Note: Descriptions are shown in the official language in which they were submitted.
N. . i
CA 02583703 2007-04-19
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CORYNEBACTERIUMGLUTAMICUM GENES ENCODING PROTEINS
INVOLVED IN MEMBRANE SYNTHESIS AND MEMBRANE TRANSPORT
This application is a divisional application of co-pending application Serial
No. 2,380,863, filed June 23, 2000.
Bac round of the Invention
Certain products and by-products of naturally-occurring metabolic processes in
cells have utility in a wide array of industries, including the food, feed,
cosmetics, and
pharmaceutical industries. These molecules, collectively termed 'fine
chemicals',
include organic acids, both proteinogenic and non-proteinogenic amino acids,
nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates,
aromatic
compounds, vitamins and cofactors, and enzymes. Their production is most
conveniently performed through the large-scale culture of bacteria developed
to produce
and secrete large quantities of one or more desired molecules. One
particularly useful
organism for this purpose is Corynebacterium glutamfcum, 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
The invention provides novel bacterial nucleic acid molecules which have a
variety of uses. These uses include the identification of microorganisms which
can be
used to produce fine chemicals, the modulation of fine chemical production in
C.
glutamicum or related bacteria, the typing or identification of C. glutamicum
or related
bacteria, as reference points for mapping the C. glutamicum genome, and as
markers for
transformation. These novel nucleic acid molecules encode proteins, referred
to herein
as membrane construction and membrane transport (MCT) proteins.
C glutamicum is a gram positive, aerobic bacterium which is commonly used in
industry for the large-scale production of a variety of fine chemicals, and
also for the
degradation of hydrocarbons (such as in petroleum spills) and for the
oxidation of
terpenoids. The MCT nucleic acid molecules of the invention, therefore, can be
used to
identify microorganisms which can be used to produce fine chemicals, e.g., by
CA 02583703 2007-04-19
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fermentation processes. Modulation of the expression of the MCT nucleic acids
of the
invention, or modification of the sequence of the MCT nucleic acid molecules
of the
invention, can be used to modulate the production of one or more fine
chemicals from a
microorganism (e.g., to improve the yield or production of one or more fine
chemicals
from a Corynebacterium or Brevibacterium species).
The MCT nucleic acids of the invention may aiso 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.
The MCT nucleic acid molecules of the invention may also serve as reference
points for mapping of the C. glutamicum genome, or of genomes of related
organisms.
Similarly, these molecules, or variants or portions thereof, may serve as
markers for
genetically engineered Corynebacterium or Brevibacterium species.
The MCT proteins encoded by- the novel nucleic acid molecules of the invention
are capable of, for example, performing a function involved in the metabolism
(e.g., the
biosynthesis or degradation) of compounds necessary for membrane biosynthesis,
or of
assisting in the transmembrane transport of one or more compounds either into
or out of
the cell. Given the availability of cloning vectors for use in Corynebacterium
glutamicum, such as those disclosed in Sinskey et al., U.S. Patent No.
4,649,119, and
techniques for genetic manipulation of C. glutamicum and the related
Brevibacterium
species (e.g., lactofermentum) (Yoshihama et al, J. Bacteriol. 162: 591-597
(1985);
Katsumata et al., J. Bacteriol. 159: 306-311 (1984); and Santamaria et al., J.
Gen.
Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of the invention
may be
utilized in the genetic engineering of this organism to make it a better or
more efficient
producer of one or more fine chemicals. This improved production or efficiency
of
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production of a fine chemical may be due to a direct effect of manipulation of
a gene of
the invention, or it may be due to an indirect effect of such manipulation.
There are a number of mechanisms by which the alteration of an MCT protein of
the invention may directly affect the yield, production, and/or efficiency of
production
of a fine chemical from a C. glutamicum strain incorporating such an altered
protein.
Those MCT proteins involved in the export of fine chemical molecules from the
cell
may be increased in number or activity such that greater quantities of these
compounds
are secreted to the extracellular medium, from which they are more readily
recovered.
Similarly, those MCT proteins involved in the import of nutrients necessary
for the
biosynthesis of one or more fine chemicals (e.g., phosphate, sulfate, nitrogen
compounds, etc.) may be increased in number or activity such that these
precursors,
cofactors, or intermediate compounds are increased in concentration within the
cell.
Further, fatty acids and lipids themselves are desirable fine chemicals; by
optimizing the
activity or increasing the number of one or more MCT proteins of the invention
which
participate in the biosynthesis of these compounds, or by impairing the
activity of one or
more MCT proteins which are involved in the degradation of these compounds, it
may
be possible to increase the yield, production, and/or efficiency of production
of fatty
acid and lipid molecules from C. glutamicum.
The mutagenesis of one or more MCT genes of the invention may also result in
MCT proteins having altered activities which indirectly impact the production
of one or
more desired fine chemicals from C.glutamicum. For example, MCT proteins of
the
invention involved in the export of waste products may be increased in number
or
activity such that the normal metabolic wastes of the cell (possibly increased
in quantity
due to the overproduction of the desired fine chemical) are efficiently
exported before
they are able to damage nucleotides and proteins within the cell (which would
decrease
the viability of the cell) or to interfere with fine chemical biosynthetic
pathways (which
would decrease the yield, production, or efficiency of production of the
desired fine
chemical). Further, the relatively large intracellular quantities of the
desired fine
chemical may in itself be toxic to the cell, so by increasing the activity or
number of
transporters able to export this compound from the cell, one may increase the
viability of
the cell in culture, in turn leading to a greater number of cells in the
culture producing
the desired fine chemical. The MCT proteins of the invention may also be
manipulated
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such that the relative amounts of different lipid and fatty acid molecules are
produced.
This may have a profound effect on the lipid composition of the membrane of
the cell.
Since each type of lipid has different physical properties, an alteration in
the lipid
composition of a membrane may significantly alter membrane fluidity. Changes
in
membrane fluidity can impact the transport of molecules across the membrane,
as well
as the integrity of the cell, both of which have a profound effect on the
production of
fine chemicals from C. glutamicum in large-scale fermentative culture.
The invention provides novel nucleic acid molecules which encode proteins,
referred to herein as MCT proteins, which are capable of, for example,
participating in
the metabolism of compounds necessary for the construction of cellular
membranes in
C. glutamicum, or in the transport of molecules across these membranes.
Nucleic acid
molecules encoding an MCT protein are referred to herein as MCT nucleic acid
molecules. In a preferred embodiment, the MCT protein participates in the
metabolism
of compounds necessary for the construction of cellular membranes in C.
glutamicum,
or in the transport of molecules across these membranes. Examples of such
proteins
include those encoded by the genes set forth in Table 1.
Accordingly, one aspect of the invention pertains to isolated nucleic acid
molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotide sequence
encoding
an MCT protein or biologically active portions thereof, as well as nucleic
acid fragments
suitable as primers or hybridization probes for the detection or amplification
of MCT-
encoding nucleic acid (e.g., DNA or mRNA). In particularly preferred
embodiments,
the isolated nucleic acid molecule comprises one of the nucleotide sequences
set forth as
the odd-numbered SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7....), forth as the odd-numbered SEQ ID NOs in
the
Sequence Listing (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7....),
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 as an odd-numbered SEQ ID NO in the Sequence
Listing
(e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7....), or a portion
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thereof. In other preferred embodiments, the isolated nucleic acid molecule
encodes one
of the amino acid sequences set forth as an even-numbered SEQ ID NO in the
Sequence
Listing (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8...). The
preferred MCT proteins of the present invention also preferably possess at
least one of
the MCT activities described herein.
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 the invention
(e.g., a
sequence having an even-numbered SEQ ID NO: in the Sequence Listing), e.g.,
sufficiently homologous to an amino acid sequence of the invention such that
the protein
or portion thereof maintains an MCT activity. Preferably; the protein or
portion thereof
encoded by the nucleic acid molecule maintains.the ability to participate in
the
metabolism of compounds necessary for the construction of cellular membranes
in C.
glutamicum, or in the transport of molecules across these membranes. In one
embodiment, the protein encoded by the nucleic acid molecule is at least about
50%,
preferably at least about 60%, and more preferably at least about 70%, 80%, or
90% and
most preferably at least about 95%, 96%, 97%, 98%, or 99% or more homologous
to an
amino acid sequence of the invention (e.g., an entire amino acid sequence
selected from
those having an even-numbered SEQ ID NO in the Sequence Listing). In another
preferred embodiment, the protein is a full length C. glutamicum protein which
is
substantially homologous to an entire amino acid sequence of the invention
(encoded by
an open reading frame shown in the corresponding odd-numbered SEQ ID NOs in
the
Sequence Listing (e.g., SEQ ID NO:I, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7....).
In another preferred embodiment, the isolated nucleic acid molecule is derived
from C. glutamicum and encodes a protein (e.g., an MCT fusion protein) which
includes
a biologically active domain which is at least about 50% or more homologous to
one of
the amino acid sequences of the invention (e.g., a sequence of one of the even-
numbered
SEQ ID NOs in the Sequence Listing) and is able to participate in the
metabolism of
compounds necessary for the construction of cellular membranes in C.
glutamicum, or in
the transport of molecules across these membranes, or has one or more of the
activities
set forth in Table 1, and which also includes heterologous nucleic acid
sequences
encoding a heterologous polypeptide or regulatory regions.
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Jn 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 the invention (e.g., a sequence
of an odd-
numbered SEQ ID NO in the Sequence Listing). Preferably, the isolated nucleic
acid
molecule corresponds to a naturally-occurring nucleic acid molecule. More
preferably,
the isolated nucleic acid encodes a naturally-occurring C. glutamicum MCT
protein, or a
biologically active portion thereof.
Another aspect of the invention pertains to vectors, e.g., recombinant
expression
vectors, containing the nucleic acid molecules of the invention, and host
cells into which
such vectors have been introduced. In one embodiment, such a host cell is used
to
produce an MCT protein by culturing the host cell in a suitable medium. The
MCT
protein can be then isolated from the medium or the host cell.
Yet another aspect of the invention pertains to a genetically altered
microorganism in which an MCT gene has been introduced or altered. In one
embodiment, the genome of the microorganism has been altered by introduction
of a
nucleic acid molecule of the invention encoding wild-type or mutated MCT
sequence as
a transgene. In another embodiment, an endogenous MCT gene within the genome
of
the microorganism has been altered, e.g., functionally disrupted, by
homologous
recombination with an altered MCT gene. In another embodiment, an endogenous
or
introduced MCT gene in a microorganism has been altered by one or more point
mutations, deletions, or inversions, but still encodes a functional MCT
protein. In still
another embodiment, one or more of the regulatory regions (e.g., a promoter,
repressor,
or inducer) of an MCT gene in a microorganism has been altered (e.g., by
deletion,
truncation, inversion, or point mutation) such that the expression of the MCT
gene is
modulated. In a preferred embodiment, the microorganism belongs to the genus
Corynebacterium or Brevibacterium, with Corynebacierium 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.
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
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sequences set forth in the Sequence Listing as SEQ ID NOs 1 through 676) in a
subject,
thereby detecting the presence or activity of Corynebacterium diphtheriae in
the subject.
Still another aspect of the invention pertains to an isolated MCT protein or a
portion, e.g., a biologically active portion, thereof. In a preferred
embodiment, the
isolated MCT protein or portion thereof can participate in the metabolism of
compounds
necessary for the construction of cellular membranes in C. glutamicum, or in
the
transport of molecules across these membranes. In another preferred
embodiment, the
isolated MCT protein or portion thereof is sufficiently homologous to an amino
acid
sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: in
the
Sequence Listing) such that the protein or portion thereof maintains the
ability to
participate in the metabolism of compounds necessary for the construction of
cellular
membranes in C. glutamicum, or in the transport of molecules across these
membranes.
The invention also provides an isolated preparation of an MCT protein. In
preferred embodiments, the MCT protein comprises an amino acid sequence of the
invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence
Listing).
In another preferred embodiment, the invention pertains to an isolated full
length protein
which is substantially homologous to an entire amino acid sequence of the
invention
(e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing)
(encoded
by an open reading frame set forth in a corresponding odd-numbered SEQ ID NO:
of the
Sequence Listing 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 the invention (e.g., a sequence of an even-
numbered
SEQ ID NO: of the Sequence Listing). In other embodiments, the isolated MCT
protein
comprises an amino acid sequence which is at least about 50% or more
homologous to
one of the amino acid sequences of the invention (e.g., a sequence of an even-
numbered
SEQ ID NO: of the Sequence Listing) and is able to participate in the
metabolism of
compounds necessary for the construction of cellular membranes in C.
glutamicum, or in
the transport of molecules across these membranes, or has one or more of the
activities
set forth in Table 1.
Alternatively, the isolated MCT protein can comprise an amino acid sequence
which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes
under
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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
one of
the even-numbered SEQ ID NOs set forth in the Sequence Listing. It is also
preferred
that the prefenred forms of MCT proteins also have one or more of the MCT
bioactivities described herein.
The MCT polypeptide, or a biologically active portion thereof, can be
operatively linked to a non-MCT polypeptide to form a fusion protein. In
preferred
embodiments, this fusion protein has an activity which differs from that of
the MCT
protein alone. In other preferred embodiments, this fusion protein participate
in the
metabolism of compounds necessary for the construction of cellular membranes
in C.
glutamicum, or in the transport of molecules across these membranes. In
particularly
preferred embodiments, integration of this fusion protein into a host cell
modulates
production of a desired compound from the cell.
In another aspect, the invention provides methods for screening molecules
which
modulate the activity of an MCT protein, either by interacting with the
protein itself or a
substrate or binding partner of the MCT protein, or by modulating the
transcription or
translation of an MCT nucleic acid molecule of the invention.
Another aspect of the invention pertains to a method for producing a fine
chemical. This method involves the culturing of a cell containing a vector
directing the
expression of an MCT nucleic acid molecule of the invention, such that a fine
chemical
is produced. In a preferred embodiment, this method further includes the step
of
obtaining a cell containing such a vector, in which a cell is transfected with
a vector
directing the expression of an MCT nucleic acid. In another preferred
embodiment, this
method further includes the step of recovering the fine chemical from the
culture. In a
particularly preferred embodiment, the cell is from the genus Corynebacterium
or
Brevibacterium, or is selected from those strains set forth in Table 3.
Another aspect of the invention pertains to methods for modulating production
of
a molecule from a microorganism. Such methods include contacting the cell with
an
agent which modulates MCT protein activity or MCT nucleic acid expression such
that a
cell associated activity is altered relative to this same activity in the
absence of the
agent. In a preferred embodiment, the cell is modulated for one or more C.
glutamicum
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metabolic pathways for cell membrane components or is modulated for the
transport of
compounds across such membranes, such that the yields or rate of production of
a
desired fine chemical by this microorganism is improved. The agent which
modulates
MCT protein activity can be an agent which stimulates MCT protein activity or
MCT
nucleic acid expression. Examples of agents which stimulate MCT protein
activity or
MCT nucleic acid expression include small molecules, active MCT proteins, and
nucleic
acids encoding MCT proteins that have been introduced into the cell. Examples
of
agents which inhibit MCT activity or expression include small molecules and
antisense
MCT nucleic acid molecules.
Another aspect of the invention pertains to methods for modulating yields of a
desired compound from a cell, involving the introduction of a wild-type or
mutant MCT
gene into a cell, either maintained on a separate plasmid or integrated into
the genome of
the host cell. If integrated into the genome, such integration can be random,
or it can
take place by homologous recombination such that the native gene is replaced
by the
introduced copy, causing the production of the desired compound from the cell
to be
modulated. In a preferred embodiment, said yields are increased. In another
preferred
embodiment, said chemical is a fine chemical. In a particularly preferred
embodiment,
said fine chemical is an amino acid. In especially preferred embodiments, said
amino
acid is L-lysine.
Detailed Description of the Invention
The present invention provides MCT nucleic acid and protein molecules which
are involved in the metabolism of cellular membrane components in C.
glutamicum or
in the transport of compounds across such membranes. The molecules of the
invention
may be utilized in the modulation of production of fine chemicals from
microorganisms,
such as C. glutamicum, either directly (e.g., where overexpression or
optimization of a
fatty acid biosynthesis protein has a direct impact on the yield, production,
and/or
efficiency of production of the fatty acid from modified C. glutamicum), or
may have an
indirect impact which nonetheless results in an increase of yield, production,
and/or
efficiency of production of the desired compound (e.g., where modulation of
the
metabolism of cell membrane components results in alterations in the yield,
production,
CA 02583703 2007-04-19
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and/or efficiency of production or the composition of the cell membrane, which
in turn
may impact the production of one or more fine chemicais). Aspects of the
invention are
further explicated below.
I. Fine Chemicals
The term 'fine chemical' is art-recognized and includes molecules produced by
an organism which have applications in various industries, such as, but not
limited to,
the pharmaceutical, agriculture, and cosmetics industries. Such compounds
include
organic acids, such as tartaric acid, itaconic acid, and diaminopimelic acid,
both
proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases,
nucleosides, and nucleotides (as described e.g. in Kuninaka, A. (1996)
Nucleotides and
related compounds, p. 561-612, in Biotechnology vol. 6, Rehm et al., eds. VCH:
Weinheim, and references contained therein), lipids, both saturated and
unsaturated fatty
acids (e.g., arachidonic acid), diols (e.g., propane diol, and butane diol),
carbohydrates
(e.g., hyaluronic acid and trehalose), aromatic compounds (e.g., aromatic
amines,
vanillin, and indigo), vitamins and cofactors (as described in Ullmann's
Encyclopedia of
Industrial Chemistry, vol. A27, "Vitamins", p. 443-613 (1996) VCH: Weinheim
and
references therein; and Ong, A.S., Niki, E. & Packer, L. (1995) "Nutrition,
Lipids,
Health, and Disease" Proceedings of the UNESCO/Confederation of Scientific and
Technological Associations in Malaysia, and the Society for Free Radical
Research -
Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes,
polyketides (Cane et al. (1998) Science 282: 63-68), and all other chemicals
described in
Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN:
0818805086 and references therein. The metabolism and uses of certain of these
fine
chemicals are further explicated below.
A. Amino Acid Metabolism and Uses
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
CA 02583703 2007-04-19
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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, 3rd 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.
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
conunonly
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.
The biosynthesis of these natural amino acids in organisms capable of
producing them, such as bacteria, has been well characterized (for review of
bacterial
CA 02583703 2007-04-19
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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
a-
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
(3-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-l-pyrophosphate, an activated sugar.
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~d 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, 3rd 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.
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B. Vitamin, Cofactor, and Nutraceutical Metabolism and Uses
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).
The biosynthesis of these molecules in organisms capable of producing them,
such as bacteria, has been largely characterized (Ullman's Encyclopedia of
Industrial
Chemistry, "Vitamins" vol. A27, p. 443-613, VCH: Weinheim, 1996; Michal, G.
(1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John
Wiley
& Sons; Ong, A.S., Niki, E. & Packer, L. (1995) "Nutrition, Lipids, Health,
and
Disease" Proceedings of the UNESCO/Confederation of Scientific and
Technological
Associations in Malaysia, and the Society for Free Radical Research - Asia,
held Sept.
1-3, 1994 at Penang, Malaysia, AOCS Press: Champaign, IL X, 374 S).
Thiamin (vitamin Bi) is produced by the chemical coupling of pyrimidine and
thiazole moieties. Riboflavin (vitamin B2) 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
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compounds collectively termed 'vitamin B6' (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-I-oxobutyl)-(3-alanine) can be
produced
either by chemical synthesis or by fermentation. The final steps in
pantothenate
biosynthesis consist of the ATP-driven condensation of 0-alanine and pantoic
acid. The
enzymes responsible for the biosynthesis steps for the conversion to pantoic
acid, to 0-
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 BS), pantetheine (and its derivatives) and coenzyme A.
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 a-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.
Corrinoids (such as the cobalamines and particularly vitamin B1Z) and
porphyrines belong to a group of chemicals characterized by a tetrapyrole ring
system.
The biosynthesis of vitamin B12 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.
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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 B6, pantothenate,
and
biotin. Only Vitamin B12 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
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 fonn nucleic acid molecules, but rather serve as
energy stores
(i.e., AMP) or as coenzymes (i.e., FAD and NAD).
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,
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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.
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
Trehalose consists of two glucose molecules, bound in a, a-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.
CA 02583703 2007-04-19
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Patent No. 5,759,610; Singer, M.A. and Lindquist, S. (1998) Trends Biotech.
16: 460-
467; Paiva, C.L.A. and Panek, A.D. (1996) Biotech. Ann. Rev. 2: 293-314; and
Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose is produced by enzymes
from
many microorganisms and is naturally released into the surrounding medium,
from
which it can be collected using methods known in the art.
II. Membrane Biosynthesis and Transmembrane Transport
Cellular membranes serve a variety of functions in a cell. First and foremost,
a
membrane differentiates the contents of a cell from the surrounding
environment, thus
giving integrity to the cell. Membranes may also serve as barriers to the
influx of
hazardous or unwanted compounds, and also to the efflux of desired compounds.
Cellular membranes are by nature impervious to the unfacilitated diffusion of
hydrophilic compounds such as proteins, water molecules and ions due to their
structure:
a bilayer of lipid molecules in which the polar head groups face outwards
(towards the
exterior and interior of the cell, respectively) and the nonpolar tails face
inwards at the
center of the bilayer, fonning a hydrophobic core (for a general review of
membrane
structure and function, see Gennis, R.B. (1989) Biomembranes, Molecular
Structure and
Function, Springer: Heidelberg). This barrier enables cells to maintain a
relatively
higher concentration of desired compounds and a relatively lower concentration
of
undesired compounds than are contained within the surrounding medium, since
the
diffusion of these compounds is effectively blocked by the membrane.
However, the membrane also presents an effective barrier to the import of
desired
compounds and the export of waste molecules. To overcome this difficulty,
cellular
membranes incorporate many kinds of transporter proteins which are able to
facilitate
the transmembrane transport of different kinds of compounds. There are two
general
classes of these transport proteins: pores or channels and transporters. The
former are
integral membrane proteins, sometimes complexes of proteins, which form a
regulated
hole through the membrane. This regulation, or 'gating' is generally specific
to the
molecules to be transported by the pore or channel, rendering these
transmembrane
constructs selectively permeable to a specific class of substrates; for
example, a
potassium channel is constructed such that only ions having a like charge and
size to that
of potassium may pass through. Channel and pore proteins tend to have discrete
CA 02583703 2007-04-19
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hydrophobic and hydrophilic domains, such that the hydrophobic face of the
protein
may associate with the interior of the membrane while the hydrophilic face
lines the
interior of the channel, thus providing a sheltered hydrophilic environment
through
which the selected hydrophilic molecule may pass. Many such pores/channels are
known in the art, including those for potassium, calcium, sodium, and chloride
ions.
This pore and channel-mediated system of facilitated diffusion is limited to
very
small molecules, such as ions, because pores or channels large enough to
permit the
passage of whole proteins by facilitated diffusion would be unable to prevent
the
passage of smaller hydrophilic molecules as well. Transport of molecules by
this process
is sometimes termed 'facilitated diffusion' since the driving force of a
concentration
gradient is required for the transport to occur. Permeases also permit
facilitated
diffusion of larger molecules, such as glucose or other sugars, into the cell
when the
concentration of these molecules on one side of the membrane is greater than
that on the
other (also called 'uniport'). In contrast to pores or channels, these
integral membrane
proteins (often having between 6-14 membrane-spanning a-helices) do not form
open
channels through the membrane, but rather bind to the target molecule at the
surface of
the membrane and then undergo a conformational shift such that the target
molecule is
released on the opposite side of the membrane.
However, cells frequently require the import or export of molecules against
the
existing concentration gradient ('active transport'), a situation in which
facilitated
diffusion cannot occur. There are two general mechanisms used by cells for
such
membrane transport: symport or antiport, and energy-coupled transport such as
that
mediated by the ABC transporters. Symport and antiport systems couple the
movement
of two different molecules across the membrane (via permeases having two
separate
binding sites for the two different molecules); in symport, both molecules are
transported in the same direction, while in antiport, one molecule is imported
while the
other is exported. This is possible energetically because one of the two
molecules
moves in accordance with a concentration gradient, and this energetically
favorable
event is permitted only upon concomitant movement of a desired compound
against the
prevailing concentration gradient. Single molecules may be transported across
the
membrane against the concentration gradient in an energy-driven process, such
as that
utilized by the ABC transporters. In this system, the transport protein
located in the
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membrane has an ATP-binding cassette; upon binding of the target molecule, the
ATP is
converted to ADP + Pi, and the resulting release of energy is used to drive
the
movement of the target molecule to the opposite face of the membrane,
facilitated by the
transporter. For more detailed descriptions of all of these transport systems,
see:
Bamberg, E. et al., (1993) "Charge transport of ion pumps on lipid bilayer
membranes",
Q. Rev. Biophys. 26: 1-25; Findlay, J.B.C. (1991) "Structure and function in
membrane
transport systems", Curr. Opin. Struct. Biol. 1:804-810; Higgins, C.F. (1992)
"ABC
transporters from microorganisms to man", Ann. Rev. Cell Biol. 8: 67-113;
Gennis, R.B.
(1989) "Pores, Channels and Transporters", in: Biomembranes, Molecular
Structure and
Function, Springer: Heidelberg, p. 270-322; and Nikaido, H. and Saier, H.
(1992)
"Transport proteins in bacteria: common themes in their design", Science 258:
936-942,
and references contained within each of these references.
The synthesis of membranes is a well-characterized process involving a number
of components, the most important of which are lipid molecules. Lipid
synthesis may
be divided into two parts: the synthesis of fatty acids and their attachment
to sn-
glycerol-3-phosphate, and the addition or modification of a polar head group.
Typical
lipids utilized in bacterial membranes include phospholipids, glycolipids,
sphingolipids,
and phosphoglycerides. Fatty acid synthesis begins with the conversion of
acetyl CoA
either to malonyl CoA by acetyl CoA carboxylase, or to acetyl-ACP by
acetyltransacylase. Following a condensation reaction, these two product
molecules
together form acetoacetyl-ACP, which is converted by a series of condensation,
reduction and dehydration reactions to yield a saturated fatty acid molecule
having a
desired chain length. The production of unsaturated fatty acids from such
molecules is
catalyzed by specific desaturases either aerobically, with the help of
molecular oxygen,
or anaerobically (for reference on fatty acid synthesis, see F.C. Neidhardt et
al. (1996)
E. coli and Salmonella. ASM Press: Washington, D.C., p. 612-636 and references
contained therein; Lengeler et al. (eds) (1999) Biology of Procaryotes.
Thieme:
Stuttgart, New York, and references contained therein; and Magnuson, K. el
al., (1993)
Microbiological Reviews 57: 522-542, and references contained therein). The
cyclopropane fatty acids (CFA) are synthesized by a specific CFA-synthase
using SAM
as a cosubstrate. Branched chain fatty acids are synthesized from branched
chain amino
acids that are deaminated to yield branched chain 2-oxo-acids (see Lengeler et
al., eds.
CA 02583703 2007-04-19
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(1999) Biology of Procaryotes. Thieme: Stuttgart, New York, and references
contained
therein). Another essential step in lipid synthesis is the transfer of fatty
acids onto the
polar head groups by, for example, glycerol-phosphate-acyltransferases. The
combination of various precursor molecules and biosynthetic enzymes results in
the
production of different fatty acid molecules, which has a profound effect on
the
composition of the membrane.
III. Elements and Methods of the Invention
The present invention is based, at least in part, on the discovery of novel
molecules, referred to herein as MCT nucleic acid and protein molecules, which
control
the production of cellular membranes in C. glutamicum and govem the movement
of
molecules across such membranes. In one embodiment, the MCT molecules
participate
in the metabolism of compounds necessary for the construction of cellular
membranes in
C. glutamicum, or in the transport of molecules across these membranes. In a
preferred
embodiment, the activity of the MCT molecules of the present invention to
regulate
membrane component production and membrane transport has an impact on the
production of a desired fine chemical by this organism. In a particularly
preferred
embodiment, the MCT molecules of the invention are modulated in activity, such
that
the C. glutamicum metabolic pathways which the MCT proteins of the invention
regulate are modulated in yield, production, and/or efficiency of production
and the
transport of compounds through the membranes is altered in efficiency, which
either
directly or indirectly modulates the yield, production, and/or efficiency of
production of
a desired fine chemical by C. glutamicum.
The language, "MCT protein" or "MCT polypeptide" includes proteins which
participate in the metabolism of compounds necessary for the construction of
cellular
membranes in C. glutamicum, or in the transport of molecules across these
membranes.
Examples of MCT proteins include those encoded by the MCT genes set forth in
Table 1
and by the odd-numbered SEQ ID NOs. The terms "MCT gene" or "MCT nucleic acid
sequence" include nucleic acid sequences encoding an MCT protein, which
consist of a
coding region and also corresponding untranslated 5' and 3' sequence regions.
Examples of MCT genes include those set forth in Table 1. The terms
"production" or
"productivity" are art-recognized and include the concentration of the
fermentation
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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.
In another embodiment, the MCT molecules of the invention are capable of
modulating the production of a desired molecule, such as a fine chemical, in a
microorganism such as C glutamicum. There are a number of mechanisms by which
the alteration of an MCT protein of the invention may directly affect the
yield,
production, and/or efficiency of production of a fine chemical from a C
glutamicum
strain incorporating such an altered protein. Those MCT proteins involved in
the export
of fine chemical molecules from the cell may be increased in number or
activity such
that greater quantities of these compounds are secreted to the extracellular
medium,
from which they are more readily recovered. Similarly, those MCT proteins
involved in
the import of nutrients necessary for the biosynthesis of one or more fine
chemicals
(e.g., phosphate, sulfate, nitrogen compounds, etc.) may be increased in
number or
CA 02583703 2007-04-19
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activity such that these precursor, cofactor, or intermediate compounds are
increased in
concentration within the cell. Further, fatty acids and lipids themselves are
desirable fine
chemicals; by optimizing the activity or increasing the number of one or more
MCT
proteins of the invention which participate in the biosynthesis of these
compounds, or by
impairing the activity of one or more MCT proteins which are involved in the
degradation of these compounds, it may be possible to increase the yield,
production,
and/or efficiency of production of fatty acid and lipid molecules from C.
glutamicum.
The mutagenesis of one or more MCT genes of the invention may also result in
MCT proteins having altered activities which indirectly impact the production
of one or
more desired fine chemicals from C.glutamicum. For example, MCT proteins of
the
invention involved in the export of waste products may be increased in number
or
activity such that the normal metabolic wastes of the cell (possibly increased
in quantity
due to the overproduction of the desired fine chemical) are efficiently
exported before
they are able to damage nucleotides and proteins within the cell (which would
decrease
the viability of the cell) or to interfere with fine chemical biosynthetic
pathways (which
would decrease the yield, production, or efficiency of production of the
desired fine
chemical). Further, the relatively large intracellular quantities of the
desired fine
chemical may in itself be toxic to the cell, so by increasing the activity or
number of
transporters able to export this compound from the cell, one may increase the
viability of
the cell in culture, in turn leading to a greater number of cells in the
culture producing
the desired fine chemical. The MCT proteins of the invention may also be
manipulated
such that the relative amounts of different lipid and fatty acid molecules are
produced.
This may have a profound effect on the lipid composition of the membrane of
the cell.
Since each type of lipid has different physical properties, an alteration in
the lipid
composition of a membrane may significantly alter membrane fluidity. Changes
in
membrane fluidity can impact the transport of molecules across the membrane,
as well
as the integrity of the cell, both of which have a profound effect on the
production of
fine chemicals from C. glutamicum in large-scale fermentative culture.
The isolated nucleic acid sequences of the invention are contained within the
genome of a Corynebacterium glutamicum strain available through the American
Type
Culture Collection, given designation ATCC 13032. The nucleotide sequence of
the
isolated C. glutamicum MCT DNAs and the predicted amino acid sequences of the
C.
CA 02583703 2007-04-19
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glutamicum MCT proteins are shown in the Sequence Listing as odd-numbered SEQ
ID
NOs and even-numbered SEQ ID NOs, respectively. Computational analyses were
performed which classified and/or identified these nucleotide sequences as
sequences
which encode proteins involved in the metabolism of cellular membrane
components or
proteins involved in the transport of compounds across such membranes.
The present invention also pertains to proteins which have an amino acid
sequence which is substantially homologous to an amino acid sequence of the
invention
(e.g., the sequence of an even-numbered SEQ ID NO of the Sequence Listing)..
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.
The MCT protein or a biologically active portion or fragment thereof of the
invention can participate in the metabolism of compounds necessary for the
construction
of cellular membranes in C. glutamicum, or in the transport of molecules
across these
membranes, or have one or more of the activities set forth in Table 1.
Various aspects of the invention are described in further detail in the
following
subsections:
A. Isolated Nucleic Acid Molecules
One aspect of the invention pertains to isolated nucleic acid molecules that
encode MCT polypeptides or biologically active portions thereof, as well as
nucleic acid
fragments sufficient for use as hybridization probes or primers for the
identification or
amplification of MCT-encoding nucleic acid (e.g., MCT DNA). As used herein,
the
term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA
or
genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA
generated using nucleotide analogs. This term also encompasses untranslated
sequence
located at both the 3' and 5' ends of the coding region of the gene: at least
about 100
nucleotides of sequence upstream from the 5' end of the coding region and at
least about
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20 nucleotides of sequence downstream from the 3'end of the coding region of
the gene.
The nucleic acid molecule can be single-stranded or double-stranded, but
preferably is
double-stranded DNA. An "isolated" nucleic acid molecule is one which is
separated
from other nucleic acid molecules which are present in the natural source of
the nucleic
acid. Preferably, an "isolated" nucleic acid is free of sequences which
naturally flank
the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic
acid) in the
genomic DNA of the organism from which the nucleic acid is derived. For
example, in
various embodiments, the isolated MCT nucleic acid molecule can contain less
than
about 5 kb, 4kb, 3kb, 2kb, 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.
A nucleic acid molecule of the present invention, e.g., a nucleic acid
molecule
having a nucleotide sequence of an odd-numbered SEQ ID NO of the Sequence
Listing,
or a portion thereof, can be isolated using standard molecular biology
techniques and the
sequence information provided herein. For example, a C. glutamicum MCT DNA can
be isolated from a C. glutamicum library using all or portion of one of the
odd-numbered
SEQ ID NO sequences of the Sequence Listing 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, NY,
1989).
Moreover, a nucleic acid molecule encompassing all or a portion of one of the
nucleic
acid sequences of the invention (e.g., an odd-numbered SEQ ID NO:) 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
nucleic acid sequences of the invention (e.g., an odd-numbered SEQ ID NO of
the
Sequence Listing) can be isolated by the polymerase chain reaction using
oligonucleotide primers designed based upon this same sequence). 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
CA 02583703 2007-04-19
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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, FL). Synthetic oligonucleotide
primers
for polymerase chain reaction amplification can be designed based upon one of
the
nucleotide sequences shown in the Sequence Listing. A nucleic acid of the
invention
can be amplified using cDNA or, alternatively, genomic DNA, as a template and
appropriate oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an appropriate
vector and
characterized by DNA sequence analysis. Furthermore, oligonucleotides
corresponding
to an MCT nucleotide sequence can be prepared by standard synthetic
techniques, e.g.,
using an automated DNA synthesizer.
In a preferred embodiment, an isolated nucleic acid molecule of the invention
comprises one of the nucleotide sequences shown in the Sequence Listing. The
nucleic
acid sequences of the invention, as set forth in the Sequence Listing
correspond to the
Corynebacterium glutamicum MCT DNAs of the invention. This DNA comprises
sequences encoding MCT proteins (i.e., the "coding region", indicated in each
odd-
numbered SEQ ID NO: in the Sequence Listing), as well as 5' untranslated
sequences
and 3' untranslated sequences, also indicated in each odd-numbered SEQ ID NO:
in the
Sequence Listing. Alternatively, the nucleic acid molecule can comprise only
the
coding region of any of the nucleic acid sequences of the Sequence Listing.
