPORTER 565 566 RXN03109 VV0082 659 6 HEMIN
TRANSPORT SYSTEM PERMEASE PROTEIN HMUU 567 568 RXN02979 VV0149 2150
2383 MERCURIC TRANSPORT PROTEIN PERIPLASMIC COMPONENT PRECURSOR 569
570 RXN02987 VV0234 527 294 MERCURIC TRANSPORT PROTEIN PERIPLASMIC
COMPONENT PRECURSOR 571 572 RXN03084 VV0048 900 1817 IRON(III)
DICITRATE-BINDING PERIPLASMIC PROTEIN PRECURSOR 573 574 RXN03183
VV0372 1 417 TREHALOSE/MALTOSE BINDING PROTEIN 575 576 RXN01139
VV0077 2776 1823 CATION EFFLUX SYSTEM PROTEIN CZCD 577 578 RXN00378
VV0223 8027 5418 Cation transport ATPases 579 580 RXN01338 VV0032 2
1903 CATION-TRANSPORTING ATPASE PACS (EC 3.6.1.--) 581 582 RXN00980
VV0149 2635 4428 CATION-TRANSPORTING P-TYPE ATPASE B (EC 3.6.1.--)
583 584 RXN00099 VV0129 18876 17704 CYANATE TRANSPORT PROTEIN CYNX
585 586 RXN02662 VV0315 1461 1724 DIPEPTIDE TRANSPORT SYSTEM
PERMEASE PROTEIN DPPC 587 588 RXN02442 VV0217 5970 6818 zinc
transport system membrane protein 589 590 RXN02443 VV0217 6818 7771
zinc-binding periplasmic protein precursor 591 592 RXN00842 VV0138
8686 7487 BRANCHED CHAIN AMINO ACID TRANSPORT SYSTEM II CARRIER
PROTEIN 593 594 F RXA00842 GR00228 3208 2009 Permeases 595 596
RXN00832 VV0180 3133 4182 CALCIUM/PROTON ANTIPORTER 597 598
RXN00466 VV0086 63271 64266 Ferrichrome transport proteins 599 600
RXN01936 VV0127 40116 41387 MACROLIDE-EFFLUX PROTEIN 601 602
RXN01995 VV0182 2139 3476 PUTATIVE 3-(3-HYDROXYPHENYL) PROPIONATE
TRANSPORT PROTEIN 603 604 RXN00661 VV0142 9718 9029 PNUC PROTEIN
Permeases 605 606 RXN02566 VV0154 11823 13031 NUCLEOSIDE PERMEASE
NUPG 607 608 F RXA02561 GR00732 664 5 NUCLEOSIDE PERMEASE NUPG 609
610 F RXA02566 GR00733 782 345 NUCLEOSIDE PERMEASE NUPG 611 612
RXA00051 GR00008 5770 7173 PROLINE-SPECIFIC PERMEASE PROY 613 614
RXA01172 GR00334 2687 4141 SULFATE PERMEASE 615 616 RXA02128
GR00637 2906 4600 SULFATE PERMEASE 617 618 RXA02634 GR00748 6045
7655 SULFATE PERMEASE 619 620 RXN02233 VV0068 6856 8142 URACIL
PERMEASE 621 622 F RXA02233 GR00653 6856 8067 URACIL PERMEASE 623
624 RXN02372 VV0213 9311 11197 XANTHINE PERMEASE 625 626 F RXA02372
GR00688 6 560 XANTHINE PERMEASE 627 628 F RXA02377 GR00689 3336
4526 XANTHINE PERMEASE 629 630 RXA02676 GR00754 2697 1309 GLUCONATE
PERMEASE 631 632 RXN00432 VV0112 14751 13267 NA(+)-LINKED D-ALANINE
GLYCINE PERMEASE 633 634 F RXA00432 GR00100 1 891 NA(+)-LINKED
D-ALANINE GLYCINE PERMEASE 635 636 F RXA00436 GR00101 45 569
NA(+)-LINKED D-ALANINE GLYCINE PERMEASE 637 638 RXA00847 GR00230
1829 381 OLIGOPEPTIDE-BINDING PROTEIN APPA PRECURSOR (permease) 639
640 RXN01382 VV0119 8670 9761 OLIGOPEPTIDE-BINDING PROTEIN OPPA
PRECURSOR 641 642 F RXA01382 GR00405 1067 6 OLIGOPEPTIDE-BINDING
PROTEIN OPPA PRECURSOR (permease) 643 644 RXA02659 GR00753 2 313
OLIGOPEPTIDE-BINDING PROTEIN OPPA PRECURSOR (permease) 645 646
RXN02933 VV0176 30042 29233 DIPEPTIDE TRANSPORT SYSTEM PERMEASE
PROTEIN DPPC 647 648 RXN02991 VV0072 618 4 GLUTAMINE TRANSPORT
SYSTEM PERMEASE PROTEIN GLNP 649 650 RXN02992 VV0072 842 621
GLUTAMINE TRANSPORT SYSTEM PERMEASE PROTEIN GLNP 651 652 RXN02996
VV0069 1980 2648 HIGH-AFFINITY BRANCHED-CHAIN AMINO ACID TRANSPORT
PERMEASE PROTEIN LIVH 653 654 RXN03126 VV0112 9894 9001 TEICHOIC
ACID TRANSLOCATION PERMEASE PROTEIN TAGG 655 656 RXN00443 VV0112
21572 20769 MOLYBDATE-BINDING PERIPLASMIC PROTEIN PRECURSOR 657 658
RXN00444 VV0112 20785 19949 MOLYBDENUM TRANSPORT SYSTEM PERMEASE
PROTEIN MODB 659 660 RXN00193 VV0371 1 594 POTENTIAL STARCH
DEGRADATION PRODUCTS TRANSPORT SYSTEM PERMEASE PROTEIN AMYD 661 662
RXN01298 VV0116 2071 1142 POTENTIAL STARCH DEGRADATION PRODUCTS
TRANSPORT SYSTEM PERMEASE PROTEIN AMYD Channel Proteins 663 664
RXA01737 GR00493 2913 3971 POTASSIUM CHANNEL PROTEIN 665 666
RXN02348 VV0078 6027 7910 KUP SYSTEM POTASSIUM UPTAKE PROTEIN 667
668 RXA02426 GR00707 2165 633 PROBABLE NA(+)/H(+) ANTIPORTER 669
670 RXN03164 VV0277 1586 2455 POTASSIUM CHANNEL BETA SUBUNIT 671
672 RXN00024 VV0127 64219 63275 POTASSIUM CHANNEL BETA SUBUNIT
Lipoprotein and Lipopolysaccharide synthesis 673 674 RXN01164
VV0117 15894 14260 DOLICHOL-PHOSPHATE MANNOSYLTRANSFERASE (EC
2.4.1.83)/ APOLIPOPROTEIN N-ACYLTRANSFERASE (EC 2.3.1.--) 675 676
RXN01168 VV0117 14224 13415 DOLICHOL-PHOSPHATE MANNOSYLTRANSFERASE
(EC 2.4.1.83)/ APOLIPOPROTEIN N-ACYLTRANSFERASE (EC 2.3.1.--)
[0169] TABLE-US-00002 TABLE 2 GENES IDENTIFIED FROM GENBANK GenBank
.TM. Accession No. Gene Name Gene Function Reference A09073 ppg
Phosphoenol pyruvate carboxylase Bachmann, B. et al. "DNA fragment
coding for phosphoenolpyruvat corboxylase, recombinant DNA carrying
said fragment, strains carrying the recombinant DNA and method for
producing L-aminino acids using said strains," Patent: EP 0358940-A
3 Mar. 21, 1990 A45579, Threonine dehydratase Moeckel, B. et al.
"Production of L-isoleucine by means of recombinant A45581,
micro-organisms with deregulated threonine dehydratase," Patent: WO
A45583, 9519442-A 5 Jul. 20, 1995 A45585 A45587 AB003132 murC;
ftsQ; ftsZ Kobayashi, M. et al. "Cloning, sequencing, and
characterization of the ftsZ gene from coryneform bacteria,"
Biochem. Biophys. Res. Commun., 236(2): 383-388 (1997) AB015023
murC; ftsQ Wachi, M. et al. "A murC gene from Coryneform bacteria,
" Appl. Microbiol. Biotechnol., 51(2): 223-228 (1999) AB018530 dtsR
Kimura, E. et al. "Molecular cloning of a novel gene, dtsR, which
rescues the detergent sensitivity of a mutant derived from
Brevibacterium lactofermentum, " Biosci. Biotechnol. Biochem.,
60(10): 1565-1570 (1996) AB018531 dtsR1; dtsR2 AB020624 murI
D-glutamate racemase AB023377 tkt transketolase AB024708 gltB; gltD
Glutamine 2-oxoglutarate aminotransferase large and small subunits
AB025424 acn aconitase AB027714 rep Replication protein AB027715
rep; aad Replication protein; aminoglycoside adenyltransferase
AF005242 argC N-acetylglutamate-5-semialdehyde dehydrogenase
AF005635 glnA Glutamine synthetase AF030405 hisF cyclase AF030520
argG Argininosuccinate synthetase AF031518 argF Ornithine
carbamolytransferase AF036932 aroD 3-dehydroquinate dehydratase
AF038548 pyc Pyruvate carboxylase AF038651 dciAE; apt; rel
Dipeptide-binding protein; adenine Wehmeier, L. et al. "The role of
the Corynebacterium glutamicum rel gene in (p)ppGpp
phosphoribosyltransferase; GTP metabolism," Microbiology, 144:
1853-1862 (1998) pyrophosphokinase AF041436 argR Arginine repressor
AF045998 impA Inositol monophosphate phosphatase AF048764 argH
Argininosuccinate lyase AF049897 argC; argJ; argB;
N-acetylglutamylphosphate reductase; argD; argF; argR; ornithine
acetyltransferase; N- argG; argH acetylglutamate kinase;
acetylornithine transminase; ornithine carbamoyltransferase;
arginine repressor; argininosuccinate synthase; argininosuccinate
lyase AF050109 inhA Enoyl-acyl carrier protein reductase AF050166
hisG ATP phosphoribosyltransferase AF051846 hisA
Phosphoribosylformimino-5-amino-1-
phosphoribosyl-4-imidazolecarboxamide isomerase AF052652 metA
Homoserine O-acetyltransferase Park, S. et al. "Isolation and
analysis of metA, a methionine biosynthetic gene encoding
homoserine acetyltransferase in Corynebacterium glutamicum," Mol.
Cells., 8(3): 286-294 (1998) AF053071 aroB Dehydroquinate
synthetase AF060558 hisH Glutamine amidotransferase AF086704 hisE
Phosphoribosyl-ATP- pyrophosphohydrolase AF114233 aroA
5-enolpyruvylshikimate 3-phosphate synthase AF116184 panD
L-aspartate-alpha-decarboxylase precursor Dusch, N. et al.
"Expression of the Corynebacterium glutamicum panD gene encoding
L-aspartate-alpha-decarboxylase leads to pantothenate
overproduction in Escherichia coli," Appl. Environ. Microbiol.,
65(4)1530-1539 (1999) AF124518 aroD; aroE 3-dehydroquinase;
shikimate dehydrogenase AF124600 aroC; aroK; aroB; Chorismate
synthase; shikimate kinase; 3- pepQ dehydroquinate synthase;
putative cytoplasmic peptidase AF145897 inhA AF145898 inhA AJ001436
ectP Transport of ectoine, glycine betaine, Peter, H. et al.
"Corynebacterium glutamicum is equipped with four secondary
carriers proline for compatible solutes: Identification,
sequencing, and characterization of the proline/ectoine uptake
system, ProP, and the ectoine/proline/glycine betaine carrier,
EctP," J. Bacteriol., 180(22): 6005-6012 (1998) AJ004934 dapD
Tetrahydrodipicolinate succinylase Wehrmann, A. et al. "Different
modes of diaminopimelate synthesis and their (incomplete.sup.i)
role in cell wall integrity: A study with Corynebacterium
glutamicum," J. Bacteriol., 180(12): 3159-3165 (1998) AJ007732 ppc;
secG; amt; ocd; Phosphoenolpyruvate-carboxylase; ?; high soxA
affinity ammonium uptake protein; putative
ornithine-cyclodecarboxylase; sarcosine oxidase AJ010319 ftsY,
glnB, glnD; srp; amtP Involved in cell division; PII protein;
Jakoby, M. et al. "Nitrogen regulation in Corynebacterium
glutamicum ; uridylyltransferase (uridylyl-removing Isolation of
genes involved in biochemical characterization of corresponding
enzmye); signal recognition particle; low proteins," FEMS
Microbiol., 173(2): 303-310 (1999) affinity ammonium uptake protein
AJ132968 cat Chloramphenicol aceteyl transferase AJ224946 mqo
L-malate: quinone oxidoreductase Molenaar, D. et al. "Biochemical
and genetic characterization of the membrane-associated malate
dehydrogenase (acceptor) from Corynebacterium glutamicum," Eur. J.
Biochem., 254(2): 395-403 (1998) AJ238250 ndh NADH dehydrogenase
AJ238703 porA Porin Lichtinger, T. et al. "Biochemical and
biophysical characterization of the cell wall porin of
Corynebacterium glutamicum: The channel is formed by a low
molecular mass polypeptide," Biochemistry, 37(43): 15024-15032
(1998) D17429 Transposable element IS31831 Vertes, A. A. et al.
"Isolation and characterization of IS31831, a transposable element
from Corynebacterium glutamicum," Mol. Microbiol., 11(4): 739-746
(1994) D84102 odhA 2-oxoglutarate dehydrogenase Usuda, Y. et al.
"Molecular cloning of the Corynebacterium glutamicum
(Brevibacterium lactofermentum AJ12036) odhA gene encoding a novel
type of 2-oxoglutarate dehydrogenase," Microbiology, 142: 3347-3354
(1996) E01358 hdh; hk Homoserine dehydrogenase; homoserine
Katsumata, R. et al. "Production of L-thereonine and L-isoleucine,"
Patent: JP kinase 1987232392-A 1 Oct. 12, 1987 E01359 Upstream of
the start codon of homoserine Katsumata, R. et al. "Production of
L-thereonine and L-isoleucine," Patent: JP kinase gene 1987232392-A
2 Oct. 12, 1987 E01375 Tryptophan operon E01376 trpL; trpE Leader
peptide; anthranilate synthase Matsui, K. et al. "Tryptophan
operon, peptide and protein coded thereby, utilization of
tryptophan operon gene expression and production of tryptophan,"
Patent: JP 1987244382-A 1 Oct. 24, 1987 E01377 Promoter and
operator regions of Matsui, K. et al. "Tryptophan operon, peptide
and protein coded thereby, tryptophan operon utilization of
tryptophan operon gene expression and production of tryptophan,"
Patent: JP 1987244382-A 1 Oct. 24, 1987 E03937 Biotin-synthase
Hatakeyama, K. et al. "DNA fragment containing gene capable of
coding biotin synthetase and its utilization," Patent: JP
1992278088-A 1 Oct. 02, 1992 E04040 Diamino pelargonic acid
aminotransferase Kohama, K. et al. "Gene coding diaminopelargonic
acid aminotransferase and desthiobiotin synthetase and its
utilization," Patent: JP 1992330284-A 1 Nov. 18, 1992 E04041
Desthiobiotinsynthetase Kohama, K. et al. "Gene coding
diaminopelargonic acid aminotransferase and desthiobiotin
synthetase and its utilization," Patent: JP 1992330284-A 1 Nov. 18,
1992 E04307 Flavum aspartase Kurusu, Y. et al. "Gene DNA coding
aspartase and utilization thereof," Patent: JP 1993030977-A 1 Feb.
09, 1993 E04376 Isocitric acid lyase Katsumata, R. et al. "Gene
manifestation controlling DNA," Patent: JP 1993056782-A 3 Mar. 09,
1993 A09073 ppg Phosphoenol pyruvate carboxylase Bachmann, B. et
al. "DNA fragment coding for phosphoenolpyruvat E04377 Isocitric
acid lyase N-terminal fragment Katsumata, R. et al. "Gene
manifestation controlling DNA," Patent: JP 1993056782-A 3 Mar. 09,
1993 E04484 Prephenate dehydratase Sotouchi, N. et al. "Production
of L-phenylalanine by fermentation," Patent: JP 1993076352-A 2 Mar.
30, 1993 E05108 Aspartokinase Fugono, N. et al. "Gene DNA coding
Aspartokinase and its use," Patent: JP 1993184366-A 1 Jul. 27, 1993
E05112 Dihydro-dipichorinate synthetase Hatakeyama, K. et al. "Gene
DNA coding dihydrodipicolinic acid synthetase and its use," Patent:
JP 1993184371-A 1 Jul. 27, 1993 E05776 Diaminopimelic acid
dehydrogenase Kobayashi, M. et al. "Gene DNA coding Diaminopimelic
acid dehydrogenase and its use," Patent: JP 1993284970-A 1 Nov. 02,
1993 E05779 Threonine synthase Kohama, K. et al. "Gene DNA coding
threonine synthase and its use," Patent: JP 1993284972-A 1 Nov. 02,
1993 E06110 Prephenate dehydratase Kikuchi, T. et al. "Production
of L-phenylalanine by fermentation method," Patent: JP 1993344881-A
1 Dec. 27, 1993 E06111 Mutated Prephenate dehydratase Kikuchi, T.
et al. "Production of L-phenylalanine by fermentation method,"
Patent: JP 1993344881-A 1 Dec. 27, 1993 E06146 Acetohydroxy acid
synthetase Inui, M. et al. "Gene capable of coding Acetohydroxy
acid synthetase and its use," Patent: JP 1993344893-A 1 Dec. 27,
1993 E06825 Aspartokinase Sugimoto, M. et al. "Mutant aspartokinase
gene," patent: JP 1994062866-A 1 Mar. 08, 1994 E06826 Mutated
aspartokinase alpha subunit Sugimoto, M. et al. "Mutant
aspartokinase gene," patent: JP 1994062866-A 1 Mar. 08, 1994 E06827
Mutated aspartokinase alpha subunit Sugimoto, M. et al. "Mutant
aspartokinase gene," patent: JP 1994062866-A 1 Mar. 08, 1994 E07701
secY Honno, N. et al. "Gene DNA participating in integration of
membraneous protein to membrane," Patent: JP 1994169780-A 1 Jun.
21, 1994 E08177 Aspartokinase Sato, Y. et al. "Genetic DNA capable
of coding Aspartokinase released from feedback inhibition and its
utilization," Patent: JP 1994261766-A 1 Sep. 20, 1994 E08178,
Feedback inhibition-released Aspartokinase Sato, Y. et al. "Genetic
DNA capable of coding Aspartokinase released from E08179, feedback
inhibition and its utilization," Patent: JP 1994261766-A 1 Sep. 20,
1994 E08180, E08181, E08182 E08232 Acetohydroxy-acid
isomeroreductase Inui, M. et al. "Gene DNA coding acetohydroxy acid
isomeroreductase," Patent: JP 1994277067-A 1 Oct. 04, 1994 E08234
secE Asai, Y. et al. "Gene DNA coding for translocation machinery
of protein," Patent: JP 1994277073-A 1 Oct. 04, 1994 E08643 FT
aminotransferase and desthiobiotin Hatakeyama, K. et al. "DNA
fragment having promoter function in synthetase promoter region
coryneform bacterium," Patent: JP 1995031476-A 1 Feb. 03, 1995
E08646 Biotin synthetase Hatakeyama, K. et al. "DNA fragment having
promoter function in coryneform bacterium," Patent: JP 1995031476-A
1 Feb. 03, 1995 E08649 Aspartase Kohama, K. et al "DNA fragment
having promoter function in coryneform bacterium," Patent: JP
1995031478-A 1 Feb. 03, 1995 E08900 Dihydrodipicolinate reductase
Madori, M. et al. "DNA fragment containing gene coding
Dihydrodipicolinate acid reductase and utilization thereof,"
Patent: JP 1995075578-A 1 Mar. 20, 1995 E08901 Diaminopimelic acid
decarboxylase Madori, M. et al. "DNA fragment containing gene
coding Diaminopimelic acid decarboxylase and utilization thereof,"
Patent: JP 1995075579-A 1 Mar.
