U.S. patent application number 11/505174 was filed with the patent office on 2007-04-12 for corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production.
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 | 20070082383 11/505174 |
Document ID | / |
Family ID | 38481749 |
Filed Date | 2007-04-12 |
United States Patent
Application |
20070082383 |
Kind Code |
A1 |
Pompejus; Markus ; et
al. |
April 12, 2007 |
Corynebacterium glutamicum genes encoding proteins involved in
carbon metabolism and energy production
Abstract
Isolated nucleic acid molecules, designated SMP nucleic acid
molecules, which encode novel SMP proteins from Corynebacterium
glutamicum are described. The invention also provides antisense
nucleic acid molecules, recombinant expression vectors containing
SMP nucleic acid molecules, and host cells into which the
expression vectors have been introduced. The invention still
further provides isolated SMP proteins, mutated SMP proteins,
fusion proteins, antigenic peptides and methods for the improvement
of production of a desired compound from C. glutamicum based on
genetic engineering of SMP 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: |
38481749 |
Appl. No.: |
11/505174 |
Filed: |
August 15, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09602740 |
Jun 23, 2000 |
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11505174 |
Aug 15, 2006 |
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60141031 |
Jun 25, 1999 |
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60143208 |
Jul 9, 1999 |
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60151572 |
Aug 31, 1999 |
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Current U.S.
Class: |
435/106 ;
435/193; 435/252.3; 435/471; 536/23.2 |
Current CPC
Class: |
C12N 9/18 20130101; C12P
19/34 20130101; C12P 7/6427 20130101; C12P 13/227 20130101; C12P
13/14 20130101; C12P 13/04 20130101; C12P 13/12 20130101; C12P
13/222 20130101; C12P 13/06 20130101; C12P 13/08 20130101; C12Y
301/01031 20130101; C12P 7/6463 20130101; C12P 13/10 20130101; C12N
9/00 20130101; C12N 9/90 20130101; C12P 13/24 20130101; C12P 19/00
20130101; C12P 7/6409 20130101; C07K 2319/00 20130101; C12P 7/6472
20130101; C12P 13/225 20130101; C12P 7/40 20130101; C12P 7/18
20130101; C12P 13/20 20130101; C07K 14/34 20130101 |
Class at
Publication: |
435/106 ;
435/193; 435/471; 435/252.3; 536/023.2 |
International
Class: |
C12P 13/04 20060101
C12P013/04; C07H 21/04 20060101 C07H021/04; C12N 9/10 20060101
C12N009/10; C12N 15/74 20060101 C12N015/74; C12N 1/21 20060101
C12N001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 9, 1999 |
DE |
19932180.9 |
Jul 9, 1999 |
DE |
19932227.9 |
Jul 9, 1999 |
DE |
19932230.9 |
Jul 14, 1999 |
DE |
19933005.0 |
Aug 27, 1999 |
DE |
19940765.7 |
Jul 8, 1999 |
DE |
19931428.4 |
Jul 8, 1999 |
DE |
19931431.4 |
Jul 8, 1999 |
DE |
19931433.0 |
Jul 8, 1999 |
DE |
19931562.0 |
Jul 14, 1999 |
DE |
19932924.9 |
Jul 14, 1999 |
DE |
19932973.7 |
Sep 3, 1999 |
DE |
19942123.4 |
Sep 3, 1999 |
DE |
19942125.0 |
Sep 3, 1999 |
DE |
19942079.3 |
Sep 3, 1999 |
DE |
19942088.2 |
Jul 8, 1999 |
DE |
19931413.6 |
Sep 3, 1999 |
DE |
19942087.4 |
Jul 8, 1999 |
DE |
19931419.5 |
Jul 8, 1999 |
DE |
19931420.9 |
Jul 8, 1999 |
DE |
19931412.8 |
Jul 8, 1999 |
DE |
19931424.1 |
Jul 8, 1999 |
DE |
19931434.9 |
Jul 8, 1999 |
DE |
19931510.8 |
Jul 8, 1999 |
DE |
19931634.1 |
Sep 3, 1999 |
DE |
19942076.9 |
Sep 3, 1999 |
DE |
19942086.6 |
Sep 3, 1999 |
DE |
19942095.5 |
Claims
1. An insolate nucleic acid molecule selected from the group
consisiting of a) an isolated nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:71, or a complement thereof; b) an
isolated nucleic acid molecule which encodes a polypeptide
comprising the amino acid sequence of SEQ ID NO:72, 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:72, 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:71, 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:71,
or a complement thereof.
2. The isolated nucleic acid molecule of claim 1, wherein said
nucleic acid molecule encodes a protein involved in the production
of a fine chemical.
3. An isolated nucleic acid molecule comprising the nucleic acid
molecule of claim 1 and a nucleotide sequence encoding a
heterologous polypeptide.
4. A vector comprising the nucleic acid molecule of claim 1.
5. The vector of claim 4, which is an expression vector.
6. A host cell transfected with the expression vector of claim
5.
7. The host cell of claim 6, wherein said cell is a
microorganism.
8. The host cell of claim 7, wherein said cell belongs to the genus
Corynebacterium or Brevibacterium.
9. A method of producing a polypeptide comprising culturing the
host cell of claim 6 in an appropriate culture medium to, thereby,
produce the polypeptide.
10. A method for producing a fine chemical, comprising culturing
the cell of claim 6 such that the fine chemical is produced.
11. The method of claim 10, wherein said method further comprises
the step of recovering the fine chemical from said culture.
12. The method of claim 10, wherein said cell belongs to the genus
Corynebacterium or Brevibacterium.
13. The method of claim 10, 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, Brevibacteriuum
ammoniagenes, Brevibacterium butanicum, Brevibacterium divaricatum,
Brevibacterium flavum, Brevibacterium healii, Brevibacterium
ketoglutamicum, Brevibacterium ketosoreductum, Brevibacterium
lactofermentum, Brevibacterium linens, Brevibacterium
paraffinolyticum, land those strains set forth in Table 3.
14. The method of claim 10, wherein expression of the nucleic acid
molecule from said vector results in modulation of production of
said fine chemical.
15. The method of claim 10, 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.
16. The method of claim 10, 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.
17. An isolated polypeptide selected from the group consisting of
a) an isolated polypeptide comprising the amino acid sequence of
SEQ ID NO:72; b) an isolated polypeptide comprising a naturally
occurring allelic variant of a polypeptide comprising the amino
acid sequence of SEQ ID NO:72; c) an isolated polypeptide which is
encoded by a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:71; 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:71; 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:72; and f) an isolated polypeptide
comprising a fragment of a polypeptide comprising the amino acid
sequence of SEQ ID NO:72, wherein said polypeptide fragment
maintains a biological activity of the polypeptide comprising the
amino sequence.
18. The isolated polypeptide of claim 17, wherein said polypeptide
is involved in the production of a fine chemical.
19. The isolated polypeptide of claim 17, further comprising
heterologous amino acid sequences.
20. 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.
21. 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 17,
thereby diagnosing the presence or activity of Corynebacterium
diphtheriae in the subject.
22. A host cell comprising a nucleic acid molecule selected from
the group consisting of a) the nucleic acid molecule of SEQ ID
NO:71, 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:71, wherein the nucleic acid molecule
comprises one or more nucleic acid modifications as compared to the
sequence of SEQ ID NO:71, 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:71, 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. 09/602,740, filed Jun. 23, 2000, which claims priority to prior
U.S. Provisional Patent Application Ser. No. 60/141,031, filed Jun.
25, 1999, U.S. Provisional Patent Application Ser. No. 60/143,208,
filed Jul. 9, 1999, and U.S. Provisional Patent Application Ser.
No. 60/151,572, filed Aug. 31, 1999. This application also claims
priority to prior German Patent Application No. 19931412.8, filed
Jul. 8, 1999, German Patent Application No. 19931413.6, filed Jul.
8, 1999, German Patent Application No. 19931419.5, filed Jul. 8,
1999, German Patent Application No. 19931420.9, filed Jul. 8, 1999,
German Patent Application No. 19931424.1, filed Jul. 8, 1999,
German Patent Application No. 19931428.4, filed Jul. 8, 1999,
German Patent Application No. 19931431.4, filed Jul. 8, 1999,
German Patent Application No. 19931433.0, filed Jul. 8, 1999,
German Patent Application No. 19931434.9, filed Jul. 8, 1999,
German Patent Application No. 19931510.8, filed Jul. 8, 1999,
German Patent Application No. 19931562.0, filed Jul. 8, 1999,
German Patent Application No. 19931634.1, filed Jul. 8, 1999,
German Patent Application No. 19932180.9, filed Jul. 9, 1999,
German Patent Application No. 19932227.9, filed Jul. 9, 1999,
German Patent Application No. 19932230.9, filed Jul. 9, 1999,
German Patent Application No. 19932924.9, filed Jul. 14, 1999,
German Patent Application No. 19932973.7, filed Jul. 14, 1999,
German Patent Application No. 19933005.0, filed Jul. 14, 1999,
German Patent Application No. 19940765.7, filed Aug. 27, 1999,
German Patent Application No. 19942076.9, filed Sep. 3, 1999,
German Patent Application No. 19942079.3, filed Sep. 3, 1999,
German Patent Application No. 19942086.6, filed Sep. 3, 1999,
German Patent Application No. 19942087.4, filed Sep. 3, 1999,
German Patent Application No. 19942088.2, filed Sep. 3, 1999,
German Patent Application No. 19942095.5, filed Sep. 3, 1999,
German Patent Application No. 19942123.4, filed Sep. 3, 1999, and
German Patent Application No. 19942125.0, filed Sep. 3, 1999. The
entire contents of all of the aforementioned application are hereby
expressly incorporated herein by this reference.
INCORPORATION OF MATERIAL SUBMITTED OF 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 "seqlistcorr" (2.79 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" (444 KB) and "Appendix B" (157 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
sugar metabolism and oxidative phosphorylation (SMP) 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 SMP 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 SMP nucleic acids of the
invention, or modification of the sequence of the SMP 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 SMP 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 SMP 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.
[0008] e.g.e.g. The SMP proteins encoded by the novel nucleic acid
molecules of the invention are capable of, for example, performing
a function involved in the metabolism of carbon compounds such as
sugars or in the generation of energy molecules by processes such
as oxidative phosphorylation in Corynebacterium glutamicum. 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.
[0009] There are a number of mechanisms by which the alteration of
an SMP 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. The
degradation of high-energy carbon molecules such as sugars, and the
conversion of compounds such as NADH and FADH.sub.2 to compounds
containing high energy phosphate bonds via oxidative
phosphorylation results in a number of compounds which themselves
may be desirable fine chemicals, such as pyruvate, ATP, NADH, and a
number of intermediate sugar compounds. Further, the energy
molecules (such as ATP) and the reducing equivalents (such as NADH
or NADPH) produced by these metabolic pathways are utilized in the
cell to drive reactions which would otherwise be energetically
unfavorable. Such unfavorable reactions include many biosynthetic
pathways for fine chemicals. By improving the ability of the cell
to utilize a particular sugar (e.g., by manipulating the genes
encoding enzymes involved in the degradation and conversion of that
sugar into energy for the cell), one may increase the amount of
energy available to permit unfavorable, yet desired metabolic
reactions (e.g., the biosynthesis of a desired fine chemical) to
occur.
[0010] The mutagenesis of one or more SMP genes of the invention
may also result in SMP proteins having altered activities which
indirectly impact the production of one or more desired fine
chemicals from C. glutamicum. For example, by increasing the
efficiency of utilization of one or more sugars (such that the
conversion of the sugar to useful energy molecules is improved), or
by increasing the efficiency of conversion of reducing equivalents
to useful energy molecules (e.g., by improving the efficiency of
oxidative phosphorylation, or the activity of the ATP synthase),
one can increase the. amount of these high-energy compounds
available to the cell to drive normally unfavorable metabolic
processes. These processes include the construction of cell walls,
transcription, translation, and the biosynthesis of compounds
necessary for growth and division of the cells (e.g., nucleotides,
amino acids, vitamins, lipids, etc.) (Lengeler et al. (1999)
Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109;
913-918; 875-899). By improving the growth and multiplication of
these engineered cells, it is possible to increase both the
viability of the cells in large-scale culture, and also to improve
their rate of division, such that a relatively larger number of
cells can survive in fermnentor culture. The yield, production, or
efficiency of production may be increased, at least due to the
presence of a greater number of viable cells, each producing the
desired fine chemical. Also, many of the degradation products
produced during sugar metabolism are utilized by the cell as
precursors or intermediates in the production of other desirable
products, such as fine chemicals. So, by increasing the ability of
the cell to metabolize sugars, the number of these degradation
products available to the cell for other processes should also be
increased.
[0011] The invention provides novel nucleic acid molecules which
encode proteins, referred to herein as SMP proteins, which are
capable of, for example, performing a function involved in the
metabolism of carbon compounds such as sugars and the generation of
energy molecules by processes such as oxidative phosphorylation in
Corynebacterium glutamicum. Nucleic acid molecules encoding an SMP
protein are referred to herein as SMP nucleic acid molecules. In a
preferred embodiment, the SMP protein participates in the
conversion of carbon molecules and degradation products thereof to
energy which is utilized by the cell for metabolic processes.
Examples of such proteins include those encoded by the genes set
forth in Table 1.
[0012] Accordingly, one aspect of the invention pertains to
isolated nucleic acid molecules (e.g., cDNAs, DNAs, or RNAs)
comprising a nucleotide sequence encoding an SMP protein or
biologically active portions thereof, as well as nucleic acid
fragments suitable as primers or hybridization probes for the
detection or amplification of SMP-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 SMP
proteins of the present invention also preferably possess at least
one of the SMP activities described herein.
[0013] 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 SMP activity.
Preferably, the protein or portion thereof encoded by the nucleic
acid molecule maintains the ability to perform a function involved
in the metabolism of carbon compounds such as sugars or the
generation of energy molecules (e.g., ATP) by processes such as
oxidative phosphorylation in Corynebacterium glutamicum. 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).
[0014] In another preferred embodiment, the isolated nucleic acid
molecule is derived from C. glutamicum and encodes a protein (e.g,
an SMP 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 perform a function
involved in the metabolism of carbon compounds such as sugars or
the generation of energy molecules (e.g., ATP) by processes such as
oxidative phosphorylation in Corynebacterium glutamicum, 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.
[0015] 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 SMP protein, or a biologically
active portion thereof.
[0016] 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 SMP protein by culturing the host cell in a suitable
medium. The SMP protein can be then isolated from the medium or the
host cell.
[0017] Yet another aspect of the invention pertains to a
genetically altered microorganism in which an SMP 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 SMP
sequence as a transgene. In another embodiment, an endogenous SMP
gene within the genome of the microorganism has been altered, e.g.,
functionally disrupted, by homologous recombination with an altered
SMP gene. In another embodiment, an endogenous or introduced SMP
gene in a microorganism has been altered by one or more point
mutations, deletions, or inversions, but still encodes a fluctional
SMP protein. In still another embodiment, one or more of the
regulatory regions (e.g., a promoter, repressor, or inducer) of an
SMP gene in a microorganism has been altered (e.g., by deletion,
truncation, inversion, or point mutation) such that the expression
of the SMP 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.
[0018] 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.
[0019] Still another aspect of the invention pertains to an
isolated SMP protein or a portion, e.g., a biologically active
portion, thereof. In a preferred embodiment, the isolated SMP
protein or portion thereof is capable of performing a function
involved in the metabolism of carbon compounds such as sugars or in
the generation of energy molecules (e.g., ATP) by processes such as
oxidative phosphorylation in Corynebacterium glutamicum. In another
preferred embodiment, the isolated SMP 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
perform a function involved in the metabolism of carbon compounds
such as sugars or in the generation of energy molecules (e.g., ATP)
by processes such as oxidative phosphorylation in Corynebacterium
glutamicum.
[0020] The invention also provides an isolated preparation of an
SMP protein. In preferred embodiments, the SMP 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 SMP 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 perform a function involved in the metabolism of
carbon compounds such as sugars or in the generation of energy
molecules (e.g., ATP) by processes such as oxidative
phosphorylation in Corynebacterium glutamicum, or has one or more
of the activities set forth in Table 1.
[0021] Alternatively, the isolated SMP 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 SMP proteins also have one or more of the SMP
bioactivities described herein.
[0022] The SMP polypeptide, or a biologically active portion
thereof, can be operatively linked to a non-SMP polypeptide to form
a fusion protein. In preferred embodiments, this fusion protein has
an activity which differs from that of the SMP protein alone. In
other preferred embodiments, this fusion protein performs a
function involved in the metabolism of carbon compounds such as
sugars or in the generation of energy molecules (e.g., ATP) by
processes such as oxidative phosphorylation in Corynebacterium
glutamicum. In particularly preferred embodiments, integration of
this fusion protein into a host cell modulates production of a
desired compound from the cell.
[0023] In another aspect, the invention provides methods for
screening molecules which modulate the activity of an SMP protein,
either by interacting with the protein itself or a substrate or
binding partner of the SMP protein, or by modulating the
transcription or translation of an SMP nucleic acid molecule of the
invention.
[0024] 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 SMP 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
SMP 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.
[0025] 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
SMP protein activity or SMP 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 carbon metabolism pathways
or for the production of energy through processes such as oxidative
phosphorylation, such that the yields or rate of production of a
desired fine chemical by this microorganism is improved. The agent
which modulates SMP protein activity can be an agent which
stimulates SMP protein activity or SMP nucleic acid expression.
Examples of agents which stimulate SMP protein activity or SMP
nucleic acid expression include small molecules, active SMP
proteins, and nucleic acids encoding SMP proteins that have been
introduced into the cell. Examples of agents which inhibit SMP
activity or expression include small molecules and antisense SMP
nucleic acid molecules.
[0026] 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 SMP 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
[0027] The present invention provides SMP nucleic acid and protein
molecules which are involved in the metabolism of carbon compounds
such as sugars and the generation of energy molecules by processes
such as oxidative phosphorylation in Corynebacterium glutamicum.
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 glycolytic pathway protein has a direct impact on
the yield, production, and/or efficiency of production of, e.g.,
pyruvate 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 proteins involved in oxidative
phosphorylation results in alterations in the amount of energy
available to perform necessary metabolic processes and other
cellular functions, such as nucleic acid and protein biosynthesis
and transcription/translation). Aspects of the invention are
further explicated below.
I. Fine Chemicals
[0028] 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
Sep. 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
[0029] 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, profine,
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.
[0030] 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.
[0031] 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.
[0032] 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 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
[0033] 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).
[0034] 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
Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press: Champaign, IL X,
374 S).
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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
[0039] 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).
[0040] 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.
[0041] 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. (1999) "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
[0042] 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. Sugar and Carbon Molecule Utilization and Oxidative
Phosphorylation
[0043] Carbon is a critically important element for the formation
of all organic compounds, and thus is a nutritional requirement not
only for the growth and division of C. glutamicum, but also for the
overproduction of fine chemicals from this microorganism. Sugars,
such as mono-, di-, or polysaccharides, are particularly good
carbon sources, and thus standard growth media typically contain
one or more of: glucose, fructose, mannose, galactose, ribose,
sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, or
cellulose (Ullmann's Encyclopedia of Industrial Chemistry (1987)
vol. A9, "Enzymes", VCH: Weinheim). Alternatively, more complex
forms of sugar may be utilized in the media, such as molasses, or
other by-products of sugar refinement. Other compounds aside from
the sugars may be used as alternate carbon sources, including
alcohols (e.g., ethanol or methanol), alkanes, sugar alcohols,
fatty acids, and organic acids (e.g., acetic acid or lactic acid).
For a review of carbon sources and their utilization by
microorganisms in culture, see: Ullman's Encyclopedia of Industrial
Chemistry (1987) vol. A9, "Enzymes", VCH: Weinheim; Stoppok, E. and
Buchholz, K. (1996) "Sugar-based raw materials for fermentation
applications" in Biotechnology (Rehm, H. J. et al., eds.) vol. 6,
VCH: Weinheim, p. 5-29; Rehm, H. J. (1980) Industrielle
Mikrobiologie, Springer: Berlin; Bartholomew, W. H., and Reiman, H.
B. (1979). Economics of Fermentation Processes, in: Peppler, H. J.
and Perlman, D., eds. Microbial Technology 2.sup.nd ed., vol. 2,
chapter 18, Academic Press: New York; and Kockova-Kratachvilova, A.
(1981) Characteristics of Industrial Microorganisms, in: Rehm, H.
J. and Reed, G., eds. Handbook of Biotechnology, vol. 1, chapter 1,
Verlag Chemie: Weinheim.
[0044] After uptake, these energy-rich carbon molecules must be
processed such that they are able to be degraded by one of the
major sugar metabolic pathways. Such pathways lead directly to
useful degradation products, such as ribose-5-phosphate and
phosphoenolpyruvate, which may be subsequently converted to
pyruvate. Three of the most important pathways in bacteria for
sugar metabolism include the Embden-Meyerhoff-Pamas (EMP) pathway
(also known as the glycolytic or fructose bisphosphate pathway),
the hexosemonophosphate (HMP) pathway (also known as the pentose
shunt or pentose phosphate pathway), and the Entner-Doudoroff (ED)
pathway (for review, see Michal, G. (1999) Biochemical Pathways: An
Atlas of Biochemistry and Molecular Biology, Wiley: New York, and
Stryer, L. (1988) Biochemistry, Chapters 13-19, Freeman: New York,
and references therein).
[0045] The EMP pathway converts hexose molecules to pyruvate, and
in the process produces 2 molecules of ATP and 2 molecules of NADH.
Starting with glucose-1-phosphate (which may be either directly
taken up from the medium, or alternatively may be generated from
glycogen, starch, or cellulose), the glucose molecule is isomerized
to fructose-6-phosphate, is phosphorylated, and split into two
3-carbon molecules of glyceraldehyde-3-phosphate. After
dehydrogenation, phosphorylation, and successive rearrangements,
pyruvate results.
[0046] The HMP pathway converts glucose to reducing equivalents,
such as NADPH, and produces pentose and tetrose compounds which are
necessary as intermediates and precursors in a number of other
metabolic pathways. In the HMP pathway, glucose-6-phosphate is
converted to ribulose-5-phosphate by two successive dehydrogenase
reactions (which also release two NADPH molecules), and a
carboxylation step. Ribulose-5-phosphate may also be converted to
xyulose-5-phosphate and ribose-5-phosphate; the former can undergo
a series of biochemical steps to glucose-6-phosphate, which may
enter the EMP pathway, while the latter is commonly utilized as an
intermediate in other biosynthetic pathways within the cell.
[0047] The ED pathway begins with the compound glucose or
gluconate, which is subsequently phosphorylated and dehydrated to
form 2-dehydro-3-deoxy-6-P-gluconate. Glucuronate and galacturonate
may also be converted to 2-dehydro-3-deoxy-6-P-gluconate through
more complex biochemical pathways. This product molecule is
subsequently cleaved into glyceraldehyde-3-P and pyruvate;
glyceraldehyde-3-P may itself also be converted to pyruvate.
[0048] The EMP and HMP pathways share many features, including
intermediates and enzymes. The EMP pathway provides the greatest
amount of ATP, but it does not produce ribose-5-phosphate, an
important precursor for, e.g., nucleic acid biosynthesis, nor does
it produce erythrose-4-phosphate, which is important for amino acid
biosynthesis. Microorganisms that are capable of using only the EMP
pathway for glucose utilization are thus not able to grow on simple
media with glucose as the sole carbon source. They are referred to
as fastidious organisms, and their growth requires inputs of
complex organic compounds, such as those found in yeast
extract.
[0049] In contrast, the HMP pathway produces all of the precursors
necessary for both nucleic acid and amino acid biosynthesis, yet
yields only half the amount of ATP energy that the EMP pathway
does. The HMP pathway also produces NADPH, which may be used for
redox reactions in biosynthetic pathways. The HMP pathway does not
directly produce pyruvate, however, and thus these microorganisms
must also possess this portion of the EMP pathway. It is therefore
not surprising that a number of microorganisms, particularly the
facultative anerobes, have evolved such that they possess both of
these pathways.
[0050] The ED pathway has thus far has only been found in bacteria.
Although this pathway is linked partly to the HMP pathway in the
reverse direction for precursor formation, the ED pathway directly
forms pyruvate by the aldolase cleavage of
3-ketodeoxy-6-phosphogluconate. The ED pathway can exist on its own
and is utilized by the majority of strictly aerobic microorganisms.
The net result is similar to that of the HMP pathway, although one
mole of ATP can be formed only if the carbon atoms are converted
into pyruvate, instead of into precursor molecules.
[0051] The pyruvate molecules produced through any of these
pathways can be readily converted into energy via the Krebs cycle
(also known as the citric acid cycle, the citrate cycle, or the
tricarboxylic acid cycle (TCA cycle)). In this process, pyruvate is
first decarboxylated, resulting in the production of one molecule
of NADH, 1 molecule of acetyl-CoA, and 1 molecule of CO.sub.2. The
acetyl group of acetyl CoA then reacts with the 4 carbon unit,
oxaolacetate, leading to the formation of citric acid, a 6 carbon
organic acid. Dehydration and two additional CO.sub.2 molecules are
released. Ultimately, oxaloacetate is regenerated and can serve
again as an acetyl acceptor, thus completing the cycle. The
electrons released during the oxidation of intermediates in the TCA
cycle are transferred to NAD.sup.+ to yield NADH.
[0052] During respiration, the electrons from NADH are transferred
to molecular oxygen or other terminal electron acceptors. This
process is catalyzed by the respiratory chain, an electron
transport system containing both integral membrane proteins and
membrane associated proteins. This system serves two basic
functions: first, to accept electrons from an electron donor and to
transfer them to an electron acceptor, and second, to conserve some
of the energy released during electron transfer by the synthesis of
ATP. Several types of oxidation-reduction enzymes and electron
transport proteins are known to be involved in such processes,
including the NADH dehydrogenases, flavin-containing electron
carriers, iron sulfur proteins, and cytochromes. The NADH
dehydrogenases are located at the cytoplasmic surface of the plasma
membrane, and transfer hydrogen atoms from NADH to flavoproteins,
in turn accepting electrons from NADH. The flavoproteins are a
group of electron carriers possessing a flavin prosthetic group
which is alternately reduced and oxidized as it accepts and
transfers electrons. Three flavins are known to participate in
these reactions: riboflavin, flavin-adenine dinucleotide (FAD) and
flavin-mononucleotide (FMN). Iron sulfur proteins contain a cluster
of iron and sulfur atoms which are not bonded to a heme group, but
which still are able to participate in dehydration and rehydration
reactions. Succinate dehydrogenase and aconitase are exemplary
iron-sulfur proteins; their iron-sulfur complexes serve to accept
and transfer electrons as part of the overall electron-transport
chain. The cytochromes are proteins containing an iron porphyrin
ring (heme). There are a number of different classes of
cytochromes, differing in their reduction potentials. Functionally,
these cytochromes form pathways in which electrons may be
transferred to other cytochromes having increasingly more positive
reduction potentials. A further class of non-protein electron
carriers is known: the lipid-soluble quinones (e.g., coenzyme Q).
These molecules also serve as hydrogen atom acceptors and electron
donors.
[0053] The action of the respiratory chain generates a proton
gradient across the cell membrane, resulting in proton motive
force. This force is utilized by the cell to synthesize ATP, via
the membrane-spanning enzyme, ATP synthase. This enzyme is a
multiprotein complex in which the transport of H.sup.+ molecules
through the membrane results in the physical rotation of the
intracellular subunits and concomitant phosphorylation of ADP to
form ATP (for review, see Fillingame, R. H. and Divall, S. (1999)
Novartis Found. Symp. 221: 218-229, 229-234).
[0054] Non-hexose carbon substrates may also serve as carbon and
energy sources for cells. Such substrates may first be converted to
hexose sugars in the gluconeogenesis pathway, where glucose is
first synthesized by the cell and then is degraded to produce
energy. The starting material for this reaction is
phosphoenolpyruvate (PEP), which is one of the key intermediates in
the glycolytic pathway. PEP may be formed from substrates other
than sugars, such as acetic acid, or by decarboxylation of
oxaloacetate (itself an intermediate in the TCA cycle). By
reversing the glycolytic pathway (utilizing a cascade of enzymes
different than those of the original glycolysis pathway),
glucose-6-phosphate may be formed. The conversion of pyruvate to
glucose requires the utilization of 6 high energy phosphate bonds,
whereas glycolysis only produces 2 ATP in the conversion of glucose
to pyruvate. However, the complete oxidation of glucose
(glycolysis, conversion of pyruvate into acetyl CoA, citric acid
cycle, and oxidative phosphorylation) yields between 36-38 ATP, so
the net loss of high energy phosphate bonds experienced during
gluconeogenesis is offset by the overall greater gain in such
high-energy molecules produced by the oxidation of glucose.
III. Elements and Methods of the Invention
[0055] The present invention is based, at least in part, on the
discovery of novel molecules, referred to herein as SMP nucleic
acid and protein molecules, which participate in the conversion of
sugars to usefuil degradation products and energy (e.g., ATP) in C.
glutamicum or which may participate in the production of useful
energy-rich molecules (e.g., ATP) by other processes, such as
oxidative phosphorylation. In one embodiment, the SMP molecules
participate in the metabolism of carbon compounds such as sugars or
the generation of energy molecules (e.g., ATP) by processes such as
oxidative phosphorylation in Corynebacterium glutamicum. In a
preferred embodiment, the activity of the SMP molecules of the
present invention to contribute to carbon metabolism or energy
production in C. glutamicum has an impact on the production of a
desired fine chemical by this organism. In a particularly preferred
embodiment, the SMP molecules of the invention are modulated in
activity, such that the C. glutamicum metabolic and energetic
pathways in which the SMP proteins of the invention participate are
modulated in yield, production, and/or efficiency of production,
which either directly or indirectly modulates the yield,
production, and/or efficiency of production of a desired fine
chemical by C. glutamicum.
[0056] The language, "SMP protein" or "SMP polypeptide" includes
proteins which are capable of performing a function involved in the
metabolism of carbon compounds such as sugars and the generation of
energy molecules by processes such as oxidative phosphorylation in
Corynebacterium glutamicum. Examples of SMP proteins include those
encoded by the SMP genes set forth in Table 1 and Appendix A. The
terms "SMP gene" or "SMP nucleic acid sequence" include nucleic
acid sequences encoding an SMP protein, which consist of a coding
region and also corresponding untranslated 5' and 3' sequence
regions. Examples of SMP 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 term "degradation
product" is art-recognized and includes breakdown products of a
compound. Such products may themselves have utility as precursor
(starting point) or intermediate molecules necessary for the
biosynthesis of other compounds by the cell. 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.
[0057] In another embodiment, the SMP 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 SMP
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. The
degradation of high-energy carbon molecules such as sugars, and the
conversion of compounds such as NADH and FADH.sub.2 to more useful
forms via oxidative phosphorylation results in a number of
compounds which themselves may be desirable fine chemicals, such as
pyruvate, ATP, NADH, and a number of intermediate sugar compounds.
Further, the energy molecules (such as ATP) and the reducing
equivalents (such as NADH or NADPH) produced by these metabolic
pathways are utilized in the cell to drive reactions which would
otherwise be energetically unfavorable. Such unfavorable reactions
include many biosynthetic pathways for fine chemicals. By improving
the ability of the cell to utilize a particular sugar (e.g., by
manipulating the genes encoding enzymes involved in the degradation
and conversion of that sugar into energy for the cell), one may
increase the amount of energy available to permit unfavorable, yet
desired metabolic reactions (e.g., the biosynthesis of a desired
fine chemical) to occur.
