JP2002535007A - Genes identified as required for the growth of Escherichia coli - Google Patents

Abstract

  (57) [Summary] E. FIG. Disclosed are nucleic acid sequences encoding proteins necessary for the growth of E. coli. The nucleic acid is used to express a protein or a part thereof, and an antibody capable of specifically binding to the expressed protein is obtained. The expressed protein is used as a screen for a rational drug delivery program. Can be isolated. Using the present nucleic acids, Homologous genes required for the growth of microorganisms other than E. coli can also be screened. The present nucleic acids can also be used to design expression and secretion vectors. The nucleic acids of the invention can also be used in various assay systems for screening genes required for the growth of other organisms, as well as for screening antibiotics.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Since the discovery of penicillin, many lives have been saved by the use of antibiotics to treat harm from bacterial infections. With the advent of these “magic bullets”, in general,
This was ultimately thought to protect humans from bacterial sources. In fact, 1980
During the 1980's and early 1990's, many large pharmaceutical companies reduced or withdrawn antibiotic research and development. It was thought that the market for new drugs would be limited, eventually overcoming bacterial infections. Unfortunately, this belief was too optimistic.

[0002] Bacteria are beginning to be of interest as drug-resistant bacteria are reported more frequently. The United States Health Service has announced that vancomycin, one of the most potent known antibiotics, cannot cure common Staphylococcus aureus (staph) infections. This organism is found in a common environment and is responsible for many nosocomial infections. The importance of this publication becomes clear in light of the many years that vancomycin has been used to treat infections caused by powerful bacterial strains (such as staph). Briefly, bacteria are becoming resistant to our most potent antibiotics. If this trend continues, it is likely that what is now considered a minor bacterial infection will become incurable.

There are many sources of plight posed by those skilled in the medical arts. Excessive medical care and improper prescribing habits by some physicians have increased the indiscriminate use of antibiotics by the public. Patients also have some responsibilities. For example, even if antibiotics are an appropriate treatment, patients may use drugs inappropriately, leading to yet another bacterial population that is resistant to classical antibiotics in whole or in part. I have.

[0004] Despite the development of current scientific tools for treating bacterial-caused diseases, the scourge of bacterial emergence in humans remains. Drug resistant bacteria are now threatening human health. There is a need to create new antibiotics that once again deal with the unresolved health harms of bacteria.

[0005] Discovery of new antibiotics [0005] As bacterial strains that are resistant to a range of available antibiotics increase, new compounds are needed. Previously, those skilled in pharmacology had to rely on traditional drug discovery methods to create new, safe and effective compounds for the treatment of disease.
Traditional drug discovery methods have included binding studies of potential drug candidate compounds, which were often randomly selected and expected that the compounds would be effective in treating some diseases . The process is meticulous and laborious, and there is no guarantee of success. The average cost of discovery and development of a new drug is about $ 500 million and the average time from lab to clinical is 15 years. By improving this process, the creation of new antimicrobials, albeit little by little, will be greatly advanced.

[0006] Newly emerging means of drug discovery utilize a number of biochemical techniques to gain an approach to pharmaceutical rather than random discovery of new drugs. For example, the gene sequences required for the growth of organisms and the proteins encoded thereby are excellent targets because exposure of bacteria to compounds active on the target inactivates the organism. Once a target has been identified, biological analysis of the target can be used to discover or design molecules that interact with and alter the function of the target. Analyzing structural and biochemical targets from compounds that interact with targets using physical and computer techniques is called rational drug design and is very promising. Thus, the emergence of drug discovery tools uses molecular modeling techniques, chemical combination approaches, and other tools for generating and screening and / or designing large numbers of candidate compounds.

[0007] Despite the clear future of this drug discovery approach, problems remain. For example, the first step in identifying a molecular target for investigation can be a very time consuming task. Designing molecules that interact with targets by using computer modeling techniques can also be difficult. Furthermore, if the function of the target is unknown or poorly understood, designing a detection assay for molecules that interact with and alter the function of the target may be difficult. Improving the speed of discovery and development of new drugs is urgently needed to identify key molecular targets in pathogenic microorganisms and to identify and interact with and alter the function of such molecular targets .

[0008] Escherichia coli is an excellent model system for understanding bacterial biochemistry and physiology. Escherichia coli (E. col
The 4288 genes scattered over 4.6 × 10 ⁶ base pairs of the chromosome in i) are very promising for understanding bacterial biochemical processes. Similarly, this knowledge will aid in the development of new tools for the diagnosis and treatment of bacterial human diseases. All E. The E. coli genome has been sequenced, and this set of information has enormous potential for the discovery and development of new antimicrobial compounds. However, despite this realization, the general function or role of many of these genes is unknown. For example, E. The genes required for growth contained within the E. coli genome are unknown, but have been variously evaluated to be approximately 200-700 (Armstrong, KA and Fan, D.E.
P. , Essential Genes in the metB-malB
Region of Escherichia coli K12, 1975,
J. Bacteriol. (26, 48-55)).