20, 1995 E12594 Serine hydroxymethyltransferase Hatakeyama, K. et
al. "Production of L-trypophan," Patent: JP 1997028391-A 1 Feb. 4,
1997 E12760, transposase Moriya, M. et al. "Amplification of gene
using artificial transposon," Patent: E12759, JP 1997070291-A Mar.
18, 1997 E12758 E12764 Arginyl-tRNA synthetase; diaminopimelic
Moriya, M. et al. "Amplification of gene using artificial
transposon," Patent: acid decarboxylase JP 1997070291-A Mar. 18,
1997 E12767 Dihydrodipicolinic acid synthetase Moriya, M. et al.
"Amplification of gene using artificial transposon," Patent: JP
1997070291-A Mar. 18, 1997 E12770 aspartokinase Moriya, M. et al.
"Amplification of gene using artificial transposon," Patent: JP
1997070291-A Mar. 18, 1997 E12773 Dihydrodipicolinic acid reductase
Moriya, M. et al. "Amplification of gene using artificial
transposon," Patent: JP 1997070291-A Mar. 18, 1997 E13655
Glucose-6-phosphate dehydrogenase Hatakeyama, K. et al.
"Glucose-6-phosphate dehydrogenase and DNA capable of coding the
same," Patent: JP 1997224661-A 1 Sep. 02, 1997 L01508 IlvA
Threonine dehydratase Moeckel, B. et al. "Functional and structural
analysis of the threonine dehydratase of Corynebacterium
glutamicum," J. Bacteriol., 174: 8065-8072 (1992) L07603 EC
4.2.1.15 3-deoxy-D-arabinoheptulosonate-7- Chen, C. et al. "The
cloning and nucleotide sequence of Corynebacterium phosphate
synthase glutamicum 3-deoxy-D-arabinoheptulosonate-7-phosphate
synthase gene," FEMS Microbiol. Lett., 107: 223-230 (1993) L09232
IlvB; ilvN; ilvC Acetohydroxy acid synthase large subunit;
Keilhauer, C. et al. "Isoleucine synthesis in Corynebacterium
glutamicum: Acetohydroxy acid synthase small subunit; molecular
analysis of the ilvB-ilvN-ilvC operon," J. Bacteriol., 175(17):
5595-5603 Acetohydroxy acid isomeroreductase (1993) L18874 PtsM
Phosphoenolpyruvate sugar Fouet, A et al. "Bacillus subtilis
sucrose-specific enzyme II of the phosphotransferase
phosphotransferase system: expression in Escherichia coli and
homology to enzymes II from enteric bacteria," PNAS USA, 84(24):
8773-8777 (1987); Lee, J. K. et al. "Nucleotide sequence of the
gene encoding the Corynebacterium glutamicum mannose enzyme II and
analyses of the deduced protein sequence," FEMS Microbiol. Lett.,
119(1-2): 137-145 (1994) L27123 aceB Malate synthase Lee, H-S. et
al. "Molecular characterization of aceB, a gene encoding malate
synthase in Corynebacterium glutamicum," J. Microbiol. Biotechnol.,
4(4): 256-263 (1994) L27126 Pyruvate kinase Jetten, M. S. et al.
"Structural and functional analysis of pyruvate kinase from
Corynebacterium glutamicum," Appl. Environ. Microbiol., 60(7):
2501-2507 (1994) L28760 aceA Isocitrate lyase L35906 dtxr
Diphtheria toxin repressor Oguiza, J. A. et al. "Molecular cloning,
DNA sequence analysis, and characterization of the Corynebacterium
diphtheriae dtxR from Brevibacterium lactofermentum," J.
Bacteriol., 177(2): 465-467 (1995) M13774 Prephenate dehydratase
Follettie, M. T. et al. "Molecular cloning and nucleotide sequence
of the Corynebacterium glutamicum pheA gene," J. Bacteriol., 167:
695-702 (1986) M16175 5S rRNA Park, Y-H. et al. "Phylogenetic
analysis of the coryneform bacteria by 56 rRNA sequences," J.
Bacteriol., 169: 1801-1806 (1987) M16663 trpE Anthranilate
synthase, 5' end Sano, K. et al. "Structure and function of the trp
operon control regions of Brevibacterium lactofermentum , a
glutamic-acid-producing bacterium," Gene, 52: 191-200 (1987) M16664
trpA Tryptophan synthase, 3'end Sano, K. et al. "Structure and
function of the trp operon control regions of Brevibacterium
lactofermentum , a glutamic-acid-producing bacterium," Gene, 52:
191-200 (1987) M25819 Phosphoenolpyruvate carboxylase O'Regan, M.
et al. "Cloning and nucleotide sequence of the Phosphoenolpyruvate
carboxylase-coding gene of Corynebacterium glutamicum ATCC13032,"
Gene, 77(2): 237-251 (1989) M85106 23S rRNA gene insertion sequence
Roller, C. et al. "Gram-positive bacteria with a high DNA G + C
content are characterized by a common insertion within their 23S
rRNA genes," J. Gen. Microbiol., 138: 1167-1175 (1992) M85107, 23S
rRNA gene insertion sequence Roller, C. et al. "Gram-positive
bacteria with a high DNA G + C content are M85108 characterized by
a common insertion within their 23S rRNA genes," J. Gen.
Microbiol., 138: 1167-1175 (1992) M89931 aecD; brnQ; yhbw Beta C-S
lyase; branched-chain amino acid Rossol, I. et al. "The
Corynebacterium glutamicum aecD gene encodes a C-S uptake carrier;
hypothetical protein yhbw lyase with alpha, beta-elimination
activity that degrades aminoethylcysteine," J. Bacteriol., 174(9):
2968-2977 (1992); Tauch, A. et al. "Isoleucine uptake in
Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene
product," Arch. Microbiol., 169(4): 303-312 (1998) S59299 trp
Leader gene (promoter) Herry, D. M. et al. "Cloning of the trp gene
cluster from a tryptophan- hyperproducing strain of Corynebacterium
glutamicum: identification of a mutation in the trp leader
sequence," Appl. Environ. Microbiol., 59(3): 791-799 (1993) U11545
trpD Anthranilate phosphoribosyltransferase O'Gara, J. P. and
Dunican, L. K. (1994) Complete nucleotide sequence of the
Corynebacterium glutamicum ATCC 21850 tpD gene." Thesis,
Microbiology Department, University College Galway, Ireland. U13922
cglIM; cglIR; clgIIR Putative type II 5-cytosoine Schafer, A. et
al. "Cloning and characterization of a DNA region encoding a
methyltransferase; putative type II stress-sensitive restriction
system from Corynebacterium glutamicum ATCC restriction
endonuclease; putative type I or 13032 and analysis of its role in
intergeneric conjugation with Escherichia type III restriction
endonuclease coli," J. Bacteriol., 176(23): 7309-7319 (1994);
Schafer, A. et al. "The Corynebacterium glutamicum cglIM gene
encoding a 5-cytosine in an McrBC- deficient Escherichia coli
strain," Gene, 203(2): 95-101 (1997) U14965 recA U31224 ppx Ankri,
S. et al. "Mutations in the Corynebacterium glutamicum proline
biosynthetic pathway: A natural bypass of the proA step," J.
Bacteriol., 178(15): 4412-4419 (1996) U31225 proC L-proline: NADP+
5-oxidoreductase Ankri, S. et al. "Mutations in the Corynebacterium
glutamicum proline biosynthetic pathway: A natural bypass of the
proA step," J. Bacteriol., 178(15): 4412-4419 (1996) U31230 obg;
proB; unkdh ?; gamma glutamyl kinase; similar to D- Ankri, S. et
al. "Mutations in the Corynebacterium glutamicum proline isomer
specific 2-hydroxyacid biosynthetic pathway: A natural bypass of
the proA step," J. Bacteriol., dehydrogenases 178(15): 4412-4419
(1996) U31281 bioB Biotin synthase Serebriiskii, I. G., "Two new
members of the bio B superfamily: Cloning, sequencing and
expression of bio B genes of Methylobacillus flagellatum and
Corynebacterium glutamicum," Gene, 175: 15-22 (1996) U35023 thtR;
accBC Thiosulfate sulfurtransferase; acyl CoA Jager, W. et al. "A
Corynebacterium glutamicum gene encoding a two-domain carboxylase
protein similar to biotin carboxylases and biotin-carboxyl-carrier
proteins," Arch. Microbiol., 166(2); 76-82 (1996) U43535 cmr
Multidrug resistance protein Jager, W. et al. "A Corynebacterium
glutamicum gene conferring multidrug resistance in the heterologous
host Escherichia coli," J. Bacteriol., 179(7): 2449-2451 (1997)
U43536 clpB Heat shock ATP-binding protein U53587 aphA-3
3'5''-aminoglycoside phosphotransferase U89648 Corynebacterium
glutamicum unidentified sequence involved in histidine
biosynthesis, partial sequence X04960 trpA; trpB; trpC; trpD; trpE;
trpG; trpL Tryptophan operon Matsui, K. et al. "Complete nucleotide
and deduced amino acid sequences of the Brevibacterium
lactofermentum tryptophan operon," Nucleic Acids Res., 14(24):
10113-10114 (1986) X07563 lys A DAP decarboxylase
(meso-diaminopimelate Yeh, P. et al. "Nucleic sequence of the lysA
gene of Corynebacterium decarboxylase, EC 4.1.1.20) glutamicum and
possible mechanisms for modulation of its expression," Mol. Gen.
Genet., 212(1): 112-119 (1988) X14234 EC 4.1.1.31
Phosphoenolpyruvate carboxylase Eikmanns, B. J. et al. "The
Phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum:
Molecular cloning, nucleotide sequence, and expression," Mol. Gen.
Genet., 218(2): 330-339 (1989); Lepiniec, L. et al. "Sorghum
Phosphoenolpyruvate carboxylase gene family: structure, function
and molecular evolution," Plant. Mol. Biol., 21 (3): 487-502 (1993)
X17313 fda Fructose-bisphosphate aldolase Von der Osten, C. H. et
al. "Molecular cloning, nucleotide sequence and fine- structural
analysis of the Corynebacterium glutamicum fda gene: structural
comparison of C. glutamicum fructose-1,6-biphosphate aldolase to
class I and class II aldolases," Mol. Microbiol., X53993 dapA
L-2,3-dihydrodipicolinate synthetase (EC Bonnassie, S. et al.
"Nucleic sequence of the dapA gene from 4.2.1.52) Corynebacterium
glutamicum," Nucleic Acids Res., 18(21): 6421 (1990) X54223
AttB-related site Cianciotto, N. et al. "DNA sequence homology
between att B-related sites of Corynebacterium diphtheriae,
Corynebacterium ulcerans, Corynebacterium glutamicum, and the attP
site of lambdacorynephage," FEMS. Microbiol, Lett., 66: 299-302
(1990) X54740 argS; lysA Arginyl-tRNA synthetase; Diaminopimelate
Marcel, T. et al. "Nucleotide sequence and organization of the
upstream region decarboxylase of the Corynebacterium glutamicum
lysA gene," Mol. Microbiol., 4(11): 1819-1830 (1990) X55994 trpL;
trpE Putative leader peptide; anthranilate Heery, D. M. et al.
"Nucleotide sequence of the Corynebacterium glutamicum synthase
component 1 trpE gene," Nucleic Acids Res., 18(23): 7138 (1990)
X56037 thrC Threonine synthase Han, K. S. et al. "The molecular
structure of the Corynebacterium glutamicum threonine synthase
gene," Mol. Microbiol., 4(10): 1693-1702 (1990) X56075 attB-related
site Attachment site Cianciotto, N. et al. "DNA sequence homology
between att B-related sites of Corynebacterium diphtheriae,
Corynebacterium ulcerans , Corynebacterium glutamicum, and the attP
site of lambdacorynephage," FEMS. Microbiol, Lett., 66: 299-302
(1990) X57226 lysC-alpha; lysC-beta; Aspartokinase-alpha subunit;
Kalinowski, J. et al. "Genetic and biochemical analysis of the
Aspartokinase asd Aspartokinase-beta subunit; aspartate beta from
Corynebacterium glutamicum," Mol. Microbiol., 5(5): 1197-1204
(1991); semialdehyde dehydrogenase Kalinowski, J. et al.
"Aspartokinase genes lysC alpha and lysC beta overlap and are
adjacent to the aspertate beta-semialdehyde dehydrogenase gene asd
in Corynebacterium glutamicum," Mol. Gen. Genet., 224(3): 317-324
(1990) X59403 gap; pgk; tpi Glyceraldehyde-3-phosphate; Eikmanns,
B. J. "Identification, sequence analysis, and expression of a
phosphoglycerate kinase; triosephosphate Corynebacterium glutamicum
gene cluster encoding the three glycolytic isomerase enzymes
glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate
kinase, and triosephosphate isomeras," J. Bacteriol., 174(19):
6076-6086 (1992) X59404 gdh Glutamate dehydrogenase Bormann, E. R.
et al. "Molecular analysis of the Corynebacterium glutamicum gdh
gene encoding glutamate dehydrogenase," Mol. Microbiol., 6(3):
317-326 (1992) X60312 lysI L-lysine permease Seep-Feldhaus, A. H.
et al. "Molecular analysis of the Corynebacterium glutamicum lysI
gene involved in lysine uptake," Mol. Microbiol., 5(12): 2995-3005
(1991) X66078 cop1 Ps1 protein Joliff, G. et al. "Cloning and
nucleotide sequence of the csp1 gene encoding PS1, one of the two
major secreted proteins of Corynebacterium glutamicum: The deduced
N-terminal region of PS1 is similar to the Mycobacterium antigen 85
complex," Mol. Microbiol., 6(16): 2349-2362 (1992) X66112 glt
Citrate synthase Eikmanns, B. J. et al. "Cloning sequence,
expression and transcriptional analysis of the Corynebacterium
glutamicum gltA gene encoding citrate synthase," Microbiol., 140:
1817-1828 (1994)
X67737 dapB Dihydrodipicolinate reductase X69103 csp2 Surface layer
protein PS2 Peyret, J. L. et al. "Characterization of the cspB gene
encoding PS2, an ordered surface-layer protein in Corynebacterium
glutamicum," Mol. Microbiol., 9(1): 97-109 (1993) X69104 IS3
related insertion element Bonamy, C. et al. "Identification of
IS1206, a Corynebacterium glutamicum IS3-related insertion sequence
and phylogenetic analysis," Mol. Microbiol., 14(3): 571-581 (1994)
X70959 leuA Isopropylmalate synthase Patek, M. et al. "Leucine
synthesis in Corynebacterium glutamicum: enzyme activities,
structure of leuA, and effect of leuA inactivation on lysine
synthesis," Appl. Environ. Microbiol., 60(1): 133-140 (1994) X71489
icd Isocitrate dehydrogenase (NADP+) Eikmanns, B. J. et al.
"Cloning sequence analysis, expression, and inactivation of the
Corynebacterium glutamicum icd gene encoding isocitrate
dehydrogenase and biochemical characterization of the enzyme," J.
Bacteriol., 177(3): 774-782 (1995) X72855 GDHA Glutamate
dehydrogenase (NADP+) X75083, mtrA 5-methyltryptophan resistance
Heery, D. M. et al. "A sequence from a tryptophan-hyperproducing
strain of X70584 Corynebacterium glutamicum encoding resistance to
5-methyltryptophan," Biochem. Biophys. Res. Commun., 201(3):
1255-1262 (1994) X75085 recA Fitzpatrick, R. et al. "Construction
and characterization of recA mutant strains of Corynebacterium
glutamicum and Brevibacterium lactofermentum," Appl. Microbiol.
Biotechnol., 42(4): 575-580 (1994) X75504 aceA; thiX Partial
Isocitrate lyase; ? Reinscheid, D. J. et al. "Characterization of
the isocitrate lyase gene from Corynebacterium glutamicum and
biochemical analysis of the enzyme," J. Bacteriol., 176(12):
3474-3483 (1994) X76875 ATPase beta-subunit Ludwig, W. et al.
"Phylogenetic relationships of bacteria based on comparative
sequence analysis of elongation factor Tu and ATP-synthase
beta-subunit genes," Antonie Van Leeuwenhoek, 64: 285-305 (1993)
X77034 tuf Elongation factor Tu Ludwig, W. et al. "Phylogenetic
relationships of bacteria based on comparative sequence analysis of
elongation factor Tu and ATP-synthase beta-subunit genes," Antonie
Van Leeuwenhoek, 64: 285-305 (1993) X77384 recA Billman-Jacobe, H.
"Nucleotide sequence of a recA gene from Corynebacterium
glutamicum," DNA Seq., 4(6): 403-404 (1994) X78491 aceB Malate
synthase Reinscheid, D. J. et al. "Malate synthase from
Corynebacterium glutamicum pta-ack operon encoding
phosphotransacetylase: sequence analysis," Microbiology, 140:
3099-3108 (1994) X80629 16S rDNA 16S ribosomal RNA Rainey, F. A. et
al. "Phylogenetic analysis of the genera Rhodococcus and Norcardia
and evidence for the evolutionary origin of the genus Norcardia
from within the radiation of Rhodococcus species," Microbiol., 141:
523-528 (1995) X81191 gluA; gluB; gluC; Glutamate uptake system
Kronemeyer, W. et al. "Structure of the gluABCD cluster encoding
the gluD glutamate uptake system of Corynebacterium glutamicum," J.
Bacteriol., 177(5): 1152-1158 (1995) X81379 dapE
Succinyldiaminopimelate desuccinylase Wehrmann, A. et al. "Analysis
of different DNA fragments of Corynebacterium glutamicum
complementing dapE of Escherichia coli," Microbiology, 40: 3349-56
(1994) X82061 16S rDNA 16S ribosomal RNA Ruimy, R. et al.
"Phylogeny of the genus Corynebacterium deduced from analyses of
small-subunit ribosomal DNA sequences," Int. J. Syst. Bacteriol.,
45(4): 740-746 (1995) X82928 asd; lysC Aspartate-semialdehyde
dehydrogenase; ? Serebrijski, I. et al. "Multicopy suppression by
asd gene and osmotic stress- dependent complementation by
heterologous proA in proA mutants," J. Bacteriol., 177(24):
7255-7260 (1995) X82929 proA Gamma-glutamyl phosphate reductase
Serebrijski, I. et al. "Multicopy suppression by asd gene and
osmotic stress- dependent complementation by heterologous proA in
proA mutants," J. Bacteriol., 177(24): 7255-7260 (1995) X84257 16S
rDNA 16S ribosomal RNA Pascual, C. et al. "Phylogenetic analysis of
the genus Corynebacterium based on 16S rRNA gene sequences," Int.
J. Syst. Bacteriol., 45(4): 724-728 (1995) X85965 aroP; dapE
Aromatic amino acid permease; ? Wehrmann, A. et al. "Functional
analysis of sequences adjacent to dapE of Corynebacterium
glutamicum proline reveals the presence of aroP, which encodes the
aromatic amino acid transporter," J. Bacteriol., 177(20): 5991-5993
(1995) X86157 argB; argC; argD; Acetylglutamate kinase;
N-acetyl-gamma- Sakanyan, V. et al. "Genes and enzymes of the
acetyl cycle of arginine argF; argJ glutamyl-phosphate reductase;
biosynthesis in Corynebacterium glutamicum: enzyme evolution in the
early acetylornithine aminotransferase; ornithine steps of the
arginine pathway," Microbiology, 142: 99-108 (1996)
carbamoyltransferase; glutamate N- acetyltransferase X89084 pta;
ackA Phosphate acetyltransferase; acetate kinase Reinscheid, D. J.
et al. "Cloning, sequence analysis, expression and inactivation of
the Corynebacterium glutamicum pta-ack operon encoding
phosphotransacetylase and acetate kinase," Microbiology, 145:
503-513 (1999) X89850 attB Attachment site Le Marrec, C. et al.
"Genetic characterization of site-specific integration functions of
phi AAU2 infecting "Arthrobacter aureus C70," J. Bacteriol.,
178(7): 1996-2004 (1996) X90356 Promoter fragment F1 Patek, M. et
al. "Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90357 Promoter fragment F2 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90358 Promoter fragment F10 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90359 Promoter fragment F13 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90360 Promoter fragment F22 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90361 Promoter fragment F34 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90362 Promoter fragment F37 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90363 Promoter fragment F45 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90364 Promoter fragment F64 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90365 Promoter fragment F75 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90366 Promoter fragment PF101 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90367 Promoter fragment PF104 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90368 Promoter fragment PF109 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X93513 amt Ammonium transport system Siewe, R. M.
et al. "Functional and genetic characterization of the (methyl)
ammonium uptake carrier of Corynebacterium glutamicum," J. Biol.