For the purposes of this application, it will be understood that each of the
nucleic
acid and amino acid sequences set forth in the Sequence Listing has an
identifying RXA,
RXN, RXS, or RXC number having the designation "RXA", "RXN", "RXS" or "RXC"
followed by 5 digits (i.e., RXA02099, RXN03097, RXS00148, or RXC01748). Each
of
the nucleic acid sequences comprises up to three parts: a 5' upstream region,
a coding
region, and a downstream region. Each of these three regions is identified by
the same
RXA, RXN, RXS, or RXC designation to eliminate confusion. The recitation "one
of
the odd-numbered sequences in of the Sequence Listing", then, refers to any of
the
nucleic acid sequences in the Sequence Listing, which may also be
distinguished by
their differing RXA, RXN, RXS, or RXC designations. The coding region of each
of
these sequences is translated into a corresponding amino acid sequence, which
is also set
forth in the Sequence Listing, as an even-numbered SEQ ID NO: immediately
following
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the corresponding nucleic acid sequence. For example, the coding region for
RXA03097 is set forth in SEQ ID NO: 1, while the amino acid sequence which it
encodes is set forth as SEQ ID NO:2. The sequences of the nucleic acid
molecules of
the invention are identified by the same RXA, RXN, RXS, or RXC designations as
the
amino acid molecules which they encode, such that they can be readily
correlated. For
example, the amino acid sequences designated RXA02099, RXN03097, RXS00148, and
RXC01748 are translations of the coding region of the nucleotide sequences of
nucleic
acid molecules RXA02099, RXN03097, RXS00148, and RXC01748, respectively. The
correspondence between the RXA, RXN, RXS, and RXC nucleotide and amino acid
sequences of the invention and their assigned SEQ ID NOs is set forth in Table
1. For
example, as set forth in Table 1, the nucleotide sequence of RXA00104 is SEQ
ID
NO:5, and the amino acid sequence of RXA00104 is SEQ ID NO:6.
Several of the genes of the invention are "F-designated genes". An F-
designated
gene includes those genes set forth in Table 1 which have an 'F' in front of
the RXA,
RXN, RXS, or RXC designation. For example, SEQ ID NO: 11, designated, as
indicated
on Table 1, as "F RXA02581 ", is an F-designated gene, as are SEQ ID NOs: 31,
33, and
43 (designated on Table 1 as "F RXA02487", "F RXA02490", and "F RXA02809",
respectively).
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.
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 of the invention (e.g., a sequence of an odd-numbered SEQ
ID
NO: of the Sequence Listing), or a portion thereof. A nucleic acid molecule
which is
complementary to one of the nucleotide sequences shown in of the invention is
one
which is sufficiently complementary to one of the nucleotide sequences shown
in the
Sequence Listing (e.g., the sequence of an odd-numbered SEQ ID NO:)such that
it can
CA 02583703 2007-04-19
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hybridize to one of the nucleotide sequences of the invention, thereby forming
a stable
duplex.
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 of
the
invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence
Listing),
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 of
the
invention, or a portion thereof.
Moreover, the nucleic acid molecule of the invention can comprise only a
portion of the coding region of the sequence of one of the odd-numbered SEQ ID
NOs
of the Sequence Listing, for example a fragment which can be used as a probe
or primer
or a fragment encoding a biologically active portion of an MCT protein. The
nucleotide
sequences determined from the cloning of the MCT genes from C. glutamicum
allows
for the generation of probes and primers designed for use in identifying
and/or cloning
MCT homologues in other cell types and organisms, as well as MCT homologues
from
other Corynebacteria or related species. The probe/primer typically comprises
substantially.purified oligonucleotide. The oligonucleotide typically
comprises a region
of nucleotide sequence that hybridizes under stringent conditions to at least
about 12,
preferably about 25, more preferably about 40, 50 or 75 consecutive
nucleotides of a
sense strand of one of the nucleotide sequences of the invention (e.g., a
sequence of one
of the odd-numbered SEQ ID NOs of the Sequence Listing), an anti-sense
sequence of
one of these sequences, or naturally occurring mutants thereof. Primers based
on a
CA 02583703 2007-04-19
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nucleotide sequence of the invention can be used in PCR reactions to clone MCT
homologues. Probes based on the MCT nucleotide sequences can be used to detect
transcripts or genomic sequences encoding the same or homologous proteins. In
preferred embodiments, the probe further comprises a label group attached
thereto, e.g.
the label group can be a radioisotope, a fluorescent compound, an enzyme, or
an enzyme
co-factor. Such probes can be used as a part of a diagnostic test kit for
identifying cells
which misexpress an MCT protein, such as by measuring a level of an MCT-
encoding
nucleic acid in a sample of cells, e.g., detecting MCT mRNA levels or
determining
whether a genomic MCT gene has been mutated or deleted.
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 the invention (e.g., a sequence of an
even-
numbered SEQ ID NO of the Sequence Listing) such that the protein or portion
thereof
maintains the ability to participate in the metabolism of compounds necessary
for the
construction of cellular membranes in C. glutamicum, or in the transport of
molecules
across these membranes. As used herein, the language "sufficiently homologous"
refers
to proteins or portions thereof which have amino acid sequences which include
a
minimum number of identical or equivalent (e.g., an amino acid residue which
has a
similar side chain as an amino acid residue in a sequence of one of the even-
numbered
SEQ ID NOs of the Sequence Listing) amino acid residues to an amino acid
sequence of
the invention such that the protein or portion thereof is able to participate
in the
metabolism of compounds necessary for the construction of cellular membranes
in C.
glutamicum, or in the transport of molecules across these membranes. Protein
members
of such membrane component metabolic pathways or membrane transport systems,
as
described herein, may play a role in the production and secretion of one or
more fine
chemicals. Examples of such activities are also described herein. Thus, "the
function of
an MCT protein" contributes either directly or indirectly to the yield,
production, and/or
efficiency of production of one or more fine chemicals. Examples of MCT
protein
activities are set forth in Table 1.
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
CA 02583703 2007-04-19
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acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID
NO: of
the Sequence Listing).
Portions of proteins encoded by the MCT nucleic acid molecules of the
invention
are preferably biologically active portions of one of the MCT proteins. As
used herein,
the term "biologically active portion of an MCT protein" is intended to
include a
portion, e.g., a domain/motif, of an MCT protein that participates in the
metabolism of
compounds necessary for the construction of cellular membranes in C.
glutamicum, or in
the transport of molecules across these membranes, or has an activity as set
forth in
Table 1. To determine whether an MCT protein or a biologically active portion
thereof
can participate in the metabolism of compounds necessary for the construction
of
cellular membranes in C. glutamicum, or in the transport of molecules across
these
membranes, an assay of enzymatic activity may be performed. Such assay methods
are
well known to those of ordinary skill in the art, as detailed in Example 8 of
the
Exemplification.
Additional nucleic acid fragments encoding biologically active portions of an
MCT protein can be prepared by isolating a portion of one of the amino acid
sequences
of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the
Sequence
Listing), expressing the encoded portion of the MCT protein or peptide (e.g.,
by
recombinant expression in vitro) and assessing the activity of the encoded
portion of the
MCT protein or peptide.
The invention further encompasses nucleic acid molecules that differ from one
of
the nucleotide sequences shown of the invention (e.g., a sequence of an odd-
numbered
SEQ ID NO: of the Sequence Listing) (and portions thereof) due to degeneracy
of the
genetic code and thus encode the same MCT protein as that encoded by the
nucleotide
sequences of the invention. 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 the Sequence Listing (e.g., an even-numbered SEQ ID NO:). 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
the invention (encoded by an open reading frame shown in an odd-numbered SEQ
ID
NO: of the Sequence Listing).
CA 02583703 2007-04-19
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It will be understood by one of ordinary skill in the art that in one
embodiment
the sequences of the invention are not meant to include the sequences of the
prior art,
such as those Genbank sequences set forth in Tables 2 or 4 which were
available prior to
the present invention. In one embodiment, the invention includes nucleotide
and amino
acid sequences having a percent identity to a nucleotide or amino acid
sequence of the
invention which is greater than that of a sequence of the prior art (e.g., a
Genbank
sequence (or the protein encoded by such a sequence) set forth in Tables 2 or
4). For
example, the invention includes a nucleotide sequence which is greater than
and/or at
least 38% identical to the nucleotide sequence designated RXA01420 (SEQ ID
NO:7), a
nucleotide sequence which is greater than and/or at least 43% identical to the
nucleotide
sequence designated RXA00104 (SEQ ID NO:5), and a nucleotide sequence which is
greater than and/or at least 45% identical to the nucleotide sequence
designated
RXA02173 (SEQ ID NO:25). One of ordinary skill in the art would be able to
calculate
the lower threshold of percent identity for any given sequence of the
invention by
examining the GAP-calculated percent identity scores set forth in Table 4 for
each of the
three top hits for the given sequence, and by subtracting the highest GAP-
calculated
percent identity from 100 percent. One of ordinary skill in the art will also
appreciate
that nucleic acid and amino acid sequences having percent identities greater
than the
lower threshold so calculated (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%,
56%,
57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%,
67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or
90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%,
97%,
98%, 99% or more identical) are also encompassed by the invention.
In addition to the C. glutamicum MCT nucleotide sequences set forth in the
Sequence Listing as odd-numbered SEQ ID NOs, it will be appreciated by one of
ordinary skill in the art that DNA sequence polymorphisms that lead to changes
in the
amino acid sequences of MCT proteins may exist within a population (e.g., the
C.
glutamicum population). Such genetic polymorphism in the MCT gene may exist
among individuals within a population due to natural variation. As used
herein, the
terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising
an
open reading frame encoding an MCT protein, preferably a C. glutamicum MCT
protein.
CA 02583703 2007-04-19
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Such natural variations can typically result in 1-5% variance in the
nucleotide sequence
of the MCT gene. Any and all such nucleotide variations and resulting amino
acid
polymorphisms in MCT that are the result of natural variation and that do not
alter the
functional activity of MCT proteins are intended to be within the scope of the
invention.
Nucleic acid molecules corresponding to natural variants and non-C. glutamicum
homologues of the C. glutamicum MCT DNA of the invention can be isolated based
on
their homology to the C. glutamicum MCT nucleic acid disclosed herein using
the C.
glutamicum DNA, or a portion thereof, as a hybridization probe according to
standard
hybridization techniques under stringent hybridization conditions.
Accordingly, in
another embodiment, an isolated nucleic acid molecule of the invention is at
least 15
nucleotides in length and hybridizes under stringent conditions to the nucleic
acid
molecule comprising a nucleotide sequence of an odd-numbered SEQ ID NO: of the
Sequence Listing. In other embodiments, the nucleic acid is at least 30, 50,
100, 250 or
more nucleotides in length. As used herein, the term "hybridizes under
stringent
conditions" is intended to describe conditions for hybridization and washing
under
which nucleotide sequences at least 60% homologous to each other typically
remain
hybridized to each other. Preferably, the conditions are such that sequences
at least
about 65%, more preferably at least about 70%, and even more preferably at
least about
75% or more homologous to each other typically remain hybridized to each
other. Such
stringent conditions are known to those of ordinary skill in the art and can
be found in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-
6.3.6.
A preferred, non-limiting example of stringent hybridization conditions are
hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 C,
followed by
one or more washes in 0.2 X SSC, 0.1% SDS at 50-65 C. Preferably, an isolated
nucleic acid molecule of the invention that hybridizes under stringent
conditions to a
nucleotide sequence of the invention corresponds to a naturally-occurring
nucleic acid
molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers
to an
RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g.,
encodes a natural protein). In one embodiment, the nucleic acid encodes a
natural C.
glutamicum MCT protein.
In addition to naturally-occurring variants of the MCT sequence that may exist
in
the population, one of ordinary skill in the art will further appreciate that
changes can be
CA 02583703 2007-04-19
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introduced by mutation into a nucleotide sequence of the invention, thereby
leading to
changes in the amino acid sequence of the encoded MCT protein, without
altering the
functional ability of the MCT protein. For example, nucleotide substitutions
leading to
amino acid substitutions at "non-essential" amino acid residues can be made in
a
nucleotide sequence of the invention. A "non-essential" amino acid residue is
a residue
that can be altered from the wild-type sequence of one of the MCT proteins
(e.g., an
even-numbered SEQ ID NO: of the Sequence Listing) without altering the
activity of
said MCT protein, whereas an "essential" amino acid residue is required for
MCT
protein activity. Other amino acid residues, however, (e.g., those that are
not conserved
or only semi-conserved in the domain having MCT activity) may not be essential
for
activity and thus are likely to be amenable to alteration without altering MCT
activity.
Accordingly, another aspect of the invention pertains to nucleic acid
molecules
encoding MCT proteins that contain changes in amino acid residues that are not
essential for MCT activity. Such MCT proteins differ in amino acid sequence
from a
sequence of an even-numbered SEQ ID NO: of the Sequence Listing yet retain at
least
one of the MCT activities described herein. In one embodiment, the isolated
nucleic
acid molecule comprises a nucleotide sequence encoding a protein, wherein the
protein
comprises an amino acid sequence at least about 50% homologous to an amino
acid
sequence of the invention and is capable of participate in the metabolism of
compounds
necessary for the construction of cellular membranes in C. glutamicum, or in
the
transport of molecules across these membranes, or has one or more activities
set forth in
Table 1. Preferably, the protein encoded by the nucleic acid molecule is at
least about
50-60% homologous to the amino acid sequence of one of the odd-numbered SEQ ID
NOs of the Sequence Listing , more preferably at least about 60-70% homologous
to one
of these sequences, even more preferably at least about 70-80%, 80-90%, 90-95%
homologous to one of these sequences, and most preferably at least about 96%,
97%,
98%, or 99% homologous to one of the amino acid sequences of the invention..
To determine the percent homology of two amino acid sequences (e.g., one of
the amino acid sequences of the invention 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
CA 02583703 2007-04-19
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amino acid positions or nucleotide positions are then compared. When a
position in one
sequence (e.g., one of the amino acid sequences of the invention) 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 amino acid sequence), then the molecules
are
homologous at that position (f.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 (f.e., % homology =# of identical positions/total # of positions x
100).
An isolated nucleic acid molecule encoding an MCT protein homologous to a
protein sequence of the invention (e.g., a sequence of an even-numbered SEQ ID
NO: of
the Sequence Listing) can be created by introducing one or more nucleotide
substitutions, additions or deletions into a nucleotide sequence of the
invention 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 nucleotide
sequences of
the invention by standard techniques, such as site-directed mutagenesis and
PCR-
mediated mutagenesis. Preferably, conservative amino acid substitutions are
made at
one or more predicted non-essential amino acid residues. A "conservative amino
acid
substitution" is one in which the amino acid residue is replaced with an amino
acid
residue having a similar side chain. Families of amino acid residues having
similar side
chains have been defined in the art. These families include amino acids with
basic side
chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic
acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine,
serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched
side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine,
phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino
acid residue
in an MCT protein is preferably replaced with another amino acid residue from
the same
side chain family. Alternatively, in another embodiment, mutations can be
introduced
randomly along all or part of an MCT coding sequence, such as by saturation
mutagenesis, and the resultant mutants can be screened for an MCT activity
described
herein to identify mutants that retain MCT activity. Following mutagenesis of
the
nucleotide sequence of one of the odd-numbered SEQ ID NOs of the Sequence
Listing,
CA 02583703 2007-04-19
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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).
In addition to the nucleic acid molecules encoding MCT proteins described
above, another aspect of the invention pertains to isolated nucleic acid
molecules which
are antisense thereto. An "antisense" nucleic acid comprises a nucleotide
sequence
which is complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the coding strand of a double-stranded cDNA molecule or
complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can
hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be
complementary to an entire MCT coding strand, or to only a portion thereof. In
one
embodiment, an antisense nucleic acid molecule is antisense to a"coding
region" of the
coding strand of a nucleotide sequence encoding an MCT protein. The term
"coding
region" refers to the region of the nucleotide sequence comprising codons
which are
translated into amino acid residues (e.g., the entire coding region of SEQ ID
NO:5
(RXA00104) comprises nucleotides 1 to 756). In another embodiment, the
antisense
nucleic acid molecule is antisense to a "noncoding region" of the coding
strand of a
nucleotide sequence encoding MCT. The term "noncoding region" refers to 5' and
3'
sequences which flank the coding region that are not translated into amino
acids (i.e.,
also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding MCT disclosed herein (e.g., the
sequences set forth as odd-numbered SEQ ID NOs in the Sequence Listing),
antisense
nucleic acids of the invention can be designed according to the rules of
Watson and
Crick base pairing. The antisense nucleic acid molecule can be complementary
to the
entire coding region of MCT mRNA, but more preferably is an oligonucleotide
which is
antisense to only a portion of the coding or noncoding region of MCT mRNA. For
example, the antisense oligonucleotide can be complementary to the region
surrounding
the translation start site of MCT mRNA. An antisense oligonucleotide can be,
for
example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
An
antisense nucleic acid of the invention can be constructed using chemical
synthesis and
enzymatic ligation reactions using procedures known in the art. For example,
an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically
synthesized
CA 02583703 2007-04-19
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using naturally occurring nucleotides or variously modified nucleotides
designed to
increase the biological stability of the molecules or to increase the physical
stability of
the duplex formed between the antisense and sense nucleic acids, e.g.,
phosphorothioate
derivatives and acridine substituted nucleotides can be used. Examples of
modified
nucleotides which can be used to generate the antisense nucleic acid include 5-
fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-
acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-
thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-
galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-
methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-
methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-
methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-
methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-
isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-
thiocytosine, 5-
methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-
oxyacetic acid
methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-
N-2-
carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the
antisense
nucleic acid can be produced biologically using an expression vector into
which a
nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from
the inserted nucleic acid will be of an antisense orientation to a target
nucleic acid of
interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered
to a cell or generated in situ such that they hybridize with or bind to
cellular mRNA
and/or genomic DNA encoding an MCT protein to thereby inhibit expression of
the
protein, e.g., by inhibiting transcription and/or translation. The
hybridization can be by
conventional nucleotide complementarity to form a stable duplex, or, for
example, in the
case of an antisense nucleic acid molecule which binds to DNA duplexes,
through
specific interactions in the major groove of the double helix. The antisense
molecule can
be modified such that it specifically binds to a receptor or an antigen
expressed on a
selected cell surface, e.g., by linking the antisense nucleic acid molecule to
a peptide or
an antibody which binds to a cell surface receptor or antigen. The antisense
nucleic acid
molecule can also be delivered to cells using the vectors described herein. To
achieve
CA 02583703 2007-04-19
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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.
In yet another embodiment, the antisense nucleic acid molecule of the
invention
is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule
forms
specific double-stranded hybrids with complementary RNA in which, contrary to
the
usual 0-units, the strands run parallel to each other (Gaultier el 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 el al. (1987) FEBSLett. 215:327-330).
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) IVature 334:585-591)) can be used
to
catalytically cleave MCT mRNA transcripts to thereby inhibit translation of
MCT
mRNA. A ribozyme having specificity for an MCT-encoding nucleic acid can be
designed based upon the nucleotide sequence of an MCT DNA disclosed herein
(i.e.,
SEQ ID NO. 5(RXA00104). For example, a derivative of a Tetrahymena L-19 IVS
RNA can be constructed in which the nucleotide sequence of the active site is
complementary to the nucleotide sequence to be cleaved in an MCT-encoding
mRNA.
See, e.g., Cech et a1. U.S. Patent No. 4,987,071 and Cech et al. U.S. Patent
No.
5,116,742. Alternatively, MCT mRNA can be used to select a catalytic RNA
having a
specific ribonuclease activity from a pool of RNA molecules. See, e.g.,
Bartel, D. and
Szostak, J.W. (1993) Science 261:1411-1418.
Alternatively, MCT gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of an MCT nucleotide sequence
(e.g.,
an MCT promoter and/or enhancers) to form triple helical structures that
prevent
transcription of an MCT gene in target cells. See generally, Helene, C. (1991)
Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N. Y. Acad.
Sci. 660:27-
36; and Maher, L.J. (1992) Bioassays 14(12):807-15.
CA 02583703 2007-04-19
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B. Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors, containing a nucleic acid encoding an MCT protein (or a portion
thereof). As
used herein, the term "vector" refers to a nucleic acid molecule capable of
transporting
another nucleic acid to which it has been linked. One type of vector is a
"plasmid",
which refers to a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein
additional
DNA segments can be ligated into the viral genome. Certain vectors are capable
of
autonomous replication in a host cell into which they are introduced (e.g.,
bacterial
vectors having a bacterial origin of replication and episomal mammalian
vectors). Other
vectors (e.g., non-episomal mammalian vectors) are integrated into the genome
of a host
cell upon introduction into the host cell, and thereby are replicated along
with the host
genome. Moreover, certain vectors are capable of directing the expression of
genes to
which they are operatively linked. Such vectors are referred to herein as
"expression
vectors". In general, expression vectors of utility in recombinant DNA
techniques are
often in the form of plasmids. In the present specification, "plasmid" and
"vector" can
be used interchangeably as the plasmid is the most commonly used form of
vector.
However, the invention is intended to include such other forms of expression
vectors,
such as viral vectors (e.g., replication defective retroviruses, adenoviruses
and adeno-
associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of
the invention in a fonn 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,
CA 02583703 2007-04-19
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Academic Press, San Diego, CA (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-, lacIq-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-,
amy, SPO2, X-PR-
or a. PL, which are used preferably in bacteria. Additional regulatory
sequences are, for
example, promoters from yeasts and fungi, such as ADC 1, MFa, AC, P-60, CYC 1,
GAPDH, TEF, rp28, ADH, promoters from plants such as CaMV/35S, SSU, OCS, lib4,
usp, STLS1, B33, nos or ubiquitin- or phaseolin-promoters. It is also possible
to use
artificial promoters. It will be appreciated by one of ordinary skill in the
art that the
design of the expression vector can depend on such factors as the choice of
the host cell
to be transformed, the level of expression of protein desired, etc. The
expression vectors
of the invention can be introduced into host cells to thereby produce proteins
or
peptides, including fusion proteins or peptides, encoded by nucleic acids as
described
herein (e.g., MCT proteins, mutant forms of MCT proteins, fusion proteins,
etc.).
The recombinant expression vectors of the invention can be designed for
expression of MCT proteins in prokaryotic or eukaryotic cells. For example,
MCT
genes can be expressed in bacterial cells such as C. glutamicum, insect cells
(using
baculovirus expression vectors), yeast and other fungal cells (see Romanos,
M.A. et al.
(1992) "Foreign gene expression in yeast: a review", Yeast 8: 423-488; van den
Hondel,
C.A.M.J.J. et al. (1991) "Heterologous gene expression in filamentous fungi"
in: More
Gene Manipulations in Fungi, J.W. Bennet & L.L. Lasure, eds., p. 396-428:
Academic
Press: San Diego; and van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene
transfer
systems and vector development for filamentous fungi, in: Applied Molecular
Genetics
of Fungi, Peberdy, J.F. et al., eds., p. 1-28, Cambridge University Press:
Cambridge),
algae and multicellular plant cells (see Schmidt, R. and Willmitzer, L. (1988)
High
efficiency Agrobacterium tumefaciens -mediated transformation of Arabidopsis
thaliana leaf and cotyledon explants" Plant Cell Rep.: 583-586), or mammalian
cells.
Suitable host cells are discussed further in Goeddel, Gene Expression
Technology:
Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
Alternatively, the
recombinant expression vector can be transcribed and translated in vitro, for
example
using T7 promoter regulatory sequences and T7 polymerase.
CA 02583703 2007-04-19
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Expression of proteins in prokaryotes is most often carried out with vectors
containing constitutive or inducible promoters directing the expression of
either fusion
or non-fusion proteins. Fusion vectors add a number of amino acids to a
protein
encoded therein, usually to the amino terminus of the recombinant protein but
also to the
C-terminus or fused within suitable regions in the proteins. Such fusion
vectors
typically serve three purposes: 1) to increase expression of recombinant
protein; 2) to
increase the solubility of the recombinant protein; and 3) to aid in the
purification of the
recombinant protein by acting as a ligand in affinity purification. Often, in
fusion
expression vectors, a proteolytic cleavage site is introduced at the junction
of the fusion
moiety and the recombinant protein to enable separation of the recombinant
protein
from the fusion moiety subsequent to purification of the fusion protein. Such
enzymes,
and their cognate recognition sequences, include Factor Xa, thrombin and
enterokinase.
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,
MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to the target
recombinant
protein. In one embodiment, the coding sequence of the MCT protein is cloned
into a
pGEX expression vector to create a vector encoding a fusion protein
comprising, from
the N-terminus to the C-terminus, GST-thrombin cleavage site-X protein. The
fusion
protein can be purified by affinity chromatography using glutathione-agarose
resin.
Recombinant MCT protein unfused to GST can be recovered by cleavage of the
fusion
protein with thrombin.
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc (Amann et al., (1988) Gene 69:301-315) pLG338, pACYC184, pBR322, pUCl8,
pUC19, pKC30, pRep4, pHSI, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-
III113-B1, kgt11, pBdCl, and pET I ld (Studier et al., Gene Expression
Technology:
Methods in Enzymology 185, Academic Press, San Diego, Califomia (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 11 d vector relies on transcription from a T7 gn 10-lac fusion promoter
mediated by
a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied
by
CA 02583703 2007-04-19
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host strains BL21(DE3) or HMS 174(DE3) from a resident k prophage harboring a
T7
gn 1 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 pUB 110, pC 194, or pBD214 are suited for
transformation
of Bacillus species. Several plasmids of use in the transfer of genetic
information into
Corynebacterium include pHM 1519, pBL 1, pSA77, or pAJ667 (Pouwels et al.,
eds.
(1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).
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, Califomia (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.
In another embodiment, the MCT protein expression vector is a yeast expression
vector. Examples of vectors for expression in yeast S. cerevisiae include
pYepSecl
(Baldari, et al., (1987) Embo J. 6:229-234), 2 , pAG-1, Yep6, Yep13,
pEMBLYe23,
pMFa (Kurjan and Herskowitz, (1982) Ce1130:933-943), pJRY88 (Schultz et al.,
(1987)
Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). 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).
Alternatively, the MCT proteins of the invention can be expressed in insect
cells
using baculovirus expression vectors. Baculovirus vectors available for
expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al.
CA 02583703 2007-04-19
.'~
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(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers
(1989)
Virology 170:31-39).
In another embodiment, the MCT proteins of the invention may be expressed in
unicellular plant cells (such as algae) or in plant cells from higher plants
(e.g., the
spermatophytes, such as crop plants). Examples of plant expression vectors
include
those detailed in: Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992)
"New
plant binary vectors with selectable markers located proximal to the left
border", Plant
Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors
for
plant transformation", 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).
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,
NY,
1989.
In another embodiment, the recombinant mammalian expression vector is
capable of directing expression of the nucleic acid preferentially in a
particular cell type
(e.g., tissue-specific regulatory elements are used to express the nucleic
acid). Tissue-
specific regulatory elements are known in the art. Non-limiting examples of
suitable
tissue-specific promoters include the albumin promoter (liver-specific;
Pinkert et al.
(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988)
Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto
and
Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983)
Cell
33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific
promoters
(e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),
CA 02583703 2007-04-19
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pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and
mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Patent 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 a-fetoprotein promoter (Campes and Tilghman
(1989)
Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a
DNA molecule of the invention cloned into the expression vector in an
antisense
orientation. That is, the DNA molecule is operatively linked to a regulatory
sequence in
a manner which allows for expression (by transcription of the DNA molecule) of
an
RNA molecule which is antisense to MCT mRNA. Regulatory sequences operatively
linked to a nucleic acid cloned in the antisense orientation can be chosen
which direct
the continuous expression of the antisense RNA molecule in a variety of cell
types, for
instance viral promoters and/or enhancers, or regulatory sequences can be
chosen which
direct constitutive, tissue, specific or cell type specific expression of
antisense RNA.
The antisense expression vector can be in the form of a recombinant plasmid,
phagemid
or attenuated virus in which antisense nucleic acids are produced under the
control of a
high efficiency regulatory region, the activity ofwhich 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.
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.
A host cell can be any prokaryotic or eukaryotic cell. For example, an MCT
protein can be expressed in bacterial cells such as C. glutamicum, insect
cells, yeast or
mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
Other
CA 02583703 2007-04-19
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suitable host cells are known to one of ordinary skill in the art.
Microorganisms related
to Corynebacterium glutamicum which may be conveniently used as host cells for
the
nucleic acid and protein molecules of the invention are set forth in Table 3.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation or transfection techniques. As used herein, the
terms
"transformation" and "transfection", "conjugation" and "transduction" are
intended to
refer to a variety of art-recognized techniques for introducing foreign
nucleic acid (e.g.,
linear DNA or RNA (e.g., a linearized vector or a gene construct alone without
a vector)
or nucleic acid in the form of a vector (e.g., a plasmid, phage, phasmid,
phagemid,
transposon or other DNA) into a host cell, including calcium phosphate or
calcium
chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection,
natural
competence, chemical-mediated transfer, or electroporation. Suitable methods
for
transforming or transfecting host cells can be found in Sambrook, et al.
(Molecular
Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory
manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may
integrate the foreign DNA into their genome. In order to identify and select
these
integrants, a gene that encodes a selectable marker (e.g., resistance to
antibiotics) is
generally introduced into the host cells along with the gene of interest.
Preferred
selectable markers include those which confer resistance to drugs, such as
G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be
introduced into a host cell on the same vector as that encoding an MCT protein
or can be
introduced on a separate vector. Cells stably transfected with the introduced
nucleic
acid can be identified by, for example, drug selection (e.g., cells that have
incorporated
the selectable marker gene will survive, while the other cells die).
To create a homologous recombinant microorganism, a vector is prepared which
contains at least a portion of an MCT gene into which a deletion, addition or
substitution
has been introduced to thereby alter, e.g., functionally disrupt, the MCT
gene.
Preferably, this MCT gene is a Corynebacterium glutamicum MCT gene, but it can
be a
homologue from a related bacterium or even from a mammalian, yeast, or insect
source.
In a preferred embodiment, the vector is designed such that, upon homologous
CA 02583703 2007-04-19
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recombination, the endogenous MCT gene is functionally disrupted (i.e., no
longer
encodes a functional protein; also referred to as a "knock out" vector).
Alternatively,
the vector can be designed such that, upon homologous recombination, the
endogenous
MCT gene is mutated or otherwise altered but still encodes functional protein
(e.g., the
upstream regulatory region can be altered to thereby alter the expression of
the
endogenous MCT protein). In the homologous recombination vector, the altered
portion
of the MCT gene is flanked at its 5' and 3' ends by additional nucleic acid of
the MCT
gene to allow for homologous recombination to occur between the exogenous MCT
gene carried by the vector and an endogenous MCT gene in a microorganism. The
additional flanking MCT nucleic acid is of sufficient length for successful
homologous
recombination with the endogenous gene. Typically, several kilobases of
flanking DNA
(both at the 5' and 3' ends) are included in the vector (see e.g., Thomas,
K.R., and
Capecchi, M.R. (1987) Cell 51: 503 for a description of homologous
recombination
vectors). The vector is introduced into a microorganism (e.g., by
electroporation) and
cells in which the introduced MCT gene has homologously recombined with the
endogenous MCT gene are selected, using art-known techniques.
In another embodiment, recombinant microorganisms can be produced which
contain selected systems which allow for regulated expression of the
introduced gene.
For example, inclusion of an MCT gene on a vector placing it under control of
the lac
operon permits expression of the MCT gene only in the presence of IPTG. Such
regulatory systems are well known in the art.
In another embodiment, an endogenous MCT gene in a host cell is disrupted
(e.g., by homologous recombination or other genetic means known in the art)
such that
expression of its protein product does not occur. In another embodiment, an
endogenous
or introduced MCT gene in a host cell has been altered by one or more point
mutations,
deletions, or inversions, but still encodes a functional MCT protein. In still
another
embodiment, one or more of the regulatory regions (e.g., a promoter,
repressor, or
inducer) of an MCT gene in a microorganism has been altered (e.g., by
deletion,
truncation, inversion, or point mutation) such that the expression of the MCT
gene is
modulated. One of ordinary skill in the art will appreciate that host cells
containing
more than one of the described MCT gene and protein modifications may be
readily
CA 02583703 2007-04-19
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produced using the methods of the invention, and are meant to be included in
the present
invention.
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture, can be used to produce (i. e., express) an MCT protein. Accordingly,
the
invention further provides methods for producing MCT proteins using the host
cells of
the invention. In one embodiment, the method comprises culturing the host cell
of
invention (into which a recombinant expression vector encoding an MCT protein
has
been introduced, or into which genome has been introduced a gene encoding a
wild-type
or altered MCT protein) in a suitable medium until MCT protein is produced. In
another
embodiment, the method further comprises isolating MCT proteins from the
medium or
the host cell.
C. Isolated MCT Proteins
Another aspect of the invention pertains to isolated MCT proteins, and
biologically active portions thereof. An "isolated" or "purified" protein or
biologically
active portion thereof is substantially free of cellular material when
produced by
recombinant DNA techniques, or chemical precursors or other chemicals when
chemically synthesized. The language "substantially free of cellular material"
includes
preparations of MCT protein in which the protein is separated from cellular
components
of the cells in which it is naturally or recombinantly produced. In one
embodiment, the
language "substantially free of cellular material" includes preparations of
MCT protein
having less than about 30% (by dry weight) of non-MCT protein (also referred
to herein
as a "contaminating protein"), more preferably less than about 20% of non-MCT
protein, still more preferably less than about 10% of non-MCT protein, and
most
preferably less than about 5% non-MCT protein. When the MCT protein or
biologically
active portion thereof is recombinantly produced, it is also preferably
substantially free
of culture medium, i.e., culture medium represents less than about 20%, more
preferably
less than about 10%, and most preferably less than about 5% of the volume of
the
protein preparation. The language "substantially free of chemical precursors
or other
chemicals" includes preparations of MCT protein in which the protein is
separated from
chemical precursors or other chemicals which are involved in the synthesis of
the
protein. In one embodiment, the language "substantially free of chemical
precursors or
CA 02583703 2007-04-19
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other chemicals" includes preparations of MCT protein having less than about
30% (by
dry weight) of chemical precursors or non-MCT chemicals, more preferably less
than
about 20% chemical precursors or non-MCT chemicals, still more preferably less
than
about 10% chemical precursors or non-MCT chemicals, and most preferably less
than
about 5% chemical precursors or non-MCT chemicals. In preferred embodiments,
isolated proteins or biologically active portions thereof lack contaminating
proteins from
the same organism from which the MCT protein is derived. Typically, such
proteins are
produced by recombinant expression of, for example, a C. glutamicum MCT
protein in a
microorganism such as C. glutamicum.
An isolated MCT protein or a portion thereof of the invention can participate
in
the metabolism of compounds necessary for the construction of cellular
membranes in
C. glutamicum, or in the transport of molecules across these membranes, or has
one or
more of the activities set forth in Table 1. In preferred embodiments, the
protein or
portion thereof comprises an amino acid sequence which is sufficiently
homologous to
an amino acid sequence of the invention (e.g., a sequence of an even-numbered
SEQ ID
NO: of the Sequence Listing) such that the protein or portion thereof
maintains the
ability participate in the metabolism of compounds necessary for the
construction of
cellular membranes in C. glutamicum, or in the transport of molecules across
these
membranes. The portion of the protein is preferably a biologically active
portion as
described herein. In another preferred embodiment, an MCT protein of the
invention
has an amino acid sequence set forth as an even-numbered SEQ ID NO: of the
Sequence
Listing.. In yet another preferred embodiment, the MCT protein has an amino
acid
sequence which is encoded by a nucleotide sequence which hybridizes, e.g.,
hybridizes
under stringent conditions, to a nucleotide sequence of the invention (e.g., a
sequence of
an odd-numbered SEQ ID NO: of the Sequence Listing). In still another
preferred
embodiment, the MCT protein has an amino acid sequence which is encoded by a
nucleotide sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%,
58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%,
68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%,
77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%,
or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%,
98%,
99% or more homologous to one of the nucleic acid sequences of the invention,
or a
CA 02583703 2007-04-19
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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
prefen:ed MCT proteins of the present invention also preferably possess at
least one of
the MCT activities described herein. For example, a preferred MCT protein of
the
present invention includes an amino acid sequence encoded by a nucleotide
sequence
which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide
sequence of
the invention, and which can participate in the metabolism of compounds
necessary for
the construction of cellular membranes in C. glutamicum, or in the transport
of
molecules across these membranes, or which has one or more of the activities
set forth
in Table 1.