[0009] With the rapid development of antibiotic resistant microorganisms, new safe and effective antimicrobial compounds are needed. However, prior to the present invention, the characterization of even one bacterial gene required a laborious multi-year effort. Accordingly, newer methods for identifying and characterizing bacterial genomic sequences that encode gene products required for growth, and for identifying molecules that interact with and alter the function of such genes and gene products, are urgently needed. is necessary.

SUMMARY OF THE INVENTION One embodiment of the present invention consists essentially of one of SEQ ID NOs: 1-81, 405-485.
A purified or isolated nucleic acid sequence, wherein the nucleic acid inhibits microbial growth. The nucleic acid sequence may be complementary to at least a portion of the coding sequence of a gene whose expression is required for microbial growth. The nucleic acid sequences are SEQ ID NOs: 1-81, 405-48
5 comprising one of SEQ ID NOs: 1-81, 405-405.
485 is selected from the group consisting of fragments comprising at least 10, at least 20, at least 25, at least 30, at least 50, or more than 50 contiguous bases. The nucleic acid sequence may be complementary to the coding sequence of a gene whose expression is required for microbial growth.

[0011] Another embodiment of the present invention is a vector comprising a promoter operably linked to a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 1-81, 405-485. Promoters include Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacter cloacae.
robacter cloacae), Helicobacter pylori, Neisseria gonorrhoeae, Enterococcus faecalis, Streptococcus pneumoniae (Streptoco)
ccus pneumoniae, Haemophilus influenzae
), Salmonella typhimurium, Saccharomyces cerevisiae, Candida albicans (
Candida albicans), Cryptococcus neoformans
formans), Aspergillus fumigatus, Klebsiella pneumoniae, Salmone chifi (Salmone)
lla typhi, Salmonella paratyphi, Salmonella cholera suis (Salmonella cholerasuis), Staphylococcus epidermidis (Staphylococcus epidermidis), Mycobacterium tuberculosis (Mycobacterium tuberculosis), mycobacterium leprae
erium leprae), Treponema pallidum, Bacillus
Bacillus anthracis, Yersinia pestis
estis), Clostridium botulinum, Campylobacter jejuni, Chlamydia trachomatus, Chlamydia pneumon
iae) or any of the above species may be active in an organism selected from the group consisting of any species within the genus.

[0012] Another embodiment of the present invention is a host cell comprising the above vector.

[0013] Another embodiment of the present invention is a purified or isolated nucleic acid consisting essentially of the coding sequence of one of SEQ ID NOs: 82-88, 90-242. One aspect of this embodiment is a fragment of a nucleic acid comprising at least 10, at least 20, at least 25, at least 30, at least 50, or more than 50 contiguous bases of one of SEQ ID NOs: 82-88, 90-242. .

[0014] Another embodiment of the present invention is a vector comprising a promoter operably linked to the nucleic acid of the above embodiment.

[0015] Another aspect of the present invention relates to an intragenic sequence in an operon encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 243-357, 359-398.
Intergenic sequences, sequences spanning at least a portion of two or more genes, 5
A purified or isolated nucleic acid comprising a nucleic acid sequence complementary to at least a portion of the 'non-coding region, or 3' non-coding region.

[0016] Another embodiment of the present invention provides that SEQ ID NOs: 1-81, 405-485, 8 as determined using BLASTN version 2.0 with default parameters.
A purified or isolated nucleic acid comprising a nucleic acid having at least 70% homology to a sequence selected from the group consisting of 2-88, 90-242 or a sequence complementary thereto. Nucleic acids are Staphylococcus aureus, Pseudo
omonas aeruginosa, Enterobacter cloac
ae, Helicobacter pylori, Neisseria gon
orrhoeae, Enterococcus faecalis, Strep
tococcus pneumoniae, Haemophilus infl
uenzae, Salmonella typhimurium, Saccha
romyces cerevisiae, Candida albicans,
Cryptococcus neoformans, Aspergillus
fumigatus, Klebsiella pneumoniae, Salm
onella typhi, Salmonella paratyphi, Sa
lmonella cholerasuis, Staphylococcus
epidermidis, Mycobacterium tuberculos
is, Mycobacterium leprae, Treponema pa
llidum, Bacillus anthracis, Yersinia p
estis, Clostridium botulinum, Campylob
acter jejuni, Chlamydia trachomatus, C
hlamydia pneumoniae, or an organism selected from the group consisting of any species within the genus to which any of the above species belongs.