Chem., 271(10): 5398-5403 (1996) X93514 betP Glycine betaine
transport system Peter, H. et al. "Isolation, characterization, and
expression of the Corynebacterium glutamicum betP gene, encoding
the transport system for the compatible solute glycine betaine," J.
Bacteriol., 178(17): 5229-5234 (1996) X95649 orf4 Patek, M. et al.
"Identification and transcriptional analysis of the dapB-ORF2-
dapA-ORF4 operon of Corynebacterium glutamicum, encoding two
enzymes involved in L-lysine synthesis," Biotechnol. Lett., 19:
1113-1117 (1997) X96471 lysE; lysG Lysine exporter protein; Lysine
export Vrljic, M. et al. "A new type of transporter with a new type
of cellular regulator protein function: L-lysine export from
Corynebacterium glutamicum," Mol. Microbiol., 22(5): 815-826 (1996)
X96580 panB; panC; xylB 3-methyl-2-oxobutanoate Sahm, H. et al.
"D-pantothenate synthesis in Corynebacterium glutamicum and
hydroxymethyltransferase; pantoate-beta- use of panBC and genes
encoding L-valine synthesis for D-pantothenate alanine ligase;
xylulokinase overproduction," Appl. Environ. Microbiol., 65(5):
1973-1979 (1999) X96962 Insertion sequence IS1207 and transposase
X99289 Elongation factor P Ramos, A. et al. "Cloning, sequencing
and expression of the gene encoding elongation factor P in the
amino-acid producer Brevibacterium lactofermentum (Corynebacterium
glutamicum ATCC 13869)," Gene, 198: 217-222 (1997) Y00140 thrB
Homoserine kinase Mateos, L. M. et al. "Nucleotide sequence of the
homoserine kinase (thrB) gene of the Brevibacterium
lactofermentum," Nucleic Acids Res., 15(9): 3922 (1987) Y00151 ddh
Meso-diaminopimelate D-dehydrogenase Ishino, S. et al. "Nucleotide
sequence of the meso-diaminopimelate D- (EC 1.4.1.16) dehydrogenase
gene from Corynebacterium glutamicum," Nucleic Acids Res., 15(9):
3917 (1987) Y00476 thrA Homoserine dehydrogenase Mateos, L. M. et
al. "Nucleotide sequence of the homoserine dehydrogenase (thrA)
gene of the Brevibacterium lactofermentum," Nucleic Acids Res.,
15(24): 10598 (1987) Y00546 hom; thrB Homoserine dehydrogenase;
homoserine Peoples, O. P. et al. "Nucleotide sequence and fine
structural analysis of the kinase Corynebacterium glutamicum
hom-thrB operon," Mol. Microbiol., 2(1): 63-72 (1988) Y08964 murC;
ftsQ/divD; ftsZ UPD-N-acetylmuramate-alanine ligase; Honrubia, M.
P. et al. "Identification, characterization, and chromosomal
division initiation protein or cell division organization of the
ftsZ gene from Brevibacterium lactofermentum," Mol. Gen. protein;
cell division protein Genet., 259(1): 97-104 (1998) Y09163 putP
High affinity proline transport system Peter, H. et al. "Isolation
of the putP gene of Corynebacterium glutamicumproline and
characterization of a low-affinity uptake system for compatible
solutes," Arch. Microbiol., 168(2): 143-151 (1997) Y09548 pyc
Pyruvate carboxylase Peters-Wendisch, P. G. et al. "Pyruvate
carboxylase from Corynebacterium glutamicum: characterization,
expression and inactivation of the pyc gene," Microbiology, 144:
915-927 (1998) Y09578 leuB 3-isopropylmalate dehydrogenase Patek,
M. et al. "Analysis of the leuB gene from Corynebacterium
glutamicum," Appl. Microbiol. Biotechnol., 50(1): 42-47 (1998)
Y12472 Attachment site bacteriophage Phi-16 Moreau, S. et al.
"Site-specific integration of corynephage Phi-16: The construction
of an integration vector," Microbiol., 145: 539-548 (1999) Y12537
proP Proline/ectoine uptake system protein Peter, H. et al.
"Corynebacterium glutamicum is equipped with four secondary
carriers for compatible solutes: Identification, sequencing, and
characterization of the proline/ectoine uptake system, ProP, and
the ectoine/proline/glycine betaine carrier, EctP," J. Bacteriol.,
180(22): 6005-6012 (1998) Y13221 glnA Glutamine synthetase I
Jakoby, M. et al. "Isolation of Corynebacterium glutamicum glnA
gene encoding glutamine synthetase I," FEMS Microbiol. Lett.,
154(1): 81-88 (1997) Y16642 lpd Dihydrolipoamide dehydrogenase
Y18059 Attachment site Corynephage 304L Moreau, S. et al. "Analysis
of the integration functions of φ 304L: An integrase module among
corynephages," Virology, 255(1): 150-159 (1999) Z21501 argS; lysA
Arginyl-tRNA synthetase; diaminopimelate Oguiza, J. A.
et al. "A gene encoding arginyl-tRNA synthetase is located in the
decarboxylase (partial) upstream region of the lysA gene in
Brevibacterium lactofermentum: Regulation of argS-lysA cluster
expression by arginine," J. Bacteriol., 175(22): 7356-7362 (1993)
Z21502 dapA; dapB Dihydrodipicolinate synthase; Pisabarro, A. et
al. "A cluster of three genes (dapA, orf2, and dapB) of
dihydrodipicolinate reductase Brevibacterium lactofermentum encodes
dihydrodipicolinate reductase, and a third polypeptide of unknown
function," J. Bacteriol., 175(9): 2743-2749 (1993) Z29563 thrC
Threonine synthase Malumbres, M. et al. "Analysis and expression of
the thrC gene of the encoded threonine synthase," Appl. Environ.
Microbiol., 60(7)2209-2219 (1994) Z46753 16S rDNA Gene for 16S
ribosomal RNA A09073 ppg Phosphoenol pyruvate carboxylase Bachmann,
B. et al. "DNA fragment coding for phosphoenolpyruvat Z49822 sigA
SigA sigma factor Oguiza, J. A. et al "Multiple sigma factor genes
in Brevibacterium lactofermentum: Characterization of sigA and
sigB," J. Bacteriol., 178(2): 550-553 (1996) Z49823 galE; dtxR
Catalytic activity UDP-galactose 4- Oguiza, J. A. et al "The galE
gene encoding the UDP-galactose 4-epimerase of epimerase;
diphtheria toxin regulatory Brevibacterium lactofermentum is
coupled transcriptionally to the dmdR protein gene," Gene, 177:
103-107 (1996) Z49824 orfl; sigB ?; SigB sigma factor Oguiza, J. A.
et al "Multiple sigma factor genes in Brevibacterium
lactofermentum: Characterization of sigA and sigB," J. Bacteriol.,
178(2): 550-553 (1996) Z66534 Transposase Correia, A. et al.
"Cloning and characterization of an IS-like element present in the
genome of Brevibacterium lactofermentum ATCC 13869," Gene, 170(1):
91-94 (1996) .sup.1A sequence for this gene was published in the
indicated reference. However, the sequence obtained by the
inventors of the present application is significantly longer than
the published version. It is believed that the published version
relied on an incorrect start codon, and thus represents only a
fragment of the actual coding region.
[0170] TABLE-US-00003 TABLE 3 Corynebacterium and Brevibacterium
Strains Which May be Used in the Practice of the Invention Genus
species ATCC FERM NRRL CECT NCIMB CBS NCTC DSMZ Brevibacterium
ammoniagenes 21054 Brevibacterium ammoniagenes 19350 Brevibacterium
ammoniagenes 19351 Brevibacterium ammoniagenes 19352 Brevibacterium
ammoniagenes 19353 Brevibacterium ammoniagenes 19354 Brevibacterium
ammoniagenes 19355 Brevibacterium ammoniagenes 19356 Brevibacterium
ammoniagenes 21055 Brevibacterium ammoniagenes 21077 Brevibacterium
ammoniagenes 21553 Brevibacterium ammoniagenes 21580 Brevibacterium
ammoniagenes 39101 Brevibacterium butanicum 21196 Brevibacterium
divaricatum 21792 P928 Brevibacterium flavum 21474 Brevibacterium
flavum 21129 Brevibacterium flavum 21518 Brevibacterium flavum
B11474 Brevibacterium flavum B11472 Brevibacterium flavum 21127
Brevibacterium flavum 21128 Brevibacterium flavum 21427
Brevibacterium flavum 21475 Brevibacterium flavum 21517
Brevibacterium flavum 21528 Brevibacterium flavum 21529
Brevibacterium flavum B11477 Brevibacterium flavum B11478
Brevibacterium flavum 21127 Brevibacterium flavum B11474
Brevibacterium healii 15527 Brevibacterium ketoglutamicum 21004
Brevibacterium ketoglutamicum 21089 Brevibacterium ketosoreductum
21914 Brevibacterium lactofermentum 70 Brevibacterium
lactofermentum 74 Brevibacterium lactofermentum 77 Brevibacterium
lactofermentum 21798 Brevibacterium lactofermentum 21799
Brevibacterium lactofermentum 21800 Brevibacterium lactofermentum
21801 Brevibacterium lactofermentum B11470 Brevibacterium
lactofermentum B11471 Brevibacterium lactofermentum 21086
Brevibacterium lactofermentum 21420 Brevibacterium lactofermentum
21086 Brevibacterium lactofermentum 31269 Brevibacterium linens
9174 Brevibacterium linens 19391 Brevibacterium linens 8377
Brevibacterium paraffinolyticum 11160 Brevibacterium spec. 717.73
Brevibacterium spec. 717.73 Brevibacterium spec. 14604
Brevibacterium spec. 21860 Brevibacterium spec. 21864
Brevibacterium spec. 21865 Brevibacterium spec. 21866
Brevibacterium spec. 19240 Corynebacterium acetoacidophilum 21476
Corynebacterium acetoacidophilum 13870 Corynebacterium
acetoglutamicum B11473 Corynebacterium acetoglutamicum B11475
Corynebacterium acetoglutamicum 15806 Corynebacterium
acetoglutamicum 21491 Corynebacterium acetoglutamicum 31270
Corynebacterium acetophilum B3671 Corynebacterium ammoniagenes 6872
2399 Corynebacterium ammoniagenes 15511 Corynebacterium fujiokense
21496 Corynebacterium glutamicum 14067 Corynebacterium glutamicum
39137 Corynebacterium glutamicum 21254 Corynebacterium glutamicum
21255 Corynebacterium glutamicum 31830 Corynebacterium glutamicum
13032 Corynebacterium glutamicum 14305 Corynebacterium glutamicum
15455 Corynebacterium glutamicum 13058 Corynebacterium glutamicum
13059 Corynebacterium glutamicum 13060 Corynebacterium glutamicum
21492 Corynebacterium glutamicum 21513 Corynebacterium glutamicum
21526 Corynebacterium glutamicum 21543 Corynebacterium glutamicum
13287 Corynebacterium glutamicum 21851 Corynebacterium glutamicum
21253 Corynebacterium glutamicum 21514 Corynebacterium glutamicum
21516 Corynebacterium glutamicum 21299 Corynebacterium glutamicum
21300 Corynebacterium glutamicum 39684 Corynebacterium glutamicum
21488 Corynebacterium glutamicum 21649 Corynebacterium glutamicum
21650 Corynebacterium glutamicum 19223 Corynebacterium glutamicum
13869 Corynebacterium glutamicum 21157 Corynebacterium glutamicum
21158 Corynebacterium glutamicum 21159 Corynebacterium glutamicum
21355 Corynebacterium glutamicum 31808 Corynebacterium glutamicum
21674 Corynebacterium glutamicum 21562 Corynebacterium glutamicum
21563 Corynebacterium glutamicum 21564 Corynebacterium glutamicum
21565 Corynebacterium glutamicum 21566 Corynebacterium glutamicum
21567 Corynebacterium glutamicum 21568 Corynebacterium glutamicum
21569 Corynebacterium glutamicum 21570 Corynebacterium glutamicum
21571 Corynebacterium glutamicum 21572 Corynebacterium glutamicum
21573 Corynebacterium glutamicum 21579 Corynebacterium glutamicum
19049 Corynebacterium glutamicum 19050 Corynebacterium glutamicum
19051 Corynebacterium glutamicum 19052 Corynebacterium glutamicum
19053 Corynebacterium glutamicum 19054 Corynebacterium glutamicum
19055 Corynebacterium glutamicum 19056 Corynebacterium glutamicum
19057 Corynebacterium glutamicum 19058 Corynebacterium glutamicum
19059 Corynebacterium glutamicum 19060 Corynebacterium glutamicum
19185 Corynebacterium glutamicum 13286 Corynebacterium glutamicum
21515 Corynebacterium glutamicum 21527 Corynebacterium glutamicum
21544 Corynebacterium glutamicum 21492 Corynebacterium glutamicum
B8183 Corynebacterium glutamicum B8182 Corynebacterium glutamicum
B12416 Corynebacterium glutamicum B12417 Corynebacterium glutamicum
B12418 Corynebacterium glutamicum B11476 Corynebacterium glutamicum
21608 Corynebacterium lilium P973 Corynebacterium nitrilophilus
21419 11594 Corynebacterium spec. P4445 Corynebacterium spec. P4446
Corynebacterium spec. 31088 Corynebacterium spec. 31089
Corynebacterium spec. 31090 Corynebacterium spec. 31090
Corynebacterium spec. 31090 Corynebacterium spec. 15954 20145
Corynebacterium spec. 21857 Corynebacterium spec. 21862
Corynebacterium spec. 21863 ATCC: American Type Culture Collection,
Rockville, MD, USA FERM: Fermentation Research Institute, Chiba,
Japan NRRL: ARS Culture Collection, Northern Regional Research
Laboratory, Peoria, IL, USA CECT: Coleccion Espanola de Cultivos
Tipo, Valencia, Spain NCIMB: National Collection of Industrial and
Marine Bacteria Ltd., Aberdeen, UK CBS: Centraalbureau voor
Schimmelcultures, Baarn, NL NCTC: National Collection of Type
Cultures, London, UK DSMZ: Deutsche Sammlung von Mikroorganismen
und Zellkulturen, Braunschweig, Germany For reference see Sugawara,
H. et al. (1993) World directory of collections of cultures of
microorganisms: Bacteria, fungi and yeasts (4.sup.th edn), World
federation for culture collections world data center on
microorganisms, Saimata, Japen.
[0171] TABLE-US-00004 TABLE 4 ALIGNMENT RESULTS % length homology
Date of ID # (NT) Genbank Hit Length Accession Name of Genbank Hit
Source of Genbank Hit (GAP) Deposit rxa00051 1527 GB_HTG3: AC009685
210031 AC009685 Homo sapiens chromosome 15 clone 91_E_13 map 15,
*** SEQUENCING IN Homo sapiens 34,247 29-Sep-99 PROGRESS ***, 27
unordered pieces. GB_HTG3: AC009685 210031 AC009685 Homo sapiens
chromosome 15 clone 91_E_13 map 15, *** SEQUENCING IN Homo sapiens
34,247 29-Sep-99 PROGRESS ***, 27 unordered pieces. GB_HTG7:
AC009511 271896 AC009511 Homo sapiens clone RP11-860B13, ***
SEQUENCING IN PROGRESS ***, 59 Homo sapiens 35,033 09-DEC-1999
unordered pieces. rxa00091 876 GB_BA1: D50453 146191 D50453
Bacillus subtilis DNA for 25-36 degree region containing the
amyE-srfA region, Bacillus subtilis 54,452 10-Feb-99 complete cds.
GB_BA1: SCI51 40745 AL109848 Streptomyces coelicolor cosmid I51.
Streptomyces coelicolor 36,806 16-Aug-99 A3(2) GB_BA1: ECOUW93
338534 U14003 Escherichia coli K-12 chromosomal region from 92.8 to
00.1 minutes. Escherichia coli 38,642 17-Apr-96 rxa00092 789
GB_BA1: SCH35 45396 AL078610 Streptomyces coelicolor cosmid H35.
Streptomyces coelicolor 49,934 4-Jun-99 GB_HTG3: AC011498_0 312343
AC011498 Homo sapiens chromosome 19 clone CIT978SKB_50L17, ***
SEQUENCING IN Homo sapiens 37,117 13-Dec-99 PROGRESS ***, 190
unordered pieces. GB_HTG3: AC011498_0 312343 AC011498 Homo sapiens
chromosome 19 clone CIT978SKB_50L17, *** SEQUENCING IN Homo sapiens
37,117 13-Dec-99 PROGRESS ***, 190 unordered pieces. rxa00104 879
GB_BA1: MTCY270 37586 Z95388 Mycobacterium tuberculosis H37Rv
complete genome; segment 96/162. Mycobacterium 36,732 10-Feb-99
tuberculosis GB_PL2: T24M8 68251 AF077409 Arabidopsis thaliana BAC
T24M8. Arabidopsis thaliana 37,150 3-Aug-98 GB_BA1: MTCY270 37586
Z95388 Mycobacterium tuberculosis H37Rv complete genome; segment
96/162. Mycobacterium 42,874 10-Feb-99 tuberculosis rxa00113 5745
GB_BA1: MAFASGEN 10520 X87822 B. ammoniagenes FAS gene.
Corynebacterium 68,381 03-OCT-1996 ammoniagenes GB_BA1: BAFASAA
10549 X64795 B. ammoniagenes FAS gene. Corynebacterium 57,259
14-OCT-1997 ammoniagenes GB_BA1: MTCY159 33818 Z83863 Mycobacterium
tuberculosis H37Rv complete genome; segment 111/162. Mycobacterium
39,870 17-Jun-98 tuberculosis rxa00164 1812 GB_HTG2: HSJ1153D9
118360 AL109806 Homo sapiens chromosome 20 clone RP5-1153D9, ***
SEQUENCING IN Homo sapiens 35,714 03-DEC-1999 PROGRESS ***, in
unordered pieces. GB_HTG2: HSJ1153D9 118360 AL109806 Homo sapiens
chromosome 20 clone RP5-1153D9, *** SEQUENCING IN Homo sapiens
35,714 03-DEC-1999 PROGRESS ***, in unordered pieces. GB_HTG2:
HSJ1153D9 118360 AL109806 Homo sapiens chromosome 20 clone
RP5-1153D9, *** SEQUENCING IN Homo sapiens 35,334 03-DEC-1999
PROGRESS ***, in unordered pieces. rxa00181 1695 GB_BA1: CGPUTP
3791 Y09163 C. glutamicum putP gene. Corynebacterium 100,000
8-Sep-97 glutamicum GB_BA2: U32814 10393 U32814 Haemophilus
influenzae Rd section 129 of 163 of the complete genome.
Haemophilus influenzae 36,347 29-MAY-1998 Rd GB_BA1: CGPUTP 3791
Y09163 C. glutamicum putP gene. Corynebacterium 37,454 8-Sep-97
glutamicum rxa00186 870 GB_PR3: AC004843 136655 AC004843 Homo
sapiens PAC clone DJ0612F12 from 7p12-p14, complete sequence. Homo
sapiens 37,315 5-Nov-98 GB_HTG2: HS745I14 133309 AL033532 Homo
sapiens chromosome 1 clone RP4-745I14 map q23.1-24.3, ***
SEQUENCING Homo sapiens 38,129 03-DEC-1999 IN PROGRESS ***, in
unordered pieces. GB_HTG2: HS745I14 133309 AL033532 Homo sapiens
chromosome 1 clone RP4-745I14 map q23.1-24.3, *** SEQUENCING Homo
sapiens 38,129 03-DEC-1999 IN PROGRESS ***, in unordered pieces.
rxa00187 474 GB_GSS10: AQ184082 506 AQ184082 HS_3216_A1_G08_T7 CIT
Approved Human Genomic Sperm Library D Homo Homo sapiens 37,297
1-Nov-98 sapiens genomic clone Plate = 3216 Col = 15 Row = M,
genomic survey sequence. GB_GSS1: CNS008ZZ 1101 AL052951 Drosophila
melanogaster genome survey sequence T7 end of BAC # BACR18L01 of
Drosophila melanogaster 34,120 3-Jun-99 RPCI-98 library from
Drosophila melanogaster (fruit fly), genomic survey sequence.