In other embodiments, the MCT protein is substantially homologous to an amino
acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID
NO: of
the Sequence Listing) and retains the functional activity of the protein of
one of the
amino acid sequences of the invention yet differs in amino acid sequence due
to natural
variation or mutagenesis, as described in detail in subsection I above.
Accordingly, in
another embodiment, the MCT protein is a protein which comprises an amino acid
sequence which is at least about 50%,.51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,
69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or
91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%,
98%,
99% or more homologous to an entire amino acid sequence of the invention and
which
has at least one of the MCT activities described herein. Ranges and identity
values
intermediate to the above-recited values, (e.g., 70-90% identical or 80-95%
identical)
are also intended to be encompassed by the present invention. For example,
ranges of
identity values using a combination of any of the above values recited as
upper and/or
lower limits are intended to be included. In another embodiment, the invention
pertains
to a full length C. glutamicum protein which is substantially homologous to an
entire
amino acid sequence of the invention.
CA 02583703 2007-04-19
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Biologically active portions of an MCT protein include peptides comprising
amino acid sequences derived from the amino acid sequence of an MCT protein,
e.g.,
the an amino acid sequence of an even-numbered SEQ ID NO: of the Sequence
Listing
or the amino acid sequence of a protein homologous to an MCT protein, which
include
fewer amino acids than a full length MCT protein or the full length protein
which is
homologous to an MCT protein, and exhibit at least one activity of an MCT
protein.
Typically, biologically active portions (peptides, e.g., peptides which are,
for example,
5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in
length) comprise
a domain or motif with at least one activity of an MCT protein. Moreover,
other
biologically active portions, in which other regions of the protein are
deleted, can be
prepared by recombinant techniques and evaluated for one or more of the
activities
described herein. Preferably, the biologically active portions of an MCT
protein include
one or more selected domains/motifs or portions thereof having biological
activity.
MCT proteins are preferably produced by recombinant DNA techniques. For
example, a nucleic acid molecule encoding the protein is cloned into an
expression
vector (as described above), the expression vector is introduced into a host
cell (as
described above) and the MCT protein is expressed in the host cell. The MCT
protein
can then be isolated from the cells by an appropriate purification scheme
using standard
protein purification techniques. Alternative to recombinant expression, an MCT
protein,
polypeptide, or peptide can be synthesized chemically using standard peptide
synthesis
techniques. Moreover, native MCT protein can be isolated from cells (e.g.,
endothelial
cells), for example using an anti-MCT antibody, which can be produced by
standard
techniques utilizing an MCT protein or fragment thereof of this invention.
The invention also provides MCT chimeric or fusion proteins. As used herein,
an MCT "chimeric protein" or "fusion protein" comprises an MCT polypeptide
operatively linked to a non-MCT polypeptide. An "MCT polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to an MCT protein,
whereas
a "non-MCT polypeptide" refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous to the MCT
protein,
e.g., a protein which is different from the MCT protein and which is derived
from the
same or a different organism. Within the fusion protein, the term "operatively
linked" is
intended to indicate that the MCT polypeptide and the non-MCT polypeptide are
fused
CA 02583703 2007-04-19
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in-frame to each other. The non-MCT polypeptide can be fused to the N-terminus
or C-
terminus of the MCT polypeptide. For example, in one embodiment the fusion
protein
is a GST-MCT fusion protein in which the MCT sequences are fused to the C-
terminus
of the GST sequences. Such fusion proteins can facilitate the purification of
recombinant MCT proteins. In another embodiment, the fusion protein is an MCT
protein containing a heterologous signal sequence at its N-terminus. In
certain host cells
(e.g., mammalian host cells), expression and/or secretion of an MCT protein
can be
increased through use of a heterologous signal sequence.
Preferably, an MCT chimeric or fusion protein of the invention is produced by
standard recombinant DNA techniques. For example, DNA fragments coding for the
different polypeptide sequences are ligated together in-frame in accordance
with
conventional techniques, for example by employing blunt-ended or stagger-ended
termini for ligation, restriction enzyme digestion to provide for appropriate
termini,
filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to
avoid
undesirable joining, and enzymatic ligation. In another embodiment, the fusion
gene
can be synthesized by conventional techniques including automated DNA
synthesizers.
Altematively, PCR amplification of gene fragments can be carried out using
anchor
primers which give rise to complementary overhangs between two consecutive
gene
fragments which can subsequently be annealed and reamplified to generate a
chimeric
gene sequence (see, for example, Current Protocols in Molecular Biology, eds.
Ausubel
et al. John Wiley & Sons: 1992). Moreover, many expression vectors are
commercially
available that already encode a fusion moiety (e.g., a GST polypeptide). An
MCT-
encoding nucleic acid can be cloned into such an expression vector such that
the fusion
moiety is linked in-frame to the MCT protein.
Homologues of the MCT protein can be generated by mutagenesis, e.g., discrete
point mutation or truncation of the MCT protein. As used herein, the term
"homologue"
refers to a variant form of the MCT protein which acts as an agonist or
antagonist of the
activity of the MCT protein. An agonist of the MCT protein can retain
substantially the
same, or a subset, of the biological activities of the MCT protein. An
antagonist of the
MCT protein can inhibit one or more of the activities of the naturally
occurring form of
the MCT protein, by, for example, competitively binding to a downstream or
upstream
member of the cell membrane component metabolic cascade which includes the MCT
CA 02583703 2007-04-19
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-51-
protein, or by binding to an MCT protein which mediates transport of compounds
across
such membranes, thereby preventing translocation from taking place.
In an al.temative embodiment, homologues of the MCT protein can be identified
by screening combinatorial libraries of mutants, e.g., truncation mutants, of
the MCT
protein for MCT protein agonist or antagonist activity. In one embodiment, a
variegated
library of MCT variants is generated by combinatorial mutagenesis at the
nucleic acid
level and is encoded by a variegated gene library. A variegated library of MCT
variants
can be produced by, for example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of potential
MCT
sequences is expressible as individual polypeptides, or alternatively, as a
set of larger
fusion proteins (e.g., for phage display) containing the set of MCT sequences
therein.
There are a variety of methods which can be used to produce libraries of
potential MCT
homologues from a degenerate oligonucleotide sequence. Chemical synthesis of a
degenerate gene sequence can be performed in an automatic DNA synthesizer, and
the
synthetic gene then ligated into an appropriate expression vector. Use of a
degenerate
set of genes allows for the provision, in one mixture, of all of the sequences
encoding
the desired set of potential MCT sequences. Methods for synthesizing
degenerate
oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983)
Tetrahedron 39:3;
Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984)
Science
198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
In addition, libraries of fragments of the MCT protein coding can be used to
generate a variegated population of MCT fragments for screening and subsequent
selection of homologues of an MCT protein. In one embodiment, a library of
coding
sequence fragments can be generated by treating a double stranded PCR fragment
of an
MCT coding sequence with a nuclease under conditions wherein nicking occurs
only
about once per molecule, denaturing the double stranded DNA, renaturing the
DNA to
form double stranded DNA which can include sense/antisense pairs from
different
nicked products, removing single stranded portions from reformed duplexes by
treatment with S 1 nuclease, and ligating the resulting fragment library into
an expression
vector. By this method, an expression library can be derived which encodes N-
terminal,
C-terminal and internal fragments of various sizes of the MCT protein.
CA 02583703 2007-04-19
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Several techniques are known in the art for screening gene products of
combinatorial libraries made by point mutations or truncation, and for
screening cDNA
libraries for gene products having a selected property. Such techniques are
adaptable for
rapid screening of the gene libraries generated by the combinatorial
mutagenesis of
MCT homologues. The most widely used techniques, which are amenable to high
through-put analysis, for screening large gene libraries typically include
cloning the
gene library into replicable expression vectors, transforming appropriate
cells with the
resulting library of vectors, and expressing the combinatorial genes under
conditions in
which detection of a desired activity facilitates isolation of the vector
encoding the gene
whose product was detected. Recursive ensemble mutagenesis (REM), a new
technique
which enhances the frequency of functional mutants in the libraries, can be
used in
combination with the screening assays to identify MCT homologues (Arkin and
Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering
6(3):327-331).
In another embodiment, cell based assays can be exploited to analyze a
variegated MCT library, using methods well known in the art.
D. Uses and Methods of the Invention
The nucleic acid molecules, proteins, protein homologues, fusion proteins,
primers, vectors, and host cells described herein can be used in one or more
of the
following methods: identification of C. glutamicum and related organisms;
mapping of
genomes of organisms related to C. glutamicum; identification and localization
of C.
glutamicum sequences of interest; evolutionary studies; determination of MCT
protein
regions required for function; modulation of an MCT protein activity;
modulation of the
metabolism of one or more cell membrane components; modulation of the
transmembrane transport of one or more compounds; and modulation of cellular
production of a desired compound, such as a fine chemical.
The MCT nucleic acid molecules of the invention have a variety of uses. First,
they may be used to identify an organism as being Corynebacterium glutamicum
or a
close relative thereof. Also, they may be used to identify the presence of C.
glutamicum
or a relative thereof in a mixed population of microorganisms. The invention
provides
the nucleic acid sequences of a number of C. glutamicum genes; by probing the
CA 02583703 2007-04-19
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extracted genomic DNA of a culture of a unique or mixed population of
microorganisms
under stringent conditions with a probe spanning a region of a C. glutamicum
gene
which is unique to this organism, one can ascertain whether this organism is
present.
Although Corynebacterium glutamicum itself is nonpathogenic, it is related to
pathogenic species, such as Corynebacterium diphtheriae. Corynebacterium
diphtheriae
is the causative agent of diphtheria, a rapidly developing, acute, febrile
infection which
involves both local and systemic pathology. In this disease, a local lesion
develops in
the upper respiratory tract and involves necrotic injury to epithelial cells;
the bacilli
secrete toxin which is disseminated through this lesion to distal susceptible
tissues of the
body. Degenerative changes brought about by the inhibition of protein
synthesis in
these tissues, which include heart, muscle, peripheral nerves, adrenals,
kidneys, liver and
spleen, result in the systemic pathology of the disease. Diphtheria continues
to have
high incidence in many parts of the world, including Africa, Asia, Eastern
Europe and
the independent states of the former Soviet Union. An ongoing epidemic of
diphtheria
in the latter two regions has resulted in at least 5,000 deaths since 1990.
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 as odd-numbered or even-numbered SEQ ID NOs, respectively,
in
the Sequence Listing) 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.
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
CA 02583703 2007-04-19
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localization of the fragment to the genome map of C. glutamicum, and, when
performed
multiple times with different enzymes, facilitates a rapid detennination 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.
The MCT nucleic acid molecules of the invention are also useful for
evolutionary and protein structural studies. The metabolic and transport
processes in
which the molecules of the invention participate are utilized by a wide
variety of
prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic
acid
molecules of the present invention to those encoding similar enzymes from
other
organisms, the evolutionary relatedness of the organisms can be assessed.
Similarly,
such a comparison permits an assessment of which regions of the sequence are
conserved and which are not, which may aid in determining those regions of the
protein
which are essential for the functioning of the enzyme. This type of
determination is of
value for protein engineering studies and may give an indication of what the
protein can
tolerate in terms of mutagenesis without losing function.
Manipulation of the MCT nucleic acid molecules of the invention may result in
the production of MCT proteins having functional differences from the wild-
type MCT
proteins. These proteins may be improved in efficiency or activity, may be
present in
greater numbers in the cell than is usual, or may be decreased in efficiency
or activity.
The invention provides methods for screening molecules which modulate the
activity of an MCT protein, either by interacting with the protein itself or a
substrate or
binding partner of the MCT protein, or by modulating the transcription or
translation of
an MCT nucleic acid molecule of the invention. In such methods, a
microorganism
expressing one or more MCT proteins of the invention is contacted with one or
more test
compounds, and the effect of each test compound on the activity or level of
expression
of the MCT protein is assessed.
There are a number of mechanisms by which the alteration of an MCT protein of
the invention may directly affect the yield, production, and/or efficiency of
production
of a fine chemical from a C. glutamicum strain incorporating such an altered
protein.
Recovery of fine chemical compounds from large-scale cultures of C. glulamicum
is
CA 02583703 2007-04-19
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significantly improved if C. glutamicum secretes the desired compounds, since
such
compounds may be readily purified from the culture medium (as opposed to
extracted
from the mass of C. glutamicum cells). By either increasing the number or the
activity
of transporter molecules which export fine chemicals from the cell, it may be
possible to
increase the amount of the produced fine chemical which is present in the
extracellular
medium, thus permitting greater ease of harvesting and purification.
Conversely, in
order to efficiently overproduce one or more fine chemicals, increased amounts
of the
cofactors, precursor molecules, and intermediate compounds for the appropriate
biosynthetic pathways are required. Therefore, by increasing the number and/or
activity
of transporter proteins involved in the import of nutrients, such as carbon
sources (i.e.,
sugars), nitrogen sources (i.e., amino acids, ammonium salts), phosphate, and
sulfur, it
may be possible to improve the production of a fine chemical, due to the
removal of any
nutrient supply limitations on the biosynthetic process. Further, fatty acids
and lipids
are themselves desirable fine chemicals, so by optimizing the activity or
increasing the
number of one or more MCT proteins of the invention which participate in the
biosynthesis of these compounds, or by impairing the activity of one or more
MCT
proteins which are involved in the degradation of these compounds, it may be
possible
to increase the yield, production, and/or efficiency of production of fatty
acid and lipid
molecules from C. glutamicum.
The engineering of one or more MCT genes of the invention may also result in
MCT proteins having altered activities which indirectly impact the production
of one or
more desired fine chemicals from C.glutamicum. For example, the normal
biochemical
processes of metabolism result in the production of a variety of waste
products (e.g.,
hydrogen peroxide and other reactive oxygen species) which may actively
interfere with
these same metabolic processes (for example, peroxynitrite is known to nitrate
tyrosine
side chains, thereby inactivating some enzymes having tyrosine in the active
site
(Groves, J.T. (1999) Curr. Opin. Chem. Biol. 3(2): 226-235). While these waste
products are typically excreted, the C. glutamicum strains utilized for large-
scale
fermentative production are optimized for the overproduction of one or more
fine
chemicals, and thus may produce more waste products than is typical for a wild-
type C.
glutamicum. By optimizing the activity of one or more MCT proteins of the
invention
which are involved in the export of waste molecules, it may be possible to
improve the
CA 02583703 2007-04-19
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viability of the cell and to maintain efficient metabolic activity. Also, the
presence of
high intracellular levels of the desired fine chemical may actually be toxic
to the cell, so
by increasing the ability of the cell to secrete these compounds, one may
improve the
viability of the cell.
Further, the MCT proteins of the invention may be manipulated such that the
relative amounts of various lipid and fatty acid molecules produced are
altered. This
may have a profound effect on the lipid composition of the membrane of the
cell. Since
each type of lipid has different physical properties, an alteration in the
lipid composition
of a membrane may significantly alter membrane fluidity. Changes in membrane
fluidity
can impact the transport of molecules across the membrane, which, as
previously
explicated, may modify the export of waste products or the produced fine
chemical or
the import of necessary nutrients. Such membrane fluidity changes may also
profoundly
affect the integrity of the cell; cells with relatively weaker membranes are
more
vulnerable in the large-scale fermentor environment to mechanical stresses
which may
damage or kill the cell. By manipulating MCT proteins involved in the
production of
fatty acids and lipids for membrane construction such that the resulting
membrane has a
membrane composition more amenable to the environmental conditions extant in
the
cultures utilized to produce fine chemicals, a greater proportion of the C.
glutamicum
cells should survive and multiply. Greater numbers of C. glutamicum cells in a
culture
should translate into greater yields, production, or efficiency of production
of the fine
chemical from the culture.
The aforementioned mutagenesis strategies for MCT proteins to result in
increased yields of a fine chemical from C. glutamicum are not meant to be
limiting;
variations on these strategies will be readily apparent to one of ordinary
skill in the art.
Using such strategies, and incorporating the mechanisms disclosed herein, the
nucleic
acid and protein molecules of the invention may be utilized to generate C.
glutamicum
or related strains of bacteria expressing mutated MCT nucleic acid and protein
molecules such that the yield, production, and/or efficiency of production of
a desired
compound is improved. This desired compound may be any natural product of C.
glutamicum, which includes the final products of biosynthesis pathways and
intermediates of naturally-occurring metabolic pathways, as well as molecules
which do
CA 02583703 2007-04-19
$7 -
not naturally occur in the metabolism of C. glutamicum, but which are produced
by a C.
glutamicum strain of the invention.
This invention is further illustrated by the following examples which should
not
be construed as limiting.
TABLE 1: GENES IN THE APPLICATION
Nucleic Acid Amino Acid Identification Code Contig. NT Start NT Stop Function
SEQ ID NO SEQ ID NO
1 2 RXN03097 W0062 3 557 AMMONIUM TRANSPORT SYSTEM
3 4 RXA02099 GR00630 6198 6470 AMMONIUM TRANSPORT SYSTEM
6 RXA00104 GR00014 15895 16650 CYSQ PROTEIN, ammonium transport protein
Polyketide Synthesis
Nucleic Acid Amino Acid Identification Code Conti9. NT Start NT Stop Function
SEQ ID NO SEQ ID NO
7 8 RXA01420 GR00416 775 17 4"-MYCAROSYL ISOVALERYL-COA TRANSFERASE (EC 2.-.-.-
)
9 10 RXN02581 W0098 30482 28623 POLYKETIDE SYNTHASE
11 12 F RXA02581 GR00741 1 1527 POLYKETIDE SYNTHASE
13 14 RXA02582 GR00741 1890 6719 PROBABLE POLYKETIDE SYNTHASE CY338.20 N
16 RXA01138 GR00318 1656 2072 ACTINORHODIN POLYKETIDE DIMERASE (EC -: .-.-)
17 18 RXA01980 GR00573 1470 838 POLYKETIDE CYCLASE w
.3
19 20 RXN01007 W0021 2572 866 FRNA 0
21 22 RXN00784 W0103 27531 28265 FRNE w
Fatty acid and lipid synthesis O
Nucleic Acid Amino Acid Identification Code Conti9. NT Start NT Stop Function
SEQ ID NO SEQ ID NO
23 24 RXA02335 GR00672 550 2322 BIOTIN CARBOXYLASE (EC 6.3.4.14)
26 RXA02173 GR00641 7473 8924 ACETYL-COENZYME A CARBOXYLASE CARBOXYL
TRANSFERASE SUBUNIT
BETA (EC 6.4.1.2)
27 28 RXA01764 GR00500 2178 3110 3-OXOACYL-(ACYL-CARRIER PROTEINJ REDUCTASE
(EC 1.1.1.100)
29 30 RXN02487 W0007 6367 4664 LONG-CHAIN-FATTY-ACID-COA LIGASE (EC 6.2.1.3)
31 32 F RXA02487 GR00718 4937 4650 LONG-CHAIN-FATTY-ACID--COA LIGASE (EC
6.2.1.3)
33 34 F RXA02490 GR00720 817 5 LONG-CHAtN-FATTY-ACID--COA LIGASE (EC 6.2.1.3)
36 RXA01467 GR00422 920 1210 ACYL CARRIER PROTEIN
37 38 RXA00796 GR00212 202 5 Acyl carrier protein phosphodiesterase
39 40 RXA01897 GR00544 617 1159 Acyl carrier protein phosphodiesterase
41 42 RXN02809 W0342 380 6 Acyl carrier protein phosphodiesterase
43 44 F RXA02809 GR00790 277 5 Acyl carrier protein phosphodiesterase
46 RXN00113 W0129 103 5724 FATTY ACID SYNTHASE (EC 2.3.1.85) [INCLUDES: EC
2.3.1.38; EC 2.3.1.39; EC
2.3.1.41;
47 48 F RXA00113 GR00017 2 3295 FATTY-ACID SYNTHASE (EC 2.3.1.85)
Table I (continued)
Nucleic Acid Amino Acid Identification Code Conti9. NT Start NT Stop Function
SEQ ID NO SEQ ID NO
49 50 RXN03111 W0084 6040 5 FATTY ACID SYNTHASE (EC 2.3.1.85) [INCLUDES: EC
2.3.1.38; EC 2.3.1.39; EC
2.3.1.41; EC 1.1.1.100; EC 4.2.1.61; EC 1.3.1.10; EC 3.1.2.141
51 52 F RXA00158 GR00024 2088 4 FATTY ACID SYNTHASE (EC 2.3.1.85)
53 54 F RXA00572 GR00155 2 3832 FATTY ACID SYNTHASE (EC 2.3.1.85)
55 56 RXA02582 GR00741 1890 6719 PROBABLE POLYKETIDE SYNTHASE CY338.20
57 58 RXA02691 GR00754 15347 14541 FATTY ACYL RESPONSIVE REGULATOR
59 60 RXA00880 GR00242 6213 8057 LONG-CHAIN-FATTY-ACID-COA LIGASE (EC 6.2.1.3)
61 62 RXA01060 GR00296 9566 10489 OMEGA-3 FATTY ACID DESATURASE (EC 1.14.99 -)
63 64 RXN01722 W0036 2938 1214 MEDIUM-CHAIN-FATTY-ACID-COA LIGASE (EC 6.2.1.-)
65 66 F RXA01722 GR00488 5746 4022 MEDIUM-CHAIN-FATTY-ACID-COA LIGASE (EC
6.2.1.-)
67 68 RXA01644 GR00456 9854 8577 CYCLOPROPANE-FATTY-ACYL-PHOSPHOLIPID SYNTHASE
(EC 2.1.1.79)
69 70 RXA02029 GRO0618 356 1669 CYCLOPROPANE-FATTY-ACYL-PHOSPHOLIPID SYNTHASE
(EC 2.1.1.79)
71 72 RXA01801 GR00509 3396 2380 ENOYL-COA HYDRATASE (EC 4.2.1.17)
73 74 RXN02512 W0171 16147 15185 LIPID A BIOSYNTHESIS LAUROYL ACYLTRANSFERASE
(EC 2.3.1.-)
75 76 F RXA02512 GR00721 3303 4259 LIPID A BIOSYNTHESIS LAUROYL
ACYLTRANSFERASE (EC 2.3.1 -)
77 78 RXA00899 GR00245 1599 2864 CARDIOLIPIN SYNTHETASE (EC 2.7.8.-)
79 80 RXN00819 W0054 18127 19455 ACYL-COA DEHYDROGENASE (EC 1.3.99.-)
o
81 82 F RXA00819 GR00221 18 1007 ACYL-COA DEHYDROGENASE (EC 1.3.99.-) Ln
83 84 F RXA01766 GR00500 4081 4371 ACYL-COA DEHYDROGENASE (EC 1.3.99.-) 00
85 86 RXN01762 W0054 15318 13783 LONG-CHAIN-FATTY-ACID-COA LIGASE (EC 6.2.1.3)
~
87 88 F RXA01762 GR00500 1272 10 LONG-CHAIN-FATTY-ACID-COA LIGASE (EC 6.2.1.3)
- W 0
89 90 RXA00681 GR00179 3405 2662 3-OXOACYL4ACYL-CARRIER PROTEIN) REDUCTASE (EC
1.1.1.100)
91 92 RXA00802 GR00214 3803 4516 3-OXOACYL4ACYL-CARRIER PROTEIN] REDUCTASE (EC
1.1.1.100)
93 94 RXA02133 GR00639 3 308 3-OXOACYL-[ACYL-CARRIER PROTEIN] REDUCTASE (EC
1.1.1.100) ~p o
95 96 RXN01114 W0182 9118 10341 3-KETOACYL-COA THIOLASE (EC 2.3.1.16) ~ = -.3
97 98 F RXA01114 GR00308 2 793 3-KETOACYL-COA THIOLASE (EC 2.3.1.16)
99 100 RXA01894 GR00542 1622 2476 PHOSPHATIDATE CYTIDYLYLTRANSFERASE (EC
2.7.7.41)
101 102 RXA02599 GR00742 3179 3655 PHOSPHATIDYLGLYCEROPHOSPHATASE B (EC
3.1.3.27)
103 104 RXN02638 W0098 54531 53656 1-ACYL-SN-GLYCEROL-3-PHOSPHATE
ACYLTRANSFERASE (EC 2.3.1.51) 1O
105 106 F RXA02638 GR00749 8 511 1-ACYLSN-GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE
(EC 2.3.1.51)
107 108 RXA00856 GR00232 720 1256 CDP-DIACYLGLYCEROL-GLYCEROL-3-PHOSPHATE 3-
PHOSPHATIDYLTRANSFERASE (EC 2.7.8.5)
109 110 RXA02511 GR00721 2621 3277 CDP-DIACYLGLYCEROL--GLYCEROL-3-PHOSPHATE 3-
PHOSPHATIDYLTRANSFERASE (EC 2.7.8.5)
111 112 RXN02836 W0102 32818 33372 KETOACYL REDUCTASE HETN (EC 1.3.1.-)
113 114 F RXA02836 GR00827 106 411 KETOACYL REDUCTASE HETN (EC 1.3.1.-)
115 116 RXA02578 GR00740 2438 3541 PUTATIVE ACYLTRANSFERASE
117 118 RXA02150 GR00639 18858 19658 1-ACYLSN-GLYCEROL-3-PHOSPHATE
ACYLTRANSFERASE (EC 2.3.1.51)
119 120 RXA00607 GR00160 1869 2249 POLY(3-HYDROXYALKANOATE) POLYMERASE (EC
2.3.1.-)
121 122 RXA02397 GR00698 1688 2683 POLY-BETA-HYDROXYBUTYRATE POLYMERASE (EC
2.3.1.-)
123 124 RXN03110 W0083 16568 17929 HYDROXYACYLGLUTATHIONE HYDROLASE (EC
3.1.2.6)
125 126 F RXA00660 GR00171 1027 5 HYDROXYACYLGLUTATHIONE HYDROLASE (EC
3.1.2.6)
127 128 RXA00801 GR00214 3138 3770 HYDROXYACYLGLUTATHIONE HYDROLASE (EC
3.1.2.6)
129 130 RXA00821 GR00221 1469 2311 HYDROXYACYLGLUTATHIONE HYDROLASE (EC
3.1.2.6)
131 132 RXN02966 VV0143 12056 13462 HYDROXYACYLGLUTATHIONE HYDROLASE (EC
3.1.2.6)
133 134 F RXA01833 GR00517 1666 260 HYDROXYACYLGLUTATHIONE HYDROLASE (EC
3.1.2.6)
135 136 RXA01853 GR00525 5561 5010 HYDROXYACYLGLUTATHIONE HYDROLASE (EC
3.1.2.6)
Nucleic Acid Amino Acid Identification Code Cont . Table I (continued)
NT Start NT Stop Function
SEQ ID NO SEQ ID NO
137 138 RXN02424 W0116 10570 11169 HYDROXYACYLGLUTATHIONE HYDROLASE (EC
3.1.2.6)
139 140 F RXA02424 GR00706 808 428 HYDROXYACYLGLUTATHIONE HYDROLASE (EC
3.1.2.6)
141 142 RXN00419 W0112 1024 266 ACETOACETYL-COA REDUCTASE (EC 1.1.1.36)
143 144 F RXA00419 GR00095 3 464 ACETOACETYL-COA REDUCTASE (EC 1.1.1.36)
145 146 F RXA00421 GR00096 565 723 ACETOACETYL-COA REDUCTASE (EC 1.1.1.36)
147 148 RXN02923 W0088 3301 2564 ACETOACETYL-COA REDUCTASE (EC 1.1.1.36)
149 150 RXN02922 W0321 11407 10328 ACYL-COA DEHYDROGENASE, SHORT-CHAIN
SPECIFIC (EC 1.3.99.2)
151 152 RXN03065 W0038 6237 6629 HOLO-[ACYL-CARRIER PROTEIN] SYNTHASE (EC
2.7.8.7)
153 154 RXN03132 W0127 39053 39472 POLY-BETA-HYDROXYBUTYRATE POLYMERASE (EC
2.3.1.-)
155 156 RXN03157 W0188 1607 1170 LIPOPOLYSACCHARIDE CORE BIOSYNTHESIS PROTEIN
KDTB
157 158 RXN00934 W0171 15181 14099 (AE000805) LPS biosynthesis RfbU related
protein [Methanobacterium
thermoautotrophicum)
159 160 RXN00792 W0321 10328 9132 ACYL-COA DEHYDROGENASE, SHORT-CHAIN SPECIFIC
(EC 1.3.99.2)
161 162 RXN00931 W0171 13011 12166 ACYL-COA THIOESTERASE II (EC 3.1.2.-)
163 164 F RXA00931 GR00253 4959 4114 thioesterase 11
165 166 RXN01421 W0122 16024 15638 ACYLTRANSFERASE (EC 2.3.1: )
167 168 RXN02342 W0078 3460 4266 BIOTIN-[ACETYL-COA-CARBOXYLASE] SYNTHETASE
(EC 6.3.4.15)
169 170 RXN00563 W0038 1 2739 FATTY ACID SYNTHASE (EC 2.3.1.85) [INCLUDES: EC
2.3.1.38; EC 2.3.1.39; EC t,n
2.3.1.41; EC 1.1.1.100; EC 4.2.1.61; EC 1.3.1.10; EC 3.1.2.141 D
171 172 RXN02168 W0100 2894 81 FATTY ACID SYNTHASE (EC 2,3.1.85) [INCLUDES: EC
2.3.1.38; EC 2.3.1.39; EC ~
2.3.1.41; EC 1.1.1.100; EC 4.2.1.61; EC 1.3.1.10; EC 3.1.2.14]
u'
173 174 RXN01090 W0155 6483 5686 KETOACYL REDUCTASE HETN (EC 1.3.1.-)
175 176 RXN02062 W0222 3159 1990 Lipopolysaccharide N-
acetylglucosaminy(transferase
177 178 RXN02148 W0300 16561 17703 Lipopolysaccharide N-
acetylglucosaminyltransferase p o
179 180 RXN02595 W0098 11098 9935 Lipopolysaccharide N-
acetylglucosaminyttransferase .3
181 182 RXS00148 W0167 9849 12059 METHYLMALONYL-COA MUTASE ALPHA-SUBUNIT (EC
5.4.99.2) o
183 184 RXS00149 W0167 7995 9842 METHYLMALONYL-COA MUTASE BETA-SUBUNIT (EC
5.4.99.2) P.
185 186 RXS02106 W0123 22649 21594 LIPOATE-PROTEIN LIGASE A (EC 6.-.-.-) 187
188 RXS01746 W0185 934 1686 LIPOATE-PROTEIN LIGASE B (EC 6.-.-.-) 1O
189 190 RXS01747 W0185 1826 2869 LIPOIC ACID SYNTHETASE
191 192 RXC01748 W0185 3001 3780 protein involved in lipid metabolism
193 194 RXC00354 W0135 33604 32792 Cytosolic Protein involved in lipid
metabolism
195 196 RXC01749 W0185 3953 5569 Membrane Spanning Protein involved in lipid
metabolism
Fatty acid degradation
NucleicAcid Amino Acid Identification Code Contig. NT Start NT Stop Function
SEQ ID NO SEQ ID NO
197 198 RXA02268 GR00655 2182 3081 LIPASE (EC 3.1.1.3)
199 200 RXA02269 GR00655 3094 4065 LIPASE (EC 3.1.1.3)
201 202 RXA01614 GR00449 8219 7197 LYSOPHOSPHOLIPASE L2 (EC 3.1.1.5)
203 204 RXA01983 GR00573 3559 3053 LIPASE (EC 3.1.1.3)
205 206 RXN02947 W0078 1319 6 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC
6.4.1.3)
207 208 F RXA02320 GR00667 593 6 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC
6.4.1.3)
209 210 F RXA02B51 GR00851 524 6 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC
6.4.1.3)
211 212 RXN02321 W0078 3291 1663 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC
6.4.1.3)
Table 1 (continued)
Nucleic Acid Amino Acid Identification Code Conti . NT Start NT Stop Function
SEQ ID NO SEQ ID NO
213 214 F RXA02321 GR00667 1380 937 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC
6.4.1.3)
215 216 F RXA02343 GR00675 1403 1816 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC
6.4.1.3)
217 218 F RXA02850 GR00850 2 493 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC
6.4.1.3)
219 220 RXA02583 GR00741 6743 8290 PROPIONYL-COA CARBOXYLASE BETA CHAIN (EC
6.4.1.3)
221 222 RXA00870 GR00239 809 2320 METHYLMALONATE-SEMIALDEHYDE DEHYDROGENASE
(ACYLATING) (EC
1.2.1.27) 2-Methyl-3-oxopropanoate:NAD+ oxidoreductase (CoA-propanoylating)
223 224 RXA01260 GR00367 2381 1200 LIPOAMIDE DEHYDROGENASE COMPONENT (E3) OF
BRANCHED-CHAIN
ALPHA-KETO ACID DEHYDROGENASE COMPLEX (EC 1.8.1.4)
225 226 RXA01261 GR00367 2607 2437 LIPOAMIDE DEHYDROGENASE COMPONENT (E3) OF
BRANCHED-CHAIN
ALPHA-KETO ACID DEHYDROGENASE COMPLEX (EC 1.8.1.4)
227 228 RXA01136 GR00318 685 1116 ISOVALERYL-COA DEHYDROGENASE (EC 1.3.99.10)
229 230 RXN00559 W0103 7568 6552 PROTEIN VDLD
231 232 F RXA00559 GR00149 218 6 PROTEIN VDLD
233 234 RXA01580 GR00440 707 6 Glycerophosphoryl diester phosphodiesterase
235 236 RXA02677 GR00754 3119 3877 GLYCEROPHOSPHORYL DIESTER PHOSPHODIESTERASE
(EC 3.1.4.46)
237 238 RXS01166 W0117 18142 16838 EXTRACELLULAR LIPASE PRECURSOR (EC 3.1.1.3)
0
U9
W
Terpenoid biosynthesis w
-.3
Nucleic Acid Amino Acid Identification Code Contig. NT Start NT Stop Function
w
SEQ ID NO SEQ ID NO
239 240 RXA00875 GR00241 2423 1857 ISOPENTENYL-DIPHOSPHATE DELTA-ISOMERASE (EC
5.3.3.2) C~ o
241 242 RXA01292 GR00373 1204 2388 PHYTOENE DEHYDROGENASE (EC 1.3.-.-) -.3
243 244 RXA01293 GR00373 2370 2696 PHYTOENE DEHYDROGENASE (EC 1.3.-.-) o
245 246 RXA02310 GR00665 1132 2394 GERANYLGERANYL HYDROGENASE
247 248 RXA02718 GR00758 18539 19585 GERANYLGERANYL PYROPHOSPHATE SYNTHASE (EC
2.5.1.1)
249 250 RXA01067 GR00298 1453 2181 undecaprenyl-diphosphate synthase (EC
2.5.1.31)
251 252 RXA01269 GR00367 20334 19894 UNDECAPRENYL-PHOSPHATE
GALACTOSEPHOSPHOTRANSFERASE (EC
2.7.8.6)
253 254 RXA01205 GR00346 3 533 PUTATIVE UNDECAPRENYL-PHOSPHATE ALPHA-N-
ACETYLGLUCOSAMINYLTRANSFERASE (EC 2.4.1.-)
255 256 RXA01576 GR00438 8053 8811 DOLICHYL-PHOSPHATE BETA-GLUCOSYLTRANSFERASE
(EC 2.4.1.117)
257 258 RXN02309 W0025 28493 29542 OCTAPRENYL-DIPHOSPHATE SYNTHASE (EC 2.5.1.-
)
259 260 F RXA02309 GR00665 978 4 OCTAPRENYL-DIPHOSPHATE SYNTHASE (EC 2.5.1.-)
261 262 RXN00477 W0086 38905 37262 PHYTOENE DEHYDROGENASE (EC 1.3.-.-)
263 264 F RXA00477 GR00119 13187 11544 PHYTOENE DEHYDROGENASE (EC 1.3.-.-)
265 266 RXA00478 GR00119 14020 13190 PHYTOENE SYNTHASE (EC 2.5.1.-)
267 268 RXA01291 GR00373 345 1277 PHYTOENE SYNTHASE (EC 2.5.1.-)
269 270 RXA00480 GR00119 17444 16329 FARNESYL DIPHOSPHATE SYNTHASE (EC
2.5.1.1) (EC 2.5.1.10)
271 272 RXS01879 VV0105 1505 573 isopentenyl-phosphate kinase (EC 2.7.4.-)
273 274 RXS02023 W0160 3234 4001 P450 cytochrome,isopentenyRransf, ferridox
275 276 RXS00948 W0107 4266 5384 12-oxophytodienoate reductase (EC 1.3.1.42)
277 278 RXS02228 W0068 1876 2778 TRNA DELTA(2)-ISOPENTENYLPYROPHOSPHATE
TRANSFERASE (EC 2.5.1.8)
279 280 RXC01971 W0105 4545 3715 Metal-Dependent Hydrolase involved in
metabolism of terpenoids
281 282 RXC02697 W0017 31257 32783 membrane protein involved in metabolism of
terpenoids
ABC-Transporter Table 1 (continued)
Nudeic Acid Amino Acid Identification Code Conti . NT Start NT Stop Function
SEQ ID NO SEQ ID NO
283 284 RXN01946 W0228 2 1276 Hypothetical ABC Transporter ATP-Binding Protein
285 286 F RXA01946 GR00559 1849 575 (AL021184) ABC transporter ATP binding
protein [Mycobacterium tuberculosis]
287 288 RXN00164 W0232 1782 94 Hypothetical ABC Transporter ATP-Binding
Protein
289 290 F RXA00164 GR00025 1782 94 P, G, R ATPase subunits of ABC transporters
291 292 RXN00243 W0057 28915 27899 , P. G. R ATPase subunits of ABC
transporters
293 294 F RXA00243 GR00037 930 4 P, G, R ATPase subunits of ABC transporters
295 296 RXA00259 GR0o039 8469 6268 , P, G, R ATPase subunits of ABC
transporters
297 298 RXN00410 VV0086 51988 51323 GLUTAMINE TRANSPORT ATP-BINDING PROTEIN
GLNQ
299 300 F RXA00410 GR00092 829 164 , P, G, R ATPase subunits of ABC
transporters
301 302 RXN00456 W0076 6780 8156 , P, G, R ATPase subunits of ABC transporters
303 304 F RXA00456 GR00114 316 5 , P, G, R ATPase subunits of ABC transporters
305 306 F RXA00459 GR00115 1231 245 , P, G, R ATPase subunits of ABC
transporters
307 308 RXN01604 W0137 8117 7470 , P, G. R ATPase subunits of ABC transporters
309 310 F RXA01604 GR00448 2 607 , P, G, R ATPase subunits of ABC transporters
311 312 RXN02547 W0057 27726 25588 , P. G, R ATPase subunits of ABC
transporters
313 314 F RXA02547 GR00726 22055 19932 P, G, R ATPase subunits of ABC
transporters oLno
315 316 RXN02571 W0101 12331 13359 MALTOSE/MALTODEXTRfN TRANSPORT ATP-BINDING
PROTEIN MALK u'
317 318 F RXA02571 GR00736 1469 2497 , P, G, R ATPase subunits of ABC
transporters o
319 320 RXN02074 W0318 12775 11153 TRANSPORT ATP-BINDING PROTEIN CYDD u'
321 322 F RXA02074 GR00628 5798 4176 , P, G, R ATPase subunits of ABC
transporters ~ rv
323 324 RXA02095 GR00629 14071 15474 , P, G, R ATPase subunits of ABC
transporters
325 326 RXA02225 GR00652 3156 2275 , P, G, R ATPase subunits of ABC
transporters
327 328 RXA02253 GR0o654 20480 21406 P, G, R ATPase subunits of ABC
transporters
329 330 RXN01881 W0105 529 95 Hypothetical ABC Transporter ATP-Binding Protein
P.