[0058] The mutagenesis of one or more SMP genes of the invention
may also result in SMP proteins having altered activities which
indirectly impact the production of one or more desired fine
chemicals from C. glutamicum. For example, by increasing the
efficiency of utilization of one or more sugars (such that the
conversion of the sugar to useful energy molecules is improved), or
by increasing the efficiency of conversion of reducing equivalents
to useful energy molecules (e.g., by improving the efficiency of
oxidative phosphorylation, or the activity of the ATP synthase),
one can increase the amount of these high-energy compounds
available to the cell to drive normally unfavorable metabolic
processes. These processes include the construction of cell walls,
transcription, translation, and the biosynthesis of compounds
necessary for growth and division of the cells (e.g., nucleotides,
amino acids, vitamins, lipids, etc.) (Lengeler et al. (1999)
Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109;
913-918; 875-899). By improving the growth and multiplication of
these engineered cells, it is possible to increase both the
viability of the cells in large-scale culture, and also to improve
their rate of division, such that a relatively larger number of
cells can survive in fermentor culture. The yield, production, or
efficiency of production may be increased, at least due to the
presence of a greater number of viable cells, each producing the
desired fine chemical. Further, a number of the degradation and
intermediate compounds produced during sugar metabolism are
necessary precursors and intermediates for other biosynthetic
pathways throughout the cell. For example, many amino acids are
synthesized directly from compounds normally resulting from
glycolysis or the TCA cycle (e.g., serine is synthesized from
3-phosphoglycerate, an intermediate in glycolysis). Thus, by
increasing the efficiency of conversion of sugars to useful energy
molecules, it is also possible to increase the amount of useful
degradation products as well.
[0059] 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 SMP DNAs and the predicted amino acid sequences of the
C. glutamicum SMP 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 having a function involved in the
metabolism of carbon compounds such as sugars or in the generation
of energy molecules by processes such as oxidative phosphorylation
in Corynebacterium glutamicum.
[0060] 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.
[0061] An SMP protein or a biologically active portion or fragment
thereof of the invention can participate in the metabolism of
carbon compounds such as sugars or in the generation of energy
molecules (e.g., ATP) by processes such as oxidative
phosphorylation in Corynebacterium glutamicum, or can have one or
more of the activities set forth in Table 1.
[0062] Various aspects of the invention are described in further
detail in the following subsections:
A. Isolated Nucleic Acid Molecules
[0063] One aspect of the invention pertains to isolated nucleic
acid molecules that encode SMP 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 SMP-encoding nucleic acid (e.g., SMP 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 SMP 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.
[0064] 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 SMP 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 SMP nucleotide
sequence can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[0065] 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 SMP DNAs of the invention. This DNA
comprises sequences encoding SMP 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.
[0066] 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, or RXS number having the designation "RXA,"
"RXN," or "RXS" followed by 5 digits (i.e., RXA00013, RXN00043, or
RXS0735). 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, or RXS
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,
or RXS 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, or RXS designations as Appendix A, such that
they can be readily correlated. For example, the amino acid
sequence in Appendix B designated RXAOO00013 is a translation of
the coding region of the nucleotide sequence of nucleic acid
molecule RXA00013 in Appendix A, and the amino acid sequence in
Appendix B designated RXN00043 is a translation of the coding
region of the nucleotide sequence of nucleic acid molecule RXN00043
in Appendix A. Each of the RXARXN and RXS nucleotide and amino acid
sequences of the invention has also been assigned a SEQ ID NO, as
indicated in Table 1.
[0067] 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 RXAdesignation. For
example, SEQ ID NO:11, designated, as indicated on Table 1, as "F
RXA01312", is an F-designated gene, as are SEQ ID NOs: 29, 33, and
39 (designated on Table 1 as "F RXA02803", "F RXA02854", and "F
RXA01365", respectively).
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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 SMP protein. The nucleotide sequences determined from
the cloning of the SMP genes from C. glutamicum allows for the
generation of probes and primers designed for use in identifying
and/or cloning SMP homologues in other cell types and organisms, as
well as SMP 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 SMP homologues. Probes based on the SMP 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 SMP
protein, such as by measuring a level of an SMP-encoding nucleic
acid in a sample of cells, e.g., detecting SMP mRNA levels or
determining whether a genomic SMP gene has been mutated or
deleted.
[0072] 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 perform a function involved in the
metabolism of carbon compounds such as sugars or in the generation
of energy molecules (e.g., ATP) by processes such as oxidative
phosphorylation in Corynebacterium glutamicum. 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 perform a function involved in the metabolism of carbon
compounds such as sugars or in the generation of energy molecules
(e.g., ATP) by processes such as oxidative phosphorylation in
Corynebacterium glutamicum. Protein members of such sugar metabolic
pathways or energy producing 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 SMP protein" contributes either directly
or indirectly to the yield, production, and/or efficiency of
production of one or more fine chemicals. Examples of SMP protein
activities are set forth in Table 1.
[0073] 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.
[0074] Portions of proteins encoded by the SMP nucleic acid
molecules of the invention are preferably biologically active
portions of one of the SMP proteins. As used herein, the term
"biologically active portion of an SMP protein" is intended to
include a portion, e.g., a domain/motif, of an SMP protein that
participates in the metabolism of carbon compounds such as sugars,
or in energy-generating pathways in C. glutamicum, or has an
activity as set forth in Table 1. To determine whether an SMP
protein or a biologically active portion thereof can participate in
the metabolism of carbon compounds or in the production of
energy-rich molecules in C. glutamicum, 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.
[0075] Additional nucleic acid fragments encoding biologically
active portions of an SMP protein can be prepared by isolating a
portion of one of the sequences in Appendix B, expressing the
encoded portion of the SMP protein or peptide (e.g., by recombinant
expression in vitro) and assessing the activity of the encoded
portion of the SMP protein or peptide.
[0076] 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 SMP 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).
[0077] 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 58% identical to the
nucleotide sequence designated RXA00014 (SEQ ID NO:41), a
nucleotide sequence which is greater than and/or at least %
identical to the nucleotide sequence designated RXA00195 (SEQ ID
NO:399), and a nucleotide sequence which is greater than and/or at
least 42% identical to the nucleotide sequence designated RXA00196
(SEQ ID NO:401). 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.
[0078] In addition to the C. glutamicum SMP nucleotide sequences
shown in Appendix A, it will be appreciated by those of ordinary
skill in the art that DNA sequence polymorphisms that lead to
changes in the amino acid sequences of SMP proteins may exist
within a population (e.g., the C. glutamicum population). Such
genetic polymorphism in the SMP 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 SMP protein,
preferably a C. glutamicum SMP protein. Such natural variations can
typically result in 1-5% variance in the nucleotide sequence of the
SMP gene. Any and all such nucleotide variations and resulting
amino acid polymorphisms in SMP that are the result of natural
variation and that do not alter the finctional activity of SMP
proteins are intended to be within the scope of the invention.
[0079] Nucleic acid molecules corresponding to natural variants and
non-C. glutamicum homologues of the C. glutamicum SMP DNA of the
invention can be isolated based on their homology to the C.
glutamicum SMP 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 SMP protein.
[0080] In addition to naturally-occurring variants of the SMP
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 SMP protein,
without altering the fumctional ability of the SMP 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 SMP proteins (Appendix B) without altering the activity of said
SMP protein, whereas an "essential" amino acid residue is required
for SMP protein activity. Other amino acid residues, however,
(e.g., those that are not conserved or only semi-conserved in the
domain having SMP activity) may not be essential for activity and
thus are likely to be amenable to alteration without altering SMP
activity.
[0081] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding SMP proteins that contain changes
in amino acid residues that are not essential for SMP activity.
Such SMP proteins differ in amino acid sequence from a sequence
contained in Appendix B yet retain at least one of the SMP
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 carbon compounds
such as sugars, or in the biosynthesis of high-energy compounds in
C. glutamicum, 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.
[0082] 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).
[0083] An isolated nucleic acid molecule encoding an SMP 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 SMP 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 SMP coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for an SMP activity described herein to
identify mutants that retain SMP 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).
[0084] In addition to the nucleic acid molecules encoding SMP
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 DNA
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 SMP
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 SMP
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 NO. 3
(RXA01626) comprises nucleotides 1 to 345). In another embodiment,
the antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding SMP.
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).
[0085] Given the coding strand sequences encoding SMP 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 SMP
mRNA, but more preferably is an oligonucleotide which is antisense
to only a portion of the coding or noncoding region of SMP mRNA.
For example, the antisense oligonucleotide can be complementary to
the region surrounding the translation start site of SMP 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).
[0086] 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 SMP 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.
[0087] 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).
[0088] 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 SMP mRNA transcripts to thereby
inhibit translation of SMP mRNA. A ribozyme having specificity for
an SMP-encoding nucleic acid can be designed based upon the
nucleotide sequence of an SMP cDNA disclosed herein (i.e., SEQ ID
NO. 3 (RXA01626) 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 SMP-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, SMP 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.
[0089] Alternatively, SMP gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of an SMP nucleotide sequence (e.g., an SMP promoter and/or
enhancers) to form triple helical structures that prevent
transcription of an SMP 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
[0090] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding
an SMP 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.
[0091] 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-, arny, SPO2, .lamda.-P.sub.R7-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
those 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., SMP proteins, mutant forms of SMP proteins,
fusion proteins, etc.).
[0092] The recombinant expression vectors of the invention can be
designed for expression of SMP proteins in prokaryotic or
eukaryotic cells. For example, SMP 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.
[0093] 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.
[0094] 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 SMP 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 SMP protein unfused to GST
can be recovered by cleavage of the fusion protein with
thrombin.
[0095] 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 gn1). 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: New York IBSN 0 444 904018).
[0096] 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.
[0097] In another embodiment, the SMP 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: New York (IBSN 0 444 904018).
[0098] Alternatively, the SMP 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., Sf 9 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).
[0099] In another embodiment, the SMP 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", Nucl. Acid. Res. 12: 8711-8721, and include
pLGV23, pGHlac+, pBIN19, pAK2004, and pDH51 (Pouwels et al., eds.
(1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).
[0100] 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 finctions 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.
[0101] 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 (Baneiji 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).
[0102] 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 SMP 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.
[0103] 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.
[0104] A host cell can be any prokaryotic or eukaryotic cell. For
example, an SMP 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.
[0105] 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.
[0106] 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 SMP 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).
[0107] To create a homologous recombinant microorganism, a vector
is prepared which contains at least a portion of an SMP gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., finctionally disrupt, the SMP gene.
Preferably, this SMP gene is a Corynebacterium glutamicum SMP 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 SMP 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 SMP 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 SMP protein). In the homologous
recombination vector, the altered portion of the SMP gene is
flanked at its 5' and 3' ends by additional nucleic acid of the SMP
gene to allow for homologous recombination to occur between the
exogenous SMP gene carried by the vector and an endogenous SMP gene
in a microorganism. The additional flanking SMP nucleic acid is of
sufficient length for successfuil 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 SMP gene has homologously recombined
with the endogenous SMP gene are selected, using art-known
techniques.
[0108] 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 SMP
gene on a vector placing it under control of the lac operon permits
expression of the SMP gene only in the presence of IPTG. Such
regulatory systems are well known in the art.
[0109] In another embodiment, an endogenous SMP 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
SMP gene in a host cell has been altered by one or more point
mutations, deletions, or inversions, but still encodes a functional
SMP protein. In still another embodiment, one or more of the
regulatory regions (e.g., a promoter, repressor, or inducer) of an
SMP gene in a microorganism has been altered (e.g., by deletion,
truncation, inversion, or point mutation) such that the expression
of the SMP gene is modulated. One of ordinary skill in the art will
appreciate that host cells containing more than one of the
described SMP gene and protein modifications may be readily
produced using the methods of the invention, and are meant to be
included in the present invention.
[0110] 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 SMP protein. Accordingly, the invention further
provides methods for producing SMP 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 SMP protein has been introduced, or into which
genome has been introduced a gene encoding a wild-type or altered
SMP protein) in a suitable medium until SMP protein is produced. In
another embodiment, the method further comprises isolating SMP
proteins from the medium or the host cell.
C. Isolated SMP Proteins
[0111] Another aspect of the invention pertains to isolated SMP
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 SMP 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 SMP protein having less than about 30% (by
dry weight) of non-SMP protein (also referred to herein as a
"contaminating protein"), more preferably less than about 20% of
non-SMP protein, still more preferably less than about 10% of
non-SMP protein, and most preferably less than about 5% non-SMP
protein. When the SMP 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 SMP 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 SMP protein
having less than about 30% (by dry weight) of chemical precursors
or non-SMP chemicals, more preferably less than about 20% chemical
precursors or non-SMP chemicals, still more preferably less than
about 10% chemical precursors or non-SMP chemicals, and most
preferably less than about 5% chemical precursors or non-SMP
chemicals. In preferred embodiments, isolated proteins or
biologically active portions thereof lack contaminating proteins
from the same organism from which the SMP protein is derived.
Typically, such proteins are produced by recombinant expression of,
for example, a C. glutamicum SMP protein in a microorganism such as
C. glutamicum.
[0112] An isolated SMP protein or a portion thereof of the
invention can participate in the metabolism of carbon compounds
such as sugars, or in the production of energy compounds (e.g., by
oxidative phosphorylation) utilized to drive unfavorable metabolic
pathways, 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 to perform a fumction
involved in the metabolism of carbon compounds such as sugars or in
the generation of energy molecules by processes such as oxidative
phosphorylation in Corynebacterium glutamicum. The portion of the
protein is preferably a biologically active portion as described
herein. In another preferred embodiment, an SMP protein of the
invention has an amino acid sequence shown in Appendix B. In yet
another preferred embodiment, the SMP 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 SMP 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 SMP
proteins of the present invention also preferably possess at least
one of the SMP activities described herein. For example, a
preferred SMP 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 perform a function involved
in the metabolism of carbon compounds such as sugars or in the
generation of energy molecules (e.g., ATP) by processes such as
oxidative phosphorylation in Corynebacterium glutamicum, or which
has one or more of the activities set forth in Table 1.
[0113] In other embodiments, the SMP 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 SMP 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 SMP 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.
[0114] Biologically active portions of an SMP protein include
peptides comprising amino acid sequences derived from the amino
acid sequence of an SMP protein, e.g., the an amino acid sequence
shown in Appendix B or the amino acid sequence of a protein
homologous to an SMP protein, which include fewer amino acids than
a full length SMP protein or the full length protein which is
homologous to an SMP protein, and exhibit at least one activity of
an SMP 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 SMP 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 SMP protein include one or more selected domains/motifs or
portions thereof having biological activity.
[0115] SMP 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 SMP protein is expressed in the host cell. The SMP
protein can then be isolated from the cells by an appropriate
purification scheme using standard protein purification techniques.
Alternative to recombinant expression, an SMP protein, polypeptide,
or peptide can be synthesized chemically using standard peptide
synthesis techniques. Moreover, native SMP protein can be isolated
from cells (e.g., endothelial cells), for example using an anti-SMP
antibody, which can be produced by standard techniques utilizing an
SMP protein or fragment thereof of this invention.
[0116] The invention also provides SMP chimeric or fusion proteins.
As used herein, an SMP "chimeric protein" or "fusion protein"
comprises an SMP polypeptide operatively linked to a non-SMP
polypeptide. An "SMP polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to an SMP protein, whereas a
"non-SMP polypeptide" refers to a polypeptide having an amino acid
sequence corresponding to a protein which is not substantially
homologous to the SMP protein, e.g., a protein which is different
from the SMP 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 SMP
polypeptide and the non-SMP polypeptide are fused in-frame to each
other. The non-SMP polypeptide can be fused to the N-terminus or
C-terninus of the SMP polypeptide. For example, in one embodiment
the fusion protein is a GST-SMP fusion protein in which the SMP
sequences are fused to the C-terminus of the GST sequences. Such
fusion proteins can facilitate the purification of recombinant SMP
proteins. In another embodiment, the fusion protein is an SMP
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 SMP protein can be increased
through use of a heterologous signal sequence.
[0117] Preferably, an SMP 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, Ausubel et aL, eds. John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). An SMP-encoding nucleic acid can be cloned into
such an expression vector such that the fusion moiety is linked
in-frame to the SMP protein.
[0118] Homologues of the SMP protein can be generated by
mutagenesis, e.g., discrete point mutation or truncation of the SMP
protein. As used herein, the term "homologue" refers to a variant
form of the SMP protein which acts as an agonist or antagonist of
the activity of the SMP protein. An agonist of the SMP protein can
retain substantially the same, or a subset, of the biological
activities of the SMP protein. An antagonist of the SMP protein can
inhibit one or more of the activities of the naturally occurring
form of the SMP protein, by, for example, competitively binding to
a downstream or upstream member of the sugar molecule metabolic
cascade or the energy-producing pathway which includes the SMP
protein.
[0119] In an alternative embodiment, homologues of the SMP protein
can be identified by screening combinatorial libraries of mutants,
e.g., truncation mutants, of the SMP protein for SMP protein
agonist or antagonist activity. In one embodiment, a variegated
library of SMP variants is generated by combinatorial mutagenesis
at the nucleic acid level and is encoded by a variegated gene
library. A variegated library of SMP variants can be produced by,
for example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of
potential SMP sequences is expressible as individual polypeptides,
or alternatively, as a set of larger fusion proteins (e.g., for
phage display) containing the set of SMP sequences therein. There
are a variety of methods which can be used to produce libraries of
potential SMP 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 SMP
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.
[0120] In addition, libraries of fragments of the SMP protein
coding can be used to generate a variegated population of SMP
fragments for screening and subsequent selection of homologues of
an SMP protein. In one embodiment, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of an SMP 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 SMP protein.
[0121] 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 SMP 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 fuinctional
mutants in the libraries, can be used in combination with the
screening assays to identify SMP homologues (Arkin and Yourvan
(1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein
Engineering 6(3):327-331).
[0122] In another embodiment, cell based assays can be exploited to
analyze a variegated SMP library, using methods well known in the
art.
D. Uses and Methods of the Invention
[0123] 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 SMP protein regions required for function;
modulation of an SMP protein activity; modulation of the metabolism
of one or more sugars; modulation of high-energy molecule
production in a cell (i.e., ATP, NADPH); and modulation of cellular
production of a desired compound, such as a fine chemical.
[0124] The SMP 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. 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.
[0125] 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.
[0126] 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
fumctional 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.
[0127] The SMP nucleic acid molecules of the invention are also
useful for evolutionary and protein structural studies. The
metabolic and energy-releasing 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.
[0128] Manipulation of the SMP nucleic acid molecules of the
invention may result in the production of SMP proteins having
functional differences from the wild-type SMP 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.
[0129] The invention provides methods for screening molecules which
modulate the activity of an SMP protein, either by interacting with
the protein itself or a substrate or binding partner of the SMP
protein, or by modulating the transcription or translation of an
SMP nucleic acid molecule of the invention. In such methods, a
microorganism expressing one or more SMP 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
SMP protein is assessed.
[0130] There are a number of mechanisms by which the alteration of
an SMP 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. The
degradation of high-energy carbon molecules such as sugars, and the
conversion of compounds such as NADH and FADH.sub.2 to more useful
forms via oxidative phosphorylation results in a number of
compounds which themselves may be desirable fine chemicals, such as
pyruvate, ATP, NADH, and a number of intermediate sugar compounds.
Further, the energy molecules (such as ATP) and the reducing
equivalents (such as NADH or NADPH) produced by these metabolic
pathways are utilized in the cell to drive reactions which would
otherwise be energetically unfavorable. Such unfavorable reactions
include many biosynthetic pathways for fine chemicals. By improving
the ability of the cell to utilize a particular sugar (e.g., by
manipulating the genes encoding enzymes involved in the degradation
and conversion of that sugar into energy for the cell), one may
increase the amount of energy available to permit unfavorable, yet
desired metabolic reactions (e.g., the biosynthesis of a desired
fine chemical) to occur.
[0131] Further, modulation of one or more pathways involved in
sugar utilization permits optimization of the conversion of the
energy contained within the sugar molecule to the production of one
or more desired fine chemicals. For example, by reducing the
activity of enzymes involved in, for example, gluconeogenesis, more
ATP is available to drive desired biochemical reactions (such as
fine chemical biosyntheses) in the cell. Also, the overall
production of energy molecules from sugars may be modulated to
ensure that the cell maximizes its energy production from each
sugar molecule. Inefficient sugar utilization can lead to excess
CO.sub.2 production and excess energy, which may result in futile
metabolic cycles. By improving the metabolism of sugar molecules,
the cell should be able to function more efficiently, with a need
for fewer carbon molecules. This should result in an improved fine
chemical product: sugar molecule ratio (improved carbon yield), and
permits a decrease in the amount of sugars that must be added to
the medium in large-scale fermentor culture of such engineered C.
glutamicum.
[0132] The mutagenesis of one or more SMP genes of the invention
may also result in SMP proteins having altered activities which
indirectly impact the production of one or more desired fine
chemicals from C. glutamicum. For example, by increasing the
efficiency of utilization of one or more sugars (such that the
conversion of the sugar to useful energy molecules is improved), or
by increasing the efficiency of conversion of reducing equivalents
to useful energy molecules (e.g., by improving the efficiency of
oxidative phosphorylation, or the activity of the ATP synthase),
one can increase the amount of these high-energy compounds
available to the cell to drive normally unfavorable metabolic
processes. These processes include the construction of cell walls,
transcription, translation, and the biosynthesis of compounds
necessary for growth and division of the cells (e.g., nucleotides,
amino acids, vitamins, lipids, etc.) (Lengeler et al. (1999)
Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109;
913-918; 875-899). By improving the growth and multiplication of
these engineered cells, it is possible to increase both the
viability of the cells in large-scale culture, and also to improve
their rate of division, such that a relatively larger number of
cells can survive in fermentor culture. The yield, production, or
efficiency of production may be increased, at least due to the
presence of a greater number of viable cells, each producing the
desired fine chemical.
[0133] Further, many of the degradation products produced during
sugar metabolism are themselves utilized by the cell as precursors
or intermediates for the production of a number of other useful
compounds, some of which are fine chemicals. For example, pyruvate
is converted into the amino acid alanine, and ribose-5-phosphate is
an integral part of, for example, nucleotide molecules. The amount
and efficiency of sugar metabolism, then, has a profound effect on
the availability of these degradation products in the cell. By
increasing the ability of the cell to process sugars, either in
terms of efficiency of existing pathways (e.g., by engineering
enzymes involved in these pathways such that they are optimized in
activity), or by increasing the availability of the enzymes
involved in such pathways (e.g., by increasing the number of these
enzymes present in the cell), it is possible to also increase the
availability of these degradation products in the cell, which
should in turn increase the production of many different other
desirable compounds in the cell (e.g., fine chemicals).
[0134] The aforementioned mutagenesis strategies for SMP 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 SMP 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 product produced by
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.
[0135] 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
[0136] 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.7H.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-I 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
[0137] 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.)
[0138] 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
[0139] 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
[0140] 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
[0141] 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).
[0142] 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. 159:306-311), 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).
[0143] 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).
[0144] 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
[0145] 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: New York), 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.
[0146] 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: New York). 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 calorimetric 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
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] 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
[0153] 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.,
Gra.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.
[0154] 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.
[0155] 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
[0156] 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.)
[0157] 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
[0158] 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 centrifugation, 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.
[0159] 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.
[0160] 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: New York (1986).
[0161] 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
[0162] 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 SMP 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 SMP 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.
[0163] 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 PAM120 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.
[0164] 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.
[0165] 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
[0166] 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).
[0167] 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).
[0168] 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).
[0169] 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.
[0170] 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).
[0171] 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)
[0172] 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.
[0173] 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.
[0174] 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, 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.
[0175] 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.
[0176] 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.