GB_GSS10: AQ184082 506 AQ184082 HS_3216_A1_G08_T7 CIT Approved
Human Genomic Sperm Library D Homo Homo sapiens 39,655 1-Nov-98
sapiens genomic clone Plate = 3216 Col = 15 Row = M, genomic survey
sequence. rxa00201 292 GB_PR3: HSJ824F16 139330 AL050325 Human DNA
sequence from clone 824F16 on chromosome 20, complete sequence.
Homo sapiens 34,520 23-Nov-99 GB_BA1: RCSECA 2724 X89411 R.
capsulatus DNA for secA gene. Rhodobacter capsulatus 38,163
6-Jan-96 GB_EST34: AV122904 242 AV122904 AV122904 Mus musculus
C57BL/6J 10-day embryo Mus musculus cDNA clone Mus musculus 38,889
1-Jul-99 2610529H07, mRNA sequence. rxa00228 714 GB_EST15: AA486042
515 AA486042 ab40c08.r1 Stratagene HeLa cell s3 937216 Homo sapiens
cDNA clone Homo sapiens 37,500 06-MAR-1998 IMAGE: 843278 5', mRNA
sequence. GB_EST15: AA486042 515 AA486042 ab40c08.r1 Stratagene
HeLa cell s3 937216 Homo sapiens cDNA clone Homo sapiens 38,816
06-MAR-1998 IMAGE: 843278 5', mRNA sequence. rxa00243 1140 GB_PR2:
CNS01DS5 101584 AL121655 BAC sequence from the SPG4 candidate
region at 2p21-2p22, complete sequence. Homo sapiens 37,001
29-Sep-99 GB_HTG3: AC011408 79332 AC011408 Homo sapiens clone
CIT978SKB_65D22, Homo sapiens 38,040 06-OCT-1999 *** SEQUENCING IN
PROGRESS ***, 10 unordered pieces. GB_HTG3: AC011408 79332 AC011408
Homo sapiens clone CIT978SKB_65D22, Homo sapiens 38,040 06-OCT-1999
*** SEQUENCING IN PROGRESS ***, 10 unordered pieces. rxa00259 2325
GB_HTG1: CEY62E10 254217 AL031580 Caenorhabditis elegans chromosome
IV clone Y62E10, *** SEQUENCING IN Caenorhabditis elegans 36,776
6-Sep-99 PROGRESS ***, in unordered pieces. GB_HTG1: CEY62E10
254217 AL031580 Caenorhabditis elegans chromosome IV clone Y62E10,
*** SEQUENCING IN Caenorhabditis elegans 36,776 6-Sep-99 PROGRESS
***, in unordered pieces. GB_PL2: YSCCHROMI 41988 L22015
Saccharomyces cerevisiae chromosome I centromere and right arm
sequence. Saccharomyces 39,260 05-MAR-1998 cerevisiae rxa00269 912
GB_HTG4: AC009974 219565 AC009974 Homo sapiens chromosome unknown
clone NH0459I19, WORKING DRAFT Homo sapiens 37,358 29-OCT-1999
SEQUENCE, in unordered pieces. GB_HTG4: AC009974 219565 AC009974
Homo sapiens chromosome unknown clone NH0459I19, WORKING DRAFT Homo
sapiens 37,358 29-OCT-1999 SEQUENCE, in unordered pieces. GB_BA1:
AB017508 32050 AB017508 Bacillus halodurans C-125 genomic DNA, 32
kb fragment, complete cds. Bacillus halodurans 44,622 14-Apr-99
rxa00281 766 GB_BA1: SCE8 24700 AL035654 Streptomyces coelicolor
cosmid E8. Streptomyces coelicolor 36,328 11-MAR-1999 GB_BA1:
SCU51332 3216 U51332 Streptomyces coelicolor histidine kinase
homolog (absA1) and response regulator Streptomyces coelicolor
39,089 14-Sep-96 homolog (absA2) genes, complete cds. GB_HTG4:
AC011122 187123 AC011122 Homo sapiens chromosome 8 clone 23_D_19
map 8, *** SEQUENCING IN Homo sapiens 38,658 14-OCT-1999 PROGRESS
***, 27 ordered pieces. rxa00298 1968 GB_BA1: CGECTP 2719 AJ001436
Corynebacterium glutamicum ectP gene. Corynebacterium 100,000
20-Nov-98 glutamicum GB_BA1: CGECTP 2719 AJ001436 Corynebacterium
glutamicum ectP gene. Corynebacterium 100,000 20-Nov-98 glutamicum
GB_EST24: AI234006 432 AI234006 EST230694 Normalized rat lung,
Bento Soares Rattus sp. cDNA clone RLUCU01 3' Rattus sp. 46,552
31-Jan-99 end, mRNA sequence. rxa00346 813 GB_BA1: SC2E9 20850
AL021530 Streptomyces coelicolor cosmid 2E9. Streptomyces
coelicolor 43,267 28-Jan-98 GB_BA1: SC9B1 24800 AL049727
Streptomyces coelicolor cosmid 9B1. Streptomyces coelicolor 44,613
27-Apr-99 GB_BA1: ECU70214 123171 U70214 Escherichia coli
chromosome minutes 4-6. Escherichia coli 39,490 21-Sep-96 rxa00368
1698 GB_BA2: AF065159 35209 AF065159 Bradyrhizobium japonicum
putative arylsulfatase (arsA), putative soluble lytic
Bradyrhizobium 40,409 27-OCT-1999 transglycosylase precursor
(sltA), dihydrodipicolinate synthase (dapA), MscL (mscL), japonicum
SmpB (smpB), BcpB (bcpB), RnpO (rnpO), RelA/SpoT homolog (relA),
PdxJ (pdxJ), and acyl carrier protein synthase AcpS (acpS) genes,
complete cds; prokaryotic type I signal peptidase SipF (sipF) gene,
sipF-sipS allele, complete cds; RNase III (rnc) gene, complete cds;
GTP-binding protein Era (era) gene, partial cds; and unknown genes.
GB_BA1: AEOCHIT1 6861 D63139 Aeromonas sp. gene for chitinase,
complete and partial cds. Aeromonas sp. 10S-24 38,577 13-Feb-99
GB_EST4: D62996 314 D62996 HUM347G01B Clontech human aorta polyA+
mRNA (#6572) Homo sapiens cDNA Homo sapiens 41,613 29-Aug-95 clone
GEN-347G01 5', mRNA sequence. rxa00369 817 GB_BA1: YP102KB 119443
AL031866 Yersinia pestis 102 kbases unstable region: from 1 to
119443. Yersinia pestis 35,396 4-Jan-99 GB_GSS8: AQ012142 501
AQ012142 8750H1A037010398 Cosmid library of chromosome II
Rhodobacter sphaeroides Rhodobacter sphaeroides 54,800 4-Jun-98
genomic clone 8750H1A037010398, genomic survey sequence. GB_HTG2:
AC005081 180096 AC005081 Homo sapiens clone RG270D13, ***
SEQUENCING IN PROGRESS ***, 18 Homo sapiens 45,786 12-Jun-98
unordered pieces. rxa00410 789 GB_BA1: ATPLOCC 8870 Z30328 A.
tumefaciens Ti plasmid pTiAch5 genes for OccR, OccQ, OccM, OccP,
OccT, Agrobacterium 46,490 10-OCT-1994 OoxB, OoxA and ornithine
cyclodeaminase. tumefaciens GB_BA2: U67591 9829 U67591
Methanococcus jannaschii section 133 of 150 of the complete genome.
Methanococcus 45,677 28-Jan-98 jannaschii GB_BA1: TIPOCCQMPJ 4350
M80607 Plasmid pTiA6 (from Agribacterium tumefaciens)
periplasmic-type octopine Plasmid pTiA6 46,490 24-Apr-96 permease
(occR, occQ, occM, occP, and occJ) and lysR-type regulatory protein
(occR) genes, complete cds. rxa00419 882 GB_BA2: MSU46844 16951
U46844 Mycobacterium smegmatis catalase-peroxidase (katG), putative
arabinosyl Mycobacterium 57,029 12-MAY-1997 transferase (embC,
embA, embB), genes complete cds and putative propionyl-coA
smegmatis carboxylase beta chain (pccB) genes, partial cds.
GB_EST28: AI513245 471 AI513245 GH13311.3prime GH Drosophila
melanogaster head pOT2 Drosophila melanogaster Drosophila
melanogaster 37,696 16-MAR-1999 cDNA clone GH13311 3prime, mRNA
sequence. GB_HTG4: AC010066 187240 AC010066 Drosophila melanogaster
chromosome 3L/72A4 clone RPCI98-25O1, *** Drosophila melanogaster
39,607 16-OCT-1999 SEQUENCING IN PROGRESS ***, 70 unordered pieces.
rxa00432 1608 GB_BA1: BSUB0015 218410 Z99118 Bacillus subtilis
complete genome (section 15 of 21): from 2795131 to 3013540.
Bacillus subtilis 49,810 26-Nov-97 GB_PL1: CAC35A5 42565 AL033396
C. albicans cosmid Ca35A5. Candida albicans 35,041 5-Nov-98
GB_EST13: AA336266 378 AA336266 EST40981 Endometrial tumor Homo
sapiens cDNA 5' end, mRNA sequence. Homo sapiens 39,733 21-Apr-97
rxa00449 1704 GB_HTG2: AC008199 124050 AC008199 Drosophila
melanogaster chromosome 3 clone BACR01K08 (D756) RPCI-98 01.K.8
Drosophila melanogaster 38,392 2-Aug-99 map 94D-94D strain y; cn bw
sp, *** SEQUENCING IN PROGRESS ***, 83 unordered pieces. GB_HTG2:
AC008199 124050 AC008199 Drosophila melanogaster chromosome 3 clone
BACR01K08 (D756) RPCI-98 01.K.8 Drosophila melanogaster 38,392
2-Aug-99 map 94D-94D strain y; cn bw sp, *** SEQUENCING IN PROGRESS
***, 83 unordered pieces. GB_RO: RATLNKP2 177 M22337 Rat link
protein gene, exon 2. Rattus sp. 40,678 27-Apr-93 rxa00456 1500
GB_GSS1: FR0030597 476 AL026966 Fugu rubripes GSS sequence, clone
091C22aF9, genomic survey sequence. Fugu rubripes 47,407 25-Jun-98
GB_GSS5: AQ786587 556 AQ786587 HS_3086_B1_H05_MR CIT Approved Human
Genomic Sperm Library D Homo Homo sapiens 38,406 3-Aug-99 sapiens
genomic clone Plate = 3086 Col = 9 Row = P, genomic survey
sequence. GB_GSS14: AQ526586 434 AQ526586 HS_5198_B1_B03_SP6E
RPCI-11 Human Male BAC Library Homo sapiens Homo sapiens 36,951
11-MAY-1999 genomic clone Plate = 774 Col = 5 Row = D, genomic
survey sequence. rxa00477 1767 GB_EST17: AA610489 407 AA610489
np93e05.s1 NCI_CGAP_Thy1 Homo sapiens cDNA clone IMAGE: 1133888
similar Homo sapiens 41,791 09-DEC-1997 to gb: M11353 HISTONE H3.3
(HUMAN);, mRNA sequence. GB_PR1: HSH33G4 1015 X05857 Human H3.3
gene exon 4. Homo sapiens 38,182 24-Jan-96 GB_EST30: AI637667 579
AI637667 tt10g11.x1 NCI_CGAP_GC6 Homo sapiens cDNA clone IMAGE:
2240420 3', Homo sapiens 35,417 27-Apr-99 mRNA sequence. rxa00478
954 GB_HTG3: AC008708 83932 AC008708 Homo sapiens chromosome 5
clone CIT978SKB_78F1, *** SEQUENCING IN Homo sapiens 38,769
3-Aug-99
PROGRESS ***, 12 unordered pieces. GB_HTG3: AC008708 83932 AC008708
Homo sapiens chromosome 5 clone CIT978SKB_78F1, *** SEQUENCING IN
Homo sapiens 38,769 3-Aug-99 PROGRESS ***, 12 unordered pieces.
GB_HTG3: AC008708 83932 AC008708 Homo sapiens chromosome 5 clone
CIT978SKB_78F1, *** SEQUENCING IN Homo sapiens 36,797 3-Aug-99
PROGRESS ***, 12 unordered pieces. rxa00480 1239 GB_HTG1: HSJ575L21
94715 AL096841 Homo sapiens chromosome 1 clone RP4-575L21, ***
SEQUENCING IN PROGRESS Homo sapiens 38,138 23-Nov-99 ***, In
unordered pieces. GB_HTG1: HSJ575L21 94715 AL096841 Homo sapiens
chromosome 1 clone RP4-575L21, *** SEQUENCING IN PROGRESS Homo
sapiens 38,138 23-Nov-99 ***, In unordered pieces. GB_RO: AC005960
158414 AC005960 Mus musculus chromosome 17 BAC cltb20h22 from the
MHC region, complete Mus musculus 38,712 01-DEC-1998 sequence.
rxa00524 433 GB_BA1: SCI51 40745 AL109848 Streptomyces coelicolor
cosmid I51. Streptomyces coelicolor 40,284 16-Aug-99 A3(2) GB_BA2:
AF082879 3434 AF082879 Yersinia enterocolitica ABC transporter
enterochelin/enterobactin gene cluster, Yersinia enterocolitica
55,634 20-OCT-1999 complete sequence. GB_BA1: BSP132617 5192
AJ132617 Burkholderia sp. P-transporter operon and flanking genes.
Burkholderia sp. 40,793 13-Jul-99 rxa00526 813 GB_BA1: BSUB0008
208230 Z99111 Bacillus subtilis complete genome (section 8 of 21):
from 1394791 to 1603020. Bacillus subtilis 54,534 26-Nov-97 GB_BA2:
AF012285 46864 AF012285 Bacillus subtilis mobA-nprE gene region.
Bacillus subtilis 54,534 1-Jul-98 GB_BA1: D90725 13796 D90725
Escherichia coli genomic DNA. (19.7-20.0 min). Escherichia coli
51,481 7-Feb-99 rxa00559 1140 GB_BA2: CAU77910 3385 U77910
Corynebacterium ammoniagenes sequence upstream of the
5-phosphoribosyl-1- Corynebacterium 39,007 1-Jan-98 pyrophosphate
amidotransferase (purF) gene. ammoniagenes GB_EST4: H34952 382
H34952 EST108261 Rat PC-12 cells, untreated Rattus sp. cDNA clone
RPCCK07 similar to Rattus sp. 39,267 2-Apr-98 NADH-ubiquinone
oxidoreductase complex I 23 kDa precursor (iron-sulfur protein),
mRNA sequence. GB_BA2: AE000963 22014 AE000963 Archaeoglobus
fulgidus section 144 of 172 of the complete genome. Archaeoglobus
fulgidus 38,338 15-DEC-1997 rxa00570 852 GB_GSS12: AQ422451 563
AQ422451 RPCI-11-185C3.TV RPCI-11 Homo sapiens genomic clone
RPCI-11-185C3, Homo sapiens 38,767 23-MAR-1999 genomic survey
sequence. GB_EST28: AI504741 568 AI504741 vl16c01.x1 Stratagene
mouse Tcell 937311 Mus musculus cDNA clone Mus musculus 37,900
11-MAR-1999 IMAGE: 972384 3' similar to gb: Z14044 M. musculus mRNA
for valosin-containing protein (MOUSE);, mRNA sequence. GB_EST18:
AA712043 68 AA712043 vu29f10.r1 Barstead mouse myotubes MPLRB5 Mus
musculus cDNA clone Mus musculus 42,647 24-DEC-1997 IMAGE: 1182091
5' similar to gb: L05093 60S RIBOSOMAL PROTEIN L18A (HUMAN);, mRNA
sequence. rxa00571 1280 GB_BA1: MTCY78 33818 Z77165 Mycobacterium
tuberculosis H37Rv complete genome; segment 145/162. Mycobacterium
38,468 17-Jun-98 tuberculosis GB_PR3: AC005788 36224 AC005788 Homo
sapiens chromosome 19, cosmid R26652, complete sequence. Homo
sapiens 36,911 06-OCT-1998 GB_PR3: AC005338 34541 AC005338 Homo
sapiens chromosome 19, cosmid R31646, complete sequence. Homo
sapiens 36,911 30-Jul-98 rxa00590 1288 GB_HTG6: AC010932 203273
AC010932 Homo sapiens chromosome 15 clone RP11-296E22 map 15, ***
SEQUENCING IN Homo sapiens 37,242 30-Nov-99 PROGRESS ***, 36
unordered pieces. GB_HTG6: AC010932 203273 AC010932 Homo sapiens
chromosome 15 clone RP11-296E22 map 15, *** SEQUENCING IN Homo
sapiens 36,485 30-Nov-99 PROGRESS ***, 36 unordered pieces. GB_BA1:
MSGB26CS 37040 L78816 Mycobacterium leprae cosmid B26 DNA sequence.
Mycobacterium leprae 39,272 15-Jun-96 rxa00591 1476 GB_IN1: CEK09E9
30098 Z79602 Caenorhabditis elegans cosmid K09E9, complete
sequence. Caenorhabditis elegans 34,092 2-Sep-99 GB_PR4: AF135802
4965 AF135802 Homo sapiens thyroid hormone receptor-associated
protein complex component Homo sapiens 36,310 9-Apr-99 TRAP170
mRNA, complete cds. GB_PR4: AF104256 4365 AF104256 Homo sapiens
transcriptional co-activator CRSP150 (CRSP150) mRNA, complete Homo
sapiens 36,617 4-Feb-99 cds. rxa00596 576 GB_PR3: AC004659 129577
AC004659 Homo sapiens chromosome 19, CIT-HSP-87m17 BAC clone,
complete sequence. Homo sapiens 34,321 02-MAY-1998 GB_PR3: AC004659
129577 AC004659 Homo sapiens chromosome 19, CIT-HSP-87m17 BAC
clone, complete sequence. Homo sapiens 35,739 02-MAY-1998 GB_PR1:
HUMCBP2 2047 D83174 Human mRNA for collagen binding protein 2,
complete cds. Homo sapiens 40,404 6-Feb-99 rxa00607 504 GB_BA1:
MTV010 3400 AL021186 Mycobacterium tuberculosis H37Rv complete
genome; segment 119/162. Mycobacterium 40,862 23-Jun-99
tuberculosis GB_BA1: MTV010 3400 AL021186 Mycobacterium
tuberculosis H37Rv complete genome; segment 119/162. Mycobacterium
38,833 23-Jun-99 tuberculosis rxa00623 1461 GB_BA1: MTCY428 26914
Z81451 Mycobacterium tuberculosis H37Rv complete genome; segment
107/162. Mycobacterium 60,552 17-Jun-98 tuberculosis GB_BA1:
RSPNGR234 34010 Z68203 Rhizobium sp. plasmid NGR234a DNA. Rhizobium
sp. 51,992 8-Aug-96 GB_BA2: AE000101 10057 AE000101 Rhizobium sp,
NGR234 plasmid pNGR234a, section 38 of 46 of the complete Rhizobium
sp. NGR234 51,992 12-DEC-1997 plasmid sequence. rxa00681 rxa00690
1269 GB_HTG5: AC008338 136685 AC008338 Drosophila melanogaster
chromosome X clone BACR30J04 (D908) RPCI-98 30.J.4 Drosophila
melanogaster 35,341 15-Nov-99 map 19C-19E strain y; cn bw sp, ***
SEQUENCING IN PROGRESS ***, 93 unordered pieces. GB_HTG4: AC009766
170502 AC009766 Homo sapiens chromosome 11 clone 404_A_03 map 11,
*** SEQUENCING IN Homo sapiens 37,984 19-OCT-1 999 PROGRESS ***, 27
unordered pieces. GB_HTG4: AC009766 170502 AC009766 Homo sapiens
chromosome 11 clone 404_A_03 map 11, *** SEQUENCING IN Homo sapiens
37,984 19-OCT-1999 PROGRESS ***, 27 unordered pieces. rxa00733 1008
GB_EST30: AU054038 245 AU054038 AU054038 Dictyostelium discoideum
SL (H. Urushihara) Dictyostelium discoideum Dictyostelium
discoideum 43,265 28-Apr-99 cDNA clone SLK472, mRNA sequence.
