U.S. patent application number 11/508783 was filed with the patent office on 2007-03-15 for corynebacterium glutamicum genes encoding stress, resistance and tolerance proteins.
This patent application is currently assigned to BASF AG. Invention is credited to Gregor Haberhauer, Hyung-Joon Kim, Burkhard Kroger, Heung-Shick Lee, Markus Pompejus, Hartwig Schroder, Oskar Zelder.
Application Number | 20070059810 11/508783 |
Document ID | / |
Family ID | 33437330 |
Filed Date | 2007-03-15 |
United States Patent
Application |
20070059810 |
Kind Code |
A1 |
Pompejus; Markus ; et
al. |
March 15, 2007 |
Corynebacterium glutamicum genes encoding stress, resistance and
tolerance proteins
Abstract
Isolated nucleic acid molecules, designated SRT nucleic acid
molecules, which encode novel SRT proteins from Corynebacterium
glutamicum are described. The invention also provides antisense
nucleic acid molecules, recombinant expression vectors containing
SRT nucleic acid molecules, and host cells into which the
expression vectors have been introduced. The invention still
further provides isolated SRT proteins, mutated SRT proteins,
fusion proteins, antigenic peptides and methods for the improvement
of production of a desired compound from C. glutamicum based on
genetic engineering of SRT 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) ; Lee;
Heung-Shick; (Seoul, KR) ; Kim; Hyung-Joon;
(Seoul, KR) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP
ONE POST OFFICE SQUARE
BOSTON
MA
02109-2127
US
|
Assignee: |
BASF AG
Ludwigshafen
DE
|
Family ID: |
33437330 |
Appl. No.: |
11/508783 |
Filed: |
August 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10703799 |
Nov 7, 2003 |
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11508783 |
Aug 22, 2006 |
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09603208 |
Jun 23, 2000 |
6822084 |
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10703799 |
Nov 7, 2003 |
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60141031 |
Jun 25, 1999 |
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60142692 |
Jul 1, 1999 |
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60151214 |
Aug 27, 1999 |
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Current U.S.
Class: |
435/106 ;
435/193; 435/252.3; 435/471; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12P 13/225 20130101;
C12P 13/222 20130101; C12N 9/18 20130101; C12P 13/04 20130101; C12P
13/08 20130101; C12P 13/12 20130101; C12P 13/14 20130101; C12P
13/24 20130101; C07K 2319/00 20130101; C12P 13/10 20130101; C12P
13/06 20130101; C12P 13/20 20130101; C07K 14/34 20130101; C12P
13/227 20130101; C12N 9/00 20130101 |
Class at
Publication: |
435/106 ;
435/069.1; 435/252.3; 435/193; 435/471; 536/023.2 |
International
Class: |
C12P 21/06 20060101
C12P021/06; C07H 21/04 20060101 C07H021/04; C12P 13/04 20060101
C12P013/04; 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 |
19932209.0 |
Jul 9, 1999 |
DE |
19932230.9 |
Aug 27, 1999 |
DE |
19940764.9 |
Jul 1, 1999 |
DE |
19930429.7 |
Jul 8, 1999 |
DE |
19931413.6 |
Jul 8, 1999 |
DE |
19931457.8 |
Jul 8, 1999 |
DE |
19931541.8 |
Jul 14, 1999 |
DE |
19932914.1 |
Aug 31, 1999 |
DE |
19941382.7 |
Claims
1. An isolated nucleic acid molecule selected from the group
consisting of a) an isolated nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:79, or a complement thereof; b) an
isolated nucleic acid molecule which encodes a polypeptide
comprising the amino acid sequence of SEQ ID NO:80, 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:80, 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:79, 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:79,
or a complement thereof.
2. An isolated nucleic acid molecule comprising the nucleic acid
molecule of claim 1 and a nucleotide sequence encoding a
heterologous polypeptide.
3. A vector comprising the nucleic acid molecule of claim 1.
4. The vector of claim 3, which is an expression vector.
5. A host cell transfected with the expression vector of claim
4.
6. The host cell of claim 5, wherein said cell is a
microorganism.
7. The host cell of claim 6, wherein said cell belongs to the genus
Corynebacterium or Brevibacterium.
8. A method of producing a polypeptide comprising culturing the
host cell of claim 5 in an appropriate culture medium to, thereby,
produce the polypeptide.
9. A method for producing a fine chemical, comprising culturing the
cell of claim 5 such that the fine chemical is produced.
10. The method of claim 9, wherein said method further comprises
the step of recovering the fine chemical from said culture.
11. The method of claim 9, wherein said cell belongs to the genus
Corynebacterium or Brevibacterium.
12. The method of claim 9, wherein said cell is selected from the
group consisting of Corynebacterium glutamicum, Corynebacterium
herculis, Corynebacterium, lilium, Corynebacterium
acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium
acetophilum, Corynebacterium ammoniagenes, Corynebacterium
fujiokense, Corynebacterium nitrilophilus, Brevibacterium
ammoniagenes, Brevibacterium butanicum, Brevibacterium divaricatum,
Brevibacterium flavum, Brevibacterium healii, Brevibacterium
ketoglutamicum, Brevibacterium ketosoreductum, Brevibacterium
lactofermentum, Brevibacterium linens, Brevibacterium
paraffinolyticum, and those strains set forth in Table 3.
13. The method of claim 9, wherein expression of the nucleic acid
molecule from said vector results in modulation of production of
said fine chemical.
14. The method of claim 9, wherein said fine chemical is selected
from the group consisting of organic acids, proteinogenic and
nonproteinogenic amino acids, purine and pyrimidine bases,
nucleosides, nucleotides, lipids, saturated and unsaturated fatty
acids, diols, carbohydrates, aromatic compounds, vitamins,
cofactors, polyketides, and enzymes.
15. The method of claim 9, wherein said fine chemical is an amino
acid selected from the group consisting of lysine, glutamate,
glutamine, alanine, aspartate, glycine, serine, threonine,
methionine, cysteine, valine, leucine, isoleucine, arginine,
proline, histidine, tyrosine, phenylalanine, and tryptophan.
16. An isolated polypeptide selected from the group consisting of
a) an isolated polypeptide comprising the amino acid sequence of
SEQ ID NO:80; b) an isolated polypeptide comprising a naturally
occurring allelic variant of a polypeptide comprising the amino
acid sequence of SEQ ID NO:80; c) an isolated polypeptide which is
encoded by a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:79; 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:79; 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:80; and f) an isolated polypeptide
comprising a fragment of a polypeptide comprising the amino acid
sequence of SEQ ID NO:80, wherein said polypeptide fragment
maintains a biological activity of the polypeptide comprising the
amino sequence.
17. The isolated polypeptide of claim 16, further comprising
heterologous amino acid sequences.
18. A method for diagnosing the presence or activity of
Corynebacterium diphtheriae in a subject, comprising detecting the
presence of at least one of the nucleic acid molecules of claim 1,
thereby diagnosing the presence or activity of Corynebacterium
diphtheriae in the subject.
19. A method for diagnosing the presence or activity of
Corynebacterium diphtheriae in a subject, comprising detecting the
presence of at least one of the polypeptide molecules of claim 16,
thereby diagnosing the presence or activity of Corynebacterium
diphtheriae in the subject.
20. A host cell comprising a nucleic acid molecule selected from
the group consisting of a) the nucleic acid molecule of SEQ ID
NO:79, 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:79, wherein the nucleic acid molecule
comprises one or more nucleic acid modifications as compared to the
sequence of SEQ ID NO:79, 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:79, wherein the regulatory
region of the nucleic acid molecule is modified relative to the
wild-type regulatory region of the molecule by at least one
technique selected from the group consisting of a point mutation, a
truncation, an inversion, a deletion, an addition, a substitution
and homologous recombination.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/703,799, filed Nov. 7, 2003, which is a continuation of U.S.
patent application Ser. No. 09/603,208, filed Jun. 23, 2000, issued
as U.S. Pat. No. 6,822,084 on Nov. 23, 2004, which, in turn, claims
priority to prior filed U.S. Provisional Patent Application Ser.
No. 60/141,031, filed Jun. 25, 1999, U.S. Provisional Patent
Application Ser. No. 60/142,692, filed Jul. 1, 1999, and also to
U.S. Provisional Patent Application Ser. No. 60/151,214, filed Aug.
27, 1999. This application also claims priority to German Patent
Application No. 19930429.7, filed Jul. 1, 1999, German Patent
Application No. 19931413.6, filed Jul. 8, 1999, German Patent
Application No. 19931457.8, filed Jul. 8, 1999, German Patent
Application No. 19931541.8, filed Jul. 8, 1999, German Patent
Application No. 19932209.0, filed Jul. 9, 1999, German Patent
Application No. 19932230.9, filed Jul. 9, 1999, German Patent
Application No. 19932914.1, filed Jul. 14, 1999, German Patent
Application No. 19940764.9, filed Aug. 27, 1999, and German Patent
Application No. 19941382.7, filed Aug. 31, 1999. The entire
contents of each of the aforementioned applications are hereby
expressly incorporated herein by this reference.
INCORPORATION OF MATERIAL SUBMITTED ON COMPACT DISCS
[0002] This application incorporates herein by reference the
material contained on the compact discs submitted herewith as part
of this application. Specifically, the file "seqlistcorrected" (992
KB) 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 Aug. 21, 2006. In addition, the files "Appendix
A" (159 KB) and "Appendix B" (55.5 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 large-scale culture of bacteria developed to produce and
secrete large quantities of a particular desired molecule. 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
stress, resistance and tolerance (SRT) 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 SRT 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 SRT nucleic acids of the
invention, or modification of the sequence of the SRT 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 SRT 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 SRT 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] The SRT proteins encoded by the novel nucleic acid molecules
of the invention are capable of, for example, permitting C.
glutamicum to survive in a setting which is either chemically or
environmentally hazardous to this microorganism. 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, through the
ability of these proteins to permit growth and multiplication of C.
glutamicum (and also continuous production of one or more fine
chemicals) under circumstances which would normally impede growth
of the organism, such as those conditions frequently encountered
during large-scale fermentative growth. For example, by
overexpressing or engineering a heat-shock induced protease
molecule such that it is optimized in activity, one may increase
the ability of the bacterium to degrade incorrectly folded proteins
when the bacterium is challenged with high temperatures. By having
fewer misfolded (and possibly misregulated or nonfunctional)
proteins to interfere with normal reaction mechanisms in the cell,
the cell is increased in its ability to function normally in such a
culture, which should in turn provide increased viability. This
overall increase in number of cells having greater viability and
activity in the culture should also result in an increase in yield,
production, and/or efficiency of production of one or more desired
fine chemicals, due at least to the relatively greater number of
cells producing these chemicals in the culture.
[0009] This invention provides novel SRT nucleic acid molecules
which encode SRT proteins which are capable of, for example,
permitting C. glutamicum to survive in a setting which is either
chemically or environmentally hazardous to this microorganism.
Nucleic acid molecules encoding an SRT protein are referred to
herein as SRT nucleic acid molecules. In a preferred embodiment,
the SRT protein participates in metabolic pathways permitting C.
glutamicum to survive in a setting which is either chemically or
environmentally hazardous to this microorganism. Examples of such
proteins include those encoded by the genes set forth in Table
1.
[0010] Accordingly, one aspect of the invention pertains to
isolated nucleic acid molecules (e.g., cDNAs, DNAs, or RNAs)
comprising a nucleotide sequence encoding an SRT protein or
biologically active portions thereof, as well as nucleic acid
fragments suitable as primers or hybridization probes for the
detection or amplification of SRT-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 SRT
proteins of the present invention also preferably possess at least
one of the SRT activities described herein.
[0011] 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 SRT activity.
Preferably, the protein or portion thereof encoded by the nucleic
acid molecule maintains the ability to increase the survival of C.
glutamicum in a setting which is either chemically or
environmentally hazardous to this microorganism. 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).
[0012] In another preferred embodiment, the isolated nucleic acid
molecule is derived from C. glutamicum and encodes a protein (e.g.,
an SRT 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 has the ability to increase the
survival of C. glutamicum in a setting which is either chemically
or environmentally hazardous to this microorganism, or possesses
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.
[0013] 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 SRT protein, or a biologically
active portion thereof.
[0014] 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 SRT protein by culturing the host cell in a suitable
medium. The SRT protein can be then isolated from the medium or the
host cell.
[0015] Yet another aspect of the invention pertains to a
genetically altered microorganism in which an SRT gene has been
introduced or altered. In one embodiment, the genome of the
microorganism has been altered by the introduction of a nucleic
acid molecule of the invention encoding wild-type or mutated SRT
sequence as a transgene. In another embodiment, an endogenous SRT
gene within the genome of the microorganism has been altered, e.g.,
functionally disrupted, by homologous recombination with an altered
SRT gene. In another embodiment, an endogenous or introduced SRT
gene in a microorganism has been altered by one or more point
mutations, deletions, or inversions, but still encodes a functional
SRT protein. In still another embodiment, one or more of the
regulatory regions (e.g., a promoter, repressor, or inducer) of a
SRT gene in a microorganism has been altered (e.g., by deletion,
truncation, inversion, or point mutation) such that the expression
of the SRT 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.
[0016] 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.
[0017] Still another aspect of the invention pertains to an
isolated SRT protein or a portion, e.g., a biologically active
portion, thereof. In a preferred embodiment, the isolated SRT
protein or portion thereof possesses the ability to increase the
survival of C. glutamicum in a setting which is either chemically
or environmentally hazardous to this microorganism. In another
preferred embodiment, the isolated SRT 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
increase the survival of C. glutamicum in a setting which is either
chemically or environmentally hazardous to this microorganism.
[0018] The invention also provides an isolated preparation of an
SRT protein. In preferred embodiments, the SRT 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 SRT 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 improve the survival rate of C. glutamicum in a
setting which is either chemically or environmentally hazardous to
this microorganism, or has one or more of the activities set forth
in Table 1.
[0019] Alternatively, the isolated SRT 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 SRT proteins also have one or more of the SRT
bioactivities described herein.
[0020] The SRT polypeptide, or a biologically active portion
thereof, can be operatively linked to a non-SRT polypeptide to form
a fusion protein. In preferred embodiments, this fusion protein has
an activity which differs from that of the SRT protein alone. In
other preferred embodiments, this fusion protein results in
increased yields, production, and/or efficiency of production of a
desired fine chemical from C. glutamicum. In particularly preferred
embodiments, integration of this fusion protein into a host cell
modulates the production of a desired compound from the cell.
[0021] In another aspect, the invention provides methods for
screening molecules which modulate the activity of an SRT protein,
either by interacting with the protein itself or a substrate or
binding partner of the SRT protein, or by modulating the
transcription or translation of an SRT nucleic acid molecule of the
invention.
[0022] 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 SRT 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
SRT 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.
[0023] 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
SRT protein activity or SRT 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 in resistance to one or more toxic chemicals or in
resistance to one or more environmental stresses, such that the
yields or rate of production of a desired fine chemical by this
microorganism is improved. The agent which modulates SRT protein
activity can be an agent which stimulates SRT protein activity or
SRT nucleic acid expression. Examples of agents which stimulate SRT
protein activity or SRT nucleic acid expression include small
molecules, active SRT proteins, and nucleic acids encoding SRT
proteins that have been introduced into the cell. Examples of
agents which inhibit SRT activity or expression include small
molecules, and antisense SRT nucleic acid molecules.
[0024] 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 SRT 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
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
[0025] The present invention provides SRT nucleic acid and protein
molecules which are involved in the survival of C. glutamicum upon
exposure of this microorganism to chemical or environmental
hazards. The molecules of the invention may be utilized in the
modulation of production of fine chemicals from microorganisms,
since these SRT proteins provide a means for continued growth and
multiplication of C. glutamicum in the presence of toxic chemicals
or hazardous environmental conditions, such as may be encountered
during large-scale fermentative growth. By increasing the growth
rate or at least maintaining normal growth in the face of poor, if
not toxic, conditions, one may increase the yield, production,
and/or efficiency of production of one or more fine chemicals from
such a culture, at least due to the relatively greater number of
cells producing the fine chemical in the culture. Aspects of the
invention are further explicated below.
I. Fine Chemicals
[0026] 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
[0027] Amino acids comprise the basic structural units of all
proteins, and as such are essential for normal cellular functioning
in all organisms. The term "amino acid" is art-recognized. The
proteinogenic amino acids, of which there are 20 species, serve as
structural units for proteins, in which they are linked by peptide
bonds, while the nonproteinogenic amino acids (hundreds of which
are known) are not normally found in proteins (see Ulmann's
Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH:
Weinheim (1985)). Amino acids may be in the D- or L-optical
configuration, though L-amino acids are generally the only type
found in naturally-occurring proteins. Biosynthetic and degradative
pathways of each of the 20 proteinogenic amino acids have been well
characterized in both prokaryotic and eukaryotic cells (see, for
example, Stryer, L. Biochemistry, 3.sup.rd edition, pages 578-590
(1988)). The `essential` amino acids (histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, threonine, tryptophan,
and valine), so named because they are generally a nutritional
requirement due to the complexity of their biosyntheses, are
readily converted by simple biosynthetic pathways to the remaining
11 `nonessential` amino acids (alanine, arginine, asparagine,
aspartate, cysteine, glutamate, glutamine, glycine, proline,
serine, and tyrosine). Higher animals do retain the ability to
synthesize some of these amino acids, but the essential amino acids
must be supplied from the diet in order for normal protein
synthesis to occur.
[0028] 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.
[0029] 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.
[0030] 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
[0031] 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).
[0032] 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).
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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
[0037] 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).
[0038] 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.
[0039] 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
[0040] 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. Resistance to Damage from Chemicals, Environmental Stress, and
Antibiotics
[0041] Production of fine chemicals is typically performed by
large-scale culture of bacteria developed to produce and secrete
large quantities of these molecules. However, this type of
large-scale fermentation results in the subjection of the
microorganisms to stresses of various kinds. These stresses include
environmental stress and chemical stress.
A. Resistance to Environmental Stress
[0042] Examples of environmental stresses typically encountered in
large-scale fermentative culture include mechanical stress, heat
stress, stress due to limited oxygen, stress due to oxygen
radicals, pH stress, and osmotic stress. The stirring mechanism
used in most large-scale fermentors to ensure aeration of the
culture produces heat, thus increasing the temperature of the
culture. Increases in temperature induce the well-characterized
heat shock response, in which a set of proteins are expressed which
not only aid in the survival of the bacterium in the face of high
temperatures, but also increase survival in response to a number of
other environmental stresses (see Neidhardt, F., eds. (1996) E.
coli and Salmonella. ASM Press: Washington, D.C., p. 1382-1399;
Wosten, M. M. (1998) FEMS Microbiology Reviews 22(3): 127-50; Bahl,
H. et al. (1995) FEMS Microbiology Reviews 17(3): 341-348;
Zimmerman, J. L., Cohill, P. R. (1991) New Biologist 3(7): 641-650;
Samali, A., and Orrenius, S. (1998) Cell. Stress Chaperones 3(4):
228-236, and references contained therein from each of these
citations). Regulation of the heat shock response in bacteria is
facilitated by specific sigma factors and other cellular regulators
of gene expression (Hecker, M., Volker, U (1998). Molecular
Microbiology 29(5): 1129-1136). One of the largest problems that
the cell encounters when exposed to high temperature is that
protein folding is impaired; nascent proteins have sufficient
kinetic energy in high temperature circumstances that it is
difficult for the growing polypeptide chain to remain in a stable
conformation long enough to fold properly. Thus, two of the key
types of proteins expressed during the heat shock response consist
of chaperones (proteins which assist in the folding or unfolding of
other proteins--see, e.g., Fink, A. L. (1999) Physiol. Rev. 79(2):
425-449), and proteases, which can destroy any improperly folded
proteins. Examples of chaperones expressed during the heat shock
response include GroEL and DNAK; proteases known to be expressed
during this cellular reaction to heat shock include Lon, FtsH, and
ClpB.