[0177] 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
[0178] 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 Identification NT ID
NO NO Code Contig. NT Start Stop Function HMP: 1 2 RXS02735 VV0074
14576 15280 6-Phosphogluconolactonase 3 4 RXA01626 GR00452 4270
3926 L-ribulose-phosphate 4-epimerase 5 6 RXA02245 GR00654 13639
14295 RIBULOSE-PHOSPHATE 3-EPIMERASE (EC 5.1.3.1) 7 8 RXA01015
GR00290 346 5 RIBOSE 5-PHOSPHATE ISOMERASE (EC 5.3.1.6) TCA: 9 10
RXN01312 VV0082 20803 18785 SUCCINATE DEHYDROGENASE FLAVOPROTEIN
SUBUNIT (EC 1.3.99.1) 11 12 F RXA01312 GR00380 2690 1614 SUCCINATE
DEHYDROGENASE FLAVOPROTEIN SUBUNIT (EC 1.3.99.1) 13 14 RXN00231
VV0083 15484 14015 SUCCINATE-SEMIALDEHYDE DEHYDROGENASE (NADP+) (EC
1.2.1.16) 15 16 RXA01311 GR00380 1611 865 SUCCINATE DEHYDROGENASE
IRON-SULFUR PROTEIN (EC 1.3.99.1) 17 18 RXA01535 GR00427 1354 2760
FUMARATE HYDRATASE PRECURSOR (EC 4.2.1.2) 19 20 RXA00517 GR00131
1407 2447 MALATE DEHYDROGENASE (EC 1.1.1.37) (EC 1.1.1.82) 21 22
RXA01350 GR00392 1844 2827 MALATE DEHYDROGENASE (EC 1.1.1.37)
EMB-Pathway 23 24 RXA02149 GR00639 17786 18754 GLUCOKINASE (EC
2.7.1.2) 25 26 RXA01814 GR00515 2571 910 PHOSPHOGLUCOMUTASE (EC
5.4.2.2)/PHOSPHOMANNOMUTASE (EC 5.4.2.8) 27 28 RXN02803 VV0086 1
657 PHOSPHOGLUCOMUTASE (EC 5.4.2.2)/PHOSPHOMANNOMUTASE (EC 5.4.2.8)
29 30 F RXA02803 GR00784 2 400 PHOSPHOGLUCOMUTASE (EC
5.4.2.2)/PHOSPHOMANNOMUTASE (EC 5.4.2.8) 31 32 RXN03076 VV0043 1624
35 PHOSPHOGLUCOMUTASE (EC 5.4.2.2)/PHOSPHOMANNOMUTASE (EC 5.4.2.8)
33 34 F RXA02854 GR10002 1588 5 PHOSPHOGLUCOMUTASE (EC
5.4.2.2)/PHOSPHOMANNOMUTASE (EC 5.4.2.8) 35 36 RXA00511 GR00129 1
513 PHOSPHOGLUCOMUTASE (EC 5.4.2.2)/PHOSPHOMANNOMUTASE (EC 5.4.2.8)
37 38 RXN01365 VV0091 1476 103 PHOSPHOGLUCOMUTASE (EC
5.4.2.2)/PHOSPHOMANNOMUTASE (EC 5.4.2.8) 39 40 F RXA01365 GR00397
897 4 PHOSPHOGLUCOMUTASE (EC 5.4.2.2)/PHOSPHOMANNOMUTASE (EC
5.4.2.8) 41 42 RXA00098 GR00014 6525 8144 GLUCOSE-6-PHOSPHATE
ISOMERASE (GPI) (EC 5.3.1.9) 43 44 RXA01989 GR00578 1 630
GLUCOSE-6-PHOSPHATE ISOMERASE A (GPI A) (EC 5.3.1.9) 45 46 RXA00340
GR00059 1549 2694 PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1) 47 48
RXA02492 GR00720 2201 2917 PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1) 49
50 RXA00381 GR00082 1451 846 PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1)
51 52 RXA02122 GR00636 6511 5813 PHOSPHOGLYCERATE MUTASE (EC
5.4.2.1) 53 54 RXA00206 GR00032 6171 5134 6-PHOSPHOFRUCTOKINASE (EC
2.7.1.11) 55 56 RXA01243 GR00359 2302 3261 1-PHOSPHOFRUCTOKINASE
(EC 2.7.1.56) 57 58 RXA01882 GR00538 1165 2154
1-PHOSPHOFRUCTOKINASE (EC 2.7.1.56) 59 60 RXA01702 GR00479 1397 366
FRUCTOSE-BISPHOSPHATE ALDOLASE (EC 4.1.2.13) 61 62 RXA02258 GR00654
26451 27227 TRIOSEPHOSPHATE ISOMERASE (EC 5.3.1.1) 63 64 RXN01225
VV0064 6382 4943 GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (EC
1.2.1.12) 65 66 F RXA01225 GR00354 5302 6741
GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE HOMOLOG 67 68 RXA02256
GR00654 23934 24935 GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (EC
1.2.1.12) 69 70 RXA02257 GR00654 25155 26369 PHOSPHOGLYCERATE
KINASE (EC 2.7.2.3) 71 72 RXA00235 GR00036 2365 1091 ENOLASE (EC
4.2.1.11) 73 74 RXA01093 GR00306 1552 122 PYRUVATE KINASE (EC
2.7.1.40) 75 76 RXN02675 VV0098 72801 70945 PYRUVATE KINASE (EC
2.7.1.40) 77 78 F RXA02675 GR00754 2 364 PYRUVATE KINASE (EC
2.7.1.40) 79 80 F RXA02695 GR00755 2949 4370 PYRUVATE KINASE (EC
2.7.1.40) 81 82 RXA00682 GR00179 5299 3401 PHOSPHOENOLPYRUVATE
SYNTHASE (EC 2.7.9.2) 83 84 RXA00683 GR00179 6440 5349
PHOSPHOENOLPYRUVATE SYNTHASE (EC 2.7.9.2) 85 86 RXN00635 VV0135
22708 20972 PYRUVATE DEHYDROGENASE (CYTOCHROME) (EC 1.2.2.2) 87 88
F RXA02807 GR00788 88 552 PYRUVATE DEHYDROGENASE (CYTOCHROME) (EC
1.2.2.2) 89 90 F RXA00635 GR00167 3 923 PYRUVATE DEHYDROGENASE
(CYTOCHROME) (EC 1.2.2.2) 91 92 RXN03044 VV0019 1391 2221 PYRUVATE
DEHYDROGENASE E1 COMPONENT (EC 1.2.4.1) 93 94 F RXA02852 GR00852 3
281 PYRUVATE DEHYDROGENASE E1 COMPONENT (EC 1.2.4.1) 95 96 F
RXA00268 GR00041 125 955 PYRUVATE DEHYDROGENASE E1 COMPONENT (EC
1.2.4.1) 97 98 RXN03086 VV0049 2243 2650 PYRUVATE DEHYDROGENASE E1
COMPONENT (EC 1.2.4.1) 99 100 F RXA02887 GR10022 411 4 PYRUVATE
DEHYDROGENASE E1 COMPONENT (EC 1.2.4.1) 101 102 RXN03043 VV0019 1
1362 PYRUVATE DEHYDROGENASE E1 COMPONENT (EC 1.2.4.1) 103 104 F
RXA02897 GR10039 1291 5 PYRUVATE DEHYDROGENASE E1 COMPONENT (EC
1.2.4.1) 105 106 RXN03083 VV0047 88 1110 DIHYDROLIPOAMIDE
DEHYDROGENASE (EC 1.8.1.4) 107 108 F RXA02853 GR10001 89 1495
DIHYDROLIPOAMIDE DEHYDROGENASE (EC 1.8.1.4) 109 110 RXA02259
GR00654 27401 30172 PHOSPHOENOLPYRUVATE CARBOXYLASE (EC 4.1.1.31)
111 112 RXN02326 VV0047 4500 5315 PYRUVATE CARBOXYLASE (EC 6.4.1.1)
113 114 F RXA02326 GR00668 5338 4523 PYRUVATE CARBOXYLASE 115 116
RXN02327 VV0047 3533 4492 PYRUVATE CARBOXYLASE (EC 6.4.1.1) 117 118
F RXA02327 GR00668 6305 5346 PYRUVATE CARBOXYLASE 119 120 RXN02328
VV0047 1842 3437 PYRUVATE CARBOXYLASE (EC 6.4.1.1) 121 122 F
RXA02328 GR00668 7783 6401 PYRUVATE CARBOXYLASE (EC 6.4.1.1) 123
124 RXN01048 VV0079 12539 11316 MALIC ENZYME (EC 1.1.1.39) 125 126
F RXA01048 GR00296 3 290 MALIC ENZYME (EC 1.1.1.39) 127 128 F
RXA00290 GR00046 4693 5655 MALIC ENZYME (EC 1.1.1.39) 129 130
RXA02694 GR00755 1879 2820 L-LACTATE DEHYDROGENASE (EC 1.1.1.27)
131 132 RXN00296 VV0176 35763 38606 D-LACTATE DEHYDROGENASE
(CYTOCHROME) (EC 1.1.2.4) 133 134 F RXA00296 GR00048 3 2837
D-LACTATE DEHYDROGENASE (CYTOCHROME) (EC 1.1.2.4) 135 136 RXA01901
GR00544 4158 5417 L-LACTATE DEHYDROGENASE (CYTOCHROME) (EC 1.1.2.3)
137 138 RXN01952 VV0105 9954 11666 D-LACTATE DEHYDROGENASE (EC
1.1.1.28) 139 140 F RXA01952 GR00562 1 216 D-LACTATE DEHYDROGENASE
(EC 1.1.1.28) 141 142 F RXA01955 GR00562 4611 6209 D-LACTATE
DEHYDROGENASE (EC 1.1.1.28) 143 144 RXA00293 GR00047 2645 1734
D-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1.1.1.95) 145 146 RXN01130
VV0157 6138 5536 D-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1.1.1.95)
147 148 F RXA01130 GR00315 2 304 D-3-PHOSPHOGLYCERATE DEHYDROGENASE
(EC 1.1.1.95) 149 150 RXN03112 VV0085 509 6 D-3-PHOSPHOGLYCERATE
DEHYDROGENASE (EC 1.1.1.95) 151 152 F RXA01133 GR00316 568 1116
D-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1.1.1.95) 153 154 RXN00871
VV0127 3127 2240 IOLB PROTEIN 155 156 F RXA00871 GR00239 2344 3207
IOLB PROTEIN: D-FRUCTOSE 1,6-BISPHOSPHATE = GLYCERONE-CC PHOSPHATE
+ D-GLYCERALDEHYDE 3-PHOSPHATE. 157 158 RXN02829 VV0354 287 559
IOLS PROTEIN 159 160 F RXA02829 GR00816 287 562 IOLS PROTEIN 161
162 RXN01468 VV0019 7474 8298 NAGD PROTEIN 163 164 F RXA01468
GR00422 1250 2074 PUTATIVE N-GLYCERALDEHYDE-2-PHOSPHOTRANSFERASE
165 166 RXA00794 GR00211 3993 2989 GLPX PROTEIN 167 168 RXN02920
VV0213 6135 5224 D-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1.1.1.95)
169 170 F RXA02379 GR00690 1390 686 D-3-PHOSPHOGLYCERATE
DEHYDROGENASE (EC 1.1.1.95) 171 172 RXN02688 VV0098 59053 58385
PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1) 173 174 RXN03087 VV0052 3216
3428 PYRUVATE CARBOXYLASE (EC 6.4.1.1) 175 176 RXN03186 VV0377 310
519 PYRUVATE DEHYDROGENASE E1 COMPONENT (EC 1.2.4.1) 177 178
RXN03187 VV0382 3 281 PYRUVATE DEHYDROGENASE E1 COMPONENT (EC
1.2.4.1) 179 180 RXN02591 VV0098 14370 12541 PHOSPHOENOLPYRUVATE
CARBOXYKINASE [GTP] (EC 4.1.1.32) 181 182 RXS01260 VV0009 3477 2296
LIPOAMIDE DEHYDROGENASE COMPONENT (E3) OF BRANCHED- CHAIN
ALPHA-KETO ACID DEHYDROGENASE COMPLEX (EC 1.8.1.4) 183 184 RXS01261
VV0009 3703 3533 LIPOAMIDE DEHYDROGENASE COMPONENT (E3) OF
BRANCHED- CHAIN ALPHA-KETO ACID DEHYDROGENASE COMPLEX (EC 1.8.1.4)
Glycerol metabolism 185 186 RXA02640 GR00749 1400 2926 GLYCEROL
KINASE (EC 2.7.1.30) 187 188 RXN01025 VV0143 5483 4488
GLYCEROL-3-PHOSPHATE DEHYDROGENASE (NAD(P)+) (EC 1.1.1.94) 189 190
F RXA01025 GR00293 939 1853 GLYCEROL-3-PHOSPHATE DEHYDROGENASE
(NAD(P)+) (EC 1.1.1.94) 191 192 RXA01851 GR00525 3515 1830 AEROBIC
GLYCEROL-3-PHOSPHATE DEHYDROGENASE (EC 1.1.99.5) 193 194 RXA01242
GR00359 1526 2302 GLYCEROL-3-PHOSPHATE REGULON REPRESSOR 195 196
RXA02288 GR00661 992 147 GLYCEROL-3-PHOSPHATE REGULON REPRESSOR 197
198 RXN01891 VV0122 24949 24086 GLYCEROL-3-PHOSPHATE-BINDING
PERIPLASMIC PROTEIN PRECURSOR 199 200 F RXA01891 GR00541 1736 918
GLYCEROL-3-PHOSPHATE-BINDING PERIPLASMIC PROTEIN PRECURSOR 201 202
RXA02414 GR00703 3808 3062 Uncharacterized protein involved in
glycerol metabolism (homolog of Drosophila rhomboid) 203 204
RXN01580 VV0122 22091 22807 Glycerophosphoryl diester
phosphodiesterase Acetate metabolism 205 206 RXA01436 GR00418 2547
1357 ACETATE KINASE (EC 2.7.2.1) 207 208 RXA00686 GR00179 8744 7941
ACETATE OPERON REPRESSOR 209 210 RXA00246 GR00037 4425 3391 ALCOHOL
DEHYDROGENASE (EC 1.1.1.1) 211 212 RXA01571 GR00438 1360 1959
ALCOHOL DEHYDROGENASE (EC 1.1.1.1) 213 214 RXA01572 GR00438 1928
2419 ALCOHOL DEHYDROGENASE (EC 1.1.1.1) 215 216 RXA01758 GR00498
3961 2945 ALCOHOL DEHYDROGENASE (EC 1.1.1.1) 217 218 RXA02539
GR00726 11676 10159 ALDEHYDE DEHYDROGENASE (EC 219 220 RXN03061
VV0034 108 437 ALDEHYDE DEHYDROGENASE (EC 1.2.1.3) 221 222 RXN03150
VV0155 10678 10055 ALDEHYDE DEHYDROGENASE (EC 1.2.1.3) 223 224
RXN01340 VV0033 3 860 ALDEHYDE DEHYDROGENASE (EC 1.2.1.3) 225 226
RXN01498 VV0008 1598 3160 ALDEHYDE DEHYDROGENASE (EC 1.2.1.3) 227
228 RXN02674 VV0315 15614 14163 ALDEHYDE DEHYDROGENASE (EC 1.2.1.3)
229 230 RXN00868 VV0127 2230 320 ACETOLACTATE SYNTHASE LARGE
SUBUNIT (EC 4.1.3.18) 231 232 RXN01143 VV0077 9372 8254
ACETOLACTATE SYNTHASE LARGE SUBUNIT (EC 4.1.3.18) 233 234 RXN01146
VV0264 243 935 ACETOLACTATE SYNTHASE LARGE SUBUNIT (EC 4.1.3.18)
235 236 RXN01144 VV0077 8237 7722 ACETOLACTATE SYNTHASE SMALL
SUBUNIT (EC 4.1.3.18) Butanediol, diacetyl and acetoin formation
237 238 RXA02474 GR00715 8082 7309 (S,S)-butane-2,3-diol
dehydrogenase (EC 1.1.1.76) 239 240 RXA02453 GR00710 6103 5351
ACETOIN(DIACETYL) REDUCTASE (EC 1.1.1.5) 241 242 RXS01758 VV0112
27383 28399 ALCOHOL DEHYDROGENASE (EC 1.1.1.1) HMP-Cycle 243 244
RXA02737 GR00763 3312 1771 GLUCOSE-6-PHOSPHATE 1-DEHYDROGENASE (EC
1.1.1.49) 245 246 RXA02738 GR00763 4499 3420 TRANSALDOLASE (EC
2.2.1.2) 247 248 RXA02739 GR00763 6769 4670 TRANSKETOLASE (EC
2.2.1.1) 249 250 RXA00965 GR00270 1232 510 6-PHOSPHOGLUCONATE
DEHYDROGENASE, DECARBOXYLATING (EC 1.1.1.44) 251 252 RXN00999
VV0106 2817 1366 6-PHOSPHOGLUCONATE DEHYDROGENASE, DECARBOXYLATING
(EC 1.1.1.44) 253 254 F RXA00999 GR00283 3012 4448
6-PHOSPHOGLUCONATE DEHYDROGENASE, DECARBOXYLATING (EC 1.1.1.44)
Nucleotide sugar conversion 255 256 RXN02596 VV0098 48784 47582
UDP-GALACTOPYRANOSE MUTASE (EC 5.4.99.9) 257 258 F RXA02596 GR00742
1 489 UDP-GALACTOPYRANOSE MUTASE (EC 5.4.99.9) 259 260 F RXA02642
GR00749 5383 5880 UDP-GALACTOPYRANOSE MUTASE (EC 5.4.99.9) 261 262
RXA02572 GR00737 2 646 UDP-GLUCOSE 6-DEHYDROGENASE (EC 1.1.1.22)
263 264 RXA02485 GR00718 2345 3445
UDP-N-ACETYLENOLPYRUVOYLGLUCOSAMINE REDUCTASE (EC 1.1.1.158) 265
266 RXA01216 GR00352 2302 1202 UDP-N-ACETYLGLUCOSAMINE
PYROPHOSPHORYLASE (EC 2.7.7.23) 267 268 RXA01259 GR00367 987 130
UTP-GLUCOSE-1-PHOSPHATE
URIDYLYLTRANSFERASE (EC 2.7.7.9) 269 270 RXA02028 GR00616 573 998
UTP-GLUCOSE-1-PHOSPHATE URIDYLYLTRANSFERASE (EC 2.7.7.9) 271 272
RXA01262 GR00367 8351 7191 GDP-MANNOSE 6-DEHYDROGENASE (EC
1.1.1.132) 273 274 RXA01377 GR00400 3935 5020 MANNOSE-1-PHOSPHATE
GUANYLTRANSFERASE (EC 2.7.7.13) 275 276 RXA02063 GR00626 3301 4527
GLUCOSE-1-PHOSPHATE ADENYLYLTRANSFERASE (EC 2.7.7.27) 277 278
RXN00014 VV0048 8848 9627 GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
(EC 2.7.7.24) 279 280 F RXA00014 GR00002 4448 5227
GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE (EC 2.7.7.24) 281 282
RXA01570 GR00438 427 1281 GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
(EC 2.7.7.24) 283 284 RXA02666 GR00753 7260 6493
D-RIBITOL-5-PHOSPHATE CYTIDYLYLTRANSFERASE (EC 2.7.7.40) 285 286
RXA00825 GR00222 222 1154 DTDP-GLUCOSE 4,6-DEHYDRATASE (EC
4.2.1.46) Inositol and ribitol metabolism 287 288 RXA01887 GR00539
4219 3209 MYO-INOSITOL 2-DEHYDROGENASE (EC 1.1.1.18) 289 290
RXN00013 VV0048 7966 8838 MYO-INOSITOL-1(OR 4)-MONOPHOSPHATASE 1
(EC 3.1.3.25) 291 292 F RXA00013 GR00002 3566 4438
MYO-INOSITOL-1(OR 4)-MONOPHOSPHATASE 1 (EC 3.1.3.25) 293 294
RXA01099 GR00306 6328 5504 INOSITOL MONOPHOSPHATE PHOSPHATASE 295
296 RXN01332 VV0273 579 4 MYO-INOSITOL 2-DEHYDROGENASE (EC
1.1.1.18) 297 298 F RXA01332 GR00388 552 4 MYO-INOSITOL
2-DEHYDROGENASE (EC 1.1.1.18) 299 300 RXA01632 GR00454 2338 3342
MYO-INOSITOL 2-DEHYDROGENASE (EC 1.1.1.18) 301 302 RXA01633 GR00454
3380 4462 MYO-INOSITOL 2-DEHYDROGENASE (EC 1.1.1.18) 303 304
RXN01406 VV0278 2999 1977 MYO-INOSITOL 2-DEHYDROGENASE (EC
1.1.1.18) 305 306 RXN01630 VV0050 48113 47037 MYO-INOSITOL
2-DEHYDROGENASE (EC 1.1.1.18) 307 308 RXN00528 VV0079 23406 22318
MYO-INOSITOL-1-PHOSPHATE SYNTHASE (EC 5.5.1.4) 309 310 RXN03057
VV0028 7017 7688 MYO-INOSITOL 2-DEHYDROGENASE (EC 1.1.1.18) 311 312
F RXA02902 GR10040 10277 10948 GLUCOSE-FRUCTOSE OXIDOREDUCTASE
PRECURSOR (EC 1.1.99.28) 313 314 RXA00251 GR00038 931 224 RIBITOL
2-DEHYDROGENASE (EC 1.1.1.56) Utilization of sugars 315 316
RXN02654 VV0090 12206 13090 GLUCOSE 1-DEHYDROGENASE (EC 1.1.1.47)
317 318 F RXA02654 GR00752 7405 8289 GLUCOSE 1-DEHYDROGENASE II (EC
1.1.1.47) 319 320 RXN01049 VV0079 9633 11114 GLUCONOKINASE (EC
2.7.1.12) 321 322 F RXA01049 GR00296 1502 492 GLUCONOKINASE (EC
2.7.1.12) 323 324 F RXA01050 GR00296 1972 1499 GLUCONOKINASE (EC
2.7.1.12) 325 326 RXA00202 GR00032 1216 275 D-RIBOSE-BINDING
PERIPLASMIC PROTEIN PRECURSOR 327 328 RXN00872 VV0127 6557 5604
FRUCTOKINASE (EC 2.7.1.4) 329 330 F RXA00872 GR00240 565 1086
FRUCTOKINASE (EC 2.7.1.4) 331 332 RXN00799 VV0009 58477 56834
PERIPLASMIC BETA-GLUCOSIDASE/BETA-XYLOSIDASE PRECURSOR (EC
3.2.1.21) (EC 3.2.1.37) 333 334 F RXA00799 GR00214 1 1584
PERIPLASMIC BETA-GLUCOSIDASE/BETA-XYLOSIDASE PRECURSOR (EC
3.2.1.21) (EC 3.2.1.37) 335 336 RXA00032 GR00003 12028 10520
MANNITOL 2-DEHYDROGENASE (EC 1.1.1.67) 337 338 RXA02528 GR00725
6880 7854 FRUCTOSE REPRESSOR 339 340 RXN00316 VV0006 7035 8180
Hypothetical Oxidoreductase (EC 1.1.1.--) 341 342 F RXA00309
GR00053 316 5 GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC
1.1.99.28) 343 344 RXN00310 VV0006 6616 7050 GLUCOSE-FRUCTOSE
OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) 345 346 F RXA00310 GR00053
735 301 GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28)
347 348 RXA00041 GR00007 1246 5 SUCROSE-6-PHOSPHATE HYDROLASE (EC
3.2.1.26) 349 350 RXA02026 GR00615 725 6 SUCROSE-6-PHOSPHATE
HYDROLASE (EC 3.2.1.26) 351 352 RXA02061 GR00626 1842 349
SUCROSE-6-PHOSPHATE HYDROLASE (EC 3.2.1.26) 353 354 RXN01369 VV0124
595 1776 MANNOSE-6-PHOSPHATE ISOMERASE (EC 5.3.1.8) 355 356 F
RXA01369 GR00398 3 503 MANNOSE-6-PHOSPHATE ISOMERASE (EC 5.3.1.8)
357 358 F RXA01373 GR00399 595 1302 MANNOSE-6-PHOSPHATE ISOMERASE
(EC 5.3.1.8) 359 360 RXA02611 GR00743 1 1752 1,4-ALPHA-GLUCAN
BRANCHING ENZYME (EC 2.4.1.18) 361 362 RXA02612 GR00743 1793 3985
1,4-ALPHA-GLUCAN BRANCHING ENZYME (EC 2.4.1.18) 363 364 RXN01884
VV0184 1 1890 GLYCOGEN DEBRANCHING ENZYME (EC 2.4.1.25) (EC
3.2.1.33) 365 366 F RXA01884 GR00539 3 1475 GLYCOGEN DEBRANCHING
ENZYME (EC 2.4.1.25) (EC 3.2.1.33) 367 368 RXA01111 GR00306 16981
17427 GLYCOGEN OPERON PROTEIN GLGX (EC 3.2.1.--) 369 370 RXN01550
VV0143 14749 16260 GLYCOGEN PHOSPHORYLASE (EC 2.4.1.1) 371 372 F
RXA01550 GR00431 3 1346 GLYCOGEN PHOSPHORYLASE (EC 2.4.1.1) 373 374
RXN02100 VV0318 2 2326 GLYCOGEN PHOSPHORYLASE (EC 2.4.1.1) 375 376
F RXA02100 GR00631 3 920 GLYCOGEN PHOSPHORYLASE (EC 2.4.1.1) 377
378 F RXA02113 GR00633 2 1207 GLYCOGEN PHOSPHORYLASE (EC 2.4.1.1)
379 380 RXA02147 GR00639 15516 16532 ALPHA-AMYLASE (EC 3.2.1.1) 381
382 RXA01478 GR00422 10517 12352 GLUCOAMYLASE G1 AND G2 PRECURSOR
(EC 3.2.1.3) 383 384 RXA01888 GR00539 4366 4923 GLUCOSE-RESISTANCE
AMYLASE REGULATOR 385 386 RXN01927 VV0127 50623 49244 XYLULOSE
KINASE (EC 2.7.1.17) 387 388 F RXA01927 GR00555 3 1118 XYLULOSE
KINASE (EC 2.7.1.17) 389 390 RXA02729 GR00762 747 4 RIBOKINASE (EC
2.7.1.15) 391 392 RXA02797 GR00778 1739 2641 RIBOKINASE (EC
2.7.1.15) 393 394 RXA02730 GR00762 1768 731 RIBOSE OPERON REPRESSOR
395 396 RXA02551 GR00729 2193 2552 6-PHOSPHO-BETA-GLUCOSIDASE (EC
3.2.1.86) 397 398 RXA01325 GR00385 5676 5005 DEOXYRIBOSE-PHOSPHATE
ALDOLASE (EC 4.1.2.4) 399 400 RXA00195 GR00030 543 1103
1-deoxy-D-xylulose 5-phosphate reductoisomerase (EC 1.1.1.--) 401
402 RXA00196 GR00030 1094 1708 1-deoxy-D-xylulose 5-phosphate
reductoisomerase (EC 1.1.1.--) 403 404 RXN01562 VV0191 1230 3137
1-DEOXYXYLULOSE-5-PHOSPHATE SYNTHASE 405 406 F RXA01562 GR00436 2
1039 1-DEOXYXYLULOSE-5-PHOSPHATE SYNTHASE 407 408 F RXA01705
GR00480 971 1573 1-DEOXYXYLULOSE-5-PHOSPHATE SYNTHASE 409 410
RXN00879 VV0099 8763 6646 4-ALPHA-GLUCANOTRANSFERASE (EC 2.4.1.25)
411 412 F RXA00879 GR00242 5927 3828 4-ALPHA-GLUCANOTRANSFERASE (EC
2.4.1.25), amylomaltase 413 414 RXN00043 VV0119 3244 2081
N-ACETYLGLUCOSAMINE-6-PHOSPHATE DEACETYLASE (EC 3.5.1.25) 415 416 F
RXA00043 GR00007 3244 2081 N-ACETYLGLUCOSAMINE-6-PHOSPHATE
DEACETYLASE (EC 3.5.1.25) 417 418 RXN01752 VV0127 35265 33805
N-ACETYLGLUCOSAMINYLTRANSFERASE (EC 2.4.1.--) 419 420 F RXA01839
GR00520 1157 510 N-ACETYLGLUCOSAMINYLTRANSFERASE (EC 2.4.1.--) 421
422 RXA01859 GR00529 1473 547 N-ACETYLGLUCOSAMINYLTRANSFERASE (EC
2.4.1.--) 423 424 RXA00042 GR00007 2037 1279
GLUCOSAMINE-6-PHOSPHATE ISOMERASE (EC 5.3.1.10) 425 426 RXA01482
GR00422 17271 15397 GLUCOSAMINE-FRUCTOSE-6-PHOSPHATE
AMINOTRANSFERASE (ISOMERIZING) (EC 2.6.1.16) 427 428 RXN03179
VV0336 2 667 URONATE ISOMERASE (EC 5.3.1.12) 429 430 F RXA02872
GR10013 675 4 URONATE ISOMERASE, Glucuronate isomerase (EC
5.3.1.12) 431 432 RXN03180 VV0337 672 163 URONATE ISOMERASE (EC
5.3.1.12) 433 434 F RXA02873 GR10014 672 163 URONATE ISOMERASE,
Glucuronate isomerase (EC 5.3.1.12) 435 436 RXA02292 GR00662 1611
2285 GALACTOSIDE O-ACETYLTRANSFERASE (EC 2.3.1.18) 437 438 RXA02666
GR00753 7260 6493 D-RIBITOL-5-PHOSPHATE CYTIDYLYLTRANSFERASE (EC
2.7.7.40) 439 440 RXA00202 GR00032 1216 275 D-RIBOSE-BINDING
PERIPLASMIC PROTEIN PRECURSOR 441 442 RXA02440 GR00709 5097 4258
D-RIBOSE-BINDING PERIPLASMIC PROTEIN PRECURSOR 443 444 RXN01569
VV0009 41086 42444 dTDP-4-DEHYDRORHAMNOSE REDUCTASE (EC 1.1.1.133)
445 446 F RXA01569 GR00438 2 427 DTDP-4-DEHYDRORHAMNOSE REDUCTASE
(EC 1.1.1.133) 447 448 F RXA02055 GR00624 7122 8042
DTDP-4-DEHYDRORHAMNOSE REDUCTASE (EC 1.1.1.133) 449 450 RXA00825
GR00222 222 1154 DTDP-GLUCOSE 4,6-DEHYDRATASE (EC 4.2.1.46) 451 452
RXA02054 GR00624 6103 7119 DTDP-GLUCOSE 4,6-DEHYDRATASE (EC
4.2.1.46) 453 454 RXN00427 VV0112 7004 6219 dTDP-RHAMNOSYL
TRANSFERASE RFBF (EC 2.--.--.--) 455 456 F RXA00427 GR00098 1591
2022 DTDP-RHAMNOSYL TRANSFERASE RFBF (EC 2.--.--.--) 457 458
RXA00327 GR00057 10263 9880 PROTEIN ARAJ 459 460 RXA00328 GR00057
11147 10656 PROTEIN ARAJ 461 462 RXA00329 GR00057 12390 11167
PROTEIN ARAJ 463 464 RXN01554 VV0135 28686 26545 GLUCAN
ENDO-1,3-BETA-GLUCOSIDASE A1 PRECURSOR (EC 3.2.1.39) 465 466
RXN03015 VV0063 289 8 UDP-GLUCOSE 6-DEHYDROGENASE (EC 1.1.1.22) 467
468 RXN03056 VV0028 6258 6935 PUTATIVE HEXULOSE-6-PHOSPHATE
ISOMERASE (EC 5.--.--.--) 469 470 RXN03030 VV0009 57006 56443
PERIPLASMIC BETA-GLUCOSIDASE/BETA-XYLOSIDASE PRECURSOR (EC
3.2.1.21) (EC 3.2.1.37) 471 472 RXN00401 VV0025 12427 11489
5-DEHYDRO-4-DEOXYGLUCARATE DEHYDRATASE (EC 4.2.1.41) 473 474
RXN02125 VV0102 23242 22442 ALDOSE REDUCTASE (EC 1.1.1.21) 475 476
RXN00200 VV0181 1679 5116 arabinosyl transferase subunit B (EC
2.4.2.--) 477 478 RXN01175 VV0017 39688 38303
PHOSPHO-2-DEHYDRO-3-DEOXYHEPTONATE ALDOLASE (EC 4.1.2.15) 479 480
RXN01376 VV0091 5610 4750 PUTATIVE GLYCOSYL TRANSFERASE WBIF 481
482 RXN01631 VV0050 47021 46143 PUTATIVE HEXULOSE-6-PHOSPHATE
ISOMERASE (EC 5.--.--.--) 483 484 RXN01593 VV0229 13274 12408 NAGD
PROTEIN 485 486 RXN00337 VV0197 20369 21418 GALACTOKINASE (EC
2.7.1.6) 487 488 RXS00584 VV0323 5516 6640
PHOSPHO-2-DEHYDRO-3-DEOXYHEPTONATE ALDOLASE (EC 4.1.2.15) 489 490
RXS02574 BETA-HEXOSAMINIDASE A PRECURSOR (EC 3.2.1.52) 491 492
RXS03215 GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28)
493 494 F RXA01915 GR00549 1 1008 GLUCOSE-FRUCTOSE OXIDOREDUCTASE
PRECURSOR (EC 1.1.99.28) 495 496 RXS03224 CYCLOMALTODEXTRINASE (EC
3.2.1.54) 497 498 F RXA00038 GR00006 1417 260 CYCLOMALTODEXTRINASE
(EC 3.2.1.54) 499 500 RXC00233 protein involved in sugar metabolism
501 502 RXC00236 Membrane Lipoprotein involved in sugar metabolism
503 504 RXC00271 Exported Protein involved in ribose metabolism 505
506 RXC00338 protein involved in sugar metabolism 507 508 RXC00362
Membrane Spanning Protein involved in metabolism of diols 509 510
RXC00412 Amino Acid ABC Transporter ATP-Binding Protein involved in
sugar metabolism 511 512 RXC00526 ABC Transporter ATP-Binding
Protein involved in sugar metabolism 513 514 RXC01004 Membrane
Spanning Protein involved in sugar metabolism 515 516 RXC01017
Cytosolic Protein involved in sugar metabolism 517 518 RXC01021
Cytosolic Kinase involved in metabolism of sugars and thiamin 519
520 RXC01212 ABC Transporter ATP-Binding Protein involved in sugar
metabolism 521 522 RXC01306 Membrane Spanning Protein involved in
sugar metabolism 523 524 RXC01366 Cytosolic Protein involved in
sugar metabolism 525 526 RXC01372 Cytosolic Protein involved in
sugar metabolism 527 528 RXC01659 protein involved in sugar
metabolism 529 530 RXC01663 protein involved in sugar metabolism
531 532 RXC01693 protein involved in sugar metabolism 533 534
RXC01703 Cytosolic Protein involved in sugar metabolism 535 536
RXC02254 Membrane Associated Protein involved in sugar metabolism
537 538 RXC02255 Cytosolic Protein involved in sugar metabolism 539
540 RXC02435 protein involved in sugar metabolism 541 542 F
RXA02435 GR00709 825 268 Uncharacterized protein involved in
glycerol metabolism (homolog of Drosophila rhomboid) 543 544
RXC03216 protein involved in sugar metabolism TCA-cycle 545 546
RXA02175 GR00641 10710 9418 CITRATE SYNTHASE (EC 4.1.3.7) 547 548
RXA02621 GR00746 2647 1829 CITRATE LYASE BETA CHAIN (EC 4.1.3.6)
549 550 RXN00519 VV0144 5585 3372 ISOCITRATE DEHYDROGENASE (NADP)
(EC 1.1.1.42) 551 552 F RXA00521 GR00133 2 1060 ISOCITRATE
DEHYDROGENASE [NADP] (EC 1.1.1.42) 553 554 RXN02209 VV0304 1 1671
ACONITATE HYDRATASE (EC 4.2.1.3) 555 556 F RXA02209 GR00648 3 1661
ACONITATE HYDRATASE (EC 4.2.1.3) 557 558 RXN02213 VV0305 1378 2151
ACONITATE HYDRATASE (EC 4.2.1.3) 559 560 F RXA02213 GR00649 1330
2046 ACONITATE HYDRATASE (EC 4.2.1.3) 561 562 RXA02056 GR00625 3
2870 2-OXOGLUTARATE DEHYDROGENASE E1 COMPONENT (EC 1.2.4.2) 563 564
RXA01745 GR00495 2 1495 DIHYDROLIPOAMIDE SUCCINYLTRANSFERASE
COMPONENT (E2) OF 2-OXOGLUTARATE DEHYDROGENASE COMPLEX (EC
2.3.1.61) 565 566 RXA00782 GR00206 3984 3103 SUCCINYL-COA
SYNTHETASE ALPHA CHAIN (EC 6.2.1.5) 567 568 RXA00783 GR00206 5280
4009 SUCCINYL-COA SYNTHETASE BETA CHAIN (EC 6.2.1.5) 569 570
RXN01695 VV0139 11307 12806 L-MALATE DEHYDROGENASE (ACCEPTOR) (EC
1.1.99.16) 571 572 F RXA01615 GR00449 8608 9546 L-MALATE
DEHYDROGENASE (ACCEPTOR) (EC 1.1.99.16) 573 574 F RXA01695 GR00474
4388 4179 L-MALATE DEHYDROGENASE (ACCEPTOR) (EC 1.1.99.16) 575 576
RXA00290 GR00046 4693 5655 MALIC ENZYME (EC 1.1.1.39) 577 578
RXN01048 VV0079 12539 11316 MALIC ENZYME (EC 1.1.1.39) 579 580 F
RXA01048 GR00296 3 290 MALIC ENZYME (EC 1.1.1.39) 581 582 F
RXA00290 GR00046 4693 5655 MALIC ENZYME (EC 1.1.1.39) 583 584
RXN03101 VV0066 2 583 DIHYDROLIPOAMIDE SUCCINYLTRANSFERASE
COMPONENT (E2) OF 2-OXOGLUTARATE DEHYDROGENASE COMPLEX (EC
2.3.1.61) 585 586 RXN02046 VV0025 15056 14640 DIHYDROLIPOAMIDE
SUCCINYLTRANSFERASE COMPONENT OF 2- OXOGLUTARATE DEHYDROGENASE
COMPLEX (EC 2.3.1.61) 587 588 RXN00389 VV0025 11481 9922
oxoglutarate semialdehyde dehydrogenase (EC 1.2.1.--) Glyoxylate
bypass 589 590 RXN02399 VV0176 19708 18365 ISOCITRATE LYASE (EC
4.1.3.1) 591 592 F RXA02399 GR00699 478 1773 ISOCITRATE LYASE (EC
4.1.3.1) 593 594 RXN02404 VV0176 20259 22475 MALATE SYNTHASE (EC
4.1.3.2) 595 596 F RXA02404 GR00700 3798 1663 MALATE SYNTHASE (EC
4.1.3.2) 597 598 RXA01089 GR00304 3209 3958 GLYOXYLATE-INDUCED
PROTEIN 599 600 RXA01886 GR00539 3203 2430 GLYOXYLATE-INDUCED
PROTEIN Methylcitrate-pathway 601 602 RXN03117 VV0092 3087 1576
2-methylisocitrate synthase (EC 5.3.3.--) 603 604 F RXA00406
GR00090 978 4 2-methylisocitrate synthase (EC 5.3.3.--) 605 606 F
RXA00514 GR00130 1983 1576 2-methylisocitrate synthase (EC
5.3.3.--) 607 608 RXA00512 GR00130 621 4 2-methylcitrate synthase
(EC 4.1.3.31) 609 610 RXA00518 GR00131 3069 2773 2-methylcitrate
synthase (EC 4.1.3.31) 611 612 RXA01077 GR00300 4647 6017
2-methylisocitrate synthase (EC 5.3.3.--) 613 614 RXN03144 VV0141 2
901 2-methylisocitrate synthase (EC 5.3.3.--) 615 616 F RXA02322
GR00668 415 5 2-methylisocitrate synthase (EC 5.3.3.--) 617 618
RXA02329 GR00669 607 5 2-methylisocitrate synthase (EC 5.3.3.--)
619 620 RXA02332 GR00671 1906 764 2-methylcitrate synthase (EC
4.1.3.31) 621 622 RXN02333 VV0141 901 1815 methylisocitrate lyase
(EC 4.1.3.30) 623 624 F RXA02333 GR00671 2120 1902 methylisocitrate
lyase (EC 4.1.3.30) 625 626 RXA00030 GR00003 9590 9979
LACTOYLGLUTATHIONE LYASE (EC 4.4.1.5) Methyl-Malonyl-CoA-Mutases
627 628 RXN00148 VV0167 9849 12059 METHYLMALONYL-COA MUTASE
ALPHA-SUBUNIT (EC 5.4.99.2) 629 630 F RXA00148 GR00023 2002 5
METHYLMALONYL-COA MUTASE ALPHA-SUBUNIT (EC 5.4.99.2) 631 632
RXA00149 GR00023 3856 2009 METHYLMALONYL-COA MUTASE BETA-SUBUNIT
(EC 5.4.99.2) Others 633 634 RXN00317 VV0197 26879 27532
PHOSPHOGLYCOLATE PHOSPHATASE (EC 3.1.3.18) 635 636 F RXA00317
GR00055 344 6 PHOSPHOGLYCOLATE PHOSPHATASE (EC 3.1.3.18) 637 638
RXA02196 GR00645 3956 3264 PHOSPHOGLYCOLATE PHOSPHATASE (EC
3.1.3.18) 639 640 RXN02461 VV0124 14236 14643 PHOSPHOGLYCOLATE
PHOSPHATASE (EC 3.1.3.18) Redox Chain 641 642 RXN01744 VV0174 2350
812 CYTOCHROME D UBIQUINOL OXIDASE SUBUNIT I (EC 1.10.3.--) 643 644
F RXA00055 GR00008 11753 11890 CYTOCHROME D UBIQUINOL OXIDASE
SUBUNIT I (EC 1.10.3.--) 645 646 F RXA01744 GR00494 2113 812
CYTOCHROME D UBIQUINOL OXIDASE SUBUNIT I (EC 1.10.3.--) 647 648
RXA00379 GR00082 212 6 CYTOCHROME C-TYPE BIOGENESIS PROTEIN CCDA
649 650 RXA00385 GR00083 773 435 CYTOCHROME C-TYPE BIOGENESIS
PROTEIN CCDA 651 652 RXA01743 GR00494 806 6 CYTOCHROME D UBIQUINOL
OXIDASE SUBUNIT II (EC 1.10.3.--) 653 654 RXN02480 VV0084 31222
29567 CYTOCHROME C OXIDASE POLYPEPTIDE I (EC 1.9.3.1) 655 656 F