[0017] Another embodiment of the present invention is directed to essentially any of SEQ ID NOs: 243-357, 359-398.
A purified or isolated nucleic acid consisting of a nucleic acid encoding a polypeptide having a sequence selected from the group consisting of:

[0018] Another embodiment of the present invention is a vector comprising a promoter operably linked to a nucleic acid encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 243-357, 359-398. .

[0019] Another embodiment of the present invention is a host cell comprising the vector of the above embodiment.

Another embodiment of the present invention is a purified or isolated polypeptide comprising the sequence of one of SEQ ID NOs: 243-357, 359-398.

Another embodiment of the present invention includes a fragment of one of the polypeptides of SEQ ID NOs: 243-357, 359-398, wherein the fragments comprise SEQ ID NOs: 243-35.
7. A purification selected from the group consisting of fragments comprising at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, or more than 60 contiguous amino acids of one of the 7,359-398 polypeptides. Or an isolated polypeptide.

Another embodiment of the present invention is an antibody that can specifically bind to the polypeptide of the above embodiment.

[0023] Another embodiment of the present invention provides a vector comprising a promoter operably linked to a nucleic acid encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 243-357, 359-398. This is a method for producing a polypeptide, which comprises a step of transferring into a cell. The method may further include isolating the protein.

[0024] Another embodiment of the present invention provides a method for inhibiting or reducing the activity of a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 243-357, 359-398, or a polypeptide as described above. Is a method for inhibiting growth, which comprises a step of inhibiting the activity of the nucleic acid encoding or reducing the amount thereof.

Another embodiment of the present invention provides a method comprising: contacting a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 243 to 357, 359 to 398 with a candidate compound; Identifying a compound that affects the activity of the polypeptide required for growth.

[0026] The activity may be an enzymatic activity. The activity may be a carbon compound catabolic activity. The activity may be a biosynthetic activity. The activity may be a transporter activity. The activity may be a transcription activity. The activity may be a DNA replication activity. The activity may be a cell division activity.

Another embodiment of the present invention is a compound identified using the method described above.

Another embodiment of the present invention is a step of obtaining a target, wherein the target comprises SEQ ID NOs: 82-82.
88, 90 to 242, comprising a coding sequence of a sequence selected from the group consisting of: 88, 90 to 242; 4 is an assay of a compound for its ability to reduce the level of activity of a polypeptide.

[0029] The target is a messenger RNA molecule transcribed from one of the coding regions of SEQ ID NOs: 82-88, 90-242, and the activity is the transcription of this messenger RNA. The target can be a single coding region of SEQ ID NOs: 82-88, 90-242, and the activity is transcription of this messenger RNA.

Another embodiment of the present invention is a compound identified using the method described above.

Another embodiment of the present invention is a method of identifying a compound that reduces the activity or level of a gene product required for cell growth, the method comprising the steps of: Expressing an antisense nucleic acid against the nucleic acid encoding the gene product to produce a sensitized cell; contacting the sensitized cell with a compound; and the compound inhibiting the growth of a non-sensitized cell. Identifying whether the compound inhibits the proliferation of the sensitized cells in a wider range than the above.

[0032] The cells can be selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells. The cells are E. coli. coli cells. The cells are Staphyloc
occus aureus, Pseudomonas aeruginosa,
Enterobacter cloacae, Helicobacter py
lori, Neisseria gonorrhoeae, Enterococ
cus faecalis, Streptococcus pneumonia
e, Haemophilus influenzae, Salmonella
typhimurium, Saccharomyces cerevisiae
, Candida albicans, Cryptococcus neofo
rmans, Aspergillus fumigatus, Klebsiel
la pneumoniae, Salmonella typhi, Salmo
nella paratyphi, Salmonella cholerasu
is, Staphylococcus epidermidis, Mycoba
cterium tuberculosis, Mycobacterium l
eprae, Treponema pallidum, Bacillus an
thracis, Yersinia pestis, Clostridium
botulinum, Campylobacter jejuni, Chlam
ydia trachomatus, Chlamydia pneumonia
e, or an organism selected from the group consisting of any species within the genus to which any of the above species belongs. Antisense nucleic acids can be transcribed from an inducible promoter. The method may further comprise contacting the cell with an inducer at a concentration that induces the antisense nucleic acid to sublethal levels. A sublethal concentration of the inducer may be that growth inhibition is 8% or more. The inducer can be isopropyl-1-thio-β-D-galactoside. Growth inhibition can be measured by monitoring the optical density of the culture growth solution. The gene product can be a polypeptide. The gene product can be RNA. Claims 243-357,35 wherein the gene product is
It may include a polypeptide having a sequence selected from the group consisting of 9-398.