[0043] Other environmental stresses besides heat may also provoke a
stress response. Though the fermentor stirring process is meant to
introduce oxygen into the culture, oxygen may remain in limited
supply, particularly when the culture is advanced in growth and the
oxygen needs of the culture are thereby increased; an insufficient
supply of oxygen is another stress for the microorganism. Cells in
fermentor cultures are also subjected to a number of osmotic
stresses, particularly when nutrients are added to the culture,
resulting in a high extracellular and low intracellular
concentration of these molecules. Further, the large quantities of
the desired molecules produced by these organisms in culture may
contribute to osmotic stress of the bacteria. Lastly, aerobic
metabolism such as that used by C. glutamicum results in carbon
dioxide as a waste product; secretion of this molecule may acidify
the culture medium due to conversion of this molecule to carboxylic
acid. Thus, bacteria in culture are also frequently subjected to
acidic pH stress. The converse may also be true--when high levels
of basic waste molecules such as ammonium are present in the
culture medium, the bacteria in culture may be subjected to basic
pH stress as well.
[0044] To combat such environmental stresses, bacteria have elegant
gene systems which are expressed upon exposure to one or more
stresses, such as the aforementioned heat shock system. Genes
expressed in response to osmotic stress, for example, encode
proteins capable of transporting or synthesizing compatible solutes
such that osmotic intake or export of a particular molecule is
slowed to manageable levels. Other examples of stress-induced
bacterial proteins are those involved in trehalose biosynthesis,
those encoding enzymes involved in ppGpp metabolism, those involved
in signal transduction, particularly those encoding two-component
systems which are sensitive to osmotic pressure, and those encoding
transcription factors which are responsive to a variety of stress
factors (e.g., RssB analogues and/or sigma factors). Many other
such genes and their protein products are known in the art.
B. Resistance to Chemical Stress
[0045] Aside from environmental stresses, cells may also experience
a number of chemical stresses. These may fall into two categories.
The first are natural waste products of metabolism and other
cellular processes which are secreted by the cell to the
surrounding medium. The second are chemicals present in the
extracellular medium which do not originate from the cell.
Generally, when cells excrete toxic waste products from the
concentrated intracellular cytoplasm into the relatively much more
dilute extracellular medium, these products dissipate such that
extracellular levels of the possibly toxic compound are quite low.
However, in large-scale fermentative culture of the bacterium, this
may not be the case: so many bacteria are grown in a relatively
small environment and at such a high metabolic rate that waste
products may accumulate in the medium to nearly toxic levels.
Examples of such wastes are carbon dioxide, metal ions, and
reactive oxygen species such as hydrogen peroxide. These compounds
may interfere with the activity or structure of cell surface
molecules, or may re-enter the cell, where they can seriously
damage proteins and nucleic acids alike. Certain other chemicals
hazardous to the normal functioning of cells may be naturally found
in the extracellular medium. For example, metal ions such as
mercury, cadmium, nickel or copper are frequently found in water
sources, and may form tight complexes with cellular enzymes which
prevent the normal functioning of these proteins.
C. Resistance to Antibiotics
[0046] Bacteriocidal proteins or antibiotics, may also be found in
the extracellular milieu, either through the intervention of the
researcher, or as a natural product from another organism, utilized
to gain a competitive advantage. Microorganisms have several
art-known mechanisms to protect themselves against antimicrobial
chemicals. Degradation, modification, and export of compounds toxic
to the cell are common methods by which microorganisms eliminate or
detoxify antibiotics. Cytoplasmic `efflux-pumps` are known in
several prokaryotes and show similarities to the so-called
`multidrug resistance` proteins from higher eukaryotes (Neyfakh, A.
A., et al. (1991) Proc. Natl. Acad. Sci. USA 88: 4781-4785).
Examples of such proteins include emrAB from E. coli (Lomovskaya,
O. and K. Lewis (1992) Proc. Natl. Acad. Sci. USA 89: 8938-8942),
ImrB from B. subtilis (Kumano, M. et al. (1997) Microbiology 143:
2775-2782), smr from S. aureus (Grinius, L. G. et al. (1992)
Plasmid 27: 119-129) or cmr from C. glutamicum (Kaidoh, K. et al.
(1997) Micro. Drug Resist. 3: 345-350). C. glutamicum itself is
non-pathogenic, in contrast to several other members of the genus
Corynebacterium, such as C. diphtheriae or C. pseudotuberculosis.
Several pathogenic Corynebacteria are known to have multiple
resistances against a variety of antibiotics, such as C. jeikeium
and C. urealyticum (Soriano, F. et al. (1995) Antimicrob. Agents
Chemother. 39: 208-214).
[0047] Lincosamides are recognized as effective antibiotics against
Corynebacterium species (Soriano, F. et al. (1995) Antimicrob.
Agents Chemother. 39: 208-214). An unexpected result of the present
invention was the identification of a gene encoding a
lincosamide-resistance protein (in particular, a
lincomycin-resistance protein). The LMRB protein from C. glutamicum
shows 40% homology to the product of the lmrB gene from B. subtilis
(see Genbank accession no. AL009126), as calculated using version
1.7 of the program CLUSTALW (Thompson, J. D., Higgins, D. G.,
Gibson, T. J. (1994) Nucl. Acids Res. 22: 4673-4680) using standard
parameters (PAIRWISE ALIGNMENT PARAMETERS: slow/accurate
alignments: Gap Open Penalty=10.00, Gap Extension Penalty=0.10,
Protein weight matrix=BLOSUM 30, DNA weight matrix=IUB,
Fast/Approximate alignments: Gap penalty=3, K-tuple (word) size=1,
No. of top diagonals=5, Window size=5, Toggle Slow/Fast pairwise
alignments=slow. Multiple alignment parameters: Gap Opening
Penalty=10.00, Gap Extension Penalty=0.05, Delay divergent
sequences=40%, DNA transitions weight=0.50, Protein weight
matrix=BLOSUM series, DNA weight matrix=IUB, Use negative
matrix=OFF).
[0048] Environmental stress, chemical stress, and antibiotic or
other antimicrobial stress may influence the behavior of the
microorganisms during fermentor culture, and may have an impact on
the production of the desired compound from these organisms. For
example, osmotic stress of a microorganism may cause inappropriate
or inappropriately rapid uptake of one or more compounds which can
ultimately lead to cellular damage or death due to osmotic shock.
Similarly, chemicals present in the culture, either exogenously
added (e.g., antimicrobial compounds intended to eliminate unwanted
microbes) or generated by the bacteria themselves (e.g., waste
compounds such as heavy metals or oxygen radicals, or even
antimicrobial compounds) may result in inhibition of fine chemical
production or even death of the organism. The genes of the
invention encode C. glutamicum proteins which act to prevent cell
damage or death, by specifically counteracting the source or effect
of the environmental or chemical stress.
III. Elements and Methods of the Invention
[0049] The present invention is based, at least in part, on the
discovery of novel molecules, referred to herein as SRT nucleic
acid and protein molecules, which increase the ability of C.
glutamicum to survive in chemically or environmentally hazardous
settings. In one embodiment, the SRT molecules function to confer
resistance to one or more environmental or chemical stresses to C.
glutamicum. In a preferred embodiment, the activity of the SRT
molecules of the present invention has an impact on the production
of a desired fine chemical by this organism. In a particularly
preferred embodiment, the SRT molecules of the invention are
modulated in activity, such that the yield, production, and/or
efficiency of production of one or more fine chemicals from C.
glutamicum is also modulated.
[0050] The language, "SRT protein" or "SRT polypeptide" includes
proteins which participate in the resistance of C. glutamicum to
one or more environmental or chemical stresses. Examples of SRT
proteins include those encoded by the SRT genes set forth in Table
1 and Appendix A. The terms "SRT gene" or "SRT nucleic acid
sequence" include nucleic acid sequences encoding an SRT protein,
which consist of a coding region and also corresponding
untranslated 5' and 3' sequence regions. Examples of SRT genes
include those set forth in Table 1. The terms "production" or
"productivity" are art-recognized and include the concentration of
the fermentation product (for example, the desired fine chemical)
formed within a given time and a given fermentation volume (e.g.,
kg product per hour per liter). The term "efficiency of production"
includes the time required for a particular level of production to
be achieved (for example, how long it takes for the cell to attain
a particular rate of output of a fine chemical). The term "yield"
or "product/carbon yield" is art-recognized and includes the
efficiency of the conversion of the carbon source into the product
(i.e., fine chemical). This is generally written as, for example,
kg product per kg carbon source. By increasing the yield or
production of the compound, the quantity of recovered molecules, or
of useful recovered molecules of that compound in a given amount of
culture over a given amount of time is increased. The terms
"biosynthesis" or a "biosynthetic pathway" are art-recognized and
include the synthesis of a compound, preferably an organic
compound, by a cell from intermediate compounds in what may be a
multistep and highly regulated process. The terms "degradation" or
a "degradation pathway" are art-recognized and include the
breakdown of a compound, preferably an organic compound, by a cell
to degradation products (generally speaking, smaller or less
complex molecules) in what may be a multistep and highly regulated
process. The language "metabolism" is art-recognized and includes
the totality of the biochemical reactions that take place in an
organism. The metabolism of a particular compound, then, (e.g., the
metabolism of an amino acid such as glycine) comprises the overall
biosynthetic, modification, and degradation pathways in the cell
related to this compound. The terms "resistance" and "tolerance"
are art-known and include the ability of a cell to not be affected
by exposure to a chemical or an environment which would otherwise
be detrimental to the normal functioning of these organisms. The
terms "stress" or "hazard" include factors which are detrimental to
the normal functioning of cells such as C. glutamicum. Examples of
stresses include "chemical stress", in which a cell is exposed to
one or more chemicals which are detrimental to the cell, and
"environmental stress" where a cell is exposed to an environmental
condition outside of those to which it is adapted. Chemical
stresses may be either natural metabolic waste products such as,
but not limited to reactive oxygen species or carbon dioxide, or
chemicals otherwise present in the environment, including, but not
limited to heavy metal ions or bacteriocidal proteins such as
antibiotics. Environmental stresses may be, but are not limited to
temperatures outside of the normal range, suboptimal oxygen
availability, osmotic pressures, or extremes of pH, for
example.
[0051] In another embodiment, the SRT 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.
Using recombinant genetic techniques, one or more of the SRT
proteins of the invention may be manipulated such that its function
is modulated. The alteration of activity of stress response,
resistance or tolerance genes such that the cell is increased in
tolerance to one or more stresses may improve the ability of that
cell to grow and multiply in the relatively stressful conditions of
large-scale fermentor culture. For example, by overexpressing or
engineering a heat-shock induced chaperone molecule such that it is
optimized in activity, one may increase the ability of the
bacterium to correctly fold proteins in the face of nonoptimal
temperature conditions. By having fewer misfolded (and possibly
misregulated or nonfunctional) proteins, the cell is increased in
its ability to function normally in such a culture, which should in
turn provide increased viability. This overall increase in number
of cells having greater viability and activity in the culture
should also result in an increase in the yield, production, and/or
efficiency of production of one or more desired fine chemicals, due
at least to the relatively greater number of cells producing these
chemicals in the culture.
[0052] 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 SRT DNAs and the predicted amino acid sequences of the
C. glutamicum SRT 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 chemical and environmental stress,
resistance, and tolerance proteins.
[0053] 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. Ranges and identity values
intermediate to the above-recited values, (e.g., 75%-80% identical,
85-87% identical, 91-92% 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.
[0054] The SRT proteins or biologically active portions or
fragments thereof of the invention can confer resistance or
tolerance to one or more chemical or environmental stresses, or may
have one or more of the activities set forth in Table 1.
[0055] Various aspects of the invention are described in further
detail in the following subsections:
A. Isolated Nucleic Acid Molecules
[0056] One aspect of the invention pertains to isolated nucleic
acid molecules that encode SRT 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 SRT-encoding nucleic acid (e.g., SRT 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 SRT 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.
[0057] 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 SRT 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 SRT nucleotide
sequence can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[0058] 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 SRT DNAs of the invention. This DNA
comprises sequences encoding SRT 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.
[0059] 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 RXSnumber having the designation "RXA", or
"RXS" followed by 5 digits (i.e., RXA01524, RXN00493, or RXS01027).
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 RXA01524 is a translation of the
coding region of the nucleotide sequence of nucleic acid molecule
RXA01524 in Appendix A, the amino acid sequence in Appendix B
designated RXN00034 is a translation of the coding region of the
nucleotide sequence of nucleic acid molecule RXN00034 in Appendix
A, and the amino acid sequence in Appendix B designated RXS00568 is
a translation of the coding region of the nucleotide sequence of
nucleic acid molecule RXS00568 in Appendix A. Each of the RXA, RXN,
and RXS nucleotide and amino acid sequences of the invention has
also been assigned a SEQ ID NO, as indicated in Table 1.
[0060] Several of the genes of the invention are "F-designated
genes". An F-designated gene includes those genes set forth in
Table 1 which have an `F` in front of the RXA, RXN, or RXS
designation. For example, SEQ ID NO:7, designated, as indicated on
Table 1, as "F RXA00498", is an F-designated gene, as are SEQ ID
NOs: 25, 33, and 37 (designated on Table 1 as "F RXA01345", "F
RXA02543", and "F RXA02282", respectively).
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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 SRT protein. The nucleotide sequences determined from
the cloning of the SRT genes from C. glutamicum allows for the
generation of probes and primers designed for use in identifying
and/or cloning SRT homologues in other cell types and organisms, as
well as SRT 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 SRT homologues. Probes based on the SRT 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 SRT
protein, such as by measuring a level of an SRT-encoding nucleic
acid in a sample of cells, e.g., detecting SRT mRNA levels or
determining whether a genomic SRT gene has been mutated or
deleted.
[0065] 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 confer resistance or tolerance of
C. glutamicum to one or more chemical or environmental stresses. 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 capable of participating in the resistance of C.
glutamicum to one or more chemical or environmental stresses.
Protein members of such metabolic pathways, as described herein,
function to increase the resistance or tolerance of C. glutamicum
to one or more environmental or chemical hazards or stresses.
Examples of such activities are also described herein. Thus, "the
function of an SRT protein" contributes to the overall resistance
of C. glutamicum to elements of its surroundings which may impede
its normal growth or functioning, and/or contributes, either
directly or indirectly, to the yield, production, and/or efficiency
of production of one or more fine chemicals. Examples of SRT
protein activities are set forth in Table 1.
[0066] 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. Ranges and identity values intermediate to
the above-recited values, (e.g., 75%-80% identical, 85-87%
identical, or 91-92% 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.
[0067] Portions of proteins encoded by the SRT nucleic acid
molecules of the invention are preferably biologically active
portions of one of the SRT proteins. As used herein, the term
"biologically active portion of an SRT protein" is intended to
include a portion, e.g., a domain/motif, of an SRT protein that is
capable of imparting resistance or tolerance to one or more
environmental or chemical stresses or hazards, or has an activity
as set forth in Table 1. To determine whether an SRT protein or a
biologically active portion thereof can increase the resistance or
tolerance of C. glutamicum to one or more chemical or environmental
stresses or hazards, 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.
[0068] Additional nucleic acid fragments encoding biologically
active portions of an SRT protein can be prepared by isolating a
portion of one of the sequences in Appendix B, expressing the
encoded portion of the SRT protein or peptide (e.g., by recombinant
expression in vitro) and assessing the activity of the encoded
portion of the SRT protein or peptide.
[0069] 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 SRT 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).
[0070] 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 39% identical to the
nucleotide sequence designated RXA00084 (SEQ ID NO:189), a
nucleotide sequence which is greater than and/or at least 56%
identical to the nucleotide sequence designated RXA00605 (SEQ ID
NO:11), and a nucleotide sequence which is greater than and/or at
least 50% identical to the nucleotide sequence designated RXA00886
(SEQ ID NO:39). 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.
[0071] In addition to the C. glutamicum SRT nucleotide sequences
shown in Appendix A, it will be appreciated by one of ordinary
skill in the art that DNA sequence polymorphisms that lead to
changes in the amino acid sequences of SRT proteins may exist
within a population (e.g., the C. glutamicum population). Such
genetic polymorphism in the SRT 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 SRT protein,
preferably a C. glutamicum SRT protein. Such natural variations can
typically result in 1-5% variance in the nucleotide sequence of the
SRT gene. Any and all such nucleotide variations and resulting
amino acid polymorphisms in SRT that are the result of natural
variation and that do not alter the functional activity of SRT
proteins are intended to be within the scope of the invention.
[0072] Nucleic acid molecules corresponding to natural variants and
non-C. glutamicum homologues of the C. glutamicum SRT DNA of the
invention can be isolated based on their homology to the C.
glutamicum SRT 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 in the art and can be found in
Ausubel et al., 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
SRT protein.
[0073] In addition to naturally-occurring variants of the SRT
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 SRT protein,
without altering the functional ability of the SRT 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 SRT proteins (Appendix B) without altering the activity of said
SRT protein, whereas an "essential" amino acid residue is required
for SRT protein activity. Other amino acid residues, however,
(e.g., those that are not conserved or only semi-conserved in the
domain having SRT activity) may not be essential for activity and
thus are likely to be amenable to alteration without altering SRT
activity.
[0074] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding SRT proteins that contain changes
in amino acid residues that are not essential for SRT activity.
Such SRT proteins differ in amino acid sequence from a sequence
contained in Appendix B yet retain at least one of the SRT
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 increasing the resistance or tolerance of C.
glutamicum to one or more environmental or chemical stresses, or
has one or more of the 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.
[0075] 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).
[0076] An isolated nucleic acid molecule encoding an SRT 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 SRT 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 SRT coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for an SRT activity described herein to
identify mutants that retain SRT 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).
[0077] In addition to the nucleic acid molecules encoding SRT
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 SRT
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 SRT
protein. The term "coding region" refers to the region of the
nucleotide sequence comprising codons which are translated into
amino acid residues (e.g., the entire coding region of SEQ ID NO.:
120 (RXA00600) comprises nucleotides 1 to 1098). In another
embodiment, the antisense nucleic acid molecule is antisense to a
"noncoding region" of the coding strand of a nucleotide sequence
encoding SRT. 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).
[0078] Given the coding strand sequences encoding SRT 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 SRT
mRNA, but more preferably is an oligonucleotide which is antisense
to only a portion of the coding or noncoding region of SRT mRNA.
For example, the antisense oligonucleotide can be complementary to
the region surrounding the translation start site of SRT 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-N-6-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).
[0079] 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 SRT 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.
[0080] 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).
[0081] 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 SRT mRNA transcripts to thereby
inhibit translation of SRT mRNA. A ribozyme having specificity for
an SRT-encoding nucleic acid can be designed based upon the
nucleotide sequence of an SRT cDNA disclosed herein (i.e., SEQ ID
NO:119 (RXA00600 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 SRT-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, SRT 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.
[0082] Alternatively, SRT gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of an SRT nucleotide sequence (e.g., an SRT promoter and/or
enhancers) to form triple helical structures that prevent
transcription of an SRT 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
[0083] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding
an SRT 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.
[0084] 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.R- or
.lamda.P.sub.L, which are used preferably in bacteria. Additional
regulatory sequences are, for example, promoters from yeasts and
fingi, such as ADC1, MF.alpha., AC, P-60, CYC1, GAPDH, TEF, rp28,
ADH, promoters from plants such as CaMV/35S, SSU, OCS, lib4, usp,
STLS1, B33, nos or ubiquitin- or phaseolin-promoters. It is also
possible to use artificial promoters. It will be appreciated by one
of ordinary skill in the art that the design of the expression
vector can depend on such factors as the choice of the host cell to
be transformed, the level of expression of protein desired, etc.
The expression vectors of the invention can be introduced into host
cells to thereby produce proteins or peptides, including fusion
proteins or peptides, encoded by nucleic acids as described herein
(e.g., SRT proteins, mutant forms of SRT proteins, fusion proteins,
etc.).
[0085] The recombinant expression vectors of the invention can be
designed for expression of SRT proteins in prokaryotic or
eukaryotic cells. For example, SRT 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.
[0086] 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.
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.
[0087] 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 SRT 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 SRT protein unfused to GST
can be recovered by cleavage of the fusion protein with
thrombin.
[0088] 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, pBdCl, 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).
[0089] 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.
[0090] In another embodiment, the SRT 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), 62.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).
[0091] Alternatively, the SRT 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).
[0092] In another embodiment, the SRT 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).
[0093] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)
EMBO J. 6:187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E.
F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd,
ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989.
[0094] 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 (Banedji 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).
[0095] 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 SRT 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.
[0096] 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.
[0097] A host cell can be any prokaryotic or eukaryotic cell. For
example, an SRT 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 those 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.