331 332 F RXA01881 GR00537 3092 3532 ATPase components of ABC transporters
with duplicated ATPase domains 1333 334 RXA00526 GR00136 1353 664 Hypothetical
ABC Transporter ATP-Binding Protein
335 336 RXN00733 W0132 1647 2531 Hypothetical ABC Transporter ATP-Binding
Protein
337 338 F RXA00733 GR00197 411 4 Hypothetical ABC Transporter ATP-Binding
Protein
339 340 RXA00735 GR00198 849 181 Hypothetical ABC TransporterATP-8inding
Protein
341 342 RXA00878 GR00242 3733 1871 Hypothetical ABC Transporter ATP-Binding
Protein
343 344 RXN01191 W0169 10478 12067 Hypothetical ABC Transporter ATP-Binding
Protein
345 346 F RXA01191 GR00341 1571 165 Hypothetical ABC Transporter ATP-Binding
Protein
347 348 RXN01212 W0169 3284 4207 Hypothetical ABC Transporter ATP-Binding
Protein
349 350 F RXA01212 GR00350 1 813 Hypothetical ABC Transporter ATP-Binding
Protein
351 352 RXA02749 GR00764 4153 5028 Hypothetical ABC Transporter ATP-Binding
Protein
353 354 RXA02224 GR00652 2271 475 Hypothetical ABC Transporter ATP-Binding
Protein
355 356 RXN01602 W0229 1109 2638 Hypothet'ical ABC Transporter ATP-Binding
Protein
357 358 RXN02515 W0087 962 1717 Hypothetical ABC Transporter ATP-Binding
Protein
359 360 RXN00525 W0079 26304 27566 Hypothetical ABC Transporter Permease
Protein
361 362 RXN02096 W0126 20444 22135 Hypothetical ABC Transporter Perrnease
Protein
363 364 RXN00412 W0086 53923 52844 Hypothetical Amino Acid ABC Transporter ATP-
Binding Protein
365 366 RXN00411 W0086 52844 52170 Hypothetical Amino Acid ABC Transporter
Pemiease Protein
367 368 RXN02614 W0313 5964 5236 TAURINE TRANSPORT ATP-BINDING PROTEIN TAUB
369 370 RXN02613 W0313 5223 4267 TAURINE-BINDING PERIPLASMIC PROTEIN PRECURSOR
Table I (continued)
Nucleic Acid Amino Acid Identification Code Contig. NT Start NT Stop Function
SEO ID NO SEQ ID NO
371 372 RXN00368 W0226 2300 726 SPERMIDINE/PUTRESCINE TRANSPORT ATP-BINDING
PROTEIN POTA
373 374 F RXA00368 GR00076 1 579 SPERMIDINE/PUTRESCINE TRANSPORT ATP-BINDING
PROTEIN POTA
375 376 F RXA00370 GR00077 6 803 SPERMIDINE/PUTRESCINE TRANSPORT ATP-BINDING
PROTEIN POTA
377 378 RXN01285 W0215 1780 1055 FERRIC ENTEROBACTIN TRANSPORT ATP-BINDING
PROTEIN FEPC
379 380 RXN00523 W0194 1363 338 FERRIC ENTEROBACTIN TRANSPORT PROTEIN FEPG
381 382 RXN01142 W0077 5805 6302 NITRATE TRANSPORT ATP-BINDING PROTEIN NRTD
383 384 RXN01141 W0077 4644 5468 NITRATE TRANSPORT PROTEIN NRTA
385 386 RXN01002 W0106 8858 8055 PHOSPHONATES TRANSPORT ATP-BINDING PROTEIN
PHNC
387 388 RXN01000 W0106 7252 6407 PHOSPHONATES TRANSPORT SYSTEM PERMEASE
PROTEIN PHNE
389 390 RXN01732 W0106 9944 8895 PHOSPHONATES-BINDING PERIPLASMIC PROTEIN
PRECURSOR
391 392 RXN03080 W0045 1670 2449 FERRIC ENTEROBACTIN TRANSPORT ATP-BINDING
PROTEIN FEPC
393 394 RXN03081 W0045 2476 2934 FERRIENTEROBACTIN-BINDING PERIPLASMIC PROTEIN
PRECURSOR
395 396 RXN03082 W0045 3131 3451 FERRIENTEROBACTIN-BINDING PERIPLASMIC PROTEIN
PRECURSOR
C)
Other transporters
U9
Nucleic Acid Amino Acid Identification Code Conti . NT Start NT Stop Function
W
SEQ ID NO SEQ ID NO -.3
397 398 RXA02261 GR00654 30936 32291 AMMONIUM TRANSPORT SYSTEM
399 400 RXA02020 GR00613 1015 5 AROMATIC AMINO ACID TRANSPORT PROTEIN AROP
401 402 RXA00281 GR00043 4721 5404 BACITRACIN TRANSPORT ATP-BINDING PROTEIN
BCRA
403 404 RXN00570 W0147 855 4 BENZOATE MEMBRANE TRANSPORT PROTEIN W o
405 406 F RXA00570 GR00153 1 498 BENZOATE MEMBRANE TRANSPORT PROTEIN
407 408 RXN00571 W0173 1298 42 BENZOATE MEMBRANE TRANSPORT PROTEIN o
409 410 F RXA00571 GR00154 2 1186 BENZOATE MEMBRANE TRANSPORT PROTEIN
411 412 RXA00962 GR00268 2 667 BENZOATE MEMBRANE TRANSPORT PROTEIN ~
413 414 RXA02811 GR00792 177 560 BENZOATE MEMBRANE TRANSPORT PROTEIN
415 416 RXA02115 GR00635 2 1198 BENZOATE MEMBRANE TRANSPORT PROTEIN
417 418 RXN00590 W0178 5043 6230 BRANCHED CHAIN AMINO ACID TRANSPORT SYSTEM II
CARRIER PROTEIN
419 420 F RXA00590 GR00157 178 564 BRANCHED CHAIN AMINO ACID TRANSPORT SYSTEM
II CARRIER PROTEIN
421 422 F RXA01538 GR00427 5040 5429 BRANCHED CHAIN AMINO ACID TRANSPORT
SYSTEM tl CARRIER PROTEIN
423 424 RXA01727 GR00489 1471 194 BRANCHED-CHAIN AMINO ACID TRANSPORT SYSTEM
CARRIER PROTEIN
425 426 RXA00623 GR00163 6525 7862 C4-DICARBOXYLATE TRANSPORT PROTEIN
427 428 RXA01584 GR00441 55 597 CHROMATE TRANSPORT PROTEIN
429 430 RXA00852 GR00231 3137 2448 COBALT TRANSPORT ATP-BINDING PROTEIN CBIO
431 432 RXA00690 GR00181 1213 68 COBALT TRANSPORT PROTEIN CBIQ
433 434 RXA00827 GR00223 1319 567 COBALT TRANSPORT PROTEIN CBIQ
435 436 RXA00851 GR00231 2448 1840 COBALT TRANSPORT PROTEIN CBIQ
437 438 RXS03220 D-XYLOSE-PROTON SYMPORT
439 440 F RXA02762 GR00768 346 630 D-XYLOSE PROTON-SYMPORTER
441 442 RXN00092 W0129 27509 26844 GLUTAMINE TRANSPORT ATP-BINOING PROTEIN
GLNQ
443 444 F RXA00092 GR00014 1 204 GLUTAMINE TRANSPORT ATP-BINDING PROTEIN GLNQ
445 446 RXN03060 W0030 6227 5376 GLUTAMINE TRANSPORT ATP-BINDING PROTEIN GLNQ
447 448 F RXA02618 GR00745 1914 2351 GLUTAMINE TRANSPORT ATP-BINDING PROTEIN
GLNQ
449 450 F RXA02900 GR10040 2979 2128 GLUTAMINE TRANSPORT ATP-BINDING PROTEIN
GLNQ
Table I (continued)
Nucleic Acid Amino Acid Identification Code 222ti9. NT Start NT Stop Function
SEQ ID NO SEQ ID NO
451 452 RXS03212 GLYCINE BETAINE TRANSPORTER BETP
453 454 F RXA01591 GR00446 3 947 GLYCINE BETAINE TRANSPORTER BETP
455 456 RXN00201 W0096 197 6 HIGH AFFINITY RIBOSE TRANSPORT PROTEIN RBSD
457 458 F RXA00201 GR00032 191 6 HIGH AFFINITY RIBOSE TRANSPORT PROTEIN RBSD
459 460 RXA01221 GR00354 2108 2833 HIGH-AFFINITY BRANCHED-CHAIN AMINO ACID
TRANSPORT ATP-BINDING
PROTEIN BRAG
461 462 RXA01222 GR00354 2844 3542 HIGH-AFFINITY BRANCHED-CHAIN AMINO ACID
TRANSPORT ATP-BINDING
PROTEIN LIVF
463 464 RXA01219 GR00354 151 1032 HIGH-AFFINITY BRANCHED-CHAIN AMINO ACID
TRANSPORT PERMEASE
PROTEIN LIVH
465 466 RXA01220 GR00354 1032 2108 HIGH-AFFINITY BRANCHED-CHAIN AMINO ACID
TRANSPORT PERMEASE
PROTEIN LIVM
467 468 RXA00091 GR00013 7762 8514 IRON(III) DICITRATE TRANSPORT ATP-BINDING
PROTEIN FECE
469 470 RXA00228 GR00032 29232 28642 IRON(II1) DICITRATE TRANSPORT ATP-BINDING
PROTEIN FECE
471 472 RXA00346 GR00064 1054 1743 IRON(III) DICITRATE TRANSPORT ATP-BINDING
PROTEIN FECE
473 474 RXA00524 GR00135 779 1111 IRON(III) DICITRATE TRANSPORT ATP-BINDING
PROTEIN FECE
475 476 RXA01823 GR00516 591 1367 IRON(III) DICITRATE TRANSPORT ATP-BINDING
PROTEIN FECE rv
477 478 RXA02767 GR00770 1032 1814 IRON(III) DICITRATE TRANSPORT ATP-BINDING
PROTEIN FECE L"
479 480 RXA02792 GR00777 8581 7829 IRON(III) DICITRATE TRANSPORT ATP-BINDING
PROTEIN FECE W
481 482 RXN02929 W0090 36837 37874 IRON(III) DICITRATE TRANSPORT SYSTEM
PERMEASE PROTEIN FECD -.3
483 484 F RXA01235 GR00358 1165 194 IRON(III) DICITRATE TRANSPORT SYSTEM
PERMEASE PROTEIN FECD ~,~
485 486 RXN02794 VV0134 10625 9552 IRON(III) DICITRATE TRANSPORT SYSTEM
PERMEASE PROTEIN FECD
487 488 F RXA01419 GR00415 888 1151 IRON(III) DICITRATE TRANSPORT SYSTEM
PERMEASE PROTEIN FECD
489 490 F RXA02794 GR00777 10172 9552 IRON(III) DICITRATE TRANSPORT SYSTEM
PERMEASE PROTEIN FECD
491 492 RXN03079 W0045 644 1660 IRON(III) DICITRATE TRANSPORT SYSTEM PERMEASE
PROTEIN FECO 493 494 F RXA02865 GR10007 3832 2816 IRON(III) DICITRATE
TRANSPORT SYSTEM PERMEASE PROTEIN FECD
495 496 RXA00181 GR00028 3954 2383 PROLINE TRANSPORT SYSTEM
497 498 RXA00591 GR00158 229 1581 PROLINE/BETAINE TRANSPORTER ~
499 500 RXA01629 GR00453 3476 1965 PROLINEBETAINE TRANSPORTER 1O
501 502 RXA02030 GR00618 3072 1687 PROLINEBETAINE TRANSPORTER
503 504 RXA00186 GR00028 12242 12988 SHORT-CHAIN FATTY ACIDS TRANSPORTER
505 506 RXA00187 GR00028 13097 13447 SHORT-CHAIN FATTY ACIDS TRANSPORTER
507 508 RXA01667 GR00464 703 1908 SODIUM/GLUTAMATE SYMPORT CARRIER PROTEIN
509 510 RXA02171 GR00641 6571 4919 SODIUM/PROLINE SYMPORTER
511 512 RXA00902 GR00245 4643 5875 SODIUM-DEPENDENT PHOSPHATE TRANSPORT
PROTEIN
513 514 RXA00941 GR00257 1999 683 sodium-dependent phosphate transport protein
515 516 RXN00449 W0112 30992 32572 Sodium-Dicarboxylate Symport Protein
517 518 F RXA00449 GR00109 2040 1036 Sodium-Dicarboxylate Symport Protein
519 520 F RXA01755 GR00498 352 5 Sodium-Dicarboxylate Symport Protein
521 522 RXA00269 GR00041 1826 1038 SPERMIDINE/PUTRESCINE TRANSPORT ATP-BINDING
PROTEIN POTA
523 524 RXA00369 GR00076 583 1299 SPERMIDINE/PUTRESCINE TRANSPORT ATP-BINDING
PROTEIN POTA
525 526 RXA02073 GR00628 4176 2647 TRANSPORT ATP-BINDING PROTEIN CYDC
527 528 RXA01399 GR00409 1 1119 TRANSPORT ATP-BINDING PROTEIN CYDD
529 530 RXA01339 GR00389 8408 7164 TYROSINE-SPECIFIC TRANSPORT PROTEIN
531 532 RXA02527 GR00725 5519 6847 2-OXOGLUTARATElMALATE TRANSLOCATOR
PRECURSOR
533 534 RXN00298 VV0176 40228 42072 HIGH-AFFINITY CHOLINE TRANSPORT PROTEIN
535 536 F RXA00298 GR00048 4459 6303 Ectoine/Proline/Glycine betaine carrier
ectP
Table I (continued)
Nucleic Acid Amino Acid Identification Code Cond9. NT Start NT Stop Function
SEQ ID NO SEQ ID NO
537 538 RXA00596 GR00159 335 787 potassium efflux system protein phaE
539 540 RXA02364 GR00686 841 215 C4-DICARBOXYLATE-BINDING PERIPLASMIC PROTEIN
PRECURSOR, transport
protein
541 542 RXN01411 W0050 26015 26779 SHIKIMATE TRANSPORTER
543 544 RXN00960 W0075 1139 105 PROTON/SODIUM-GLUTAMATE SYMPORT PROTEIN
545 546 RXN02447 W0107 14297 13203 GALACTOSE-PROTON SYMPORT
547 548 RXN02395 W0176 16747 14858 GLYCINE BETAINE TRANSPORTER BETP
549 550 RXN02348 W0078 6027 7910 KUP SYSTEM POTASSIUM UPTAKE PROTEIN
551 552 RXN00297 W0176 38630 39541 Hypothetical Malonate Transporter
553 554 RXN03103 W0070 845 1087 GLUTAMATE-BINDING PROTEIN PRECURSOR
555 556 RXN02993 W0071 736 65 GLUTAMINE-BINDING PROTEIN
557 558 RXN00349 W0135 35187 36653 Hypothetical Trehalose Transport Protein
559 560 RXN03095 W0057 4056 4424 CADMIUM EFFLUX SYSTEM ACCESSORY PROTEIN
HOMOLOG
561 562 RXN03160 W0189 5150 5617 CHROMATE TRANSPORT PROTEIN
563 564 RXN02955 W0176 8666 9187 DICARBOXYLATE TRANSPORTER
565 566 RXN03109 W0082 659 6 HEMIN TRANSPORT SYSTEM PERMEASE PROTEIN HMUU
567 568 RXN02979 W0149 2150 2383 MERCURIC TRANSPORT PROTEIN PERIPLASMIC
COMPONENT PRECURSOR N
569 570 RXN02987 W0234 527 294 MERCURIC TRANSPORT PROTEIN PERIPLASMIC
COMPONENT PRECURSOR Ln
571 572 RXN03084 W0048 900 1817 IRON(III) DICITRATE-BINDING PERIPLASMIC
PROTEIN PRECURSOR co
573 574 RXN03183 W0372 1 417 TREHALOSE/MALTOSE BINDING PROTEIN w
.3
575 576 RXN01139 W0077 2776 1823 CATION EFFLUX SYSTEM PROTEIN CZCD 0
577 578 RXN00378 W0223 8027 5418 Cation transport ATPases u'
579 580 RXN01338 W0032 2 1903 CATION-TRANSPORTING ATPASE PACS (EC 3.6.1 -)
581 582 RXN00980 W0149 2635 4428 CATION-TRANSPORTING P-TYPE ATPASE B (EC
3.6.1.-) o
583 584 RXN00099 W0129 18876 17704 CYANATE TRANSPORT PROTEIN CYNX -.3
585 586 RXN02662 W0315 1461 1724 DIPEPTIDE TRANSPORT SYSTEM PERMEASE PROTEIN
DPPC
587 588 RXN02442 W0217 5970 6818 zinc transport system membrane protein
589 590 RXN02443 W0217 6818 7771 zinc-binding periplasmic protein precursor
591 592 RXN00842 W0138 8686 7487 BRANCHED CHAIN AMINO ACID TRANSPORT SYSTEM II
CARRIER PROTEIN
593 594 F RXA00842 GR00228 3208 2009 Permeases
595 596 RXN00832 W0180 3133 4182 CALCIUM/PROTON ANTIPORTER
597 598 RXN00466 W0086 63271 64266 Ferrichrome transport proteins
599 600 RXN01936 W0127 40116 41387 MACROLIDE-EFFLUX PROTEIN
601 602 RXN01995 W0182 2139 3476 PUTATIVE 3-(3-HYDROXYPHENYL) PROPIONATE
TRANSPORT PROTEIN
603 604 RXN00661 W0142 9718 9029 PNUC PROTEIN
Permeases
Nucleic Acid Amino Acid Identification Code Contig. NT Start NT Stop Function
SEQ ID NO SEQ ID NO
605 606 RXN02566 W0154 11823 13031 NUCLEOSIDE PERMEASE NUPG
607 608 F RXA02561 GR00732 664 5 NUCLEOSIDE PERMEASE NUPG
609 610 F RXA02566 GR00733 782 345 NUCLEOSIDE PERMEASE NUPG
611 612 RXA00051 GR00008 5770 7173 PROLINE-SPECIFIC PERMEASE PROY
613 614 RXA01172 GR00334 2687 4141 SULFATE PERMEASE
615 616 RXA02128 GR00637 2906 4600 SULFATE PERMEASE
Table 1 (continued)
Nucleic Acid Amino Acid Identification Code Contig. NT Start NT Stop Function
SEQ ID NO SEQ ID NO
617 618 RXA02634 GR00748 6045 7655 SULFATE PERMEASE
619 620 RXN02233 W0068 6856 8142 URACIL PERMEASE
621 622 F RXA02233 GR00653 6856 8067 URACIL PERMEASE
623 624 RXN02372 W0213 9311 11197 XANTHINE PERMEASE
625 626 F RXA02372 GR00688 6 560 XANTHINE PERMEASE
627 628 F RXA02377 GR00689 3336 4526 XANTHINE PERMEASE
629 630 RXA02676 GR00754 2697 1309 GLUCONATE PERMEASE
631 632 RXN00432 W0112 14751 13267 NA(+)-LINKED D-ALANINE GLYCINE PERMEASE
633 634 F RXA00432 GR00100 1 891 NA(+)-LINKED D-ALANINE GLYCINE PERMEASE
635 636 F RXA00436 GR00101 45 569 NA(+)-LINKED D-ALANINE GLYCINE PERMEASE
637 638 RXA00847 GR00230 1829 381 OLIGOPEPTIDE-BINDING PROTEIN APPA PRECURSOR
(permease)
639 640 RXN01382 W0119 8670 9761 OLIGOPEPTIDE-BINDING PROTEIN OPPA PRECURSOR
641 642 F RXA01382 GR00405 1067 6 OLIGOPEPTIDE-BINDING PROTEIN OPPA PRECURSOR
(pennease)
643 644 RXA02659 GR00753 2 313 OLIGOPEPTIDE-BINDING PROTEIN OPPA PRECURSOR
(permease)
645 646 RXN02933 W0176 30042 29233 DIPEPTIDE TRANSPORT SYSTEM PERMEASE PROTEIN
DPPC
647 648 RXN02991 W0072 618 4 GLUTAMINE TRANSPORT SYSTEM PERMEASE PROTEIN GLNP
649 650 RXN02992 W0072 842 621 GLUTAMINE TRANSPORT SYSTEM PERMEASE PROTEIN
GLNP o
651 652 RXN02996 W0069 1980 2648 HIGH-AFFINITY BRANCHED-CHAIN AMINO ACID
TRANSPORT PERMEASE Ln
PROTEIN LIVH 00
653 654 RXN03126 W0112 9894 9001 TEICHOIC ACID TRANSLOCATION PERMEASE PROTEIN
TAGG
655 656 RXN00443 W0112 21572 20769 MOLYBDATE-BINDING PERIPLASMIC PROTEIN
PRECURSOR = o
657 658 RXN00444 W0112 20785 19949 MOLYBDENUM TRANSPORT SYSTEM PERMEASE
PROTEIN MODB w
659 660 RXN00193 W0371 1 594 POTENTIAL STARCH DEGRADATION PRODUCTS TRANSPORT
SYSTEM
PERMEASE PROTEIN AMYD p~ o
661 662 RXN01298 W0116 2071 1142 POTENTIAL STARCH DEGRADATION PRODUCTS
TRANSPORT SYSTEM - 1 .3
PERMEASE PROTEIN AMYD o
Channel Proteins ~
Nudeic Acid Amino Acid Identification Code Conti9. NT Start NT Stop Function
SEQ ID NO SEQ ID NO
663 664 RXA01737 GR00493 2913 3971 POTASSIUM CHANNEL PROTEIN
665 666 RXN02348 W0078 6027 7910 KUP SYSTEM POTASSIUM UPTAKE PROTEIN
667 668 RXA02426 GR00707 2165 633 PROBABLE NA(+)/H(+) ANTIPORTER
669 670 RXN03164 W0277 1586 2455 POTASSIUM CHANNEL BETA SUBUNIT
671 672 RXN00024 W0127 64219 63275 POTASSIUM CHANNEL BETA SUBUNIT
Lipoprotein and Lipopolysaccharide synthesis
Nucleic Acid Amino Acid Identification Code Con ". NT Start NT Stop Function
SEQ ID NO SEQ ID NO
673 674 RXN01164 W0117 15894 14260 DOLICHOL-PHOSPHATE MANNOSYLTRANSFERASE (EC
2.4.1.83) /
APOLIPOPROTEIN N-ACYLTRANSFERASE (EC 2.3.1.-)
675 676 RXN01168 W0117 14224 13415 DOLICHOL-PHOSPHATE MANNOSYLTRANSFERASE (EC
2.4.1.83) !
APOLIPOPROTEIN N-ACYLTRANSFERASE (EC 2.3.1.-)
~ =
TABLE 2 - Excluded Genes
GenBankT" Gene Name Gene Function Reference
Accession No.
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 03/21/90
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 07/20/95
A45585
A45587
AB003132 murC; ftsQ; ftsZ Kobayashi, M. et al. "Cloning, sequencing, and
characterization of the ftsZ
gene from coryneform bacteria," Biochem. Biophys. Res. Commun.,
236(2):383-388 (1997)
AB015023 murC; ftsQ Wachi, M. et al. "A murC gene from Coryneform bacteria,"
Appl. Microbiol. Ln
Biotechnol., 51(2):223-228 (1999) co
AB018530 dtsR Kimura, E. et al. "Molecular cloning of a novel gene, dtsR,
which rescues the
detergent sensitivity of a mutant derived from Brevibacterium w
lactojermentum," Biosci. Biotechnol. Biochem., 60(10):1565-1570 (1996)
AB018531 dtsR 1; dtsR2
AB020624 murl D-glutamate racemase - 1 .3
AB023377 tkt transketolase
AB024708 gltB; g1tD Glutamine 2-oxoglutarate aminotransferase
large and small subunits 1O
AB025424 acn aconitase
AB027714 rep Replication protein
AB027715 rep; aad Replication protein; aminoglycoside
adenyltransferase
AF005242 argC N-acetylglutamate-5-semialdehyde
dehydrogenase
AF005635 ginA Glutamine synthetase
AF030405 hisF cyclase
AF030520 argG Argininosuccinate synthetase
AF031518 argF Ornithine carbamolytransferase
AF036932 aroD 3-dehydroquinate dehydratase
AF038548 pyc Pyruvate carboxylase
Table 2 (continued)
AF038651 dciAE; apt; rel Dipeptide-binding protein; adenine Wehmeier, L. et
al. "The role of the Corynebacterium glutamicum rel gene in
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; argJ; argB; N-acetylglutamylphosphate reductase;
argD; argF; argR; omithine acetyltransferase; N-
argG; argH acetylglutamate kinase; acetylornithine
transminase; ornithine
carbamoyltransferase; arginine repressor;
argininosuccinate synthase;
argininosuccinate lyase
AF050109 inhA Enoyl-acyl carrier protein reductase
AF050166 hisG ATP phosphoribosyltransferase
A 51846 hisA Phosphoribosylformimino-5-amino-l- L,
00
p osphoribosyl-4-imida2olecarboxamide i,,
isomerase -.3
0
AF052652 metA Homoserine 0-acetyltransferase Park, S. et al. "Isolation and
analysis of metA, a methionine biosynthetic gene w
encoding homoserine acetyltransferase in Corynebacterium glutamicum," Mol. o
Cells., 8(3):286-294 (1998) O0 0
-.3
AF053071 aroB Dehydroquinate synthetase
AF060558 hisH Glutamine amidotransferase
AF086704 hisE Phosphoribosyl-ATP-
pyrophosphohydrolase
AF114233 aroA 5-enolpyruvylshikimate 3-phosphate
synthase
AF 116184 panD L-aspartate-alpha-decarboxylase precursor Dusch, N. et al.
"Expression of the Corynebacterium glutamicum panD gene
encoding L-aspartate-alpha-decarboxylase leads to pantothenate
overproduction in Escherichia coli," Appl. Environ. Microbiol., 65(4)1530-
1539(1999)
AF124518 aroD; aroE 3-dehydroquinase; shikimate
dehydrogenase
AF124600 aroC; aroK; aroB; Chorismate synthase; shikimate kinase; 3-
pepQ dehydroquinate synthase; putative
cytoplasmic peptidase
AF145897 inhA
AF145898 inhA
Table 2 (continued)
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, I80(22):6005-6012 (1998)
AJ004934 dapD Tetrahydrodipicolinate succinylase Wehrmann, A. et al.
"Different modes of diaminopimelate synthesis and their
(incomplete ) role in cell wall integrity: A study with Corynebacterium
glutamicum," J.
Bacteriol., 180(12):3159-3165 (1998)
AJ007732 ppc; secG; amt; ocd; Phosphoenolpyruvate-carboxylase; ?; high
soxA affinity ammonium uptake protein; putative
ornithine-cyclodecarboxylase; sarcosine
oxidase
AJ010319 ftsY, glnB, glnD; srp; Involved in cell division; Pll protein;
Jakoby, M. et al. "Nitrogen regulation in Corynebacterium glutamicum;
amtP uridylyltransferase (uridylyl-removing Isolation of genes involved in
biochemical characterization of corresponding
enzmye); signal recognition particle; low proteins," FEMS Microbiol.,
173(2):303-310 (1999)
affinity ammonium uptake protein
AJ132968 cat Chloramphenicol aceteyl transferase
cn
AJ224946 mqo L-malate: quinone oxidoreductase Molenaar, D. et al. "Biochemical
and genetic characterization of the ~ D,,
membrane-associated malate dehydrogenase (acceptor) from Corynebacterium o
glutamicum," Eur. J. Biochem., 254(2):395-403 (1998) w
AJ238250 ndh NADH dehydrogenase OS o
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 o
molecular mass polypeptide," Biochemistry, 37(43):15024-15032 (1998)
D17429 Transposable element IS31831 Vertes 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 AJ 12036) 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 10/12/87
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 10/12/87
E01375 Tryptophan operon
E01376 trpL; trpE Leader peptide; anthranilate synthase Matsui, K. et al.
"Tryptophan operon, peptide and protein coded thereby,
utilization of tryptophan operon gene expression and production of
tryptophan," Patent: JP 1987244382-A 1 10/24/87
Table 2 continued
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 10/24/87
E03937 Biotin-synthase Hatakeyama, K. et al. "DNA fragment containing gene
capable of coding
biotin synthetase and its utilization," Patent: JP 1992278088-A 1 10/02/92
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
11/18/92
E04041 Desthiobiotinsynthetase Kohama, K. et al. "Gene coding
diaminopelargonic acid aminotransferase and
desthiobiotin synthetase and its utilization," Patent: JP 1992330284-A 1
11/18/92
E04307 Flavum aspartase Kurusu, Y. et al. "Gene DNA coding aspartase and
utilization thereof," Patent:
JP 1993030977-A 1 02/09/93
E04376 Isocitric acid lyase Katsumata, R. et al. "Gene manifestation
controlling DNA," Patent: JP
1993056782-A 3 03/09/93
E04377 Isocitric acid lyase N-terminal fragment Katsumata, R. et al. "Gene
manifestation controlling DNA," Patent: JP Ln
1993056782-A 3 03/09/93 w
-.3
E04484 Prephenate dehydratase Sotouchi, N. et al. "Production of L-
phenylalanine by fermentation," Patent: JP o
1993076352-A 2 03/30/93 w
E05108 Aspartokinase Fugono, N. et al. "Gene DNA coding Aspartokinase and its
use," Patent: JP ~ o
1993184366-A 1 07/27/93
E05112 Dihydro-dipichorinate synthetase Hatakeyama, K. et al. "Gene DNA coding
dihydrodipicolinic acid synthetase
and its use," Patent: JP 1993184371-A 1 07/27/93
E05776 Diaminopimelic acid dehydrogenase Kobayashi, M. et al. "Gene DNA coding
Diaminopimelic acid dehydrogenase
and its use," Patent: JP 1993284970-A I 11/02/93
E05779 Threonine synthase Kohama, K. et al. "Gene DNA coding threonine
synthase and its use," Patent:
JP 1993284972-A 1 11/02/93
E06110 Prephenate dehydratase Kikuchi, T. et al. "Production of L-
phenylalanine by fennentation method,"
Patent: JP 1993344881-A 1 12/27/93
E06111 Mutated Prephenate dehydratase Kikuchi, T. et al. "Production of L-
phenylalanine by fermentation method,"
Patent: JP 1993344881-A 1 12/27/93
E06146 Acetohydroxy acid synthetase lnui, M. et al. "Gene capable of coding
Acetohydroxy acid synthetase and its
use," Patent: JP 1993344893-A 1 12/27/93
E06825 Aspartokinase Sugimoto, M. et al. "Mutant aspartokinase gene," patent:
JP 1994062866-A I
03/08/94
E06826 Mutated aspartokinase alpha subunit Sugimoto, M. et al. "Mutant
aspartokinase gene," patent: JP 1994062866-A I
03/08/94
7t
Table 2 continued
E06827 Mutated aspartokinase alpha subunit Sugimoto, M. et al. "Mutant
aspartokinase gene," patent: JP 1994062866-A 1
03/08/94
E07701 secY Honno, N. et al. "Gene DNA participating in integration of
membraneous
protein to membrane," Patent: JP 1994169780-A I 06/21/94
E08177 Aspartokinase Sato, Y. et al. "Genetic DNA capable of coding
Aspartokinase released from
feedback inhibition and its utilization," Patent: JP 1994261766-A 1 09/20/94
E08178, Feedback inhibition-released Aspartokinase Sato, Y. et al. "Genetic
DNA capable of coding Aspartokinase released from
E08179, feedback inhibition and its utilization," Patent: JP 1 99426 1 766-A 1
09/20/94
E08180,
E08181,
E08182
E08232 Acetohydroxy-acid isomeroreductase lnui, M. et al. "Gene DNA coding
acetohydroxy acid isomeroreductase,"
Patent: JP 1994277067-A 1 10/04/94
E08234 secE Asai, Y. et al. "Gene DNA coding for translocation machinery of
protein,"
Patent: JP 1994277073-A I 10/04/94
E08643 FT aminotransferase and desthiobiotin Hatakeyama, K. et al. "DNA
fragment having promoter function in ci,
synthetase promoter region coryneform bacterium," Patent: JP 1995031476-A 1
02/03/95 w
E08646 Biotin synthetase Hatakeyama, K. et al. "DNA fragment having promoter
function in o
coryneform bacterium," Patent: JP 1995031476-A 1 02/03/95 w
E08649 Aspartase Kohama, K. et al "DNA fragment having promoter function in
coryneform -j o
bacterium," Patent: JP 1995031478-A 1 02/03/95
E08900 Dihydrodipicolinate reductase Madori, M. et al. "DNA fragment
containing gene coding Dihydrodipicolinate
acid reductase and utilization thereof," Patent: JP 1995075578-A I 03/20/95
E08901 Diaminopimelic acid decarboxylase Madori, M. et al. "DNA fragment
containing gene coding Diaminopimelic acid
decarboxylase and utilization thereof," Patent: JP 1995075579-A I 03/20/95
E12594 Serine hydroxymethyltransferase Hatakeyama, K. et al. "Production of L-
trypophan," Patent: JP 1997028391-A
1 02/04/97
E 12760, transposase Moriya, M. et al. "Amplification of gene using artificial
transposon," Patent:
E12759, JP 1997070291-A 03/18/97
E12758
E12764 Arginyl-tRNA synthetase; diaminopimelic Moriya, M. et al.