Another embodiment of the present invention is a compound identified using the method described above.

Another embodiment of the present invention provides transferring a compound having activity against a gene corresponding to one of SEQ ID NOs: 82-88, 90-242, or having activity against said gene product into a cell population expressing the gene. And a method for inhibiting cell proliferation. The compound can be an antisense oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 1-81, 405-485, or a growth inhibiting portion thereof. One growth inhibiting portion of SEQ ID NOs: 1-81, 405-485 comprises SEQ ID NOs: 1-81, 4
It may be a fragment comprising at least 10, at least 20, at least 25, at least 30, at least 50, or more than 50 contiguous bases of one of 05-485. The compound can be a triple helix oligonucleotide.

[0035] Another embodiment of the present invention provides a method comprising providing SEQ ID NOs: 1-81 in a pharmaceutically acceptable carrier.
, 405-485, or a preparation comprising an effective concentration of an antisense oligonucleotide, or a growth-inhibiting portion thereof, comprising a sequence selected from the group consisting of: SEQ ID NO: 1
-81, 405-485 are SEQ ID NOs: 1-81, 405-485.
One of the 485 may comprise at least 10, at least 20, at least 25, at least 30, at least 50, or more than 50 contiguous bases.

Another embodiment of the present invention is a method of inhibiting gene expression in an operon required for growth, comprising contacting a cell in a cell population with an antisense nucleic acid, wherein the cell expresses the gene. Corresponds to one of SEQ ID NOs: 82 to 88 and 90 to 242, wherein the antisense nucleic acid comprises at least a growth-inhibitory portion of the operon in an antisense orientation that effectively inhibits gene expression. The antisense nucleic acid has SEQ ID NO: 82 to
88, 90-242.
The antisense nucleic acid can be one of SEQ ID NOs: 405-485, or a portion thereof. The cells can be contacted with the antisense nucleic acid by transferring a plasmid expressing the antisense nucleic acid to a cell population. By transferring a phage expressing the antisense nucleic acid to a cell population, the cells can be contacted with the antisense nucleic acid. Transfer of the sequence encoding the antisense nucleic acid into the chromosomes of the cells in the cell population allows the cells to be contacted with the antisense nucleic acids. Cells can be contacted with the antisense nucleic acid by transfer of a retron expressing the antisense nucleic acid to the cell population. The cells can be contacted with the antisense nucleic acid by transfer of the ribozyme to a cell population, wherein the binding portion of the ribozyme is complementary to the antisense oligonucleotide. Transfer of the liposome containing the antisense oligonucleotide to the cell allows the cell to be contacted with the antisense nucleic acid. Cells can be contacted with the antisense nucleic acid by electroporation. The antisense nucleic acid can be a fragment comprising at least 10, at least 20, at least 25, at least 30, at least 50, or more than 50 contiguous bases of one of SEQ ID NOs: 82-88, 90-242. Antisense nucleic acids can be oligonucleotides.

Another embodiment of the present invention provides a method for obtaining a sample comprising a bacterial species, comprising the steps of: providing a sample having a sequence selected from the group consisting of SEQ ID NOs: 1-81, 405-485, 82-88, 90-242. Identifying the bacterial species using a specific probe.

Another embodiment of the present invention provides a method comprising: (a) identifying an inhibitory nucleic acid that inhibits the activity of a gene or gene product required for growth of the first microorganism; and (b) inhibiting the second microorganism and the inhibitor. Contacting the nucleic acid; (c) identifying whether the inhibitory nucleic acid derived from the first microorganism inhibits the growth of the second microorganism; and (d) the second microorganism inhibited by the inhibitory nucleic acid. And a step of identifying a gene in the microorganism.

Another embodiment of the present invention provides a method comprising: (a) identifying a gene or gene product required for growth of a first microorganism; and (b) a homolog of said gene or gene product in a second microorganism. (C) identifying an inhibitory nucleic acid sequence that inhibits the activity of a homolog of the second microorganism; and (d) contacting the second microorganism with a growth-inhibiting amount of the inhibitory nucleic acid to form a second nucleic acid. (E) contacting the compound with the sensitizing microorganism of step (d) above; and (f) sensitizing the compound more than the compound inhibits the growth of non-sensitizing microorganisms. Identifying whether the degree of inhibition of the growth of the cropping microorganisms is higher.

The step of identifying a gene associated with the growth of the first microorganism is a step of introducing a nucleic acid containing a random genomic fragment from the first microorganism operably linked to a promoter, wherein the random genomic fragment is Presenting in the antisense orientation, comparing the growth of a first microorganism that transcribes the random genomic fragment at a first level with the growth of a first microorganism that transcribes a lower level of the random genomic fragment. Wherein the difference in growth may comprise the step of random genomic fragments comprising genes involved in growth.