[0098] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" 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, 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.
[0099] 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 SRT protein or can be introduced on a separate
vector. Cells stably transfected with the introduced nucleic acid
can be identified by drug selection (e.g., cells that have
incorporated the selectable marker gene will survive, while the
other cells die).
[0100] To create a homologous recombinant microorganism, a vector
is prepared which contains at least a portion of an SRT gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the SRT gene.
Preferably, this SRT gene is a Corynebacterium glutamicum SRT 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 SRT 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 SRT 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 SRT protein). In the homologous
recombination vector, the altered portion of the SRT gene is
flanked at its 5' and 3' ends by additional nucleic acid of the SRT
gene to allow for homologous recombination to occur between the
exogenous SRT gene carried by the vector and an endogenous SRT gene
in a microorganism. The additional flanking SRT nucleic acid is of
sufficient length for successful homologous recombination with the
endogenous gene. Typically, several kilobases of flanking DNA (both
at the 5' and 3' ends) are included in the vector (see e.g.,
Thomas, K. R., and Capecchi, M. R. (1987) Cell 51: 503 for a
description of homologous recombination vectors). The vector is
introduced into a microorganism (e.g., by electroporation) and
cells in which the introduced SRT gene has homologously recombined
with the endogenous SRT gene are selected, using art-known
techniques.
[0101] 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 SRT
gene on a vector placing it under control of the lac operon permits
expression of the SRT gene only in the presence of IPTG. Such
regulatory systems are well known in the art.
[0102] In another embodiment, an endogenous SRT 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
SRT gene in a host cell has been altered by one or more point
mutations, deletions, or inversions, but still encodes a functional
SRT protein. In still another embodiment, one or more of the
regulatory regions (e.g., a promoter, repressor, or inducer) of an
SRT gene in a microorganism has been altered (e.g., by deletion,
truncation, inversion, or point mutation) such that the expression
of the SRT gene is modulated. One of ordinary skill in the art will
appreciate that host cells containing more than one of the
described SRT gene and protein modifications may be readily
produced using the methods of the invention, and are meant to be
included in the present invention.
[0103] 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 SRT protein. Accordingly, the invention further
provides methods for producing SRT 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 SRT protein has been introduced, or into which
genome has been introduced a gene encoding a wild-type or altered
SRT protein) in a suitable medium until SRT protein is produced. In
another embodiment, the method further comprises isolating SRT
proteins from the medium or the host cell.
C. Isolated SRT Proteins
[0104] Another aspect of the invention pertains to isolated SRT
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 SRT 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 SRT protein having less than about 30% (by
dry weight) of non-SRT protein (also referred to herein as a
"contaminating protein"), more preferably less than about 20% of
non-SRT protein, still more preferably less than about 10% of
non-SRT protein, and most preferably less than about 5% non-SRT
protein. When the SRT 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 SRT 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 SRT protein
having less than about 30% (by dry weight) of chemical precursors
or non-SRT chemicals, more preferably less than about 20% chemical
precursors or non-SRT chemicals, still more preferably less than
about 10% chemical precursors or non-SRT chemicals, and most
preferably less than about 5% chemical precursors or non-SRT
chemicals. In preferred embodiments, isolated proteins or
biologically active portions thereof lack contaminating proteins
from the same organism from which the SRT protein is derived.
Typically, such proteins are produced by recombinant expression of,
for example, a C. glutamicum SRT protein in a microorganism such as
C. glutamicum.
[0105] An isolated SRT protein or a portion thereof of the
invention can contribute to the resistance or tolerance of C.
glutamicum to one or more chemical or environmental stresses or
hazards, 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 mediate the resistance or
tolerance of C. glutamicum to one or more chemical or environmental
stresses or hazards. The portion of the protein is preferably a
biologically active portion as described herein. In another
preferred embodiment, an SRT protein of the invention has an amino
acid sequence shown in Appendix B. In yet another preferred
embodiment, the SRT 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 SRT 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 SRT proteins of the present invention
also preferably possess at least one of the SRT activities
described herein. For example, a preferred SRT 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 increase the resistance or tolerance of C. glutamicum to
one or more environmental or chemical stresses, or which has one or
more of the activities set forth in Table 1.
[0106] In other embodiments, the SRT 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 SRT 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 SRT 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.
[0107] Biologically active portions of an SRT protein include
peptides comprising amino acid sequences derived from the amino
acid sequence of an SRT protein, e.g., an amino acid sequence shown
in Appendix B or the amino acid sequence of a protein homologous to
an SRT protein, which include fewer amino acids than a full length
SRT protein or the full length protein which is homologous to an
SRT protein, and exhibit at least one activity of an SRT 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 SRT 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 SRT protein
include one or more selected domains/motifs or portions thereof
having biological activity.
[0108] SRT 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 SRT protein is expressed in the host cell. The SRT
protein can then be isolated from the cells by an appropriate
purification scheme using standard protein purification techniques.
Alternative to recombinant expression, an SRT protein, polypeptide,
or peptide can be synthesized chemically using standard peptide
synthesis techniques. Moreover, native SRT protein can be isolated
from cells (e.g., endothelial cells), for example using an anti-SRT
antibody, which can be produced by standard techniques utilizing an
SRT protein or fragment thereof of this invention.
[0109] The invention also provides SRT chimeric or fusion proteins.
As used herein, an SRT "chimeric protein" or "fusion protein"
comprises an SRT polypeptide operatively linked to a non-SRT
polypeptide. An "SRT polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to SRT, whereas a "non-SRT
polypeptide" refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous to
the SRT protein, e.g., a protein which is different from the SRT
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 SRT polypeptide and the non-SRT
polypeptide are fused in-frame to each other. The non-SRT
polypeptide can be fused to the N-terminus or C-terminus of the SRT
polypeptide. For example, in one embodiment the fusion protein is a
GST-SRT fusion protein in which the SRT sequences are fused to the
C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant SRT proteins. In another
embodiment, the fusion protein is an SRT 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 SRT protein can be increased through use of a heterologous
signal sequence.
[0110] Preferably, an SRT chimeric or fusion protein of the
invention is produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, for example by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al. John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). An SRT-encoding nucleic acid can be cloned into
such an expression vector such that the fusion moiety is linked
in-frame to the SRT protein.
[0111] Homologues of the SRT protein can be generated by
mutagenesis, e.g., discrete point mutation or truncation of the SRT
protein. As used herein, the term "homologue" refers to a variant
form of the SRT protein which acts as an agonist or antagonist of
the activity of the SRT protein. An agonist of the SRT protein can
retain substantially the same, or a subset, of the biological
activities of the SRT protein. An antagonist of the SRT protein can
inhibit one or more of the activities of the naturally occurring
form of the SRT protein, by, for example, competitively binding to
a downstream or upstream member of the SRT system which includes
the SRT protein. Thus, the C. glutamicum SRT protein and homologues
thereof of the present invention may increase the tolerance or
resistance of C. glutamicum to one or more chemical or
environmental stresses.
[0112] In an alternative embodiment, homologues of the SRT protein
can be identified by screening combinatorial libraries of mutants,
e.g., truncation mutants, of the SRT protein for SRT protein
agonist or antagonist activity. In one embodiment, a variegated
library of SRT variants is generated by combinatorial mutagenesis
at the nucleic acid level and is encoded by a variegated gene
library. A variegated library of SRT variants can be produced by,
for example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of
potential SRT sequences is expressible as individual polypeptides,
or alternatively, as a set of larger fusion proteins (e.g., for
phage display) containing the set of SRT sequences therein. There
are a variety of methods which can be used to produce libraries of
potential SRT 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 SRT
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.
[0113] In addition, libraries of fragments of the SRT protein
coding can be used to generate a variegated population of SRT
fragments for screening and subsequent selection of homologues of
an SRT protein. In one embodiment, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of an SRT 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 SRT protein.
[0114] 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 SRT homologues. The most widely used techniques,
which are amenable to high through-put analysis, for screening
large gene libraries typically include cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates isolation of the vector encoding the
gene whose product was detected. Recursive ensemble mutagenesis
(REM), a new technique which enhances the frequency of functional
mutants in the libraries, can be used in combination with the
screening assays to identify SRT homologues (Arkin and Yourvan
(1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein
Engineering 6(3):327-331).
[0115] In another embodiment, cell based assays can be exploited to
analyze a variegated SRT library, using methods well known in the
art.
D. Uses and Methods of the Invention
[0116] 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 SRT protein regions required for function;
modulation of an SRT protein activity; modulation of the activity
of an SRT pathway; and modulation of cellular production of a
desired compound, such as a fine chemical.
[0117] The SRT 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.
[0118] 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.
[0119] 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.
[0120] The nucleic acid and protein molecules of the invention may
also serve as markers for specific regions of the genome. This has
utility not only in the mapping of the genome, but also for
functional studies of C. glutamicum proteins. For example, to
identify the region of the genome to which a particular C.
glutamicum DNA-binding protein binds, the C. glutamicum genome
could be digested, and the fragments incubated with the DNA-binding
protein. Those which bind the protein may be additionally probed
with the nucleic acid molecules of the invention, preferably with
readily detectable labels; binding of such a nucleic acid molecule
to the genome fragment enables the localization of the fragment to
the genome map of C. glutamicum, and, when performed multiple times
with different enzymes, facilitates a rapid determination of the
nucleic acid sequence to which the protein binds. Further, the
nucleic acid molecules of the invention may be sufficiently
homologous to the sequences of related species such that these
nucleic acid molecules may serve as markers for the construction of
a genomic map in related bacteria, such as Brevibacterium
lactofermentum.
[0121] The SRT nucleic acid molecules of the invention are also
useful for evolutionary and protein structural studies. The
resistance processes in which the molecules of the invention
participate are utilized by a wide variety of 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.
[0122] The genes of the invention, e.g., the gene encoding LMRB
(SEQ ID NO:1) or other gene of the invention encoding a chemical or
environmental resistance or tolerance protein (e.g., resistance
against one or more antibiotics), may be used as genetic markers
for the genetic transformation of (e.g., the transfer of additional
genes into or disruption of preexisting genes of) organisms such as
C. glutamicum or other bacterial species. Use of these nucleic acid
molecules permits efficient selection of organisms which have
incorporated a given transgene cassette (e.g., a plasmid, phage,
phasmid, phagemid, transposon, or other nucleic acid element),
based on a trait which permits the survival of the organism in an
otherwise hostile or toxic environment (e.g., in the presence of an
antimicrobial compound). By employing one or more of the genes of
the invention as genetic markers, the speed and ease with which
organisms having desirable transformed traits (e.g., modulated fine
chemical production) are engineered and isolated are improved.
While it is advantageous to use the genes of the invention for
selection of transformed C. glutamicum and related bacteria, it is
possible, as described herein, to use homologs (e.g., homologs from
other organisms), allelic variants or fragments of the gene
retaining desired activity. Furthermore, 5' and 3' regulatory
elements of the genes of the invention may be modified as described
herein (e.g., by nucleotide substitution, insertion, deletion, or
replacement with a more desirable genetic element) to modulate the
transcription of the gene. For example, an LMRB variant in which
the nucleotide sequence in the region from -1 to -200 5' to the
start codon has been altered to modulate (preferably increase) the
transcription and/or translation of LMRB may be employed, as can
constructs in which a gene of the invention (e.g., the LMRB gene
(SEQ ID NO:1)) is functionally coupled to one or more regulatory
signals (e.g., inducer or repressor binding sequences) which can be
used for modulating gene expression. Similarly, more than one copy
of a gene (functional or inactivated) of the invention may be
employed.
[0123] An additional application of the genes of the invention
(e.g., the gene encoding LMRB (SEQ ID NO:1) or other drug- or
antibiotic-resistance gene) is in the discovery of new antibiotics
which are active against Corynebacteria and/or other bacteria. For
example, a gene of the invention may be expressed (or
overexpressed) in a suitable host to generate an organism with
increased resistance to one or more drugs or antibiotics (in the
case of LMRB, lincosamides in particular, especially lincomycin).
This type of resistant host can subsequently be used to screen for
chemicals with bacteriostatic and/or bacteriocidal activity, such
as novel antibiotic compounds. It is possible, in particular, to
use the genes of the invention (e.g., the LMRB gene) to identify
new antibiotics which are active against those microorganisms which
are already resistant to standard antibiotic compounds.
[0124] The invention provides methods for screening molecules which
modulate the activity of an SRT protein, either by interacting with
the protein itself or a substrate or binding partner of the SRT
protein, or by modulating the transcription or translation of a SRT
nucleic acid molecule of the invention. In such methods, a
microorganism expressing one or more SRT 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
SRT protein is assessed.
[0125] Manipulation of the SRT nucleic acid molecules of the
invention may result in the production of SRT proteins having
functional differences from the wild-type SRT 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. The goal of such manipulations is to
increase the viability and activity of the cell when the cell is
exposed to the environmental and chemical stresses and hazards
which frequently accompany large-scale fermentative culture. Thus,
by increasing the activity or copy number of a heat-shock-regulated
protease, one may increase the ability of the cell to destroy
incorrectly folded proteins, which may otherwise interfere with
normal cellular functioning (for example, by continuing to bind
substrates or cofactors although the protein lacks the activity to
act on these molecules appropriately). The same is true for the
overexpression or optimization of activity of one or more chaperone
molecules induced by heat or cold shock. These proteins aid in the
correct folding of nascent polypeptide chains, and thus their
increased activity or presence should increase the percentage of
correctly folded proteins in the cell, which in turn should
increase the overall metabolic efficiency and viability of the
cells in culture. The overexpression or optimization of the
transporter molecules activated by osmotic shock should result in
an increased ability on the part of the cell to maintain
intracellular homeostasis, thereby increasing the viability of
these cells in culture. Similarly, the overproduction or increase
in activity by mutagenesis of proteins involved in the development
of cellular resistance to chemical stresses of various kinds
(either by transport of the offending chemical out of the cell or
by modification of the chemical to a less hazardous substance)
should increase the fitness of the organism in the environment
containing the hazardous substance (i.e., large-scale fermentative
culture), and thereby may permit relatively larger numbers of cells
to survive in such a culture. The net effect of all of these
mutagenesis strategies is to increase the quantity of
fine-chemical-producing compounds in the culture, thereby
increasing the yield, production, and/or efficiency of production
of one or more desired fine chemicals from the culture.
[0126] This aforementioned list of mutagenesis strategies for SRT
proteins to result in increased yields of a desired compound is not
meant to be limiting; variations on these mutagenesis strategies
will be readily apparent to one of ordinary skill in the art. By
these mechanisms, the nucleic acid and protein molecules of the
invention may be utilized to generate C. glutamicum or related
strains of bacteria expressing mutated SRT nucleic acid and protein
molecules such that the yield, production, and/or efficiency of
production of a desired compound is improved. This desired compound
may be any natural product of C. glutamicum, which includes the
final products of biosynthesis pathways and intermediates of
naturally-occurring metabolic pathways, as well as molecules which
do not naturally occur in the metabolism of C. glutamicum, but
which are produced by a C. glutamicum strain of the invention.
[0127] 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
[0128] 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.7H.sub.2O, 3
mg/l MnCl.sub.2.times.4H.sub.2O, 30 mg/l H.sub.3BO.sub.3 20 mg/l
CoCl.sub.2.times.6H.sub.2O, 1 mg/l NiCl.sub.2.times.6H.sub.2O, 3
mg/l Na.sub.2MoO.sub.4.times.2H.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 .alpha.-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
[0129] 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.)
[0130] 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
[0131] Genomic libraries as described in Example 2 were used for
DNA sequencing according to standard methods, in particular by the
chain termination method using AB1377 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
[0132] 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.sub.--7: 32-34.
Example 5
DNA Transfer Between Escherichia coli and Corynebacterium
glutamicum
[0133] 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).
[0134] 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 vector into strains of Corynebacterium
glutamicum. Transformation of C. glutamicum can be achieved by
protoplast transformation (Kastsumata, R. et al. (1984) J.
Bacteriol. 159306-311), electroporation (Liebl, E. et al. (1989)
FEMS Microbiol. Letters, 53:399-303) and in cases where special
vectors are used, also by conjugation (as described e.g. in
Schafer, A et al. (1990) J. Bacteriol. 172:1663-1666). It is also
possible to transfer the shuttle vectors for C. glutamicum to E.
coli by preparing plasmid DNA from C. glutamicum (using standard
methods well-known in the art) and transforming it into E. coli.
This transformation step can be performed using standard methods,
but it is advantageous to use an Mcr-deficient E. coli strain, such
as NM522 (Gough & Murray (1983) J. Mol. Biol. 166:1-19).
[0135] 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).
[0136] 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
[0137] 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.
[0138] 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
[0139] 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; U.S. Pat.
No. 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.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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
[0145] 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. Academic Press: New
York; Bisswanger, H., (1994) Enzymkinetik, 2.sup.nd ed. VCH:
Weinheim (ISBN 3527300325); Bergmeyer, H. U., Bergmeyer, J.,
Gra.beta.1, 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.
[0146] 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.
[0147] 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
[0148] 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.)
[0149] 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 yield, production, and/or 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
[0150] 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.
[0151] 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.
[0152] 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).
[0153] 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
Cloning of a Corynebacterium glutamicum Gene Involved in Lincomycin
Resistance Using a Reporter Gene Approach
A. Identification of the Gene Encoding the LMRB Protein
[0154] Plasmid pSL130 was constructed by ligation of the aceB
promoter region (paceB) of C. glutamicum (Kim, H. J. et al. (1997)
J. Microbiol. Biotechnol. 7: 287-292) into the polylinker of the
lac operon fusion vector pRS415, which lacks a promoter (Simon, R.
W. et al. (1987) Gene 53: 85-96). Plasmid pSL145 was constructed by
ligating the resulting paceB-lac region into the E. coli cloning
vector pACYC184. E. coli DH5.alpha.F' was transformed with pSL145
and the resulting strain was used as a host for screening of a
genomic C. glutamicum library (in pSL109).
[0155] Transformants were screened by growth on agar medium
containing 5-bromo-4-chloro-3-indolyl-beta-D-glalactopyranoside
(X-Gal). A white colony, containing DNA influencing lacZ
expression, was selected for further analysis. This clone was found
to contain a 4 kB fragment from the gene library. Subdlones were
constructed in pSL109 and a subclone which retained the white
phenotype on X-Gal plates was identified. This subclone was found
to contain a 2.6 kB BamHI-XhoI fragment (plasmid pSL149-5). The
fragment was sequenced and identified as a membrane
protein-encoding gene (LMRB gene).
[0156] The 1442 nucleotides of the coding sequence of the LMRB gene
encode a polypeptide of 481 amino acid residues with a high
percentage of hydrophobic amino acids. A Genbank search determined
that the LMRB protein is 40% identical to the protein product of
the ImrB gene from Bacillus subtilis (Genbank Accession AL009126,
TREMBL Accession P94422), as determined using a CLUSTAL W analysis
(using standard parameters).
[0157] The LMRN protein contains a sequence pattern:
158-A-P-A-L-G-P-T-L-S-G-167 (SEQ ID NO:301), which resembles the
known multi-drug-resistance-protein consensus motif
G-X-X-X-G-P-X-X-G-G (SEQ ID NO:302) (Paulsen, I. T., and Skurray,
R. A. (1993) Gene 124: 1-11). Therefore, the LMRB protein was
classified as a drug resistance protein.
B. In vivo Analysis of lmrB Function
[0158] The lmrB gene was overexpressed in C. glutamicum ASO19E12
(Kim, H. J. et al. (1997) J. Microbiol. Biotechnol. 7: 287-292)
using the plasmid pSL149-5, described above.
[0159] Disruption of the LMRB gene was accomplished by use of the
vector pSL18-lmrB. This vector was constructed as follows: an
internal fragment of the LMRB gene was amplified by PCR under
standard conditions using primers 5'-CTCCAGGATTGCTCCGAAGG-3' (SEQ
ID NO:303) and 5'-CACAGTGGTTGACCACTGGC-3' (SEQ ID NO:304). The
resulting PCR product was treated with T7 DNA polymerase and T7
polynucleotide kinase, and was cloned into the SmaI site of plasmid
pSL18 (Kim, Y. H. and H.-S. Lee (1996) J. Microbiol. Biotechnol. 6:
315-320). The disruption of the LMRB gene in C. glutamicum ASO19E12
was performed by conjugation, as previously described (Schwarzer
and Puhler (1991) Bio/Technology 9:84-87).