"Amplification of gene using artificial transposon," Patent:
acid decarboxylase JP 199707029I-A 03/18/97
E12767 Dihydrodipicolinic acid synthetase Moriya, M. et al. "Amplification of
gene using artificial transposon," Patent:
JP 1997070291-A 03/18/97
E12770 aspartokinase Moriya, M. et al. "Amplification of gene using artificial
transposon," Patent:
JP 1997070291-A 03/18/97
E12773 Dihydrodipicolinic acid reductase Moriya, M. et al. "Amplification of
gene using artificial transposon," Patent:
JP 1997070291-A 03/18/97
Table 2 continued
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 09/02/97
L01508 IIvA Threonine dehydratase Moeckel, B. et al. "Functional and
structural analysis of the threonine
dehydratase of Corynebacterium glutamicum," J. Bacteriol., 174:8065-8072
(1992)
L07603 EC 4.2.1.15 3-deoxy-D-arabinoheptulosonate-7- Chen, C. et al. "The
cloning and nucleotide sequence of Corynebacterium
phosphate synthase glutamicum 3-deoxy-D-arabinoheptulosonate-7-phosphate
synthase gene,"
FEMS Microbiol. Lett., 107:223-230 (1993)
L09232 llvB; i1vN; ilvC Acetohydroxy acid synthase large subunit; Keilhauer,
C. et al. "Isoleucine synthesis in Corynebacterium glutamicum:
Acetohydroxy acid synthase small subunit; molecular analysis of the ilvB-ilvN-
i1vC operon," J. Bacteriol., 175(17):5595-
Acetohydroxy acid isomeroreductase 5603 (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 11 from enteric bacteria," PNAS USA, 84(24):8773-8777 (1987); Lee,
J.K. et al. "Nucleotide sequence of the gene encoding the Corynebacterium o
glutamicum mannose enzyme 11 and analyses of the deduced protein Lõ
sequence," FBMSMicrobiol. Lett., 119(1-2):137-145 (1994) D
w
L27123 aceB Malate synthase Lee, H-S. et al. "Molecular characterization of
aceB, a gene encoding malate o
synthase in Corynebacterium glutamicum," J. Microbiol. Biotechnol., w
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., I77(2):465-467 (1995)
M 13774 Prephenate dehydratase Follettie, M.T. et al. "Molecular cloning and
nucleotide sequence of the
Corynebacterium glutamicum pheA gene," J. Bacteriol., 167:695-702 (1986)
M 16175 5S rRNA Park, Y-H. et al. "Phylogenetic analysis of the coryneform
bacteria by 56
rRNA sequences," J. Bacteriol., 169:1801-1806,(1987)
M 16663 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)
M 16664 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)
Table 2 continued
M25819 Phosphoenolpyruvate carboxylase O'Regan, M. et al. "Cloning and
nucleotide sequence of the
Phosphoenolpyruvate carboxylase-coding gene of Corynebacterium
glutamicum ATCC 13032," Gene, 77(2):237-251 (1989)
M85106 23S rRNA gene insertion sequence Roller, C. et al. "Gram-positive
bacteria with a high DNA G+C content are
characterized by a common insertion within their 23S rRNA genes," J. Gen.
MicrobioL, 138:1167-1175 (1992)
M85107, 23S rRNA gene insertion sequence Roller, C. et al. "Gram-positive
bacteria with a high DNA G+C content are
M85108 characterized by a common insertion within their 23S rRNA genes," J.
Gen.
Microbiol., 138:1167-1175 (1992)
M89931 aecD; brnQ; yhbw Beta C-S lyase; branched-chain amino acid Rossol, I.
et al. "The Corynebacterium glutamicum aecD gene encodes a C-S
uptake carrier; hypothetical protein yhbw lyase with alpha, beta-elimination
activity that degrades aminoethylcysteine,"
J. BacterioL, 174(9):2968-2977 (1992); Tauch, A. et al. "Isoleucine uptake in
Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene
product," Arch. Microbiol., 169(4):303-312 (1998)
S59299 trp Leader gene (promoter) Henry, D.M. et al. "Cloning of the trp gene
cluster from a tryptophan-
hyperproducing
strain of Corynebacterium glutamicum: identification of a cn
mutation in the trp leader sequence," Appl. Environ. Microbiol., 59(3):791-799
~ D.,
(1993) -.3
U11545 trpD Anthranilate phosphoribosyltransferase O'Gara, J.P. and Dunican,
L.K. (1994) Complete nucleotide sequence of the w
Corynebacterium glutamicum ATCC 21850 tpD gene." Thesis, Microbiology
Department, University College Galway, Ireland. -.3
U 13922 cg1IM; cg11R; clglIR Putative type II 5-cytosoine Schafer, A. et al.
"Cloning and characterization of a DNA region encoding a
methyltransferase; putative type II stress-sensitive restriction system from
Corynebacterium glutamicum ATCC
restriction endonuclease; putative type I or 13032 and analysis of its role in
intergeneric conjugation with Escherichia
type IlI restriction endonuclease coli," J. Bacteriol., 176(23):7309-7319
(1994); Schafer, A. et al. "The 1O
Corynebacterium glutamicum cg1iM gene encoding a 5-cytosine in an McrBC-
deficient Escherichia coli strain," Gene, 203(2):95-101 (1997)
U 14965 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; proB; unkdh ?;gamma glutamyl kinase;similar to D- Ankri, S. et al.
"Mutations in the Corynebacterium glutamicumproline
isomer specific 2-hydroxyacid biosynthetic pathway: A natural bypass of the
proA step," J. BaclerioL,
dehydrogenases 178(15):4412-4419 (1996)
Table 2 (continued)
U31281 bioB Biotin synthase Serebriiskii, I.G., "Two new members of the bio B
superfamily: Cloning,
sequencing and expression of bio B genes of Methylobacillus flagellatum and
Corynebacterium glutamicum," Gene, 175:15-22 (1996)
U35023 thtR; accBC Thiosulfate sulfurtransferase; acyl CoA Jager, W. et al. "A
Corynebacterium glutamicum gene encoding a two-domain
carboxylase protein similar to biotin carboxylases and biotin-carboxyl-carrier
proteins,"
Arch. MicrobioL, 166(2);76-82 (1996)
U43535 cmr Multidrug resistance protein Jager, W. et al. "A Corynebacterium
glutamicum gene conferring multidrug
resistance in the heterologous host Escherichia coli," J. Bacteriol.,
179(7):2449-2451 (1997)
U43536 cipB Heat shock ATP-binding protein
U53587 aphA-3 3'5"-aminoglycoside phosphotransferase
U89648 Corynebacterium glutamicum unidentified
sequence involved in histidine biosynthesis,
partial sequence
X04960 trpA; trpB; trpC; trpD; Tryptophan operon Matsui, K. et al. "Complete
nucleotide and deduced amino acid sequences of
trpE; trpG; trpL the Brevibacterium lactofermentum tryptophan operon," Nucleic
Acids Res., L"
00
14(24):10113-10114(1986) w
-.3
X07563 lys A DAP decarboxylase (meso-diaminopimelate Yeh, P. et al. "Nucleic
sequence of the lysA gene of Corynebacterium o
decarboxylase, EC 4.1.1.20) glutamicum and possible mechanisms for modulation
of its expression," Mol. w
Gen. Genet., 212(l):112-119 (1988) v o
X14234 EC 4.1.1.31 Phosphoenolpyruvate carboxylase Eikmanns, B.J. et al. "The
Phosphoenolpyruvate carboxylase gene of
Corynebacterium glutamicum: Molecular cloning, nucleotide sequence, and
expression," Mol. Gen. Genet., 218(2):330-339 (1989); Lepiniec, L. et al.
"Sorghum Phosphoenolpyruvate carboxylase gene family: structure, function
and molecular evolution," Plant. Mol. Blol., 21 (3):487-502 (1993)
X 17313 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-I, 6-biphosphate aldolase to class I and
class II aldolases," Mol. MicrobioL,
X53993 dapA L-2, 3-dihydrodipicolinate synthetase (EC Bonnassie, S. et al.
"Nucleic sequence of the dapA gene from
4.2.1.52) Corynebacterium glutamicum," Nucleic Acids Res., 18(21):6421 (1990)
X54223 AttB-related site Cianciotto, N. et al. "DNA sequence homology between
att B-related sites of
Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium
glutamicum , and the attP site of lambdacorynephage," FEMS. Microbiol,
Lett., 66:299-302 (1990)
X54740 argS; lysA Arginyl-tRNA synthetase; Diaminopimelate Marcel, T. et al.
"Nucleotide sequence and organization of the upstream region
decarboxylase of the Corynebacterium glutamicum lysA gene," Mol. Microbiol.,
4(l 1):1819-
1830 (1990)
Table 2 continued
X55994 trpL; trpE Putative leader peptide; anthranilate Heery, D.M. et al.
"Nucleotide sequence of the Corynebacterium glutamicum
synthase component I trpE gene," Nucleic Acids Res., 18(23):7138 (1990)
X56037 thrC Threonine synthase Han, K.S. et al. "The molecular structure of
the Corynebacterium glutamicum
threonine synthase gene," Mol. Microbiol., 4(10):1693-1702 (1990)
X56075 attB-related site Attachment site Cianciotto, N. et al. "DNA sequence
homology between att B-related sites of
Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium
glutamicum , and the attP site of lambdacorynephage," FEMS. Microbiol,
Lett., 66:299-302 (1990)
X57226 lysC-alpha; lysC-beta; Aspartokinase-alpha subunit; Kalinowski, J. et
al. "Genetic and biochemical analysis of the Aspartokinase
asd Aspartokinase-beta subunit; aspartate beta from Corynebacterium
glutamicum," Mol. Microbiol., 5(5):1197-1204 (1991);
semialdehyde dehydrogenase Kalinowski, J. et al. "Aspartokinase genes lysC
alpha and IysC beta overlap
and are adjacent to the aspertate beta-semialdehyde dehydrogenase gene asd in
Corynebacterium glutamicum," Mol. Gen. Genet., 224(3):317-324 (1990)
X59403 gap;pgk; tpi Glyceraldehyde-3-phosphate; Eikmanns, B.J.
"Identification, sequence analysis, and expression of a
phosphoglycerate kinase; triosephosphate Corynebacterium glutamicum gene
cluster encoding the three glycolytic
isomerase enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate
Ln
kinase, and triosephosphate isomeras," J. Bacteriol., 174(19):6076-6086 w
(1992) 0
X59404 gdh Glutamate dehydrogenase Bormann, E.R. et al. "Molecular analysis of
the Corynebacterium glutamicum w
gdh gene encoding glutamate dehydrogenase," Mol. Microbiol., 6(3):317-326 o
(1992) v~ o X60312 lysl L-lysine permease Seep-Feldhaus, A.H. et al.
"Molecular analysis of the Corynebacterium o
glutamicum lysl gene involved in lysine uptake," Mol. Microbiol., 5(12):2995-
3005(1991)
X66078 copl Psl protein Joliff, G. et al. "Cloning and nucleotide sequence of
the cspl gene encoding
PS 1, one of the two major secreted proteins of Corynebacterium glutamicum:
The deduced N-terminal region of PSI 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 IS
1206, a Corynebacterium glutamicum
IS3-related insertion sequence and phylogenetic analysis," Mol. Microbiol.,
14(3):571-581(1994)
Table 2 continued
X70959 IeuA Isopropylmalate synthase Patek, M. et al. "Leucine synthesis in
Corynebacterium glutamicum: enzyme
activities, structure of IeuA, and effect of leuA inactivation on lysine
synthesis," Appl. Environ. Microbiol., 60( l):133-140 (1994)
X71489 icd Isocitrate dehydrogenase (NADP+) Eikmanns, B.J. et al. "Cloning
sequence analysis, expression, and inactivation
of the Corynebacterium glutamicum icd gene encoding isocitrate
dehydrogenase and biochemical characterization of the enzyme," J. Bacteriol.,
177(3):774-782(1995)
X72855 GDHA Glutamate dehydrogenase (NADP+)
X75083, mtrA 5-methyltryptophan resistance Heery, D.M. et al. "A sequence from
a tryptophan-hyperproducing strain of
X70584 Corynebacterium glutamicum encoding resistance to 5-methyltryptophan,"
Biochem. Biophys. Res. Commun., 201(3):1255-1262 (1994)
X75085 recA Fitzpatrick, R. et al. "Construction and characterization of recA
mutant strains
of Corynebacterium glutamicum and Brevibacterium lactofermentum," Appl.
Microbiol. Biotechnol., 42(4):575-580 (1994)
X75504 aceA; thiX Partial Isocitrate lyase; ? Reinscheid, D.J. et al.
"Characterization of the isocitrate lyase gene from
Corynebacterium glutamicum and biochemical analysis of the enzyme," J. Ln
Bacteriol., 176(12):3474-3483 (1994) w
X76875 ATPase beta-subunit Ludwig, W. et al. "Phylogenetic relationships of
bacteria based on comparative o
sequence analysis of elongation factor Tu and ATP-synthase beta-subunit W
genes," Antonie Van Leeuwenhoek, 64:285-305 (1993) o
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 ~ o
genes," Antonie Van Leeuwenhoek, 64:285-305 (1993)
X77384 recA Billman-Jacobe, H. "Nucleotide sequence of a recA gene from
Corynebacterium glutamicum," DNA Seq., 4(6):403-404 (1994)
X78491 aceB Malate synthase Reinscheid, D.J. et al. "Malate synthase from
Corynebacterium glutamicum
pta-ack operon encoding phosphotransacetylase: sequence analysis,"
Microbiology, 140:3099-3108 (1994)
X80629 16S rDNA l6S ribosomal RNA Rainey, F.A. et al. "Phylogenetic analysis
of the genera Rhodococcus and
Norcardia and evidence for the evolutionary origin of the genus Norcardia
from within the radiation of Rhodococcus species," Microbiol., 141:523-528
(1995)
X81191 gluA; gluB; gluC; Glutamate uptake system Kronemeyer, W. et al.
"Structure of the gIuABCD cluster encoding the
gluD glutamate uptake system of Corynebacterium glutamicum," J. BacterioL,
177(5):1152-1158 (1995)
X81379 dapE Succinyldiaminopimelate desuccinylase Wehrmann, A. et al.
"Analysis of different DNA fragments of
Corynebacterium glutamicum complementing dapE of Escherichia coli,"
Microbiology, 40:3349-56 (1994)
Table 2 continued
X82061 16S rDNA 16S ribosomal RNA Ruimy, R. et al. "Phylogeny of the genus
Corynebacterium deduced from
analyses of small-subunit ribosomal DNA sequences," Int. J. Syst. Bacteriol.,
45(4):740-746 (1995)
X82928 asd; lysC Aspartate-semialdehyde dehydrogenase; ? Serebrijski, I. et
al. "Multicopy suppression by asd gene and osmotic stress-
dependent complementation by heterologous proA in proA mutants," J.
Bacteriol., 177(24):7255-7260 (1995)
X82929 proA Gamma-glutamyl phosphate reductase Serebrijski, 1. et al.
"Multicopy suppression by asd gene and osmotic stress-
dependent complementation by heterologous proA in proA mutants," J.
Bacteriol., 177(24):7255-7260 (1995)
X84257 16S rDNA 16S ribosomal RNA Pascual, C. et al. "Phylogenetic analysis of
the genus Corynebacterium based
on 16S rRNA gene sequences," Int. J. Syst. Bacteriol., 45(4):724-728 (1995)
X85965 aroP; dapE Aromatic amino acid permease; ? Wehrmann et al. "Functional
analysis of sequences adjacent to dapE of C.
glutamicum proline reveals the presence of aroP, which encodes the aromatic
amino acid transporter," J. Bacteriol., 177(20):5991-5993 (1995)
X86157 argB; argC; argD; Acetylglutamate kinase; N-acetyl-gamma- Sakanyan, V.
et al. "Genes and enzymes of the acetyl cycle of arginine
argF; argJ glutamyl-phosphate reductase; biosynthesis in Corynebacterium
glutamicum: enzyme evolution in the early cn
acetylornithine aminotransferase; ornithine steps of the arginine pathway,"
Microbiology, 142:99-108 (1996) w
carbamoyltransferase; glutamate N- o
acetyltransferase W
X89084 pta; ackA Phosphate acetyltransferase; acetate kinase Reinscheid, D.J.
et al. "Cloning, sequence analysis, expression and inactivation o
of the Corynebacterium glutamicum pta-ack operon encoding
phosphotransacetylase and acetate kinase," Microbiology, 145:503-513 (1999)
0
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) 1O
X90356 Promoter fragment Fl Patek, M. et al. "Promoters from Corynebacterium
glutamicum: cloning,
molecular analysis and search for a consensus motif," Microbiology,
142:1297-1309 (1996)
X90357 Promoter fragment F2 Patek, M. et al. "Promoters from Corynebacterium
glutamicum: cloning,
molecular analysis and search for a consensus motif," Microbiology,
142:1297-1309(1996)
X90358 Promoter fragment F10 Patek, M. et al. "Promoters from Corynebacterium
glutamicum: cloning,
molecular analysis and search for a consensus motif," Microbiology,
142:1297-1309 (1996)
X90359 Promoter fragment F 13 Patek, M. et al. "Promoters from Corynebacterium
glutamicum: cloning,
molecular analysis and search for a.consensus motif," Microbiolosy,
142:1297-1309(1996)
Table 2 continued
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 a1. "Promoters from C. 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, o
molecular analysis and search for a consensus motif," Microbiology, Lõ
142:1297-1309 (1996) W
X90366 Promoter fragment PF 101 Patek, M. et al. "Promoters from
Corynebacterium glutamicum: cloning, o
molecular analysis and search for a consensus motif," Microbiology, W
142:1297-1309 (1996) X90367 Promoter fragment PF104 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning, O
molecular analysis and search for a consensus motif," Microbiology,
142:1297-1309 (1996) X90368 Promoter fragment PF 109 Patek, M. et al.
"Promoters from Corynebacterium glutamicum: cloning,
molecular analysis and search for a consensus motif," Microbiology,
142:1297-1309 (1996)
X93513 amt Ammonium transport system Siewe, R.M. et al. "Functional and
genetic characterization of the (methyl)
ammonium uptake carrier of Corynebacterium glutamicum," J. Biol. Chem.,
271(10):5398-5403(1996)
X93514 betP Glycine betaine transport system Peter, H. et al. "Isolation,
characterization, and expression of the
Corynebacterium glutamicum betP gene, encoding the transport system for the
compatible solute glycine betaine," J. Bacteriol., 178(17):5229-5234 (1996)
X95649 orf4 Patek, M. et al. "Identification and transcriptional analysis of
the dapB-ORF2-
dapA-ORF4 operon of Corynebacterium glutamicum, encoding two enzymes
involved in L-lysine synthesis," Biotechnol. Lett., 19:1113-1117 (1997)
X96471 lysE; lysG Lysine exporter protein; Lysine export Vrljic, M. et at. "A
new type of transporter with a new type of cellular
regulator protein function: L-tysine export from Corynebacterium glutamicum,"
Mol.
Microbiol., 22(5):815-826 (1996)
Table 2 (continued)
X96580 panB; panC; xylB 3-methyl-2-oxobutanoate Sahm, H. et al. "D-
pantothenate synthesis in Corynebacterium glutamicum and
hydroxymethyltransferase; pantoate-beta- use of panBC and genes encoding L-
valine synthesis for D-pantothenate
alanine ligase; xylulokinase overproduction," Appl. Environ. Microbiol.,
65(5):1973-1979 (1999)
X96962 Insertion sequence IS 1207 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., W
-.3
15(24):10598 (1987) . 0
Y00546 hom; thrB Homoserine dehydrogenase; homoserine Peoples, O.P. et al.
"Nucleotide sequence and fine structural analysis of the
kinase Corynebacterium glutamicum hom-thrB operon," Mol. Microbiol., 2(1):63-
72
(1988) -.3
Y08964 murC; ftsQ/divD; ftsZ UPD-N-acetylmuramate-alanine ligase; Honrubia,
M.P. et al. "Identification, characterization, and chromosomal
division initiation protein or cell division organizatiQn of the ftsZ gene
from Brevibacterium lactofermentum," MoL Gen.
protein; cell division protein Genet., 259(1):97-104 (1998)
Y09163 putP High affinity proline transport system Peter, H. et al. "Isolation
of the putP gene of Corynebacterium
glutamicumproline and characterization of a low-afftnity 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(l):42-47 (1998)
Y12472 Attachment site bacteriophage Phi-l6 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)
Table 2 continued
Y13221 ginA Glutamine synthetase I Jakoby, M. et al. "Isolation of
Corynebacterium glutamicum g1nA gene
encoding glutamine synthetase I," FEMS Microbiol. Lett., 154(1):81-88 (1997)
Y16642 lpd Dihydrolipoamide dehydrogenase
Y 18059 Attachment site Corynephage 304L Moreau, S. et al. "Analysis of the
integration functions of φ304L: An
integrase module among corynephages," Virology, 255(l):150-159 (1999)
Z21501 argS; lysA Arginyl-tRNA synthetase; diaminopimelate Oguiza, J.A. et al.
"A gene encoding arginyl-tRNA synthetase is located in the
decarboxylase (partial) upstream region of the lysA gene in Brevibacterium
lactofenmentum:
Regulation of argS-lysA cluster expression by arginine," J.
Bacteriol.,175(22):7356-7362 (1993)
Z21502 dapA; dapB Dihydrodipicolinate synthase; Pisabarro, A. et al. "A
cluster of three genes (dapA, orf2, and dapB) of
dihydrodipicolinate reductase Brevibacterium lactofermentum encodes
dihydrodipicolinate reductase, and a
third polypeptide of unknown function," J. Bacterrol., 175(9):2743-2749
(1993)
Z29563 thrC Threonine synthase Malumbres, M. et al. "Analysis and expression
of the thrC gene of the encoded
threonine synthase," Appl. Environ. Microbiol., 60(7)2209-2219 (1994)
Z46753 16S rDNA Gene for 16S ribosomal RNA Lõ
Z49822 sigA SigA sigma factor Oguiza, J.A. et al "Multiple sigma factor genes
in Brevibacterium w
lactofermentum: Characterization of sigA and sigB," J. BacterioL, 178(2):550-
o
553 (1996) w
Z49823 galE; dtxR Catalytic activity UDP-galactose 4- Oguiza, J.A. et al "The
galE gene encoding the UDP-galactose 4-epimerase of oo No
epimerase; diphtheria toxin regulatory Brevibacterium lactofermentum is
coupled transcriptionally to the dmdR o
-.3
protein gene," Gene, 177:103-107 (1996)
Z49824 orfl; sigB ?; SigB sigma factor Oguiza, J.A. et al "Multiple sigma
factor genes in Brevibacterium
lactofermentum: Characterization of sigA and sigB," J. BacterioL, 178(2):550-
553 (1996)
Z66534 Transposase Correia, A. et al. "Cloning and characterization of an IS-
like element present in
the genome of Brevibacterium lactofermentum ATCC 13869," Gene,
170(1):91-94(1996)
A 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.
CA 02583703 2007-04-19
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TABLE 3: Corynebacterium and Brevibacterium Strains Which May be Used in
the Practice of the Invention
Gep . ;., species ATC Im-gM NRIt CECT NCIMB DSJWZ
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 B 11474
Brevibacterium flavum B 11472
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 B 11478
Brevibacterium flavum 21127
Brevibacterium flavum B 11474
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 B 11470
Brevibacterium lactofetmentum B 11471
CA 02583703 2007-04-19
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G~euus ;~ ~ : ; speeies ~ +C F~RM I: ~CE,CT~ NGINB ;t~m NW PSMZ;
, ~.. ~., .. . -
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 B 11473
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
CA 02583703 2007-04-19
-83-
Cenus .:; A~; species ~',1TCGV 1FERM :NRRL e1" CT_'1!TGIMB ; CBS ' NCTC DS1NZ
x
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 B 12416
Corynebacterium glutamicum B12417
CA 02583703 2007-04-19
-84-
Genus . . -:; . _ .: s"4~,ecies FERM C~C~T NCIMB =: CBS,~ NCTC :DSMZ~
_.
Corynebacterium glutamicum B 12418
Corynebacterium glutamicum B 11476
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 Sammiung 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'" edn), World federation for
culture collections world
data center on microorganisms, Saimata, Japen.
Table 4: Alianment Results
ID # length Genbank Hit LRnath Accession Name of Genbank Hit Source of Genbank
Hit % homoloav Date of
(NT) (GAPI Deposit
nca00051 1527 GB_HTG3:AC009685 210031 AC009685 Homo sapiens chromosome 15
clone 91 E 13 map 15, SEQUENCING IN Homo sapiens 34,247 29Sep-99
PROGRESS =", 27 unordered pieces.
GB_HTG3:AC009685 210031 AC009685 Homo sapiens chromosome 15 clone 91_E_13 map
15, SEQUENCING IN Homo sapiens 34,247 29-Sep-99
PROGRESS "', 27 unordered pieces.
GB HTG7:AC009511 271896 AC009511 Homo sapiens clone RP11-860B13, "= SEQUENCING
IN PROGRESS "', 59 Homo sapiens 35,033 09-DEC-1999
unordered pieces,
nca00091 876 GB BA1:D50453 146191 D50453 Bacillus subtilis DNA for 25-36
degree region containing the amyE-srfA region, Bacillus subtilis 54,452 10-Feb-
99
complete cds.
GB BAI:SCI51 40745 AL109848 Streptomyces coelicolor cosmid 151. Streptomyces
coelicolor 36,806 16-Aug-99
A3(2)
GB_BA1:ECOUW93 338534 U14003 Escherichia coli K-12 chromosomal region from
92.8 to 00.1 minutes. Escherichia coli 38,642 17-Apr-96
rxa00092 789 GB_BAI:SCH35 45396 AL078610 Streptomyces coelicolor cosmid H35.
Streptomyces coelicolor 49,934 4-Jun-99
Ln
GB_HTG3:AC011498_ 312343 AC011498 Homo sapiens chromosome 19 clone CIT978SK6
50L17, '=' SEQUENCING IN Homo sapiens 37,117 13-Dec-99 W
0 PROGRESS "', 190 unordered pieces. .3
GB HTG3:AC011498_ 312343 AC011498 Homo sapiens chromosome 19 clone CIT978SKB
50L17, SEQUENCING IN Homo sapiens 37,117 13-Dec-99 0
w
0 PROGRESS "', 190 unordered pieces.
nca00104 879 GB_BA1:MTCY270 37586 Z95388 Mycobacterium tuberculosis H37Rv
complete genome; segment 96/162. Mycobacterium 36,732 10-Feb-99 o
tuberculosis
.3
GB PL2:T24M8 68251 AF077409 Arabidopsis thaliana BAC T24M8. Arabidopsis
thaliana 37,150 3-Aug-98 = 1
GB BAI:MTCY270 37586 Z95388 Mycobacterium tuberculosis H37Rv complete genome;
segment 96/162. Mycobacterium 42,874 10-Feb-99 r.
tuberculosis 1
r
rxa00113 5745 GB BA1:MAFASGEN 10520 X87822 B.ammoniagenes FAS gene.
Corynebacterium 68.381 03-OCT-1996
ammoniagenes
GB BA1:BAFASAA 10549 X64795 B.ammoniagenes FAS gene. Corynebacterium 57,259 14-
OCT-1997
ammoniagenes
GB BA1:MTCY159 33818 Z83863 Mycobacterium tuberculosis H37Rv complete genome;
segment 111/162. Mycobacterium 39,870 17-Jun-98
tuberculosis
rxa00164 1812 GB_HTG2:HSJ1153D9 118360 AL109806 Homo sapiens chromosome 20
clone RP5-1153D9, SEQUENCING IN Homo sapiens 35,714 03-DEC-1999
PROGRESS "', in unordered pieces.
GB HTG2:HSJ1153D9 118360 AL109806 Homo sapiens chromosome 20 clone RP5-1153D9,
SEQUENCING IN Homo sapiens 35,714 03-DEC-1999
PROGRESS "', in unordered pieces.
GB_HTG2:HSJ115309 118360 AL109806 Homo sapiens chromosome 20 clone RP5-1153D9,
SEQUENCING IN Homo sapiens 35,334 03-DEC-1999
PROGRESS "', in unordered pieces. _
nca00181 1695 GB BAI:CGPUTP 3791 Y09163 C.glutamicum putP gene.
Corynebacterium 100,000 8-Sep-97
glutamicum
GB BA2:U32814 10393 U32814 Haemophilus influenzae Rd section 129 of 163 of the
complete genome. Haemophilus influenzae 36,347 29-MAY-1998
Rd
GB_BAI:CGPUTP 3791 Y09163 C.glutamicum putP gene. Corynebacterium 37,454 8-Sep-
97
glutamicum
nca00186 870 GB PR3:AC004843 136655 AC004843 Homo sapiens PAC clone DJ0612F12
from 7p12-p14, complete sequence. Homo sapiens 37,315 5-Nov-98
Table 4 (continued)
GB_HTG2:HS745114 133309 AL033532 Homo sapiens chromosome 1 clone RP4-745114
map q23.1-24.3, '=' Homo sapiens 38,129 03-DEC-1999
SEQUENCING IN PROGRESS ""', in unordered pieces.
GB HTG2:HS745t14 133309 AL033532 Homo sapiens chromosome 1 clone RP4-745114
map q23.1-24.3, Homo sapiens 38,129 03-DEC-1999
SEQUENCING IN PROGRESS "', in unordered pieces.
nca00187 474 GB_GSS10:AQ184082 506 AQ184082 HS_3216 A1 G08_T7 CIT Approved
Human Genomic Sperm Library D Homo Homo sapiens 37,297 1-Nov-98
sapiens genomic clone Plate=3216 Col=15 Row=M, genomic survey sequence.
GB GSS1:CNS008ZZ 1101 AL052951 Drosophila melanogaster genome survey sequence
T7 end of BAC # Drosophila melanogaster 34,120 3-Jun-99
BACR18L01 of RPCI-98 library from Drosophila melanogaster (fruR fly), genomic
survey sequence.
GB GSS10:AQ184082 506 AQ184082 HS_3216 A1_G08_T7 CIT Approved Human Genomic
Sperm Library D Homo Homo sapiens 39,655 1-Nov-98
sapiens genomic clone Plate=3216 Col=15 Row=M, genomic survey sequence.
nca00201 292 GB_PR3:HSJ824F16 139330 AL050325 Human DNA sequence from clone
824F16 on chromosome 20, complete Homo sapiens 34,520 23-Nov-99
sequence.
GB BA1:RCSECA 2724 X89411 R.capsulatus DNA for secA gene. Rhodobacter
capsulatus 38,163 6-Jan-96
GB EST34:AV122904 242 AV122904 AV122904 Mus musculus C57BU6J 10-day embryo Mus
musculus cDNA clone Mus musculus 38,889 1-Ju1-99
2610529H07, mRNA sequence. Li,
rxa00228 714 GB_EST15:AA486042 515 AA486042 ab40c08.r1 Stratagene HeLa cell s3
937216 Homo sapiens cDNA clone Homo sapiens 37,500 06-MAR-1998 D
IMAGE:843278 5', mRNA sequence.
GB EST15:AA486042 515 AA486042 ab40c08.r1 Stratagene HeLa cell s3 937216 Homo
sapiens cDNA ctone Homo sapiens 38,816 06-MAR-1998 Co w
IMAGE:843278 5', mRNA sequence. N
0
0
rxa00243 1140 GB_PR2:CNSO1DS5 101584 AL121655 BAC sequence from the SPG4
candidate region at 2p21-2p22, complete Homo sapiens 37,001 29-Sep-99
GB HTG3:AC011408 79332 AC011408 Homo sapiens clone CIT978SKB 65D22, SEQUENCING
IN PROGRESS Homo sapiens 38,040 06-OCT-1999 0
unordered pieces. r.
GB_HTG3:AC011408 79332 AC011408 Homo sapiens clone CIT978SKB_65D22, SEQUENCING
IN PROGRESS Homo sapiens 38.040 06-OCT-1999
10 unordered pieces.
nca00259 2325 GB HTGt:CEY62E10 254217 AL031580 Caenorhabditis elegans
chromosome IV clone Y62E10, "= SEQUENCING IN Caenorhabditis elegans 36,776 6-
Sep-99
PROGRESS ===, in unordered pieces.
GB_HTG1:CEY62E10 254217 AL031580 CaenorhabdRis elegans chromosome IV clone
Y62E10, SEQUENCING IN Caenorhabditis elegans 36,776 6-Sep-99
PROGRESS "=, in unordered pieces.
GB PL2:YSCCHROMt 41988 L22015 Saccharomyces cerevisiae chromosome I centromere
and right arm sequence. Saccharomyces 39.260 05-MAR-1998
cerevisiae
rxa00269 912 GB_HTG4:AC009974 219565 AC009974 Homo sapiens chromosome unknown
clone NH0459119, WORKING DRAFT Homo sapiens 37,358 29-OCT-1999
SEQUENCE, in unordered pieces.
GB HTG4:AC009974 219565 AC009974 Homo sapiens chromosome unknown clone
NH0459119, WORKING DRAFT Homo sapiens 37,358 29-OCT-1999
SEQUENCE, in unordered pieces.
GB BA1:AB017508 32050 AB017508 Bacillus halodurans C-125 genomic DNA, 32 kb
fragment, complete cds. Bacillus halodurans 44,622 14-Apr-99 -
nca00281 766 GB_BA1:SCE8 24700 AL035654 Streptomyces coelicolor cosmid E8.
Streptomyces coelicolor 36,328 11-MAR-1999
GB BAI:SCU51332 3216 U51332 Streptomyces coelicolor histidine kinase homolog
(absAl) and response Streptomyces coelicolor 39,089 14-Sep-96
regulator homolog (absA2) genes, complete cds.
GB HTG4:AC011122 187123 AC011122 Homo sapiens chromosome 8 clone 23_D_19 map
8, *** SEQUENCING IN Homo sapiens 38,658 14-OCT-1999
PROGRESS ="', 27 ordered pieces.
Table 4 (continued)
rxa00298 1968 GB_BAI:CGECTP 2719 AJ001436 Corynebacterium glutamicum ectP
gene. Corynebacterium 100,000 20-Nov-98
glutamicum
GB BA1:CGECTP 2719 AJ001436 Corynebacterium glutamicum ectP gene.
Corynebacterium 100,000 20-Nov-98
glutamicum
GB EST24:AI234006 432 A1234006 EST230694 Normalized rat lung, Bento Soares
Rattus sp. cONA clone Rattus sp. 46,552 31-Jan-99
RLUCU01 3' end, mRNA sequence.
rxa00346 813 GB BA1:SC2E9 20850 AL021530 Streptomyces coelicolor cosmid 2E9.