The step of identifying the homologue of the gene of the second microorganism comprises BLASTN version 2.0 using default parameters and FA using default parameters.
The method can include identifying a homologous nucleic acid or a nucleic acid encoding a homologous polypeptide in a database using an algorithm selected from the group consisting of the STA version 3.0t78 algorithm. The step of identifying a homolog of the gene of the second microorganism can include the step of identifying a nucleic acid encoding a homologous nucleic acid or a homologous polypeptide by identifying a nucleic acid that hybridizes to the first gene. Second
Identifying a homologue of a gene of the microbe may comprise expressing a nucleic acid that inhibits growth of the first microbe in the second microbe. The inhibitory nucleic acid can be an antisense nucleic acid. Inhibitory nucleic acids can include antisense nucleic acids to some of the homologs. Inhibitory nucleic acids can include antisense nucleic acids to a portion of the operon that encodes a homolog. The step of contacting the second microorganism with the inhibitory amount of the nucleic acid sequence comprises a second step.
Contacting the microorganism with the nucleic acid directly. Contacting the second microorganism with a growth inhibiting amount of the nucleic acid sequence can include expressing an antisense nucleic acid against a homolog in the second microorganism.

Another embodiment of the present invention is a compound identified using the method described above.

Another embodiment of the present invention provides a method comprising: (a) identifying an inhibitory nucleic acid that inhibits the activity of a gene or gene product required for growth of a first microorganism; and (b) growing with a second microorganism. (C) bringing the compound into contact with the growth-inhibiting microorganism of step (b), and contacting the compound with the compound; Identifying whether the compound inhibits the growth of a sensitizing microorganism to a greater extent than does the growth of a second microorganism in the crop. It is.

[0044] The inhibitory nucleic acid can be an antisense nucleic acid that inhibits growth of the first microorganism. The inhibitory nucleic acid can include a portion of an antisense nucleic acid that inhibits growth of the first microorganism. The inhibitory nucleic acid may include an antisense molecule for the entire coding region of the gene associated with the growth of the first microorganism. The inhibitory nucleic acid can include an antisense nucleic acid to a portion of the operon that encodes a gene associated with growth of the first microorganism.

Another embodiment of the present invention is a compound identified using the method described above.

Another embodiment of the present invention provides a method of sensitizing a cell by expressing an antisense nucleic acid against a nucleic acid encoding a gene product required for growth in the cell to reduce the activity or amount of the gene product. Contacting the sensitized cells with the compound, and identifying whether the compound inhibits the growth of the sensitized cells more than the compound inhibits the growth of the non-sensitized cells. , Assays for compounds for activity on biological pathways required for growth.

[0047] The cells may be selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells. The cells are E. coli. coli cells. The cells are Staphyloc
occus aureus, Pseudomonas aeruginosa,
Enterobacter cloacae, Helicobacter py
lori, Neisseria gonorrhoeae, Enterococ
cus faecalis, Streptococcus pneumonia
e, Haemophilus influenzae, Salmonella
typhimurium, Saccharomyces cerevisiae
, Candida albicans, Cryptococcus neofo
rmans, Aspergillus fumigatus, Klebsiel
la pneumoniae, Salmonella typhi, Salmo
nella paratyphi, Salmonella cholerasu
is, Staphylococcus epidermidis, Mycoba
cterium tuberculosis, Mycobacterium l
eprae, Treponema pallidum, Bacillus an
thracis, Yersinia pestis, Clostridium
botulinum, Campylobacter jejuni, Chlam
ydia trachomatus, Chlamydia pneumonia
e, or an organism selected from the group consisting of any species within the genus to which any of the above species belongs. Antisense nucleic acids can be transcribed from an inducible promoter. The method may further include contacting the cell with an agent that induces the expression of an antisense nucleic acid derived from an inducible promoter, wherein the antisense nucleic acid is expressed at a sublethal level. Sublethal levels of antisense nucleic acids can inhibit proliferation by 8% or more.
The agent can be isopropyl-1-thio-β-D-galactoside (IPTG). Growth inhibition can be measured by monitoring the optical density of the liquid culture. The gene product can include a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 243-357, 359-398.

Another embodiment of the present invention is a compound identified using the method described above.

Another embodiment of the present invention is an assay for the ability of a compound to inhibit cell growth, comprising contacting a cell with an agent that reduces the activity or level of a gene product required for the growth of the cell. Contacting the cell with the compound; identifying whether the compound reduces inhibition more than the compound reduces proliferation of cells not contacted with the agent. It is a method including.