[0160] C. glutamicum cells transformed with pSL149-5 displayed
similar resistances as untransformed cells against erythromycin,
penicillin G, tetracycline, chloramphenicol, spectinomycin,
nalidixic acid, gentamycin, streptomycin, ethidium bromide,
carbonyl cyanide m-chlorophenylhydrazone (CCCP), and sodium dodecyl
sulfate. Significant differences were observed, however, in the
resistance of transformed and untransformed cells to
lincomycin.
[0161] LMRB-overexpressing C. glutamicum cells were found to be
able to grow in the presence of 20 .mu.g/ml lincomycin. In
contrast, cells which do not overexpress LMRB (or cells carrying a
LMRB disruption) were not able to grow on agar media containing 5
.mu.g/ml lincomycin. This effect was clearly visible in liquid
culture. LMRB overexpression led to a 9-fold increased resistance
(compared to wild-type) against lincomycin and LMRB disruption
resulted in a decreased resistance (28% of wild-type) to this
antibiotic.
Example 12
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 SRT 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 SRT 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 13
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 14
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 Included in
the Application Nucleic Amino Acid Acid SEQ SEQ Identification NT
NT ID NO ID NO Code Contig. Start Stop Function 1 2 RXA01524
GR00424 29041 30483 Lincomycine RESISTANCE PROTEIN 3 4 RXA00497
GR00124 52 348 10 KD CHAPERONIN 5 6 RXN00493 VV0086 14389 16002 60
KD CHAPERONIN 7 8 F RXA00498 GR00124 363 1601 60 KD CHAPERONIN 9 10
RXA01217 GR00353 802 203 GENERAL STRESS PROTEIN CTC 11 12 RXA00605
GR00159 7412 5865 CATALASE (EC 1.11.1.6) 13 14 RXA00404 GR00089
2909 594 CARBON STARVATION PROTEIN A 15 16 RXN03119 VV0098 86877
87008 SUPEROXIDE DISMUTASE [MN] (EC 1.15.1.1) 17 18 RXN03120 VV0098
87351 87476 SUPEROXIDE DISMUTASE [MN] (EC 1.15.1.1) 19 20 RXN00575
VV0323 14716 15252 PHOSPHINOTHRICIN-RESISTANCE PROTEIN 21 22 F
RXA00575 GR00156 2130 1648 PHOSPHINOTHRICIN-RESISTANCE PROTEIN
Chaperones 23 24 RXN01345 VV0123 4883 3432 Moleculares chaperon
(HSP70/DnaK family) 25 26 F RXA01345 GR00391 1172 6 Molecular
chaperones (HSP70/DnaK family) 27 28 RXA02541 GR00726 13657 12473
DNAJ PROTEIN 29 30 RXA02542 GR00726 14518 13865 GRPE PROTEIN 31 32
RXN02543 VV0057 22031 20178 DNAK PROTEIN 33 34 F RXA02543 GR00726
16375 14522 DNAK PROTEIN 35 36 RXN02280 VV0152 1849 26 TRAP1 37 38
F RXA02282 GR00659 1145 1480 Molecular chaperone, HSP90 family 39
40 RXA00886 GR00242 12396 13541 DNAJ PROTEIN 41 42 RXS00568 VV0251
2928 1582 TRIGGER FACTOR 43 44 RXN03038 VV0017 42941 43666 PS1
PROTEIN VORLAUFER 45 46 RXN03039 VV0018 2 631 PS1 PROTEIN VORLAUFER
47 48 RXN03040 VV0018 761 1069 PS1 PROTEIN VORLAUFER 49 50 RXN03051
VV0022 2832 3566 PS1 PROTEIN VORLAUFER 51 52 RXN03054 VV0026 1906
3486 PS1 PROTEIN VORLAUFER 53 54 RXN02949 VV0025 31243 31575
PREPROTEIN TRANSLOKASE SECE UNTEREINHEIT 55 56 RXN02462 VV0124
11932 13749 PREPROTEIN TRANSLOKASE SECA UNTEREINHEIT 57 58 RXN01559
VV0171 7795 5954 PROTEIN-EXPORT MEMBRANE PROTEIN SECD 59 60
RXN00046 VV0119 5363 6058 Signal Erkennung particle GTPase 61 62
RXN01863 VV0206 1172 24 /O/C Thioredoxin-ahnliche oxidoreductase 63
64 RXN00833 VV0180 8039 8533 THIOL PEROXIDASE (EC 1.11.1.--) 65 66
RXN01676 VV0179 12059 11304 THIOL: DISULFIDE AUSTAUSCH PROTEIN DSBD
67 68 RXN00380 VV0223 836 216 THIOL: DISULFIDE AUSTAUSCH PROTEIN
TLPA 69 70 RXN00937 VV0079 42335 42706 THIOREDOXIN 71 72 RXN02325
VV0047 5527 6393 THIOREDOXIN 73 74 RXN01837 VV0320 7103 7879
PEPTIDYL-PROLYL CIS-TRANS ISOMERASE (EC 5.2.1.8) 75 76 RXN01926
VV0284 1 741 PEPTID KETTE RELEASE FACTOR 3 77 78 RXN02002 VV0111
141 518 PEPTID KETTE RELEASE FACTOR 3 79 80 RXN02736 VV0074 13600
14556 PUTATIVES OXPPCYCLE PROTEIN OPCA 81 82 RXS03217 SMALL
COLD-SHOCK PROTEIN 83 84 F RXA01917 GR00549 3465 3665 SMALL
COLD-SHOCK PROTEIN Proteins involved in stress responses 85 86
RXA02184 GR00641 19628 19248 COLD SHOCK-LIKE PROTEIN CSPC 87 88
RXA00810 GR00218 792 992 SMALL COLD-SHOCK PROTEIN 89 90 RXA01674
GR00467 1878 2771 PROBABLE HYDROGEN PEROXIDE-INDUCIBLE GENES
ACTIVATOR 91 92 RXA02431 GR00708 2 1192 damage-inducible protein P
93 94 RXA02446 GR00709 11640 11206 OSMOTICALLY INDUCIBLE PROTEIN C
95 96 RXA02861 GR10006 551 1633 probable metallothionein u0308aa
--Mycobacterium leprae 97 98 RXA00981 GR00276 3388 4017 GTP
PYROPHOSPHOKINASE (EC 2.7.6.5) 99 100 RXN00786 VV0321 1680 706 LYTB
PROTEIN 101 102 RXS01027 VV0143 5761 6768 DIADENOSINE
5',5'''-P1,P4-TETRAPHOSPHATE HYDROLASE (EC 3.6.1.17) 103 104
RXS01528 VV0050 17276 16749 DIADENOSINE
5',5'''-P1,P4-TETRAPHOSPHATE HYDROLASE (EC 3.6.1.17) 105 106
RXS01716 VV0319 3259 2774 EXOPOLYPHOSPHATASE (EC 3.6.1.11) 107 108
RXS01835 VV0143 10575 10045 GUANOSINE-3',5'-BIS(DIPHOSPHATE)
3'-PYROPHOSPHOHYDROLASE (EC 3.1.7.2) 109 110 RXS02497 VV0007 15609
16535 EXOPOLYPHOSPHATASE (EC 3.6.1.11) 111 112 RXS02972 VV0319 2763
2353 EXOPOLYPHOSPHATASE (EC 3.6.1.11) Resistance and tolerance 113
114 RXA02159 GR00640 6231 6743 ARGININE HYDROXIMATE RESISTANCE
PROTEIN 115 116 RXA02201 GR00646 5837 6199 ARSENATE REDUCTASE 117
118 RXA00599 GR00159 1843 1457 ARSENICAL-RESISTANCE PROTEIN ACR3
119 120 RXA00600 GR00159 2940 1843 ARSENICAL-RESISTANCE PROTEIN
ACR3 121 122 RXA02200 GR00646 4651 5760 ARSENICAL-RESISTANCE
PROTEIN ACR3 123 124 RXA02202 GR00646 6278 6916
ARSENICAL-RESISTANCE PROTEIN ACR3 125 126 RXA02205 GR00646 9871
8993 BACITRACIN RESISTANCE PROTEIN (PUTATIVE UNDECAPRENOL KINASE)
(EC 2.7.1.66) 127 128 RXA00900 GR00245 4052 3201 BICYCLOMYCIN
RESISTANCE PROTEIN 129 130 RXN00901 VV0140 8581 8168 BICYCLOMYCIN
RESISTANCE PROTEIN 131 132 F RXA00901 GR00245 4357 3980
BICYCLOMYCIN RESISTANCE PROTEIN 133 134 RXA00289 GR00046 3263 4438
CHLORAMPHENICOL RESISTANCE PROTEIN 135 136 RXN01984 VV0056 1515
1811 CHLORAMPHENICOL RESISTANCE PROTEIN 137 138 F RXA01984 GR00574
282 4 CHLORAMPHENICOL RESISTANCE PROTEIN 139 140 RXA00109 GR00015
1176 565 COPPER RESISTANCE PROTEIN C PRECURSOR 141 142 RXA00109
GR00015 1176 565 COPPER RESISTANCE PROTEIN C PRECURSOR 143 144
RXA00996 GR00283 1763 1023 DAUNORUBICIN RESISTANCE ATP-BINDING
PROTEIN DRRA 145 146 RXN00829 VV0180 7950 5611 DAUNORUBICIN
RESISTANCE PROTEIN 147 148 F RXA00829 GR00224 2 256 DAUNORUBICIN
RESISTANCE PROTEIN 149 150 F RXA00834 GR00225 463 2025 DAUNORUBICIN
RESISTANCE PROTEIN 151 152 RXA00995 GR00283 1023 283 DAUNORUBICIN
RESISTANCE TRANSMEMBRANE PROTEIN 153 154 RXN00803 VV0009 53858
52629 METHYLENOMYCIN A RESISTANCE PROTEIN 155 156 F RXA00803
GR00214 4560 5162 METHYLENOMYCIN A RESISTANCE PROTEIN 157 158
RXA01407 GR00410 3918 3028 METHYLENOMYCIN A RESISTANCE PROTEIN 159
160 RXA01408 GR00410 4384 4184 METHYLENOMYCIN A RESISTANCE PROTEIN
161 162 RXN01922 VV0020 2031 3182 METHYLENOMYCIN A RESISTANCE
PROTEIN 163 164 F RXA01922 GR00552 3 1109 METHYLENOMYCIN A
RESISTANCE PROTEIN 165 166 RXA02060 GR00626 1 339
MYCINAMICIN-RESISTANCE PROTEIN MYRA 167 168 RXN01936 VV0127 40116
41387 MACROLIDE-EFFLUX PROTEIN 169 170 F RXA01936 GR00555 9796 8975
NICKEL RESISTANCE PROTEIN 171 172 F RXA01937 GR00555 10246 9821
NICKEL RESISTANCE PROTEIN 173 174 RXN01010 VV0209 3776 4894
QUINOLONE RESISTANCE NORA PROTEIN 175 176 F RXA01010 GR00288 774 4
QUINOLONE RESISTANCE NORA PROTEIN 177 178 RXN03142 VV0136 5754 4612
QUINOLONE RESISTANCE NORA PROTEIN 179 180 F RXA01150 GR00323 3807
2917 QUINOLONE RESISTANCE NORA PROTEIN 181 182 RXN02964 VV0102 7931
6714 QUINOLONE RESISTANCE NORA PROTEIN 183 184 F RXA02116 GR00636
911 6 QUINOLONE RESISTANCE NORA PROTEIN 185 186 RXA00858 GR00233
1680 2147 TELLURIUM RESISTANCE PROTEIN TERC 187 188 RXA02305
GR00663 2921 2070 DAUNOMYCIN C-14 HYDROXYLASE 189 190 RXA00084
GR00013 2367 1543 VIBRIOBACTIN UTILIZATION PROTEIN VIUB 191 192
RXA00843 GR00228 3236 3580 ARSENATE REDUCTASE 193 194 RXA01052
GR00296 3398 3706 MERCURIC REDUCTASE (EC 1.16.1.1) 195 196 RXA01053
GR00296 3772 4191 MERCURIC REDUCTASE (EC 1.16.1.1) 197 198 RXA01054
GR00296 4229 4717 MERCURIC REDUCTASE (EC 1.16.1.1) 199 200 RXN03123
VV0106 808 1245 HEAVY METAL TOLERANCE PROTEIN PRECURSOR 201 202 F
RXA00993 GR00282 641 6 HEAVY METAL TOLERANCE PROTEIN PRECURSOR 203
204 RXA01051 GR00296 3298 2690 VANZ PROTEIN teicoplanin resistance
protein 205 206 RXN01873 VV0248 2054 819 Hypothetical Drug
Resistance Protein 207 208 F RXA01873 GR00535 855 1946 Hypothetical
Drug Resistance Protein 209 210 RXN00034 VV0020 16933 18381
MULTIDRUG RESISTANCE PROTEIN B 211 212 F RXA02273 GR00655 8058 9002
Hypothetical Drug Resistance Protein 213 214 RXN03075 VV0042 2491
3216 Hypothetical Drug Transporter 215 216 F RXA02907 GR10044 1395
2120 Hypothetical Drug Transporter 217 218 RXA00479 GR00119 16290
14101 Hypothetical Drug Transporter 219 220 RXN03124 VV0108 4 963
Hypothetical Drug Transporter 221 222 F RXA01180 GR00336 4 765
Hypothetical Drug Transporter 223 224 RXA02586 GR00741 10296 10027
Hypothetical Drug Transporter 225 226 RXA02587 GR00741 12343 10253
Hypothetical Drug Transporter 227 228 RXN03042 VV0018 2440 1835
Hypothetical Drug Transporter 229 230 F RXA02893 GR10035 1841 1236
Hypothetical Drug Transporter 231 232 RXA01616 GR00450 1684 203
MULTIDRUG EFFLUX PROTEIN QACB 233 234 RXA01666 GR00463 2307 3683
MULTIDRUG RESISTANCE PROTEIN 235 236 RXA00062 GR00009 13252 11855
MULTIDRUG RESISTANCE PROTEIN B 237 238 RXA00215 GR00032 13834 15294
MULTIDRUG RESISTANCE PROTEIN B 239 240 RXN03064 VV0038 4892 6223
MULTIDRUG RESISTANCE PROTEIN B 241 242 F RXA00565 GR00151 4892 5884
MULTIDRUG RESISTANCE PROTEIN B 243 244 F RXA02878 GR10016 1837 1481
MULTIDRUG RESISTANCE PROTEIN B 245 246 RXA00648 GR00169 2713 1304
MULTIDRUG RESISTANCE PROTEIN B 247 248 RXN01320 VV0082 13146 11500
MULTIDRUG RESISTANCE PROTEIN B 249 250 F RXA01314 GR00382 744 4
MULTIDRUG RESISTANCE PROTEIN B 251 252 F RXA01320 GR00383 1979 1200
MULTIDRUG RESISTANCE PROTEIN B 253 254 RXN02926 VV0082 11497 9866
MULTIDRUG RESISTANCE PROTEIN B 255 256 F RXA01319 GR00383 1197 4
MULTIDRUG RESISTANCE PROTEIN B 257 258 RXA01578 GR00439 1423 29
MULTIDRUG RESISTANCE PROTEIN B 259 260 RXA02087 GR00629 7076 5730
MULTIDRUG RESISTANCE PROTEIN B 261 262 RXA02088 GR00629 8294 7080
MULTIDRUG RESISTANCE PROTEIN B 263 264 RXA00764 GR00204 3284 2169
BMRU PROTEIN Bacillus subtilis bmrU, multidrug efflux transporter
265 266 RXN03125 VV0108 972 1142 Hypothetical Drug Transporter 267
268 RXN01553 VV0135 25201 26520 Hypothetical Drug Permease 269 270
RXN00535 VV0219 5155 5871 Hypothetical Drug Resistance Protein 271
272 RXN00453 VV0076 1173 3521 Hypothetical Drug Transporter 273 274
RXN00932 VV0171 13120 13593 Hypothetical Drug Transporter 275 276
RXN03022 VV0002 65 511 MULTIDRUG RESISTANCE PROTEIN B 277 278
RXN03151 VV0163 489 4 MYCINAMICIN-RESISTANCE PROTEIN MYRA 279 280
RXN02832 VV0358 547 5 LYSOSTAPHIN IMMUNITY FACTOR 281 282 RXN00165
VV0232 3275 1860 MULTIDRUG RESISTANCE-LIKE ATP-BINDING PROTEIN MDL
283 284 RXN01190 VV0169 8992 10338 MULTIDRUG RESISTANCE-LIKE
ATP-BINDING PROTEIN MDL 285 286 RXN01102 VV0059 6128 4884 QUINOLONE
RESISTANCE NORA PROTEIN 287 288 RXN00788 VV0321 3424 3648
CHLORAMPHENICOL RESISTANCE PROTEIN 289 290 RXN02119 VV0102 11242
9602 A201A-RESISTANCE ATP-BINDING PROTEIN 291 292 RXN01605 VV0137
7124 5610 DAUNORUBICIN RESISTANCE TRANSMEMBRANE PROTEIN 293 294
RXN01091 VV0326 567 4 MAZG PROTEIN 295 296 RXS02979 VV0149 2150
2383 MERCURIC TRANSPORT PROTEIN PERIPLASMIC COMPONENT PRECURSOR 297
298 RXS02987 VV0234 527 294 MERCURIC TRANSPORT PROTEIN PERIPLASMIC
COMPONENT PRECURSOR 299 300 RXS03095 VV0057 4056 4424 CADMIUM
EFFLUX SYSTEM ACCESSORY PROTEIN HOMOLOG
[0179] TABLE-US-00002 TABLE 2 GenBank .TM. Accession Gene No. Name
Gene Function Reference A09073 ppg Phosphoenol pyruvate carboxylase
Bachmann, B. et al. "DNA fragment coding for phosphoenolpyruvat
corboxylase, recombinant DNA carrying said fragment, strains
carrying the recombinant DNA and method for producing L-aminino
acids using said strains," Patent: EP 0358940-A 3 Mar. 21, 1990
A45579, Threonine dehydratase Moeckel, B. et al. "Production of
L-isoleucine by means of recombinant A45581, micro-organisms with
deregulated threonine dehydratase," Patent: WO A45583, 9519442-A 5
Jul. 20, 1995 A45585 A45587 AB003132 murC; Kobayashi, M. et al.
"Cloning, sequencing, and characterization of the ftsZ ftsQ; gene
from coryneform bacteria," Biochem. Biophys. Res. Commun., ftsZ
236(2): 383-388 (1997) AB015023 murC; Wachi, M. et al. "A murC gene
from Coryneform bacteria," Appl. Microbiol. ftsQ 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; Glutamine 2-oxoglutarate
gltD 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; Dipeptide-binding
protein; adenine Wehmeier, L. et al. "The role of the
Corynebacterium glutamicum rel gene in apt; rel
phosphoribosyltransferase; GTP (p)ppGpp metabolism," Microbiology,
144: 1853-1862 (1998) pyrophosphokinase AF041436 argR Arginine
repressor AF045998 impA Inositol monophosphate phosphatase AF048764
argH Argininosuccinate lyase AF049897 argC;
N-acetylglutamylphosphate reductase; argJ; ornithine
acetyltransferase; N- argB; acetylglutamate kinase; acetylornithine
argD; transminase; ornithine argF; carbamoyltransferase; arginine
repressor; argR; argininosuccinate synthase; argG;
argininosuccinate lyase argH AF050109 inhA Enoyl-acyl carrier
protein reductase AF050166 hisG ATP phosphoribosyltransferase
AF051846 hisA Phosphoribosylformimino-5-amino-1-
phosphoribosyl-4-imidazolecarboxamide isomerase AF052652 metA
Homoserine O-acetyltransferase Park, S. et al. "Isolation and
analysis of metA, a methionine biosynthetic gene encoding
homoserine acetyltransferase in Corynebacterium glutamicum," Mol.
Cells., 8(3): 286-294 (1998) AF053071 aroB Dehydroquinate
synthetase AF060558 hisH Glutamine amidotransferase AF086704 hisE
Phosphoribosyl-ATP- pyrophosphohydrolase AF114233 aroA
5-enolpyruvylshikimate 3-phosphate synthase AF116184 panD
L-aspartate-alpha-decarboxylase precursor Dusch, N. et al.