RXA01919 GR00550 288 4 CYTOCHROME C OXIDASE SUBUNIT I (EC 1.9.3.1)
657 658 F RXA02480 GR00717 1449 601 CYTOCHROME C OXIDASE
POLYPEPTIDE I (EC 1.9.3.1) 659 660 F RXA02481 GR00717 1945 1334
CYTOCHROME C OXIDASE POLYPEPTIDE I (EC 1.9.3.1) 661 662 RXA02140
GR00639 7339 8415 CYTOCHROME C OXIDASE POLYPEPTIDE II (EC 1.9.3.1)
663 664 RXA02142 GR00639 9413 10063 CYTOCHROME C OXIDASE
POLYPEPTIDE I (EC 1.9.3.1) 665 666 RXA02144 GR00639 11025 12248
RIESKE IRON-SULFUR PROTEIN 667 668 RXA02740 GR00763 7613 8542
PROBABLE CYTOCHROME C OXIDASE ASSEMBLY FACTOR 669 670 RXA02743
GR00763 13534 12497 CYTOCHROME AA3 CONTROLLING PROTEIN 671 672
RXA01227 GR00355 1199 1519 FERREDOXIN 673 674 RXA01865 GR00532 436
122 FERREDOXIN 675 676 RXA00680 GR00179 2632 2315 FERREDOXIN VI 677
678 RXA00679 GR00179 2302 1037 FERREDOXIN-NAD(+) REDUCTASE (EC
1.18.1.3) 679 680 RXA00224 GR00032 24965 24015 ELECTRON TRANSFER
FLAVOPROTEIN ALPHA-SUBUNIT 681 682 RXA00225 GR00032 25783 24998
ELECTRON TRANSFER FLAVOPROTEIN BETA-SUBUNIT 683 684 RXN00606 VV0192
11299 9026 NADH DEHYDROGENASE I CHAIN L (EC 1.6.5.3) 685 686 F
RXA00606 GR00160 121 1869 NADH DEHYDROGENASE I CHAIN L (EC 1.6.5.3)
687 688 RXN00595 VV0192 8642 7113 NADH DEHYDROGENASE I CHAIN M (EC
1.6.5.3) 689 690 F RXA00608 GR00160 2253 3017 NADH DEHYDROGENASE I
CHAIN M (EC 1.6.5.3) 691 692 RXA00913 GR00249 3 2120 NADH
DEHYDROGENASE I CHAIN L (EC 1.6.5.3) 693 694 RXA00909 GR00247 2552
3406 NADH DEHYDROGENASE I CHAIN L (EC 1.6.5.3) 695 696 RXA00700
GR00182 846 43 NADH-UBIQUINONE OXIDOREDUCTASE CHAIN 2 697 698
RXN00483 VV0086 44824 46287 NADH-UBIQUINONE OXIDOREDUCTASE 39 KD
SUBUNIT PRECURSOR (EC 1.6.5.3) (EC 1.6.99.3) 699 700 F RXA00483
GR00119 19106 20569 NADH-UBIQUINONE OXIDOREDUCTASE 39 KD SUBUNIT
PRECURSOR (EC 1.6.5.3) (EC 1.6.99.3) 701 702 RXA01534 GR00427 1035
547 NADH-DEPENDENT FMN OXYDOREDUCTASE 703 704 RXA00288 GR00046 2646
1636 QUINONE OXIDOREDUCTASE (EC 1.6.5.5) 705 706 RXA02741 GR00763
9585 8620 QUINONE OXIDOREDUCTASE (EC 1.6.5.5) 707 708 RXN02560
VV0101 9922 10788 NADPH-FLAVIN OXIDOREDUCTASE (EC 1.6.99.--) 709
710 F RXA02560 GR00731 6339 7160 NADPH-FLAVIN OXIDOREDUCTASE (EC
1.6.99.--) 711 712 RXA01311 GR00380 1611 865 SUCCINATE
DEHYDROGENASE IRON-SULFUR PROTEIN (EC 1.3.99.1) 713 714 RXN03014
VV0058 1273 368 NADH DEHYDROGENASE I CHAIN M (EC 1.6.5.3) 715 716 F
RXA00910 GR00248 3 1259 Hydrogenase subunits 717 718 RXN01895
VV0117 955 5 NADH DEHYDROGENASE (EC 1.6.99.3) 719 720 F RXA01895
GR00543 2 817 DEHYDROGENASE 721 722 RXA00703 GR00183 2556 271
FORMATE DEHYDROGENASE ALPHA CHAIN (EC 1.2.1.2) 723 724 RXN00705
VV0005 6111 5197 FDHD PROTEIN 725 726 F RXA00705 GR00184 1291 407
FDHD PROTEIN 727 728 RXN00388 VV0025 2081 3091 CYTOCHROME C
BIOGENESIS PROTEIN CCSA 729 730 F RXA00388 GR00085 969 667
essential protein similar to cytochrome c 731 732 F RXA00386
GR00084 514 5 RESC PROTEIN, essential protein similar to cytochrome
c biogenesis protein 733 734 RXA00945 GR00259 1876 2847 putative
cytochrome oxidase 735 736 RXN02556 VV0101 5602 6759
FLAVOHEMOPROTEIN/DIHYDROPTERIDINE REDUCTASE (EC 1.6.99.7) 737 738 F
RXA02556 GR00731 2019 3176 FLAVOHEMOPROTEIN 739 740 RXA01392
GR00408 2297 3373 GLUTATHIONE S-TRANSFERASE (EC 2.5.1.18) 741 742
RXA00800 GR00214 2031 3134 GLUTATHIONE-DEPENDENT FORMALDEHYDE
DEHYDROGENASE (EC 1.2.1.1) 743 744 RXA02143 GR00639 10138 11025
QCRC PROTEIN, menaquinol: cytochrome c oxidoreductase 745 746
RXN03096 VV0058 405 4 NADH DEHYDROGENASE I CHAIN M (EC 1.6.5.3) 747
748 RXN02036 VV0176 32683 33063 NADH-UBIQUINONE OXIDOREDUCTASE
CHAIN 4 (EC 1.6.5.3) 749 750 RXN02765 VV0317 3552 2794 Hypothetical
Oxidorductase 751 752 RXN02206 VV0302 1784 849 Hypothetical
Oxidoreductase 753 754 RXN02554 VV0101 4633 4010 Hypothetical
Oxidoreductase (EC 1.1.1.--) ATP-Synthase 755 756 RXN01204 VV0121
1270 461 ATP SYNTHASE A CHAIN (EC 3.6.1.34) 757 758 F RXA01204
GR00345 394 1155 ATP SYNTHASE A CHAIN (EC 3.6.1.34) 759 760
RXA01201 GR00344 675 2315 ATP SYNTHASE ALPHA CHAIN (EC 3.6.1.34)
761 762 RXN01193 VV0175 5280 3832 ATP SYNTHASE BETA CHAIN (EC
3.6.1.34) 763 764 F RXA01193 GR00343 15 755 ATP SYNTHASE BETA CHAIN
(EC 3.6.1.34) 765 766 F RXA01203 GR00344 3355 3993 ATP SYNTHASE
BETA CHAIN (EC 3.6.1.34) 767 768 RXN02821 VV0121 324 85 ATP
SYNTHASE C CHAIN (EC 3.6.1.34) 769 770 F RXA02821 GR00802 139 318
ATP SYNTHASE C CHAIN (EC 3.6.1.34) 771 772 RXA01200 GR00344 2 610
ATP SYNTHASE DELTA CHAIN (EC 3.6.1.34) 773 774 RXA01194 GR00343 770
1141 ATP SYNTHASE EPSILON CHAIN (EC 3.6.1.34) 775 776 RXA01202
GR00344 2375 3349 ATP SYNTHASE GAMMA CHAIN (EC 3.6.1.34) 777 778
RXN02434 VV0090 4923 3274 ATP-BINDING PROTEIN Cytochrome metabolism
779 780 RXN00684 VV0005 29864 28581 CYTOCHROME P450 116 (EC
1.14.--.--) 781 782 RXN00387 VV0025 1150 2004 Hypothetical
Cytochrome c Biogenesis Protein
[0179] TABLE-US-00002 TABLE 2 GENES IDENTIFIED FROM GENBANK GenBank
.TM. Accession No. Gene Name Gene Function Reference A09073 ppg
Phosphoenol Bachmann, B. et al. "DNA fragment coding for
phosphoenolpyruvat pyruvate corboxylase, recombinant DNA carrying
said fragment, strains carrying the carboxylase 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 Wehmeier, L. et al. "The role of the
Corynebacterium glutamicum rel gene in protein; adenine (p)ppGpp
metabolism," Microbiology, 144: 1853-1862 (1998)
phosphoribosyltransferase; GTP pyrophosphokinase AF041436 argR
Arginine repressor AF045998 impA Inositol monophosphate phosphatase
AF048764 argH Argininosuccinate lyase AF049897 argC; argJ; argB;
N-acetylglutamylphosphate argD; argF; argR; reductase; ornithine
argG; argH acetyltransferase; N- 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 Park, S.
et al. "Isolation and analysis of metA, a methionine biosynthetic
gene O-acetyltransferase 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- Dusch, N. et al.
"Expression of the Corynebacterium glutamicum panD gene alpha-
encoding L-aspartate-alpha-decarboxylase leads to pantothenate
decarboxylase overproduction in Escherichia coli," Appl. Environ.
Microbiol., 65(4)1530-1539 precursor (1999) AF124518 aroD; aroE
3-dehydroquinase; shikimate dehydrogenase AF124600 aroC; aroK;
aroB; Chorismate synthase; pepQ shikimate kinase; 3- dehydroquinate
synthase; putative cytoplasmic peptidase AF145897 inhA AF145898
inhA AJ001436 ectP Transport of ectoine, Peter, H. et al.
"Corynebacterium glutamicum is equipped with four secondary glycine
betaine, carriers for compatible solutes: Identification,
sequencing, and characterization proline 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 Wehrmann, A. et al. "Different modes of
diaminopimelate synthesis and their succinylase role in cell wall
integrity: A study with Corynebacterium glutamicum," J.
(incomplete.sup.1) Bacteriol., 180(12): 3159-3165 (1998) AJ007732
ppc; secG; Phosphoenolpyruvate- amt; ocd; soxA carboxylase; ?; high
affinity ammonium uptake protein; putative ornithine-
cyclodecarboxylase; sarcosine oxidase AJ010319 ftsY, glnB, Involved
in cell division; Jakoby, M. et al. "Nitrogen regulation in
Corynebacterium glutamicum; glnD; srp; amtP PII protein; Isolation
of genes involved in biochemical characterization of corresponding
uridylyltransferase (uridylyl- proteins," FEMS Microbiol., 173(2):
303-310 (1999) removing enzmye); signal recognition particle; low
affinity ammonium uptake protein AJ132968 cat Chloramphenicol
aceteyl transferase AJ224946 mqo L-malate: Molenaar, D. et al.
"Biochemical and genetic characterization of the quinone
oxidoreductase 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) DI7429 Transposable Vertes, A. A. et al.
"Isolation and characterization of IS31831, a transposable element
IS31831 element from Corynebacterium glutamicum," Mol. Microbiol.,
11(4): 739-746 (1994) D84102 odhA 2-oxoglutarate Usuda, Y. et al.
"Molecular cloning of the Corynebacterium glutamicum dehydrogenase
(Brevibacterium lactofermentum AJ12036) odhA gene encoding a novel
type of 2-oxoglutarate dehydrogenase," Microbiology, 142: 3347-3354
(1996) E01358 hdh; hk Homoserine Katsumata, R. et al. "Production
of L-thereonine and L-isoleucine," Patent: JP dehydrogenase;
1987232392-A 1 Oct. 12, 1987 homoserine kinase E01359 Upstream of
the start Katsumata, R. et al. "Production of L-thereonine and
L-isoleucine," Patent: JP codon of homoserine 1987232392-A 2 Oct.
12, 1987 kinase gene E01375 Tryptophan operon E01376 trpL; trpE
Leader peptide; Matsui, K. et al. "Tryptophan operon, peptide and
protein coded thereby, anthranilate synthase utilization of
tryptophan operon gene expression and production of tryptophan,"
Patent: JP 1987244382-A 1 Oct. 24, 1987 E01377 Promoter and Matsui,
K. et al. "Tryptophan operon, peptide and protein coded thereby,
operator regions of utilization of tryptophan operon gene
expression and production of tryptophan operon 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 Kohama, K. et al. "Gene coding
diaminopelargonic acid aminotransferase and acid aminotransferase
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 E04377 Isocitric acid lyase Katsumata,
R. et al. "Gene manifestation controlling DNA," Patent: JP
N-terminal fragment 1993056782-A 3 Mar. 09, 1993 E04484 Prephenate
Sotouchi, N. et al. "Production of L-phenylalanine by
fermentation," Patent: JP dehydratase 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 Hatakeyama, K. et al. "Gene DNA coding
dihydrodipicolinic acid synthetase synthetase and its use," Patent:
JP 1993184371-A 1 Jul. 27, 1993 E05776 Diaminopimelic acid
Kobayashi, M. et al. "Gene DNA coding Diaminopimelic acid
dehydrogenase 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 Kikuchi, T. et al. "Production of
L-phenylalanine by fermentation method," dehydratase Patent: JP
1993344881-A 1 Dec. 27, 1993 E06111 Mutated Prephenate Kikuchi, T.
et al. "Production of L-phenylalanine by fermentation method,"
dehydratase Patent: JP 1993344881-A 1 Dec. 27, 1993 E06146
Acetohydroxy acid Inui, M. et al. "Gene capable of coding
Acetohydroxy acid synthetase and its synthetase 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 Sugimoto, M. et al. "Mutant
aspartokinase gene," patent: JP 1994062866-A 1 alpha subunit Mar.
08, 1994 E06827 Mutated aspartokinase Sugimoto, M. et al. "Mutant
aspartokinase gene," patent: JP 1994062866-A 1 alpha subunit 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- Sato, Y. et al. "Genetic DNA capable of coding
Aspartokinase released from released Aspartokinase E08179, feedback
inhibition and its utilization," Patent: JP 1994261766-A 1 Sep. 20,
1994 E08180, E08181, E08182 E08232 Acetohydroxy-acid Inui, M. et
al. "Gene DNA coding acetohydroxy acid isomeroreductase,"
isomeroreductase Patent: JP 1994277067-A 1 Oct. 04, 94 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 Hatakeyama, K. et al. "DNA fragment having
promoter function in and desthiobiotin coryneform bacterium,"
Patent: JP 1995031476-A 1 Feb. 03, 1995 synthetase promoter region
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 Madori, M.
et al. "DNA fragment containing gene coding Dihydrodipicolinate
reductase acid reductase and utilization thereof," Patent: JP
1995075578-A 1 Mar. 20, 1995 E08901 Diaminopimelic acid Madori, M.
et al. "DNA fragment containing gene coding Diaminopimelic acid
decarboxylase decarboxylase and utilization thereof," Patent: JP
1995075579-A 1 Mar. 20, 1995 E12594 Serine Hatakeyama, K. et al.
"Production of L-trypophan," Patent: JP 1997028391-A
hydroxymethyltransferase 1 Feb. 04, 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 Moriya, M. et al. "Amplification of gene using
artificial transposon," Patent: synthetase; diaminopimelic JP
1997070291-A Mar. 18, 1997 acid decarboxylase E12767
Dihydrodipicolinic Moriya, M. et al. "Amplification of gene using
artificial transposon," Patent: acid synthetase 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 Moriya, M. et al. "Amplification
of gene using artificial transposon," Patent: acid reductase JP
1997070291-A Mar. 18, 1997 E13655 Glucose-6-phosphate Hatakeyama,
K. et al. "Glucose-6-phosphate dehydrogenase and DNA capable
dehydrogenase 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- Chen, C. et al. "The cloning and
nucleotide sequence of Corynebacterium arabinoheptulosonate-7-
glutamicum 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase
gene," phosphate synthase FEMS Microbiol. Lett., 107: 223-230
(1993) L09232 IlvB; ilvN; ilvC Acetohydroxy acid Keilhauer, C. et
al. "Isoleucine synthesis in Corynebacterium glutamicum: synthase
large subunit; molecular analysis of the ilvB-ilvN-ilvC operon," J.
Bacteriol., 175(17): 5595-5603 Acetohydroxy acid (1993) synthase
small subunit; Acetohydroxy acid isomeroreductase L18874 PtsM
Phosphoenolpyruvate Fouet, A et al. "Bacillus subtilis
sucrose-specific enzyme II of the sugar phosphotransferase system:
expression in Escherichia coli and homology to phosphotransferase
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 Oguiza, J. A. et al. "Molecular cloning, DNA
sequence analysis, and repressor characterization of the
Corynebacterium diphtheriae dtxR from Brevibacterium
lactofermentum," J. Bacteriol., 177(2): 465-467 (1995) M13774
Prephenate Follettie, M. T. et al. "Molecular cloning and
nucleotide sequence of the dehydratase 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, Sano, K. et al. "Structure and function of
the trp operon control regions of 5' end Brevibacterium
lactofermentum, a glutamic-acid-producing bacterium," Gene, 52:
191-200 (1987) M16664 trpA Tryptophan synthase, Sano, K. et al.
"Structure and function of the trp operon control regions of 3'end
Brevibacterium lactofermentum, a glutamic-acid-producing
bacterium," Gene, 52: 191-200 (1987) M25819 Phosphoenolpyruvate
O'Regan, M. et al. "Cloning and nucleotide sequence of the
carboxylase Phosphoenolpyruvate carboxylase-coding gene of
Corynebacterium glutamicum ATCC13032," Gene, 77(2): 237-251 (1989)
M85106 23S rRNA gene Roller, C. et al. "Gram-positive bacteria with
a high DNA G + C content are insertion sequence characterized by a
common insertion within their 23S rRNA genes," J. Gen. Microbiol.,
138: 1167-1175 (1992) M85107, 23S rRNA gene Roller, C. et al.
"Gram-positive bacteria with a high DNA G + C content are M85108
insertion sequence characterized by a common insertion within their
23S rRNA genes," J. Gen. Microbiol., 138: 1167-1175 (1992) M89931
aecD; brnQ; Beta C-S lyase; Rossol, I. et al. "The Corynebacterium
glutamicum aecD gene encodes a C-S yhbw branched-chain lyase with
alpha, beta-elimination activity that degrades aminoethylcysteine,"
amino acid uptake J. Bacteriol., 174(9): 2968-2977 (1992); Tauch,
A. et al. "Isoleucine uptake in carrier; hypothetical
Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene
protein yhbw product," Arch. Microbial., 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. Microbial., 59(3): 791-799 (1993)
U11545 trpD Anthranilate O'Gara, J. P. and Dunican, L. K. (1994)
Complete nucleotide sequence of the phosphoribosyltransferase
Corynebacterium glutamicum ATCC 21850 tpD gene." Thesis,
Microbiology Department, University College Galway, Ireland. U13922
cglIM; cglIR; Putative type II Schafer, A. et al. "Cloning and
characterization of a DNA region encoding a clgIIR 5-cytosoine
stress-sensitive restriction system from Corynebacterium glutamicum
ATCC methyltransferase; putative 13032 and analysis of its role in
intergeneric conjugation with Escherichia type II restriction
coli," J. Bacteriol., 176(23): 7309-7319 (1994); Schafer, A. et al.
"The endonuclease; Corynebacterium glutamicum cglIM gene encoding a
5-cytosine in an McrBC- putative type I or type deficient
Escherichia coli strain," Gene, 203(2): 95-101 (1997) III
restriction endonuclease U14965 recA U31224 ppx Ankri, S. et al.
"Mutations in the Corynebacterium glutamicumproline biosynthetic
pathway: A natural bypass of the proA step," J. Bacteriol.,
178(15): 4412-4419 (1996) U31225 proC L-proline: Ankri, S. et al.
"Mutations in the Corynebacterium glutamicumproline NADP+ 5-
biosynthetic pathway: A natural bypass of the proA step," J.
Bacteriol., oxidoreductase 178(15): 4412-4419 (1996) U31230 obg;
proB; unkdh ?; gamma Ankri, S. et al. "Mutations in the
Corynebacterium glutamicumproline glutamyl kinase; similar to
biosynthetic pathway: A natural bypass of the proA step," J.
Bacteriol., D-isomer specific 178(15): 4412-4419 (1996)
2-hydroxyacid dehydrogenases 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 Jager, W. et al.
"A Corynebacterium glutamicum gene encoding a two-domain
sulfurtransferase; acyl protein similar to biotin carboxylases and
biotin-carboxyl-carrier proteins," CoA carboxylase Arch. Microbiol,
166(2); 76-82 (1996) U43535 cmr Multidrug resistance Jager, W. et
al. "A Corynebacterium glutamicum gene conferring multidrug protein
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; Tryptophan operon Matsui, K. et al.
"Complete nucleotide and deduced amino acid sequences of trpD;
trpE; the Brevibacterium lactofermentum tryptophan operon," Nucleic
Acids Res., trpG; trpL 14(24): 10113-10114 (1986) X07563 lys A DAP
decarboxylase Yeh, P. et al. "Nucleic sequence of the lysA gene of
Corynebacterium (meso-diaminopimelate glutamicum and possible
mechanisms for modulation of its expression," Mol. decarboxylase,
EC 4.1.1.20) Gen. Genet., 212(1): 112-119 (1988) X14234 EC 4.1.1.31
Phosphoenolpyruvate Eikmanns, B. J. et al. "The Phosphoenolpyruvate
carboxylase gene of carboxylase 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 Von der Osten, C. H. et al.
"Molecular cloning, nucleotide sequence and fine- aldolase
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 Bonnassie, S. et al. "Nucleic
sequence of the dapA gene from synthetase Corynebacterium
glutamicum," Nucleic Acids Res., 18(21): 6421 (1990) (EC 4.2.1.52)
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 Marcel, T. et al.
"Nucleotide sequence and organization of the upstream region
synthetase; of the Corynebacterium glutamicum lysA gene," Mol.
Microbiol., 4(11): 1819-1830 Diaminopimelate (1990) decarboxylase
X55994 trpL; trpE Putative leader Heery, D. M. et al. "Nucleotide
sequence of the Corynebacterium glutamicum peptide; anthranilate
trpE gene," Nucleic Acids Res., 18(23): 7138 (1990) synthase
component 1 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 attB-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; Aspartokinase-alpha
Kalinowski, J. et al. "Genetic and biochemical analysis of the
Aspartokinase lysC-beta; subunit; from Corynebacterium glutamicum,"
Mol. Microbiol., 5(5): 1197-1204 (1991); asd Aspartokinase-beta
Kalinowski, J. et al. "Aspartokinase genes lysC alpha and lysC beta
overlap subunit; aspartate and are adjacent to the aspertate
beta-semialdehyde dehydrogenase gene asd in beta semialdehyde
Corynebacterium glutamicum," Mol. Gen. Genet., 224(3): 317-324
(1990) dehydrogenase X59403 gap; pgk; tpi Glyceraldehyde-3-
Eikmanns, B. J. "Identification, sequence analysis, and expression
of a phosphate; Corynebacterium glutamicum gene cluster encoding
the three glycolytic phosphoglycerate enzymes
glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate
kinase; kinase, and triosephosphate isomeras," J. Bacteriol.,
174(19): 6076-6086 triosephosphate (1992) isomerase X59404 gdh
Glutamate Bormann, E. R. et al; "Molecular analysis of the
Corynebacterium glutamicum dehydrogenase gdh gene encoding
glutamate dehydrogenase," Mol. Microbiol., 6(3): 317-326 (1992)
X60312 lysl L-lysine permease Seep-Feldhaus, A. H. et al.
"Molecular analysis of the Corynebacterium glutamicum lysl 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 Peyret, J. L. et al. "Characterization
of the cspB gene encoding PS2, an ordered PS2 surface-layer protein
in Corynebacterium glutamicum," Mol. Microbiol., 9(1): 97-109
(1993) X69104 IS3 related insertion Bonamy, C. et al.
"Identification of IS1206, a Corynebacterium glutamicum element
IS3-related insertion sequence and phylogenetic analysis," Mol.
Microbiol., 14(3): 571-581 (1994) X70959 leuA Isopropylmalate
Patek, M. et al. "Leucine synthesis in Corynebacterium glutamicum:
enzyme synthase activities, structure of leuA, and effect of leuA
inactivation on lysine synthesis," Appl. Environ. Microbiol.,
60(1): 133-140 (1994) X71489 icd Isocitrate Eikmanns, B. J. et al.
"Cloning sequence analysis, expression, and inactivation
dehydrogenase of the Corynebacterium glutamicum icd gene encoding
isocitrate (NADP+) dehydrogenase and biochemical characterization
of the enzyme," J. Bacteriol., 177(3): 774-782 (1995) X72855 GDHA
Glutamate dehydrogenase (NADP+) X75083, mtrA 5-methyltryptophan
Heery, D. M. et al. "A sequence from a tryptophan-hyperproducing
strain of resistance 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 Reinscheid, D. J. et al. "Characterization
of the isocitrate lyase gene from lyase; ? 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
Kronemeyer, W. et al. "Structure of the gluABCD cluster encoding
the gluD system glutamate uptake system of Corynebacterium
glutamicum," J. Bacteriol., 177(5): 1152-1158 (1995) X81379 dapE
Succinyldiaminopimelate Wehrmann, A. et al. "Analysis of different
DNA fragments of desuccinylase 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
Serebrijski, I. et al. "Multicopy suppression by asd gene and
osmotic stress- dehydrogenase; ? dependent complementation by
heterologous proA in proA mutants," J. Bacteriol., 177(24):
7255-7260 (1995) X82929 proA Gamma-glutamyl Serebrijski, I. et al.
"Multicopy suppression by asd gene and osmotic stress- phosphate
reductase 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 Wehrmann, A. et al. "Functional analysis of sequences
adjacent to dapE of permease;? Corynebacterium glutamicumproline
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; Sakanyan, V. et al. "Genes and
enzymes of the acetyl cycle of arginine argF; argJ N-acetyl-gamma-
biosynthesis in Corynebacterium glutamicum: enzyme evolution in the
early glutamyl-phosphate steps of the arginine pathway,"
Microbiology, 142: 99-108 (1996) reductase;acetylornithine
aminotransferase; ornithine carbamoyltransferase; glutamate N-
acetyltransferase X89084 pta; ackA Phosphate Reinscheid, D. J. et
al. "Cloning, sequence analysis, expression and inactivation
acetyltransferase; of the Corynebacterium glutamicum pta-ack operon
encoding acetate kinase 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 Siewe, R. M. et al. "Functional and genetic
characterization of the (methyl) system ammonium uptake carrier of
Corynebacterium glutamicum," J. Biol. Chem., 271(10): 5398-5403
(1996) X93514 betP Glycine betaine Peter, H. et al. "Isolation,
characterization, and expression of the transport system
Corynebacterium glutamicumbetP 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 Vrljic, M. et al. "A new type of
transporter with a new type of cellular protein; Lysine export
function: L-lysine export from Corynebacterium glutamicum," Mol.
regulator protein Microbiol., 22(5): 815-826 (1996) X96580 panB;
panC; xylB 3-methyl-2- Sahm, H. et al. "D-pantothenate synthesis in
Corynebacterium glutamicum and oxobutanoate use of panBC and genes
encoding L-valine synthesis for D-pantothenate
hydroxymethyltransferase; overproduction," Appl. Environ.
Microbial., 65(5): 1973-1979 (1999) pantoate-beta-alanine ligase;
xylulokinase 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 Ishino, S. et al. "Nucleotide sequence of the
meso-diaminopimelate D- D-dehydrogenase dehydrogenase gene from
Corynebacterium glutamicum," Nucleic Acids Res., (EC 1.4.1.16)
15(9): 3917 (1987) Y00476 thrA Homoserine Mateos, L. M. et al.
"Nucleotide sequence of the homoserine dehydrogenase dehydrogenase
(thrA) gene of the Brevibacterium lactofermentum," Nucleic Acids
Res., 15(24): 10598 (1987) Y00546 hom; thrB Homoserine Peoples, O.
P. et al. "Nucleotide sequence and fine structural analysis of the
dehydrogenase; Corynebacterium glutamicum hom-thrB operon," Mol.
Microbiol., 2(1): 63-72 homoserine kinase (1988) Y08964 murC;
UPD-N-acetylmuramate- Honrubia, M. P. et al. "Identification,
characterization, and chromosomal ftsQ/divD; ftsZ alanine ligase;
organization of the ftsZ gene from Brevibacterium lactofermentum,"
Mol. Gen. division initiation protein Genet., 259(1): 97-104 (1998)
or cell division protein; cell division protein Y09163 putP High
affinity proline Peter, H. et al. "Isolation of the putP gene of
Corynebacterium transport system 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 Patek, M. et al.
"Analysis of the leuB gene from Corynebacterium dehydrogenase
glutamicum," Appl. Microbial. Biotechnol., 50(1): 42-47 (1998)
Y12472 Attachment site Moreau, S. et al. "Site-specific integration
of corynephage Phi-16: The bacteriophage Phi-16 construction of an
integration vector," Microbial., 145: 539-548 (1999) Y12537 proP
Proline/ectoine Peter, H. et al. "Corynebacterium glutamicum is
equipped with four secondary uptake system protein 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 Moreau, S. et al. "Analysis of the
integration functions of φ 304L: An Corynephage 304L integrase
module among corynephages," Virology, 255(1): 150-159 (1999) Z21501
argS; lysA Arginyl-tRNA Oguiza, J. A. et al. "A gene encoding
arginyl-tRNA synthetase is located in the synthetase;
diaminopimelate upstream region of the lysA gene in Brevibacterium
lactofermentum: decarboxylase (partial) Regulation of argS-lysA
cluster expression by arginine," J. Bacteriol., 175(22): 7356-7362
(1993) Z21502 dapA; dapB Dihydrodipicolinate Pisabarro, A. et al.
"A cluster of three genes (dapA, orf2, and dapB) of synthase;
Brevibacterium lactofermentum encodes dihydrodipicolinate
reductase, and a dihydrodipicolinate third polypeptide of unknown
function," J. Bacteriol., 175(9): 2743-2749 reductase (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 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 Oguiza,
J. A. et al "The galE gene encoding the UDP-galactose 4-epimerase
of UDP-galactose 4- Brevibacterium lactofermentum is coupled
transcriptionally to the dmdR epimerase; diphtheria gene," Gene,
177: 103-107 (1996) toxin regulatory protein Z49824 orf1; 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.