Agents that reduce the activity or level of a gene product that is required for cell growth can include an antisense nucleic acid to a gene or operon that is required for growth. Agents that reduce the activity or level of a gene product required for cell growth can include antibiotics. The cell may contain a temperature-sensitive mutation that reduces the activity or level of a gene product that is required for the growth of the cell. The antisense nucleic acid can be directed to a nucleic acid that encodes the same functional domain as the gene product required for growth of the cell to which the antisense nucleic acid is directed. An antisense nucleic acid can be directed to a nucleic acid that encodes a functional domain of a gene product required for the growth of the cell that differs from the functional domain to which the antisense nucleic acid is directed.

Another embodiment of the present invention is a compound identified using the method described above.

Another embodiment of the present invention is a method of identifying a pathway in which a nucleic acid required for growth or a gene product thereof is present, comprising a sublethal level of antisense directed to the nucleic acid required for growth in a cell. Expressing a nucleic acid, contacting the cell with an antibiotic, wherein the biological pathway on which the antibiotic acts is known, expressing a sublethal level of the antisense nucleic acid Identifying whether the cells are substantially more susceptible to the antibiotic than cells without.

Another embodiment of the present invention is a method of identifying a pathway in which a test compound acts, comprising: (a) expressing a sublethal level of an antisense nucleic acid directed to a nucleic acid required for growth in a cell. The steps of: (b) contacting said cells with said test compound; and (c) sub-lethal levels of said anti- Identifying whether the cells are substantially more sensitive to the test compound than cells that do not express the sense nucleic acid.

The method comprises the step of: (d) expressing a sublethal level of a second antisense nucleic acid directed to the nucleic acid required for the second growth in the cell, wherein the second antisense nucleic acid is required for the second growth. The nucleic acid is in a different biological pathway than the nucleic acid required for propagation in step (a) above,
And (e) identifying whether the cells are substantially more sensitive to the test compound than cells that do not express the sublethal level of the second antisense nucleic acid. .

Another embodiment of the present invention is a purified or isolated nucleic acid consisting essentially of one of SEQ ID NOs: 358, 399-402.

Another embodiment of the present invention provides SEQ ID NOs: 1-81, 405-485, 82-88,
A purified or isolated nucleic acid comprising a sequence selected from the group consisting of 90-242, 358, 399-402.

Another embodiment of the present invention provides a method of inhibiting the growth of SEQ ID NOs: 82-88, 90-
242 is a compound that interacts with a gene or gene product of a nucleic acid comprising one of the sequences.

Another embodiment of the present invention provides a method of inhibiting the growth of SEQ ID NOs: 243-357,3
A compound that interacts with a polypeptide comprising one of the compounds 59-398.

Another embodiment of the present invention provides a method of inhibiting growth of SEQ ID NOs: 358, 399-4
02 is a compound that interacts with a nucleic acid that comprises one of the two.

Definitions “Biological pathway” means any individual cell function or step performed by a gene product or subset of gene products. Biological pathways include enzymatic, biochemical, and metabolic pathways, as well as those involved in the production of cellular structures such as cell walls. Generally, the biological pathways required for microbial growth include cell division, DNA synthesis and replication, RNA synthesis (transcription), protein synthesis (translation), protein processing, protein transport, fatty acid biosynthesis, cell wall synthesis, and cell membrane synthesis. And maintenance, but are not limited to these.

“Inhibiting activity on a gene or gene product” has the ability to interfere with the function of a gene or gene product in such a way as to reduce gene expression or the level of activity of the gene product. Means that. Agents having activity on a gene include agents that inhibit gene transcription and agents that inhibit translation of mRNA transcribed from the gene. In microorganisms, an agent having activity on a gene can act to reduce the expression of an operon that alters the processing of the operon RNA where the gene is present or reduces the level or activity of the gene product. The gene product can be untranslated RNA, such as ribosomal RNA, translated RNA (mRNA), or a protein obtained from translation of a gene mRNA. A particular use of the present invention is for antisense RNAs that have activity on specifically hybridizing operons or genes.

“Activity against a gene product” refers to inhibiting the function of a gene product in a cell,
Alternatively, it means having the ability to reduce its level or activity.

“Activity against a protein” refers to inhibiting the function of a protein in a cell,
Alternatively, it means having the ability to reduce its level or activity.

By “activity against a nucleic acid” is meant having the ability to inhibit the function of, or reduce the level or activity of, a nucleic acid in a cell.