"Expression of the Corynebacterium glutamicum panD gene encoding
L-aspartate-alpha-decarboxylase leads to pantothenate
overproduction in Escherichia coli," Appl. Environ. Microbiol.,
65(4)1530-1539 (1999) AF124518 aroD; 3-dehydroquinase; shikimate
aroE dehydrogenase AF124600 aroC; Chorismate synthase; shikimate
kinase; 3- aroK; dehydroquinate synthase; putative aroB;
cytoplasmic peptidase pepQ AF145897 inhA AF145898 inhA AJ001436
ectP Transport of ectoine, glycine betaine, Peter, H. et al.
"Corynebacterium glutamicum is equipped with four secondary proline
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) AJ004934 dapD Tetrahydrodipicolinate
succinylase Wehrmann, A. et al. "Different modes of diaminopimelate
synthesis and their (incomplete.sup.i) role in cell wall integrity:
A study with Corynebacterium glutamicum," J. Bacteriol., 180(12):
3159-3165 (1998) AJ007732 ppc; Phosphoenolpyruvate-carboxylase; ?;
high secG; affinity ammonium uptake protein; amt; putative
ornithine-cyclodecarboxylase; ocd; sarcosine oxidase soxA AJ010319
ftsY, Involved in cell division; PII protein; Jakoby, M. et al.
"Nitrogen regulation in Corynebacterium glutamicum; glnB,
uridylyltransferase (uridylyl-removing Isolation of genes involved
in biochemical characterization of corresponding glnD; enzmye);
signal recognition particle; low proteins," FEMS Microbiol.,
173(2): 303-310 (1999) srp; affinity ammonium uptake protein amtP
AJ132968 cat Chloramphenicol aceteyl transferase AJ224946 mqo
L-malate: quinone oxidoreductase Molenaar, D. et al. "Biochemical
and genetic characterization of the membrane-associated malate
dehydrogenase (acceptor) from Corynebacterium glutamicum," Eur. J.
Biochem., 254(2): 395-403 (1998) AJ238250 ndh NADH dehydrogenase
AJ238703 porA Porin Lichtinger, T. et al. "Biochemical and
biophysical characterization of the cell wall porin of
Corynebacterium glutamicum: The channel is formed by a low
molecular mass polypeptide," Biochemistry, 37(43): 15024-15032
(1998) D17429 Transposable element IS31831 Vertes, A. A. et al.
"Isolation and characterization of IS31831, a transposable element
from Corynebacterium glutamicum," Mol. Microbiol., 11(4): 739-746
(1994) D84102 odhA 2-oxoglutarate dehydrogenase Usuda, Y. et al.
"Molecular cloning of the Corynebacterium glutamicum
(Brevibacterium lactofermentum AJ12036) odhA gene encoding a novel
type of 2-oxoglutarate dehydrogenase," Microbiology, 142: 3347-3354
(1996) E01358 hdh; hk Homoserine dehydrogenase; homoserine
Katsumata, R. et al. "Production of L-thereonine and L-isoleucine,"
Patent: JP kinase 1987232392-A 1 Oct. 12, 1987 E01359 Upstream of
the start codon of homoserine Katsumata, R. et al. "Production of
L-thereonine and L-isoleucine," Patent: JP kinase gene 1987232392-A
2 Oct. 12, 1987 E01375 Tryptophan operon E01376 trpL; Leader
peptide; anthranilate synthase Matsui, K. et al. "Tryptophan
operon, peptide and protein coded thereby, trpE utilization of
tryptophan operon gene expression and production of tryptophan,"
Patent: JP 1987244382-A 1 Oct. 24, 1987 E01377 Promoter and
operator regions of Matsui, K. et al. "Tryptophan operon, peptide
and protein coded thereby, tryptophan operon utilization of
tryptophan operon gene expression and production of tryptophan,"
Patent: JP 1987244382-A 1 Oct. 24, 1987 E03937 Biotin-synthase
Hatakeyama, K. et al. "DNA fragment containing gene capable of
coding biotin synthetase and its utilization," Patent: JP
1992278088-A 1 Oct. 02, 1992 E04040 Diamino pelargonic acid
aminotransferase Kohama, K. et al. "Gene coding diaminopelargonic
acid aminotransferase and desthiobiotin synthetase and its
utilization," Patent: JP 1992330284-A 1 Nov. 18, 1992 E04041
Desthiobiotinsynthetase Kohama, K. et al. "Gene coding
diaminopelargonic acid aminotransferase and desthiobiotin
synthetase and its utilization," Patent: JP 1992330284-A 1 Nov. 18,
1992 E04307 Flavum aspartase Kurusu, Y. et al. "Gene DNA coding
aspartase and utilization thereof," Patent: JP 1993030977-A 1 Feb.
09, 1993 E04376 Isocitric acid lyase Katsumata, R. et al. "Gene
manifestation controlling DNA," Patent: JP 1993056782-A 3 Mar. 09,
1993 E04377 Isocitric acid lyase N-terminal fragment Katsumata, R.
et al. "Gene manifestation controlling DNA," Patent: JP
1993056782-A 3 Mar. 09, 1993 E04484 Prephenate dehydratase
Sotouchi, N. et al. "Production of L-phenylalanine by
fermentation," Patent: JP 1993076352-A 2 Mar. 30, 1993 E05108
Aspartokinase Fugono, N. et al. "Gene DNA coding Aspartokinase and
its use," Patent: JP 1993184366-A 1 Jul. 27, 1993 E05112
Dihydro-dipichorinate synthetase Hatakeyama, K. et al. "Gene DNA
coding dihydrodipicolinic acid synthetase and its use," Patent: JP
1993184371-A 1 Jul. 27, 1993 E05776 Diaminopimelic acid
dehydrogenase Kobayashi, M. et al. "Gene DNA coding Diaminopimelic
acid dehydrogenase and its use," Patent: JP 1993284970-A 1 Nov. 02,
1993 E05779 Threonine synthase Kohama, K. et al. "Gene DNA coding
threonine synthase and its use," Patent: JP 1993284972-A 1 Nov. 02,
1993 E06110 Prephenate dehydratase Kikuchi, T. et al. "Production
of L-phenylalanine by fermentation method," Patent: JP 1993344881-A
1 Dec. 27, 1993 E06111 Mutated Prephenate dehydratase Kikuchi, T.
et al. "Production of L-phenylalanine by fermentation method,"
Patent: JP 1993344881-A 1 Dec. 27, 1993 E06146 Acetohydroxy acid
synthetase Inui, M. et al. "Gene capable of coding Acetohydroxy
acid synthetase and its use," Patent: JP 1993344893-A 1 Dec. 27,
1993 E06825 Aspartokinase Sugimoto, M. et al. "Mutant aspartokinase
gene," patent: JP 1994062866-A 1 Mar. 08, 1994 E06826 Mutated
aspartokinase alpha subunit Sugimoto, M. et al. "Mutant
aspartokinase gene," patent: JP 1994062866-A 1 Mar. 08, 1994 E06827
Mutated aspartokinase alpha subunit Sugimoto, M. et al. "Mutant
aspartokinase gene," patent: JP 1994062866-A 1 Mar. 08, 1994 E07701
secY Honno, N. et al. "Gene DNA participating in integration of
membraneous protein to membrane," Patent: JP 1994169780-A 1 Jun.
21, 1994 E08177 Aspartokinase Sato, Y. et al. "Genetic DNA capable
of coding Aspartokinase released from feedback inhibition and its
utilization," Patent: JP 1994261766-A 1 Sep. 20, 1994 E08178,
Feedback inhibition-released Sato, Y. et al. "Genetic DNA capable
of coding Aspartokinase released from E08179, Aspartokinase
feedback inhibition and its utilization," Patent: JP 1994261766-A 1
Sep. 20, 1994 E08180, E08181, E08182 E08232 Acetohydroxy-acid
isomeroreductase Inui, M. et al. "Gene DNA coding acetohydroxy acid
isomeroreductase," Patent: JP 1994277067-A 1 Oct. 04, 1994 E08234
secE Asai, Y. et al. "Gene DNA coding for translocation machinery
of protein," Patent: JP 1994277073-A 1 Oct. 04, 1994 E08643 FT
aminotransferase and desthiobiotin Hatakeyama, K. et al. "DNA
fragment having promoter function in synthetase promoter region
coryneform bacterium," Patent: JP 1995031476-A 1 Feb. 03, 1995
E08646 Biotin synthetase Hatakeyama, K. et al. "DNA fragment having
promoter function in coryneform bacterium," Patent: JP 1995031476-A
1 Feb. 03, 1995 E08649 Aspartase Kohama, K. et al "DNA fragment
having promoter function in coryneform bacterium," Patent: JP
1995031478-A 1 Feb. 03, 1995 E08900 Dihydrodipicolinate reductase
Madori, M. et al. "DNA fragment containing gene coding
Dihydrodipicolinate acid reductase and utilization thereof,"
Patent: JP 1995075578-A 1 Mar. 20, 1995 E08901 Diaminopimelic acid
decarboxylase Madori, M. et al. "DNA fragment
containing gene coding Diaminopimelic acid decarboxylase and
utilization thereof," Patent: JP 1995075579-A 1 Mar. 20, 1995
E12594 Serine hydroxymethyltransferase Hatakeyama, K. et al.
"Production of L-trypophan," Patent: JP 1997028391-A 1 Feb. 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 synthetase; diaminopimelic
Moriya, M. et al. "Amplification of gene using artificial
transposon," Patent: acid decarboxylase JP 1997070291-A Mar. 18,
1997 E12767 Dihydrodipicolinic acid synthetase Moriya, M. et al.
"Amplification of gene using artificial transposon," Patent: JP
1997070291-A Mar. 18, 1997 E12770 aspartokinase Moriya, M. et al.
"Amplification of gene using artificial transposon," Patent: JP
1997070291-A Mar. 18, 1997 E12773 Dihydrodipicolinic acid reductase
Moriya, M. et al. "Amplification of gene using artificial
transposon," Patent: JP 1997070291-A Mar. 18, 1997 E13655
Glucose-6-phosphate dehydrogenase Hatakeyama, K. et al.
"Glucose-6-phosphate dehydrogenase and DNA capable of coding the
same," Patent: JP 1997224661-A 1 Sep. 02, 1997 L01508 IlvA
Threonine dehydratase Moeckel, B. et al. "Functional and structural
analysis of the threonine dehydratase of Corynebacterium
glutamicum," J. Bacteriol., 174: 8065-8072 (1992) L07603 EC
3-deoxy-D-arabinoheptulosonate-7- Chen, C. et al. "The cloning and
nucleotide sequence of Corynebacterium 4.2.1.15 phosphate synthase
glutamicum 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase
gene," FEMS Microbiol. Lett., 107: 223-230 (1993) L09232 IlvB;
Acetohydroxy acid synthase large subunit; Keilhauer, C. et al.
"Isoleucine synthesis in Corynebacterium glutamicum: ilvN;
Acetohydroxy acid synthase small subunit; molecular analysis of the
ilvB-ilvN-ilvC operon," J. Bacteriol., 175(17): 5595-5603 ilvC
Acetohydroxy acid isomeroreductase (1993) L18874 PtsM
Phosphoenolpyruvate sugar Fouet, A et al. "Bacillus subtilis
sucrose-specific enzyme II of the phosphotransferase
phosphotransferase system: expression in Escherichia coli and
homology to enzymes II from enteric bacteria," PNAS USA, 84(24):
8773-8777 (1987); Lee, J. K. et al. "Nucleotide sequence of the
gene encoding the Corynebacterium glutamicum mannose enzyme II and
analyses of the deduced protein sequence," FEMS Microbiol. Lett.,
119(1-2): 137-145 (1994) L27123 aceB Malate synthase Lee, H-S. et
al. "Molecular characterization of aceB, a gene encoding malate
synthase in Corynebacterium glutamicum," J. Microbiol. Biotechnol.,
4(4): 256-263 (1994) L27126 Pyruvate kinase Jetten, M. S. et al.
"Structural and functional analysis of pyruvate kinase from
Corynebacterium glutamicum," Appl. Environ. Microbiol., 60(7):
2501-2507 (1994) L28760 aceA Isocitrate lyase L35906 dtxr
Diphtheria toxin repressor Oguiza, J. A. et al. "Molecular cloning,
DNA sequence analysis, and characterization of the Corynebacterium
diphtheriae dtxR from Brevibacterium lactofermentum," J.
Bacteriol., 177(2): 465-467 (1995) M13774 Prephenate dehydratase
Follettie, M. T. et al. "Molecular cloning and nucleotide sequence
of the Corynebacterium glutamicum pheA gene," J. Bacteriol., 167:
695-702 (1986) M16175 5S Park, Y-H. et al. "Phylogenetic analysis
of the coryneform bacteria by 56 rRNA rRNA sequences," J.
Bacteriol., 169: 1801-1806 (1987) M16663 trpE Anthranilate
synthase, 5' end Sano, K. et al. "Structure and function of the trp
operon control regions of Brevibacterium lactofermentum, a
glutamic-acid-producing bacterium," Gene, 52: 191-200 (1987) M16664
trpA Tryptophan synthase, 3'end Sano, K. et al. "Structure and
function of the trp operon control regions of Brevibacterium
lactofermentum, a glutamic-acid-producing bacterium," Gene, 52:
191-200 (1987) M25819 Phosphoenolpyruvate carboxylase O'Regan, M.
et al. "Cloning and nucleotide sequence of the Phosphoenolpyruvate
carboxylase-coding gene of Corynebacterium glutamicum ATCC13032,"
Gene, 77(2): 237-251 (1989) M85106 23S rRNA gene insertion sequence
Roller, C. et al. "Gram-positive bacteria with a high DNA G + C
content are characterized by a common insertion within their 23S
rRNA genes," J. Gen. Microbiol., 138: 1167-1175 (1992) M85107, 23S
rRNA gene insertion sequence Roller, C. et al. "Gram-positive
bacteria with a high DNA G + C content are M85108 characterized by
a common insertion within their 23S rRNA genes," J. Gen.
Microbiol., 138: 1167-1175 (1992) M89931 aecD; Beta C-S lyase;
branched-chain amino Rossol, I. et al. "The Corynebacterium
glutamicum aecD gene encodes a C-S brnQ; acid uptake carrier;
hypothetical protein lyase with alpha, beta-elimination activity
that degrades aminoethylcysteine," yhbw yhbw J. Bacteriol., 174(9):
2968-2977 (1992); Tauch, A. et al. "Isoleucine uptake in
Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene
product," Arch. Microbiol., 169(4): 303-312 (1998) S59299 trp
Leader gene (promoter) Herry, D. M. et al. "Cloning of the trp gene
cluster from a tryptophan- hyperproducing strain of Corynebacterium
glutamicum: identification of a mutation in the trp leader
sequence," Appl. Environ. Microbiol., 59(3): 791-799 (1993) U11545
trpD Anthranilate phosphoribosyltransferase O'Gara, J. P. and
Dunican, L. K. (1994) Complete nucleotide sequence of the
Corynebacterium glutamicum ATCC 21850 tpD gene." Thesis,
Microbiology Department, University College Galway, Ireland. U13922
cglIM; Putative type II 5-cytosoine Schafer, A. et al. "Cloning and
characterization of a DNA region encoding a cglIR;
methyltransferase; putative type II stress-sensitive restriction
system from Corynebacterium glutamicum ATCC clgIIR restriction
endonuclease; putative type I or 13032 and analysis of its role in
intergeneric conjugation with Escherichia type III restriction
endonuclease coli," J. Bacteriol., 176(23): 7309-7319 (1994);
Schafer, A. et al. "The Corynebacterium glutamicum cglIM gene
encoding a 5-cytosine in an McrBC- deficient Escherichia coli
strain," Gene, 203(2): 95-101 (1997) U14965 recA U31224 ppx Ankri,
S. et al. "Mutations in the Corynebacterium glutamicumproline
biosynthetic pathway: A natural bypass of the proA step," J.
Bacteriol., 178(15): 4412-4419 (1996) U31225 proC L-proline: NADP+
5-oxidoreductase Ankri, S. et al. "Mutations in the Corynebacterium
glutamicumproline biosynthetic pathway: A natural bypass of the
proA step," J. Bacteriol., 178(15): 4412-4419 (1996) U31230 obg; ?;
gamma glutamyl kinase; similar to D- Ankri, S. et al. "Mutations in
the Corynebacterium glutamicumproline proB; isomer specific
2-hydroxyacid biosynthetic pathway: A natural bypass of the proA
step," J. Bacteriol., unkdh dehydrogenases 178(15): 4412-4419
(1996) U31281 bioB Biotin synthase Serebriiskii, I. G., "Two new
members of the bio B superfamily: Cloning, sequencing and
expression of bio B genes of Methylobacillus flagellatum and
Corynebacterium glutamicum," Gene, 175: 15-22 (1996) U35023 thtR;
Thiosulfate sulfurtransferase; acyl CoA Jager, W. et al. "A
Corynebacterium glutamicum gene encoding a two-domain accBC
carboxylase protein similar to biotin carboxylases and
biotin-carboxyl-carrier proteins," Arch. Microbiol., 166(2); 76-82
(1996) U43535 cmr Multidrug resistance protein Jager, W. et al. "A
Corynebacterium glutamicum gene conferring multidrug resistance in
the heterologous host Escherichia coli," J. Bacteriol., 179(7):
2449-2451 (1997) U43536 clpB Heat shock ATP-binding protein U53587
aphA-3 3'5''-aminoglycoside phosphotransferase U89648
Corynebacterium glutamicum unidentified sequence involved in
histidine biosynthesis, partial sequence X04960 trpA; Tryptophan
operon Matsui, K. et al. "Complete nucleotide and deduced amino
acid sequences of trpB; the Brevibacterium lactofermentum
tryptophan operon," Nucleic Acids Res., trpC; 14(24): 10113-10114
(1986) trpD; trpE; trpG; trpL X07563 lys A DAP decarboxylase (meso-
Yeh, P. et al. "Nucleic sequence of the lysA gene of
Corynebacterium diaminopimelate decarboxylase, glutamicum and
possible mechanisms for modulation of its expression," Mol. EC
4.1.1.20) Gen. Genet., 212(1): 112-119 (1988) X14234 EC
Phosphoenolpyruvate carboxylase Eikmanns, B. J. et al. "The
Phosphoenolpyruvate carboxylase gene of 4.1.1.31 Corynebacterium
glutamicum: Molecular cloning, nucleotide sequence, and
expression," Mol. Gen. Genet., 218(2): 330-339 (1989); Lepiniec, L.
et al. "Sorghum Phosphoenolpyruvate carboxylase gene family:
structure, function and molecular evolution," Plant. Mol. Biol., 21
(3): 487-502 (1993) X17313 fda Fructose-bisphosphate aldolase Von
der Osten, C. H. et al. "Molecular cloning, nucleotide sequence and
fine- structural analysis of the Corynebacterium glutamicum fda
gene: structural comparison of C. glutamicum fructose-1,
6-biphosphate aldolase to class I and class II aldolases," Mol.
Microbiol., X53993 dapA L-2, 3-dihydrodipicolinate synthetase (EC
Bonnassie, S. et al. "Nucleic sequence of the dapA gene from
4.2.1.52) Corynebacterium glutamicum," Nucleic Acids Res., 18(21):
6421 (1990) X54223 AttB-related site Cianciotto, N. et al. "DNA
sequence homology between att B-related sites of Corynebacterium
diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum,
and the attP site of lambdacorynephage," FEMS. Microbiol, Lett.,
66: 299-302 (1990) X54740 argS; Arginyl-tRNA synthetase; Marcel, T.
et al. "Nucleotide sequence and organization of the upstream region
lysA Diaminopimelate decarboxylase of the Corynebacterium
glutamicum lysA gene," Mol. Microbiol., 4(11): 1819-1830 (1990)
X55994 trpL; Putative leader peptide; anthranilate Heery, D. M. et
al. "Nucleotide sequence of the Corynebacterium glutamicum trpE
synthase component 1 trpE gene," Nucleic Acids Res., 18(23): 7138
(1990) X56037 thrC Threonine synthase Han, K. S. et al. "The
molecular structure of the Corynebacterium glutamicum threonine
synthase gene," Mol. Microbiol., 4(10): 1693-1702 (1990) X56075
attB- Attachment site Cianciotto, N. et al. "DNA sequence homology
between att B-related sites of related Corynebacterium diphtheriae,
Corynebacterium ulcerans, Corynebacterium site glutamicum, and the
attP site of lambdacorynephage," FEMS. Microbiol, Lett., 66:
299-302 (1990) X57226 lysC- Aspartokinase-alpha subunit;
Kalinowski, J. et al. "Genetic and biochemical analysis of the
Aspartokinase alpha; Aspartokinase-beta subunit; aspartate beta
from Corynebacterium glutamicum," Mol. Microbiol., 5(5): 1197-1204
(1991); lysC- semialdehyde dehydrogenase Kalinowski, J. et al.