GB_EST30: AU054038 245 AU054038 AU054038 Dictyostelium discoideum
SL (H. Urushihara) Dictyostelium discoideum Dictyostelium
discoideum 43,265 28-Apr-99 cDNA clone SLK472, mRNA sequence.
rxa00735 692 GB_BA1: MTCY50 36030 Z77137 Mycobacterium tuberculosis
H37Rv complete genome; segment 55/162. Mycobacterium 36,819
17-Jun-98 tuberculosis GB_BA1: D90904 150894 D90904 Synechocystis
sp. PCC6803 complete genome, 6/27, 630555-781448. Synechocystis sp.
52,585 7-Feb-99 GB_BA1: D90904 150894 D90904 Synechocystis sp.
PCC6803 complete genome, 6/27, 630555-781448. Synechocystis sp.
39,699 7-Feb-99 rxa00796 298 GB_GSS14: AQ579838 651 AQ579838
T135342b shotgun sub-library of BAC clone 31P06 Medicago truncatula
genomic Medicago truncatula 37,153 27-Sep-99 clone 31-P-06-C-054,
genomic survey sequence. GB_PR4: AC007625 174701 AC007625 Genomic
sequence of Homo sapiens clone 2314F2 from chromosome 18, complete
Homo sapiens 38,014 30-Jun-99 sequence. GB_EST14: AA427576 580
AA427576 zw54b04.s1 Soares_total_fetus_Nb2HF8_9w Homo sapiens cDNA
clone Homo sapiens 42,731 16-OCT-1997 IMAGE: 773839 3' similar to
gb: M86852 PEROXISOME ASSEMBLY FACTOR-1 (HUMAN);, mRNA sequence.
rxa00801 756 GB_BA1: MTV022 13025 AL021925 Mycobacterium
tuberculosis H37Rv complete genome; segment 100/162. Mycobacterium
59,350 17-Jun-98 tuberculosis GB_RO: AC002109 160048 AC002109
Genomic sequence from Mouse 9, complete sequence. Mus musculus
39,398 9-Sep-97 GB_BA1: MTV022 13025 AL021925 Mycobacterium
tuberculosis H37Rv complete genome; segment 100/162. Mycobacterium
36,842 17-Jun-98 tuberculosis rxa00802 837 GB_GSS14: AQ563349 642
AQ563349 HS_5335_B2_A09_T7A RPCI-11 Human Male BAC Library Homo
Homo sapiens 37,649 29-MAY-1999 sapiens genomic clone Plate = 911
Col = 18 Row = B, genomic survey sequence. GB_BA1: DIHCLPBA 2441
M32229 B. nodosus clpB gene encoding a regulatory subunit of
ATP-dependent protease. Dichelobacter nodosus 41,140 26-Apr-93
GB_GSS3: B61538 698 B61538 T17M17TR TAMU Arabidopsis thaliana
genomic clone T17M17, genomic survey Arabidopsis thaliana 36,946
21-Nov-97 sequence. rxa00819 1452 GB_HTG3: AC008691_1 110000
AC008691 Homo sapiens chromosome 5 clone CIT978SKB_63A22, ***
SEQUENCING IN Homo sapiens 38,270 3-Aug-99 PROGRESS ***, 253
unordered pieces. GB_HTG3: AC008691_1 110000 AC008691 Homo sapiens
chromosome 5 clone CIT978SKB_63A22, *** SEQUENCING IN Homo sapiens
38,270 3-Aug-99 PROGRESS ***, 253 unordered pieces. GB_HTG3:
AC009127 186591 AC009127 Homo sapiens chromosome 16 clone
RPCI-11_498D10, *** SEQUENCING IN Homo sapiens 38,947 3-Aug-99
PROGRESS ***, 49 unordered pieces. rxa00821 966 GB_HTG1: HS32B1
271488 AL023693 Homo sapiens chromosome 6 clone RP1-32B1, ***
SEQUENCING IN PROGRESS Homo sapiens 36,565 23-Nov-99 ***, in
unordered pieces. GB_HTG1: HS32B1 271488 AL023693 Homo sapiens
chromosome 6 clone RP1-32B1, *** SEQUENCING IN PROGRESS Homo
sapiens 36,565 23-Nov-99 ***, in unordered pieces. GB_PR3: AC004919
75547 AC004919 Homo sapiens PAC clone DJ0895B23 from UL, complete
sequence. Homo sapiens 34,346 19-Sep-98 rxa00827 876 GB_EST6:
W06539 300 W06539 T2367 MVAT4 bloodstream form of serodeme
WRATat1.1 Trypanosoma brucei Trypanosoma brucei 40,000 12-Aug-96
rhodesiense cDNA 5', mRNA sequence. rhodesiense GB_PR4: AC008179
181745 AC008179 Homo sapiens clone NH0576F01, complete sequence.
Homo sapiens 35,903 28-Sep-99 GB_EST18: AA710415 533 AA710415
vt53f08.r1 Barstead mouse irradiated colon MPLRB7 Mus musculus cDNA
clone Mus musculus 41,562 24-DEC-1997 IMAGE: 1166823 5', mRNA
sequence. rxa00842 1323 GB_PR2: AC002379 118595 AC002379 Human BAC
clone GS165I04 from 7q21, complete sequence. Homo sapiens 36,321
23-Jul-97 GB_PR2: AC002379 118595 AC002379 Human BAC clone GS165I04
from 7q21, complete sequence. Homo sapiens 37,284 23-Jul-97 GB_IN1:
CEF02D8 31624 Z78411 Caenorhabditis elegans cosmid F02D8, complete
sequence. Caenorhabditis elegans 38,163 23-Nov-98 rxa00847 1572
GB_OV: XELRDS38A 1209 L79915 Xenopus laevis rds/peripherin (rds38)
mRNA, complete cds. Xenopus laevis 36,044 30-Jul-97 GB_HTG4:
AC007920 234529 AC007920 Homo sapiens chromosome 3q27 clone
RPCI11-208N14, *** SEQUENCING IN Homo sapiens 33,742 21-OCT-1999
PROGRESS ***, 51 unordered pieces. GB_HTG4: AC007920 234529
AC007920 Homo sapiens chromosome 3q27 clone RPCI11-208N14, ***
SEQUENCING IN Homo sapiens 33,742 21-OCT-1999 PROGRESS ***, 51
unordered pieces. rxa00851 732 GB_HTG2: AC004064 185000 AC004064
Homo sapiens chromosome 4, *** SEQUENCING IN PROGRESS ***, 10
unordered Homo sapiens 39,833 9-Jul-98 pieces. GB_HTG2: AC004064
185000 AC004064 Homo sapiens chromosome 4, *** SEQUENCING IN
PROGRESS ***, 10 unordered Homo sapiens 39,833 9-Jul-98 pieces.
GB_PR3: HSJ824F16 139330 AL050325 Human DNA sequence from clone
824F16 on chromosome 20, complete sequence. Homo sapiens 39,833
23-Nov-99 rxa00852 813 GB_HTG3: AC010120 121582 AC010120 Drosophila
melanogaster chromosome 3 clone BACR22N13 (D1061) RPCI-98
Drosophila melanogaster 36,855 24-Sep-99 22.N.13 map 96F-96F strain
y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 83 unordered pieces.
GB_HTG3: AC010120 121582 AC010120 Drosophila melanogaster
chromosome 3 clone BACR22N13 (D1061) RPCI-98 Drosophila
melanogaster 36,855 24-Sep-99 22.N.13 map 96F-96F strain y; cn bw
sp, *** SEQUENCING IN PROGRESS ***, 83 unordered pieces. GB_HTG2:
AC006898 299308 AC006898 Caenorhabditis elegans clone Y73B6x, ***
SEQUENCING IN PROGRESS ***, 9 Caenorhabditis elegans 36,768
24-Feb-99 unordered pieces. rxa00856 rxa00870 1635 GB_BA1: STMMSDA
3986 L48550 Streptomyces coelicolor methylmalonic acid semialdehyde
dehydrogenase (msdA) Streptomyces coelicolor 63,743 09-MAY-1996
gene, complete cds. GB_PAT: I92043 713 I92043 Sequence 10 from
patent U.S. Pat. No. 5726299. Unknown. 38,850 01-DEC-1998 GB_PAT:
I78754 713 I78754 Sequence 10 from patent U.S. Pat. No. 5693781.
Unknown. 38,850 3-Apr-98 rxa00875 690 GB_BA2: AF119715 549 AF119715
Escherichia coli isopentyl diphosphate isomerase (idi) gene,
complete cds. Escherichia coli 54,827 22-Apr-99 GB_BA2: AE000372
12144 AE000372 Escherichia coli K-12 MG1655 section 262 of 400 of
the complete genome. Escherichia coli 51,416 12-Nov-98 GB_BA1:
ECU28375 55175 U28375 Escherichia coli K-12 genome; approximately
64 to 65 minutes. Escherichia coli 51,416 08-DEC-1995 rxa00878 1986
GB_HTG2: AC007472 114003 AC007472 Drosophila melanogaster
chromosome 2 clone BACR30D19 (D587) RPCI-98 Drosophila melanogaster
36,592 2-Aug-99 30.D.19 map 49E-49F strain y; cn bw sp, ***
SEQUENCING IN PROGRESS ***, 79 unordered pieces. GB_HTG2: AC007472
114003 AC007472 Drosophila melanogaster chromosome 2 clone
BACR30D19 (D587) RPCI-98 Drosophila melanogaster 36,592 2-Aug-99
30.D.19 map 49E-49F strain y; cn bw sp, *** SEQUENCING IN PROGRESS
***, 79 unordered pieces. GB_HTG2: AC006798 207370 AC006798
Caenorhabditis elegans clone Y51F8,
*** SEQUENCING IN PROGRESS ***, 30 Caenorhabditis elegans 36,699
25-Feb-99 unordered pieces. rxa00880 1968 GB_EST4: H22888 468
H22888 ym54e12.r1 Soares infant brain 1NIB Home sapiens cDNA clone
IMAGE: 52158 5', Homo sapiens 37,179 6-Jul-95 mRNA sequence.
GB_GSS13: AQ426858 516 AQ426858 CITBI-E1-2578F1.TF CITBI-E1 Home
sapiens genomic clone 2578F1, genomic Homo sapiens 38,447
24-MAR-1999 survey sequence. GB_PR1: AB002335 6289 AB002335 Human
mRNA for KIAA0337 gene, complete cds. Homo sapiens 35,799 13-Feb-99
rxa00899 1389 GB_BA1: NGU58849 2401 U58849 Neisseria gonorrhoeae
pilS6 silent pilus locus. Neisseria gonorrhoeae 40,623 20-Jun-96
GB_BA1: PLPDHOS 3119 L06822 Plasmid pSa (from Escherichia coli)
dihydropteroate synthase gene, 3' end. Plasmid pSa 38,966
20-MAR-1996 GB_BA1: PDGINTORF 6747 L06418 Integron In7 (from
Plasmid pDGO100 from Escherichia coli) integrase (int), Plasmid
pDGO100 38,966 20-MAR-1996 aminoglycoside adenylyltransferase
(aad), quaternary ammonium compound- resistance protein,
dihydrofolate reductase (dhfrX), and dihydropteroate synthase
(sull) genes. rxa00902 1333 GB_GSS15: AQ606873 581 AQ606873
HS_5404_B2_H05_T7A RPCI-11 Human Male BAC Library Homo sapiens Homo
sapiens 37,900 10-Jun-99 genomic clone Plate = 980 Col = 10 Row =
P, genomic survey sequence. GB_GSS9: AQ163442 658 AQ163442
nbxb0007A07f CUGI Rice BAC Library Oryza sativa genomic clone
nbxb0007A07f, Oryza sativa 41,885 12-Sep-98 genomic survey
sequence. GB_PL1: PSST70 4974 X69213 P. sativum Psst70 gene for
heat-shock protein. Pisum sativum 36,866 3-Jul-96 rxa00931 969
GB_GSS1: FR0025208 612 AL018047 F. rubripes GSS sequence, clone
145D10aA8, genomic survey sequence. Fugu rubripes 37,815
10-DEC-1997 GB_GSS1: FR0021844 252 AL014715 F. rubripes GSS
sequence, clone 069K22aG5, genomic survey sequence. Fugu rubripes
37,698 10-DEC-1997 GB_GSS12: AQ403344 593 AQ403344
HS_2257_B1_B03_T7C CIT Approved Human Genomic Sperm Library D Homo
Homo sapiens 31,552 13-MAR-1999 sapiens genomic clone Plate = 2257
Col = 5 Row = D, genomic survey sequence. rxa00941 1440 GB_BA1:
MTCY180 44201 Z97193 Mycobacterium tuberculosis H37Rv complete
genome; segment 85/162. Mycobacterium 37,902 17-Jun-98 tuberculosis
GB_BA1: MTCY180 44201 Z97193 Mycobacterium tuberculosis H37Rv
complete genome; segment 85/162. Mycobacterium 39,140 17-Jun-98
tuberculosis GB_BA2: MSGKATG 1745 L14268 Mycobacterium tuberculosis
ethyl methane sulphonate resistance protein (katG) Mycobacterium
42,517 26-Aug-99 gene, 3'end. tuberculosis rxa00962 689 GB_HTG6:
AC010998 144338 AC010998 Homo sapiens clone RP11-95I16, ***
SEQUENCING IN PROGRESS ***, 17 Homo sapiens 39,497 08-DEC-1999
unordered pieces. GB_GSS1: GGA340111 990 AJ232089 Gallus gallus
anonymous sequence from Cosmid mapping to chromosome 2 Gallus
gallus 37,970 25-Aug-98 (Cosmid 34 - Contig 15), genomic survey
sequence. GB_HTG6: AC010998 144338 AC010998 Homo sapiens clone
RP11-95I16, *** SEQUENCING IN PROGRESS ***, 17 Homo sapiens 38,226
08-DEC-1999 unordered pieces. rxa01060 1047 GB_BA1: ECTTN7 2280
AJ001816 Escherichia coli left end of transposon Tn7 including type
2 Integron. Escherichia coli 38,822 4-Nov-97 GB_IN2: AF176377 8220
AF176377 Caenorhabditis briggsae CES-1 (ces-1) gene, complete cds;
and CPN-1 (cpn-1) Caenorhabditis briggsae 39,921 09-DEC-1999 gene,
partial cds. GB_GSS10: AQ196728 429 AQ196728 CIT-HSP-2381F4.TR
CIT-HSP Homo sapiens genomic clone 2381F4, genomic Homo sapiens
39,019 16-Sep-98 survey sequence. rxa01067 852 GB_BA1: U00016 42931
U00016 Mycobacterium leprae cosmid B1937. Mycobacterium leprae
58,303 01-MAR-1994 GB_BA1: SYCGROESL 3256 D12677 Synechocystis sp.
groES and groEL genes. Synechocystis sp. 34,593 3-Feb-99 GB_BA1:
D90905 139467 D90905 Synechocystis sp. PCC6803 complete genome,
7/27, 781449-920915. Synechocystis sp. 34,593 7-Feb-99 rxa01114
1347 GB_BA1: PSEFAOAB 3480 D10390 P. fragi faoA and faoB genes,
complete cds. Pseudomonas fragi 51,919 2-Feb-99 GB_BA1: AB014757
6057 AB014757 Pseudomonas sp. 61-3 genes for PhbR, acetoacetyl-CoA
reductase, beta- Pseudomonas sp. 61-3 50,573 26-DEC-1998
ketothiolase and PHB synthase, complete cds. GB_BA1: SC8D9 38681
AL035569 Streptomyces coelicolor cosmid 8D9. Streptomyces
coelicolor 42,200 26-Feb-99 rxa01136 555 GB_EST11: AA244557 379
AA244557 mx07a01.r1 Soares mouse NML Mus musculus cDNA clone IMAGE:
679464 5', Mus musculus 39,050 10-MAR-1997 mRNA sequence. GB_EST14:
AA407673 306 AA407673 EST01834 Mouse 7.5 dpc embryo ectoplacental
cone cDNA library Mus musculus Mus musculus 38,562 26-Aug-98 cDNA
clone C0014F02 3', mRNA sequence. GB_EST26: AI390328 604 AI390328
mx07a01.y1 Soares mouse NML Mus musculus cDNA clone IMAGE: 679464
5', Mus musculus 33,136 2-Feb-99 mRNA sequence. rxa01138 540 GB_OV:
XLXINT1 1278 X13138 Xenopus laevis int-1 mRNA for int-1 protein.
Xenopus laevis 40,038 31-MAR-1995 GB_PR4: AC006054 143738 AC006054
Homo sapiens Xq28 BAC RPCI11-382P7 (Roswell Park Cancer Institute
Human Homo sapiens 37,996 1-Apr-99 BAC Library) complete sequence.
GB_PR4: AC006054 143738 AC006054 Homo sapiens Xq28 BAC RPCI11-382P7
(Roswell Park Cancer Institute Human Homo sapiens 36,053 1-Apr-99
BAC Library) complete sequence. rxa01172 1578 GB_BA1: SCE39 23550
AL049573 Streptomyces coelicolor cosmid E39. Streptomyces
coelicolor 62,357 31-MAR-1999 GB_BA1: MSU50335 5193 U50335
Mycobacterium smegmatis phage resistance (mpr) gene, complete cds.
Mycobacterium 37,853 1-Feb-97 smegmatis GB_BA1: BACTHRTRNA 15467
D84213 Bacillus subtilis genome, trnl-feuABC region. Bacillus
subtilis 53,807 6-Feb-99 rxa01191 1713 GB_PR2: HS1191B2 60828
AL022237 Human DNA sequence from clone 1191B2 on chromosome
22q13.2-13.3. Contains Homo sapiens 38,366 23-Nov-99 part of the
BIK (NBK, BP4, BIP1) gene for BCL2-interacting killer (apoptosis-
inducing), a 40S Ribososmal Protein S25 pseudogene and part of an
alternatively spliced novel Acyl Transferase gene similar to C.
elegans C50D2.7. Contains ESTs, STSs, GSSs, two putative CpG
islands and genomic marker D22S1151, complete sequence. GB_PR2:
HS1191B2 60828 AL022237 Human DNA sequence from clone 1191B2 on
chromosome 22q13.2-13.3. Contains Homo sapiens 39,595 23-Nov-99
part of the BIK (NBK, BP4, BIP1) gene for BCL2-interacting killer
(apoptosis- inducing), a 40S Ribososmal Protein S25 pseudogene and
part of an alternatively spliced novel Acyl Transferase gene
similar to C. elegans C50D2.7. Contains ESTs, STSs, GSSs, two
putative CpG islands and genomic marker D22S1151, complete
sequence. rxa01205 554 GB_BA1: MTCY373 35516 Z73419 Mycobacterium
tuberculosis H37Rv complete genome; segment 57/162. Mycobacterium
57,762 17-Jun-98 tuberculosis GB_PL1: ATY12776 38483 Y12776
Arabidopsis thaliana DNA, 40 kb surrounding ACS1 locus. Arabidopsis
thaliana 32,971 7-Sep-98 GB_PL2: ATT6K21 99643 AL021889 Arabidopsis
thaliana DNA chromosome 4, BAC clone T6K21 (ESSA project).
Arabidopsis thaliana 35,273 16-Aug-99 rxa01212 1047 GB_BA2: SCD25
41622 AL118514 Streptomyces coelicolor cosmid D25. Streptomyces
coelicolor 39,654 21-Sep-99 A3(2) GB_BA1: SLGLYUB 2576 X65556 S.
lividans tRNA-GlyU beta gene. Streptomyces lividans 54,493
20-DEC-1993 GB_BA1: SCH10 39524 AL049754 Streptomyces coelicolor
cosmid H10. Streptomyces coelicolor 44,638 04-MAY-1999 rxa01219
1005 GB_PAT: A68024 520 A68024 Sequence 19 from Patent WO9743409.
unidentified 42,553 05-MAY-1999 GB_PAT: A68025 193 A68025 Sequence
20 from Patent WO9743409. unidentified 43,229 05-MAY-1999 GB_PAT:
A68027 193 A68027 Sequence 22 from Patent WO9743409. unidentified
38,342 05-MAY-1999 rxa01220 1200 GB_PR3: HS512B11 64356 AL031058
Human DNA sequence from clone 512B11 on chromosome 6p24-25.