As used herein, “sublethal” refers to a concentration of an agent that is less than that required to inhibit whole cell proliferation.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides E. coli required for growth and / or proliferation. E. coli gene groups and gene families are described. A gene or gene family that is required for growth is one in which the growth or viability of the microorganism is reduced or eliminated in the absence of gene transcription and / or gene product. Thus, as used herein, the term "required for growth" refers to sequences whose cell growth is completely abolished due to the absence of gene transcripts and / or gene products, and gene transcripts and / or genes Include sequences that only reduce cell growth due to the absence of product. The genes required for these growths can be used as potential targets for the production of new antimicrobial agents. To this end, the invention also includes novel analytical assays for genes required for growth and assays for identifying compounds that interfere with the gene product of the gene required for growth. In addition, the present invention contemplates the purification of proteins identified as genes required for gene expression and propagation and encoded by the nucleic acid sequences reported herein. Using the purified protein to make a drug,
And small molecule libraries or other candidate compounds for compounds that can be further developed to obtain new antimicrobial compounds can be screened. The present invention also relates to E.I. A method for identifying a homologous gene in an organism other than E. coli is described.

The present invention provides E. coli required for growth. Utilizes a novel method for identifying E. coli sequences. Generally, a nucleic acid library from a given source is subcloned or inserted into an inducible expression vector to form an expression library. Although the insert nucleic acid may be derived from the chromosome of the organism into which the expression vector is transferred, the insert nucleic acid is an exogenous nucleic acid of interest for purposes of the present discussion since the insert is not present at its natural chromosomal location. . The term "expression" is defined as the production of an RNA molecule from a gene, gene fragment, genomic fragment, or operon. Expression can also be used to indicate a step in peptide or polypeptide synthesis. An expression vector is defined as a vehicle in which a ribonucleic acid (RNA) sequence is transcribed from a nucleic acid sequence retained in the expression vehicle. Expression vectors may also have the feature of being able to translate protein products from transcribed RNA messages expressed from exogenous nucleic acid sequences carried on the expression vector. Thus, an expression vector is capable of producing an RNA molecule such that its sole product or expression vector can produce an RNA molecule that is ultimately translated into a protein product.

Once created, an expression library containing exogenous nucleic acid sequences was used to search for genes required for bacterial growth. E. coli population. The molecules of the library are E. coli. The expression vector and the nucleic acid segments contained therein are considered to be exogenous nucleic acids since they are foreign to the E. coli population.

Subsequently, E. coli containing an expression vector library was used. Activate expression of an exogenous nucleic acid fragment in a test population of E. coli. Activation of the expression vector comprises subjecting the cell containing the vector to conditions under which the exogenous nucleic acid sequence carried by the expression vector library is expressed. Then, E. A test population of E. coli cells is assayed to identify the effect of exogenous nucleic acid fragment expression on the test population of cells. Upon activation and expression, These expression vectors, which negatively affect the growth of the E. coli screen population, were purified for identification, isolation, and further study.

Various assays are included to identify nucleic acid sequences whose growth negatively affects expression. In one embodiment, E. coli expressing an exogenous nucleic acid sequence. The growth of an E. coli culture is compared to that of a culture that does not express these sequences. Proliferation measurements are assayed by testing the range of proliferation by measuring optical density. Alternatively, bacterial growth rates can be measured using enzymatic assays to identify the exogenous nucleic acid sequence of interest. Colony size, colony morphology, and cell morphology are additional factors used to assess host cell growth. Identify media that do not grow or grow with reduced efficiency under expression conditions, including expression vectors encoding nucleic acid fragments that negatively affect genes required for growth.

[0071] Once the exogenous nucleic acid sequences of interest have been identified, they are analyzed. The first step of the analysis requires the nucleic acid sequence of the nucleic acid fragment of interest. To achieve the objective, the insert in these expression vectors containing the sequence of interest is sequenced using standard techniques well known in the art. The next step is to identify the source of the nucleic acid sequence.

Identification of the sequence source is performed by comparing the obtained sequence data with sequences known from various gene databases. Search these gene databases using the identified sequences. The result of this procedure is a list of exogenous nucleic acid sequences that correspond to the list containing novel bacterial genes required for growth, as well as genes previously identified as required for growth.

The number of DNA and protein sequences available in database systems has increased exponentially over the years. For example, at the end of 1998, Cae
norhabditis elegans, Saccharomyces ce
revisiae, and E. The complete sequence of 19 bacterial genomes, including E. coli, was available. This sequence information is obtained from GenBank (the Nationa).
l Center for Biotechnology Information
on (NCBI)) and are publicly searchable.