"Aspartokinase genes lysC alpha and lysC beta overlap beta; and are
adjacent to the aspertate beta-semialdehyde dehydrogenase gene asd
in asd Corynebacterium glutamicum," Mol. Gen. Genet., 224(3):
317-324 (1990) X59403 gap; Glyceraldehyde-3-phosphate; Eikmanns, B.
J. "Identification, sequence analysis, and expression of a pgk;
phosphoglycerate kinase; triosephosphate Corynebacterium glutamicum
gene cluster encoding the three glycolytic tpi isomerase enzymes
glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate
kinase, and triosephosphate isomeras," J. Bacteriol., 174(19):
6076-6086 (1992) X59404 gdh Glutamate dehydrogenase Bormann, E. R.
et al. "Molecular analysis of the Corynebacterium glutamicum gdh
gene encoding glutamate dehydrogenase," Mol. Microbiol., 6(3):
317-326 (1992) X60312 lysI L-lysine permease Seep-Feldhaus, A. H.
et al. "Molecular analysis of the Corynebacterium glutamicum 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 PS2 Peyret, J. L. et
al. "Characterization of the cspB gene encoding PS2, an ordered
surface-layer protein in Corynebacterium glutamicum," Mol.
Microbiol., 9(1): 97-109 (1993) X69104 IS3 related insertion
element Bonamy, C. et al. "Identification of IS1206, a
Corynebacterium glutamicum IS3-related insertion sequence and
phylogenetic analysis," Mol. Microbiol., 14(3): 571-581 (1994)
X70959 leuA Isopropylmalate synthase Patek, M. et al. "Leucine
synthesis in Corynebacterium glutamicum: enzyme activities,
structure of leuA, and effect of leuA inactivation on lysine
synthesis," Appl. Environ. Microbiol., 60(1): 133-140 (1994) X71489
icd Isocitrate dehydrogenase (NADP+) Eikmanns, B. J. et al.
"Cloning sequence analysis, expression, and inactivation of the
Corynebacterium glutamicum icd gene encoding isocitrate
dehydrogenase and biochemical characterization of the enzyme," J.
Bacteriol., 177(3): 774-782 (1995) X72855 GDHA Glutamate
dehydrogenase (NADP+) X75083, mtrA 5-methyltryptophan resistance
Heery, D. M. et al. "A sequence from a tryptophan-hyperproducing
strain of X70584 Corynebacterium glutamicum encoding resistance to
5-methyltryptophan," Biochem. Biophys. Res. Commun., 201(3):
1255-1262 (1994) X75085 recA Fitzpatrick, R. et al. "Construction
and characterization of recA mutant strains of Corynebacterium
glutamicum and Brevibacterium lactofermentum," Appl. Microbiol.
Biotechnol., 42(4): 575-580 (1994) X75504 aceA; Partial Isocitrate
lyase; ? Reinscheid, D. J. et al. "Characterization of the
isocitrate lyase gene from thiX 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 16S ribosomal RNA Rainey, F. A. et al.
"Phylogenetic analysis of the genera Rhodococcus and rDNA 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; Glutamate uptake system Kronemeyer, W.
et al. "Structure of the gluABCD cluster encoding the gluB;
glutamate uptake system of Corynebacterium glutamicum," J.
Bacteriol., gluC; 177(5): 1152-1158 (1995) gluD X81379 dapE
Succinyldiaminopimelate desuccinylase Wehrmann, A. et al. "Analysis
of different DNA fragments of Corynebacterium glutamicum
complementing dapE of Escherichia coli," Microbiology, 40: 3349-56
(1994) X82061 16S 16S ribosomal RNA Ruimy, R. et al. "Phylogeny of
the genus Corynebacterium deduced from rDNA analyses of
small-subunit ribosomal DNA sequences," Int. J. Syst. Bacteriol.,
45(4): 740-746 (1995) X82928 asd; Aspartate-semialdehyde
dehydrogenase; ? Serebrijski, I. et al. "Multicopy suppression by
asd gene and osmotic stress- lysC dependent complementation by
heterologous proA in proA mutants," J. Bacteriol., 177(24):
7255-7260 (1995) X82929 proA Gamma-glutamyl phosphate reductase
Serebrijski, I. et al. "Multicopy suppression by asd gene and
osmotic stress- dependent complementation by heterologous proA in
proA mutants," J. Bacteriol., 177(24): 7255-7260 (1995) X84257 16S
16S ribosomal RNA Pascual, C. et al. "Phylogenetic analysis of the
genus Corynebacterium based rDNA on 16S rRNA gene sequences," Int.
J. Syst. Bacteriol., 45(4): 724-728 (1995) X85965 aroP; Aromatic
amino acid permease; ? Wehrmann, A. et al. "Functional analysis of
sequences adjacent to dapE of dapE Corynebacterium glutamicum
proline reveals the presence of aroP, which encodes the aromatic
amino acid transporter," J. Bacteriol., 177(20): 5991-5993 (1995)
X86157 argB; Acetylglutamate kinase; N-acetyl-gamma- Sakanyan, V.
et al. "Genes and enzymes of the acetyl cycle of arginine argC;
glutamyl-phosphate reductase; biosynthesis in Corynebacterium
glutamicum: enzyme evolution in the early argD; acetylornithine
aminotransferase; ornithine steps of the arginine pathway,"
Microbiology, 142: 99-108 (1996) argF; carbamoyltransferase;
glutamate N- argJ acetyltransferase X89084 pta; Phosphate
acetyltransferase; acetate kinase Reinscheid, D. J. et al.
"Cloning, sequence analysis, expression and inactivation ackA of
the Corynebacterium glutamicum pta-ack operon encoding
phosphotransacetylase and acetate kinase," Microbiology, 145:
503-513 (1999) X89850 attB Attachment site Le Marrec, C. et al.
"Genetic characterization of site-specific integration functions of
phi AAU2 infecting "Arthrobacter aureus C70," J. Bacteriol.,
178(7): 1996-2004 (1996) X90356 Promoter fragment F1 Patek, M. et
al. "Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90357 Promoter fragment F2 Patek, M. et al.
"Promoters from Corynebacterium glutainicum: 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 glutaniicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90359 Promoter fragment F13 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90360 Promoter fragment F22 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90361 Promoter fragment F34 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90362 Promoter fragment F37 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90363 Promoter fragment F45 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90364 Promoter fragment F64 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90365 Promoter fragment F75 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90366 Promoter fragment PF101 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90367 Promoter fragment PF104 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X90368 Promoter fragment PF109 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, molecular
analysis and search for a consensus motif," Microbiology, 142:
1297-1309 (1996) X93513 amt Ammonium transport system Siewe, R. M.
et al. "Functional and genetic characterization of the (methyl)
ammonium uptake carrier of Corynebacterium glutamicum," J. Biol.
Chem., 271(10): 5398-5403 (1996) X93514 betP Glycine betaine
transport system Peter, H. et al. "Isolation, characterization, and
expression of the Corynebacterium glutamicum betP gene, encoding
the transport system for the compatible solute glycine betaine," J.
Bacteriol., 178(17): 5229-5234 (1996) X95649 orf4 Patek, M. et al.
"Identification and transcriptional analysis of the dapB-ORF2-
dapA-ORF4 operon of Corynebacterium glutamicum, encoding two
enzymes involved in L-lysine synthesis," Biotechnol. Lett., 19:
1113-1117 (1997) X96471 lysE; Lysine exporter protein; Lysine
export Vrljic, M. et al. "A new type of transporter with a new type
of cellular lysG regulator protein function: L-lysine export from
Corynebacterium glutamicum," Mol. Microbiol., 22(5): 815-826 (1996)
X96580 panB; 3-methyl-2-oxobutanoate Sahm, H. et al.
"D-pantothenate synthesis in Corynebacterium glutamicum and panC;
hydroxymethyltransferase; pantoate-beta- use of panBC and genes
encoding L-valine synthesis for D-pantothenate xylB alanine ligase;
xylulokinase overproduction," Appl. Environ. Microbiol., 65(5):
1973-1979 (1999) X96962 Insertion sequence IS1207 and transposase
X99289 Elongation factor P Ramos, A. et al. "Cloning, sequencing
and expression of the gene encoding elongation factor P in the
amino-acid producer Brevibacterium lactofermentum (Corynebacterium
glutamicum ATCC 13869)," Gene, 198: 217-222 (1997) Y00140 thrB
Homoserine kinase Mateos, L. M. et al. "Nucleotide sequence of the
homoserine kinase (thrB) gene of the Brevibacterium
lactofermentum," Nucleic Acids Res., 15(9): 3922 (1987) Y00151 ddh
Meso-diaminopimelate D-dehydrogenase Ishino, S. et al. "Nucleotide
sequence of the meso-diaminopimelate D- (EC 1.4.1.16) dehydrogenase
gene from Corynebacterium glutamicum," Nucleic Acids Res., 15(9):
3917 (1987) Y00476 thrA Homoserine dehydrogenase Mateos, L. M. et
al. "Nucleotide sequence of the homoserine dehydrogenase (thrA)
gene of the Brevibacterium lactofermentum," Nucleic Acids Res.,
15(24): 10598 (1987) Y00546 hom; Homoserine dehydrogenase;
homoserine Peoples, O. P. et al. "Nucleotide sequence and fine
structural analysis of the thrB kinase Corynebacterium glutamicum
hom-thrB operon," Mol. Microbiol., 2(1): 63-72 (1988) Y08964 murC;
UPD-N-acetylmuramate-alanine ligase; Honrubia, M. P. et al.
"Identification, characterization, and chromosomal ftsQ/ division
initiation protein or cell division organization of the ftsZ gene
from Brevibacterium lactofermentum," Mol. Gen. divD; protein; cell
division protein Genet., 259(1): 97-104 (1998) ftsZ Y09163 putP
High affinity proline transport system Peter, H. et al. "Isolation
of the putP gene of Corynebacterium glutamicum proline and
characterization of a low-affinity uptake system for compatible
solutes," Arch. Microbiol., 168(2): 143-151 (1997) Y09548 pyc
Pyruvate carboxylase Peters-Wendisch, P. G. et al. "Pyruvate
carboxylase from Corynebacterium glutamicum: characterization,
expression and inactivation of the pyc gene," Microbiology, 144:
915-927 (1998) Y09578 leuB 3-isopropylmalate dehydrogenase Patek,
M. et al. "Analysis of the leuB gene from Corynebacterium
glutamicum," Appl. Microbiol. Biotechnol., 50(1): 42-47 (1998)
Y12472 Attachment site bacteriophage Phi-16 Moreau, S. et al.
"Site-specific integration of corynephage Phi-16: The construction
of an integration vector," Microbiol., 145: 539-548 (1999) Y12537
proP Proline/ectoine uptake system protein Peter, H. et al.
"Corynebacterium glutamicum is equipped with four secondary
carriers for compatible solutes: Identification, sequencing, and
characterization
of the proline/ectoine uptake system, ProP, and the
ectoine/proline/glycine betaine carrier, EctP," J. Bacteriol.,
180(22): 6005-6012 (1998) Y13221 glnA Glutamine synthetase 1
Jakoby, M. et al. "Isolation of Corynebacterium glutamicum glnA
gene encoding glutamine synthetase I," FEMS Microbiol. Lett.,
154(1): 81-88 (1997) Y16642 lpd Dihydrolipoamide dehydrogenase
Y18059 Attachment site Corynephage 304L Moreau, S. et al. "Analysis
of the integration functions of φ 304L: An integrase module among
corynephages," Virology, 255(1): 150-159 (1999) Z21501 argS;
Arginyl-tRNA synthetase; Oguiza, J. A. et al. "A gene encoding
arginyl-tRNA synthetase is located in the lysA diaminopimelate
decarboxylase (partial) upstream region of the lysA gene in
Brevibacterium lactofermentum: Regulation of argS-lysA cluster
expression by arginine," J. Bacteriol., 175(22): 7356-7362 (1993)
Z21502 dapA; Dihydrodipicolinate synthase; Pisabarro, A. et al. "A
cluster of three genes (dapA, orf2, and dapB) of dapB
dihydrodipicolinate reductase Brevibacterium lactofermentum encodes
dihydrodipicolinate reductase, and a third polypeptide of unknown
function," J. Bacteriol., 175(9): 2743-2749 (1993) Z29563 thrC
Threonine synthase Malumbres, M. et al. "Analysis and expression of
the thrC gene of the encoded threonine synthase," Appl. Environ.
Microbiol., 60(7)2209-2219 (1994) Z46753 16S Gene for 16S ribosomal
RNA rDNA 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; Catalytic activity UDP-galactose 4- Oguiza, J.
A. et al "The galE gene encoding the UDP-galactose 4-epimerase of
dtxR epimerase; diphtheria toxin regulatory Brevibacterium
lactofermentum is coupled transcriptionally to the dmdR protein
gene," Gene, 177: 103-107 (1996) Z49824 orfl; ?; SigB sigma factor
Oguiza, J. A. et al "Multiple sigma factor genes in Brevibacterium
sigB 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 CECT NCIMB CBS NCTC DSMZ Brevibacterium
ammoniagenes 21054 Brevibacterium ammoniagenes 19350 Brevibacterium
ammoniagenes 19351 Brevibacterium ammoniagenes 19352 Brevibacterium
ammoniagenes 19353 Brevibacterium ammoniagenes 19354 Brevibacterium
ammoniagenes 19355 Brevibacterium ammoniagenes 19356 Brevibacterium
ammoniagenes 21055 Brevibacterium ammoniagenes 21077 Brevibacterium
ammoniagenes 21553 Brevibacterium ammoniagenes 21580 Brevibacterium
ammoniagenes 39101 Brevibacterium butanicum 21196 Brevibacterium
divaricatum 21792 P928 Brevibacterium flavum 21474 Brevibacterium
flavum 21129 Brevibacterium flavum 21518 Brevibacterium flavum
B11474 Brevibacterium flavum B11472 Brevibacterium flavum 21127
Brevibacterium flavum 21128 Brevibacterium flavum 21427
Brevibacterium flavum 21475 Brevibacterium flavum 21517
Brevibacterium flavum 21528 Brevibacterium flavum 21529
Brevibacterium flavum B11477 Brevibacterium flavum B11478
Brevibacterium flavum 21127 Brevibacterium flavum B11474
Brevibacterium healii 15527 Brevibacterium ketoglutamicum 21004
Brevibacterium ketoglutamicum 21089 Brevibacterium ketosoreductum
21914 Brevibacterium lactofermentum 70 Brevibacterium
lactofermentum 74 Brevibacterium lactofermentum 77 Brevibacterium
lactofermentum 21798 Brevibacterium lactofermentum 21799
Brevibacterium lactofermentum 21800 Brevibacterium lactofermentum
21801 Brevibacterium lactofermentum B11470 Brevibacterium
lactofermentum B11471 Brevibacterium lactofermentum 21086
Brevibacterium lactofermentum 21420 Brevibacterium lactofermentum
21086 Brevibacterium lactofermentum 31269 Brevibacterium linens
9174 Brevibacterium linens 19391 Brevibacterium linens 8377
Brevibacterium paraffinolyticum 11160 Brevibacterium spec. 717.73
Brevibacterium spec. 717.73 Brevibacterium spec. 14604
Brevibacterium spec. 21860 Brevibacterium spec. 21864
Brevibacterium spec. 21865 Brevibacterium spec. 21866
Brevibacterium spec. 19240 Corynebacterium acetoacidophilum 21476
Corynebacterium acetoacidophilum 13870 Corynebacterium
acetoglutamicum B11473 Corynebacterium acetoglutamicum B11475
Corynebacterium acetoglutamicum 15806 Corynebacterium
acetoglutamicum 21491 Corynebacterium acetoglutamicum 31270
Corynebacterium acetophilum B3671 Corynebacterium ammoniagenes 6872
2399 Corynebacterium ammoniagenes 15511 Corynebacterium fujiokense
21496 Corynebacterium glutamicum 14067 Corynebacterium glutamicum
39137 Corynebacterium glutamicum 21254 Corynebacterium glutamicum
21255 Corynebacterium glutamicum 31830 Corynebacterium glutamicum
13032 Corynebacterium glutamicum 14305 Corynebacterium glutamicum
15455 Corynebacterium glutamicum 13058 Corynebacterium glutamicum
13059 Corynebacterium glutamicum 13060 Corynebacterium glutamicum
21492 Corynebacterium glutamicum 21513 Corynebacterium glutamicum
21526 Corynebacterium glutamicum 21543 Corynebacterium glutamicum
13287 Corynebacterium glutamicum 21851 Corynebacterium glutamicum
21253 Corynebacterium glutamicum 21514 Corynebacterium glutamicum
21516 Corynebacterium glutamicum 21299 Corynebacterium glutamicum
21300 Corynebacterium glutamicum 39684 Corynebacterium glutamicum
21488 Corynebacterium glutamicum 21649 Corynebacterium glutamicum
21650 Corynebacterium glutamicum 19223 Corynebacterium glutamicum
13869 Corynebacterium glutamicum 21157 Corynebacterium glutamicum
21158 Corynebacterium glutamicum 21159 Corynebacterium glutamicum
21355 Corynebacterium glutamicum 31808 Corynebacterium glutamicum
21674 Corynebacterium glutamicum 21562 Corynebacterium glutamicum
21563 Corynebacterium glutamicum 21564 Corynebacterium glutamicum
21565 Corynebacterium glutamicum 21566 Corynebacterium glutamicum
21567 Corynebacterium glutamicum 21568 Corynebacterium glutamicum
21569 Corynebacterium glutamicum 21570 Corynebacterium glutamicum
21571 Corynebacterium glutamicum 21572 Corynebacterium glutamicum
21573 Corynebacterium glutamicum 21579 Corynebacterium glutamicum
19049 Corynebacterium glutamicum 19050 Corynebacterium glutamicum
19051 Corynebacterium glutamicum 19052 Corynebacterium glutamicum
19053 Corynebacterium glutamicum 19054 Corynebacterium glutamicum
19055 Corynebacterium glutamicum 19056 Corynebacterium glutamicum
19057 Corynebacterium glutamicum 19058 Corynebacterium glutamicum
19059 Corynebacterium glutamicum 19060 Corynebacterium glutamicum
19185 Corynebacterium glutamicum 13286 Corynebacterium glutamicum
21515 Corynebacterium glutamicum 21527 Corynebacterium glutamicum
21544 Corynebacterium glutamicum 21492 Corynebacterium glutamicum
B8183 Corynebacterium glutamicum B8182 Corynebacterium glutamicum
B12416 Corynebacterium glutamicum B12417 Corynebacterium glutamicum
B12418 Corynebacterium glutamicum B11476 Corynebacterium glutamicum
21608 Corynebacterium lilium P973 Corynebacterium nitrilophilus
21419 11594 Corynebacterium spec. P4445 Corynebacterium spec. P4446
Corynebacterium spec. 31088 Corynebacterium spec. 31089
Corynebacterium spec. 31090 Corynebacterium spec. 31090
Corynebacterium spec. 31090 Corynebacterium spec. 15954 20145
Corynebacterium spec. 21857 Corynebacterium spec. 21862
Corynebacterium spec. 21863 ATCC: American Type Culture Collection,
Rockville, MD, USA FERM: Fermentation Research Institute, Chiba,
Japan NRRL: ARS Culture Collection, Northern Regional Research
Laboratory, Peoria, IL, USA CECT: Coleccion Espanola de Cultivos
Tipo, Valencia, Spain NCIMB: National Collection of Industrial and
Marine Bacteria Ltd., Aberdeen, UK CBS: Centraalbureau voor
Schimmelcultures, Baarn, NL NCTC: National Collection of Type
Cultures, London, UK DSMZ: Deutsche Sammlung von Mikroorganismen
und Zellkulturen, Braunschweig, Germany For reference see Sugawara,
H. et al. (1993) World directory of collections of cultures of
microorganisms: Bacteria, fungi and yeasts (4.sup.th edn), World
federation for culture collections world data center on
microorganisms, Saimata, Japen.