Streptomyces coelicolor 43,267 28-Jan-98
GB_BA1:SC9B1 24800 AL049727 Streptomyces coelicolor cosmid 9B1. Streptomyces
coelicolor 44,613 27-Apr-99
GB_BAI:ECU70214 123171 U70214 Escherichia coli chromosome minutes 4-6.
Escherichia coli 39,490 21-Sep-96
rxa00368 1698 GB_BA2:AF065159 35209 AF065159 Bradyrhizobium japonicum putative
arylsulfatase (arsA), putative soluble lytic Bradyrhizobium 40,409 27-OCT-1999
transgtycosylase precursor (sltA), dihydrodipicolinate synthase (dapA), MscL
japonicum
(mscL), SmpB (smpB), BcpB (bcpB), RnpO (mpO), ReIAlSpoT homolog (relA),
PdxJ (pdxJ), and acyl carrier protein synthase AcpS (acpS) genes, complete
cds;
prokaryotic type I signal peptidase SipF (sipF) gene, sipF-sipS allele,
complete
cds; RNase III (mc) gene, complete cds; GTP-binding protein Era (era) gene,
partial cds; and unknown genes. Ln
GB_BA1:AEOCHIT1 6861 D63139 Aeromonas sp. gene for chitinase, complete and
partial cds. Aeromonas sp. 10S-24 38,577 13-Feb-99 ~ D,,
GB_EST4:D62996 314 D62996 HUM347G01B Clontech human aorta polyA+ mRNA (#6572)
Homo sapiens Homo sapiens 41,613 29-Aug-95 -.3
cDNA done GEN-347G01 5', mRNA sequence. w
nca00369 817 GB BA1:YP102KB 119443 AL031866 Yersinia pestis 102 kbases
unstable region: from 1 to 119443. Yersinia pestis 35,396 4-Jan-99 ~ rv
GB_GSS8:AQ012142 501 AQ012142 8750H1A037010398 Cosmid library of chromosome II
Rhodobacter sphaeroides Rhodobacter 54,800 4-Jun-98
genomic clone 8750H1A037010398, genomic survey sequence. sphaeroides
GB HTG2:AC005081 180096 AC005081 Homo sapiens clone RG270D13, *** SEQUENCING
IN PROGRESS "', 18 Homo sapiens 45,786 12-Jun-98 0
unordered pieces.
rxa00410 789 GB BA1:ATPLOCC 8870 Z30328 A.tumefaciens Ti plasmid pTiAch5 genes
for OccR, OccQ, OccM, OccP, OccT, Agrobacterium 46,490 10-OCT-1994
OoxB, OoxA and ornithine cyclodeaminase. tumefaciens
GB BA2:U67591 9829 U67591 Methanococcus jannaschii section 133 of 150 of the
complete genome. Methanococcus 45,677 28-Jan-98
jannaschii
GB BA1:TIPOCCQMPJ 4350 M80607 Plasmid pTiA6 (from Agribacterium tumefaciens)
periplasmic-type octopine Plasmid pTiA6 46,490 24-Apr-96
permease (occR, occQ, occM, occP, and occJ) and IysR-type regulatory protein
(occR) genes, complete cds.
rxa00419 882 GB_BA2:MSU46844 16951 U46844 Mycobacterium smegmatis catalase-
peroxidase (katG), putative arabinosyl Mycobacterium 57,029 12-MAY-1997
transferase (embC, embA, embB), genes complete cds and putative propionyl-
smegmatis
coA carboxylase beta chain (pccB) genes, partial cds.
GB_EST28:AI513245 471 A1513245 GH13311.3prime GH Drosophila melanogaster head
pOT2 Drosophila Drosophila melanogaster 37,696 16-MAR-1999
melanogaster cDNA clone GH13311 3prime, mRNA sequence.
GB_HTG4:AC010066 187240 AC010066 Drosophila melanogaster chromosome 3U72A4
clone RPCI98-2501, Drosophila melanogaster 39,607 16-OCT-1999 -
SEQUENCING IN PROGRESS "', 70 unordered pieces.
rxa00432 1608 GB_BAI:BSUB0015 218410 Z99118 Bacillus subtilis complete genome
(section 15 of 21): from 2795131 to 3013540. Bacillus subtilis 49,810 26-Nov-
97
GBPLI:CAC35A5 42565 AL033396 C.albicans cosmid Ca35A5. Candida albicans 35,041
5-Nov-98
GB__EST13:AA336266 378 AA336266 EST40981 Endometrial tumor Homo sapiens cDNA
5' end, mRNA sequence. Homo sapiens 39,733 21-Apr-97
Table 4 (continued)
nca00449 1704 GB_HTG2:AC008199 124050 AC008199 Drosophila melanogaster
chromosome 3 clone BACR01 K08 (D756) RPCI-98 Drosophila melanogaster 38,392 2-
Aug-99
01.K.8 map 94D-94D strain y; cn bw sp, =" SEQUENCING IN PROGRESS
83 unordered pieces.
GB_HTG2:AC008199 124050 AC008199 Drosophiia melanogaster chromosome 3 clone
BACR01 K08 (D756) RPCI-98 Drosophila melanogaster 38,392 2-Aug-99
01.K.8 map 94D-94D strain y; cn bw sp, "' SEQUENCING IN PROGRESS '=',
83 unordered pieces.
GB RO:RATLNKP2 177 M22337 Rat link protein gene, exon 2. Rattus sp. 40,678 27-
Apr-93
rxa00456 1500 GB GSS1:FR0030597 476 AL026966 Fugu rubripes GSS sequence, clone
091C22aF9, genomic survey sequence. Fugu rubripes 47,407 25-Jun-98
GB GSS5AQ786587 556 AQ786587 HS_3086_B1_H05_MR CIT Approved Human Genomic
Sperm Library D Homo Homo sapiens 38,406 3-Aug-99
sapiens genomic clone Plate=3086 Col=9 Row=P, genomic survey sequence.
GB_GSS14:AQ526586 434 AQ526586 HS 5198_B1 B03_SP6E RPCI-11 Human Male BAC
Library Homo sapiens Homo sapiens 36,951 11-MAY-1999
genomic clone Plate=774 Col=5 Row=D, genomic survey sequence.
nca00477 1767 GB EST17:AA610489 407 AA610489 np93e05.s1 NCI_CGAP Thy1 Homo
sapiens cONA clone IMAGE:1133888 Homo sapiens 41,791 09-DEC-1997
similar to gb:M11353 HISTONE H3.3 (HUMAN);, mRNA sequence.
GB PRI:HSH33G4 1015 X05857 Human H3.3 gene exon 4. Homo sapiens 38,182 24-Jan-
96 GB EST30:AI637667 579 AI637667 tt10g11.x1 NCI CGAP GC6 Homo sapiens cDNA
clone IMAGE:2240420 3', Homo sapiens 35,417 27-Apr-99 L"
mRNA sequence. D
w
nca00478 954 GB HTG3:AC008708 83932 AC008708 Homo sapiens chromosome 5 clone
CIT978SKB_78F1, SEQUENCING IN Homo sapiens 38.769 3-Aug-99 0
PROGRESS "', 12 unordered pieces. w
GB_HTG3:AC008708 83932 AC008708 Homo sapiens chromosome 5 clone CIT978SKB
78F1, '== SEQUENCING IN Homo sapiens 38,769 3-Aug-99 c~o
PROGRESS "=, 12 unordered pieces. o
GB HTG3:AC008708 83932 AC008708 Homo sapiens chromosome 5 clone CIT978SKB
78F1, SEQUENCING IN Homo sapiens 36,797 3-Aug-99 - .3
PROGRESS "', 12 unordered pieces.
rxa00480 1239 GB HTGI:HSJ575L21 94715 AL096841 Homo sapiens chromosome 1 clone
RP4-575L21, SEQUENCING IN Homo sapiens 38,138 23-Nov-99
PROGRESS "', in unordered pieces.
GB HTG1:HSJ575L21 94715 AL096841 Homo sapiens chromosome 1 clone RP4-575L21,
SEQUENCING IN Homo sapiens 38,138 23-Nov-99
PROGRESS "', in unordered pieces.
GB_RO:AC005960 158414 AC005960 Mus musculus chromosome 17 BAC citb20h22 from
the MHC region, complete Mus musculus 38,712 01-DEC-1998
sequence.
rca00524 433 GB_BAI:SCI51 40745 AL109848 Streptomyces coelicolor cosmid 151.
Streptomyces coelicolor 40,284 16-Aug-99
A3(2)
GB BA2:AF082879 3434 AF082879 Yersinia enterocolitica ABC transporter
enterochelin/enterobactin gene cluster, Yersinia enterocolitica 55,634 20-OCT-
1999
complete sequence.
GB BA1:BSP132617 5192 AJ132617 Burkholderia sp. P-transporter operon and
flanking genes. Burkholderia sp. 40,793 13-Jul-99
nca00526 813 GBBA1:BSUB0008 208230 Z99111 Bacillus subtilis complete genome
(section 8 of 21): from 1394791 to 1603020. Bacillus subtilis 54,534 26-Nov-97
GB BA2:AF012285 46864 AF012285 Bacillus subtilis mobA-nprE gene region.
Bacillus subtilis 54,534 1-Jul-98
GB_BA1:D90725 13796 D90725 Escherichia coli genomic DNA. (19.7 - 20.0 min).
Escherichia coli 51,481 7-Feb-99
rxa00559 1140 GB BA2:CAU77910 3385 U77910 Corynebacterium ammoniagenes
sequence upstream of the 5-phosphoribosyl-l- Corynebacterium 39,007 1-Jan-98
pyrophosphate amidotransferase (purF) gene. ammoniagenes
GB EST4:H34952 382 H34952 EST108261 Rat PC-12 cells, untreated Rattus sp. cDNA
clone RPCCK07 similar Rattus sp. 39,267 2-Apr-98
to NADH-ubiquinone oxidoreductase complex 123 kDa precursor (iron-sulfur
protein), mRNA sequence.
GB BA2:AE000963 22014 AE000963 Archaeoglobus fulgidus section 144 of 172 of
the complete genome. Archaeoglobus fulgidus 38,338 15-DEC-1997
Table 4 (continued)
nca00570 852 GB GSS12AQ422451 563 A0422451 RPCI-11-185C3.TV RPCI-11 Homo
sapiens genomic clone RPCI-11-185C3, Homo sapiens 38,767 23-MAR-1999
genomic survey sequence.
GB EST28:AI504741 568 A1504741 vI16c01.x1 Stratagene mouse Tce{I 937311 Mus
musculus cDNA clone Mus musculus 37,900 11-MAR-1999
IMAGE:972384 3' similar to gb:Z14044 M.musculus mRNA for valosin-containing
protein (MOUSE);, mRNA sequence.
GB ESTI8:AA712043 68 AA712043 vu29f10.0 Barstead mouse myotubes MPLRB5 Mus
musculus cDNA clone Mus musculus 42,647 24DEC-1997
IMAGE:1182091 5' similar to gb:L05093 60S RIBOSOMAL PROTEIN L18A
(HUMAN);, mRNA sequence.
rxa00571 1280 GB BA1:MTCY78 33818 Z77165 Mycobacterium tuberculosis H37Rv
complete genome; segment 145/162. Mycobaderium 38,468 17-Jun-98
tuberculosis
GB PR3:AC005788 36224 AC0057B8 Homo sapiens chromosome 19, cosmid R26652,
complete sequence. Homo sapiens 36,911 06-OCT-1998
GB PR3:AC005338 34541 AC005338 Homo sapiens chromosome 19, cosmid R31646,
complete sequence. Homo sapiens 36,911 30-Jul-98
rxa00590 1288 GB_HTG6:AC010932 203273 AC010932 Homo sapiens chromosome 15
clone RP1 1-296E22 map 15, SEQUENCING Homo sapiens 37,242 30-Nov-99
IN PROGRESS ='", 36 unordered pieces.
GB HTG6:AC010932 203273 AC010932 Homo sapiens chromosome 15 clone RP1 1-296E22
map 15, SEQUENCING Homo sapiens 36,485 30-Nov-99
IN PROGRESS ="', 36 unordered pieces. Ln
GB_BA1:MSGB26CS 37040 L78816 Mycobacterium leprae cosmid B26 DNA sequence.
Mycobacterium leprae 39,272 15-Jun-96 w
nca00591 1476 GB INI:CEK09E9 30098 Z79602 Caenorhabditis elegans cosmid K09E9,
complete sequence. Caenorhabditis elegans 34,092 2-Sep-99 0
GB PR4:AF135802 4965 AF135802 Homo sapiens thyroid hormone receptor-associated
protein complex component Homo sapiens 36,310 9-Apr-99 po w
TRAP170 mRNA, complete cds. rv
GB_PR4:AF104256 4365 AF104256 Homo sapiens transcriptional co-activator
CRSP150 (CRSP150) mRNA, Homo sapiens 36,617 4-Feb-99
complete cds.
rxa00596 576 GB_PR3:AC004659 129577 AC004659 Homo sapiens chromosome 19, CIT-
HSP-87m17 BAC clone, complete Homo sapiens 34,321 02-MAY-1998
GBPR3:AC004659 129577 AC004659 Homo sapiens chromosome 19, CIT-HSP-87m17 BAC
clone, complete Homo sapiens 35,739 02-MAY-1998
GB_PR1:HUMCBP2 2047 D83174 Human mRNA for collagen binding protein 2, complete
cds. Homo sapiens 40,404 6-Feb-99
nca00607 504 GB BA1:MTV010 3400 AL021186 Mycobacterium tuberculosis H37Rv
complete genome; segment 119/162. Mycobacterium 40,862 23-Jun-99
tuberculosis
GB BA1:MTV010 3400 AL021186 Mycobacterium tuberculosis H37Rv complete genome;
segment 119/162. Mycobacterium 38,833 23-Jun-99
tuberculosis
nca00623 1461 GB BA1:MTCY428 26914 Z81451 Mycobacterium tuberculosis H37Rv
complete genome; segment 107/162. Mycobacterium 60,552 17-Jun-98
tuberculosis
GB_BA1:RSPNGR234 34010 Z68203 Rhizobium sp. plasmid NGR234a DNA. Rhizobium sp.
51,992 8-Aug-96
GB_BA2:AE000101 10057 AE000101 Rhi2obium sp. NGR234 plasmid pNGR234a, section
38 of 46 of the complete Rhizobium sp. NGR234 51,992 12-DEC-1997
plasmid sequence.
rxa00681
nca00690 1269 GB HTG5:AC008338 136685 AC008338 Drosophila melanogaster
chromosome X clone BACR30JO4 (D908) RPCI-98 Drosophila melanogaster 35,341 15-
Nov-99
30.J.4 map 19C-19E strain y; cn bw sp, "' SEQUENCING IN PROGRESS
93 unordered pieces.
GB HTG4:AC009766 170502 AC009766 Homo sapiens chromosome 11 clone 404 A 03 map
11, "' SEQUENCING IN Homo sapiens 37,984 19-OCT-1999
PROGRESS "', 27 unordered pieces.
Table 4(continued)
GB HTG4:AC009766 170502 AC009766 Homo sapiens chromosome 11 clone 404 A 03 map
11, "' SEQUENCING IN Homo sapiens 37,984 19-OCT-1999
PROGRESS'=', 27 unordered pieces.
rxa00733 1008 GB EST30:AU054038 245 AU054038 AU054038 Dictyostelium discoideum
SL (Ii.Urushihara) Dictyostelium Dictyostelium discoideum 43,265 28-Apr-99
discoideum cDNA clone SLK472, mRhtA sequence.
GB_EST30:AU054038 245 AU054038 AU054038 Dictyostelium discoideum SL
(H.Urushihara) Dictyostelium Dictyostelium discoideum 43,265 28-Apr-99
discoideum cDNA clone SLK472, mRNA sequence.
rxa00735 692 GB_BA1:MTCY50 36030 Z77137 Mycobacterium tuberculosis H37Rv
complete genome; segment 551162. Mycobacterium 36,819 17-Jun-98
tuberculosis
GBBA1:D90904 150894 D90904 Synechocystis sp. PCC6803 complete genome, 6/27,
630555-781448. Synechocystis sp. 52,585 7-Feb-99
GB__BA1:D90904 150894 D90904 Synechocystis sp. PCC6803 complete genome, 6/27,
630555-781448. Synechocystis sp. 39,699 7-Feb-99
rxa00796 298 GB_GSS14:AQ579838 651 AQ579838 T135342b shotgun sub-library of
BAC clone 31 P06 Medicago truncatula Medicago truncatula 37,153 27-Sep-99
genomic clone 31-P-06-C-054, genomic survey sequence.
G8 PR4:AC007625 174701 AC007625 Genomic sequence of Homo sapiens clone 2314F2
from chromosome 18, Homo sapiens 38,014 30-Jun-99
complete sequence.
GB EST14:AA427576 580 AA427576 zw54b04.s1 Soares total fetus_Nb2HF8_9w Homo
sapiens cDNA clone Homo sapiens 42,731 16-OCT-1997 Ln
00
IMAGE:773839 3' similar to gb:M86852 PEROXISOME ASSEMBLY FACTOR-1 w
.3
(HUMAN);, mRNA sequence.
nca00B01 756 GB_BAI:MTV022 13025 AL021925 Mycobacterium tuberculosis H37Rv
complete genome; segment 100/162. Mycobacterium 59,350 17-Jun-98 w
tuberculosis
GB_RO:AC002109 160048 AC002109 Genomic sequence from Mouse 9, complete
sequenoe. Mus musculus 39,398 9-Sep-97 p o
GB_BA1:MTV022 13025 AL021925 Mycobacterium tuberculosis H37Rv complete genome;
segment 100/162. Mycobacterium 36,842 17-Jun-98 - 1 .3
tuberculosis
rxa00802 837 GB_GSS14:AQ563349 642 AQ563349 HS 5335_B2 A09_T7A RPCI-11 Human
Male BAC Library Homo sapiens Homo sapiens 37,649 29-MAY-1999
genomic clone Plate=911 Co1=18 Row=B, genomic survey sequence.
GB BA1:DIHCLPBA 2441 M32229 B.nodosus clpB gene encoding a regulatory subunit
of ATP-dependent protease. Dichetobacter nodosus 41,140 26-Apr-93
GB GSS3:B61538 698 B61538 T17M17TR TAMU Arabidopsis thaliana genomic clone
T17M17, genomic survey Arabidopsis thaliana 36,946 21-Nov-97
sequence.
rxa0UB19 1452 GB HTG3AC008691_ 110000 AC008691 Homo sapiens chromosome 5 clone
CIT978SK6 63A22, '== SEQUENCING IN Homo sapiens 38,270 3-Aug-99
1 PROGRESS "=, 253 unordered pieces.
GB_HTG3:AC008691_ 110000 AC008691 Homo sapiens chromosome 5 clone CIT978SKB
63A22, SEQUENCING IN Homo sapiens 38,270 3-Aug-99
1 PROGRESS "', 253 unordered pieces.
GB HTG3:AC009127 186591 AC009127 Homo sapiens chromosome 16 clone RPCI-
11_498D10, SEQUENCING IN Homo sapiens 38,947 3-Aug-99
PROGRESS "', 49 unordered pieces.
nca00821 966 GB_HTGI:HS32B1 271488 AL023693 Homo sapiens chromosome 6 clone
RP1-32B1, '== SEQUENCING IN Homo sapiens 36,565 23-Nov-99
PROGRESS "', in unordered pieces.
GB_HTG1:HS32B1 271488 AL023693 Homo sapiens chromosome 6 clone RP1-32B1,
SEQUENCING IN Homo sapiens 36,565 23-Nov-99
PROGRESS "', in unordered pieces.
GB PR3:AC004919 75547 AC004919 Homo sapiens PAC clone DJ0895B23 from UL,
complete sequence. Homo sapiens 34,346 19-Sep-98
rxa00827 876 GB_EST6:W06539 300 W06539 T2367 MVAT4 bloodstream form of
serodeme WRATat1.1 Trypanosoma brucei Trypanosoma bnicei 40,000 12-Aug-96
rhodesiense cDNA 5', mRNA sequence. rhodesiense
GB_PR4:AC008179 181745 AC008179 Homo sapiens clone NH0576F01, complete
sequence. Homo sapiens 35,903 28-Sep-99
Table 4 (continued)
GB_EST18:AA710415 533 AA710415 vt53f08.r1 Barstead mouse irradiated colon
MPLRB7 Mus musculus cDNA clone Mus musculus 41,562 24-DEC-1997
IMAGE:1166823 5', mRNA sequence.
rxa00842 1323 GBPR2:AC002379 118595 AC002379 Human BAC clone GS165104 from
7q21, complete sequence. Homo sapiens 36,321 23-Jul-97
GB__PR2:AC002379 118595 AC002379 Human BAC clone GS165104 from 7q21, complete
sequence. Homo sapiens 37,284 23-Jul-97
GB INI:CEF02D8 31624 Z78411 Caenorhabditis elegans cosmid F02D8, complete
sequence. Caenorhabditis elegans 38,163 23-Nov-98
rxa00847 1572 GB OV:XELRDS38A 1209 L79915 Xenopus laevis rds/peripherin
(rds38) mRNA, complete cds. Xenopus laevis 36,D44 30-Jul-97
GB_HTG4:AC007920 234529 AC007920 Homo sapiens chromosome 3q27 clone RPCI11-
208N14, ='= SEQUENCING IN Homo sapiens 33,742 21-OCT-1999
PROGRESS "=, 51 unordered pieces.
GB HTG4:AC007920 234529 AC007920 Homo sapiens chromosome 3q27 clone RPCI11-
208N14, SEQUENCING IN Homo sapiens 33,742 21-OCT-1999
PROGRESS "', 51 unordered pieces.
nca00851 732 GB_HTG2:AC004064 185000 AC004064 Homo sapiens chromosome 4, "=
SEQUENCING IN PROGRESS 10 Homo sapiens 39,833 9-Jul-98
unordered pieces.
GB HTG2:AC004064 185000 AC004064 Homo sapiens chromosome 4, "' SEQUENCING IN
PROGRESS 10 Homo sapiens 39,833 9-Jul-98
unordered pieces.
GB PR3:HSJ824F16 139330 AL050325 Human DNA sequence from clone 824F16 on
chromosome 20, complete Homo sapiens 39,833 23-Nov-99
sequence. Ln
rxa00852 813 GB_HTG3:AC010120 121582 AC010120 Drosophila melanogaster
chromosome 3 clone BACR22N13 (D1061) RPCI-98 Drosophila melanogaster 36,855 24-
Sep-99 ~ D,,
22.N.13 map 96F-96F strain y; cn bw sp, *** SEQUENCING IN PROGRESS -.3
83 unordered pieces. ,
GB HTG3:AC010120 121582 AC010120 Drosophila melanogaster chromosome 3 clone
BACR22N1 3 (D1061) RPCI-98 Drosophila melanogaster 36,855 24-Sep-99
22.N.13 map 96F-96F strain y; cn bw sp, '=' SEQUENCING IN PROGRESS
83 unordered pieces.
GB HTG2:AC006898 299308 AC006898 Caenorhabditis elegans clone Y73B6x, ""
SEQUENCING IN PROGRESS "=, 9 Caenorhabditis elegans 36,768 24-Feb-99 0
unordered pieces.
rxa00856
rxa00870 1635 GB BAI:STMMSDA 3986 L48550 Streptomyces coelicolor methylmalonic
acid semialdehyde dehydrogenase Streptomyces coelicolor 63,743 09-MAY-1996
(msdA) gene, complete cds.
GB_PAT:192043 713 192043 Sequence 10 from patent US 5726299. Unknown. 38,850
01-DEC-1998
GB PAT:178754 713 178754 Sequence 10 from patent US 5693781. Unknown. 38,850 3-
Apr-98
rxa00875 690 GB BA2:AF1 19715 549 AF119715 Escherichia coli isopentyl
diphosphate isomerase (idi) gene, complete cds. Escherichia coli 54,827 22-Apr-
99
GB_BA2:AE000372 12144 AE000372 Escherichia coli K-12 MG1655 section 262 of 400
of the complete genome. Escherichia coli 51,416 12-Nov-98
GB_13A1:ECU28375 55175 U28375 Escherichia coli K-12 genome; approximately 64
to 65 minutes. Escherichia coli 51,416 08-DEC-1995
nca00878 1986 GB_HTG2:AC007472 114003 AC007472 Drosophila melanogaster
chromosome 2 clone BACR30D19 (D587) RPCI-98 Drosophila melanogaster 36,592 2-
Aug-99
30.D.19 map 49E-49F strain y; cn bw sp, "' SEQUENCING IN PROGRESS =",
79 unordered pieces. _
GB_HTG2:AC007472 114003 AC007472 Drosophila melanogaster chromosome 2 clone
BACR30D19 (D587) RPCI-98 Drosophila melanogaster 36,592 2-Aug-99
30.D.19 map 49E-49F strain y; cn bw sp, *** SEQUENCING IN PROGRESS
79 unordered pieces.
GB HTG2:AC006798 207370 AC006798 Caenorhabditis elegans clone Y51 F8, ***
SEQUENCING IN PROGRESS =", 30 Caenorhabditis elegans 36,699 25-Feb-99
unordered pieces.
Table 4 (continued)
nca00880 1968 GB_EST4:H22888 468 H22888 ym54e12.r1 Soares infant brain 1NIB
Homo sapiens cDNA clone IMAGE:52158 Homo sapiens 37,179 6-Jul-95
5', mRNA sequence.
GB GSS13:AQ426858 516 AQ426858 CITBI-E1-2578F1.TF CITBI-El Homo sapiens
genomic clone 2578F1, genomic Homo sapiens 38,447 24-MAR-1999
survey sequence.
GB PR1:A8002335 6289 AB002335 Human mRNA for KIAA0337 gene, complete cds. Homo
sapiens 35,799 13-Feb-99
nca00899 1389 GB BAI:NGU58849 2401 U58849 Neisseria gonorrhoeae piIS6 silent
pilus locus. Neisseria gonorrhoeae 40,623 20-Jun-96
GB BA1:PLPDHOS 3119 L06822 Plasmid pSa (from Escherichia coli) dihydropteroate
synthase gene, 3' end. Plasmid pSa 38,966 20-MAR-1996
GB BA1:PDGINTORF 6747 L06418 Integron In7 (from Plasmid pDGO100 from
Escherichia coli) integrase (int), Plasmid pDGO100 38,966 20-MAR-1996
aminoglycoside adenylyltransferase (aad), quaternary ammonium compound-
resistance protein, dihydrofofate reductase (dhfrX), and dihydropteroate
synthase (sull) genes.
nca00902 1333 GB_GSS15:AQ606873 581 AQ606873 HS 5404_B2_H05_T7A RPCI-1 1 Human
Mate BAC Library Homo sapiens Homo sapiens 37,900 10-Jun-99
genomic clone Plate=980 CoI=10-Row=P, genomic survey sequence.
GB GSS9:AQ163442 658 AQ163442 nbxb0o07A07f CUGI Rice BAC Library Oryza sativa
genomic clone Oryza sativa 41,885 12-Sep-98
nbxb0007A07f, genomic survey sequence.
GB PLI:PSST70 4974 X69213 P.sativum Psst70 gene for heat-shock protein. Pisum
sativum 36,866 3-Jul-96 ci,
nca00931 969 GB GSSI:FR0025208 612 AL018047 F.rubripes GSS sequence, clone
145D10aA8, genomic survey sequence. Fugu rubripes 37,815 10-DEC-1997 W
GB_GSS1:FR0021844 252 AL014715 F.rubripes GSS sequence, clone 069K22aG5,
genomic survey sequence. Fugu rubripes 37,698 10-DEC-1997 -.3
GB_GSS12:AQ403344 593 AQ403344 HS_2257 B1_B03 T7C CIT Approved Human Genomic
Sperm Library D Homo Homo sapiens 31,552 13-MAR-1999 w
sapiens genomic clone Plate=2257 CoI=5 Row=D, genomic survey sequence. tv rv
rxa00941 1440 GB BA1:MTCY180 44201 Z97193 Mycobacterium tuberculosis H37Rv
complete genome; segment 85/162. Mycobacterium 37,902 17-Jun-98
tuberculosis
GB_BAI:MTCY180 44201 Z97193 Mycobacterium tuberculosis H37Rv complete genome;
segment 85/162. Mycobacterium 39,140 17-Jun-90
o
tuberculosis p
GB BA2:MSGKATG 1745 L14268 Mycobacterium tuberculosis ethyl methane sulphonate
resistance protein (katG) Mycobacterium 42,517 26-Aug-99
gene, 3'end. tuberculosis
rxa00962 689 GB HTG6:AC010998 144338 AC010998 Homo sapiens clone RP11-95116,
=" SEQUENCING IN PROGRESS ***, 17 Homo sapiens 39,497 08-DEC-1999
unordered pieces.
GB GSS1:GGA340111 990 AJ232089 Gallus gallus anonymous sequence from Cosmid
mapping to chromosome 2 Gallus gallus 37,970 25-Aug-98
(Cosmid 34 - Contig 15), genomic survey sequence.
GB HTG6:AC010998 144338 AC010998 Homo sapiens clone RP11-95116, "' SEQUENCING
IN PROGRESS "', 17 Homo sapiens 38,226 08-DEC-1999
unordered pieces.
rxa01060 1047 GB BA1:ECTTN7 2280 AJ001816 Escherichia coli left end of
transposon Tn7 including type 2 integron. Escherichia coli 38,822 4-Nov-97
GBIN2:AF176377 8220 AF176377 Caenorhabditis briggsae CES-1 (ces-1) gene,
complete cds; and CPN-1 (cpn-1) Caenorhabditis briggsae 39,921 09-DEC-1999
gene, partial cds.
G8_GSS10:AQ196728 429 AQ196728 CIT-HSP-2381F4.TR CIT-HSP Homo sapiens genomic
clone 2381 F4, genomic Homo sapiens 39,019 16-Sep-98
survey sequence. -
nca01067 852 GB_BA1:U00016 42931 U00016 Mycobacterium leprae cosmid B1937.
Mycobacterium leprae 58,303 01-MAR-1994
GB_BAI:SYCGROESL 3256 D12677 Synechocystis sp. groES and groEL genes.
Synechocystis sp. 34,593 3-Feb-99
GB_BA1:D90905 139467 D90905 Synechocystis sp. PCC6803 complete genome, 7/27,
781449-920915. Synechocystis sp. 34,593 7-Feb-99
rxa01114 1347 GB_BA1:PSEFAOAB 3480 D10390 P. fragi faoA and faoB genes,
complete cds. Pseudomonas fragi 51,919 2-Feb-99
Table 4 (continued)
GB_BA1:AB014757 6057 AB014757 Pseudomonas sp. 61-3 genes for PhbR, acetoacetyl-
CoA reductase, beta- Pseudomonas sp. 61-3 50,573 26-DEC-1998
ketothiolase and PHB synthase, complete cds.
GB_BA1:SC8D9 38681 AL035569 Streptomyces coelicolor cosmid 8D9. Streptomyces
coelicolor 42,200 26-Feb-99
rxa01136 555 GB ESTI1:AA244557 379 AA244557 mx07a01.r1 Soares mouse NML Mus
musculus cDNA clone IMAGE:679464 5', Mus musculus 39,050 10-MAR-1997
mRNA sequence.
GB_EST14:AA407673 306 AA407673 EST01834 Mouse 7.5 dpc embryo ectoplacental
cone cDNA library Mus Mus musculus 38,562 26-Aug-98
musculus cDNA clone C0014F02 3', mRNA sequence.
GB EST26:AI390328 604 A1390328 mx07a01.y1 Soares mouse NML Mus musculus cDNA
clone IMAGE:679464 5', Mus musculus 33,136 2-Feb-99
mRNA sequence.
nca01138 540 GB OV:XLXINT1 1278 X13138 Xenopus laevis int-1 mRNA for int-1
protein. Xenopus laevis 40,03B 31-MAR-1995
GB_PR4:AC006054 143738 AC006054 Homo sapiens Xq28 BAC RPCI11-382P7 (Roswell
Park Cancer Institute Human Homo sapiens 37,996 1-Apr-99
BAC Library) complete sequence.
GB PR4:AC006054 143738 AC006054 Homo sapiens Xq28 BAC RPCIt 1-382P7 (Roswell
Park Cancer Institute Human Homo sapiens 36,053 1-Apr-99
BAC Library) complete sequence.
rxa01172 1578 GB BA1:SCE39 23550 AL049573 Streptomyces coelicolor cosmid E39.
Streptomyoes coelicolor 62,357 31-MAR-1999
GB_BA1:MSU50335 5193 U50335 Mycobacterium smegmatis phage resistance (mpr)
gene, complete cds. Mycobacterium 37,853 1-Feb-97 Ln
smegmatis w
GB BA1:BACTHRTRN 15467 D84213 Bacillus subtilis genome, trnl-feuABC region.
Bacillus subtilis 53,807 6-Feb-99 0
A w
nca01191 1713 GB_PR2:HS1191B2 60828 AL022237 Human DNA sequence from clone
1191B2 on chromosome 22q13.2-13.3. Homo sapiens 38,366 23-Nov-99 W
Contains part of the BIK (NBK, BP4, BIPt ) gene for BCL2-interacting killer
0
(apoptosis-inducing), a 40S Ribososmal Protein S25 pseudogene and .3
part of an alternatively spliced novel Acyl Transferase gene similar to C.
eiegans
C50D2.7. Contains ESTs, STSs, GSSs, two putative CpG islands and genomic .r.
marker D22S1151, complete sequence. ~
GB_PR2:HS1191B2 60828 AL022237 Human DNA sequence from clone 1191B2 on
chromosome 22q13.2-13.3. Homo sapiens 39,595 23-Nov-99
Contains part of the BIK (NBK, BP4, BIP1) gene for BCL2-interacting killer
(apoptosis-inducing), a 40S Ribososmal Protein S25 pseudogene and
part of an altematively spliced novel Acyl Transferase gene similar to C.
elegans
C50D2.7. Contains ESTs, STSs, GSSs, two putative CpG islands and genomic
marker D22S1 151, complete sequence.
rxa01205 554 GB_BA1:MTCY373 35516 Z73419 Mycobacterium tuberculosis H37Rv
complete genome; segment 57/162. Mycobacterium 57,762 17-Jun-98
tuberculosis
GB_PLI:ATY12776 38483 Y12776 Arabidopsis thaliana DNA, 40 kb surrounding ACS1
locus. Arabidopsis thaliana 32,971 7-Sep-98
GB_PL2:ATT6K21 99643 AL021889 Arabidopsis thaliana DNA chromosome 4, BAC clone
T6K21 (ESSA project). Arabidopsis thaliana 35.273 16-Aug-99
rxa01212 1047 GB BA2:SCD25 41622 AL118514 Streptomyces coelicolor cosmid D25.
Streptomyces coelicolor 39,654 21-Sep-99
A3(2)
GB_BA1:SLGLYUB 2576 X65556 S.lividans tRNA-GlyU beta gene. Streptomyces
lividans 54,493 20-DEC-1993
GB_BA1:SCH10 39524 AL049754 Streptomyces coelicolor cosmid H10. Streptomyces
coelicolor 44,638 04-MAY-1999
rxa01219 1005 GB PAT:A68024 520 A68024 Sequence 19 from Patent W09743409.
unidentified 42,553 05-MAY-1999
GB PAT:A68025 193 A68025 Sequence 20 from Patent W09743409. unidentified
43,229 05-MAY-1999
GB PAT:A68027 193 A68027 Sequence 22 from Patent W09743409. unidentified
38,342 05-MAY-1999
Table 4 (continued~
nca01220 1200 GB_PR3:HS512B11 64356 AL031058 Human DNA sequence from cione
512B11 on chromosome 6p24-25. Contains Homo sapiens 35,478 23-Nov-99
the Desmoplakin I (DPI) gene, ESTs, STSs and GSSs, complete sequence.
GB EST6:N99239 424 N99239 zb76h11.s1 Soares_senescent fibroblasts_NbHSF Homo
sapiens cDNA clone Homo sapiens 39,623 20-Aug-96
IMAGE:309573 3', mRNA sequence.