Contains the Homo sapiens 35,478 23-Nov-99 Desmoplakin I (DPI)
gene, ESTs, STSs and GSSs, complete sequence. GB_EST6: N99239 424
N99239 zb76h11.s1 Soares_senescent_fibroblasts_NbHSF Homo sapiens
cDNA clone Homo sapiens 39,623 20-Aug-96 IMAGE: 309573 3', mRNA
sequence. GB_EST16: AA554268 400 AA554268 nk36c09.s1 NCI_CGAP_GC2
Homo sapiens cDNA clone IMAGE: 1015600 Homo sapiens 36,111 8-Sep-97
3' similar to gb: X01677 GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE,
LIVER (HUMAN);, mRNA sequence. rxa01221 849 GB_PR4: AF179633 96371
AF179633 Homo sapiens chromosome 16 map 16q23.3-q24.1 sequence.
Homo sapiens 40,199 5-Sep-99 GB_VI: EHVU20824 184427 U20824 Equine
herpesvirus 2, complete genome. Equine herpesvirus 2 37,001
2-Feb-96 GB_BA2: AE000407 10601 AE000407 Escherichia coli K-12
MG1655 section 297 of 400 of the complete genome. Escherichia coli
39,471 12-Nov-98 rxa01222 822 GB_PAT: AR068625 28804 AR068625
Sequence 1 from patent U.S. Pat. No. 5854034. Unknown. 40,574
29-Sep-99 GB_BA2: SSU51197 28804 U51197 Sphingomonas S88 sphingan
polysaccharide synthesis (spsG), (spsS), (spsR), Sphingomonas sp.
S88 40,574 16-MAY-1996 glycosyl transferase (spsQ), (spsl),
glycosyl transferase (spsK), glycosyl transferase (spsL), (spsJ),
(spsF), (spsD), (spsC), (spsE), Urf 32, Urf 26, ATP-binding
cassette transporter (atrD), ATP-binding cassette transporter
(atrB), glucosyl- isoprenylphosphate transferase (spsB),
glucose-1-phosphate thymidylyltransferase (rhsA),
dTDP-6-deoxy-D-glucose-3,5-epimerase (rhsC) dTDP-D-glucose-4,6-
dehydratase (rhsB), dTDP-6-deoxy-L-mannose-dehydrogenase (rhsD),
Urf 31, and Urf 34 genes, complete cds. GB_IN1: BBU44918 2791
U44918 Babesia bovis ATP-binding protein (babc) mRNA, complete cds.
Babesia bovis 39,228 9-Aug-97 rxa01260 1305 GB_BA1: CGLPD 1800
Y16642 Corynebacterium glutamicum lpd gene, complete CDS.
Corynebacterium 99,923 1-Feb-99 glutamicum GB_BA1: MTV038 16094
AL021933 Mycobacterium tuberculosis H37Rv complete genome; segment
24/162. Mycobacterium 59,056 17-Jun-98 tuberculosis GB_PR3:
AC005618 176714 AC005618 Homo sapiens chromosome 5, BAC clone 249h5
(LBNL H149), complete sequence. Homo sapiens 36,270 5-Sep-98
rxa01261 294 GB_BA1: CGLPD 1800 Y16642 Corynebacterium glutamicum
lpd gene, complete CDS. Corynebacterium 100,000 1-Feb-99 glutamicum
GB_HTG4: AC010045 164829 AC010045 Drosophila melanogaster
chromosome 3L/75A1 clone RPCI98-17C17, *** Drosophila melanogaster
50,512 16-OCT-1999 SEQUENCING IN PROGRESS ***, 50 unordered pieces.
GB_HTG4: AC010045 164829 AC010045 Drosophila melanogaster
chromosome 3L/75A1 clone RPCI98-17C17, *** Drosophila melanogaster
50,512 16-OCT-1999 SEQUENCING IN PROGRESS ***, 50 unordered pieces.
rxa01269 564 GB_BA2: AF125164 26443 AF125164 Bacteroides fragilis
638R polysaccharide B (PS B2) biosynthesis locus, complete
Bacteroides fragilis 56,071 01-DEC-1999 sequence; and unknown
genes. GB_BA1: AB002668 24907 AB002668 Actinobacillus
actinomycetemcomitans DNA for glycosyltransferase, lytic
Actinobacillus 46,679 21-Feb-98 transglycosylase, dTDP-4-rhamnose
reductase, complete cds. actinomycetemcomitans GB_BA1: AB010415
23112 AB010415 Actinobacillus actinomycetemcomitans gene cluster
for 6-deoxy-L-talan synthesis, Actinobacillus 46,679 13-Feb-99
complete cds. actinomycetemcomitans rxa01291 1056 GB_STS: AU027820
238 AU027820 Rattus norvegicus, OTSUKA clone, OT78.02/918b07,
microsatellite sequence, Rattus norvegicus 34,874 02-MAR-1999
sequence tagged site. GB_STS: AU027820 238 AU027820 Rattus
norvegicus, OTSUKA clone, OT78.02/918b07, microsatellite sequence,
Rattus norvegicus 34,874 02-MAR-1999 sequence tagged site. GB_HTG3:
AC006445 174547 AC006445 Homo sapiens chromosome 4, *** SEQUENCING
IN PROGRESS ***, 7 unordered Homo sapiens 34,812 15-Sep-99 pieces.
rxa01292 1308 GB_BA1: BSUB0017 217420 Z99120 Bacillus subtilis
complete genome (section 17 of 21): from 3197001 to 3414420.
Bacillus subtilis 37,802 26-Nov-97 GB_HTG3: AC010580 121119
AC010580 Drosophila melanogaster chromosome 3 clone BACR48J06
(D1102) RPCI-98 48.J.6 Drosophila melanogaster 35,637 01-OCT-1999
map 96F-96F strain y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 71
unordered pieces. GB_HTG3: AC010580 121119 AC010580 Drosophila
melanogaster chromosome 3 clone BACR48J06 (D1102) RPCI-98 48.J.6
Drosophila melanogaster 35,637 01-OCT-1999 map 96F-96F strain y; cn
bw sp, *** SEQUENCING IN PROGRESS ***, 71 unordered pieces.
rxa01293 450 GB_GSS8: AQ001809 705 AQ001809 CIT-HSP-2290D17.TF
CIT-HSP Homo sapiens genomic clone 2290D17, genomic Homo sapiens
42,021 26-Jun-98 survey sequence. GB_GSS8: AQ001809 705 AQ001809
CIT-HSP-2290D17.TF CIT-HSP Homo sapiens genomic clone 2290D17,
genomic Homo sapiens 40,323 26-Jun-98 survey sequence. rxa01339
1111 GB_PL1: MGU60290 4614 U60290 Magnaporthe grisea nitrogen
regulatory protein (NUT1) gene, complete cds. Magnaporthe grisea
38,707 3-Jul-96
GB_HTG3: AC011371 189187 AC011371 Homo sapiens chromosome 5 clone
CIT978SKB_107C20, *** SEQUENCING IN Homo sapiens 39,741 06-OCT-1999
PROGRESS ***, 31 unordered pieces. GB_HTG3: AC011371 189187
AC011371 Homo sapiens chromosome 5 clone CIT978SKB_107C20, ***
SEQUENCING IN Homo sapiens 39,741 06-OCT-1999 PROGRESS ***, 31
unordered pieces. rxa01382 1192 GB_HTG4: AC009892 138122 AC009892
Homo sapiens chromosome 19 clone CIT978SKB_83J4, *** SEQUENCING IN
Homo sapiens 40,154 31-OCT-1999 PROGRESS ***, 6 ordered pieces.
GB_HTG4: AC009892 138122 AC009892 Homo sapiens chromosome 19 clone
CIT978SKB_83J4, SEQUENCING IN Homo sapiens 40,154 31-OCT-1999
PROGRESS ***, 6 ordered pieces. GB_PR3: AC002416 128915 AC002416
Human Chromosome X, complete sequence. Homo sapiens 37,521
29-Jan-98 rxa01399 1142 GB_EST9: AA096601 524 AA096601 mo03b09.r1
Stratagene mouse lung 937302 Mus musculus cDNA clone Mus musculus
40,525 15-Feb-97 IMAGE: 552473 5' similar to gb: L06505 60S
RIBOSOMAL PROTEIN L12 (HUMAN); gb: L04280 Mus musculus ribosomal
protein (MOUSE);, mRNA sequence. GB_EST37: AI982114 626 AI982114
pat.pk0074.e9.f chicken activated T cell cDNA Gallus gallus cDNA
clone Gallus gallus 37,785 15-Sep-99 pat.pk0074.e9.f 5' similar to
H-ATPase B subunit, mRNA sequence. GB_OV: GGU20766 1645 U20766
Gallus gallus vacuolar H+-ATPase B subunit gene, complete cds.
Gallus gallus 38,244 07-DEC-1995 rxa01420 1065 GB_HTG2: AC005690
193424 AC005690 Homo sapiens chromosome 4, *** SEQUENCING IN
PROGRESS ***, 7 unordered Homo sapiens 37,464 11-Apr-99 pieces.
GB_HTG2: AC005690 193424 AC005690 Homo sapiens chromosome 4, ***
SEQUENCING IN PROGRESS ***, 7 unordered Homo sapiens 37,464
11-Apr-99 pieces. GB_HTG2: AC006637 22092 AC006637 Caenorhabditis
elegans clone F41B4, *** SEQUENCING IN PROGRESS ***, 1
Caenorhabditis elegans 37,488 23-Feb-99 unordered pieces. rxa01467
414 GB_HTG1: CEY102G3_21 10000 AL020985 Caenorhabditis elegans
chromosome V clone Y102G3, *** SEQUENCING IN Caenorhabditis elegans
35,437 3-Dec-98 GB_HTG1: CEY102G3_21 10000 AL020985 Caenorhabditis
elegans chromosome V clone Y102G3, *** SEQUENCING IN Caenorhabditis
elegans 35,437 3-Dec-98 GB_HTG1: CEY113G7_41 10000 AL031113
Caenorhabditis elegans chromosome V clone Y113G7, *** SEQUENCING IN
Caenorhabditis elegans 35,437 12-Jan-99 rxa01576 882 GB_BA2:
AF030975 2511 AF030975 Aeromonas salmonicida chaperonin GroES and
chaperonin GroEL genes, complete Aeromonas salmonicida 41,516
2-Apr-98 cds. GB_BA2: AF030975 2511 AF030975 Aeromonas salmonicida
chaperonin GroES and chaperonin GroEL genes, complete Aeromonas
salmonicida 38,171 2-Apr-98 cds. GB_EST22: AI068560 965 AI068560
mgae0003aC11f Magnaporthe grisea Appressorium Stage cDNA Library
Pyricularia Pyricularia grisea 40,073 09-DEC-1999 grisea cDNA clone
mgae0003aC11f5', mRNA sequence. rxa01580 840 GB_GSS14: AQ554460 681
AQ554460 RPCI-11-419F2.TV RPCI-11 Homo sapiens genomic clone
RPCI-11-419F2, genomic Homo sapiens 36,522 28-MAY-1999 survey
sequence. GB_IN2: AC005449 85518 AC005449 Drosophila melanogaster,
chromosome 2R, region 44C4-44C5, P1 clone DS06765, Drosophila
melanogaster 36,609 23-DEC-1998 complete sequence. GB_IN2: AC005449
85518 AC005449 Drosophila melanogaster, chromosome 2R, region
44C4-44C5, P1 clone DS06765, Drosophila melanogaster 33,612
23-DEC-1998 complete sequence. rxa01584 rxa01604 771 GB_HTG3:
AC011352 160167 AC011352 Homo sapiens chromosome 5 clone
CIT-HSPC_327F10, *** SEQUENCING IN Homo sapiens 33,688 06-OCT-1999
PROGRESS ***, 15 unordered pieces. GB_HTG3: AC011352 160167
AC011352 Homo sapiens chromosome 5 clone CIT-HSPC_327F10, ***
SEQUENCING IN Homo sapiens 33,688 06-OCT-1999 PROGRESS ***, 15
unordered pieces. GB_HTG3: AC011402 168868 AC011402 Homo sapiens
chromosome 5 clone CIT978SKB_38B5, *** SEQUENCING IN Homo sapiens
33,688 06-OCT-1999 PROGRESS ***, 7 unordered pieces. rxa01614 1146
GB_BA1: CGA224946 2408 AJ224946 Corynebacterium glutamicum DNA for
L-Malate:quinone oxidoreductase. Corynebacterium 42,284 11-Aug-98
glutamicum GB_EST17: AA608825 439 AA608825 af03g07.s1
Soares_testis_NHT Homo sapiens cDNA clone IMAGE: 1030620 3' Homo
sapiens 40,092 02-MAR-1998 similar to TR: G976083 G976083 HISTONE
H2A RELATED.;, mRNA sequence. GB_PR4: AC005377 102311 AC005377 Homo
sapiens PAC clone DJ1136G02 from 7q32-q34, complete sequence. Homo
sapiens 37,811 28-Apr-99 rxa01629 1635 GB_BA1: CGPROPGEN 2936
Y12537 C. glutamicum proP gene. Corynebacterium 100,000 17-Nov-98
glutamicum GB_BA1: CGPROPGEN 2936 Y12537 C. glutamicum proP gene.
Corynebacterium 100,000 17-Nov-98 glutamicum GB_PR4: AF191071 88481
AF191071 Homo sapiens chromosome 8 clone BAC 388D06, complete
sequence. Homo sapiens 35,612 11-OCT-1999 rxa01644 1401 GB_BA1:
MSGB577COS 37770 L01263 M. leprae genomic dna sequence, cosmid
b577. Mycobacterium leprae 55,604 14-Jun-96 GB_BA1: MLCB2407 35615
AL023596 Mycobacterium leprae cosmid B2407. Mycobacterium leprae
36,416 27-Aug-99 GB_BA1: MTV025 121125 AL022121 Mycobacterium
tuberculosis H37Rv complete genome; segment 155/162. Mycobacterium
55,844 24-Jun-99 tuberculosis rxa01667 1329 GB_BA1: CGU43536 3464
U43536 Corynebacterium glutamicum heat shock, ATP-binding protein
(clpB) gene, complete Corynebacterium 100,000 13-MAR-1997 cds.
glutamicum GB_HTG4: AC009841 164434 AC009841 Drosophila
melanogaster chromosome 3L/77E1 clone RPCI98-13F11, *** Drosophila
melanogaster 33,205 16-OCT-1999 SEQUENCING IN PROGRESS ***, 70
unordered pieces. GB_HTG4: AC009841 164434 AC009841 Drosophila
melanogaster chromosome 3L/77E1 clone RPCI98-13F11, *** Drosophila
melanogaster 33,205 16-OCT-1999 SEQUENCING IN PROGRESS ***, 70
unordered pieces. rxa01722 1848 GB_GSS1: FR0022586 522 AL015452 F.
rubripes GSS sequence, clone 077P23aB10, genomic survey sequence.
Fugu rubripes 40,192 10-DEC-1997 GB_GSS1: FR0022584 485 AL015450 F.
rubripes GSS sequence, clone 077P23aB11, genomic survey sequence.
Fugu rubripes 35,876 10-DEC-1997 GB_IN1: CET26H2 37569 Z82055
Caenorhabditis elegans cosmid T26H2, complete sequence.
Caenorhabditis elegans 34,759 19-Nov-99 rxa01727 1401 GB_BA2:
CORCSLYS 2821 M89931 Corynebacterium glutamicum beta C-S lyase
(aecD) and branched-chain amino acid Corynebacterium 99,929
4-Jun-98 uptake carrier (brnQ) genes, complete cds, and
hypothetical protein Yhbw (yhbw) glutamicum gene, partial cds.
GB_HTG6: AC011037 167849 AC011037 Homo sapiens clone RP11-7F18,
WORKING DRAFT SEQUENCE, 19 unordered Homo sapiens 36,903 30-Nov-99
pieces. GB_HTG6: AC011037 167849 AC011037 Homo sapiens clone
RP11-7F18, WORKING DRAFT SEQUENCE, 19 unordered Homo sapiens 35,642
30-Nov-99 pieces. rxa01737 1182 GB_BA1: SCGD3 33779 AL096822
Streptomyces coelicolor cosmid GD3. Streptomyces coelicolor 38,054
8-Jul-99 GB_HTG1: CNS01DSB 222193 AL121768 Homo sapiens chromosome
14 clone R-976B16, *** SEQUENCING IN PROGRESS Homo sapiens 35,147
05-OCT-1999 ***, in ordered pieces. GB_HTG1: CNS01DSB 222193
AL121768 Homo sapiens chromosome 14 clone R-976B16, *** SEQUENCING
IN PROGRESS Homo sapiens 35,147 05-OCT-1999 ***, in ordered pieces.
rxa01762 1659 GB_BA1: MTCI28 36300 Z97050 Mycobacterium
tuberculosis H37Rv complete genome; segment 10/162. Mycobacterium
49,574 23-Jun-98 tuberculosis GB_BA1: SC6G10 36734 AL049497
Streptomyces coelicolor cosmid 6G10. Streptomyces coelicolor 44,049
24-MAR-1999 GB_BA1: SCE29 26477 AL035707 Streptomyces coelicolor
cosmid E29. Streptomyces coelicolor 40,246 12-MAR-1999 rxa01764
1056 GB_PL2: SPAC343 42947 AL109739 S. pombe chromosome I cosmid
c343. Schizosaccharomyces 37,084 6-Sep-99 pombe GB_PL2: SPAC343
42947 AL109739 S. pombe chromosome I cosmid c343.
Schizosaccharomyces 34,890 6-Sep-99 pombe rxa01801 1140 GB_EST38:
AW066306 334 AW066306 687009D03.y1 687 Early embryo from Delaware
Zea mays cDNA, mRNA Zea mays 46,108 12-OCT-1999 sequence. GB_GSS13:
AQ484750 375 AQ484750 RPCI-11-248N4.TV RPCI-11 Homo sapiens genomic
clone RPCI-11-248N4, Homo sapiens 32,000 24-Apr-99 genomic survey
sequence. GB_GSS13: AQ489971 252 AQ489971 RPCI-11-247N23.TV RPCI-11
Homo sapiens genomic clone RPCI-11-247N23, Homo sapiens 36,111
24-Apr-99 genomic survey sequence. rxa01823 900 GB_BA1: SCI51 40745
AL109848 Streptomyces coelicolor cosmid I51. Streptomyces
coelicolor 35,779 16-Aug-99 A3(2) GB_BA1: ECU82598 136742 U82598
Escherichia coli genomic sequence of minutes 9 to 12. Escherichia
coli 39,211 15-Jan-97 GB_BA1: BSUB0018 209510 Z99121 Bacillus
subtilis complete genome (section 18 of 21): from 3399551 to
3609060. Bacillus subtilis 36,999 26-Nov-97 rxa01853 675 GB_BA1:
MTCY227 35946 Z77724 Mycobacterium tuberculosis H37Rv complete
genome; segment 114/162. Mycobacterium 37,612 17-Jun-98
tuberculosis GB_HTG3: AC010189 265962 AC010189 Homo sapiens clone
RPCI11-296K13, *** SEQUENCING IN PROGRESS ***, 80 Homo sapiens
39,006 16-Sep-99 unordered pieces. GB_HTG3: AC010189 265962
AC010189 Homo sapiens clone RPCI11-296K13, *** SEQUENCING IN
PROGRESS ***, 80 Homo sapiens 39,006 16-Sep-99 unordered pieces.
rxa01881 558 GB_HTG4: AC011117 148447 AC011117 Homo sapiens
chromosome 4 clone 173_C_09 map 4, *** SEQUENCING IN Homo sapiens
39,130 14-OCT-1999 PROGRESS ***, 10 ordered pieces. GB_HTG4:
AC011117 148447 AC011117 Homo sapiens chromosome 4 clone 173_C_09
map 4, *** SEQUENCING IN Homo sapiens 39,130 14-OCT-1999 PROGRESS
***, 10 ordered pieces. GB_BA1: MTCY2B12 20431 Z81011 Mycobacterium
tuberculosis H37Rv complete genome; segment 61/162. Mycobacterium
37,893 18-Jun-98 tuberculosis rxa01894 978 GB_BA1: MTCY274 39991
Z74024 Mycobacterium tuberculosis H37Rv complete genome; segment
126/162. Mycobacterium 37,229 19-Jun-98 tuberculosis GB_IN1:
CELF46H5 38886 U41543 Caenorhabditis elegans cosmid F46H5.