[0181] TABLE-US-00004 TABLE 4 ALIGNMENT RESULTS % homo- length logy
Date of ID # (NT) Genbank Hit Length Accession Name of Genbank Hit
Source of Genbank Hit (GAP) Deposit rxa00062 1521 GB_HTG2: AC007366
185001 AC007366 Homo sapiens clone NH0501G22, *** SEQUENCING IN
PROGRESS ***, 3 unordered Homo sapiens 39,080 5-Jun-99 pieces.
rxa00084 948 GB_PR3: HSU80741 912 U80741 Homo sapiens CAGH44 mRNA,
partial cds. Homo sapiens 39,264 18-DEC-1997 GB_PL1: BNDNATRNA 1732
X89901 B. nigra DNA for tRNA like gene. Brassica nigra 36,725
6-Feb-97 GB_PR3: HSU80741 912 U80741 Homo sapiens CAGH44 mRNA,
partial cds. Homo sapiens 38,957 18-DEC-1997 rxa00109 735 GB_GSS9:
AQ163721 388 AQ163721 HS_2245_A1_F07_MF CIT Approved Human Genomic
Sperm Library D Homo sapiens 45,066 16-OCT-1998 Homo sapiens
genomic clone Plate = 2245 Col = 13 Row = K, genomic survey
sequence. GB_HTG4: AC007054 171979 AC007054 Drosophila melanogaster
chromosome 2 clone BACR45O18 (D527) RPCI-98 45.O.18 Drosophila
melanogaster 36,589 13-OCT-1999 map 41E-41E strain y; cn bw sp, ***
SEQUENCING IN PROGRESS***, 13 unordered pieces. GB_HTG4: AC007054
171979 AC007054 Drosophila melanogaster chromosome 2 clone
BACR45O18 (D527) RPCI-98 45.O.18 Drosophila melanogaster 36,589
13-OCT-1999 map 41E-41E strain y; cn bw sp, *** SEQUENCING IN
PROGRESS***, 13 unordered pieces. rxa00215 1449 GB_BA1: SC9C7 31360
AL035161 Streptomyces coelicolor cosmid 9C7. Streptomyces
coelicolor 44,444 12-Jan-99 GB_BA1: SCE94 38532 AL049628
Streptomyces coelicolor cosmid E94. Streptomyces coelicolor 36,313
12-Apr-99 GB_BA2: AF110185 20302 AF110185 Burkholderia pseudomallei
strain 1026b DbhB (dbhB), general secretory pathway Burkholderia
44,159 2-Aug-99 protein D (gspD), general secretory pathway protein
E (gspE), general secretory pseudomallei pathway protein F (gspF),
GspC (gspC), general secretory pathway protein G (gspG), general
secretory pathway protein H (gspH), general secretory pathway
protein I (gspI), general secretory pathway protein J (gspJ),
general secretory pathway protein K (gspK), general secretory
pathway protein L (gspL), general secretory pathway protein M
(gspM), and general secretory pathway protein N (gspN) genes,
complete cds; and unknown genes. rxa00289 1299 GB_EST6: N80167 384
N80167 za65g02.s1 Soares fetal liver spleen 1NFLS Homo sapiens cDNA
clone IMAGE: 297458 Homo sapiens 40,420 29-MAR-1996 3', mRNA
sequence. GB_STS: G37084 384 G37084 SHGC-56832 Human Homo sapiens
STS genomic, sequence tagged site. Homo sapiens 40,420 30-MAR-1998
GB_STS: G37084 384 G37084 SHGC-56832 Human Homo sapiens STS
genomic, sequence tagged site. Homo sapiens 40,420 30-MAR-1998
rxa00404 2439 GB_BA1: MTCY22D7 31859 Z83866 Mycobacterium
tuberculosis H37Rv complete genome; segment 133/162. Mycobacterium
60,271 17-Jun-98 tuberculosis GB_BA1: ECU82598 136742 U82598
Escherichia coli genomic sequence of minutes 9 to 12. Escherichia
coli 54,256 15-Jan-97 GB_BA2: AE000165 12003 AE000165 Escherichia
coli K-12 MG1655 section 55 of 400 of the complete genome.
Escherichia coli 54,256 12-Nov-98 rxa00479 2313 GB_BA1: SCF43A
35437 AL096837 Streptomyces coelicolor cosmid F43A. Streptomyces
coelicolor 36,245 13-Jul-99 A3(2) GB_GSS2: CNS015U4 1036 AL105910
Drosophila melanogaster genome survey sequence SP6 end of BAC
BACN14G08 of Drosophila melanogaster 37,573 26-Jul-99 DrosBAC
library from Drosophila melanogaster (fruit fly), genomic survey
sequence. GB_PR3: HSA494O16 50502 AL117328 Human DNA sequence from
clone 494O16 on chromosome 22, complete sequence. Homo sapiens
36,475 23-Nov-99 rxa00497 420 GB_BA1: MTCY78 33818 Z77165
Mycobacterium tuberculosis H37Rv complete genome; segment 145/162.
Mycobacterium 40,250 17-Jun-98 tuberculosis GB_BA2: AF079544 817
AF079544 Mycobacterium avium GroESL operon, partial sequence.
Mycobacterium avium 64,439 16-Aug-98 GB_BA1: MTGROEOP 2987 X60350
M. tuberculosis groE gene for KCS and 10-kDa products.
Mycobacterium 62,857 23-Apr-92 tuberculosis rxa00575 rxa00599 510
GB_GSS10: AQ199703 439 AQ199703 RPCI11-46O13.TJ RPCI-11 Homo
sapiens genomic clone RPCI-11-46O13, genomic Homo sapiens 42,657
20-Apr-99 survey sequence. GB_PR2: AC002127 144165 AC002127 Human
BAC clone RG305H12 from 7q21, complete sequence. Homo sapiens
37,052 27-MAY-1997 GB_STS: G51234 439 G51234 SHGC-80708 Human Homo
sapiens STS genomic, sequence tagged site. Homo sapiens 42,657
25-Jun-99 rxa00600 1221 GB_BA1: MTCY441 35187 Z80225 Mycobacterium
tuberculosis H37Rv complete genome; segment 118/162. Mycobacterium
56,183 18-Jun-98 tuberculosis GB_BA1: MSGY223 42061 AD000019
Mycobacterium tuberculosis sequence from clone y223. Mycobacterium
37,217 10-DEC-1996 tuberculosis GB_BA1: BSUB0014 213420 Z99117
Bacillus subtilis complete genome (section 14 of 21): from 2599451
to 2812870. Bacillus subtilis 36,553 26-Nov-97 rxa00605 1603
GB_BA2: AF069070 2776 AF069070 Endosymbiont of Onchocerca volvulus
catalase gene, complete cds. endosymbiont of 55,396 25-Nov-98
Onchocerca volvulus GB_BA1: OVCAT 1845 X82176 Onchocerca volvulus
endobacterial mRNA for catalase. endosymbiont of 55,396 26-Nov-98
Onchocerca volvulus GB_BA1: SC2G5 38404 AL035478 Streptomyces
coelicolor cosmid 2G5. Streptomyces coelicolor 39,530 11-Jun-99
rxa00648 1533 GB_HTG1: HS74O16 169401 AL110119 Homo sapiens
chromosome 21 clone RPCIP704O1674 map 21q21, *** SEQUENCING Homo
sapiens 36,327 27-Aug-99 IN PROGRESS ***, in unordered pieces.
GB_HTG1: HS74O16 169401 AL110119 Homo sapiens chromosome 21 clone
RPCIP704O1674 map 21q21, *** SEQUENCING Homo sapiens 36,327
27-Aug-99 IN PROGRESS ***, in unordered pieces. GB_HTG1: HS74O16
169401 AL110119 Homo sapiens chromosome 21 clone RPCIP704O1674 map
21q21, *** SEQUENCING Homo sapiens 35,119 27-Aug-99 IN PROGRESS
***, in unordered pieces. rxa00764 1239 GB_EST36: AI898007 609
AI898007 EST267450 tomato ovary, TAMU Lycopersicon esculentum cDNA
clone cLED31K22, Lycopersicon esculentum 34,323 27-Jul-99 mRNA
sequence. GB_BA2: PAU93274 8008 U93274 Pseudomonas aeruginosa YafE
(yafE), LeuB (leuB), Asd (asd), FimV (fimV), and HisT Pseudomonas
aeruginosa 35,895 23-Jun-98 (hisT) genes, complete cds; TrpF (trpF)
gene, partial cds; and unknown gene. GB_BA2: PAU93274 8008 U93274
Pseudomonas aeruginosa YafE (yafE), LeuB (leuB), Asd (asd), FimV
(fimV), and HisT Pseudomonas aeruginosa 41,417 23-Jun-98 (hisT)
genes, complete cds; TrpF (trpF) gene, partial cds; and unknown
gene. rxa00803 1353 GB_IN2: CELH34C03 27748 AF100662 Caenorhabditis
elegans cosmid H34C03. Caenorhabditis elegans 34,152 28-OCT-1998
GB_HTG2: AC007905 100722 AC007905 Homo sapiens chromosome 16q24.3
clone PAC 754F23, *** SEQUENCING IN Homo sapiens 37,472 24-Jun-99
PROGRESS ***, 33 unordered pieces. GB_HTG2: AC007905 100722
AC007905 Homo sapiens chromosome 16q24.3 clone PAC 754F23, ***
SEQUENCING IN Homo sapiens 37,472 24-Jun-99 PROGRESS ***, 33
unordered pieces. rxa00810 324 GB_BA1: MTY15C10 33050 Z95436
Mycobacterium tuberculosis H37Rv complete genome; segment 154/162.
Mycobacterium 34,615 17-Jun-98 tuberculosis GB_BA1: MLCB2548 38916
AL023093 Mycobacterium leprae cosmid B2548. Mycobacterium leprae
34,615 27-Aug-99 GB_BA1: ECOUW76 225419 U00039 E. coli chromosomal
region from 76.0 to 81.5 minutes. Escherichia coli 52,997 7-Nov-96
rxa00829 2463 GB_BA1: SC5C7 41906 AL031515 Streptomyces coelicolor
cosmid 5C7. Streptomyces coelicolor 65,269 7-Sep-98 GB_BA1: SC5F2A
40105 AL049587 Streptomyces coelicolor cosmid 5F2A. Streptomyces
coelicolor 37,490 24-MAY-1999 GB_BA1: STMDRRC 3374 L76359
Streptomyces peucetius daunorubicin resistance protein (drrC) gene,
complete cds. Streptomyces peucetius 55,279 24-DEC-1996 rxa00843
468 GB_BA1: MTCY9C4 15916 Z77250 Mycobacterium tuberculosis H37Rv
complete genome; segment 113/162. Mycobacterium 40,000 17-Jun-98
tuberculosis GB_BA1: MTCY9C4 15916 Z77250 Mycobacterium
tuberculosis H37Rv complete genome; segment 113/162. Mycobacterium
37,773 17-Jun-98 tuberculosis rxa00858 568 GB_BA1: SCC54 30753
AL035591 Streptomyces coelicolor cosmid C54. Streptomyces
coelicolor 39,602 11-Jun-99 GB_EST18: N96610 547 N96610 21285
Lambda-PRL1 Arabidopsis thaliana cDNA clone F10G3T7, mRNA sequence.
Arabidopsis thaliana 37,801 5-Jan-98 GB_EST18: T45493 436 T45493
8756 Lambda-PRL2 Arabidopsis thaliana cDNA clone 133C14T7, mRNA
sequence. Arabidopsis thaliana 34,194 4-Aug-98 rxa00886 1269
GB_BA1: SYCSLLLH 132106 D64006 Synechocystis sp. PCC6803 complete
genome, 25/27, 3138604-3270709. Synechocystis sp. 37,459 13-Feb-99
GB_BA1: SCDNAJ 5611 X77458 S. coelicolor dnaK, grpE and dnaJ genes.
Streptomyces coelicolor 49.744 21-Nov-96 GB_BA1: STMDNAK 4648
L46700 Streptomyces coelicolor (strain A3(2)) dnaK operon encoding
molecular chaperones Streptomyces coelicolor 49,583 22-Nov-96
(dnaK, dnaJ), grpE and hspR genes, complete cds's. rxa00900 975
GB_BA2: ECOUW67_0 110000 U18997 Escherichia coli K-12 chromosomal
region from 67.4 to 76.0 minutes. Escherichia coli 38,314 U18997
GB_BA2: ECOUW67_0 110000 U18997 Escherichia coli K-12 chromosomal
region from 67.4 to 76.0 minutes. Escherichia coli 37,759 U18997
GB_BA2: AE000393 10516 AE000393 Escherichia coli K-12 MG1655
section 283 of 400 of the complete genome. Escherichia coli 38,314
12-Nov-98 rxa00901 537 GB_HTG3: AC010757 175571 AC010757 Homo
sapiens chromosome 18 clone 128_C_18 map 18, *** SEQUENCING IN Homo
sapiens 34,857 22-Sep-99 PROGRESS ***, 20 unordered pieces.
GB_HTG3: AC010757 175571 AC010757 Homo sapiens chromosome 18 clone
128_C_18 map 18, *** SEQUENCING IN Homo sapiens 34,857 22-Sep-99
PROGRESS ***, 20 unordered pieces. GB_HTG3: AC011283 87295 AC011283
Homo sapiens clone MS2016A09, *** SEQUENCING IN PROGRESS ***, 1
unordered Homo sapiens 35,448 07-OCT-1999 pieces. rxa00981 753
GB_OV: GGA245664 512 AJ245664 Gallus gallus partial mRNA for
ATP-citrate lyase (ACL gene). Gallus gallus 37,538 28-Sep-99
GB_PL2: AC007887 159434 AC007887 Genomic sequence for Arabidopsis
thaliana BAC F15O4 from chromosome I, complete Arabidopsis thaliana
37,600 04-OCT-1999 sequence. GB_GSS1: CNS00RNW 542 AL087338
Arabidopsis thaliana genome survey sequence T7 end of BAC F14D7 of
IGF Arabidopsis thaliana 41,264 28-Jun-99 library from strain
Columbia of Arabidopsis thaliana, genomic survey sequence. rxa00995
864 GB_EST29: AI553951 450 AI553951 te54d01.x1 Soares_NFL_T_GBC_S1
Homo sapiens cDNA clone IMAGE: 2090497 3' Homo sapiens 42,627
13-Apr-99 similar to gb: X02067 H. sapiens mRNA for 7SL RNA
pseudogene (HUMAN);, mRNA sequence. GB_PR3: AC003029 139166
AC003029 Homo sapiens Chromosome 12q24 PAC RPCI3-462E2 (Roswell
Park Cancer Institute Homo sapiens 38,915 17-Sep-98 Human PAC
library) complete sequence. GB_BA1: EAY14603 4479 Y14603 Erwinia
amylovora srlA, srlE, srlB, srlD, srlM and srlR genes. Erwinia
amylovora 37,694 6-Jan-98 rxa00996 864 GB_BA2: AE001001 10730
AE001001 Archaeoglobus fulgidus section 106 of 172 of the complete
genome. Archaeoglobus fulgidus 41,078 15-DEC-1997 GB_EST30:
AV018764 242 AV018764 AV018764 Mus musculus 18-day embryo C57BL/6J
Mus musculus cDNA clone Mus musculus 39,669 28-Aug-99 1190006M16,
mRNA sequence. GB_GSS3: B24189 377 B24189 F19E16TF IGF Arabidopsis
thaliana genomic clone F19E16, genomic survey sequence. Arabidopsis
thaliana 44,385 10-OCT-1997 rxa01010 1242 GB_OV: AF007068 356
AF007068 Coturnix coturnix arylalkylamine N-acetyltransferase mRNA,
partial cds. Coturnix coturnix 46,629 12-Jul-97 GB_EST10: AA166324
514 AA166324 ms50c09.r1 Life Tech mouse embryo 13 5dpc 10666014 Mus
musculus cDNA clone Mus musculus 38,677 19-DEC-1996 IMAGE: 614992
5' similar to SW: NEST_RAT P21263 NESTIN.;, mRNA sequence. GB_EST7:
W89968 46 W89968 mf64g11.r1 Soares mouse embryo NbME13.5 14.5 Mus
musculus cDNA clone Mus musculus 58,696 12-Sep-96 IMAGE: 419108 5'
similar to SW: NEST_RAT P21263 NESTIN. [1];, mRNA sequence.
rxa01051 732 GB_GSS12: AQ381423 579 AQ381423 RPCI11-135F10.TJ
RPCI-11 Homo sapiens genomic clone RPCI-11-135F10, genomic Homo
sapiens 37,651 21-MAY-1999 survey sequence. GB_HTG6: AC010901
206121 AC010901 Homo sapiens clone RP11-544J22, WORKING DRAFT
SEQUENCE, 1 unordered Homo sapiens 36,011 04-DEC-1999 pieces.
GB_GSS5: AQ746932 837 AQ746932 HS_5538_A1_A11_T7A RPCI-11 Human
Male BAC Library Homo sapiens genomic Homo sapiens 38,640 19-Jul-99
clone Plate = 1114 Col = 21 Row = A, genomic survey sequence.
rxa01052 432 GB_IN1: CELC13D9 43487 AF016420 Caenorhabditis elegans
cosmid
C13D9. Caenorhabditis elegans 39,344 2-Aug-97 GB_IN1: CELC13D9
43487 AF016420 Caenorhabditis elegans cosmid C13D9. Caenorhabditis
elegans 38,780 2-Aug-97 rxa01053 543 GB_OV: CHKMAFG1 1316 D28601
Chicken novel maf-related gene mafG encoding bZip nuclear protein
MafG, promoter Gallus gallus 39,205 7-Feb-99 region and exon 1.
GB_HTG6: AC010765 146468 AC010765 Homo sapiens clone RP11-115N6,
*** SEQUENCING IN PROGRESS ***, Homo sapiens 32,961 07-DEC-1999 26
unordered pieces. GB_HTG6: AC010765 146468 AC010765 Homo sapiens
clone RP11-115N6, *** SEQUENCING IN PROGRESS ***, 26 Homo sapiens
38,476 07-DEC-1999 unordered pieces. rxa01054 612 GB_PL1: PHNPNGLP
962 D45425 Pharbitis nil mRNA for Pharbitis nil Germin-like protein
precursor, complete cds. Ipomoea nil 42,925 10-Feb-99 GB_HTG2:
HSJ402N21 170302 AL049553 Homo sapiens chromosome 6 clone
RP3-402N21 map p21.1-21.31, ***SEQUENCING Homo sapiens 36,825
03-DEC-1999 IN PROGRESS ***, in unordered pieces. GB_HTG2:
HSJ402N21 170302 AL049553 Homo sapiens chromosome 6 clone
RP3-402N21 map p21.1-21.31, ***SEQUENCING Homo sapiens 36,825
03-DEC-1999 IN PROGRESS ***, in unordered pieces. rxa01217 723
GB_IN2: CELF18A12 29784 AF016688 Caenorhabditis elegans cosmid
F18A12. Caenorhabditis elegans 35,794 08-OCT-1999 GB_IN2: CELF18A12
29784 AF016688 Caenorhabditis elegans cosmid F18A12. Caenorhabditis
elegans 40,625 08-OCT-1999 GB_RO: MUSMCFTR 6304 M60493 Mouse cystic
fibrosis transmembrane conductance regulator (CFTR) mRNA, complete
Mus musculus 37,793 10-Jun-94 cds. rxa01320 1770 GB_BA2: AF031037
1472 AF031037 Neisseria meningitidis chloramphenicol
acetyltransferase gene, complete cds. Neisseria meningitidis 35,014
21-Apr-98 GB_HTG1: PFMAL13PA 80518 AL109815 Plasmodium falciparum
chromosome 13 strain 3D7, *** SEQUENCING IN PROGRESS Plasmodium
falciparum 17,697 19-Aug-99 ***, in unordered pieces. GB_HTG1:
PFMAL13PA 80518 AL109815 Plasmodium falciparum chromosome 13 strain
3D7, *** SEQUENCING IN PROGRESS Plasmodium falciparum 17,697
19-Aug-99 ***, in unordered pieces. rxa01345 1575 GB_PR3: AC005224
166687 AC005224 Homo sapiens chromosome 17, clone hRPK.214_O_1,
complete sequence. Homo sapiens 38,195 14-Aug-98 GB_PR3: AC005224
166687 AC005224 Homo sapiens chromosome 17, clone hRPK.214_O_1,
complete sequence. Homo sapiens 36,611 14-Aug-98 GB_HTG3:
AC011500_1 300851 AC011500 Homo sapiens chromosome 19 clone
CIT978SKB_60E11, *** SEQUENCING IN Homo sapiens 36,446 AC011500
PROGRESS ***, 246 unordered pieces. rxa01407 1014 GB_HTG3: AC010831
70233 AC010831 Homo sapiens clone 6_L_24, LOW-PASS SEQUENCE
SAMPLING. Homo sapiens 35,764 23-Sep-99 GB_HTG3: AC010831 70233
AC010831 Homo sapiens clone 6_L_24, LOW-PASS SEQUENCE SAMPLING.