GB_EST16:AA554268 400 AA554268 nk36c.09.s1 NCI_CGAP GC2 Homo sapiens cDNA
clone IMAGE:1015600 3' Homo sapiens 36,111 8-Sep-97
similar to gb:X01677 GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE,
LIVER (HUMAN);, mRNA sequence.
nca01221 849 GB PR4:AF1 79633 96371 AF179633 Homo sapiens chromosome 16 map
16q23.3-q24.1 sequence. Homo sapiens 40,199 5-Sep-99
GB_VI:EHVU20824 184427 U20824 Equine herpesvirus 2, complete genome. Equine
herpesvirus 2 37,001 2-Feb-96
GB_BA2:AE000407 10601 AE000407 Escherichia coli K-12 MG1655 section 297 of 400
of the complete genome. Escherichia coli 39,471 12-Nov-98
rxa01222 822 GB PAT:AR068625 28804 AR068625 Sequence 1 from patent US 5854034.
Unknown. 40,574 29-Sep-99
GB BA2:SSU51197 28804 U51197 Sphingomonas S88 sphingan polysaccharide
synthesis (spsG), (spsS), (spsR), Sphingomonas sp. S88 40,574 16-MAY-1996
glycosyl transferase (spsQ), (spsi), glycosyl transferase (spsK), glycosyl
transferase (spsL), (spsJ), (spsF), (spsD), (spsC), (spsE), Urf 32, Urf 26,
ATP-binding cassette transporter (atrD), ATP-binding cassette transporter
(atrB), glucosyl-isoprenylphosphate transferase (spsB), glucose-t-phosphate
cNn
thymidylyltransferase (rhsA), dTDP-6-deoxy-D-glucose -3,5-epimerase (rhsC) 00
w
dTDP-D-glucose-4,6-dehydratase (rhsB), dTDP-6-deoxy-L-mannose- -.3
dehydrogenase (rhsD), Urf 31, and Urf 34 genes, complete cds. w
GB_INI:BBU44918 2791 U44918 Babesia bovis ATP-binding protein (babc) mRNA,
complete cds. Babesia bovis 39,228 9-Aug-97 A
nca01260 1305 GB BA1:CGLPD 1800 Y16642 Corynebacterium glutamicum lpd gene,
complete CDS. Corynebacterium 99,923 1-Feb-99
glutamicum -.3
GBBA1:MTV038 16094 AL021933 Mycobacterium tuberculosis H37Rv complete genome;
segment 24/162. Mycobacterium 59,056 17-Jun-98 0
tuberculosis
GB_PR3:AC005618 176714 AC005618 Homo sapiens chromosome 5, BAC clone 249h5
(LBNL H149), complete Homo sapiens 36,270 5-Sep-98 sequenoe. 1O
rxa01261 294 GB_BAI:CGLPD 1800 Y16642 Corynebacterium glutamicum lpd gene,
complete CDS. Corynebacterium 100,000 1-Feb-99
glutamicum
GB HTG4:AC010045 164829 AC010045 Drosophila melanogaster chromosome 3Ll75A1
clone RPCI98-17C17, Drosophila melanogaster 50,512 16-OCT-1999
SEQUENCING IN PROGRESS ***, 50 unordered pieces.
GB HTG4:AC010045 164829 AC010045 Drosophila melanogaster chromosome 3U75A1
clone RPCI98-17C17, =" Drosophila melanogaster 50,512 16-OCT-1999
SEQUENCING IN PROGRESS "', 50 unordered pieces.
rxa01269 564 GB BA2:AF125164 26443 AF125164 Bacteroides fragilis 638R
polysaccharide B(PS B2) biosynthesis locus, complete Bacteroides fragilis
56,071 01-DEC-1999
sequence; and unknown genes.
GB BA1:AB002668 24907 AB002668 Actinobacillus actinomycetemcomitans DNA for
glycosyttransferase, lytic Actinobacillus 46,679 21-Feb-98
transglycosylase, dTDP-4-rhamnose reductase, complete cds.
actinomycetemcomitans
GB BA1:AB010415 23112 AB010415 Actinobacillus actinomycetemcomitans gene
cluster for 6-deoxy-L-talan Actinobacillus 46,679 13-Feb-99
synthesis, complete cds. actinomycetemcomftans
rxa01291 1056 GB STS:AU027820 238 AU027820 Rattus norvegicus, OTSUKA clone,
OT78.02/918b07, microsatellite sequence, Rattus norvegicus 34,874 02-MAR-1999
sequence tagged site.
GB_STS:AU027820 238 AU027820 Rattus norvegicus, OTSUKA clone, OT78.021918b07,
microsatellite sequence, Rattus norvegicus 34,874 02-MAR-1999
sequence tagged site.
Table 4 (continued)
GB HTG3:AC006445 174547 AC006445 Homo sapiens chromosome 4, '"' SEQUENCING IN
PROGRESS "==, 7 Homo sapiens 34,812 15-Sep-99
unordered pieces.
rxa01292 1308 GB BAI:BSUB0017 217420 Z99120 Bacillus subtilis complete genome
(section 17 of 21): from 3197001 to 3414420. Bacillus subtilis 37,802 26-Nov-
97
GB HTG3:AC010580 121119 AC010580 Drosophila melanogaster chromosome 3 clone
BACR48J06 (D1102) RPCI-98 Drosophila melanogaster 35,637 01-OCT-1999
48J.6 map 96F-96F strain y; cn bw sp, '== SEQUENCING IN PROGRESS '=',
71 unordered pieces.
GB_HTG3:AC010580 121119 AC010580 Drosophila melanogaster chromosome 3 clone
BACR48JO6 (D1102) RPCI-98 Drosophila melanogaster 35,637 01-OCT-1999
48.J.6 map 96F-96F strain y; cn bw sp, "' SEQUENCING IN PROGRESS
71 unordered pieces.
rxa01293 450 GB GSS8:AQ001809 705 AQ001809 CIT-HSP-2290D173F CIT-HSP Homo
sapiens genomic clone 2290D17, Homo sapiens 42,021 26-Jun-98
genomic survey sequence.
GB_GSS8:AQ001809 705 A0001809 CIT-HSP-2290D17.TF CIT-HSP Homo sapiens genomic
clone 2290D17, Homo sapiens 40,323 26-Jun-98
genomic survey sequence.
rxa01339 1111 GB PLI:MGU60290 4614 U60290 Ma na orthe nitr en re ulato
9 P grisea 09 g ry protein (NUT7) gene, complete cds. Magnaporthe grisea
38,707 3-Jul-96 GB HTG3:AC011371 189187 AC011371 Homo sapiens chromosome 5
clone CIT978SKB_107C20, SEQUENCING IN Homo sapiens 39.741 06-OCT-1999 L"
PROGRESS =", 31 unordered pieces. W
GB HTG3:AC011371 189187 AC011371 Homo sapiens chromosome 5 clone CIT978SKB
107C20, SEQUENCING IN Homo sapiens 39,741 06-OCT-1999 0
PROGRESS "', 31 unordered pieces. W
nca01382 1192 GB_HTG4:AC009892 138122 AC009892 Homo sapiens chromosome 19
clone CIT978SKB_83J4, === SEQUENCING IN Homo sapiens 40,154 31-OCT-1999 CO
PROGRESS "', 6 ordered pieces. o
GB_HTG4:AC009892 138122 AC009892 Homo sapiens chromosome 19 clone CIT978SKB
83J4, ==' SEQUENCING IN Homo sapiens 40,154 31-OCT-1999
PROGRESS "=, 6 ordered pieces. o
GB_PR3:AC002416 128915 AC002416 Human Chromosome X, complete sequence. Homo
sapiens 37,521 29-Jan-98
rxa01399 1142 GB EST9:AA096601 524 AA096601 mo03b09.r1 Stratagene mouse lung
937302 Mus musculus cDNA clone Mus musculus 40,525 15-Feb-97
IMAGE:552473 5' similar to gb:L06505 60S RIBOSOMAL PROTEIN L12
(HUMAN); gb:L04280 Mus musculus ribosomal protein (MOUSE);, mRNA
GB EST37:At982114 626 AI982114 pat.pk0074.e9.f chicken activated T cell cDNA
Gallus gallus cDNA clone Gallus gallus 37,785 15-Sep-99
pat.pk0074.e9.f 5' similar to H-ATPase B subunit, mRNA sequence.
GB_OV:GGU20766 1645 U20766 Galius gallus vacuolar H+-ATPase B subunit gene,
complete cds. Gallus gallus 38,244 07-DEC-1995
nca01420 1065 GB_HTG2:AC005690 193424 AC005690 Homo sapiens chromosome 4, '='
SEQUENCING IN PROGRESS 7 Homo sapiens 37,464 11-Apr-99
unordered pieces.
GB_HTG2:AC005690 193424 AC005690 Homo sapiens chromosome 4, "= SEQUENCING IN
PROGRESS 7 Homo sapiens 37,464 11-Apr-99
unordered pieces.
GB HTG2:AC006637 22092 AC006637 Caenorhabditis elegans clone F41B4, "=
SEQUENCING IN PROGRESS ', 1 Caenorhabditis elegans 37,488 23-Feb-99
unordered pieces.
rxa01467 414 GBHTGI:CEY102G3_ 110000 AL020985 Caenorhabditis elegans
chromosome V clone Y102G3, SEQUENCING IN Caenorhabditis elegans 35,437 3-Dec-
98
GB__HTG1:CEY102G3_ 110000 AL020985 Caenorhabditis elegans chromosome V clone
Y102G3, ==' SEQUENCING IN Caenorhabditis elegans 35,437 3-Dec-98
GB_HTG1:CEY113G7_ 110000 AL031113 Caenorhabditis elegans chromosome V clone
Y113G7, SEQUENCING IN Caenorhabditis elegans 35,437 12-Jan-99
rxa01576 882 GB_BA2:AF030975 2511 AF030975 Aeromonas salmonicida chaperonin
GroES and chaperonin GroEL genes, Aeromonas salmonicida 41,516 2-Apr-98
complete cds.
GB BA2:AF030975 2511 AF030975 Aeromonas salmonicida chaperonin GroES and
chaperonin GroEL genes, Aeromonas salmonicida 38,171 2-Apr-98
complete cds.
Table 4 (continued)
GB EST22:AI068560 965 A1068560 mgae0003aC11f Magnaporthe grisea Appressorium
Stage cDNA Library Pyricularia grisea 40,073 09-DEC-1999
Pyricularia grisea cDNA clone mgae0003aC11f 5', mRNA sequence.
nca01580 840 GB_GSS14:AQ554460 681 AQ554460 RPCI-11-419F2.TV RPCI-1 1 Homo
sapiens genomic clone RPCI-1 1-419F2, Homo sapiens 36,522 28-MAY-1999
genomic survey sequence.
GB IN2:AC005449 85518 AC005449 Drosophila melanogaster, chromosome 2R, region
44C4-44C5, P1 clone Drosophila melanogaster 36,609 23-DEC-1998
DS06765, complete sequence.
GB_IN2:AC005449 85518 AC005449 Drosophila melanogaster, chromosome 2R, region
44C4-44C5, Pt clone Drosophila melanogaster 33,612 23-DEC-1998
DS06765, complete sequence.
nca01584
rxa01604 771 GB HTG3:AC011352 160167 AC011352 Homo sapiens chromosome 5 clone
CIT-HSPC_327F10, SEQUENCING IN Homo sapiens 33,688 06-OCT-1999
PROGRESS "', 15 unordered pieces.
GB HTG3:AC011352 160167 AC011352 Homo sapiens chromosome 5 clone CIT-HSPC
327F10, SEQUENCING IN Homo sapiens 33,688 06-OCT-1999
PROGRESS "', 15 unordered pieces.
GB HTG3:AC011402 168868 AC011402 Homo sapiens chromosome 5 Gone
CIT978SKB_38B5, "' SEQUENCING IN Homo sapiens 33,688 06-OCT-1999 ci,
PROGRESS "', 7 unordered pieces. D
w
rxa01614 1146 GB BA1:CGA224946 2408 AJ224946 Corynebacterium glutamicum DNA
for L-Malate:quinone oxidoreductase. Corynebacterium 42,284 11-Aug-98 -.3
glutamicum tC - w
GB ESTI7:AA608825 439 AA608825 af03g07.s1 Soares_testis_NHT Homo sapiens cDNA
clone IMAGE: 1030620 3' Homo sapiens 40,092 02-MAR-1998 ~ rv
similar to TR:G976083 G976083 HISTONE H2A RELATED. ;, mRNA sequence.
GB_PR4:AC005377 102311 AC005377 Homo sapiens PAC clone DJ1136G02 from 7q32-
q34, compiete sequence. Homo sapiens 37,811 28-Apr-99
rxa01629 1635 GB BA1:CGPROPGEN 2936 Y12537 C.glutamicum proP gene.
Corynebacterium 100,000 17-Nov-98 0
glutamicum
GB BA1:CGPROPGEN 2936 Y12537 C.glutamicum proP gene. Corynebacterium 100,000
17-Nov-98
glutamicum
GB_PR4:AF191071 88481 AF191071 Homo sapiens chromosome 8 clone BAC 388D06,
complete sequence. Homo sapiens 35,612 11-OCT-1999
rxa01644 1401 GB_BAI:MSGB577CO 37770 L01263 M. leprae genomic dna sequence,
cosmid b577. Mycobacterium leprae 55,604 14-Jun-96
S
GB_BA1:MLCB2407 35615 AL023596 Mycobacterium leprae cosmid B2407.
Mycobacterium leprae 36,416 27-Aug-99
GB_BA1:MTV025 121125 AL022121 Mycobacterium tuberculosis H37Rv complete
genome; segment 155/162. Mycobacterium 55,844 24-Jun-99
tuberculosis
rxa01667 1329 GB_BA1:CGU43536 3464 U43536 Corynebacterium glutamicum heat
shock, ATP-binding protein (cIpB) gene, Corynebacterium 100,000 13-MAR-1997
complete cds. glutamicum
GB_HTG4:AC009841 164434 AC009841 Drosophila melanogaster chromosome 3Ll77E1
clone RPCI98-13F11, Drosophila melanogaster 33,205 16-OCT-1999
SEQUENCING IN PROGRESS "', 70 unordered pieces.
GB HTG4:AC009841 164434 AC009841 Drosophila melanogaster chromosome 3L177E1
clone RPC198-13F11, Drosophila melanogaster 33,205 16-OCT-1999 -
SEQUENCING IN PROGRESS ***, 70 unordered pieces.
nca01722 1848 GB GSSI:FR0022586 522 AL015452 F.rubripes GSS sequence, clone
077P23aB10, genomic survey sequence. Fugu rubripes 40,192 10-DEC-1997
GB GSS1:FR0022584 485 AL015450 F.rubripes GSS sequence, clone 077P23aB1 1,
genomic survey sequence. Fugu rubripes 35,876 10-DEC-1997
GB_INI:CET26H2 37569 Z82055 Caenorhabditis elegans cosmid T26H2, complete
sequence. Caenorhabditis elegans 34,759 19-Nov-99
Table 4 (continued)
rxa01727 1401 GB BA2:CORCSLYS 2821 M89931 Corynebacterium glutamicum beta C-S
lyase (aecD) and branched-chain amino Corynebacterium 99,929 4-Jun-98
acid uptake carrier (bmQ) genes, complete cds, and hypothetical protein Yhbw
glutamicum
(yhbw) gene, partial cds.
GB_HTG6:AC011037 167849 AC011037 Horno sapiens clone RP11-7F18, WORKING DRAFT
SEQUENCE, 19 Homo sapiens 36,903 30-Nov-99
unordered pieces.
GB HTG6:AC011037 167849 AC011037 Homo sapiens clone RP11-7F18, WORKING DRAFT
SEQUENCE, 19 Homo sapiens 35,642 30-Nov-99
unordered pieces.
rxa01737 1182 GB BAI:SCGD3 33779 AL096822 Streptomyces coelicolor cosmid GD3.
Streptomyces coelicolor 38,054 8-Jul-99
GB HTG1:CNSOIDSB 222193 AL121768 Homo sapiens chromosome 14 clone R-976B16,
SEQUENCING IN Homo sapiens 35,147 05-OCT-1999
PROGRESS "', in ordered pieces.
GB_HTG1:CNS0IDSB 222193 AL121768 Homo sapiens chromosome 14 clone R-976B16,
SEQUENCING IN Homo sapiens 35,147 05-OCT-1999
PROGRESS "', in ordered pieces.
rxa01762 1659 GB_BA1:MTCI28 36300 Z97050 Mycobacterium tuberculosis H37Rv
complete genome; segment 10/162. Mycobacterium 49,574 23-Jun-98
tuberculosis
GB BA1:SC6G10 36734 AL049497 Streptomyces coelicolor cosmid 6G10. Streptomyces
coelicolor 44,049 24-MAR-1999
GB BA1:SCE29 26477 AL035707 Streptomyces coelicolor cosmid E29. Streptomyces
coelicolor 40,246 12-MAR-1999 L''
rxa01764 1056 GB PL2:SPAC343 42947 AL109739 S.pombe chromosome I cosmid c343.
Schizosaccharomyces 37,084 6-Sep-99 W
.3
pombe 0
GB_PL2:SPAC343 42947 AL109739 S.pombe chromosome I cosmid c343.
Schizosaccharomyces 34,890 6-Sep-99 w
pombe
rxa01801 1140 GB_EST38:AW066306 334 AW066306 687009D03.y1 687 - Eary embryo
from Delaware Zea mays cDNA, mRNA Zea mays 46,108 12-OCT-1999 .3
sequence.
r.
GB GSS13:AQ484750 375 AQ484750 RPCI-11-248N4.TV RPCI-1 1 Homo sapiens genomic
clone RPCI-1 1-248N4, Homo sapiens 32,000 24-Apr-99
N
genomic survey sequence.
GB_GSS13:AQ489971 252 AQ489971 RPCI-11-247N23.TV RPCI-11 Homo sapiens genomic
clone RPCI-11-247N23, Homo sapiens 36,111 24-Apr-99
genomic survey sequence.
nca01823 900 GB_BAI:SCI51 40745 AL109848 Streptomyces coelicolor cosmid 151.
Streptomyces coelicolor 35,779 16-Aug-99
A3(2)
GB_BA1:ECU82598 136742 U82598 Escherichia coli genomic sequence of minutes 9
to 12. Escherichia coli 39,211 15-Jan-97
GB BAI:BSUB0018 209510 Z99121 Bacillus subtilis complete genome (section 18 of
21): from 3399551 to 3609060. Bacillus subtilis 36,999 26-Nov-97
nca01853 675 GB BA1:MTCY227 35946 Z77724 Mycobacterium tuberculosis H37Rv
complete genome; segment 114/162. Mycobacterium 37,612 17-Jun-98
tuberculosis
GB HTG3:AC010189 265962 AC010189 Homo sapiens clone RPCI11-296K13, SEQUENCING
IN PROGRESS 80 Homo sapiens 39,006 16-Sep-99
unordered pieces.
GB HTG3:AC010189 265962 AC010189 Homo sapiens clone RPCI11-296K13, SEQUENCING
IN PROGRESS 80 Homo sapiens 39,006 16-Sep-99
unordered pieces.
nca01881 558 GB_HTG4:AC011117 148447 AC011117 Homo sapiens chromosome 4 clone
173 C_09 map 4, SEQUENCING IN Homo sapiens 39,130 14-OCT-1999
PROGRESS "', 10 ordered pieces.
GB_HTG4:AC011117 148447 AC011117 Homo sapiens chromosome 4 clone 173 C 09 map
4, SEQUENCING IN Homo sapiens 39,130 14-OCT-1999
PROGRESS "=, 10 ordered pieces.
GB_BA1:MTCY2B12 20431 Z81011 Mycobacterium tuberculosis H37Rv complete genome;
segment 61/162. Mycobacterium 37,893 18-Jun-98
tuberculosis
Table 4 (continued)
nca01894 978 GB BAt :MTCY274 39991 Z74024 Mycobacterium tuberculosis H37Rv
complete genome; segment 126/162. Mycobacterium 37,229 19-Jun-98
tuberculosis
GB_INI:CELF46H5 38886 U41543 Caenorhabditis elegans cosmid F46H5.
Caenorhabditis elegans 38,525 29-Nov-96
GB_HTG3:AC009204 115633 AC009204 Drosophila melanogaster chromosome 2 clone
BACR03E19 (D1033) RPCI-98 Drosophila melanogaster 31,579 18-Aug-99
03.E.19 map 36E-37C strain y; cn bw sp, "' SEQUENCING IN PROGRESS
94 unordered pieces.
rxa01897 666 GB_HTG1:CEY48B6 293827 AL021151 Caenorhabditis elegans chromosome
II clone Y48B6, SEQUENCING IN Caenorhabditis elegans 34,703 1-Apr-99
PROGRESS "", in unordered pieces.
GB HTG1:CEY48B6 293827 AL021151 Caenorhabditis elegans chromosome II clone
Y48B6, SEQUENCING IN Caenorhabditis elegans 34,703 1-Apr-99
PROGRESS "', in unordered pieces.
GB HTG1:CEY53F4_2 110000 Z92860 Caenorhabditis elegans chromosome 11 clone
Y53F4, SEQUENCING IN Caenorhabditis elegans 33,333 15-Oct-99
PROGRESS "", in unordered pieces.
nca01946 1298 GB BA1:M7V007 32806 AL021184 Mycobacterium tuberculosis H37Rv
complete genome; segment 64/162. Mycobacterium 65,560 17-Jun-98
tuberculosis
GB_BA1:SC5F2A 40105 AL049587 Streptomyces coelicolor cosmid 5F2A. Streptomyces
coelicolor 50,648 24-MAY-1999
GB_BA1:SCARDIGN 2321 X84374 S.capreolus ardl gene. Streptomyces capreolus
44,973 23-Aug-95 Ln
rxa01980 756 GB_Pl2:AC008262 99698 AC008262 Genomic sequence for Arabidopsis
thaliana BAC F4N2 from chromosome I. Arabidopsis thaliana 35.310 21-Aug-99 W
complete sequence. -.3
GB_PL1:AB013388 73428 AB013388 Arabidopsis thaliana genomic DNA, chromosome 5,
TAC clone: K19E1, Arabidopsis thaliana 35,505 20-Nov-99 W
complete sequence. ce rv
GB PL1:AB013388 73428 AB013388 Arabidopsis thaliana genomic DNA, chromosome 5,
TAC clone: K19E1, Arabidopsis thaliana 39,973 20-Nov-99
complete sequence.
rxa01983 630 GB HTG4:AC006467 175695 AC006467 Drosophila melanogaster
chromosome 2 clone BACR03L08 (0532) RPCI-98 Drosophila melanogaster 36,672 27-
OCT-1999 0
03.L.8 map 40A-40C strain y; cn bw sp, "' SEQUENCING IN PROGRESS "', 9 p
unordered pieces.
GB HTG4:AC006467 175695 AC006467 Drosophila melanogaster chromosome 2 clone
BACR03L08 (D532) RPCI-98 Drosophila melanogaster 36,672 27-OCT-1999
03.L.8 map 40A-40C strain y; cn bw sp, "' SEQUENCING IN PROGRESS "', 9
unordered pieces.
GB HTG4:AC006467 175695 AC006467 Drosophila melanogaster chromosome 2 clone
BACR03L08 (D532) RPCI-98 Drosophila melanogaster 32,367 27-OCT-1999
03.L.8 map 40A-40C strain y; cn bw sp, "' SEQUENCING IN PROGRESSo
9 unordered pieces.
rxa02020 1111 GB BAI:CGDNAAROP 2612 X85965 C.glutamicum ORF3 and aroP gene.
Corynebacterium 100,000 30-Nov-97
glutamicum
GB_PAT:A58887 1612 A58887 Sequence 1 from Patent W09701637. unidentified
100,000 06-MAR-1998
GB_BA1:STYCARABA 4378 M95047 Salmonella typhimurium transport protein,
complete cds, and transfer RNA-Arg. Salmonella typhimurium 50,547 13-MAR-1996
rxa02029 1437 GB HTG2:AC003023 104768 AC003023 Homo sapiens chromosome 11
clone pDJ363p2, SEQUENCING IN Homo sapiens 35,820 21-OCT-1997 -
PROGRESS "', 22 unordered pieces.
GB HTG2:AC003023 104768 AC003023 Homo sapiens chromosome 11 clone pDJ363p2,
SEQUENCING IN Homo sapiens 35,820 21-OCT-1997
PROGRESS "", 22 unordered pieces.
GS HTG2:HS118B18 104729 AL034344 Homo sapiens chromosome 6 clone RP1-118B18
map p24.1-25.3, "=' Homo sapiens 34,355 03-DEC-1999
SEQUENCING IN PROGRESS "", in unordered pieces.
Table 4 (continued)
nca02030 1509 GB_PR4:AC007695 63247 AC007695 Homo sapiens 12q24 BAC RPCI11-
124N23 (Roswell Park Cancer Institute Homo sapiens 38,681 1-Sep-99
Human BAC Library) complete sequence.
GBPR4:AC006464 99908 AC006464 Homo sapiens BAC clone NH0436C12 from 2,
complete sequence. Homo sapiens 35,445 22-OCT-1999
GB_PR4:AC006464 99908 AC006464 Homo sapiens BAC clone NH0436C12 from 2,
complete sequence. Homo sapiens 35,968 22-OCT-1999
rxa02073 1653 GB BA1:CGGDHA 2037 X72855 C.glutamicum GDHA gene.
Corynebacterium 39,655 24-MAY-1993
glutamicum
GB BAI:CGGDH 2037 X59404 Corynebacterium glutamicum, gdh gen for glutamate
dehydrogenase. Corynebacterium 44,444 30-Jul-99
glutamicum
GB BA2:SC2H4 25970 AL031514 Streptomyces coelicolor cosmid 2H4. Streptomyces
coelicolor 38,452 19-OCT-1999
A3(2)
rxa02074
rxa02095 1527 GB_EST18:AA703380 471 AA703380 zj12b06.s1 Soares fetal_liver
spleen 1NFLS_S1 Homo sapiens cDNA clone Homo sapiens 36,518 24DEC-1997
IMAGE:450035 3' similar to contains LTR5.t3 LTR5 repetitive element ;, mRNA
sequence. cn
GB_HTG6:AC009769 122911 AC009769 Homo sapiens chromosome 8 clone RP1 1-202112
map 8, LOW-PASS Homo sapiens 35,473 07-DEC-1999 w
SEQUENCE SAMPLING. .3
0
GB_EST7:W70175 436 W70175 zd52c02.r1 Soares_fetal heart_NbHH19W Homo sapiens
cDNA clone Homo sapiens 34,174 16-OCT-1996 W
IMAGE:344258 5' similar to contains LTR5.b2 LTR5 repetitive element;, mRNA
sequence.
0
rxa02099 373 GB BA1:CAJ10319 5368 AJ010319 Corynebacterium glutamicum amtP,
glnB, glnD genes and partial ftsY and srp Corynebacterium 100,000 14-MAY-1999
genes. glutamicum
GB_HTG3:AC011509 111353 AC011509 Homo sapiens chromosome 19 clone CITB-
H1_2189E23, SEQUENCING IN Homo sapiens 33,423 07-OCT-1999
PROGRESS "', 35 unordered pieces.
ro
GB HTG3:AC011509 111353 AC011509 Homo sapiens chromosome 19 clone CITB-H1
2189E23, SEQUENCING IN Homo sapiens 33,423 07-OCT-1999
PROGRESS ', 35 unordered pieces.
nca02115 1197 GB HTG5:AC010126 175986 AC010126 Homo sapiens clone GS502B02,
SEQUENCING IN PROGRESS 3 Homo sapiens 36,717 13-Nov-99
unordered pieces.
GB_HTG5:AC010126 175986 AC010126 Homo sapiens clone GS502B02, SEQUENCING IN
PROGRESS 3 Homo sapiens 36,092 13-Nov-99
unordered pieces.
GB_PRI:HUMHM145 2214 D10925 Human mRNA for HM145. Homo sapiens 39,171 3-Feb-99
rxa02128 1818 GB_BAI:MTCY190 34150 Z70283 Mycobacterium tuberculosis H37Rv
complete genome; segment 98/162. Mycobacterium 38,682 17-Jun-98
tuberculosis
GB_BAI:MTCY190 34150 Z70283 Mycobacterium tuberculosis H37Rv complete genome;
segment 98/162. Mycobacterium 35,746 17-Jun-98
tuberculosis
GB_GSS10:AQ161109 738 AQ161109 nbxb0006D03r CUGI Rice BAC Library Oryza sativa
genomic clone Oryza sativa 38,482 12-Sep-98
nbxb0006D03r, genomic survey sequence.
rxa02133 329 GB BA2:MPAE000058 28530 AE000058 Mycoplasma pneumoniae section 58
of 63 of the complete genome. Mycoplasma 32,317 18-Nov-96
pneumoniae
GB_HTG4:AC008308 151373 AC008308 Drosophila melanogaster chromosome 3 clone
BACR10M16 (D743) RPCI-98 Drosophila melanogaster 34,579 20-OCT-1999
10.M.16 map 93C-93D strain y; cn bw sp, "' SEQUENCING IN PROGRESS
186 unordered pieces.
Table 4 (continued)
GB_HTG4:AC008308 151373 AC008308 Drosophila melanogaster chromosome 3 clone
BACR10M16 (D743) RPCI-98 Drosophila melanogaster 34,579 20-OCT-1999
10.M.16 map 93C-93D strain y; cn bw sp, "' SEQUENCING IN PROGRESS
186 unordered pieces.
rxa02150 924 GB EST37:AW012260 358 AW012260 um06e09.y1 Sugano mouse kidney
mkia Mus musculus cDNA clone Mus musculus 39,385 10-Sep-99
IMAGE:2182312 5' similar to SW:AMPLBOVIN P00727 CYTOSOL
AMINOPEPTIDASE ;, mRNA sequence.
GB GSS3:B87734 389 B87734 RPCI11-30D24.TP RPCI-11 Homo sapiens genomic clone
RPCI-1 1-30D24, Homo sapiens 37,629 9-Apr-99
genomic survey sequence.
GB PR4:AC005042 192218 AC005042 Homo sapiens clone NH0552E01, complete
sequence. Homo sapiens 36,901 14-Jan-99
nca02171 1776 GB_BA2:AF010496 189370 AF010496 Rhodobacter capsulatus strain
SB1003, partial genome. Rhodobacter capsulatus 53,714 12-MAY-1998
GB_EST24:AI170522 367 AI170522 EST216450 Normalized rat lung, Bento Soares
Rattus sp. cDNA clone Rattus sp. 44,186 20-Jan-99
RLUC075 3' end, mRNA sequence.
GB PLI:PHVDLECA 1441 K03288 P.vulgaris phytohemagglutinin gene encoding
erythroagglutinating Phaseolus vulgaris 39,103 27-Apr-93
phytohemagglutinin (PHA-E), complete cds.
nca02173 1575 GB_BA1:CGGLTG 3013 X66112 C.glutamicum glt gene for citrate
synthase and ORF. Corynebacterium 44,118 17-Feb-95
glutamicum cn
GB BA1:CGGLTG 3013 X66112 C.glutamicum gft gene for citrate synthase and ORF.
Corynebacterium 36,189 17-Feb-95 ~ D,
glutamicum -.3
GB BA2:AE000104 10146 AE000104 Rhizobium sp. NGR234 plasmid pNGR234a, section
41 of 46 of the complete Rhizobium sp. NGR234 38.487 12-DEC-1997 W
plasmid sequence: ~ N
rxa02224 1920 GB_BA2:CXU21300 8990 U21300 Corynebacterium striatum
hypothetical protein YbhB gene, partial cds; ABC Corynebacterium 37,264 9-Apr-
99 p o
transporter TetB (tet8), ABC transporter TetA (tetA), transposase, 23S rRNA
striatum ~
methykransferase, and transposase genes, complete cds; and unknown o
genes. p
GB HTG3:AC009185 87184 AC009185 Homo sapiens chromosome 5 clone CIT-
HSPC_248019, SEQUENCING IN Homo sapiens 36,459 07-OCT-1999 4
to
PROGRESS "', 2 ordered pieces.
GB HTG3:AC009185 87184 AC009185 Homo sapiens chromosome 5 clone CIT-HSPC
248019, SEQUENCING IN Homo sapiens 36,459 07-OCT-1999
PROGRESS "', 2 ordered pieces.
rxa02225 905 GB BA2:MPAE000058 28530 AE000058 Mycoplasma pneumoniae section 58
of 63 of the complete genome. Mycoplasma 35,498 18-Nov-96
pneumoniae
GB_EST26:AI337275 618 A1337275 tb96h11.x1 NCI_CGAP_Co16 Homo sapiens cDNA
clone IMAGE:2062245 3' Homo sapiens 35,589 18-MAR-1999
similar to TR:Q15392 015392 ORF, COMPLETE CDS. ;, mRNA sequence.
GB_EST26:AI337275 618 A1337275 tb96h11.x1 NCI CGAP_Co16 Homo sapiens eDNA
clone IMAGE:2062245 3' Homo sapiens 42,786 18-MAR-1999
similar to TR:Q15392 015392 ORF, COMPLETE CDS. ;, mRNA sequence.
rxa02233 1410 GB BA1:ERWPNLB 1291 M65057 Erwinia carotovora pectin lyase (pnl)
gene, complete cds. Erwinia carotovora 37,780 26-Apr-93
GB_EST30:AV021947 313 AV021947 AV021947 Mus musculus 18-day embryo C57BU6J Mus
musculus cDNA clone Mus musculus 39,423 28-Aug-99
11 90024M23, mRNA sequence.
GB_EST33:AV087117 251 AV087117 AV087117 Mus musculus tongue C57BU6J adult Mus
musculus cDNA clone Mus musculus 47,410 25-Jun-99
2310028C15, mRNA sequence.
rxa02253 1050 GB EST11:AA250210 532 AA250210 mx79g10.r1 Soares mouse NML Mus
musculus cDNA clone IMAGE:692610 5' Mus musculus 36,136 12-MAR-1997
similar to TR:E236517 E236517 F44G4.1 ;, mRNA sequence.
GB_EST11:AA250210 532 AA250210 mx79g10.r1 Soares mouse NML Mus musculus cDNA
clone IMAGE:692610 5' Mus musculus 36,202 12-MAR-1997
similar to TR:E236517 E236517 F44G4.1 ;, mRNA sequence.
Table 4 (continued)
nca02261 1479 GB BAI:CGL007732 4460 AJ007732 Corynebacterium glutamicum 3' ppc
gene, secG gene, amt gene, ocd gene and Corynebacterium 100,000 7-Jan-99
5' soxA gene. glutamicum
GB BAI:CGAMTGENE 2028 X93513 C.glutamicum amt gene. Corynebacterium 100,000 29-
MAY-1996
glutamicum
GB_BA1:CORPEPC 4885 M25819 C.gtutamicum phosphoenolpyruvate carboxylase gene,
complete cds. Corynebacterium 100,000 15-DEC-1995
glutamicum
nca02268 1023 GB PL2:AF087130 3478 AF087130 Neurospora crassa siderophore
regulation protein (sre) gene, complete cds. Neurospora crassa 39,268 22-OCT-
1998
GB EST30:AI663709 408 A1663709 ud47a06.y1 Soares mouse mammary gland NbMMG Mus
musculus cDNA clone Mus musculus 41,523 10-MAY-1999
IMAGE:1449010 5' similar to TR:O75585 075585 MITOGEN- AND STRESS-
ACTIVATED PROTEIN KINASE-2 ;, mRNA sequence.
GB_RO:AF074714 3120 AF074714 Mus musculus mitogen- and stress-activated
protein kinase-2 (mMSK2) mRNA, Mus musculus 38,347 24-OCT-1998
complete cds.
rxa02269 1095 GB_GSS4:AQ742825 847 AQ742825 HS 5482_B2 A04_T7A RPCI-11 Human
Male BAC Library Homo sapiens Homo sapiens 37,703 16-Jul-99
genomic clone Plate=1058 Col=8 Row=B, genomic survey sequence.