Caenorhabditis elegans 38,525 29-Nov-96 GB_HTG3: AC009204 115633
AC009204 Drosophila melanogaster chromosome 2 clone BACR03E19
(D1033) RPCI-98 Drosophila melanogaster 31,579 18-Aug-99 03.E.19
map 36E-37C strain y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 94
unordered pieces. rxa01897 666 GB_HTG1: CEY48B6 293827 AL021151
Caenorhabditis elegans chromosome II clone Y48B6, *** SEQUENCING IN
Caenorhabditis elegans 34,703 1-Apr-99 PROGRESS ***, in unordered
pieces. GB_HTG1: CEY48B6 293827 AL021151 Caenorhabditis elegans
chromosome II clone Y48B6, *** SEQUENCING IN Caenorhabditis elegans
34,703 1-Apr-99 PROGRESS ***, in unordered pieces. GB_HTG1:
CEY53F4_2 110000 Z92860 Caenorhabditis elegans chromosome II clone
Y53F4, *** SEQUENCING IN Caenorhabditis elegans 33,333 15-Oct-99
PROGRESS ***, in unordered pieces. rxa01946 1298 GB_BA1: MTV007
32806 AL021184 Mycobacterium tuberculosis H37Rv complete genome;
segment 64/162. Mycobacterium 65,560 17-Jun-98 tuberculosis GB_BA1:
SC5F2A 40105 AL049587 Streptomyces coelicolor cosmid 5F2A.
Streptomyces coelicolor 50,648 24-MAY-1999 GB_BA1: SCARD1GN 2321
X84374 S. capreolus ard1 gene. Streptomyces capreolus 44,973
23-Aug-95 rxa01980 756 GB_PL2: AC008262 99698 AC008262 Genomic
sequence for Arabidopsis thaliana BAC F4N2 from chromosome I,
Arabidopsis thaliana 35,310 21-Aug-99 complete sequence. GB_PL1:
AB013388 73428 AB013388 Arabidopsis thaliana genomic DNA,
chromosome 5, TAC clone: K19E1, complete Arabidopsis thaliana
35,505 20-Nov-99 sequence. GB_PL1: AB013388 73428 AB013388
Arabidopsis thaliana genomic DNA, chromosome 5, TAC clone: K19E1,
complete Arabidopsis thaliana 39,973 20-Nov-99 sequence. rxa01983
630 GB_HTG4: AC006467 175695 AC006467 Drosophila melanogaster
chromosome 2 clone BACR03L08 (D532) RPCI-98 03.L.8 Drosophila
melanogaster 36,672 27-OCT-1999 map 40A-40C strain y; cn bw sp, ***
SEQUENCING IN PROGRESS ***, 9 unordered pieces. GB_HTG4: AC006467
175695 AC006467 Drosophila melanogaster chromosome 2 clone
BACR03L08 (D532) RPCI-98 03.L.8 Drosophila melanogaster 36,672
27-OCT-1999 map 40A-40C strain y; cn bw sp, *** SEQUENCING IN
PROGRESS ***, 9 unordered pieces. GB_HTG4: AC006467 175695 AC006467
Drosophila melanogaster chromosome 2 clone BACR03L08 (D532) RPCI-98
03.L.8 Drosophila melanogaster 32,367 27-OCT-1999 map 40A-40C
strain y; cn bw sp, *** SEQUENCING IN PROGRESSo ***, 9 unordered
pieces. rxa02020 1111 GB_BA1: CGDNAAROP 2612 X85965 C. glutamicum
ORF3 and aroP gene. Corynebacterium 100,000 30-Nov-97 glutamicum
GB_PAT: A58887 1612 A58887 Sequence 1 from Patent WO9701637.
unidentified 100,000 06-MAR-1998 GB_BA1: STYCARABA 4378 M95047
Salmonella typhimurium transport protein, complete cds, and
transfer RNA-Arg. Salmonella typhimurium 50,547 13-MAR-1996
rxa02029 1437 GB_HTG2: AC003023 104768 AC003023 Homo sapiens
chromosome 11 clone pDJ363p2, *** SEQUENCING IN PROGRESS Homo
sapiens 35,820 21-OCT-1997
****, 22 unordered pieces. GB_HTG2: AC003023 104768 AC003023 Homo
sapiens chromosome 11 clone pDJ363p2, *** SEQUENCING IN PROGRESS
Homo sapiens 35,820 21-OCT-1997 ***, 22 unordered pieces. GB_HTG2:
HS118B18 104729 AL034344 Homo sapiens chromosome 6 clone RP1-118B18
map p24.1-25.3, *** Homo sapiens 34,355 03-DEC-1999 SEQUENCING IN
PROGRESS ***, in unordered pieces. rxa02030 1509 GB_PR4: AC007695
63247 AC007695 Homo sapiens 12q24 BAC RPCI11-124N23 (Roswell Park
Cancer Institute Human Homo sapiens 38,681 1-Sep-99 BAC Library)
complete sequence. GB_PR4: AC006464 99908 AC006464 Homo sapiens BAC
clone NH0436C12 from 2, complete sequence. Homo sapiens 35,445
22-OCT-1999 GB_PR4: AC006464 99908 AC006464 Homo sapiens BAC clone
NH0436C12 from 2, complete sequence. Homo sapiens 35,968
22-OCT-1999 rxa02073 1653 GB_BA1: CGGDHA 2037 X72855 C. glutamicum
GDHA gene. Corynebacterium 39,655 24-MAY-1993 glutamicum GB_BA1:
CGGDH 2037 X59404 Corynebacterium glutamicum, gdh gen for glutamate
dehydrogenase. Corynebacterium 44,444 30-Jul-99 glutamicum GB_BA2:
SC2H4 25970 AL031514 Streptomyces coelicolor cosmid 2H4.
Streptomyces coelicolor 38,452 19-OCT-1999 A3(2) rxa02074 rxa02095
1527 GB_EST18: AA703380 471 AA703380 zj12b06.s1
Soares_fetal_liver_spleen_1NFLS_S1 Home sapiens cDNA clone Home
sapiens 36,518 24-DEC-1997 IMAGE: 450035 3' similar to contains
LTR5.t3 LTR5 repetitive element:, mRNA sequence. GB_HTG6: AC009769
122911 AC009769 Homo sapiens chromosome 8 clone RP11-202I12 map 8,
LOW-PASS SEQUENCE Homo sapiens 35,473 07-DEC-1999 SAMPLING.
GB_EST7: W70175 436 W70175 zd52c02.r1 Soares_fetal_heart_NbHH19W
Homo sapiens cDNA clone Homo sapiens 34,174 16-OCT-1996 IMAGE:
344258 5' similar to contains LTR5.b2 LTR5 repetitive element;,
mRNA sequence. rxa02099 373 GB_BA1: CAJ10319 5368 AJ010319
Corynebacterium glutamicum amtP, glnB, glnD genes and partial ftsY
and srp genes. Corynebacterium 100,000 14-MAY-1999 glutamicum
GB_HTG3: AC011509 111353 AC011509 Homo sapiens chromosome 19 clone
CITB-H1_2189E23, *** SEQUENCING IN Homo sapiens 33,423 07-OCT-1999
PROGRESS ***, 35 unordered pieces. GB_HTG3: AC011509 111353
AC011509 Homo sapiens chromosome 19 clone CITB-H1_2189E23, ***
SEQUENCING IN Homo sapiens 33,423 07-OCT-1999 PROGRESS ***, 35
unordered pieces. rxa02115 1197 GB_HTG5: AC010126 175986 AC010126
Homo sapiens clone GS502B02, *** SEQUENCING IN PROGRESS ***, Homo
sapiens 36,717 13-Nov-99 3 unordered pieces. GB_HTG5: AC010126
175986 AC010126 Homo sapiens clone GS502B02, *** SEQUENCING IN
PROGRESS ***, Homo sapiens 36,092 13-Nov-99 3 unordered pieces.
GB_PR1: HUMHM145 2214 D10925 Human mRNA for HM145. Homo sapiens
39,171 3-Feb-99 rxa02128 1818 GB_BA1: MTCY190 34150 Z70283
Mycobacterium tuberculosis H37Rv complete genome; segment 98/162.
Mycobacterium 38,682 17-Jun-98 tuberculosis GB_BA1: MTCY190 34150
Z70283 Mycobacterium tuberculosis H37Rv complete genome; segment
98/162. Mycobacterium 35,746 17-Jun-98 tuberculosis GB_GSS10:
AQ161109 738 AQ161109 nbxb0006D03r CUGI Rice BAC Library Oryza
sativa genomic clone nbxb0006D03r, Oryza sativa 38,482 12-Sep-98
genomic survey sequence. rxa02133 329 GB_BA2: MPAE000058 28530
AE000058 Mycoplasma pneumoniae section 58 of 63 of the complete
genome. Mycoplasma pneumoniae 32,317 18-Nov-96 GB_HTG4: AC008308
151373 AC008308 Drosophila melanogaster chromosome 3 clone
BACR10M16 (D743) RPCI-98 Drosophila melanogaster 34,579 20-OCT-1999
10.M.16 map 93C-93D strain y; cn bw sp, *** SEQUENCING IN PROGRESS
***, 186 unordered pieces. GB_HTG4: AC008308 151373 AC008308
Drosophila melanogaster chromosome 3 clone BACR10M16 (D743) RPCI-98
Drosophila melanogaster 34,579 20-OCT-1999 10.M.16 map 93C-93D
strain y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 186 unordered
pieces. rxa02150 924 GB_EST37: AW012260 358 AW012260 um06e09.y1
Sugano mouse kidney mkia Mus musculus cDNA clone Mus musculus
39,385 10-Sep-99 IMAGE: 2182312 5' similar to SW: AMPL_BOVIN P00727
CYTOSOL AMINOPEPTIDASE;, mRNA sequence. GB_GSS3: B87734 389 B87734
RPCI11-30D24.TP RPCI-11 Homo sapiens genomic clone RPCI-11-30D24,
genomic Homo sapiens 37,629 9-Apr-99 survey sequence. GB_PR4:
AC005042 192218 AC005042 Homo sapiens clone NH0552E01, complete
sequence. Homo sapiens 36,901 14-Jan-99 rxa02171 1776 GB_BA2:
AF010496 189370 AF010496 Rhodobacter capsulatus strain SB1003,
partial genome. Rhodobacter capsulatus 53,714 12-MAY-1998 GB_EST24:
AI170522 367 AI170522 EST216450 Normalized rat lung, Bento Soares
Rattus sp. cDNA clone RLUCO75 3' Rattus sp. 44,186 20-Jan-99 end,
mRNA sequence. GB_PL1: PHVDLECA 1441 K03288 P. vulgaris
phytohemagglutinin gene encoding erythroagglutinating Phaseolus
vulgaris 39,103 27-Apr-93 phytohemagglutinin (PHA-E), complete cds.
rxa02173 1575 GB_BA1: CGGLTG 3013 X66112 C. glutamicum glt gene for
citrate synthase and ORF. Corynebacterium 44,118 17-Feb-95
glutamicum GB_BA1: CGGLTG 3013 X66112 C. glutamicum glt gene for
citrate synthase and ORF. Corynebacterium 36,189 17-Feb-95
glutamicum GB_BA2: AE000104 10146 AE000104 Rhizobium sp. NGR234
plasmid pNGR234a, section 41 of 46 of the complete Rhizobium sp.
NGR234 38,487 12-DEC-1997 plasmid sequence. rxa02224 1920 GB_BA2:
CXU21300 8990 U21300 Corynebacterium striatum hypothetical protein
YbhB gene, partial cds; ABC Corynebacterium 37,264 9-Apr-99
transporter TetB (tetB), ABC transporter TetA (tetA), transposase,
23S rRNA striatum methyltransferase, and transposase genes,
complete cds; and unknown genes. GB_HTG3: AC009185 87184 AC009185
Homo sapiens chromosome 5 clone CIT-HSPC_248O19, *** SEQUENCING IN
Homo sapiens 36,459 07-OCT-1999 PROGRESS ***, 2 ordered pieces.
GB_HTG3: AC009185 87184 AC009185 Homo sapiens chromosome 5 clone
CIT-HSPC_248O19, *** SEQUENCING IN Homo sapiens 36,459 07-OCT-1999
PROGRESS ***, 2 ordered pieces. rxa02225 905 GB_BA2: MPAE000058
28530 AE000058 Mycoplasma pneumoniae section 58 of 63 of the
complete genome. Mycoplasma pneumoniae 35,498 18-Nov-96 GB_EST26:
AI337275 618 AI337275 tb96h11.x1 NCI_CGAP_Co16 Homo sapiens cDNA
clone IMAGE: Homo sapiens 35,589 18-MAR-1999 2062245 3' similar to
TR: Q15392 Q15392 ORF, COMPLETE CDS.;, mRNA sequence. GB_EST26:
AI337275 618 AI337275 tb96h11.x1 NCI_CGAP_Co16 Homo sapiens cDNA
clone IMAGE: Homo sapiens 42,786 18-MAR-1999 2062245 3' similar to
TR: Q15392 Q15392 ORF, COMPLETE CDS.;, mRNA sequence. rxa02233 1410
GB_BA1: ERWPNLB 1291 M65057 Erwinia carotovora pectin lyase (pnl)
gene, complete cds. Erwinia carotovora 37,780 26-Apr-93 GB_EST30:
AV021947 313 AV021947 AV021947 Mus musculus 18-day embryo C57BL/6J
Mus musculus cDNA clone Mus musculus 39,423 28-Aug-99 1190024M23,
mRNA sequence. GB_EST33: AV087117 251 AV087117 AV087117 Mus
musculus tongue C57BL/6J adult Mus musculus cDNA clone Mus musculus
47,410 25-Jun-99 2310028C15, mRNA sequence. rxa02253 1050 GB_EST11:
AA250210 532 AA250210 mx79g10.r1 Soares mouse NML Mus musculus cDNA
clone IMAGE: 692610 5' Mus musculus 36,136 12-MAR-1997 similar to
TR: E236517 E236517 F44G4.1;, mRNA sequence. GB_EST11: AA250210 532
AA250210 mx79g10.r1 Soares mouse NML Mus musculus cDNA clone IMAGE:
692610 5' Mus musculus 36,202 12-MAR-1997 similar to TR: E236517
E236517 F44G4.1;, mRNA sequence. rxa02261 1479 GB_BA1: CGL007732
4460 AJ007732 Corynebacterium glutamicum 3' ppc gene, secG gene,
amt gene, ocd gene and 5' Corynebacterium 100,000 7-Jan-99 soxA
gene. glutamicum GB_BA1: CGAMTGENE 2028 X93513 C. glutamicum amt
gene. Corynebacterium 100,000 29-MAY-1996 glutamicum GB_BA1:
CORPEPC 4885 M25819 C. glutamicum phosphoenolpyruvate carboxylase
gene, complete cds. Corynebacterium 100,000 15-DEC-1995 glutamicum
rxa02268 1023 GB_PL2: AF087130 3478 AF087130 Neurospora crassa
siderophore regulation protein (sre) gene, complete cds. Neurospora
crassa 39,268 22-OCT-1998 GB_EST30: AI663709 408 AI663709
ud47a06.y1 Soares mouse mammary gland NbMMG Mus musculus cDNA clone
Mus musculus 41,523 10-MAY-1999 IMAGE: 1449010 5' similar to TR:
O75585 O75585 MITOGEN- AND STRESS- ACTIVATED PROTEIN KINASE-2;,
mRNA sequence. GB_RO: AF074714 3120 AF074714 Mus musculus mitogen-
and stress-activated protein kinase-2 (mMSK2) mRNA, Mus musculus
38,347 24-OCT-1998 complete cds. rxa02269 1095 GB_GSS4: AQ742825
847 AQ742825 HS_5482_B2_A04_T7A RPCI-11 Human Male BAC Library Homo
sapiens Homo sapiens 37,703 16-Jul-99 genomic clone Plate = 1058
Col = 8 Row = B, genomic survey sequence. GB_HTG3: AC009293 162944
AC009293 Homo sapiens chromosome 18 clone 53_I_06 map 18, ***
SEQUENCING IN Homo sapiens 37,006 13-Aug-99 PROGRESS ***, 15
unordered pieces. GB_HTG3: AC009293 162944 AC009293 Homo sapiens
chromosome 18 clone 53_I_06 map 18, *** SEQUENCING IN Homo sapiens
37,006 13-Aug-99 PROGRESS ***, 15 unordered pieces. rxa02309 1173
GB_BA1: MTY25D10 40838 Z95558 Mycobacterium tuberculosis H37Rv
complete genome; segment 28/162. Mycobacterium 52,344 17-Jun-98
tuberculosis GB_BA1: MSGY224 40051 AD000004 Mycobacterium
tuberculosis sequence from clone y224. Mycobacterium 52,344
03-DEC-1996 tuberculosis GB_HTG2: AC007163 186618 AC007163 Homo
sapiens clone NH0091M05, *** SEQUENCING IN PROGRESS ***, 1 Homo
sapiens 37,263 23-Apr-99 unordered pieces. rxa02310 1386 GB_BA1:
MTY25D10 40838 Z95558 Mycobacterium tuberculosis H37Rv complete
genome; segment 28/162. Mycobacterium 36,861 17-Jun-98 tuberculosis
GB_BA1: MSGY224 40051 AD000004 Mycobacterium tuberculosis sequence
from clone y224. Mycobacterium 36,861 03-DEC-1996 tuberculosis
GB_PR3: HS279N11 169998 Z98255 Human DNA sequence from PAC 279N11
on chromosome Xq11.2-13.3. Homo sapiens 34,516 23-Nov-99 rxa02321
1752 GB_BA1: AB018531 4961 AB018531 Corynebacterium glutamicum
dtsR1 and dtsR2 genes, complete cds. Corynebacterium 99,030
19-OCT-1998 glutamicum GB_PAT: E17019 4961 E17019 Brevibacterium
lactofermentum dtsR and dtsR2 genes. Corynebacterium 98,973
28-Jul-99 glutamicum GB_BA1: AB018530 2855 AB018530 Corynebacterium
glutamicum dtsR gene, complete cds. Corynebacterium 99,030
19-OCT-1998 glutamicum rxa02335 1896 GB_BA1: CGU35023 3195 U35023
Corynebacterium glutamicum thiosulfate sulfurtransferase (thtR)
gene, partial cds, Corynebacterium 99,947 16-Jan-97 acyl CoA
carboxylase (accBC) gene, complete cds. glutamicum GB_BA1: U00012
33312 U00012 Mycobacterium leprae cosmid B1308. Mycobacterium
leprae 40,247 30-Jan-96 GB_BA1: MTCY71 42729 Z92771 Mycobacterium
tuberculosis H37Rv complete genome; segment 141/162. Mycobacterium
67,568 10-Feb-99 tuberculosis rxa02364 750 GB_BA1: AP000006 319000
AP000006 Pyrococcus horikoshii OT3 genomic DNA, 1166001-1485000 nt.
position (6/7). Pyrococcus horikoshii 36,130 8-Feb-99 GB_BA1:
AP000006 319000 AP000006 Pyrococcus horikoshii OT3 genomic DNA,
1166001-1485000 nt. position (6/7). Pyrococcus horikoshii 34,543
8-Feb-99 rxa02372 2010 GB_HTG3: AC011461 100974 AC011461 Homo
sapiens chromosome 19 clone CIT-HSPC_429L19, *** SEQUENCING IN Homo
sapiens 36,138 07-OCT-1999 PROGRESS ***, 4 ordered pieces. GB_HTG3:
AC011461 100974 AC011461 Homo sapiens chromosome 19 clone
CIT-HSPC_429L19, *** SEQUENCING IN Homo sapiens 36,138 07-OCT-1999
PROGRESS ***, 4 ordered pieces. GB_EST21: AA992021 279 AA992021
ot36c01.s1 Soares_testis_NHT Homo sapiens cDNA clone IMAGE: 1618848
3', Homo sapiens 41,219 3-Jun-98 mRNA sequence. rxa02397 1119
GB_HTG4: AC009273 76175 AC009273 Arabidopsis thaliana chromosome 1
clone T1N6, *** SEQUENCING IN PROGRESS Arabidopsis thaliana 38,566
12-OCT-1999 ***, 2 ordered pieces. GB_HTG4: AC009273 76175 AC009273
Arabidopsis thaliana chromosome 1 clone T1N6, *** SEQUENCING IN
PROGRESS Arabidopsis thaliana 38,566 12-OCT-1999 ***, 2 ordered
pieces. GB_BA1: D90826 19493 D90826 E. coli genomic DNA, Kohara
clone #335(40.9-41.3 min.). Escherichia coli 39,600 21-MAR-1997
rxa02424 723 GB_EST13: AA334108 275 AA334108 EST38262 Embryo, 9
week Homo sapiens cDNA 5' end, mRNA sequence. Homo sapiens 38,603
21-Apr-97 GB_PR3: AC005224 166687 AC005224 Homo sapiens chromosome
17, clone hRPK.214_O_1, complete sequence. Homo sapiens 36,111
14-Aug-98 GB_PR3: AC005224 166687 AC005224 Homo sapiens chromosome
17, clone hRPK.214_O_1, complete sequence. Homo sapiens 33,427
14-Aug-98 rxa02426 1656 GB_PAT: A06664 1350 A06664 B.
stearothermophilus lct gene. Bacillus 39,936 29-Jul-93
stearothermophilus GB_PAT: A04115 1361 A04115 B. stearothermophilus
recombinant lct gene. synthetic construct 40,042 17-Feb-97 GB_BA1:
BACLDHL 1361 M14788 B. stearothermophilus lct gene encoding
L-lactate dehydrogenase, complete cds. Bacillus 40,338 26-Apr-93
stearothermophilus rxa02487 1827 GB_BA2: AF007101 32870 AF007101
Streptomyces hygroscopicus putative pteridine-dependent
dioxygenase, PKS Streptomyces 43,298 13-Jan-98 modules 1, 2, 3 and
4, and putative regulatory protein genes, complete cds and
hygroscopicus putative hydroxylase gene, partial cds.