Homo sapiens 35,764 23-Sep-99 GB_PR3: AC004058 38400 AC004058 Homo
sapiens chromosome 4 clone B241P19 map 4q25, complete sequence.
Homo sapiens 40,778 30-Sep-98 rxa01408 324 GB_PR4: AF152365 246546
AF152365 Homo sapiens constitutive fragile region FRA3B sequence.
Homo sapiens 41,234 1-Aug-99 GB_HTG3: AC007890 121256 AC007890
Drosophila melanogaster chromosome 3 clone BACR02G21 (D722) RPCI-98
02.G.21 Drosophila melanogaster 39,432 3-Sep-99 map 90E-91A strain
y; cn bw sp, *** SEQUENCING IN PROGRESS***, 89 unordered pieces.
GB_HTG3: AC007890 121256 AC007890 Drosophila melanogaster
chromosome 3 clone BACR02G21 (D722) RPCI-98 02.G.21 Drosophila
melanogaster 39,432 3-Sep-99 map 90E-91A strain y; cn bw sp, ***
SEQUENCING IN PROGRESS***, 89 unordered pieces. rxa01524 1566
GB_BA1: BSUB0015 218410 Z99118 Bacillus subtilis complete genome
(section 15 of 21): from 2795131 to 3013540. Bacillus subtilis
38,201 26-Nov-97 GB_HTG2: AC008260 107439 AC008260 Drosophila
melanogaster chromosome 2 clone BACR13J10 (D924) RPCI-98 13.J.10
Drosophila melanogaster 38,302 2-Aug-99 map 47B-47C strain y; cn bw
sp, *** SEQUENCING IN PROGRESS***, 82 unordered pieces. GB_HTG2:
AC008260 107439 AC008260 Drosophila melanogaster chromosome 2 clone
BACR13J10 (D924) RPCI-98 13.J.10 Drosophila melanogaster 38,302
2-Aug-99 map 47B-47C strain y; cn bw sp, *** SEQUENCING IN PROGRESS
***, 82 unordered pieces. rxa01578 1510 GB_PR4: AF111170 148083
AF111170 Homo sapiens 14q32 Jagged2 gene, complete cds; and unknown
gene. Homo sapiens 37,873 14-Jul-99 GB_PR4: AF111170 148083
AF111170 Homo sapiens 14q32 Jagged2 gene, complete cds; and unknown
gene. Homo sapiens 40,220 14-Jul-99 GB_BA1: AEY13732 6740 Y13732
Alcaligenes eutrophus genes for ureases, ureD1, ureD2, ureA, ureB,
and ORF1, ORF2. Ralstonia eutropha 42,960 23-Sep-97 rxa01616 1605
GB_BA2: AF088857 2908 AF088857 Vogesella indigofera indigoidine
biosynthesis regulatory locus, complete sequence. Vogesella
indigofera 37,626 10-Sep-99 GB_IN1: CEM04D8 21552 Z32682
Caenorhabditis elegans cosmid M04D8, complete sequence.
Caenorhabditis elegans 37,237 23-Nov-98 GB_EST25: AI281910 276
AI281910 qt82d04.x1 NCI_CGAP_Co14 Homo sapiens cDNA clone IMAGE:
1961767 3', mRNA Homo sapiens 38,406 21-DEC-1998 sequence. rxa01666
1500 GB_BA1: CGU43535 2531 U43535 Corynebacterium glutamicum
multidrug resistance protein (cmr) gene, complete cds.
Corynebacterium 99,933 9-Apr-97 glutamicum GB_HTG3: AC009213 114735
AC009213 Drosophila melanogaster chromosome 3 clone BACR09F18
(D812) RPCI-98 09.F.18 Drosophila melanogaster 36,111 23-Aug-99 map
98D-98D strain y; cn bw sp, *** SEQUENCING IN PROGRESS ***, 109
unordered pieces. GB_HTG3: AC009213 114735 AC009213 Drosophila
melanogaster chromosome 3 clone BACR09F18 (D812) RPCI-98 09.F.18
Drosophila melanogaster 36,111 23-Aug-99 map 98D-98D strain y; cn
bw sp, *** SEQUENCING IN PROGRESS***, 109 unordered pieces.
rxa01674 1017 GB_PL1: AB017159 1859 AB017159 Daucus carota mRNA for
citrate synthase, complete cds. Daucus carota 39,537 01-MAY-1999
GB_PR1: HUMGNOS48 23142 D26607 Homo sapiens endothelial nitric
oxide synthase gene, complete cds. Homo sapiens 36,419 13-Jul-99
GB_HTG3: AC011234 154754 AC011234 Homo sapiens clone NH0166D23, ***
SEQUENCING IN PROGRESS ***, 7 unordered Homo sapiens 36,317
04-OCT-1999 pieces. rxa01873 1359 GB_HTG3: AC009450 124337 AC009450
Homo sapiens chromosome 9 clone 30_C_23 map 9, *** SEQUENCING IN
Homo sapiens 35,303 22-Aug-99 PROGRESS ***, 20 unordered pieces.
GB_HTG3: AC009450 124337 AC009450 Homo sapiens chromosome 9 clone
30_C_23 map 9, *** SEQUENCING IN Homo sapiens 35,303 22-Aug-99
PROGRESS ***, 20 unordered pieces. GB_HTG3: AC009919 134724
AC009919 Homo sapiens clone 115_I_23, LOW-PASS SEQUENCE SAMPLING.
Homo sapiens 35,409 8-Sep-99 rxa01922 1275 GB_BA1: ECONEUC 1676
M84026 E. coli protein p7 (neu C) gene, complete cds. Escherichia
coli 35,189 26-Apr-93 GB_HTG2: AC007853 116280 AC007853 Drosophila
melanogaster chromosome 3 clone BACR03L02 (D766) RPCI-98 03.L.2
Drosophila melanogaster 34,365 2-Aug-99 map 96B-96C strain y; cn bw
sp, *** SEQUENCING IN PROGRESS ***, 80 unordered pieces. GB_HTG2:
AC007853 116280 AC007853 Drosophila melanogaster chromosome 3 clone
BACR03L02 (D766) RPCI-98 03.L.2 Drosophila melanogaster 34,365
2-Aug-99 map 96B-96C strain y; cn bw sp, *** SEQUENCING IN
PROGRESS***, 80 unordered pieces. rxa01936 1395 GB_HTG4: AC010037
166249 AC010037 Drosophila melanogaster chromosome 3L/66B6 clone
RPCI98-6E4, *** SEQUENCING Drosophila melanogaster 38,534
16-OCT-1999 IN PROGRESS ***, 52 unordered pieces. GB_HTG4: AC010037
166249 AC010037 Drosophila melanogaster chromosome 3L/66B6 clone
RPCI98-6E4, *** SEQUENCING Drosophila melanogaster 38,534
16-OCT-1999 IN PROGRESS ***, 52 unordered pieces. GB_PR4: AC005552
167228 AC005552 Homo sapiens chromosome 17, clone hRPK.212_E_8,
complete sequence. Homo sapiens 36,249 26-Nov-98 rxa01984 420
GB_PR1: HS169C8F 245 Z57239 H. sapiens CpG island DNA genomic Mse1
fragment, clone 169c8, forward read Homo sapiens 45,679 18-OCT-1995
cpg169c8.ft1a. GB_BA1: SERATTBXIS 3255 L11597 Saccharopolyspora
erythraea excisionase (xis) gene, integrase (int) gene, complete
Saccharopolyspora 36,232 6-Jul-94 cds's and attB site. erythraea
GB_EST7: W97557 267 W97557 mf98a09.r1 Soares mouse embryo NbME13.5
14.5 Mus musculus cDNA clone Mus musculus 42,969 16-Jul-96 IMAGE:
422296 5', mRNA sequence. rxa02060 rxa02087 1470 GB_PR3: AC005544
169045 AC005544 Homo sapiens chromosome 17, clone hRPK.349_A_8,
complete sequence. Homo sapiens 35,724 25-Sep-98 GB_PL1: ATF20B18
104738 AL049483 Arabidopsis thaliana DNA chromosome 4, BAC clone
F20B18 (ESSA project). Arabidopsis thaliana 35,890 24-MAR-1999
GB_PL2: ATT25K17 89904 AL049171 Arabidopsis thaliana DNA chromosome
4, BAC clone (ESSA project). Arabidopsis thaliana 38,128 27-Aug-99
rxa02088 1338 GB_HTG3: AC008697 167932 AC008697 Homo sapiens
chromosome 5 clone CIT978SKB_70D3, *** SEQUENCING IN Homo sapiens
36,662 3-Aug-99 PROGRESS ***, 54 unordered pieces. GB_HTG3:
AC008697 167932 AC008697 Homo sapiens chromosome 5 clone
CIT978SKB_70D3, *** SEQUENCING IN Homo sapiens 36,662 3-Aug-99
PROGRESS ***, 54 unordered pieces. GB_HTG3: AC008703 213971
AC008703 Homo sapiens chromosome 5 clone CIT978SKB_76P12, ***
SEQUENCING IN Homo sapiens 34,768 3-Aug-99 PROGRESS ***, 54
unordered pieces. rxa02159 636 GB_BA2: AF049897 9196 AF049897
Corynebacterium glutamicum N-acetylglutamylphosphate reductase
(argC), ornithine Corynebacterium 99,843 1-Jul-98 acetyltransferase
(argJ), N-acetylglutamate kinase (argB), acetylornithine
transaminase glutamicum (argD), ornithine carbamoyltransferase
(argF), arginine repressor (argR), argininosuccinate synthase
(argG), and argininosuccinate lyase (argH) genes, complete cds.
GB_BA2: AF031518 2045 AF031518 Corynebacterium glutamicum ornithine
carbamolytransferase (argF) gene, complete cds. Corynebacterium
88,679 5-Jan-99 glutamicum GB_BA2: AF041436 516 AF041436
Corynebacterium glutamicum arginine repressor (argR) gene, complete
cds. Corynebacterium 100,000 5-Jan-99 glutamicum rxa02184 504
GB_BA1: BSZ92953 8164 Z92953 B. subtilis yws[A, B, C] genes and
rbs[A, C, D, K, R] genes. Bacillus subtilis 38,951 24-Jun-98
GB_EST36: AI878071 593 AI878071 fc57a12.y1 Zebrafish WashU MPIMG
EST Danio rerio cDNA 5' similar to TR: Q13151 Danio rerio 36,774
21-Jul-99 Q13151 HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN A0;, mRNA
sequence. GB_EST37: AI958166 641 AI958166 fc91f01.y1 Zebrafish
WashU MPIMG EST Danio rerio cDNA 5' similar to TR: Q13151 Danio
rerio 36,774 20-Aug-99 Q13151 HETEROGENEOUS NUCLEAR
RIBONUCLEOPROTEIN A0;, mRNA sequence. rxa02200 1233 GB_PR3:
HSA494O16 50502 AL117328 Human DNA sequence from clone 494O16 on
chromosome 22, complete sequence. Homo sapiens 38,648 23-Nov-99
GB_HTG2: AC008161 158440 AC008161 Mus musculus clone 182_H_5, ***
SEQUENCING IN PROGRESS ***, 29 unordered Mus musculus 35,938
28-Jul-99 pieces. GB_HTG2: AC008161 158440 AC008161 Mus musculus
clone 182_H_5, *** SEQUENCING IN PROGRESS ***, 29 unordered Mus
musculus 35,938 28-Jul-99 pieces. rxa02201 486 GB_EST4: H16949 465
H16949 ym34a11.r1 Soares infant brain 1NIB Homo sapiens cDNA clone
IMAGE: 50010 5', Homo sapiens 38,267 29-Jun-95 mRNA sequence.
GB_EST4: H16949 465 H16949 ym34a11.r1 Soares infant brain 1NIB Homo
sapiens cDNA clone IMAGE: 50010 5', Homo sapiens 36,552 29-Jun-95
mRNA sequence. rxa02202 762 GB_IN1: CELC41A3 37149 U41541
Caenorhabditis elegans cosmid C41A3. Caenorhabditis elegans 41,678
08-DEC-1995 GB_EST33: AV080151 236 AV080151 AV080151 Mus musculus
stomach C57BL/6J adult Mus musculus cDNA clone Mus musculus 43,348
25-Jun-99 2210413B04, mRNA sequence. GB_GSS5: AQ766877 545 AQ766877
HS_2017_B2_B08_MR CIT Approved Human Genomic Sperm Library D Homo
sapiens 35,568 28-Jul-99 Homo sapiens genomic clone Plate = 2017
Col = 16 Row = D, genomic survey sequence. rxa02205 1002 GB_HTG2:
AC005959 127587 AC005959 Homo sapiens, *** SEQUENCING IN PROGRESS
***, 2 ordered pieces. Homo sapiens 40,310 11-Nov-98 GB_HTG2:
AC005959 127587 AC005959 Homo sapiens, *** SEQUENCING IN PROGRESS
***, 2 ordered pieces. Homo sapiens 40,310 11-Nov-98 GB_IN1:
BRPTUBBA 4571 M36380 B. pahangi beta-tubulin gene, complete cds.
Brugla pahangi 37.703 26-Apr-93 rxa02305 975 GB_RO: MUSPAFR 1140
D50872 Mouse gene for platelet activating factor receptor, complete
cds. Mus musculus 38,420 10-Feb-99 GB_PR3: HUMARL1A 1008 L28997
Homo sapiens ARL1 mRNA, complete cds. Homo sapiens 42.188 13-Jan-95
GB_BA1: MLCB2533 40245 AL035310 Mycobacterium leprae cosmid B2533.
Mycobacterium leprae 42 27-Aug-99 rxa02431 899 GB_EST4: H35255 407
H35255 EST111890 Rat PC-12 cells, NGF-treated (9 days) Rattus sp.
cDNA clone RPNCO03, Rattus sp. 39,098 2-Apr-98 mRNA sequence.
GB_HTG1: HS791K14 155318 AL035685 Homo sapiens chromosome 20
clone
RP4-791K14, *** SEQUENCING IN PROGRESS Homo sapiens 39,456
23-Nov-99 ***, in unordered pieces. GB_HTG1: HS791K14 155318
AL035685 Homo sapiens chromosome 20 clone RP4-791K14, ***
SEQUENCING IN PROGRESS Homo sapiens 39,456 23-Nov-99 ***, in
unordered pieces. rxa02446 558 GB_BA2: AF036166 895 AF036166
Xanthomonas campestris organic hydroperoxide resistance protein
(ohr) gene, complete Xanthomonas campestris 49.369 19-MAY-1998 cds.
GB_EST5: N25122 620 N25122 yx19d10.r1 Soares melanocyte 2NbHM Homo
sapiens cDNA clone IMAGE: 262195 5', Homo sapiens 35,417
28-DEC-1995 mRNA sequence. GB_EST5: N25122 620 N25122 yx19d10.r1
Soares melanocyte 2NbHM Homo sapiens cDNA clone IMAGE: 262195 5',
Homo sapiens 37,172 28-DEC-1995 mRNA sequence. rxa02541 1308
GB_BA2: DPU93358 1267 U93358 Deinococcus proteolyticus 40 kDa heat
shock chaperone protein (dnaJ) gene, complete Deinococcus 42,115
17-Jan-98 cds. proteolyticus GB_EST30: AI658096 343 AI658096
fc14c09.y1 Zebrafish WashU MPIMG EST Danio rerio cDNA 5' similar to
Danio rerio 52,059 06-MAY-1999 SW: DNJ2_HUMAN P31689 DNAJ PROTEIN
HOMOLOG 2.;, mRNA sequence. GB_EST37: AI959242 545 AI959242
fd25h11.y1 Zebrafish WashU MPIMG EST Danio rerio cDNA 5' similar to
Danio rerio 45,438 20-Aug-99 SW: DNJ2_HUMAN P31689 DNAJ PROTEIN
HOMOLOG 2.;, mRNA sequence. rxa02542 777 EM_PAT: E10832 1856 E10832
DNA encoding Dnak protein which is one of heat shock protein from
Corynebacterium 99,000 08-OCT-1997 glutamicum (Rel. 52, Created)
GB_EST24: Z82017 396 Z82017 SSZ82017 Porcine small intestine cDNA
library Sus scrofa cDNA clone c12c06 Sus scrofa 37,067 30-Apr-99 5'
similar to eukaryotic initiation factor 4 gamma, mRNA sequence.
GB_OM: CATERYTHRO 681 L10606 Cat erythropoietin mRNA, 3' end. Felis
catus 39,409 14-OCT-1993 rxa02543 1977 EM_PAT: E10832 1856 E10832
DNA encoding Dnak protein which is one of heat shock protein from
Corynebacterium 97,306 08-OCT-1997 glutamicum (Rel. 52, Created)
GB_BA1: MPHSP70 2179 X59437 M. paratuberculosis gene for 70 kD heat
shock protein. Mycobacterium avium 73,404 23-Apr-92 subsp.
paratuberculosis GB_BA1: MTY13E10 35019 Z95324 Mycobacterium
tuberculosis H37Rv complete genome; segment 18/162. Mycobacterium
72,028 17-Jun-98 tuberculosis rxa02586 393 GB_IN2: AC006472 156362
AC006472 Drosophila melanogaster, chromosome 2R, region 45E1-46A2,
BAC clone Drosophila melanogaster 37,958 30-Jan-99 BACR48G21,
complete sequence. GB_HTG4: AC010020 106541 AC010020 Drosophila
melanogaster chromosome 3L/66D10 clone RPCI98-26I3, *** Drosophila
melanogaster 37,333 16-OCT-1999 SEQUENCING IN PROGRESS ***, 55
unordered pieces. GB_HTG4: AC010020 106541 AC010020 Drosophila
melanogaster chromosome 3L/66D10 clone RPCI98-26I3, *** Drosophila
melanogaster 37,333 16-OCT-1999 SEQUENCING IN PROGRESS ***, 55
unordered pieces. rxa02587 2214 GB_BA1: MLCL622 42498 Z95398
Mycobacterium leprae cosmid L622. Mycobacterium leprae 39,848
24-Jun-97 GB_RO: AF074879 3316 AF074879 Rattus norvegicus
testis-specific protein TSPY gene, complete cds. Rattus norvegicus
35,830 6-Jul-99 GB_RO: RNJ001380 2641 AJ001380 Rattus norvegicus
Tspy partial genomic sequence, exons 1-6. Rattus norvegicus 37,702
29-Jun-98 rxs03217 331 GB_BA1: MLCB2548 38916 AL023093
Mycobacterium leprae cosmid B2548. Mycobacterium leprae 37,888
27-Aug-99 GB_HTG2: HSJ662M14 174772 AL079336 Homo sapiens
chromosome 20 clone RP4-662M14, *** SEQUENCING IN PROGRESS Homo
sapiens 36,420 4-Feb-00 ***, 10 unordered pieces. GB_HTG2:
HSJ662M14 174772 AL079336 Homo sapiens chromosome 20 clone
RP4-662M14, *** SEQUENCING IN PROGRESS Homo sapiens 35,962 4-Feb-00
***, 10 unordered pieces.
[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=US20070059810A1).
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=US20070059810A1).
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