[0180] TABLE-US-00003 TABLE 3 Corynebacterium and Brevibacterium
Strains Which May be Used in the Practice of the Invention Genus
species ATCC FERM NRRL GECT 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.
[0181] TABLE-US-00004 TABLE 4 ALIGNMENT RESULTS % length homology
ID # (NT) Genbank Hit Length Accession Name of Genbank Hit Source
of Genbank Hit (GAP) Date of Deposit rxa00013 996 GB_GSS4: AQ713475
581 AQ713475 HS_5402_B2_A12_T7A RPCI-11 Human Male BAC Homo sapiens
37,148 13-Jul-99 Library Homo sapiens genomic clone Plate = 978 Col
= 24 Row = B, genomic survey sequence. GB_HTG3: AC007420 130583
AC007420 Drosophila melanogaster chromosome 2 clone BACR07M10
(D630) RPCI-98 Drosophila melanogaster 34,568 20-Sep-99 07.M.10 map
24A-24D strain y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 83
unordered pieces. GB_HTG3: AC007420 130583 AC007420 Drosophila
melanogaster chromosome 2 clone BACR07M10 (D630) RPCI-98 Drosophila
melanogaster 34,568 20-Sep-99 07.M.10 map 24A-24D strain y; cn bw
sp, *** SEQUENCING IN PROGRESS***, 83 unordered pieces. rxa00014
903 GB_BA1: MTCY3A2 25830 Z83867 Mycobacterium tuberculosis H37Rv
complete genome; segment 136/162. Mycobacterium tuberculosis 58,140
17-Jun-98 GB_BA1: MLCB1779 43254 Z98271 Mycobacterium leprae cosmid
B1779. Mycobacterium leprae 57,589 8-Aug-97 GB_BA1: SAPURCLUS 9120
X92429 S. alboniger napH, pur7, pur10, pur6, pur4, Streptomyces
anulatus 55,667 28-Feb-96 pur5 and pur3 genes. rxa00030 513
GB_EST21: C89713 767 C89713 C89713 Dictyostelium discoideum
Dictyostelium discoideum 45,283 20-Apr-98 SS (H. Urushihara)
Dictyostelium discoideum cDNA clone SSG229, mRNA sequence.
GB_EST28: AI497294 484 AI497294 fb63g03.y1 Zebrafish WashU MPIMG
EST Danio rerio cDNA 5' similar to Danio rerio 42,991 11-MAR-1999
SW: AFP4_MYOOC P80961 ANTIFREEZE PROTEIN LS-12.;, mRNA sequence.
GB_EST21: C92167 637 C92167 C92167 Dictyostelium discoideum SS (H.
Urushihara) Dictyostelium discoideum 44,444 12-Jul-99 Dictyostelium
discoideum cDNA clone SSD179, mRNA sequence. rxa00032 1632 GB_BA2:
AF010496 189370 AF010496 Rhodobacter capsulatus strain SB1003,
partial genome. Rhodobacter capsulatus 39,689 12-MAY-1998 GB_BA2:
AF018073 9810 AF018073 Rhodobacter sphaeroides operon regulator
(smoC), periplasmic sorbitol-binding Rhodobacter sphaeroides 48,045
22-OCT-1997 protein (smoE), sorbitol/mannitol transport inner
membrane protein (smoF), sorbitol/mannitol transport inner membrane
protein (smoG), sorbitol/mannitol transport ATP-binding transport
protein (smoK), sorbitol dehydrogenase (smoS), mannitol
dehydrogenase (mtlK), and periplasmic mannitol-binding protein
(smoM) genes, complete cds. GB_BA2: AF045245 5930 AF045245
Klebsiella pneumoniae D-arabinitol transporter Klebsiella
pneumoniae 38,514 16-Jul-98 (dalT), D-arabinitol kinase (dalK), D-
arabinitol dehydrogenase (dalD), and repressor (dalR) genes,
complete cds. rxa00041 1342 EM_PAT: E11760 6911 E11760 Base
sequence of sucrase gene. Corynebacterium glutamicum 99,031
08-OCT-1997 (Rel. 52, Created) GB_PAT: I26124 6911 I26124 Sequence
4 from patent U.S. Pat. No. 5556776. Unknown. 99,031 07-OCT-1996
GB_IN1: LMFL5883 31934 AL117384 Leishmania major Friedlin
chromosome 23 cosmid L5883, complete sequence. Leishmania major
43,663 21-OCT-1999 rxa00042 882 EM_PAT: E11760 6911 E11760 Base
sequence of sucrase gene. Corynebacterium glutamicum 94,767
08-OCT-1997 (Rel. 52, Created) GB_PAT: I26124 6911 I26124 Sequence
4 from patent U.S. Pat. No. 5556776. Unknown. 94,767 07-OCT-1996
GB_IN1: CEU33051 4899 U33051 Caenorhabditis elegans sur-2 mRNA,
complete cds. Caenorhabditis elegans 40,276 23-Jan-96 rxa00043 1287
GB_PAT: I26124 6911 I26124 Sequence 4 from patent U.S. Pat. No.
5556776. Unknown. 97,591 07-OCT-1996 EM_PAT: E11760 6911 E11760
Base sequence of sucrase gene. Corynebacterium glutamicum 97,591
08-OCT-1997 (Rel. 52, Created) GB_PR3: AC005174 39769 AC005174 Homo
sapiens clone UWGC: g1564a012 from 7p14-15, complete sequence. Homo
sapiens 35,879 24-Jun-98 rxa00098 1743 GB_BA1: MSU88433 1928 U88433
Mycobacterium smegmatis phosphoglucose isomerase gene, complete
cds. Mycobacterium smegmatis 62,658 19-Apr-97 GB_BA1: SC5A7 40337
AL031107 Streptomyces coelicolor cosmid 5A7. Streptomyces
coelicolor 37,638 27-Jul-98 GB_BA1: MTCY10D7 39800 Z79700
Mycobacterium tuberculosis H37Rv complete genome; segment 44/162.
Mycobacterium tuberculosis 36,784 17-Jun-98 rxa00148 2334 GB_BA1:
MTCY277 38300 Z79701 Mycobacterium tuberculosis H37Rv complete
genome; segment 65/162. Mycobacterium tuberculosis 67,457 17-Jun-98
GB_BA1: MSGY456 37316 AD000001 Mycobacterium tuberculosis sequence
from clone y456. Mycobacterium tuberculosis 40,883 03-DEC-1996
GB_BA1: MSGY175 18106 AD000015 Mycobacterium tuberculosis sequence
from clone y175. Mycobacterium tuberculosis 67,457 10-DEC-1996
rxa00149 1971 GB_BA1: MSGY456 37316 AD000001 Mycobacterium
tuberculosis sequence from clone y456. Mycobacterium tuberculosis
35,883 03-DEC-1996 GB_BA1: MSGY175 18106 AD000015 Mycobacterium
tuberculosis sequence from clone y175. Mycobacterium tuberculosis
51,001 10-DEC-1996 GB_BA1: MTCY277 38300 Z79701 Mycobacterium
tuberculosis H37Rv complete genome; segment 65/162. Mycobacterium
tuberculosis 51,001 17-Jun-98 rxa00195 684 GB_BA1: MTCY274 39991
Z74024 Mycobacterium tuberculosis H37Rv complete genome; segment
126/162. Mycobacterium tuberculosis 35,735 19-Jun-98 GB_BA1:
MSGB1529CS 36985 L78824 Mycobacterium leprae cosmid B1529 DNA
sequence. Mycobacterium leprae 57,014 15-Jun-96 GB_BA1: MTCY274
39991 Z74024 Mycobacterium tuberculosis H37Rv complete genome;
segment 126/162. Mycobacterium tuberculosis 41,892 19-Jun-98
rxa00196 738 GB_BA1: MTCY274 39991 Z74024 Mycobacterium
tuberculosis H37Rv complete genome; segment 126/162. Mycobacterium
tuberculosis 41,841 19-Jun-98 GB_BA1: MTCY274 39991 Z74024
Mycobacterium tuberculosis H37Rv complete genome; segment 126/162.
Mycobacterium tuberculosis 36,599 19-Jun-98 GB_RO: RATCBRQ 10752
M55532 Rat carbohydrate binding receptor gene, complete cds. Rattus
norvegicus 36,212 27-Apr-93 rxa00202 1065 GB_EST11: AA253618 313
AA253618 mw95c10.r1 Soares mouse NML Mus musculus 38,816
13-MAR-1997 Mus musculus cDNA clone IMAGE: 678450 5', mRNA
sequence. GB_EST26: AI390284 490 AI390284 mw96a03.y1 Soares mouse
NML Mus musculus Mus musculus 42,239 2-Feb-99 cDNA clone IMAGE:
678508 5' similar to TR: O09171 O09171 BETAINE-HOMOCYSTEINE
METHYLTRANSFERASE;, mRNA sequence. GB_EST26: AI390280 467 AI390280
mw95c10.y1 Soares mouse NML Mus musculus 37,307 2-Feb-99 Mus
musculus cDNA clone IMAGE: 678450 5', mRNA sequence. rxa00206 1161
GB_BA1: MLCB637 44882 Z99263 Mycobacterium leprae cosmid B637.
Mycobacterium leprae 58,312 17-Sep-97 GB_BA1: MTV012 70287 AL021287
Mycobacterium tuberculosis H37Rv complete genome; segment 132/162.
Mycobacterium tuberculosis 36,632 23-Jun-99 GB_BA1: SC6E10 23990
AL109661 Streptomyces coelicolor cosmid 6E10. Streptomyces
coelicolor A3(2) 38,616 5-Aug-99 rxa00224 1074 GB_BA1: BJU32230
1769 U32230 Bradyrhizobium japonicum electron transfer
Bradyrhizobium japonicum 48,038 25-MAY-1996 flavoprotein small
subunit (etfS) nd large subunit (etfL) genes, complete cds. GB_BA1:
PDEETFAB 2440 L14864 Paracoccus denitrificans electron transfer
Paracoccus denitrificans 48,351 27-OCT-1993 flavoprotein alpha and
beta subunit genes, complete cds's. GB_HTG3: AC009689 177954
AC009689 Homo sapiens chromosome 4 clone 104_F_7 map 4, Homo
sapiens 38,756 28-Aug-99 LOW-PASS SEQUENCE SAMPLING. rxa00225 909
GB_RO: AF060178 2057 AF060178 Mus musculus heparan sulfate
2-sulfotransferase (Hs2st) mRNA, complete cds. Mus musculus 39,506
18-Jun-98 GB_GSS11: AQ325043 734 AQ325043 mgxb0020J01r CUGI Rice
Blast BAC Library Magnaporthe grisea 38,333 8-Jan-99 Magnaporthe
grisea genomic clone mgxb0020J01r, genomic survey sequence.
GB_EST31: AI676413 551 AI676413 etmEST0167 EtH1 Eimeria tenella
cDNA clone etmc074 5', mRNA sequence. Eimeria tenella 35,542
19-MAY-1999 rxa00235 1398 GB_BA1: MTCY10G2 38970 Z92539
Mycobacterium tuberculosis H37Rv complete genome; segment 47/162.
Mycobacterium tuberculosis 65,759 17-Jun-98 GB_BA2: AF061753 3721
AF061753 Nitrosomonas europaea CTP synthase (pyrG) gene,
Nitrosomonas europaea 58,941 31-Aug-98 partial cds; and enolase
(eno) gene, complete cds. GB_BA2: AF086791 37867 AF086791 Zymomonas
mobilis strain ZM4 clone 67E10 Zymomonas mobilis 61,239 4-Nov-98
carbamoylphosphate synthetase small 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. rxa00246 1158 GB_BA2:
AF012550 2690 AF012550 Acinetobacter sp. BD413 ComP (comP) gene,
complete cds. Acinetobacter sp. BD413 53,726 27-Sep-99 GB_PAT:
E03856 1506 E03856 gDNA encoding alcohol dehydrogenase. Bacillus
stearothermophilus 51,688 29-Sep-97 GB_BA1: BACADHT 1688 D90421 B.
stearothermophilus adhT gene for alcohol dehydrogenase. Bacillus
stearothermophilus 51,602 7-Feb-99 rxa00251 831 GB_BA1: MTCY20G9
37218 Z77162 Mycobacterium tuberculosis H37Rv complete genome;
segment 25/162. Mycobacterium tuberculosis 42,875 17-Jun-98 GB_BA1:
MTV004 69350 AL009198 Mycobacterium tuberculosis H37Rv complete
genome; segment 144/162. Mycobacterium tuberculosis 40,380
18-Jun-98 GB_BA1: MTV004 69350 AL009198 Mycobacterium tuberculosis
H37Rv complete genome; segment 144/162. Mycobacterium tuberculosis
41,789 18-Jun-98 rxa00288 1134 GB_BA2: AF050114 1038 AF050114
Pseudomonas sp. W7 alginate lyase gene, complete cds. Pseudomonas
sp. W7 49,898 03-MAR-1999 GB_GSS3: B16984 469 B16984 344A14.TVC
CIT978SKA1 Homo sapiens genomic clone A-344A14, Homo sapiens 39,355
4-Jun-98 genomic survey sequence. GB_IN2: AF144549 7887 AF144549
Aedes albopictus ribosomal protein L34 (rpl34) gene, complete cds.
Aedes albopictus 36,509 3-Jun-99 rxa00293 1035 GB_EST1: T28483 313
T28483 EST46182 Human Kidney Homo sapiens cDNA 3' end similar to
Homo sapiens 42,997 6-Sep-95 flavin-containing monooxygenase 1 (HT:
1956), mRNA sequence. GB_PR1: HUMFMO1 2134 M64082 Human
flavin-containing monooxygenase (FMO1) mRNA, complete cds. Homo
sapiens 37,915 8-Nov-94 GB_EST32: AI734238 512 AI734238 zb73c05.y5
Soares_fetal_lung_NbHL19W Homo sapiens cDNA clone Homo sapiens
41,502 14-Jun-99 IMAGE: 309224 5' similar to gb: M64082
DIMETHYLANILINE MONOOXYGENASE (HUMAN):, mRNA sequence. rxa00296
2967 GB_HTG6: AC011069 168266 AC011069 Drosophila melanogaster
chromosome X clone BACR11H20 (D881) RPCI-98 Drosophila melanogaster
33,890 02-DEC-1999 11.H.20 map 12B-12C strain y; cn bw sp, ***
SEQUENCING IN PROGRESS ***, 92 unordered pieces. GB_EST15: AA531468
414 AA531468 nj63d12.s1 NCI_CGAP_Pr10 Homo sapiens cDNA clone Homo
sapiens 40,821 20-Aug-97 IMAGE: 997175, mRNA sequence. GB_HTG6:
AC011069 168266 AC011069 Drosophila melanogaster chromosome X clone
BACR11H20 (D881) RPCI-98 Drosophila melanogaster 30,963 02-DEC-1999
11.H.20 map 12B-12C strain y; cn bw sp, *** SEQUENCING IN PROGRESS
***, 92 unordered pieces. rxa00310 558 GB_VI: VMVY16780 186986
Y16780 variola minor virus complete genome. variola minor virus
35,883 2-Sep-99 GB_VI: VARCG 186103 L22579 Variola major virus
(strain Bangladesh-1975) complete genome. Variola major virus
34,664 12-Jan-95 GB_VI: VVCGAA 185578 X69198 Variola virus DNA
complete genome. Variola virus 36,000 13-DEC-1996 rxa00317 777
GB_HTG3: AC009571 159648 AC009571 Homo sapiens chromosome 4 clone
57_A_22 map 4, *** SEQUENCING IN Homo sapiens 36,988 29-Sep-99
PROGRESS ***, 8 unordered pieces. GB_HTG3: AC009571 159648 AC009571
Homo sapiens chromosome 4 clone 57_A_22 map 4, *** SEQUENCING IN
Homo sapiens 36,988 29-Sep-99 PROGRESS ***, 8 unordered pieces.
GB_PR3: AC005697 174503 AC005697 Homo sapiens chromosome 17, clone
hRPK.138_P_22, complete sequence. Homo sapiens 36,340 09-OCT-1998
rxa00327 507 GB_BA1: LCATPASEB 1514 X64542 L. casei gene for ATPase
beta-subunit. Lactobacillus casel 34,664 11-DEC-1992 GB_BA1:
LCATPASEB 1514 X64542 L. casei gene for ATPase beta-subunit.
Lactobacillus casei 39,308 11-DEC-1992 rxa00328 615 GB_BA1:
STYPUTPE 1887 L01138 Salmonella (S2980) proline permease (putP)
gene, 5' end. Salmonella sp. 39,623 09-MAY-1996 GB_BA1: STYPUTPF
1887 L01139 Salmonella (S2983) proline permease (putP) gene, 5'
end. Salmonella sp. 39,623 09-MAY-1996 GB_BA1: STYPUTPI 1889 L01142
Salmonella (S3015) proline permease (putP) gene, 5' end. Salmonella
sp. 42,906 09-MAY-1996 rxa00329 1347 GB_PR3: AC004691 141990
AC004691 Homo sapiens PAC clone DJ0740D02 from 7p14-p15, complete
sequence. Homo sapiens 38,142 16-MAY-1998 GB_PR4: AC004916 129014
AC004916 Homo sapiens clone DJ0891L14, complete sequence. Homo
sapiens 38,549 17-Jul-99 GB_PR3: AC004691 141990 AC004691 Homo
sapiens PAC clone DJ0740D02 from 7p14-p15, complete sequence. Homo
sapiens 35,865 16-MAY-1998 rxa00340 1269 GB_BA1: MTCY427 38110
Z70692 Mycobacterium tuberculosis
H37Rv complete genome; segment 99/162. Mycobacterium tuberculosis
38,940 24-Jun-99 GB_GSS12: AQ412290 238 AQ412290 RPCI-11-195H2.TV
RPCI-11 Homo sapiens genomic clone RPCI-11-195H2, Homo sapiens
36,555 23-MAR-1999 genomic survey sequence. GB_PL2: AF112871 2394
AF112871 Astasia longa small subunit ribosomal RNA gene, complete
sequence. Astasia longa 36,465 28-Jun-99 rxa00379 307 GB_HTG1:
CEY56A3 224746 AL022280 Caenorhabditis elegans chromosome III clone
Y56A3, *** SEQUENCING IN Caenorhabditis elegans 35,179 6-Sep-99
PROGRESS ***, in unordered pieces. GB_HTG1: CEY56A3 224746 AL022280
Caenorhabditis elegans chromosome III clone Y56A3, *** SEQUENCING
IN Caenorhabditis elegans 35,179 6-Sep-99 PROGRESS ***, in
unordered pieces. GB_PR2: HS134O19 86897 AL034555 Human DNA
sequence from clone 134O19 on chromosome 1p36.11-36.33, Homo
sapiens 40,604 23-Nov-99 complete sequence. rxa00381 729 GB_GSS4:
AQ730532 416 AQ730532 HS_2149_A1_C06_T7C CIT Approved Human Genomic
Homo sapiens 35,766 15-Jul-99 Sperm Library D Homo sapiens genomic
clone Plate = 2149 Col = 11 Row = E, genomic survey sequence.
GB_EST23: AI120939 561 AI120939 ub74f05.r1 Soares mouse mammary
gland NMLMG Mus musculus 41,113 2-Sep-98 IMAGE: 1383489 5' similar
to gb: J04046 CALMODULIN (HUMAN); gb: M19381 Mouse calmodulin
(MOUSE);, mRNA sequence. GB_EST23: AI120939 561 AI120939 ub74f05.r1
Soares mouse mammary gland NMLMG Mus musculus 41,113 2-Sep-98 Mus
musculus cDNA clone IMAGE: 1383489 5' similar to gb: J04046
CALMODULIN (HUMAN); gb: M19381 Mouse calmodulin (MOUSE);, mRNA
sequence. rxa00385 362 GB_EST32: AI726450 565 AI726450 BNLGHI5857
Six-day Cotton fiber Gossypium hirsutum cDNA 5' similar to
Gossypium hirsutum 41,152 11-Jun-99 (AF015913) Skb1Hs [Homo
sapiens], mRNA sequence. GB_GSS4: AQ740856 768 AQ740856
HS_2274_A2_A07_T7C CIT Approved Human Genomic Sperm Homo sapiens
41,360 16-Jul-99 Library D Homo sapiens genomic clone Plate = 2274
Col = 14 Row = A, genomic survey sequence. GB_PR1: HSPAIP 1587
X91809 H. sapiens mRNA for GAIP protein. Homo sapiens 36,792
29-MAR-1996 rxa00388 1134 GB_BA1: MTY25D10 40838 Z95558
Mycobacterium tuberculosis H37Rv complete genome; segment 28/162.
Mycobacterium tuberculosis 51,852 17-Jun-98 GB_BA1: MSGY224 40051
AD000004 Mycobacterium tuberculosis sequence from clone y224.
Mycobacterium tuberculosis 51,852 03-DEC-1996 GB_HTG1: AP000471
72466 AP000471 Homo sapiens chromosome 21 clone B2308H15 map
21q22.3, Homo sapiens 36,875 13-Sep-99 *** SEQUENCING IN PROGRESS
***, in unordered pieces. rxa00427 909 GB_BA1: MSGY126 37164
AD000012 Mycobacterium tuberculosis sequence from clone y126.
Mycobacterium tuberculosis 60,022 10-DEC-1996 GB_BA1: MTY13D12
37085 Z80343 Mycobacterium tuberculosis H37Rv complete genome;
segment 156/162. Mycobacterium tuberculosis 60,022 17-Jun-98
GB_HTG1: CEY48C3 270193 Z92855 Caenorhabditis elegans chromosome II
clone Y48C3, *** SEQUENCING IN Caenorhabditis elegans 28,013
29-MAY-1999 PROGRESS ***, in unordered pieces. rxa00483 1587
GB_PR2: HSAF001550 173882 AF001550 Homo sapiens chromosome 16 BAC
clone Homo sapiens 38,226 22-Aug-97 CIT987SK-334D11 complete
sequence. GB_BA1: LLCPJW565 12828 Y12736 Lactococcus lactis
cremoris plasmid pJW565 DNA, abiiM, abiiR Lactococcus lactis subsp.
37,492 01-MAR-1999 genes and orfX. cremoris GB_HTG2: AC006754
206217 AC006754 Caenorhabditis elegans clone Y40B10, *** SEQUENCING
Caenorhabditis elegans 36,648 23-Feb-99 IN PROGRESS ***, 5
unordered pieces. rxa00511 615 GB_PR3: HSE127C11 38423 Z74581 Human
DNA sequence from cosmid E127C11 on chromosome 22q11.2-qter Homo
sapiens 39,831 23-Nov-99 contains STS. GB_PR3: HSE127C11 38423
Z74581 Human DNA sequence from cosmid E127C11 on chromosome
22q11.2-qter Homo sapiens 36,409 23-Nov-99 contains STS. rxa00512
718 GB_BA1: MTCY22G8 22550 Z95585 Mycobacterium tuberculosis H37Rv
complete genome; segment 49/162. Mycobacterium tuberculosis 56,232
17-Jun-98 GB_BA1: MSGLTA 1776 X60513 M. smegmatis gltA gene for
citrate synthase. Mycobacterium smegmatis 56,143 20-Sep-91 GB_BA2:
ECU73857 128824 U73857 Escherichia coli chromosome minutes 6-8.
Escherichia coli 48,563 14-Jul-99 rxa00517 1164 GB_HTG2: AC006911
298804 AC006911 Caenorhabditis elegans clone Y94H6x, *** SEQUENCING
Caenorhabditis elegans 37,889 24-Feb-99 IN PROGRESS ***, 15
unordered pieces. GB_HTG2: AC006911 298804 AC006911 Caenorhabditis
elegans clone Y94H6x, *** SEQUENCING Caenorhabditis elegans 37,889
24-Feb-99 IN PROGRESS ***, 15 unordered pieces. GB_EST29: AI602158
481 AI602158 UI-R-AB0-vy-a-01-0-UI.s2 UI-R-AB0 Rattus norvegicus
cDNA Rattus norvegicus 40,833 21-Apr-99 clone UI-R-AB0-vy-a-01-0-UI
3', mRNA sequence. rxa00518 320 GB_BA2: ECU73857 128824 U73857
Escherichia coli chromosome minutes 6-8. Escherichia coli 49,688
14-Jul-99 GB_BA2: STU51879 8371 U51879 Salmonella typhimurium
propionate catabolism operon: RpoN activator protein Salmonella
typhimurium 50,313 5-Aug-99 homolog (prpR),
carboxyphosphonoenolpyruvate phosphonomutase homolog (prpB),
citrate synthase homolog (prpC), prpD and prpE genes, complete cds.
GB_BA2: AE000140 12498 AE000140 Escherichia coli K-12 MG1655
section 30 of 400 of the complete genome. Escherichia coli 49,688
12-Nov-98 rxa00606 2378 GB_EST32: AU068253 376 AU068253 AU068253
Rice callus Oryza sativa cDNA clone C12658_9A, Oryza sativa 41,333
7-Jun-99 mRNA sequence. GB_EST13: AA363046 329 AA363046 EST72922
Ovary II Homo sapiens cDNA 5' end, mRNA sequence. Homo sapiens
34,347 21-Apr-97 GB_EST32: AU068253 376 AU068253 AU068253 Rice
callus Oryza sativa cDNA clone C12658_9A, Oryza sativa 41,899
7-Jun-99 mRNA sequence. rxa00635 1860 GB_BA1: PAORF1 1440 X13378
Pseudomonas amyloderamosa DNA for ORF 1. Pseudomonas amyloderamosa
53,912 14-Jul-95 GB_BA1: PAORF1 1440 X13378 Pseudomonas
amyloderamosa DNA for ORF 1. Pseudomonas amyloderamosa 54,422
14-Jul-95 rxa00679 1389 GB_PL2: AC010871 80381 AC010871 Arabidopsis
thaliana chromosome III BAC T16O11 genomic Arabidopsis thaliana
38,244 13-Nov-99 sequence, complete sequence. GB_PL1: AT81KBGEN
81493 X98130 A. thaliana 81 kb genomic sequence. Arabidopsis
thaliana 36,091 12-MAR-1997 GB_PL2: AC010871 80381 AC010871
Arabidopsis thaliana chromosome III BAC T16O11 genomic Arabidopsis
thaliana 37,135 13-Nov-99 sequence, complete sequence. rxa00680 441
GB_PR3: AC004058 38400 AC004058 Homo sapiens chromosome 4 clone
B241P19 map 4q25, complete sequence. Homo sapiens 36,165 30-Sep-98
GB_PL1: AT81KBGEN 81493 X98130 A. thaliana 81 kb genomic sequence.
Arabidopsis thaliana 38,732 12-MAR-1997 GB_PL1: AB026648 43481
AB026648 Arabidopsis thaliana genomic DNA, chromosome 3, P1
Arabidopsis thaliana 38,732 07-MAY-1999 clone: MLJ15, complete
sequence. rxa00682 2022 GB_HTG3: AC010325 197110 AC010325 Homo
sapiens chromosome 19 clone CITB-E1_2568A17, Homo sapiens 37,976
15-Sep-99 *** SEQUENCING IN PROGRESS ***, 40 unordered pieces.
GB_HTG3: AC010325 197110 AC010325 Homo sapiens chromosome 19 clone
CITB-E1_2568A17, Homo sapiens 37,976 15-Sep-99 *** SEQUENCING IN
PROGRESS ***, 40 unordered pieces. GB_PR4: AC008179 181745 AC008179
Homo sapiens clone NH0576F01, complete sequence. Homo sapiens
37,143 28-Sep-99 rxa00683 1215 GB_BA2: AE000896 10707 AE000896
Methanobacterium thermoautotrophicum from bases Methanobacterium
38,429 15-Nov-97 1189349 to 1200055 (section 102 of 148) of the
complete genome. thermoautotrophicum GB_IN1: DMBR7A4 212734
AL109630 Drosophila melanogaster clone BACR7A4. Drosophila
melanogaster 36,454 30-Jul-99 GB_EST35: AV163010 273 AV163010
AV163010 Mus musculus head C57BL/6J 13-day embryo Mus musculus
41,758 8-Jul-99 Mus musculus cDNA clone 3110006J22, mRNA sequence.
rxa00686 927 GB_HTG2: HSDJ137K2 190223 AL049820 Homo sapiens
chromosome 6 clone RP1-137K2 map q25.1-25.3, Homo sapiens 38,031
03-DEC-1999 *** SEQUENCING IN PROGRESS ***, in unordered pieces.
GB_HTG2: HSDJ137K2 190223 AL049820 Homo sapiens chromosome 6 clone
RP1-137K2 map q25.1-25.3, Homo sapiens 38,031 03-DEC-1999 ***
SEQUENCING IN PROGRESS ***, in unordered pieces. GB_EST12: AA284399
431 AA284399 zs57b04.r1 NCI_CGAP_GCB1 Homo sapiens cDNA clone Homo
sapiens 39,205 14-Aug-97 IMAGE: 701551 5', mRNA sequence. rxa00700
927 GB_EST34: AI785570 454 AI785570 uj44d03.x1 Sugano mouse liver
mlia Mus musculus cDNA Mus musculus 41,943 2-Jul-99 clone IMAGE:
1922789 3' similar to gb: Z28407 60S RIBOSOMAL PROTEIN L8 (HUMAN);,
mRNA sequence. GB_EST25: AI256147 684 AI256147 ui95e12.x1 Sugano
mouse liver mlia Mus musculus cDNA clone Mus musculus 40,791
12-Nov-98 IMAGE: 1890190 3' similar to gb: Z28407 60S RIBOSOMAL
PROTEIN L8 (HUMAN);, mRNA sequence. GB_BA1: CARCG12 2079 X14979 C.
aurantiacus reaction center genes 1 and 2. Chloroflexus aurantiacus
37,721 23-Apr-91 rxa00703 2409 GB_BA1: SC7H2 42655 AL109732
Streptomyces coelicolor cosmid 7H2. Streptomyces coelicolor A3(2)
56,646 2-Aug-99 GB_BA1: MTCY274 39991 Z74024 Mycobacterium
tuberculosis H37Rv complete genome; segment 126/162. Mycobacterium
tuberculosis 37,369 19-Jun-98 GB_BA2: REU60056 2520 U60056
Ralstonia eutropha formate dehydrogenase-like protein (cbbBc)
Ralstonia eutropha 51,087 16-OCT-1996 gene, complete cds. rxa00705
1038 GB_GSS15: AQ604477 505 AQ604477 HS_2116_B1_G07_MR CIT Approved
Human Genomic Homo sapiens 39,617 10-Jun-99 Sperm Library D Homo
sapiens genomic clone Plate = 2116 Col = 13 Row = N, genomic survey
sequence. GB_EST11: AA224340 443 AA224340 zr14e07.s1 Stratagene hNT
neuron (#937233) Homo sapiens cDNA clone Homo sapiens 35,129
11-MAR-1998 IMAGE: 648804 3', mRNA sequence. GB_EST5: N30648 291
N30648 yw77b02.s1 Soares_placenta_8to9weeks_2NbHP8to9W Homo sapiens
43,986 5-Jan-96 Homo sapiens cDNA clone IMAGE: 258219 3', mRNA
sequence. rxa00782 1005 GB_BA1: MTCY10D7 39800 Z79700 Mycobacterium
tuberculosis H37Rv complete genome; segment 44/162. Mycobacterium
tuberculosis 63,327 17-Jun-98 GB_BA1: MLCL373 37304 AL035500
Mycobacterium leprae cosmid L373. Mycobacterium leprae 62,300
27-Aug-99 GB_BA2: AF128399 2842 AF128399 Pseudomonas aeruginosa
succinyl-CoA synthetase beta subunit Pseudomonas aeruginosa 53,698
25-MAR-1999 (sucC) and succinyl CoA synthetase alpha subunit (sucD)
genes, complete cds. rxa00783 1395 GB_HTG2: AC008158 118792
AC008158 Homo sapiens chromosome 17 clone hRPK.42_F_20 map 17, Homo
sapiens 35,135 28-Jul-99 *** SEQUENCING IN PROGRESS ***, 14
unordered pieces. GB_HTG2: AC008158 118792 AC008158 Homo sapiens
chromosome 17 clone hRPK.42_F_20 map 17, Homo sapiens 35,135
28-Jul-99 *** SEQUENCING IN PROGRESS ***, 14 unordered pieces.