GB_BA1: MTCI364 29540 Z93777 Mycobacterium tuberculosis H37Rv
complete genome; segment 52/162. Mycobacterium 44,352 17-Jun-98
tuberculosis GB_BA2: AF119621 15986 AF119621 Pseudomonas
abietaniphila BKME-9 Ditl (ditl), dioxygenase DitA oxygenase
Pseudomonas 43,611 28-Apr-99 component small subunit (ditA2),
dioxygenase DitA oxygenase component large abietaniphila subunit
(ditA1), DitH (ditH), DitG (ditG), DitF (ditF), DitR (ditR), DitE
(ditE), DitD (ditD), aromatic diterpenoid extradiol ring-cleavage
dioygenase (ditC), DitB (ditB), and dioxygenase DitA ferredoxin
component (ditA3) genes, complete cds; and unknown genes. rxa02511
780 GB_PR4: AC002470 235395 AC002470 Homo sapiens Chromosome
22q11.2 BAC Clone b135h6 in BCRL2-GGT Region, Homo sapiens 37,971
30-Nov-99 complete sequence. GB_PR4: AC002472 147100 AC002472 Homo
sapiens Chromosome 22q11.2 PAC Clone p_n5 in BCRL2-GGT Region, Homo
sapiens 38,239 13-Sep-99 complete sequence. GB_EST34: AI806938 118
AI806938 wf24b07.x1 Soares_NFL_T_GBC_S1 Homo sapiens cDNA clone
IMAGE: Homo sapiens 38,983 7-Jul-99 2356501 3' similar to SW:
PLZF_HUMAN Q05516 ZINC FINGER PROTEIN PLZF;, mRNA sequence.
rxa02512 1086 GB_BA1: MTCY1A10 25949 Z95387 Mycobacterium
tuberculosis H37Rv complete genome; segment 117/162. Mycobacterium
37,407 17-Jun-98 tuberculosis GB_BA1: MLCL581 36225 Z96801
Mycobacterium leprae cosmid L581. Mycobacterium leprae 43,193
24-Jun-97 GB_OV: GGU43396 2738 U43396 Gallus gallus tropomyosin
receptor kinase A (ctrkA) mRNA, complete cds. Gallus gallus 38,789
18-Jan-96 rxa02527 1452 GB_BA2: AF008220 220060 AF008220 Bacillus
subtilis rrnB-dnaB genomic region. Bacillus subtilis 37,395
4-Feb-98 GB_BA2: AF008220 220060 AF008220 Bacillus subtilis
rrnB-dnaB genomic region. Bacillus subtilis 36,218 4-Feb-98
GB_HTG2: AC005861 112369 AC005861 Arabidopsis thaliana clone
F23B24, *** SEQUENCING IN PROGRESS ***, 6 Arabidopsis thaliana
38,407 29-Apr-99 unordered pieces. rxa02547 2262 GB_PL1: AB006530
7344 AB006530 Citrullus lanatus Sat gene for serine
acetyltransferase, complete cds and 5'-flanking Citrullus lanatus
35,449 20-Aug-97 region. GB_PL1: CNASA 5729 D85624 Citrullus
vulgaris serine acetyltransferase (Sat) DNA, complete cds.
Citrullus lanatus 35,449 6-Feb-99 GB_PL1: AB006530 7344 AB006530
Citrullus lanatus Sat gene for serine acetyltransferase, complete
cds and 5'-flanking Citrullus lanatus 34,646 20-Aug-97 region.
rxa02566 1332 GB_EST32: AI727189 619 AI727189 BNLGHI7498 Six-day
Cotton fiber Gossypium hirsutum cDNA 5' similar to Gossypium
hirsutum 35,099 11-Jun-99 (AB020715) KIAA0908 protein [Homo
sapiens], mRNA sequence. GB_BA1: CGPUTP 3791 Y09163 C. glutamicum
putP gene. Corynebacterium 38,562 8-Sep-97 glutamicum GB_PL2:
SPAC13G6 33481 Z54308 S. pombe chromosome I cosmid c13G6.
Schizosaccharomyces 35,774 18-OCT-1999 pombe rxa02571 1152 GB_BA1:
CGU43535 2531 U43535 Corynebacterium glutamicum multidrug
resistance protein (cmr) gene, complete cds. Corynebacterium 41,872
9-Apr-97 glutamicum GB_EST35: AI857385 488 AI857385 wl55e03.x1
NCI_CGAP_Brn25 Homo sapiens cDNA clone IMAGE: 2428828 3', Homo
sapiens 39,139 26-Aug-99 mRNA sequence. GB_BA1: CGU43535 2531
U43535 Corynebacterium glutamicum multidrug resistance protein
(cmr) gene, complete cds. Corynebacterium 38,552 9-Apr-97
glutamicum rxa02578 1227 GB_PL1: AB016871 79109 AB016871
Arabldopsis thaliana genomic DNA, chromosome 5, TAC clone: K16L22,
complete Arabidopsis thaliana 34,213 20-Nov-99 sequence. GB_PL1:
AB025602 55790 AB025602 Arabidopsis thaliana genomic DNA,
chromosome 5, BAC clone: F14A1, complete Arabidopsis thaliana
36,461 20-Nov-99 sequence. GB_IN1: CELF36H9 35985 AF016668
Caenorhabditis elegans cosmid F36H9. Caenorhabditis elegans 35,977
8-Aug-97 rxa02581 1983 GB_BA1: MTV005 37840 AL010186 Mycobacterium
tuberculosis H37Rv complete genome; segment 51/162. Mycobacterium
38,517 17-Jun-98 tuberculosis GB_BA1: MTV005 37840 AL010186
Mycobacterium tuberculosis H37Rv complete genome; segment 51/162.
Mycobacterium 39,173 17-Jun-98 tuberculosis rxa02582 4953 GB_BA1:
MTV026 23740 AL022076 Mycobacterium tuberculosis H37Rv complete
genome; segment 157/162. Mycobacterium 38,548 24-Jun-99
tuberculosis GB_BA1: MTCY338 29372 Z74697 Mycobacterium
tuberculosis H37Rv complete genome; segment 127/162. Mycobacterium
46,263 17-Jun-98 tuberculosis GB_BA1: SEERYABS 20444 X62569 S.
erythraea eryA gene for 6-deoxyerythronolyde B synthase II &
III. Saccharopolyspora 45,053 28-Feb-92 erythraea rxa02583 1671
GB_BA2: AF113605 1593 AF113605 Streptomyces coelicolor
proplonyl-CoA carboxylase complex B subunit (pccB) gene,
Streptomyces coelicolor 58,397 08-DEC-1999 complete cds. GB_BA1:
SC1C2 42210 AL031124 Streptomyces coelicolor cosmid 1C2.
Streptomyces coelicolor 52.916 15-Jan-99 GB_BA1: AB018531 4961
AB018531 Corynebacterium glutamicum dtsR1 and dtsR2 genes, complete
cds. Corynebacterium 58,809 19-OCT-1998 glutamicum rxa02599 600
GB_BA1: AEMML 2585 X99639 Ralstonia eutropha mmlH, mmlI & mmlJ
genes. Ralstonia eutropha 35,264 22-Jan-98 GB_EST15: AA508926 422
AA508926 MBAFCW1C08T3 Brugia malayi adult female cDNA
(SAW96MLW-BmAF) Brugia Brugia malayi 43,377 8-Jul-97 malayi cDNA
clone AFCW1C08 5', mRNA sequence. GB_BA1: AEMML 2585 X99639
Ralstonia eutropha mmlH, mmlI & mmlJ genes. Ralstonia eutropha
41,148 22-Jan-98 rxa02634 1734 GB_BA1: SYNPOO 1964 X17439
Synechocystis ndhC, psbG genes for NDH-C, PSII-G and ORF157.
Synechocystis PCC6803 38,145 10-Feb-99 GB_GSS9: AQ101527 184
AQ101527 HS_2265_A1_E11_MF CIT Approved Human Genomic Sperm Library
D Homo Homo sapiens 38,798 27-Aug-98 sapiens genomic clone Plate =
2265 Col = 21 Row = l, genomic survey sequence. GB_IN1: MNE133341
399 AJ133341 Melarhaphe neritoides partial caM gene, exons 1-2.
Melarhaphe neritoides 39,098 2-Jun-99 rxa02638 999 GB_BA2 AE001756
10938 AE001756 Thermotoga maritima section 68 of 136 of the
complete genome. Thermotoga maritima 40,104 2-Jun-99 GB_GSS12:
AQ423878 689 AQ423878 CITBI-E1-2575E20.TF CITBI-E1 Homo sapiens
genomic clone 2575E20, genomic Homo sapiens 36,451 23-MAR-1999
survey sequence. GB_HTG2: AC006765 274498 AC006765 Caenorhabditis
elegans clone Y43H11, *** SEQUENCING IN PROGRESS***, 7
Caenorhabditis elegans 39,072 23-Feb-99 unordered pieces. rxa02659
335 GB_EST36: AI900317 436 AI900317 sc04a02.y1 Gm-c1012 Glycine max
cDNA clone GENOME SYSTEMS CLONE Glycine max 41,566 06-DEC-1999 ID:
Gm-c1012-1155 5' similar to SW: PRS6_SOLTU P54778 26S PROTEASE
REGULATORY SUBUNIT 6B HOMOLOG.;, mRNA sequence. GB_GSS12: AQ342831
683 AQ342831 RPCI11-122K17.TJ RPCI-11 Homo sapiens genomic clone
RPCI-11-122K17, Homo sapiens 34,762 07-MAY-1999 genomic survey
sequence. GB_EST36: AI900856 779 AI900856 sb95c11.y1 Gm-c1012
Glycine max cDNA clone GENOME SYSTEMS CLONE ID: Glycine max 39,063
06-DEC-1999 Gm-c1012-429 5' similar to SW: PRS6_SOLTU P54778 26S
PROTEASE REGULATORY SUBUNIT 6B HOMOLOG.;, mRNA sequence. rxa02676
1512 GB_IN2: CELB0213 39134 AF039050 Caenorhabditis elegans cosmid
B0213. Caenorhabditis elegans 35,814 2-Jun-99 GB_GSS1: CNS00PZB 364
AL085157 Arabidopsis thaliana genome survey sequence SP6 end of BAC
F10D11 of IGF Arabidopsis thaliana 38,462 28-Jun-99 library from
strain Columbia of Arabidopsis thaliana, genomic survey sequence.
GB_RO: RNITPR2R 10708 X61677 Rat ITPR2 gene for type 2 inositol
triphosphate receptor. Rattus norvegicus 37,543 21-OCT-1991
rxa02677 882 GB_RO: D89728 5002 D89728 Mus musculus mRNA for LOK,
complete cds. Mus musculus 38,829 7-Feb-99 GB_GSS8: AQ062004 362
AQ062004 CIT-HSP-2346O14, TR CIT-HSP Homo sapiens genomic clone
2346O14, genomic Homo sapiens 36,565 31-Jul-98 survey sequence.
GB_GSS14: AQ555818 462 AQ555818 HS_5230_B1_G06_SP6E RPCI-11 Human
Male BAC Library Homo sapiens Homo sapiens 36,534 29-MAY-1999
genomic clone Plate = 806 Col = 11 Row = N, genomic survey
sequence. rxa02691 930 GB_IN1: DME9736 7411 AJ009736 Drosophila
melanogaster idefix retroelement: gag, pol and env genes, partial.
Drosophila melanogaster 36,522 19-Jan-99 GB_PR4: AC004801 193561
AC004801 Homo sapiens 12q13.1 PAC RPCI1-228P16 (Roswell Park Cancer
Institute Human Homo sapiens 39,341 2-Feb-99 PAC Library) complete
sequence. GB_PR4: AC004801 193561 AC004801 Homo sapiens 12q13.1 PAC
RPCI1-228P16 (Roswell Park Cancer Institute Human Homo sapiens
37,037 2-Feb-99 PAC Library) complete sequence. rxa02718 1170
GB_EST34: AV132028 258 AV132028 AV132028 Mus musculus C57BL/6J
11-day embryo Mus musculus cDNA clone Mus musculus 43,529 1-Jul-99
2700087F01, mRNA sequence. GB_GSS10: AQ240654 452 AQ240654
CIT-HSP-2385D24.TFB.1 CIT-HSP Homo sapiens genomic clone 2385D24,
genomic Homo sapiens 40,044 30-Sep-98 survey sequence. GB_GSS11:
AQ309500 576 AQ309500 CIT-HSP-2384D24.TFD CIT-HSP Homo sapiens
genomic clone 2384D24, genomic Homo sapiens 38,869 22-DEC-1998
survey sequence. rxa02749 999 GB_BA2: AF086791 37867 AF086791
Zymomonas mobilis strain ZM4 clone 67E10 carbamoylphosphate
synthetase small Zymomonas mobilis 39,024 4-Nov-98 subunit (carA),
carbamoylphosphate synthetase large subunit (carB), transcription
elongation factor (greA), enolase (eno), pyruvate dehydrogenase
alpha subunit (pdhA), pyruvate dehydrogenase beta subunit (pdhB),
ribonuclease H (mh), homoserine kinase homolog, alcohol
dehydrogenase II (adhB), and excinuclease ABC subunit A (uvrA)
genes, complete cds; and unknown genes. GB_BA1: SYCSLRB 146271
D64000 Synechocystis sp. PCC6803 complete genome, 19/27,
2392729-2538999. Synechocystis sp. 34,573 13-Feb-99 GB_BA2:
AE001306 13316 AE001306 Chlamydia trachomatis section 33 of 87 of
the complete genome. Chlamydia trachomatis 38,940 2-Sep-98 rxa02767
906 GB_BA2: AF126953 1638 AF126953 Corynebacterium glutamicum
cystathionine gamma-synthase (metB) gene, complete Corynebacterium
100,000 10-Sep-99 cds. glutamicum GB_BA1: SCI5 6661 AL079332
Streptomyces coelicolor cosmid I5. Streptomyces coelicolor 37,486
16-Jun-99 GB_PR3: HS90L6 190837 Z97353 Human DNA sequence from
clone 90L6 on chromosome 22q11.21-11.23. Contains Homo sapiens
34,149 23-Nov-99 an RPL15 (60S Ribosomal Protein L15) pseudogene,
ESTs, STSs and GSSs, complete sequence. rxa02792 876 GB_BA2:
AF099015 5000 AF099015 Streptomyces coelicolor strain A3(2)
integrase (int), Fe-containing superoxide Streptomyces coelicolor
36,721 1-Jun-99 dismutase II (sodF2), Fe uptake system permease
(ftrE), and Fe uptake system integral membrane protein (ftrD)
genes, complete cds. GB_BA1: ECOUW93 338534 U14003 Escherichia coli
K-12 chromosomal region from 92.8 to 00.1 minutes. Escherichia coli
38,787 17-Apr-96 GB_HTG3: AC011361 186148 AC011361 Homo sapiens
chromosome 5 clone CIT-HSPC_482N19, *** SEQUENCING IN Homo sapiens
43,577 06-OCT-1999 PROGRESS ***, 69 unordered pieces. rxa02794 1197
GB_PR4: AC005998 96556 AC005998 Homo sapiens clone DJ0622E21,
complete sequence. Homo sapiens 37,298 29-Jul-99 GB_PR4: AC006008
57554 AC006008 Homo sapiens clone DJ0820A21, complete sequence.
Homo sapiens 36,638 17-Jun-99 GB_PR3: HSDJ73H14 95556 AL080272
Human DNA sequence from clone 73H14 on chromosome Xq26.3-28,
complete Homo sapiens 39,726 23-Nov-99 sequence. rxa02809 375
GB_RO: MUSSPCTLT 3172 M22527 Mouse cytotoxic T lymphocyte-specific
serine protease CCPII gene, complete cds. Mus musculus 47,518
19-Jan-96 GB_RO: MUSGRC 894 M18459 Mouse granzyme C serine esterase
mRNA, complete cds. Mus musculus 44,939 12-Jun-93 GB_RO: RNU57062
880 U57062 Rattus norvegicus natural killer cell protease 4
(RNKP-4) mRNA, complete cds. Rattus norvegicus 41,554 31-Jul-96
rxa02811 484 GB_GSS6: AQ832862 476 AQ832862 HS_5261_A2_E10_SP6E
RPCI-11 Human Male BAC Library Homo sapiens Homo sapiens 35,610
27-Aug-99 genomic clone Plate = 837 Col = 20 Row = I, genomic
survey sequence. GB_GSS5: AQ784593 515 AQ784593 HS_3248_A2_F02_T7C
CIT Approved Human Genomic Sperm Library D Homo Homo sapiens 38,956
3-Aug-99 sapiens genomic clone Plate = 3248 Col = 4 Row = K,
genomic survey sequence. GB_GSS13: AQ473140 397 AQ473140
CITBI-E1-2589G6.TF CITBI-E1 Homo sapiens genomic clone 2589G6,
genomic Homo sapiens 34,761 23-Apr-99 survey sequence. rxa02836 678
GB_EST18: AA696785 316 AA696785 GM08392.5prime GM Drosophila
melanogaster ovary BlueScript Drosophila Drosophila melanogaster
40,604 28-Nov-98 melanogaster cDNA clone GM08392 5prime, mRNA
sequence. GB_EST18: AA696785 316 AA696785 GM08392.5prime GM
Drosophila melanogaster ovary BlueScript Drosophila Drosophila
melanogaster 38,281 28-Nov-98 melanogaster cDNA clone GM08392
5prime, mRNA sequence. rxs03212 1452 GB_BA1: CGBETPGEN 2339 X93514
C. glutamicum betP gene. Corynebacterium 99,931 8-Sep-97 glutamicum
GB_BA1: SC5F2A 40105 AL049587 Streptomyces coelicolor cosmid 5F2A.
Streptomyces coelicolor 57,557 24-MAY-1999
A3(2) GB_BA2: AF008220 220060 AF008220 Bacillus subtilis rrnB-dnaB
genomic region. Bacillus subtilis 40,000 4-Feb-98 rxs03220 725
GB_PL1: CKHUP2 2353 X66855 C. kessleri HUP2 mRNA. Chlorella
kessleri 45,328 17-Feb-97 GB_EST38: AW048153 383 AW048153
UI-M-BH1-alq-h-05-0-UI.s1 NIH_BMAP_M_S2 Mus musculus cDNA Mus
musculus 41,758 18-Sep-99 clone UI-M-BH1-alq-h-05-0-UI 3', mRNA
sequence. GB_PL1: CKHUP2 2353 X66855 C. kessleri HUP2 mRNA.
Chlorella kessleri 38,106 17-Feb-97
[0172]
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070111230A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070111230A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References