GB_HTG3:AC009293 162944 AC009293 Homo sapiens chromosome 18 clone 53_I_06 map
18, SEQUENCING IN Homo sapiens 37,006 13-Aug-99 ci,
00
PROGRESS ' , 15 unordered pieces. u,
GB_HTG3:AC009293 162944 AC009293 Homo sapiens chromosome 18 clone 53 1 06 map
18, SEQUENCING IN Homo sapiens 37,006 13-Aug-99 -.3
PROGRESS "', 15 unordered pieces. w
nca02309 1173 GB_BA1:MTY25D10 40838 Z95558 Mycobacterium tuberculosis H37Rv
complete genome; segment 281162. Mycobacterium 52,344 17-Jun-98 o
tuberculosis
GB_BA1:MSGY224 40051 AD000004 Mycobacterium tuberculosis sequence from clone
y224. Mycobacterium 52,344 03-DEC-1996
tuberculosis o
GB HTG2:AC007163 186618 AC007163 Homo sapiens clone NH0091M05, "' SEQUENCING
IN PROGRESS "', 1 Homo sapiens 37,263 23-Apr-99 r.
unordered pieces.
nca02310 1386 GB_BAI:MTY25D10 40838 Z95558 Mycobacterium tuberculosis H37Rv
complete genome; segment 28/162. Mycobacterium 36,861 17-Jun-98
tuberculosis
GB_BAI:MSGY224 40051 AD000004 Mycobacterium tuberculosis sequence from clone
y224. Mycobacterium 36,861 03-DEC-1996
tuberculosis
GB PR3:HS279N11 169998 Z98255 Human DNA sequence from PAC 279N11 on chromosome
Xq11.2-13.3. Homo sapiens 34,516 23-Nov-99
rxa02321 1752 GB BA1:AB018531 4961 AB018531 Corynebacterium glutamicum dtsR1
and dtsR2 genes, complete cds. Corynebacterium 99,030 19-OCT-1998
glutamicum
GB_PAT:E17019 4961 E17019 Brevibacterium lactofermentum dtsR and dtsR2 genes.
Corynebacterium 98,973 28-Jul-99
glutamicum
GB BA1:AB018530 2855 AB018530 Corynebacterium glutamicum dtsR gene, complete
cds. Corynebacterium 99,030 19-OCT-1998
glutamicum
rxa02335 1896 GB_BA1:CGU35023 3195 U35023 Corynebacterium glutamicum
thiosulfate sulfurtransferase (thtR) gene, partial Corynebacterium 99,947 16-
Jan-97
cds, acyl CoA carboxylase (accBC) gene, complete cds. glutamicum
GB_BA1:U00012 33312 U00012 Mycobacterium leprae cosmid B1308. Mycobacterium
leprae 40,247 30-Jan-96
GB BAI:MTCY71 42729 Z92771 Mycobacterium tuberculosis H37Rv complete genome;
segment 141/162. Mycobacterium 67,568 10-Feb-99
tuberculosis
rxa02364 750 GB_BA1:AP000006 319000 AP000006 Pyrococcus horikoshii OT3 genomic
DNA, 1166001-1485000 nt. position (6/7). Pyrococcus horikoshii 36,130 8-Feb-99
GB BA1:AP000006 319000 AP000006 Pyrococcus horikoshii OT3 genomic DNA, 1166001-
1485000 nt. position (6/7). Pyrococcus horikoshii 34,543 8-Feb-99
Table 4 (continued)
nca02372 2010 GB_HTG3:AC011461 100974 AC011461 Homo sapiens chromosome 19
clone CIT-HSPC_429L19, SEQUENCING IN Homo sapiens 36,138 07-OCT-1999
PROGRESS ***, 4 ordered pieces.
GB_HTG3:AC011461 100974 AC011461 Homo sapiens chromosome 19 clone CIT-HSPC
429L19, SEQUENCING IN Homo sapiens 36,138 07-OCT-1999
PROGRESS "', 4 ordered pieces.
GB EST21:AA992021 279 AA992021 ot36c01.s1 Soares testis NHT Homo sapiens cDNA
clone IMAGE:1618848 3', Homo sapiens 41,219 3-Jun-98
mRNA sequence.
nca02397 1119 GB_HTG4:AC009273 76175 AC009273 Arabidopsis thaliana chromosome
1 clone T1 N6, SEQUENCING IN Arabidopsis thaliana 38,566 12-OCT-1999
PROGRESS =", 2 ordered pieces.
GB HTG4:AC009273 76175 AC009273 Arabidopsis thaliana chromosome 1 clone T1 N6,
SEQUENCING IN Arabidopsis thaliana 38,566 12-OCT-1999
PROGRESS "', 2 ordered pieces.
GB_BA1:D90826 19493 D90826 E.coli genomic DNA, Kohara clone #335(40.9-41.3
min.). Escherichia coli 39,600 21-MAR-1997
rxa02424 723 GB_ESTI3:AA334108 275 AA334108 EST38262 Embryo, 9 week Homo
sapiens cDNA 5' end, mRNA sequence. Homo sapiens 38,603 21-Apr-97
GB PR3:AC005224 166687 AC005224 Homo sapiens chromosome 17, done hRPK.214 0_1,
complete sequence. Homo sapiens 36,111 14-Aug-98
GB_PR3:AC005224 166687 AC005224 Homo sapiens chromosome 17, clone
hRPK.214_O_1, complete sequence. Homo sapiens 33,427 14-Aug-98
rxa02426 1656 GB PAT:A06664 1350 A06664 B.stearothermophilus Ict gene.
Bacillus 39,936 29-Jul-93 0
stearothermophilus
GB_PAT:A04115 1361 A041 15 B.stearothermophilus recombinant Ict gene.
synthetic construct 40,042 17-Feb-97
GB_BAI:BACLDHL 1361 M14788 B.stearothermophilus Ict gene encoding L-lactate
dehydrogenase, complete cds. Bacillus 40,338 26-Apr-93
stearothermophilus
rxa02487 1827 GB BA2:AF007101 32870 AF007101 Streptomyces hygroscopicus
putative pteridine-dependent dioxygenase, PKS Streptomyces 43,298 13-Jan-98
modules 1,2,3 and 4, and putative regulatory protein genes, complete cds and
hygroscopicus o 0
putative hydroxylase gene, partial cds. -.3
GB BAI:MTCI364 29540 Z93777 Mycobacterium tuberculosis H37Rv complete genome;
segment 52/162. Mycobacterium 44,352 17-Jun-98
tuberculosis r.
GB_BA2:AF119621 15986 AF119621 Pseudomonas abietaniphila BKME-9 Ditl (ditl),
dioxygenase DitA oxygenase Pseudomonas 43,611 28-Apr-99 F_
component small subunit (ditA2), dioxygenase DitA oxygenase component large
abietaniphila 1O
subunit (ditAl), DitH (ditH), DitG (ditG), DitF (ditF), DitR (ditR), DitE
(ditE), DitD
(dkD), aromatic diterpenoid extradiol ring-cleavage dioygenase (ditC), DitB
(ditB), and dioxygenase DitA ferredoxin component (ditA3) genes, complete cds;
and unknown genes.
nca02511 780 GB PR4:AC002470 235395 AC002470 Homo sapiens Chromosome 22q11.2
BAC Clone b135h6 In BCRL2-GGT Homo sapiens 37,971 30-Nov-99
Region, complete sequence.
GB PR4:AC002472 147100 AC002472 Homo sapiens Chromosome 22q11.2 PAC Clone p_n5
In BCRL2-GGT Region, Homo sapiens 38,239 13-Sep-99
complete sequence.
GB EST34:AI806938 118 A1806938 wf24b07.x1 Soares_NFL T GBC S1 Homo sapiens
cDNA cione Homo sapiens 38,983 7-Jul-99
IMAGE:2356501 3' similar to SW:PLZF_HUMAN 005516 ZINC FINGER
PROTEIN PLZF;, mRNA sequence.
rxa02512 1086 GB_BA1:MTCY1A10 25949 Z95387 Mycobacterium tuberculosis H37Rv
complete genome; segment 117/162. Mycobacterium 37,407 17-Jun-98
tuberculosis
GB_BAI:MLCL581 36225 Z96801 Mycobacterium leprae cosmid L581. Mycobacterium
leprae 43,193 24-Jun-97
GB_OV:GGU43396 2738 U43396 Gallus gallus tropomyosin receptor kinase A (ctrkA)
mRNA, complete cds. Gallus gallus 38,789 18-Jan-96
rxa02527 1452 GB BA2:AF008220 220060 AF008220 Bacillus subtilis rmB-dnaB
genomic region. Bacillus subtilis 37,395 4-Feb-98
Table 4 (continued)
GB_BA2:AF008220 220060 AF008220 Bacillus subtilis rmB-dnaB genomic region.
Bacillus subtilis 36,218 4-Feb-98
GB_HTG2:AC005861 112369 AC005861 Arabidopsis thaliana clone F23B24, "'
SEQUENCING IN PROGRESS "", 6 Arabidopsis thaliana 38,407 29-Apr-99
unordered pieces.
rxa02547 2262 GB PL1:AB006530 7344 AB006530 Citrutlus lanatus Sat gene for
serine acetyltransferase, complete cds and 5'- Citrultus lanatus 35,449 20-Aug-
97
flanking region.
GB PLI:CNASA 5729 D85624 Citrullus vulgaris serine acetyltransferase (Sat)
DNA, complete cds. Citrullus lanatus 35,449 6-Feb-99
GB PL1:AB006530 7344 AB006530 Citrullus lanatus Sat gene for serine
acetyltransferase, complete cds and 5'- Citrullus lanatus 34,646 20-Aug-97
flanking region.
rxa02566 1332 GB_EST32:AI727189 619 A1727189 BNLGHi7498 Six-day Cotton fiber
Gossypium hirsutum cDNA 5' similar to Gossypium hirsutum 35,099 11-Jun-99
(AB020715) KIAA0908 protein [Homo sapiens], mRNA sequence.
GB BAI:CGPUTP 3791 Y09163 C.glutamicum putP gene. Corynebacterium 38,562 8-Sep-
97
glutamicum
GB PL2:SPAC13G6 33481 Z54308 S.pombe chromosome I cosmid c13G6.
Schizosaccharomyces 35,774 18-OCT-1999
pombe
rxa02571 1152 GB_BA1:CGU43535 2531 U43535 Corynebacterium glutamicum multidrug
resistance protein (cmr) gene, complete Corynebacterium 41,872 9-Apr-97
cds. glutamicum o
GB EST35:AI857385 488 A1857385 w155e03.x1 NCI CGAP_Brn25 Homo sapiens cDNA
clone IMAGE:2428828 3', Homo sapiens 39,139 26-Aug-99
mRNA sequence.
GB BAI:CGU43535 2531 U43535 Corynebacterium glutamicum multidrug resistance
protein (cmr) gene, complete Corynebacterium 38,552 9-Apr-97
cds. glutamicum o
w
rxa02578 1227 GB_PL1:AB016871 79109 AB016871 Arabidopsis thaliana genomic DNA,
chromosome 5, TAC clone: K16L22, Arabidopsis thaliana 34,213 20-Nov-99 I
complete sequence. F, o
GB_PL1:AB025602 55790 AB025602 Arabidopsis thatiana genomic DNA, chromosome 5,
BAC clone:F14A1, complete Arabidopsis thaliana 36,461 20-Nov-99 (:)
sequence. j
GBINI:CELF36H9 35985 AF016668 Caenorhabdftis elegans cosmid F36H9.
Caenorhabditis elegans 35,977 8-Aug-97
nca02581 1983 GB_BA1:MTV005 37840 AL010186 Mycobacterium tuberculosis H37Rv
complete genome; segment 51/162. Mycobacterium 38,517 17-Jun-98 F~.
tuberculosis
GB BA1:MTV005 37840 AL010186 Mycobacterium tuberculosis H37Rv complete genome;
segment 51/162. Mycobacterium 39,173 17-Jun-98
tuberculosis
rxa02582 4953 GB_BA1:MTV026 23740 AL022076 Mycobacterium tuberculosis H37Rv
complete genome; segment 157/162. Mycobacterium 38,548 24-Jun-99
tuberculosis
GB BA1:MTCY338 29372 Z74697 Mycobacterium tuberculosis H37Rv complete genome;
segment 127/162. Mycobacterium 46,263 17-Jun-98
tuberculosis
GB BA1:SEERYABS 20444 X62569 S.erythraea eryA gene for 6-deoxyerythronolyde B
synthase tl & III. Saccharopolyspora 45,053 28-Feb-92
erythraea
rxa02583 1671 GB_BA2:AF113605 1593 AF113605 Streptomyces coelicolor propionyl-
CoA carboxylase complex B subunit (pccB) Streptomyces coelicolor 58,397 08-DEC-
1999
gene, complete cds.
GB BA1:SC1 C2 42210 AL031124 Streptomyces coelicolor cosmid 1 C2. Streptomyces
coelicolor 52,916 15-Jan-99
GB BA1:AB018531 4961 AB018531 Corynebacterium glutamicum dtsR1 and dtsR2
genes, complete cds. Corynebacterium 58,809 19-OCT-1998
glutamicum
rxa02599 600 GB_BAI:AEMML 2585 X99639 Ralstonia eutropha mmIH, mmll & mmlJ
genes. Raistonia eutropha 35,264 22-Jan-98
Table 4 (continued)
GB EST15:AA508926 422 AA508926 MBAFCW1 C08T3 Brugia malayi adult female cDNA
(SAW96MLW-BmAF) Brugia malayi 43,377 8-Jul-97
Brugia malayi cDNA clone AFCW1C08 5', mRNA sequence.
GB BA1:AEMML 2585 X99639 Ralstonia eutropha mmlH, mmll & mmlJ genes. Raistonia
eutropha 41,148 22-Jan-98
rxa02634 1734 GB BA1:SYNPOO 1964 X17439 Synechocystis ndhC, psbG genes for NDH-
C, PSII-G and ORF157. Synechoeystis PCC6803 38,145 10-Feb-99
GB_GSS9:AQ101527 184 AQ101527 HS_2265_A1_E11_MF CIT Approved Human Genomic
Sperm Library D Homo Homo sapiens 38,798 27-Aug-98
sapiens genomic clone Plate=2265 CoI=21 Row=1, genomic survey sequence.
GB_INI:MNE133341 399 AJ133341 Melarhaphe neritoides partial caM gene, exons 1-
2. Melarhaphe neritoides 39,098 2-Jun-99
nca02638 999 GB_BA2:AE001756 10938 AE001756 Thermotoga maritima section 68 of
136 of the complete genome. Thennotoga maritima 40,104 2-Jun-99
GB_GSS12:AQ423878 689 AQ423878 CITBI-E1-2575E20.TF CITBI-El Homo sapiens
genomic clone 2575E20. Homo sapiens 36,451 23-MAR-1999
genomic survey sequence.
GB HTG2:AC006765 274498 AC006765 Caenorhabditis elegans clone Y43H1 1, "'
SEQUENCING IN PROGRESS"", 7 Caenorhabditis elegans 39,072 23-Feb-99
unordered pieces.
rxa02659 335 GB EST36:Ai900317 436 A1900317 sc04a02.y1 Gm-c1012 Glycine max
cDNA clone GENOME SYSTEMS CLONE Glycine max 41,566 06-DEC-1999
ID:Gm-c1012-1155 5' similar to SW:PRS6SOLTU P54778 26S PROTEASE
REGULATORY SUBUNIT 6B HOMOLOG_;, mRNA sequence. rv
GB GSS12:AQ342831 683 AQ342831 RPCI11-122K17.TJ RPCI-11 Homo sapiens genomic
clone RPCI-11-122K17, Homo sapiens 34,762 07-MAY-1999 L"
00
genomic survey sequence. w
GB EST36:A1900856 779 A1900856 sb95c11.y1 Gm-c1012 Glycine max cDNA Gone
GENOME SYSTEMS CLONE Glycine max 39,063 06-DEC-1999 0
ID: Gm-c1012-429 5' similar to SW:PRS6_SOLTU P54778 26S PROTEASE w
REGULATORY SUBUNIT 68 HOMOLOG. ;, mRNA sequence. 1 0
nca02676 1512 GB IN2:CELB0213 39134 AF039050 Caenorhabditis elegans cosmid
B0213. Caenorhabditis elegans 35,814 2-Jun-99 1-- o
GB GSSI:CNSOOPZB 364 AL085157 Arabidopsis thaliana genome survey sequence SP6
end of BAC F10D11 of IGF Arabidopsis thaliana 38,462 28-Jun-99 p -.3
library from strain Columbia of Arabidopsis thaliana, genomic survey sequence.
1
r.
GBRO:RNITPR2R 10708 X61677 Rat ITPR2 gene for type 2 inositol triphosphate
receptor. Rattus norvegicus 37,543 21-OCT-1991 I rxa02677 882 GB_RO:D89728
5002 D89728 Mus musculus mRNA for LOK, complete cds. Mus musculus 38,829 7-Feb-
99 ~
GB GSS8:AQ062004 362 AQ062004 CIT-HSP-2346014.TR CIT-HSP Homo sapiens genomic
clone 2346014, Homo sapiens 36,565 31-Jul-98
genomic survey sequence.
GB_GSS14:AQ555818 462 AQ555818 HS 5236 B1_G06_SP6E RPCI-11 Human Male BAC
Library Homo sapiens Homo sapiens 36,534 29-MAY-1999
genomic clone Plate=806 Col=11 Row=N, genomic survey sequence.
nca02691 930 GB INI:DME9736 7411 AJ009736 Drosophila rnelanogaster ldefix
retroelement: gag, pol and env genes, partial. Drosophila melanogaster 36,522
19-Jan-99
GB PR4:AC004801 193561 AC004801 Homo sapiens 12q13.1 PAC RPCI1-228P16 (Roswell
Park Cancer Institute Homo sapiens 39,341 2-Feb-99
Human PAC Library) complete sequence.
G8 PR4:AC004801 193561 AC004801 Homo sapiens 12q13.t PAC RPCI1-228P16 (Roswell
Park Cancer Institute Homo sapiens 37,037 2-Feb-99
Human PAC Library) complete sequence.
rxa02718 1170 GB EST34:AV132028 258 AV132028 AV132028 Mus musculus C57BU6J 11-
day embryo Mus musculus cDNA clone Mus musculus 43,529 1-Jul-99
2700087F01, mRNA sequence.
GB GSS10:AQ240654 452 AQ240654 CIT-HSP-2385D24.TFB.1 CIT-HSP Homo sapiens
genomic clone 2385D24, Homo sapiens 40,044 30-Sep-98
genomic survey sequence.
GB GSSI1:AQ309500 576 AQ309500 CIT-HSP-2384D24.TFD CIT-HSP Homo sapiens
genomic clone 2384D24, Homo sapiens 38,869 22-DEC-1998
genomic survey sequence.
Table 4 (continued)
rxa02749 999 GB BA2:AF086791 37867 AF086791 Zymomonas mobilis strain ZM4 clone
67E10 carbamoylphosphate synthetase Zymomonas mobilis 39,024 4-Nov-98
small subunit (carA), carbamoylphosphate synthetase large subunit (carB),
transcription elongation factor (greA), enolase (eno), pyruvate
dehydrogenase alpha subunit (pdhA), pyruvate dehydrogenase beta subunit
(pdhB), ribonuclease H (mh), homoserine kinase homolog, alcohol
dehydrogenase II (adhB), and excinuclease ABC subunit A (uvrA) genes,
complete cds; and unknown genes.
GB_BA1:SYCSLRB 146271 D64000 Synechocystis sp. PCC6803 complete genome, 19/27,
2392729-2538999. Synechocystis sp. 34,573 13-Feb-99
GB_BA2:AE001306 13316 AE001306 Chlamydia trachomatis section 33 of 87 of the
complete genome. Chiamydia trachomatis 38,940 2-Sep-98
rxa02767 906 GB BA2:AF126953 1638 AF126953 Corynebacterium glutamicum
cystathionine gamma-synthase (metB) gene, Corynebacterium 100,000 10-Sep-99
complete cds. glutamicum
GB_BA1:SCI5 6661 AL079332 Streptomyces coelicolor cosmid 15. Streptomyces
coelicolor 37,486 16-Jun-99
GB PR3:HS90L6 190837 Z97353 Human DNA sequence from clone 90L6 on chromosome
22q11.21-11.23. Homo sapiens 34,149 23-Nov-99
Contains an RPL1 5 (60S Ribosomal Protein L15) pseudogene, ESTs, STSs and
GSSs, complete sequence.
rxa02792 876 GB BA2:AF099015 5000 AF099015 Streptomyces coelicolor strain
A3(2) integrase (int), Fe-containing superoxide Streptomyces coelicolor 36,721
1-Jun-99
dismutase II (sodF2), Fe uptake system permease (ftrE), and Fe uptake system
integral membrane protein (ftrD) genes, complete cds. W
GB BA1:ECOUW93 338534 U14003 Escherichia coli K-12 chromosomal region from
92.8 to 00.1 minutes. Escherichia coli 38,787 17-Apr-96 0
GB_HTG3:AC011361 186148 AC011361 Homo sapiens chromosome 5 clone CIT-
HSPC_482N19, "' SEQUENCING IN Homo sapiens 43,577 06-OCT-1999 w
PROGRESS "', 69 unordered pieces.
I o
rxa02794 1197 GB_PR4:AC005998 96556 AC005998 Homo sapiens clone OJ0622E21,
complete sequence. Homo sapiens 37,298 29-Jul-99 N o
GB_PR4:AC006008 57554 AC006008 Homo sapiens clone DJ0820A21, complete
sequence. Homo sapiens 36,638 17-Jun-99 ~
GB_PR3:HSDJ73H14 95556 AL080272 Human DNA sequence from clone 73H14 on
chromosome Xq26.3-28, complete Homo sapiens 39,726 23-Nov-99 1
sequence.
rxa02809 375 GB_RO:MUSSPCTLT 3172 M22527 Mouse cytotoxic T lymphocyte-specific
serine protease CCPII gene, complete Mus musculus 47,518 19-Jan-96
GB_RO:MUSGRC 894 M18459 Mouse granzyme C serine esterase mRNA, complete cds.
Mus musculus 44,939 12-Jun-93
GB_RO:RNU57062 880 U57062 Rattus norvegicus natural killer cell protease
4(RNKP-4) mRNA, complete cds. Rattus norvegicus 41,554 31-Jul-96
rxa02811 484 GB GSS6:AQ832862 476 AQ832862 HS_5261 A2_E10_SP6E RPCI-11 Human
Male BAC Library Homo sapiens Homo sapiens 35,610 27-Aug-99
genomic clone Plate=837 Col=20 Row=I, genomic survey sequence.
GB GSS5:AQ784593 515 AQ784593 HS_3248_A2_F02_T7C CIT Approved Human Genomic
Sperm Library D Homo Homo sapiens 38,956 3-Aug-99
sapiens genomic clone Plate=3248 Col=4 Row=K, genomic survey sequence.
GB GSS13:AQ473140 397 AQ473140 CITBI-E1-2589G6.TF CITBI-E1 Homo sapiens
genomic clone 2589G6, genomic Homo sapiens 34,761 23-Apr-99
survey sequence.
rxa02836 678 GB_EST18:AA696785 316 AA696785 GM08392.5prime GM Drosophila
melanogaster ovary BlueScript Drosophila Drosophila melanogaster 40,604 28-Nov-
98
melanogaster cDNA clone GM08392 5prime, mRNA sequence.
GB_EST18:AA696785 316 AA696785 GM08392.5prime GM Drosophila melanogaster ovary
BlueScript Drosophila Drosophila melanogaster 38,281 28-Nov-98
melanogaster eDNA clone GM08392 5prime, mRNA sequence.
rxs03212 1452 GB BA1:CGBETPGEN 2339 X93514 C.glutamicum betP gene.
Corynebacterium 99,931 8-Sep-97
glutamicum
GB_BA1:SC5F2A 40105 AL049587 Streptomyces coelicolor cosmid 5F2A. Streptomyces
coelicolor 57,557 24-MAY-1999
A3(2)
Table 4 (continued)
GB BA2:AF008220 220060 AF008220 Bacillus subtilis rrnB-dnaB genomic region.
Bacillus subtilis 40,000 4-Feb-98
rxs03220 725 GB PLI:CKHUP2 2353 X66855 C.kessleri HUP2 mRNA. Chlorella
kessleri 45,328 17-Feb-97
GB EST38:AW048153 383 AW048153 UI-M-BH1-alq-h-05-0-UI.s1 NIH BMAP M_S2 Mus
musculus cDNA clone Ul-M- Mus musculus 41,758 18Sep-99
BH1-alq-h-05-0-UI 3', mRNA sequence.
GB PLI:CKHUP2 2353 X66855 C.kessleri HUP2 mRNA. Chlorella kessleri 38,106 17-
Feb-97
C)
0
U9
W
W
J
0
W
1 N
O 0
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v
CA 02583703 2007-04-19
- 107 -
Exemplification
Example 1: Preparation of total genomic DNA of Corynebacterium glutamicum
ATCC 13032
A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight
at 30 C with vigorous shaking in BHI medium (Difco). The cells were harvested
by
centrifugation, the supematant 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/1
sucrose,
2.46 g/1 MgSO, x 7HaO, 10 ml/1 KHZPO, solution (100 g/l, adjusted to pH 6.7
with
KOH), 50 ml/1 M12 concentrate (10 g/1(NH,)=SO4, 1 g/l NaCI, 2 g/l MgSO4 x
7H2O,
0.2 g/1 CaCI2, 0.5 g/l yeast extract (Difco), 10 ml/1 trace-elements-mix (200
mg/1 FeSO,,
x HaO, 10 mg/1 ZnSO4 x 7 H2O, 3 mg/1 MnCIZ x 4 HZO, 30 mg/1 H3BO3 20 mg/1
CoClZ x
6 H2O, I mg/1 NiC12 x 6 H2O, 3 mg/1 Na2MoO4 x 2 HZO, 500 mg/1 complexing agent
(EDTA or critic acid), 100 ml/1 vitamins-mix (0.2 mg/1 biotin, 0.2 mg/t folic
acid, 20
mg/i p-amino benzoic acid, 20 mg/1 riboflavin, 40 mg/1 ca-panthothenate, 140
mg/1
nicotinic acid, 40 mg/1 pyridoxole hydrochloride, 200 mg/1 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 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
NaCI solution (5 M) are added. After adding of proteinase K to a final
concentration of
200 g/ml, the suspension is incubated for ca.18 h at 37 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 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
g/ml RNaseA and dialysed at 4 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 LiCI and 0.8 ml of ethanol are added.
After a 30
CA 02583703 2007-04-19
- 108 -
min incubation at -20 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 southem
blotting or
construction of genomic libraries.
Example 2: Construction of genomic libraries in Escherichia coli of
Corynebacterium
glutamicum ATCC13032.
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.)
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); pACYC 177
(Change &
Cohen (1978) J. Bacteriol 134:1141-1156), plasmids of the pBS series (pBSSK+,
pBSSK- and
others; Stratagene, LaJolla, USA), or cosmids as SuperCosl (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 pSL 109
(Lee, H.-S. and
A. J. Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).
Example 3: DNA Sequencing and Computational Functional Analysis
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' (SEQ ID No:677) or
5'-GTAAAACGACGGCCAGT-3' (SEQ ID No:678).
Example 4: In vivo Mutagenesis
In vivo mutagenesis of Corynebacterium glutamicum can be performed by passage
of
plasmid (or other vector) DNA tluough E. coli or other microorganisms (e.g.
Bacillus spp. or
yeasts such as Saccharomyces cerevisiae) which are impaired in their
capabilities to maintain
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the integrity of their genetic information. Typical mutator strains have
mutations in the genes
for the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see
Rupp, W.D.
(1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277-
2294, ASM:
Washington.) Such strains are well known to those of ordinary skill in the
art. The use of such
strains is illustrated, for example, in Greener, A. and Callahan, M. (1994)
Strategies 7: 32-34.
Example 5: DNA Transfer Between Escherichia coli and Corynebacterium
glutamicum
Several Corynebacterium and Brevibacterium species contain endogenous
plasmids (as e.g., pHM1519 orpBLl) 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 glulamicum 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 chioramphenicol (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).
Using standard methods, it is possible to clone a gene of interest into one of
the shuttle
vectors described above and to introduce such a hybrid vectors into strains of
Corynebacterium glutamicum. Transfonrnation of C. glulamicum can be achieved
by
protoplast transformation (Kastsumata, R. et al. (1984) J. Bacteriol. 1 59306-
3 1 1),
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,
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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).
Genes may be overexpressed in C. glutamicum strains using plasmids which
comprise pCGI (U.S. Patent 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 pSL 109 (Lee, H.-S. and
A. J.
Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).
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
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
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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.
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 colorimetric label which may be readily detected. The
presence and
quantity of label observed indicates the presence and quantity of the desired
mutant
protein present in the cell.
Example 7: Growth of Genetically Modified Corynebacterium glutamicum - Media
and Culture Conditions
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., 32:205-2
10; von der
Osten et al. (1998) Biotechnology Letters, 11:11-16; Patent DE 4,120,867;
Liebl (1992)
"The Genus Corynebacterium, in: The Procaryotes, Volume II, Balows, A. et al.,
eds.
Springer-Verlag). These media consist of one or more carbon sources, nitrogen
sources,
inorganic salts, vitamins and trace elements. Preferred carbon sources are
sugars, such as
mono-, di-, or polysaccharides. For example, glucose, fructose, mannose,
galactose,
ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or
cellulose serve as
very good carbon sources. It is also possible to supply sugar to the media via
complex
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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 NH4CI or (NH,)ZSO,, NH4OH, nitrates, urea, amino acids
or
complex nitrogen sources like corn steep liquor, soy bean flour, soy bean
protein, yeast
extract, meat extract and others.
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.
All medium components are sterilized, either by heat (20 minutes at 1.5 bar
and
121 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.
Culture conditions are defined separately for each experiment. The temperature
should be in a range between I5 C and 45 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
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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
NH4OH 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.
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.
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 OD600 of 0.5 - 1.5 using cells grown on agar plates, such
as CM plates
(10 g/I glucose, 2,5 g/l NaCl, 2 g/1 urea, 10 g/1 polypeptone, 5 g/1 yeast
extract, 5 g/1 meat
extract, 22 g/1 NaCl, 2 g/I urea, 10 g/1 polypeptone, 5 g/1 yeast extract, 5
g/1 meat extract,
22 g/l agar, pH 6.8 with 2M NaOH) that had been incubated at 30 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
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,
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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, 3d ed. Academic Press: New
York; Bisswanger, H., (1994) Enzymkinetik, 2"d ed. VCH: Weinheim (ISBN
3527300325); Bergmeyer, H.U., Bergmeyer, J., Gra(3l, M., eds. (1983-1986)
Methods of
Enzymatic Analysis, 3rd ed., vol. I-XII, Verlag Chemie: Weinheim; and
Ullmann's
Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes". VCH: Weinheim,
p.
352-363.
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.
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
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
CA 02583703 2007-04-19
-115-
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.)
In addition to the measurement of the final product of fermentation, it is
also
possible to analyze other components of the metabolic pathways utilized for
the
production of the desired compound, such as intermediates and side-products,
to .
determine the overall efficiency of production of the compound. Analysis
methods
include measurements of nutrient levels in the medium (e.g., sugars,
hydrocarbons,
nitrogen sources, phosphate, and other ions), measurements of biomass
composition and
growth, analysis of the production of common metabolites of biosynthetic
pathways, and
measurement of gasses produced during fermentation. Standard methods for these
measurements are outlined in Applied Microbial Physiology, A Practical
Approach,
P.M. Rhodes and P.F. Stanbury, eds., IRL Press, p. 103-129; 131-163; and 165-
192
(ISBN: 0199635773) and references cited therein.
Example 10: Purification of the Desired Product from C. glutamicum Culture
Recovery of the desired product from the C. glutamicum cells or supematant 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 supematant fraction containing the soluble proteins is retained for
further
purification of the desired compound. If the product is secreted from the C.
glutamicum
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cells, then the cells are removed from the culture by low-speed
centrifugation, and the
supernate fraction is retained for further purification.
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.
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).
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 el al. (1996) Biotekhnologiya
11: 27-
32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's
Encyclopedia
of Industrial Chemistry, (1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540,
p. 540-
547, p. 559-566, 575-581 and p. 581-587; Michal, G. (1999) Biochemical
Pathways: An
Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A.
et al.
(1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in
Biochemistry and Molecular Biology, vol. 17.
Example 11: Analysis of the Gene Sequences of the Invention
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. Scl.
USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.
Sci. USA
CA 02583703 2007-04-19
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90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST
programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
BLAST
nucleotide searches can be performed with the NBLAST program, score = 100,
wordlength = 12 to obtain nucleotide sequences homologous to MCT nucleic acid
molecules of the invention. BLAST protein searches can be performed with the
XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences
homologous to MCT protein molecules of the invention. To obtain gapped
alignments
for comparison purposes, Gapped BLAST can be utilized as described in Altschul
et al.,
(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped
BLAST programs, one of ordinary skill in the art will know how to optimize the
parameters of the program (e.g., XBLAST and NBLAST) for the specific sequence
being analyzed.
Another example of a mathematical algorithm utilized for the comparison of
sequences is the algorithm of Meyers and Miller ((1988) Comput. Appl. Biosci.
4: 11-
17). Such an algorithm is incorporated into the ALIGN program (version 2.0)
which is
part of the GCG sequence alignment software package. When utilizing the ALIGN
program for comparing amino acid sequences, a PAM 120 weight residue table, a
gap
length penalty of 12, and a gap penalty of 4 can be used. Additional
algorithms for
sequence analysis are known in the art, and include ADVANCE and ADAM.
described
in Torelli and Robotti (1994) Comput. Appl. Biosci. 10:3-5; and FASTA,
described in
Pearson and Lipman (1988) P.N.A.S. 85:2444-8.
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.
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
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were compared to genes present in Genbank in a three-step process. In a first
step, a
BLASTN analysis (e.g., a local alignment analysis) was performed for each of
the
sequences of the invention against the nucleotide sequences present in
Genbank, and the
top 500 hits were retained for further analysis. A subsequent FASTA search
(e.g., a
combined local and global alignment analysis, in which limited regions of the
sequences
are aligned) was performed on these 500 hits. Each gene sequence of the
invention was
subsequently globally aligned to each of the top three FASTA hits, using the
GAP
program in the GCG software package (using standard parameters). In order to
obtain
correct results, the length of the sequences extracted from Genbank were
adjusted to the
length of the query sequences by methods well-known in the art. The results of
this
analysis are set forth in Table 4. The resulting data is identical to that
which would have
been obtained had a GAP (global) analysis alone been performed on each of the
genes of
the invention in comparison with each of the references in Genbank, but
required
significantly reduced computational time as compared to such a database-wide
GAP
(global) analysis. Sequences of the invention for which no alignments above
the cutoff
values were obtained are indicated on Table 4 by the absence of alignment
information.
It will further be understood by one of ordinary skill in the art that the GAP
alignment
homology percentages set forth in Table 4 under the heading "% homology (GAP)"
are
listed in the European numerical format, wherein a',' represents a decimal
point.. For
example, a value of "40,345" in this column represents "40.345%".
Example 12: Construction and Operation of DNA Microarrays
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).
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
CA 02583703 2007-04-19
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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).
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. el al. (1995)
Science 270:
467-470).
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.
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
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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).
The application of the sequences of the invention to DNA microarray
technology, as described above, permits comparative analyses of different
strains of C.
glutamicum or other Corynebacteria. For example, studies of inter-strain
variations
based on individual transcript profiles and the identification of genes that
are important
for specific and/or desired strain properties such as pathogenicity,
productivity and
stress tolerance are facilitated by nucleic acid array methodologies. Also,
comparisons
of the profile of expression of genes of the invention during the course of a
fermentation
reaction are possible using nucleic acid array technology.
Example 13: Analysis of the Dynamics of Cellular Protein Populations
(Proteomics)
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.
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:
CA 02583703 2007-04-19
' . .
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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.
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., 35S-methionine, 35S-cysteine, 14C-labelled amino acids, 15N-
amino
acids, "NO3 or "NH4+ or 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.
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.
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.
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.
CA 02583703 2007-04-19
w = .
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Equivalents
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.
CA 02583703 2007-04-19
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