GB_PR3: AC005017 137176 AC005017 Homo sapiens BAC clone GS214N13
from 7p14-p15, complete sequence. Homo sapiens 35,864 8-Aug-98
rxa00794 1128 GB_BA1: MTV017 67200 AL021897 Mycobacterium
tuberculosis H37Rv complete genome; segment 48/162. Mycobacterium
tuberculosis 40,331 24-Jun-99 GB_BA1: MLCB1222 34714 AL049491
Mycobacterium leprae cosmid B1222. Mycobacterium leprae 61,170
27-Aug-99 GB_PR2: HS151B14 128942 Z82188 Human DNA sequence from
clone 151B14 on chromosome 22 Contains Homo sapiens 37,455
16-Jun-99 SOMATOSTATIN RECEPTOR TYPE 3 (SS3R) gene, pseudogene
similar to ribosomal protein L39, RAC2 (RAS-RELATED C3 BOTULINUM
TOXIN SUBSTRATE 2 (P21-RAC2)) gene ESTs, STSs, GSSs and CpG
islands, complete sequence. rxa00799 1767 GB_PL2: AF016327 616
AF016327 Hordeum vulgare Barperm1 (perm1) mRNA, partial cds.
Hordeum vulgare 41,311 01-OCT-1997 GB_HTG2: HSDJ319M7 128208
AL079341 Homo sapiens chromosome 6 clone RP1-319M7 map p21.1-21.3,
Homo sapiens 36,845 30-Nov-99 *** SEQUENCING IN PROGRESS ***, in
unordered pieces. GB_HTG2: HSDJ319M7 128208 AL079341 Homo sapiens
chromosome 6 clone RP1-319M7 map p21.1-21.3, Homo sapiens 36,845
30-Nov-99 *** SEQUENCING IN PROGRESS ***, in unordered pieces.
rxa00800 1227 GB_BA1: MTV022 13025 AL021925 Mycobacterium
tuberculosis H37Rv complete genome; segment 100/162. Mycobacterium
tuberculosis 63,101 17-Jun-98 GB_BA1: AB019513 4417 AB019513
Streptomyces coelicolor genes for alcohol dehydrogenase and ABC
transporter, Streptomyces coelicolor 41,312 13-Nov-98 complete cds.
GB_PL1: SCSFAARP 7008 X68020 S. cerevisiae SFA and ARP genes.
Saccharomyces cerevisiae 36,288 29-Nov-94 rxa00825 1056 GB_BA1:
MTY15C10 33050 Z95436 Mycobacterium tuberculosis H37Rv complete
genome; segment 154/162. Mycobacterium tuberculosis 39,980
17-Jun-98 GB_BA1: MLCB2548 38916 AL023093 Mycobacterium leprae
cosmid B2548. Mycobacterium leprae 39,435 27-Aug-99 GB_BA2:
AF169031 1141 AF169031 Xanthomonas oryzae pv. oryzae putative sugar
nucleotide Xanthomonas oryzae pv. 46,232 14-Sep-99
epimerase/dehydratase gene, partial cds. oryzae rxa00871 rxa00872
1077 GB_IN1: CEF23H12 35564 Z74472 Caenorhabditis elegans cosmid
F23H12, complete sequence. Caenorhabditis elegans 34,502
08-OCT-1999 GB_HTG2: AC007263 167390 AC007263 Homo sapiens
chromosome 14 clone BAC Homo sapiens 35,714 24-MAY-1999 79J20 map
14q31, *** SEQUENCING IN PROGRESS ***, 5 ordered pieces. GB_HTG2:
AC007263 167390 AC007263 Homo sapiens chromosome 14 clone BAC 79J20
Homo sapiens 35,714 24-MAY-1999 map 14q31, *** SEQUENCING IN
PROGRESS ***, 5 ordered pieces. rxa00879 2241 GB_BA1: MTV049 40360
AL022021 Mycobacterium tuberculosis H37Rv complete genome; segment
81/162. Mycobacterium tuberculosis 36,981 19-Jun-98 GB_PL2:
CDU236897 1827 AJ236897 Candida dubliniensis ACT1 gene, exons 1-2.
Candida dubliniensis 38,716 1-Sep-99 GB_PL1: CAACT1A 3206 X16377
Candida albicans act1 gene for actin. Candida albicans 36,610
10-Apr-93 rxa00909 955 GB_BA2: AF010496 189370 AF010496 Rhodobacter
capsulatus strain SB1003, partial genome. Rhodobacter capsulatus
51,586 12-MAY-1998 GB_BA1: RMPHA 7888 X93358 Rhizobium meliloti
pha[A, B, C, D, E, F, G] genes. Sinorhizobium meliloti 48,367
12-MAR-1999 GB_EST16: C23528 317 C23528 C23528 Japanese flounder
spleen Paralichthys olivaceus cDNA clone HB5(2), Paralichthys
olivaceus 41,640 28-Sep-99
mRNA sequence. rxa00913 2118 GB_HTG2: AC007734 188267 AC007734 Homo
sapiens chromosome 18 clone hRPK.44_O_1 Homo sapiens 34,457
5-Jun-99 map 18, *** SEQUENCING IN PROGRESS ***, 18 unordered
pieces. GB_HTG2: AC007734 188267 AC007734 Homo sapiens chromosome
18 clone hRPK.44_O_1 Homo sapiens 34,457 5-Jun-99 map 18, ***
SEQUENCING IN PROGRESS ***, 18 unordered pieces. GB_EST18: AA709478
406 AA709478 vv34a05.r1 Stratagene mouse heart (#937316) Mus
musculus cDNA clone Mus musculus 42,065 24-DEC-1997 IMAGE: 1224272
5', mRNA sequence. rxa00945 1095 GB_HTG4: AC010351 220710 AC010351
Homo sapiens chromosome 5 clone CITB-H1_2022B6, Homo sapiens 36,448
31-OCT-1999 *** SEQUENCING IN PROGRESS ***, 68 unordered pieces.
GB_HTG4: AC010351 220710 AC010351 Homo sapiens chromosome 5 clone
CITB-H1_2022B6, Homo sapiens 36,448 31-OCT-1999 *** SEQUENCING IN
PROGRESS ***, 68 unordered pieces. GB_BA1: MTCY05A6 38631 Z96072
Mycobacterium tuberculosis H37Rv complete genome; segment 120/162.
Mycobacterium tuberculosis 36,218 17-Jun-98 rxa00965 rxa00999 1575
GB_PAT: E13660 1916 E13660 gDNA encoding 6-phosphogluconate
dehydrogenase. Corynebacterium glutamicum 98,349 24-Jun-98 GB_BA1:
MTCY359 36021 Z83859 Mycobacterium tuberculosis H37Rv complete
genome; segment 84/162. Mycobacterium tuberculosis 38,520 17-Jun-98
GB_BA1: MLCB1788 39228 AL008609 Mycobacterium leprae cosmid B1788.
Mycobacterium leprae 64,355 27-Aug-99 rxa01015 442 GB_BA1: MTV008
63033 AL021246 Mycobacterium tuberculosis H37Rv complete genome;
segment 108/162. Mycobacterium tuberculosis 39,860 17-Jun-98
GB_BA1: MTV008 63033 AL021246 Mycobacterium tuberculosis H37Rv
complete genome; segment 108/162. Mycobacterium tuberculosis 39,120
17-Jun-98 rxa01025 1119 GB_BA1: SC7A1 32039 AL034447 Streptomyces
coelicolor cosmid 7A1. Streptomyces coelicolor 55,287 15-DEC-1998
GB_BA1: MSGB1723CS 38477 L78825 Mycobacterium leprae cosmid B1723
DNA sequence. Mycobacterium leprae 56,847 15-Jun-96 GB_BA1: MLCB637
44882 Z99263 Mycobacterium leprae cosmid B637. Mycobacterium leprae
56,676 17-Sep-97 rxa01048 1347 GB_BA2: AF017444 3067 AF017444
Sinorhizobium meliloti NADP-dependent malic Sinorhizobium meliloti
53,660 2-Nov-97 enzyme (tme) gene, complete cds. GB_BA1: BSUB0013
218470 Z99116 Bacillus subtilis complete genome (section 13 of 21):
Bacillus subtilis 37,255 26-Nov-97 from 2395261 to 2613730. GB_VI:
HSV2HG52 154746 Z86099 Herpes simplex virus type 2 (strain HG52),
complete genome, human herpesvirus 2 38,081 04-DEC-1998 rxa01049
1605 GB_HTG2: AC002518 131855 AC002518 Homo sapiens chromosome X
clone bWXD20, Homo sapiens 35,647 2-Sep-97 *** SEQUENCING IN
PROGRESS ***, 11 unordered pieces. GB_HTG2: AC002518 131855
AC002518 Homo sapiens chromosome X clone bWXD20, Homo sapiens
35,647 2-Sep-97 *** SEQUENCING IN PROGRESS ***, 11 unordered
pieces. GB_HTG2: AC002518 131855 AC002518 Homo sapiens chromosome X
clone bWXD20, Homo sapiens 26,180 2-Sep-97 *** SEQUENCING IN
PROGRESS ***, 11 unordered pieces. GB_HTG2: AC002518 131855
AC002518 Homo sapiens chromosome X clone bWXD20, Homo sapiens
26,180 2-Sep-97 *** SEQUENCING IN PROGRESS ***, 11 unordered
pieces. rxa01077 1494 GB_PR3: HSDJ653C5 85237 AL049743 Human DNA
sequence from clone 653C5 on Homo sapiens 36,462 23-Nov-99
chromosome 1p21.3-22.3 Contains CA repeat(D1S435), STSs and GSSs,
complete sequence. GB_BA1: ECU29579 72221 U29579 Escherichia coli
K-12 genome; approximately 61 to 62 minutes. Escherichia coli
41,808 1-Jul-95 GB_BA1: ECU29579 72221 U29579 Escherichia coli K-12
genome; approximately 61 to 62 minutes. Escherichia coli 36,130
1-Jul-95 rxa01089 873 GB_GSS8: AQ044021 387 AQ044021
CIT-HSP-2318C18.TR CIT-HSP Homo sapiens Homo sapiens 36,528
14-Jul-98 genomic clone 2318C18, genomic survey sequence. GB_GSS8:
AQ042907 392 AQ042907 CIT-HSP-2318D17.TR CIT-HSP Homo sapiens Homo
sapiens 35,969 14-Jul-98 genomic clone 2318D17, genomic survey
sequence. GB_GSS8: AQ044021 387 AQ044021 CIT-HSP-2318C18.TR CIT-HSP
Homo sapiens Homo sapiens 44,545 14-Jul-98 genomic clone 2318C18,
genomic survey sequence. rxa01093 1554 GB_BA1: CORPYKI 2795 L27126
Corynebacterium pyruvate kinase gene, complete cds. Corynebacterium
glutamicum 100,000 07-DEC-1994 GB_BA1: MTCY01B2 35938 Z95554
Mycobacterium tuberculosis H37Rv complete genome; segment 72/162.
Mycobacterium tuberculosis 63,771 17-Jun-98 GB_BA1: MIU65430 1439
U65430 Mycobacterium intracellulare pyruvate kinase (pykF) gene,
complete cds. Mycobacterium intracellulare 67,061 23-DEC-1996
rxa01099 948 GB_BA2: AF045998 780 AF045998 Corynebacterium
glutamicum inositol Corynebacterium glutamicum 99,615 19-Feb-98
monophosphate phosphatase (impA) gene, complete cds. GB_BA2:
AF051846 738 AF051846 Corynebacterium glutamicum
phosphoribosylformimino-5- Corynebacterium glutamicum 100,000
12-MAR-1998 amino-1-phosphoribosyl-4-imidazolecarboxamide isomerase
(hisA) gene, complete cds. GB_GSS1: FR0005503 619 Z89313 F.
rubripes GSS sequence, clone 079B16aE8, genomic survey sequence.
Fugu rubripes 37,785 01-MAR-1997 rxa01111 541 GB_PR3: AC004063
177014 AC004063 Homo sapiens chromosome 4 clone B32I8, complete
sequence. Homo sapiens 35,835 10-Jul-98 GB_PR3: HS1178I21 62268
AL109852 Human DNA sequence from clone RP5-1178I21 on chromosome X,
complete Homo sapiens 37,873 01-DEC-1999 sequence. GB_HTG3:
AC009301 163369 AC009301 Homo sapiens clone NH0062F14, ***
SEQUENCING IN PROGRESS ***, 5 Homo sapiens 37,240 13-Aug-99
unordered pieces. rxa01130 687 GB_HTG3: AC009444 164587 AC009444
Homo sapiens clone 1_O_3, *** SEQUENCING Homo sapiens 38,416
22-Aug-99 IN PROGRESS ***, 8 unordered pieces. GB_HTG3: AC009444
164587 AC009444 Homo sapiens clone 1_O_3, *** SEQUENCING IN Homo
sapiens 38,416 22-Aug-99 PROGRESS ***, 8 unordered pieces. GB_IN1:
DMC66A1 34127 AL031227 Drosophila melanogaster cosmid 66A1.
Drosophila melanogaster 38,416 05-OCT-1998 rxa01193 1572 GB_BA1:
CGASO19 1452 X76875 C. glutamicum (ASO 19) ATPase beta-subunit
gene. Corynebacterium glutamicum 99,931 27-OCT-1994 EM_PAT: E09634
1452 E09634 Brevibacterium flavum UncD gene whose gene product is
involved in Corynebacterium glutamicum 99,242 07-OCT-1997 (Rel. 52,
Created) GB_BA1: MLU15186 36241 U15186 Mycobacterium leprae cosmid
L471. Mycobacterium leprae 39,153 09-MAR-1995 rxa01194 495 EM_PAT:
E09634 1452 E09634 Brevibacterium flavum UncD gene whose gene
product is involved in Corynebacterium glutamicum 100,000
07-OCT-1997 (Rel. 52, Created) GB_BA1: CGASO19 1452 X76875 C.
glutamicum (ASO 19) ATPase beta-subunit gene. Corynebacterium
glutamicum 100,000 27-OCT-1994 GB_VI: HEPCRE4B 414 X60570 Hepatitis
C genomic RNA for putative envelope protein (RE4B isolate).
Hepatitis C virus 36,769 5-Apr-92 rxa01200 rxa01201 1764 GB_BA1:
SLATPSYNA 8560 Z22606 S. lividans i protein and ATP synthase genes.
Streptomyces lividans 66,269 01-MAY-1995 GB_BA1: MTCY373 35516
Z73419 Mycobacterium tuberculosis H37Rv complete genome; segment
57/162. Mycobacterium tuberculosis 65,437 17-Jun-98 GB_BA1:
MLU15186 36241 U15186 Mycobacterium leprae cosmid L471.
Mycobacterium leprae 39,302 09-MAR-1995 rxa01202 1098 GB_BA1:
SLATPSYNA 8560 Z22606 S. lividans i protein and ATP synthase genes.
Streptomyces lividans 57,087 01-MAY-1995 GB_BA1: SLATPSYNA 8560
Z22606 S. lividans i protein and ATP synthase genes. Streptomyces
lividans 38,298 01-MAY-1995 GB_BA1: MCSQSSHC 5538 Y09978 M.
capsulatus orfx, orfy, orfz, sqs and shc genes. Methylococcus
capsulatus 37,626 26-MAY-1998 rxa01204 933 GB_PL1: AP000423 154478
AP000423 Arabidopsis thaliana chloroplast genomic DNA, complete
sequence, Chloroplast Arabidopsis 38,395 15-Sep-99 strain:
Columbia. thaliana GB_HTG6: AC009762 164070 AC009762 Homo sapiens
clone RP11-114I16, *** SEQUENCING Homo sapiens 35,459 04-DEC-1999
IN PROGRESS ***, 39 unordered pieces. GB_HTG6: AC009762 164070
AC009762 Homo sapiens clone RP11-114I16, *** SEQUENCING Homo
sapiens 36,117 04-DEC-1999 IN PROGRESS ***, 39 unordered pieces.
rxa01216 1124 GB_BA1: MTCY10G2 38970 Z92539 Mycobacterium
tuberculosis H37Rv complete genome; segment 47/162. Mycobacterium
tuberculosis 39,064 17-Jun-98 GB_BA2: AF017435 4301 AF017435
Methylobacterium extorquens methanol oxidation genes,
Methylobacterium extorquens 42,671 10-MAR-1998 glmU-like gene,
partial cds, and orfL2, orfL1, orfR genes, complete cds. GB_BA1:
CCRFLBDBA 4424 M69228 C. crescentus flagellar gene promoter region.
Caulobacter crescentus 41,054 26-Apr-93 rxa01225 1563 GB_BA2:
AF058302 25306 AF058302 Streptomyces roseofulvus frenolicin
biosynthetic gene Streptomyces roseofulvus 36,205 2-Jun-98 cluster,
complete sequence. GB_HTG3: AC007301 165741 AC007301 Drosophila
melanogaster chromosome 2 clone BACR04B09 Drosophila melanogaster
39,922 17-Aug-99 (D576) RPCI-98 04.B.9 map 43E12-44F1 strain y; cn
bw sp, *** SEQUENCING IN PROGRESS ***, 150 unordered pieces.
GB_HTG3: AC007301 165741 AC007301 Drosophila melanogaster
chromosome 2 clone BACR04B09 (D576) Drosophila melanogaster 39,922
17-Aug-99 RPCI-98 04.B.9 map 43E12-44F1 strain y; cn bw sp, ***
SEQUENCING IN PROGRESS ***, 150 unordered pieces. rxa01227 444
GB_BA1: SERFDXA 3869 M61119 Saccharopolyspora erythraea ferredoxin
(fdxA) gene, complete cds. Saccharopolyspora erythraea 64,908
13-MAR-1996 GB_BA1: MTV005 37840 AL010186 Mycobacterium
tuberculosis H37Rv complete genome; segment 51/162. Mycobacterium
tuberculosis 62,838 17-Jun-98 GB_BA1: MSGY348 40056 AD000020
Mycobacterium tuberculosis sequence from clone y348. Mycobacterium
tuberculosis 61,712 10-DEC-1996 rxa01242 900 GB_PR3: AC005697
174503 AC005697 Homo sapiens chromosome 17, clone hRPK.138_P_22,
complete sequence. Homo sapiens 35,373 09-OCT-1998 GB_HTG3:
AC010722 160723 AC010722 Homo sapiens clone NH0122L09, ***
SEQUENCING IN PROGRESS ***, 2 Homo sapiens 39,863 25-Sep-99
unordered pieces. GB_HTG3: AC010722 160723 AC010722 Homo sapiens
clone NH0122L09, *** SEQUENCING IN PROGRESS ***, 2 Homo sapiens
39,863 25-Sep-99 unordered pieces. rxa01243 1083 GB_GSS10: AQ255057
583 AQ255057 mgxb0008N01r CUGI Rice Blast BAC Library Magnaporthe
Magnaporthe grisea 38,722 23-OCT-1998 grisea genomic clone
mgxb0008N01r, genomic survey sequence. GB_IN1: CEK05D4 19000 Z92804
Caenorhabditis elegans cosmid K05D4, complete sequence.
Caenorhabditis elegans 35,448 23-Nov-98 GB_IN1: CEK05D4 19000
Z92804 Caenorhabditis elegans cosmid K05D4, complete sequence.
Caenorhabditis elegans 35,694 23-Nov-98 rxa01259 981 GB_BA1: CGLPD
1800 Y16642 Corynebacterium glutamicum lpd gene, complete CDS.
Corynebacterium glutamicum 100,000 1-Feb-99 GB_HTG4: AC010567
143287 AC010567 Drosophila melanogaster chromosome 3L/69C1 clone
RPCI98-11N6, *** Drosophila melanogaster 37,178 16-OCT-1999
SEQUENCING IN PROGRESS ***, 70 unordered pieces. GB_HTG4: AC010567
143287 AC010567 Drosophila melanogaster chromosome 3L/69C1 clone
RPCI98-11N6, Drosophila melanogaster 37,178 16-OCT-1999
***SEQUENCING IN PROGRESS ***, 70 unordered pieces. rxa01262 1284
GB_BA2: AF172324 14263 AF172324 Escherichia coli GalF (galF) gene,
partial cds; Escherichia coli 59,719 29-OCT-1999 O-antigen repeat
unit transporter Wzx (wzx), WbnA (wbnA), O-antigen polymerase Wzy
(wzy), WbnB (wbnB), WbnC (wbnC), WbnD (wbnD), WbnE (wbnE),
UDP-Glc-4-epimerase GalE (galE), 6- phosphogluconate dehydrogenase
Gnd (gnd), UDP-Glc-6-dehydrogenase Ugd (ugd), and WbnF (wbnF)
genes, complete cds; and chain length determinant Wzz (wzz) gene,
partial cds. GB_BA2: ECU78086 4759 U78086 Escherichia coli
hypothetical uridine-5'-diphosphoglucose Escherichia coli 59,735
5-Nov-97 dehydrogenase (ugd) and O-chain length regulator (wzz)
genes, complete cds. GB_BA1: D90841 20226 D90841 E. coli genomic
DNA, Kohara clone #351(45.1-45.5 min.). Escherichia coli 37,904
21-MAR-1997 rxa01311 870 GB_PR3: AC004103 144368 AC004103 Homo
sapiens Xp22 BAC GS-619J3 Homo sapiens 37,340 18-Apr-98 (Genome
Systems Human BAC library) complete sequence. GB_HTG3: AC007383
215529 AC007383 Homo sapiens clone NH0310K15, Homo sapiens 36,385
25-Sep-99 *** SEQUENCING IN PROGRESS ***, 4 unordered pieces.
GB_HTG3: AC007383 215529 AC007383 Homo sapiens clone NH0310K15,
Homo sapiens 36,385 25-Sep-99 *** SEQUENCING IN PROGRESS ***, 4
unordered pieces. rxa01312 2142 GB_BA2: AE000487 13889 AE000487
Escherichia coli K-12 MG1655 section 377 of 400 of the Escherichia
coli 39,494 12-Nov-98 complete genome. GB_BA1: MTV016 53662
AL021841 Mycobacterium tuberculosis H37Rv complete genome; segment
143/162. Mycobacterium tuberculosis 46,252 23-Jun-99 GB_BA1: U00022
36411 U00022 Mycobacterium leprae cosmid L308. Mycobacterium leprae
46,368 01-MAR-1994 rxa01325 795 GB_HTG4: AC009245 215767 AC009245
Homo sapiens chromosome 7, Homo sapiens 36,016 2-Nov-99 ***
SEQUENCING IN PROGRESS ***, 24 unordered pieces. GB_HTG4: AC009245
215767 AC009245 Homo sapiens chromosome 7, Homo sapiens 36,016
2-Nov-99 *** SEQUENCING IN PROGRESS ***, 24 unordered pieces.
GB_HTG4: AC009245 215767 AC009245 Homo sapiens chromosome 7, Homo
sapiens 39,618 2-Nov-99 *** SEQUENCING IN PROGRESS ***, 24
unordered pieces. rxa01332 576 GB_HTG6: AC007186 225851 AC007186
Drosophila melanogaster chromosome 2 Drosophila melanogaster 35,366
07-DEC-1999 clone BACR03D06 (D569) RPCI-98 03.D.6 map 32A-32A
strain y; cn bw sp, *** SEQUENCING IN PROGRESS***, 91 unordered
pieces. GB_HTG6: AC007147 202291 AC007147 Drosophila melanogaster
chromosome 2 Drosophila melanogaster 35,366 07-DEC-1999 clone
BACR19N18 (D572) RPCI-98 19.N.18 map 32A-32A strain y; cn bw sp,
*** SEQUENCING IN PROGRESS ***, 22 unordered pieces. GB_HTG3:
AC010207 207890 AC010207 Homo sapiens clone RPCI11-375I20, Homo
sapiens 34,821 16-Sep-99 *** SEQUENCING IN PROGRESS ***, 25
unordered pieces.
rxa01350 1107 GB_BA2: AF109682 990 AF109682 Aquaspirillum arcticum
malate dehydrogenase (MDH) Aquaspirillum arcticum 58,487
19-OCT-1999 gene, complete cds. GB_HTG2: AC006759 103725 AC006759
Caenorhabditis elegans clone Y40G12, Caenorhabditis elegans 37,963
25-Feb-99 *** SEQUENCING IN PROGRESS***, 8 unordered pieces.
GB_HTG2: AC006759 103725 AC006759 Caenorhabditis elegans clone
Y40G12, Caenorhabditis elegans 37,963 25-Feb-99 *** SEQUENCING IN
PROGRESS***, 8 unordered pieces. rxa01365 1497 GB_BA1: MTY20B11
36330 Z95121 Mycobacterium tuberculosis H37Rv complete
Mycobacterium tuberculosis 38,011 17-Jun-98 genome; segment
139/162. GB_BA1: XANXANAB 3410 M83231 Xanthomonas campestris
phosphoglucomutase Xanthomonas campestris 47,726 26-Apr-93 and
phosphomannomutase (xanA) and phosphomannose isomerase and
GDP-mannose pyrophosphorylase (xanB) genes, complete cds. GB_GSS10:
AQ194038 697 AQ194038 RPCI11-47D24.TJ RPCI-11 Homo sapiens genomic
Homo sapiens 36,599 20-Apr-99 clone RPCI-11-47D24, genomic survey
sequence. rxa01369 1305 GB_BA1: MTY20B11 36330 Z95121 Mycobacterium
tuberculosis H37Rv Mycobacterium tuberculosis 36,940 17-Jun-98
complete genome; segment 139/162. GB_GSS3: B10037 974 B10037
T27A19-T7 TAMU Arabidopsis thaliana genomic Arabidopsis thaliana
35,284 14-MAY-1997 clone T27A19, genomic survey sequence. GB_GSS3:
B09549 1097 B09549 T21A19-T7.1 TAMU Arabidopsis thaliana genomic
Arabidopsis thaliana 38,324 14-MAY-1997 clone T21A19, genomic
survey sequence. rxa01377 1209 GB_BA1: MTCY71 42729 Z92771
Mycobacterium tuberculosis H37Rv Mycobacterium tuberculosis 39,778
10-Feb-99 complete genome; segment 141/162. GB_HTG5: AC007547
262181 AC007547 Homo sapiens clone RP11-252O18, Homo sapiens 32,658
16-Nov-99 WORKING DRAFT SEQUENCE, 121 unordered pieces. GB_HTG5:
AC007547 262181 AC007547 Homo sapiens clone RP11-252O18, WORKING
DRAFT SEQUENCE, 121 Homo sapiens 38,395 16-Nov-99 unordered pieces.
rxa01392 1200 GB_BA2: AF072709 8366 AF072709 Streptomyces lividans
amplifiable element AUD4: putative transcriptional Streptomyces
lividans 55,221 8-Jul-98 regulator, putative ferredoxin, putative
cytochrome P450 oxidoreductase, and putative oxidoreductase genes,
complete cds; and unknown genes. GB_BA1: CGLYSEG 2374 X96471 C.
glutamicum lysE and lysG genes. Corynebacterium glutamicum 100,000
24-Feb-97 GB_PR4: AC005906 185952 AC005906 Homo sapiens 12p13.3 BAC
RPCI11-429A20 (Roswell Park Cancer Institute Homo sapiens 36,756
30-Jan-99 Human BAC Library) complete sequence. rxa01436 1314
GB_BA1: CGPTAACKA 3657 X89084 C. glutamicum pta gene and ackA gene.
Corynebacterium glutamicum 100,000 23-MAR-1999 GB_BA1: D90861 14839
D90861 E. coli genomic DNA, Kohara clone #405(52.0-52.3 min.).
Escherichia coli 53,041 29-MAY-1997 GB_PAT: E02087 1200 E02087 DNA
encoding acetate kinase protein form Escherichia coli. Escherichia
coli 54,461 29-Sep-97 rxa01468 948 GB_GSS1: HPU60627 280 U60627
Helicobacter pylori feoB-like DNA sequence, genomic survey
sequence. Helicobacter pylori 39,286 9-Apr-97 GB_EST31: AI701691
349 AI701691 we81c04.x1 Soares_NFL_T_GBC_S1 Homo sapiens cDNA clone
Homo sapiens 39,412 3-Jun-99 IMAGE: 2347494 3' similar to gb:
L19686_rna1 MACROPHAGE MIGRATION INHIBITORY FACTOR (HUMAN);, mRNA
sequence. GB_EST15: AA480256 389 AA480256 ne31f04.s1 NCI_CGAP_Co3
Homo sapiens cDNA clone IMAGE: 898975 Homo sapiens 39,574 14-Aug-97
3' similar to gb: L19686_rna1 MACROPHAGE MIGRATION INHIBITORY
FACTOR (HUMAN);, mRNA sequence. rxa01478 1959 GB_BA1: SCI51 40745
AL109848 Streptomyces coelicolor cosmid I51. Streptomyces
coelicolor A3(2) 54,141 16-Aug-99 GB_BA1: SCE36 12581 AL049763
Streptomyces coelicolor cosmid E36. Streptomyces coelicolor 38,126
05-MAY-1999 GB_BA1: CGU43535 2531 U43535 Corynebacterium glutamicum
multidrug resistance protein (cmr) gene, Corynebacterium glutamicum
41,852 9-Apr-97 complete cds. rxa01482 1998 GB_BA1: SC6G4 41055
AL031317 Streptomyces coelicolor cosmid 6G4. Streptomyces
coelicolor 62,149 20-Aug-98 GB_BA1: U00020 36947 U00020
Mycobacterium leprae cosmid B229. Mycobacterium leprae 38,303
01-MAR-1994 GB_BA1: MTCY77 22255 Z95389 Mycobacterium tuberculosis
H37Rv complete genome; segment 146/162. Mycobacterium tuberculosis
38,179 18-Jun-98 rxa01534 rxa01535 1530 GB_BA1: MLCB1222 34714
AL049491 Mycobacterium leprae cosmid B1222. Mycobacterium leprae
66,208 27-Aug-99 GB_BA1: MTV017 67200 AL021897 Mycobacterium
tuberculosis H37Rv complete genome; segment 48/162. Mycobacterium
tuberculosis 38,553 24-Jun-99 GB_BA1: PAU72494 4368 U72494
Pseudomonas aeruginosa fumarase (fumC) and Mn superoxide
Pseudomonas aeruginosa 52,690 23-OCT-1996 dismutase (sodA) genes,
complete cds. rxa01550 1635 GB_BA1: D90907 132419 D90907
Synechocystis sp. PCC6803 complete genome, 9/27, 1056467-1188885.
Synechocystis sp. 56,487 7-Feb-99 GB_IN2: AF073177 9534 AF073177
Drosophila melanogaster glycogen phosphorylase (GlyP) gene,
complete cds. Drosophila melanogaster 55,100 1-Jul-99 GB_IN2:
AF073179 3159 AF073179 Drosophila melanogaster glycogen
phosphorylase (Glp1) mRNA, Drosophila melanogaster 56,708 27-Apr-99
complete cds. rxa01562 rxa01569 1482 GB_BA1: D78182 7836 D78182
Streptococcus mutans DNA for dTDP-rhamnose synthesis pathway,
Streptococcus mutans 44,050 5-Feb-99 complete cds. GB_BA2: AF079139
4342 AF079139 Streptomyces venezuelae plkCD operon, complete
sequence. Streptomyces venezuelae 38,587 28-OCT-1998 GB_BA2:
AF087022 1470 AF087022 Streptomyces venezuelae cytochrome P450
monooxygenase (picK) Streptomyces venezuelae 38,621 15-OCT-1998
gene, complete cds. rxa01570 978 GB_BA1: MTCY63 38900 Z96800
Mycobacterium tuberculosis H37Rv complete genome; segment 16/162.
Mycobacterium tuberculosis 59,035 17-Jun-98 GB_BA2: AF097519 4594
AF097519 Klebsiella pneumoniae dTDP-D-glucose 4,6 dehydratase
(rmlB), glucose-1- Klebsiella pneumoniae 59,714 4-Nov-98 phosphate
thymidylyl transferase (rmlA), dTDP-4-keto-L-rhamnose reductase
(rmlD), dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase (rmlC), and
rhamnosyl transferase (wbbL) genes, complete cds. GB_BA2: NGOCPSPS
8905 L09189 Neisseria meningitidis dTDP-D-glucose 4,6-dehydratase
(rfbB), Neisseria meningitidis 58,384 30-Jul-96 glucose-1-phosphate
thymidyl transferase (rfbA) and rfbC genes, complete cds and
UPD-glucose-4-epimerase (galE) pseudogene. rxa01571 723 GB_BA1:
AB011413 12070 AB011413 Streptomyces griseus genes for Orf2, Orf3,
Orf4, Orf5, AfsA, Orf8, partial and Streptomyces griseus 57,500
7-Aug-98 complete cds. GB_BA1: AB011413 12070 AB011413 Streptomyces
griseus genes for Orf2, Orf3, Orf4, Orf5, AfsA, Orf8, partial and
Streptomyces griseus 35,655 7-Aug-98 complete cds. rxa01572 615
GB_BA1: AB011413 12070 AB011413 Streptomyces griseus genes for
Orf2, Orf3, Orf4, Orf5, AfsA, Orf8, partial and Streptomyces
griseus 57,843 7-Aug-98 complete cds. GB_BA1: AB011413 12070
AB011413 Streptomyces griseus genes for Orf2, Orf3, Orf4, Orf5,
AfsA, Orf8, partial and Streptomyces griseus 38,119 7-Aug-98
complete cds. rxa01606 2799 GB_VI: CFU72240 4783 U72240
Choristoneura fumiferana nuclear polyhedrosis virus ETM protein
Choristoneura fumiferana 37,115 29-Jan-99 homolog, 79 kDa protein
homolog, 15 kDa protein homolog and GTA nucleopolyhedrovirus
protein homolog genes, complete cds. GB_GSS10: AQ213248 408
AQ213248 HS_3249_B1_A02_MR CIT Approved Human Genomic Sperm Homo
sapiens 34,559 18-Sep-98 Library D Homo sapiens genomic clone Plate
= 3249 Col = 3 Row = B, genomic survey sequence. GB_GSS8: AQ070145
285 AQ070145 HS_3027_B1_H02_MR CIT Approved Human Genomic Sperm
Homo sapiens 40,351 5-Aug-98 Library D Homo sapiens genomic clone
Plate = 3027 Col = 3 Row = P, genomic survey sequence. rxa01626 468
GB_PR4: AF152510 2490 AF152510 Homo sapiens protocadherin gamma A3
short form protein (PCDH-gamma-A3) Homo sapiens 34,298 14-Jul-99
variable region sequence, complete cds. GB_PR4: AF152323 4605
AF152323 Homo sapiens protocadherin gamma A3 (PCDH-gamma-A3) Homo
sapiens 34,298 22-Jul-99 mRNA, complete cds. GB_PR4: AF152509 2712
AF152509 Homo sapiens PCDH-gamma-A3 gene, aberrantly spliced, mRNA
sequence. Homo sapiens 34,298 14-Jul-99 rxa01632 1128 GB_HTG4:
AC006590 127171 AC006590 Drosophila melanogaster chromosome 2 clone
BACR13N02 (D543) Drosophila melanogaster 33,812 19-OCT-1999 RPCI-98
13.N.2 map 36E-36E strain y; cn bw sp, *** SEQUENCING IN
PROGRESS***, 101 unordered pieces. GB_HTG4: AC006590 127171
AC006590 Drosophila melanogaster chromosome 2 clone BACR13N02
(D543) Drosophila melanogaster 33,812 19-OCT-1999 RPCI-98 13.N.2
map 36E-36E strain y; cn bw sp, *** SEQUENCING IN PROGRESS***, 101
unordered pieces. GB_GSS8: B99182 415 B99182 CIT-HSP-2280I13.TR
CIT-HSP Homo sapiens genomic clone Homo sapiens 36,111 26-Jun-98
2280I13, genomic survey sequence. rxa01633 1206 GB_BA1: BSUB0009
208780 Z99112 Bacillus subtilis complete genome (section 9 of 21):
from 1598421 to 1807200. Bacillus subtilis 36,591 26-Nov-97 GB_BA1:
BSUB0009 208780 Z99112 Bacillus subtilis complete genome (section 9
of 21): from 1598421 to 1807200. Bacillus subtilis 34,941 26-Nov-97
GB_HTG2: AC006247 174368 AC006247 Drosophila melanogaster
chromosome 2 clone BACR48I10 (D505) Drosophila melanogaster 37,037
2-Aug-99 RPCI-98 48.I.10 map 49E6-49F8 strain y; cn bw sp, ***
SEQUENCING IN PROGRESS ***, 17 unordered pieces. rxa01695 1623
GB_BA1: CGA224946 2408 AJ224946 Corynebacterium glutamicum DNA for
L-Malate:quinone oxidoreductase. Corynebacterium glutamicum 100,000
11-Aug-98 GB_BA1: MTCY24A1 20270 Z95207 Mycobacterium tuberculosis
H37Rv complete genome; segment 124/162. Mycobacterium tuberculosis
38,626 17-Jun-98 GB_IN1: DMU15974 2994 U15974 Drosophila
melanogaster kinesin-like protein (klp68d) mRNA, complete cds.
Drosophila melanogaster 36,783 18-Jul-95 rxa01702 1155 GB_BA1:
CGFDA 3371 X17313 Corynebacterium glutamicum fda gene for
fructose-bisphosphate aldolase (EC Corynebacterium glutamicum
99,913 12-Sep-93 4.1.2.13). GB_BA1: MTY13E10 35019 Z95324
Mycobacterium tuberculosis H37Rv complete genome; segment 18/162.
Mycobacterium tuberculosis 38,786 17-Jun-98 GB_BA1: MLCB4 36310
AL023514 Mycobacterium leprae cosmid B4. Mycobacterium leprae
38,238 27-Aug-99 rxa01743 901 GB_IN2: CELC27H5 35840 U14635
Caenorhabditis elegans cosmid C27H5. Caenorhabditis elegans 35,334
13-Jul-95 GB_EST24: AI167112 579 AI167112 xylem.est.878 Poplar
xylem Lambda ZAPII library Populus balsamifera subsp. Populus
balsamifera subsp. 39,222 03-DEC-1998 trichocarpa cDNA 5', mRNA
sequence. trichocarpa GB_GSS9: AQ102635 347 AQ102635
HS_3048_B1_F08_MF CIT Approved Human Genomic Sperm Homo sapiens
40,653 27-Aug-98 Library D Homo sapiens genomic clone Plate = 3048
Col = 15 Row = L, genomic survey sequence. rxa01744 1662 GB_BA1:
MTCY01B2 35938 Z95554 Mycobacterium tuberculosis H37Rv complete
genome; segment 72/162. Mycobacterium tuberculosis 36,650 17-Jun-98
GB_GSS1: AF009226 665 AF009226 Mycobacterium tuberculosis
cytochrome D oxidase subunit I (appC) Mycobacterium tuberculosis
63,438 31-Jul-97 gene, partial sequence, genomic survey sequence.
GB_BA1: SCD78 36224 AL034355 Streptomyces coelicolor cosmid D78.
Streptomyces coelicolor 53,088 26-Nov-98 rxa01745 836 GB_BA1:
MTCY190 34150 Z70283 Mycobacterium tuberculosis H37Rv complete
genome; segment 98/162. Mycobacterium tuberculosis 62,081 17-Jun-98
GB_BA1: MLCB22 40281 Z98741 Mycobacterium leprae cosmid B22.
Mycobacterium leprae 61,364 22-Aug-97 GB_BA2: AE000175 15067
AE000175 Escherichia coli K-12 MG1655 section 65 of 400 of the
complete genome. Escherichia coli 52,323 12-Nov-98 rxa01758 1140
GB_PR3: HS57G9 113872 Z95116 Human DNA sequence from BAC 57G9 on
chromosome 22q12.1 Homo sapiens 39,209 23-Nov-99 Contains ESTs, CA
repeat, GSS. GB_PL2: YSCH9666 39057 U10397 Saccharomyces cerevisiae
chromosome VIII cosmid 9666. Saccharomyces cerevisiae 40,021
5-Sep-97 GB_PL2: YSCH9986 41664 U00027 Saccharomyces cerevisiae
chromosome VIII cosmid 9986. Saccharomyces cerevisiae 34,375
29-Aug-97 rxa01814 1785 GB_BA1: ABCCELB 2058 L24077 Acetobacter
xylinum phosphoglucomutase (celB) gene, complete cds. Acetobacter
xylinus 62,173 21-Sep-94 GB_BA1: MTCY22D7 31859 Z83866
Mycobacterium tuberculosis H37Rv complete genome; segment 133/162.
Mycobacterium tuberculosis 39,749 17-Jun-98 GB_BA1: MTCY22D7 31859
Z83866 Mycobacterium tuberculosis H37Rv complete genome; segment
133/162. Mycobacterium tuberculosis 40,034 17-Jun-98 rxa01851 1809
GB_GSS9: AQ142579 529 AQ142579 HS_2222_B1_H03_MR CIT Approved Human
Genomic Sperm Homo sapiens 38,068 24-Sep-98 Library D Homo sapiens
genomic clone Plate = 2222 Col = 5 Row = P, genomic survey
sequence. GB_IN2: AC005889 108924 AC005889 Drosophila melanogaster,
chromosome 2L, region 30A3-30A6, P1 Drosophila melanogaster 36,557
30-OCT-1998 clones DS06958 and DS03097, complete sequence. GB_GSS1:
AG008814 637 AG008814 Homo sapiens genomic DNA, 21q region, clone:
B137B7BB68, Homo sapiens 35,316 7-Feb-99 genomic survey sequence.
rxa01859 1050 GB_BA2: AF183408 63626 AF183408 Microcystis
aeruginosa DNA polymerase III beta subunit (dnaN) gene, Microcystis
aeruginosa 36,364 03-OCT-1999 partial cds; microcystin synthetase
gene cluster, complete sequence; Uma1 (uma1), Uma2 (uma2), Uma3
(uma3), Uma4 (uma4), and Uma5 (uma5) genes,
complete cds; and Uma6 (uma6) gene, partial cds. GB_HTG5: AC008031
158889 AC008031 Trypanosoma brucei chromosome II clone
RPCI93-25N14, Trypanosoma brucei 35,334 15-Nov-99 *** SEQUENCING IN
PROGRESS ***, 2 unordered pieces. GB_BA2: AF183408 63626 AF183408
Microcystis aeruginosa DNA polymerase III beta subunit (dnaN) gene,
Microcystis aeruginosa 36,529 03-OCT-1999 partial cds; microcystin
synthetase gene cluster, complete sequence; Uma1 (uma1), Uma2
(uma2), Uma3 (uma3), Uma4 (uma4), and Uma5 (uma5) genes, complete
cds; and Uma6 (uma6) gene, partial cds. rxa01865 438 GB_BA1:
SERFDXA 3869 M61119 Saccharopolyspora erythraea ferredoxin (fdxA)
gene, complete cds. Saccharopolyspora erythraea 59,862 13-MAR-1996
GB_BA1: MTV005 37840 AL010186 Mycobacterium tuberculosis H37Rv
complete genome; segment 51/162. Mycobacterium tuberculosis 61,949
17-Jun-98 GB_BA1: MSGY348 40056 AD000020 Mycobacterium tuberculosis
sequence from clone y348. Mycobacterium tuberculosis 59,908
10-DEC-1996 rxa01882 1113 GB_PR1: HUMADRA2C 1491 J03853 Human
kidney alpha-2-adrenergic receptor mRNA, complete cds. Homo sapiens
36,899 27-Apr-93 GB_PR4: HSU72648 4850 U72648 Homo sapiens
alpha2-C4-adrenergic receptor gene, complete cds. Homo sapiens
36,899 23-Nov-98 GB_GSS3: B42200 387 B42200 HS-1055-B1-A03-MR.abi
CIT Human Genomic Sperm Library C Homo sapiens 34,805 18-OCT-1997
Homo sapiens genomic clone Plate = CT 777 Col = 5 Row = B, genomic
survey sequence. rxa01884 1913 GB_BA1: MTCY48 35377 Z74020
Mycobacterium tuberculosis H37Rv complete genome; segment 69/162.
Mycobacterium tuberculosis 37,892 17-Jun-98 GB_BA1: SCO001206 9184
AJ001206 Streptomyces coelicolor A3(2), glycogen metabolism cluster
II. Streptomyces coelicolor 40,413 29-MAR-1999 GB_BA1: D90908
122349 D90908 Synechocystis sp. PCC6803 complete genome, 10/27,
1188886-1311234. Synechocystis sp. 47,792 7-Feb-99 rxa01886 897
GB_GSS9: AQ116291 572 AQ116291 RPCI11-49P6.TK.1 RPCI-11 Homo
sapiens genomic clone RPCI-11-49P6, Homo sapiens 43,231 20-Apr-99
genomic survey sequence. GB_BA2: AE001721 17632 AE001721 Thermotoga
maritima section 33 of 136 of the complete genome. Thermotoga
maritima 39,306 2-Jun-99 GB_EST16: AA567090 596 AA567090
GM01044.5prime GM Drosophila melanogaster ovary BlueScript
Drosophila melanogaster 42,807 28-Nov-98 Drosophila melanogaster
cDNA clone GM01044 5prime, mRNA sequence. rxa01887 1134 GB_HTG6:
AC008147 303147 AC008147 Homo sapiens clone RP3-405J10, ***
SEQUENCING IN Homo sapiens 36,417 03-DEC-1999 PROGRESS ***, 102
unordered pieces. GB_HTG6: AC008147 303147 AC008147 Homo sapiens
clone RP3-405J10, *** SEQUENCING IN Homo sapiens 37,667 03-DEC-1999
PROGRESS ***, 102 unordered pieces. GB_BA2: ALW243431 26953
AJ243431 Acinetobacter lwoffii wzc, wzb, wza, weeA, weeB, wceC,
wzx, wzy, Acinetobacter lwoffii 39,640 01-OCT-1999 weeD, weeE,
weeF, weeG, weeH, weeI, weeJ, weeK, galU, ugd, pgi, galE, pgm
(partial) and mip (partial) genes (emulsan biosynthetic gene
cluster), strain RAG-1. rxa01888 658 GB_HTG2: AC008197 125235
AC008197 Drosophila melanogaster chromosome 3 clone BACR02L12
(D753) Drosophila melanogaster 32,969 2-Aug-99 RPCI-98 02.L.12 map
94B-94C strain y; cn bw sp, *** SEQUENCING IN PROGRESS***, 113
unordered pieces. GB_HTG2: AC008197 125235 AC008197 Drosophila
melanogaster chromosome 3 clone BACR02L12 (D753) RPCI-98 Drosophila
melanogaster 32,969 2-Aug-99 02.L.12 map 94B-94C strain y; cn bw
sp, *** SEQUENCING IN PROGRESS ***, 113 unordered pieces. GB_EST36:
AI881527 598 AI881527 606070C09.y1 606 - Ear tissue cDNA library
from Schmidt lab Zea mays 43,617 21-Jul-99 Zea mays cDNA, mRNA
sequence. rxa01891 887 GB_VI: HIV232971 621 AJ232971 Human
immunodeficiency virus type 1 subtype C nef gene, patient MP83.
Human immunodeficiency virus 40,040 05-MAR-1999 type 1 GB_PL1:
AFCHSE 6158 Y09542 A. fumigatus chsE gene. Aspergillus fumigatus
37,844 1-Apr-97 GB_PR3: AF064858 193387 AF064858 Homo sapiens
chromosome 21q22.3 BAC 28F9, complete sequence. Homo sapiens 37,136
2-Jun-98 rxa01895 1051 GB_BA1: CGL238250 1593 AJ238250
Corynebacterium glutamicum ndh gene. Corynebacterium glutamicum
100,000 24-Apr-99 GB_BA2: AF038423 1376 AF038423 Mycobacterium
smegmatis NADH dehydrogenase (ndh) gene, complete cds.
Mycobacterium smegmatis 65,254 05-MAY-1998 GB_BA1: MTCY359 36021
Z83859 Mycobacterium tuberculosis H37Rv complete genome; segment
84/162. Mycobacterium tuberculosis 40,058 17-Jun-98 rxa01901 1383
GB_BA1: MSGB38COS 37114 L01095 M. leprae genomic DNA sequence,
cosmid B38 bfr gene, complete cds. Mycobacterium leprae 59,551
6-Sep-94 GB_BA1: SCE63 37200 AL035640 Streptomyces coelicolor
cosmid E63. Streptomyces coelicolor 39,468 17-MAR-1999 GB_PR3:
AF093117 147216 AF093117 Homo sapiens chromosome 7qtelo BAC E3,
complete sequence. Homo sapiens 39,291 02-OCT-1998 rxa01927 1503
GB_BA1: CGPAN 2164 X96580 C. glutamicum panB, panC & xylB
genes. Corynebacterium glutamicum 38,384 11-MAY-1999 GB_BA1: ASXYLA
1905 X59466 Arthrobacter Sp. N.R.R.L. B3728 xylA gene for
D-xylose(D-glucose) Arthrobacter sp. 56,283 04-MAY-1992 isomerase.
GB_HTG3: AC009500 176060 AC009500 Homo sapiens clone NH0511A20, ***
SEQUENCING IN PROGRESS ***, 6 Homo sapiens 37,593 24-Aug-99
unordered pieces. rxa01952 1836 GB_BA2: AE000739 13335 AE000739
Aquifex aeolicus section 71 of 109 of the complete genome. Aquifex
aeolicus 36,309 25-MAR-1998 GB_EST28: AI519629 612 AI519629
LD39282.5prime LD Drosophila melanogaster embryo pOT2 Drosophila
Drosophila melanogaster 41,941 16-MAR-1999 melanogaster cDNA clone
LD39282 5prime, mRNA sequence. GB_EST21: AA949396 767 AA949396
LD28277.5prime LD Drosophila melanogaster embryo pOT2 Drosophila
Drosophila melanogaster 39,855 25-Nov-98 melanogaster cDNA clone
LD28277 5prime, mRNA sequence. rxa01989 630 GB_BA1: BSPGIA 1822
X16639 Bacillus stearothermophilus pgiA gene for
phosphoglucoisomerase Bacillus stearothermophilus 66,292 20-Apr-95
isoenzyme A (EC 5.3.1.9). GB_BA1: BSUB0017 217420 Z99120 Bacillus
subtilis complete genome (section 17 of 21): from Bacillus subtilis
37,255 26-Nov-97 3197001 to 3414420. GB_BA2: AF132127 8452 AF132127
Streptococcus mutans sorbitol phosphoenolpyruvate:sugar
phosphotransferase Streptococcus mutans 63,607 28-Sep-99 operon,
complete sequence and unknown gene. rxa02026 720 GB_BA1: SXSCRBA
3161 X67744 S. xylosus scrB and scrR genes. Staphylococcus xylosus
67,778 28-Nov-96 GB_BA1: BSUB0020 212150 Z99123 Bacillus subtilis
complete genome (section 20 of 21): from Bacillus subtilis 35,574
26-Nov-97 3798401 to 4010550. GB_BA1: BSGENR 97015 X73124 B.
subtilis genomic region (325 to 333). Bacillus subtilis 51,826
2-Nov-93 rxa02028 526 GB_BA1: MTCI237 27030 Z94752 Mycobacterium
tuberculosis H37Rv complete genome; segment 46/162. Mycobacterium
tuberculosis 54,476 17-Jun-98 GB_PL2: SCE9537 66030 U18778
Saccharomyces cerevisiae chromosome V cosmids 9537, 9581, 9495,
Saccharomyces cerevisiae 36,100 1-Aug-97 9867, and lambda clone
5898. GB_GSS13: AQ501177 767 AQ501177 V26G9 mTn-3xHA/lacZ Insertion
Library Saccharomyces cerevisiae Saccharomyces cerevisiae 32,039
29-Apr-99 genomic 5', genomic survey sequence. rxa02054 1140
GB_BA1: MLCB1222 34714 AL049491 Mycobacterium leprae cosmid B1222.
Mycobacterium leprae 61,896 27-Aug-99 GB_BA1: MTY13E12 43401 Z95390
Mycobacterium tuberculosis H37Rv complete genome; segment 147/162.
Mycobacterium tuberculosis 59,964 17-Jun-98 GB_BA1: MTU43540 3453
U43540 Mycobacterium tuberculosis rfbA, rhamnose biosynthesis
protein (rfbA), Mycobacterium tuberculosis 59,659 14-Aug-97 and
rmlC genes, complete cds. rxa02056 2891 GB_PAT: E14601 4394 E14601
Brevibacterium lactofermentum gene for alpha-ketoglutaric acid
Corynebacterium glutamicum 98,928 28-Jul-99 dehydrogenase. GB_BA1:
D84102 4394 D84102 Corynebacterium glutamicum DNA for
2-oxoglutarate dehydrogenase, Corynebacterium glutamicum 98,928
6-Feb-99 complete cds. GB_BA1: MTV006 22440 AL021006 Mycobacterium
tuberculosis H37Rv complete genome; Mycobacterium tuberculosis
39,265 18-Jun-98 segment 54/162. rxa02061 1617 GB_HTG7: AC005883
211682 AC005883 Homo sapiens chromosome 17 clone RP11-958E11 map
17, Homo sapiens 37,453 08-DEC-1999 *** SEQUENCING IN PROGRESS ***,
2 ordered pieces. GB_PL2: ATAC003033 84254 AC003033 Arabidopsis
thaliana chromosome II BAC T21L14 genomic sequence, Arabidopsis
thaliana 37,711 19-DEC-1997 complete sequence. GB_PL2: ATAC002334
75050 AC002334 Arabidopsis thaliana chromosome II BAC F25I18
genomic sequence, Arabidopsis thaliana 37,711 04-MAR-1998 complete
sequence. rxa02063 1350 GB_BA1: SCGLGC 1518 X89733 S. coelicolor
DNA for glgC gene. Streptomyces coelicolor 56,972 12-Jul-99
GB_GSS4: AQ687350 786 AQ687350 nbxb0074H11r CUGI Rice BAC Library
Oryza sativa genomic clone Oryza sativa 40,696 1-Jul-99
nbxb0074H11r, genomic survey sequence. GB_EST38: AW028530 444
AW028530 wv27f10.x1 NCI_CGAP_Kid11 Homo sapiens cDNA clone Homo
sapiens 36,795 27-OCT-1999 IMAGE: 2530795 3' similar to WP:
T03G11.6 CE04874;, mRNA sequence. rxa02100 2348 GB_BA1: MSGY151
37036 AD000018 Mycobacterium tuberculosis sequence from clone y151.
Mycobacterium tuberculosis 40,156 10-DEC-1996 GB_BA1: MTCY130 32514
Z73902 Mycobacterium tuberculosis H37Rv complete genome; segment
59/162. Mycobacterium tuberculosis 55,218 17-Jun-98 GB_BA1:
SCO001205 9589 AJ001205 Streptomyces coelicolor A3(2) glycogen
metabolism clusterl. Streptomyces coelicolor 38,475 29-MAR-1999
rxa02122 822 GB_BA1: D90858 13548 D90858 E. coli genomic DNA,
Kohara clone #401(51.3-51.6 min.). Escherichia coli 38,586
29-MAY-1997 GB_EST37: AI948595 469 AI948595 wq07d12.x1
NCI_CGAP_Kid12 Homo sapiens cDNA clone Homo sapiens 37,259 6-Sep-99
IMAGE: 2470583 3', mRNA sequence. GB_HTG3: AC010387 220665 AC010387
Homo sapiens chromosome 5 clone CITB-H1_2074D8, Homo sapiens 38,868
15-Sep-99 *** SEQUENCING IN PROGRESS ***, 77 unordered pieces.
rxa02140 1200 GB_BA1: MSGB1551CS 36548 L78813 Mycobacterium leprae
cosmid B1551 DNA sequence. Mycobacterium leprae 51,399 15-Jun-96
GB_BA1: MSGB1554CS 36548 L78814 Mycobacterium leprae cosmid B1554
DNA sequence. Mycobacterium leprae 51,399 15-Jun-96 GB_RO: AF093099
2482 AF093099 Mus musculus transcription factor TBLYM (Tblym) mRNA,
complete cds. Mus musculus 36,683 01-OCT-1999 rxa02142 774 GB_BA1:
MTCY190 34150 Z70283 Mycobacterium tuberculosis H37Rv complete
genome; segment 98/162. Mycobacterium tuberculosis 57,292 17-Jun-98
GB_BA1: SC6G10 36734 AL049497 Streptomyces coelicolor cosmid 6G10.
Streptomyces coelicolor 35,058 24-MAR-1999 GB_BA1: AB016787 5550
AB016787 Pseudomonas putida genes for cytochrome o ubiquinol
oxidase A-E and Pseudomonas putida 47,403 5-Aug-99 2 ORFs, complete
cds. rxa02143 1011 GB_BA1: MTCY190 34150 Z70283 Mycobacterium
tuberculosis H37Rv complete genome; segment 98/162. Mycobacterium
tuberculosis 57,317 17-Jun-98 GB_BA1: MSGB1551CS 36548 L78813
Mycobacterium leprae cosmid B1551 DNA sequence. Mycobacterium
leprae 38,159 15-Jun-96 GB_BA1: MSGB1554CS 36548 L78814
Mycobacterium leprae cosmid B1554 DNA sequence. Mycobacterium
leprae 38,159 15-Jun-96 rxa02144 1347 GB_BA1: MTCY190 34150 Z70283
Mycobacterium tuberculosis H37Rv complete genome; segment 98/162.
Mycobacterium tuberculosis 55,530 17-Jun-98 GB_HTG3: AC011500_0
300851 AC011500 Homo sapiens chromosome 19 clone CIT978SKB_60E11,
Homo sapiens 39,659 18-Feb-00 *** SEQUENCING IN PROGRESS ***, 246
unordered pieces. GB_HTG3: AC011500_0 300851 AC011500 Homo sapiens
chromosome 19 clone CIT978SKB_60E11, Homo sapiens 39,659 18-Feb-00
*** SEQUENCING IN PROGRESS ***, 246 unordered pieces. rxa02147 1140
GB_EST28: AI492095 485 AI492095 tg07a01.x1 NCI_CGAP_CLL1 Homo
sapiens cDNA clone Homo sapiens 39,798 30-MAR-1999 IMAGE: 2108040
3', mRNA sequence. GB_EST10: AA157467 376 AA157467 zo50e01.r1
Stratagene endothelial cell 937223 Homo sapiens cDNA clone Homo
sapiens 36,436 11-DEC-1996 IMAGE: 590328 5', mRNA sequence.
GB_EST10: AA157467 376 AA157467 zo50e01.r1 Stratagene endothelial
cell 937223 Homo sapiens cDNA clone Homo sapiens 36,436 11-DEC-1996
IMAGE: 590328 5', mRNA sequence. rxa02149 1092 GB_PR3: HSBK277P6
61698 AL117347 Human DNA sequence from clone 277P6 on chromosome
1q25.3-31.2, Homo sapiens 36,872 23-Nov-99 complete sequence.
GB_BA2: EMB065R075 360 AF116423 Rhizobium etli mutant MB045
RosR-transcriptionally regulated sequence. Rhizobium etli 43,175
06-DEC-1999 GB_EST34: AI789323 574 AI789323 uk53g05.y1 Sugano mouse
kidney mkia Mus musculus cDNA clone Mus musculus 39,715 2-Jul-99
IMAGE: 1972760 5' similar to WP: K11H12.8 CE12160;, mRNA sequence.
rxa02175 1416 GB_BA1: CGGLTG 3013 X66112 C. glutamicum glt gene for
citrate synthase and ORF. Corynebacterium glutamicum 100,000
17-Feb-95 GB_BA1: MTCY31 37630 Z73101 Mycobacterium tuberculosis
H37Rv complete genome; segment 41/162. Mycobacterium tuberculosis
64,331 17-Jun-98 GB_BA1: MLCB57 38029 Z99494 Mycobacterium leprae
cosmid B57. Mycobacterium leprae 62,491 10-Feb-99 rxa02196 816
GB_RO: RATDAPRP 2819 M76426 Rattus norvegicus dipeptidyl
aminopeptidase-related protein (dpp6) mRNA, Rattus norvegicus
38,791 31-MAY-1995 complete cds. GB_GSS8: AQ012162 763 AQ012162
127PB037070197 Cosmid library of chromosome II Rhodobacter
sphaeroides Rhodobacter sphaeroides 40,044 4-Jun-98 genomic clone
127PB037070197, genomic survey sequence. GB_RO: RATDAPRP 2819
M76426 Rattus norvegicus dipeptidyl aminopeptidase-related protein
(dpp6) mRNA, Rattus norvegicus 37,312 31-MAY-1995 complete cds.
rxa02209 1694 GB_BA1: AB025424 2995 AB025424 Corynebacterium
glutamicum gene for aconitase, partial cds. Corynebacterium
glutamicum 99,173
3-Apr-99 GB_BA2: AF002133 15437 AF002133 Mycobacterium avium strain
GIR10 transcriptional regulator (mav81) gene, Mycobacterium avium
40,219 26-MAR-1998 partial cds, aconitase (acn), invasin 1 (inv1),
invasin 2 (inv2), transcriptional regulator (moxR),
ketoacyl-reductase (fabG), enoyl-reductase (inhA) and
ferrochelatase (mav272) genes, complete cds. GB_BA1: MTV007 32806
AL021184 Mycobacterium tuberculosis H37Rv complete genome; segment
64/162. Mycobacterium tuberculosis 38,253 17-Jun-98 rxa02213 874
GB_BA1: AB025424 2995 AB025424 Corynebacterium glutamicum gene for
aconitase, partial cds. Corynebacterium glutamicum 99,096 3-Apr-99
GB_BA1: MTV007 32806 AL021184 Mycobacterium tuberculosis H37Rv
complete genome; segment 64/162. Mycobacterium tuberculosis 34,937
17-Jun-98 GB_BA2: AF002133 15437 AF002133 Mycobacterium avium
strain GIR10 transcriptional regulator (mav81) gene, Mycobacterium
avium 36,885 26-MAR-1998 partial cds, aconitase (acn), invasin 1
(inv1), invasin 2 (inv2), transcriptional regulator (moxR),
ketoacyl-reductase (fabG), enoyl-reductase (inhA) and
ferrochelatase (mav272) genes, complete cds. rxa02245 780 GB_BA2:
RCU23145 5960 U23145 Rhodobacter capsulatus Calvin cycle carbon
dioxide fixation operon: Rhodobacter capsulatus 48,701 28-OCT-1997
fructose-1,6-/sedoheptulose-1,7-bisphosphate aldolase (cbbA) gene,
partial cds, Form II ribulose-1,5-bisphosphate
carboxylase/oxygenase (cbbM) gene, complete cds, and Calvin cycle
operon: pentose-5-phosphate-3-epimerase (cbbE), phosphoglycolate
phosphatase (cbbZ), and cbbY genes, complete cds. GB_BA1: ECU82664
139818 U82664 Escherichia coli minutes 9 to 11 genomic sequence.
Escherichia coli 39,119 11-Jan-97 GB_HTG2: AC007922 158858 AC007922
Homo sapiens chromosome 18 clone hRPK.178_F_10 map 18, Homo sapiens
33,118 26-Jun-99 *** SEQUENCING IN PROGRESS ***, 11 unordered
pieces. rxa02256 1125 GB_BA1: CGGAPPGK 3804 X59403 C. glutamicum
gap, pgk and tpi genes for glyceraldehyde-3-phosphate,
Corynebacterium glutamicum 99,289 05-OCT-1992 phosphoglycerate
kinase and triosephosphate isomerase. GB_BA1: SCC54 30753 AL035591
Streptomyces coelicolor cosmid C54. Streptomyces coelicolor 36,951
11-Jun-99 GB_BA1: MTCY493 40790 Z95844 Mycobacterium tuberculosis
H37Rv complete genome; segment 63/162. Mycobacterium tuberculosis
64,196 19-Jun-98 rxa02257 1338 GB_BA1: CGGAPPGK 3804 X59403 C.
glutamicum gap, pgk and tpi genes for glyceraldehyde-3-phosphate,
Corynebacterium glutamicum 98,873 05-OCT-1992 phosphoglycerate
kinase and triosephosphate isomerase. GB_BA1: MTCY493 40790 Z95844
Mycobacterium tuberculosis H37Rv complete genome; segment 63/162.
Mycobacterium tuberculosis 61,273 19-Jun-98 GB_BA2: MAU82749 2530
U82749 Mycobacterium avium glyceraldehyde-3-phosphate dehydrogenase
homolog Mycobacterium avium 61,772 6-Jan-98 (gapdh) gene, complete
cds; and phosphoglycerate kinase gene, partial cds. rxa02258 900
GB_BA1: CGGAPPGK 3804 X59403 C. glutamicum gap, pgk and tpi genes
for glyceraldehyde-3-phosphate, Corynebacterium glutamicum 99,667
05-OCT-1992 phosphoglycerate kinase and triosephosphate isomerase.
GB_BA1: CORPEPC 4885 M25819 C. glutamicum phosphoenolpyruvate
carboxylase gene, complete cds. Corynebacterium glutamicum 100,000
15-DEC-1995 GB_PAT: A09073 4885 A09073 C. glutamicum ppg gene for
phosphoenol pyruvate carboxylase. Corynebacterium glutamicum
100,000 25-Aug-93 rxa02259 2895 GB_BA1: CORPEPC 4885 M25819 C.
glutamicum phosphoenolpyruvate carboxylase gene, complete cds,
Corynebacterium glutamicum 100,000 15-DEC-1995 GB_PAT: A09073 4885
A09073 C. glutamicum ppg gene for phosphoenol pyruvate carboxylase.
Corynebacterium glutamicum 100,000 25-Aug-93 GB_BA1: CGPPC 3292
X14234 Corynebacterium glutamicum phosphoenolpyruvate carboxylase
gene Corynebacterium glutamicum 99,827 12-Sep-93 (EC 4.1.1.31).
rxa02288 969 GB_PR3: HSDJ94E24 243145 AL050317 Human DNA sequence
from clone RP1-94E24 on chromosome 20q12, Homo sapiens 36,039
03-DEC-1999 complete sequence. GB_HTG3: AC010091 159526 AC010091
Homo sapiens clone NH0295A01, *** SEQUENCING IN PROGRESS ***, 4
Homo sapiens 35,331 11-Sep-99 unordered pieces. GB_HTG3: AC010091
159526 AC010091 Homo sapiens clone NH0295A01, *** SEQUENCING IN
PROGRESS ***, 4 Homo sapiens 35,331 11-Sep-99 unordered pieces.
rxa02292 798 GB_BA2: AF125164 26443 AF125164 Bacteroides fragilis
638R polysaccharide B (PS B2) biosynthesis locus, Bacteroides
fragilis 39,747 01-DEC-1999 complete sequence; and unknown genes.
GB_GSS5: AQ744695 827 AQ744695 HS_5505_A2_C06_SP6 RPCI-11 Human
Male BAC Library Homo sapiens 39,185 16-Jul-99 Homo sapiens genomic
clone Plate = 1081 Col = 12 Row = E, genomic survey sequence.
GB_EST14: AA381925 309 AA381925 EST95058 Activated T-cells I Homo
sapiens cDNA 5' end, mRNA sequence. Homo sapiens 35,922 21-Apr-97
rxa02322 511 GB_BA1: MTCY22G8 22550 Z95585 Mycobacterium
tuberculosis H37Rv complete genome; segment 49/162. Mycobacterium
tuberculosis 57,677 17-Jun-98 GB_BA1: MTCY22G8 22550 Z95585
Mycobacterium tuberculosis H37Rv complete genome; segment 49/162.
Mycobacterium tuberculosis 37,143 17-Jun-98 rxa02326 939 GB_BA1:
CGPYC 3728 Y09548 Corynebacterium glutamicum pyc gene,
Corynebacterium glutamicum 100,000 08-MAY-1998 GB_BA2: AF038548
3637 AF038548 Corynebacterium glutamicum pyruvate carboxylase (pyc)
gene, complete cds. Corynebacterium glutamicum 100,000 24-DEC-1997
GB_BA1: MTCY349 43523 Z83018 Mycobacterium tuberculosis H37Rv
complete genome; segment 131/162. Mycobacterium tuberculosis 37,363
17-Jun-98 rxa02327 1083 GB_BA1: CGPYC 3728 Y09548 Corynebacterium
glutamicum pyc gene. Corynebacterium glutamicum 99,259 08-MAY-1998
GB_BA2: AF038548 3637 AF038548 Corynebacterium glutamicum pyruvate
carboxylase (pyc) gene, complete cds. Corynebacterium glutamicum
99,259 24-DEC-1997 GB_BA1: MTCY349 43523 Z83018 Mycobacterium
tuberculosis H37Rv complete genome; segment 131/162. Mycobacterium
tuberculosis 41,317 17-Jun-98 rxa02328 1719 GB_BA1: CGPYC 3728
Y09548 Corynebacterium glutamicum pyc gene. Corynebacterium
glutamicum 100,000 08-MAY-1998 GB_BA2: AF038548 3637 AF038548
Corynebacterium glutamicum pyruvate carboxylase (pyc) gene,
complete cds. Corynebacterium glutamicum 100,000 24-DEC-1997
GB_PL2: AF097728 3916 AF097728 Aspergillus terreus pyruvate
carboxylase (Pyc) mRNA, complete cds. Aspergillus terreus 52,248
29-OCT-1998 rxa02332 1266 GB_BA1: MSGLTA 1776 X60513 M. smegmatis
gltA gene for citrate synthase. Mycobacterium smegmatis 58,460
20-Sep-91 GB_BA2: ABU85944 1334 U85944 Antarctic bacterium DS2-3R
citrate synthase (cisy) gene, complete cds. Antarctic bacterium
DS2-3R 57,154 23-Sep-97 GB_BA2: AE000175 15067 AE000175 Escherichia
coli K-12 MG1655 section 65 of 400 of the complete genome.
Escherichia coli 38,164 12-Nov-98 rxa02333 1038 GB_BA1: MSGLTA 1776
X60513 M. smegmatis gltA gene for citrate synthase. Mycobacterium
smegmatis 58,929 20-Sep-91 GB_PR4: HUAC002299 171681 AC002299 Homo
sapiens Chromosome 16 BAC clone Homo sapiens 33,070 23-Nov-99
CIT987-SKA-113A6 .about.complete genomic sequence, complete
sequence. GB_HTG2: AC007889 127840 AC007889 Drosophila melanogaster
chromosome 3 clone BACR48E12 (D695) RPCI-98 Drosophila melanogaster
34,897 2-Aug-99 48.E.12 map 87A-87B strain y; cn bw sp, ***
SEQUENCING IN PROGRESS***, 86 unordered pieces. rxa02399 1467
GB_BA1: CGACEA 2427 X75504 C. glutamicum aceA gene and thiX genes
(partial). Corynebacterium glutamicum 100,000 9-Sep-94 GB_BA1:
CORACEA 1905 L28760 Corynebacterium glutamicum isocitrate lyase
(aceA) gene. Corynebacterium glutamicum 100,000 10-Feb-95 GB_PAT:
I13693 2135 I13693 Sequence 3 from patent U.S. Pat. No. 5439822.
Unknown. 99,795 26-Sep-95 rxa02404 2340 GB_BA1: CGACEB 3024 X78491
C. glutamicum (ATCC 13032) aceB gene. Corynebacterium glutamicum
99,914 13-Jan-95 GB_BA1: CORACEB 2725 L27123 Corynebacterium
glutamicum malate synthase (aceB) gene, complete cds.
Corynebacterium glutamicum 99,786 8-Jun-95 GB_BA1: PFFC2 5588
Y11998 P. fluorescens FC2.1, FC2.2, FC2.3c, FC2.4 and FC2.5c open
reading frames. Pseudomonas fluorescens 63,539 11-Jul-97 rxa02414
870 GB_PR4: AC007102 176258 AC007102 Homo sapiens chromosome 4
clone C0162P16 map 4p16, complete sequence. Homo sapiens 35,069
2-Jun-99 GB_HTG3: AC011214 183414 AC011214 Homo sapiens clone
5_C_3, LOW-PASS SEQUENCE SAMPLING. Homo sapiens 36,885 03-OCT-1999
GB_HTG3: AC011214 183414 AC011214 Homo sapiens clone 5_C_3,
LOW-PASS SEQUENCE SAMPLING. Homo sapiens 36,885 03-OCT-1999
rxa02435 681 GB_BA2: AF101055 7457 AF101055 Clostridium
acetobutylicum atp operon, complete sequence. Clostridium
acetobutylicum 39,605 03-MAR-1999 GB_OM: RABPKA 4441 J03247 Rabbit
phosphorylase kinase (alpha subunit) mRNA, complete cds.
Oryctolagus cuniculus 36,061 27-Apr-93 GB_OM: RABPLASISM 4458
M64656 Oryctolagus cuniculus phosphorylase kinase alpha subunit
mRNA, Oryctolagus cuniculus 36,000 22-Jun-98 complete cds. rxa02440
963 GB_EST14: AA417723 374 AA417723 zv01b12.s1 NCI_CGAP_GCB1 Homo
sapiens cDNA clone IMAGE: 746207 Homo sapiens 38,770 16-OCT-1997 3'
similar to contains Alu repetitive element; contains element L1
repetitive element;, mRNA sequence. GB_EST11: AA215428 303 AA215428
zr95a07.s1 NCI_CGAP_GCB1 Homo sapiens cDNA clone IMAGE: 683412 Homo
sapiens 39,934 13-Aug-97 3' similar to contains Alu repetitive
element;, mRNA sequence. GB_BA1: MTCY77 22255 Z95389 Mycobacterium
tuberculosis H37Rv complete genome; segment 146/162. Mycobacterium
tuberculosis 38,889 18-Jun-98 rxa02453 876 GB_EST14: AA426336 375
AA426336 zv53g02.s1 Soares_testis_NHT Homo sapiens cDNA clone
IMAGE: Homo sapiens 38,043 16-OCT-1997 757394 3', mRNA sequence.
GB_BA1: STMAACC8 1353 M55426 S. fradiae aminoglycoside
acetyltransferase (aacC8) gene, complete cds. Streptomyces fradiae
37,097 05-MAY-1993 GB_PR3: AC004500 77538 AC004500 Homo sapiens
chromosome 5, P1 clone 1076B9 (LBNL H14), Homo sapiens 33,256
30-MAR-1998 complete sequence. rxa02474 897 GB_BA1: AB009078 2686
AB009078 Brevibacterium saccharolyticum gene for L-2.3-butanediol
dehydrogenase, Brevibacterium saccharolyticum 96,990 13-Feb-99
complete cds. GB_OM: BTU71200 877 U71200 Bos taurus acetoin
reductase mRNA, complete cds. Bos taurus 51,659 8-Oct-97 GB_EST2:
F12685 287 F12685 HSC3DA031 normalized infant brain cDNA Homo
sapiens cDNA Homo sapiens 41,509 14-Mar-95 clone c-3da03, mRNA
sequence rxa02480 1779 GB_BA1: MTV012 70287 AL021287 Mycobacterium
tuberculosis H37Rv complete genome; segment 132/162. Mycobacterium
tuberculosis 36,737 23-Jun-99 GB_BA1: SC6G10 36734 AL049497
Streptomyces coelicolor cosmid 6G10. Streptomyces coelicolor 35,511
24-MAR-1999 GB_BA1: AP000060 347800 AP000060 Aeropyrum pernix
genomic DNA, section 3/7. Aeropyrum pemix 48,014 22-Jun-99 rxa02485
rxa02492 840 GB_BA1: STMPGM 921 M83661 Streptomyces coelicolor
phosphoglycerate mutase (PGM) gene, complete cds. Streptomyces
coelicolor 65,672 26-Apr-93 GB_BA1: MTCY20G9 37218 Z77162
Mycobacterium tuberculosis H37Rv complete genome; segment 25/162.
Mycobacterium tuberculosis 61,436 17-Jun-98 GB_BA1: U00018 42991
U00018 Mycobacterium leprae cosmid B2168. Mycobacterium leprae
37,893 01-MAR-1994 rxa02528 1098 GB_PR2: HS161N10 56075 AL008707
Human DNA sequence from PAC 161N10 on chromosome Xq25. Homo sapiens
37,051 23-Nov-99 Contains EST. GB_HTG2: AC008235 136017 AC008235
Drosophila melanogaster chromosome 3 clone BACR15B19 (D995) RPCI-98
Drosophila melanogaster 36,822 2-Aug-99 15.B.19 map 94F-95A strain
y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 125 unordered pieces.
GB_HTG2: AC008235 136017 AC008235 Drosophila melanogaster
chromosome 3 clone BACR15B19 (D995) RPCI-98 Drosophila melanogaster
36,822 2-Aug-99 15.B.19 map 94F-95A strain y; cn bw sp, ***
SEQUENCING IN PROGRESS***, 125 unordered pieces. rxa02539 1641
GB_BA2: RSU17129 17425 U17129 Rhodococcus erythropolis ThcA (thcA)
gene, complete cds; and Rhodococcus erythropolis 66,117 16-Jul-99
unknown genes. GB_BA1: MTV038 16094 AL021933 Mycobacterium
tuberculosis H37Rv complete genome; segment 24/162. Mycobacterium
tuberculosis 65,174 17-Jun-98 GB_BA2: AF068264 3152 AF068264
Pseudomonas aeruginosa quinoprotein ethanol dehydrogenase
Pseudomonas aeruginosa 65,448 18-MAR-1999 (exaA)gene, partial cds;
cytochrome c550 precursor (exaB), NAD+ dependent acetaldehyde
dehydrogenase (exaC), and pyrroloquinoline quinone synthesis A
(pqqA) genes, complete cds; and pyrroloquinoline quinone synthesis
B (pqqB) gene, partial cds. rxa02551 483 GB_BA1: BACHYPTP 17057
D29985 Bacillus subtilis wapA and orf genes for wall-associated
protein Bacillus subtilis 53,602 7-Feb-99 and hypothetical
proteins. GB_BA1: BACHUTWAPA 28954 D31856 Bacillus subtilis genome
containing the hut and wapA loci. Bacillus subtilis 53,602 7-Feb-99
GB_BA1: BSGBGLUC 4290 Z34526 B. subtilis (Marburg 168) genes for
beta-glucoside permease Bacillus subtilis 53,602 3-Jul-95 and
beta-glucosidase. rxa02556 1281 GB_HTG3: AC008128 335761 AC008128
Homo sapiens, *** SEQUENCING IN PROGRESS ***, 106 unordered pieces.
Homo sapiens 34,022 22-Aug-99 GB_HTG3: AC008128 335761 AC008128
Homo sapiens, *** SEQUENCING IN PROGRESS ***, 106 unordered pieces.
Homo sapiens 34,022 22-Aug-99 GB_PL2: AC005292 99053 AC005292
Genomic sequence for Arabidopsis thaliana BAC F26F24, complete
sequence. Arabidopsis thaliana 33,858 16-Apr-99 rxa02560 990
GB_IN1: CEF07A11 35692 Z66511 Caenorhabditis elegans cosmid F07A11,
complete sequence. Caenorhabditis elegans 36,420 2-Sep-99 GB_EST32:
AI731605 566 AI731605 BNLGHi10201 Six-day Cotton fiber Gossypium
hirsutum cDNA 5' similar to Gossypium hirsutum 38,095 11-Jun-99
(AC004684) hypothetical protein [Arabidopsis thaliana], mRNA
sequence. GB_IN1: CEF07A11 35692 Z66511 Caenorhabditis elegans
cosmid F07A11, complete sequence. Caenorhabditis elegans 33,707
2-Sep-99 rxa02572 668 GB_BA1: MTCY63 38900 Z96800 Mycobacterium
tuberculosis H37Rv
complete genome; segment 16/162. Mycobacterium tuberculosis 61,677
17-Jun-98 GB_BA1: MTCY63 38900 Z96800 Mycobacterium tuberculosis
H37Rv complete genome; segment 16/162. Mycobacterium tuberculosis
37,170 17-Jun-98 GB_HTG1: HS24H01 46989 AL121632 Homo sapiens
chromosome 21 clone LLNLc116H0124 map 21q21, *** Homo sapiens
19,820 29-Sep-99 SEQUENCING IN PROGRESS ***, in unordered pieces.
rxa02596 1326 GB_BA1: MTV026 23740 AL022076 Mycobacterium
tuberculosis H37Rv complete genome; segment 157/162. Mycobacterium
tuberculosis 36,957 24-Jun-99 GB_BA2: AF026540 1778 AF026540
Mycobacterium tuberculosis UDP-galactopyranose mutase (glf) gene,
Mycobacterium tuberculosis 67,627 30-OCT-1998 complete cds. GB_BA2:
MTU96128 1200 U96128 Mycobacterium tuberculosis UDP-galactopyranose
mutase (glf) gene, Mycobacterium tuberculosis 70,417 25-MAR-1998
complete cds. rxa02611 1775 GB_BA1: MTCY130 32514 Z73902
Mycobacterium tuberculosis H37Rv complete genome; segment 59/162.
Mycobacterium tuberculosis 38,532 17-Jun-98 GB_BA1: MSGY151 37036
AD000018 Mycobacterium tuberculosis sequence from clone y151.
Mycobacterium tuberculosis 60,575 10-DEC-1996 GB_BA1: U00014 36470
U00014 Mycobacterium leprae cosmid B1549. Mycobacterium leprae
57,486 29-Sep-94 rxa02612 2316 GB_BA1: MTCY130 32514 Z73902
Mycobacterium tuberculosis H37Rv complete genome; segment 59/162.
Mycobacterium tuberculosis 38,018 17-Jun-98 GB_BA1: MSGY151 37036
AD000018 Mycobacterium tuberculosis sequence from clone y151.
Mycobacterium tuberculosis 58,510 10-DEC-1996 GB_BA1: STMGLGEN 2557
L11647 Streptomyces aureofaciens glycogen branching enzyme (glgB)
Streptomyces aureofaciens 57,193 25-MAY-1995 gene, complete cds.
rxa02621 942 GB_BA1: CGL133719 1839 AJ133719 Corynebacterium
glutamicum yjcc gene, amtR gene and citE gene, partial.
Corynebacterium glutamicum 36,858 12-Aug-99 GB_IN1: CEM106 39973
Z46935 Caenorhabditis elegans cosmid M106, complete sequence.
Caenorhabditis elegans 37,608 2-Sep-99 GB_EST29: AI547662 377
AI547662 UI-R-C3-sz-h-03-0-UI.s1 UI-R-C3 Rattus norvegicus cDNA
Rattus norvegicus 50,667 3-Jul-99 clone UI-R-C3-sz-h-03-0-UI 3',
mRNA sequence. rxa02640 1650 GB_BA1: MTV025 121125 AL022121
Mycobacterium tuberculosis H37Rv complete genome; segment 155/162.
Mycobacterium tuberculosis 39,187 24-Jun-99 GB_BA1: PAU49666 4495
U49666 Pseudomonas aeruginosa (orfX), glycerol diffusion
facilitator Pseudomonas aeruginosa 59,273 18-MAY-1997 (glpF),
glycerol kinase (glpK), and Glp repressor (glpR) genes, complete
cds, and (orfK) gene, partial cds. GB_BA1: AB015974 1641 AB015974
Pseudomonas tolaasii glpK gene for glycerol kinase, complete cds.
Pseudomonas tolaasii 58,339 28-Aug-99 rxa02654 1008 GB_EST6: N65787
512 N65787 20827 Lambda-PRL2 Arabidopsis thaliana cDNA Arabidopsis
thaliana 39,637 5-Jan-98 clone 232B7T7, mRNA sequence. GB_PL2:
T17H3 65839 AC005916 Arabidopsis thaliana chromosome 1 BAC T17H3
Arabidopsis thaliana 33,735 5-Aug-99 sequence, complete sequence.
GB_RO: MMU58105 88871 U58105 Mus musculus Btk locus,
alpha-D-galactosidase A Mus musculus 35,431 13-Feb-97 (Ags),
ribosomal protein (L44L), and Bruton's tyrosine kinase (Btk) genes,
complete cds. rxa02666 891 GB_PR3: AC004643 43411 AC004643 Homo
sapiens chromosome 16, cosmid clone Homo sapiens 38,851 01-MAY-1998
363E3 (LANL), complete sequence. GB_PR3: AC004643 43411 AC004643
Homo sapiens chromosome 16, cosmid clone 363E3 Homo sapiens 41,599
01-MAY-1998 (LANL), complete sequence. GB_BA2: AF049897 9196
AF049897 Corynebacterium glutamicum N-acetylglutamylphosphate
Corynebacterium glutamicum 40,413 1-Jul-98 reductase (argC),
ornithine acetyltransferase (argJ), N-acetylglutamate kinase
(argB), acetylornithine transaminase (argD), ornithine
carbamoyltransferase (argF), arginine repressor (argR),
argininosuccinate synthase (argG), and argininosuccinate lyase
(argH) genes, complete cds. rxa02675 1980 GB_BA1: PDENQOURF 10425
L02354 Paracoccus denitrificans NADH dehydrogenase (URF4),
Paracoccus denitrificans 40,735 20-MAY-1993 (NQO8), (NQO9), (URF5),
(URF6), (NQO10), (NQO11), (NQO12), (NQO13), and (NQO14) genes,
complete cds's; biotin [acetyl-CoA carboxyl] ligase (birA) gene,
complete cds. GB_BA1: MTCY339 42861 Z77163 Mycobacterium
tuberculosis H37Rv complete genome; segment 101/162. Mycobacterium
tuberculosis 36,471 17-Jun-98 GB_BA1: MXADEVRS 2452 L19029
Myxococcus xanthus devR and devS genes, complete cds's. Myxococcus
xanthus 38,477 27-Jan-94 rxa02694 1065 GB_BA1: BACLDH 1147 M19394
B. caldolyticus lactate dehydrogenase (LDH) gene, complete cds.
Bacillus caldolyticus 57,371 26-Apr-93 GB_BA1: BACLDHL 1361 M14788
B. stearothermophilus lct gene encoding L-lactate Bacillus
stearothermophilus 57,277 26-Apr-93 dehydrogenase, complete cds.
GB_PAT: A06664 1350 A06664 B. stearothermophilus lct gene. Bacillus
stearothermophilus 57,277 29-Jul-93 rxa02729 844 GB_EST15: AA494626
121 AA494626 fa09d04.r1 Zebrafish ICRFzfis Danio rerio cDNA clone
11A22 5' similar to Danio rerio 50,746 27-Jun-97 TR: G1171163
G1171163 G/T-MISMATCH BINDING PROTEIN.;, mRNA sequence. GB_EST15:
AA494626 121 AA494626 fa09d04.r1 Zebrafish ICRFzfis Danio rerio
cDNA clone 11A22 5' similar to Danio rerio 36,364 27-Jun-97 TR:
G1171163 G1171163 G/T-MISMATCH BINDING PROTEIN.;, mRNA sequence.
rxa02730 1161 GB_EST19: AA758660 233 AA758660 ah67d06.s1
Soares_testis_NHT Homo sapiens cDNA Homo sapiens 37,059 29-DEC-1998
clone 1320683 3', mRNA sequence. GB_EST15: AA494626 121 AA494626
fa09d04.r1 Zebrafish ICRFzfis Danio rerio cDNA clone 11A22 5'
similar to Danio rerio 42,149 27-Jun-97 TR: G1171163 G1171163
G/T-MISMATCH BINDING PROTEIN.;, mRNA sequence. GB_PR4: AC006285
150172 AC006285 Homo sapiens, complete sequence. Homo sapiens
37,655 15-Nov-99 rxa02737 1665 GB_PAT: E13655 2260 E13655 gDNA
encoding glucose-6-phosphate dehydrogenase. Corynebacterium
glutamicum 99,580 24-Jun-98 GB_BA1: MTCY493 40790 Z95844
Mycobacterium tuberculosis H37Rv complete genome; segment 63/162.
Mycobacterium tuberculosis 38,363 19-Jun-98 GB_BA1: SC5A7 40337
AL031107 Streptomyces coelicolor cosmid 5A7. Streptomyces
coelicolor 39,444 27-Jul-98 rxa02738 1203 GB_PAT: E13655 2260
E13655 gDNA encoding glucose-6-phosphate dehydrogenase.
Corynebacterium glutamicum 98,226 24-Jun-98 GB_BA1: SCC22 22115
AL096839 Streptomyces coelicolor cosmid C22. Streptomyces
coelicolor 60,399 12-Jul-99 GB_BA1: SC5A7 40337 AL031107
Streptomyces coelicolor cosmid 5A7. Streptomyces coelicolor 36,426
27-Jul-98 rxa02739 2223 GB_BA1: AB023377 2572 AB023377
Corynebacterium glutamicum tkt gene for transketolase, complete
cds. Corynebacterium glutamicum 99,640 20-Feb-99 GB_BA1: MLCL536
36224 Z99125 Mycobacterium leprae cosmid L536. Mycobacterium leprae
61,573 04-DEC-1998 GB_BA1: U00013 35881 U00013 Mycobacterium leprae
cosmid B1496. Mycobacterium leprae 61,573 01-MAR-1994 rxa02740 1053
GB_HTG2: AC006247 174368 AC006247 Drosophila melanogaster
chromosome 2 clone BACR48I10 Drosophila melanogaster 37,105
2-Aug-99 (D505) RPCI-98 48.I.10 map 49E6-49F8 strain y; cn bw sp,
*** SEQUENCING IN PROGRESS ***, 17 unordered pieces. GB_HTG2:
AC006247 174368 AC006247 Drosophila melanogaster chromosome 2 clone
BACR48I10 (D505) Drosophila melanogaster 37,105 2-Aug-99 RPCI-98
48.I.10 map 49E6-49F8 strain y; cn bw sp, *** SEQUENCING IN
PROGRESS ***, 17 unordered pieces. GB_HTG3: AC007150 121474
AC007150 Drosophila melanogaster chromosome 2 clone BACR16P13
(D597) RPCI-98 Drosophila melanogaster 38,728 20-Sep-99 16.P.13 map
49E-49F strain y; cn bw sp, *** SEQUENCING IN PROGRESS***, 87
unordered pieces. rxa02741 1089 GB_HTG2: AC004951 129429 AC004951
Homo sapiens clone DJ1022I14, *** SEQUENCING IN PROGRESS ***, 14
Homo sapiens 33,116 12-Jun-98 unordered pieces. GB_HTG2: AC004951
129429 AC004951 Homo sapiens clone DJ1022I14, *** SEQUENCING IN
PROGRESS ***, 14 Homo sapiens 33,116 12-Jun-98 unordered pieces.
GB_IN1: AB006546 931 AB006546 Ephydatia fluviatilis mRNA for G
protein a subunit 4, partial cds. Ephydatia fluviatilis 36,379
23-Jun-99 rxa02743 1161 GB_BA1: MLCL536 36224 Z99125 Mycobacterium
leprae cosmid L536. Mycobacterium leprae 48,401 04-DEC-1998 GB_BA1:
U00013 35881 U00013 Mycobacterium leprae cosmid B1496.
Mycobacterium leprae 48,401 01-MAR-1994 GB_HTG2: AC007401 83657
AC007401 Homo sapiens clone NH0501O07, *** SEQUENCING IN PROGRESS
***, 3 Homo sapiens 37,128 26-Jun-99 unordered pieces. rxa02797
1026 GB_BA1: CGBETPGEN 2339 X93514 C. glutamicum betP gene.
Corynebacterium glutamicum 38,889 8-Sep-97 GB_GSS9: AQ148714 405
AQ148714 HS_3136_A1_A03_MR CIT Approved Human Genomic Sperm Homo
sapiens 34,321 08-OCT-1998 Library D Homo sapiens genomic clone
Plate = 3136 Col = 5 Row = A, genomic survey sequence. GB_BA1:
BFU64514 3837 U64514 Bacillus firmus dppABC operon, dipeptide
transporter Bacillus firmus 38,072 1-Feb-97 protein dppA gene,
partial cds, and dipeptide transporter proteins dppB and dppC
genes, complete cds. rxa02803 680 GB_BA1: U00020 36947 U00020
Mycobacterium leprae cosmid B229. Mycobacterium leprae 34,462
01-MAR-1994 GB_BA2: PSU85643 4032 U85643 Pseudomonas syringae pv.
syringae putative dihydropteroate Pseudomonas syringae pv. 50,445
9-Apr-97 synthase gene, partial cds, regulatory protein MrsA
(mrsA), triose phosphate isomerase (tpiA), transport syringae
protein SecG (secG), tRNA-Leu, tRNA-Met, and 15 kDa protein genes,
complete cds. GB_BA1: SC6G4 41055 AL031317 Streptomyces coelicolor
cosmid 6G4. Streptomyces coelicolor 59,314 20-Aug-98 rxa02821 363
GB_HTG2: AC008105 91421 AC008105 Homo sapiens chromosome 17 clone
2020_K_17 map 17, Homo sapiens 37,607 22-Jul-99 *** SEQUENCING IN
PROGRESS ***, 12 unordered pieces. GB_HTG2: AC008105 91421 AC008105
Homo sapiens chromosome 17 clone 2020_K_17 map 17, Homo sapiens
37,607 22-Jul-99 *** SEQUENCING IN PROGRESS ***, 12 unordered
pieces. GB_EST33: AV117143 222 AV117143 AV117143 Mus musculus
C57BL/6J 10-day embryo Mus musculus Mus musculus 40,157 30-Jun-99
cDNA clone 2610200J17, mRNA sequence. rxa02829 373 GB_HTG1: HSU9G8
48735 AL008714 Homo sapiens chromosome X clone LL0XNC01-9G8, ***
SEQUENCING IN Homo sapiens 41,595 23-Nov-99 PROGRESS ***, in
unordered pieces. GB_HTG1: HSU9G8 48735 AL008714 Homo sapiens
chromosome X clone LL0XNC01-9G8, *** SEQUENCING IN Homo sapiens
41,595 23-Nov-99 PROGRESS ***, in unordered pieces. GB_PR3: HSU85B5
39550 Z69724 Human DNA sequence from cosmid U85B5, between markers
Homo sapiens 41,595 23-Nov-99 DXS366 and DXS87 on chromosome X.
rxc03216 1141 GB_HTG3: AC008184 151720 AC008184 Drosophila
melanogaster chromosome 2 clone BACR04D05 (D540) Drosophila
melanogaster 39,600 2-Aug-99 RPCI-98 04.D.5 map 36E5-36F2 strain y;
cn bw sp, *** SEQUENCING IN PROGRESS ***, 27 unordered pieces.
GB_EST15: AA477537 411 AA477537 zu36g12.r1 Soares ovary tumor NbHOT
Homo sapiens Homo sapiens 37,260 9-Nov-97 cDNA clone IMAGE: 740134
5' similar to contains Alu repetitive element; contains element HGR
repetitive element;, mRNA sequence. GB_EST26: AI330662 412 AI330662
fa91d08.y1 zebrafish fin day1 regeneration Danio rerio Danio rerio
37,805 28-DEC-1998 cDNA 5', mRNA sequence. rxs03215 1038 GB_BA1:
SC3F9 19830 AL023862 Streptomyces coelicolor cosmid 3F9.
Streptomyces coelicolor A3(2) 48,657 10-Feb-99 GB_BA1: SLLINC 36270
X79146 S. lincolnensis (78-11) Lincomycin production genes.
Streptomyces lincolnensis 39,430 15-MAY-1996 GB_HTG5: AC009660
204320 AC009660 Homo sapiens chromosome 15 clone RP11-424J10 map
15, Homo sapiens 35,151 04-DEC-1999 *** SEQUENCING IN PROGRESS ***,
41 unordered pieces. rxs03224 1288 GB_PR3: AC004076 41322 AC004076
Homo sapiens chromosome 19, cosmid R30217, complete sequence. Homo
sapiens 37,788 29-Jan-98 GB_PL2: SPAC926 23193 AL110469 S. pombe
chromosome I cosmid c926. Schlzosaccharomyces pombe 38,474 2-Sep-99
GB_BA2: AE001081 11473 AE001081 Archaeoglobus fulgidus section 26
of 172 of the complete genome. Archaeoglobus fulgidus 35,871
15-DEC-1997
[0182]
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=US20070082383A1).
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=US20070082383A1).
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