Various computer programs are available to assist in the analysis of sequences stored in these databases. FastA (WR Pearson
, 1990, "Rapid and Sensitive Sequence.
Comparison with FASTP and FASTA ", Met
hods in Enzymology, 183, 63-98), sequence search system (SRS) (Etzold & Argos, "SRS and index"
ing and retrieval tool for flat file
data libraries, Compt. Appl. Biosci. ,
9, 49-57, 1993) which can be used to analyze the sequence of interest.
Two exemplary computer programs. In one embodiment of the invention, the nucleic acid sequences are analyzed using a BLAST family computer program including BLASTN version 2.0 with default parameters, or BLASTX version 2.0 with default parameters.

“Basic Local Alignment Search Tool”
BLAST is a family of database similarity search programs. BLAST family programs include BLASTN (nucleotide sequence database search program), BLASTX (protein data search program when the input is a nucleic acid sequence), and BLASTP (protein database search program). The BLAST program implements sequence matches, rigorous statistical methods for determining the significance of matches, and various options for program adjustment in special circumstances. Instructions for using the program, e-mail b
last @ ncbi. nlm. nih. gov.

[0076] Bacterial genes may be transcribed in a polycistronic group. These groups include genes and operons that are collections of sequences within a gene. The operon genes are co-transcribed and may be functionally related. Given the nature of the screening protocol, the identified exogenous nucleic acid sequence may contain upstream or downstream contiguous non-coding sequences from the actual sequence required for bacterial growth, intragenic sequences (ie,
Sequences within genes), intergenic sequences (ie, intergenic sequences), sequences that span at least a portion of two or more genes, 5 'non-coding sequences, or 3'
It is possible to correspond to a gene or a part thereof that includes or does not include a noncoding region. Thus, it may be desirable to identify which genes encoded within the operon are individually required for growth.

In one embodiment of the invention, the operons are analyzed to identify which genes are required for growth. For example, the website http: // www. ci
fn. unam. mx. / Computational_Biology / re
gulondb /, which can also be found in Huerta et al. , Nucl
. Acids Res. , 26, 55-59, 1998.
The nDB database can be used to identify the boundaries of the operon encoded within the bacterial genome. The operon can be analyzed using a number of techniques well known in the art. In one aspect of this embodiment, gene disruption by homologous recombination is used to individually inactivate operon genes that are thought to contain genes required for growth.

Several gene disruption techniques have been described for the replacement of a functional gene with a mutated non-functional (null) allele. Generally, these techniques involve the use of homologous recombination. Link et al. J. Bacteriol. , 199
7, 179, 6228, which is incorporated by reference herein in its entirety. It is provided as an excellent example of these methods available for gene disruption in E. coli. This technique uses cross-PCR to create null alleles in which the coding region of the target gene has been deleted in frame. Invalid alleles are constructed in such a way that sequences flanking the wild-type gene (about 500 bp) are retained. These homologous sequences around the deletion-invalid allele provide a target for homologous recombination. The wild-type gene on the E. coli chromosome can be replaced with a constructionally invalid allele.

The cross-PCR amplification product was subcloned into the vector pK03, which features
Includes chloramphenicol resistance gene, counter-selectable marker sacB, and temperature-sensitive autonomous growth function. E. coli with such vectors. After transformation of the E. coli cell population, selection of cells that had undergone homologous recombination of the vector into the chromosome was performed by growth on chloramphenicol at a nonpermissive temperature of 43 ° C. Under these conditions, only when the chloramphenicol resistance gene is integrated into the chromosome, autonomous replication of the plasmid does not occur and the cells show chloramphenicol resistance.
Usually, E. One crossover is responsible for this integration so that the E. coli chromosome contains a tandem duplication of the target gene consisting of one wild-type allele and one invalid allele separated by the vector sequence.

This new E. coli with tandem duplication E. coli strains can be maintained at permissive temperatures in the presence of drug selection (chloramphenicol). The cells of this new strain are then cultured at the permissive temperature (30 ° C.) without drug selection. Under these conditions, the chromosomes of some cells in the population undergo internal homologous recombination to remove plasmid sequences. Thereafter, strains cultured in a growth medium lacking chloramphenicol but containing sucrose are used to select such a recombinant resolution. In the presence of the counter selection marker sacB, sucrose becomes a toxic metabolite. Thus, cells that survive this counterselection have lost both chromosomal-derived plasmid sequences and autonomously replicating plasmids that are a byproduct of recombination.

There are two possible consequences of the above recombination transformation by homologous recombination. Either the wild-type copy of the target gene is maintained on the chromosome, or the mutant null allele is maintained on the chromosome. In the case of essential genes, one copy of the invalid allele is lethal, and if applied to the essential gene, such cells (including the invalid allele) should not be obtained by the above procedure. If it is not an essential gene, cells containing approximately the same number of invalid alleles and cells containing the wild-type allele should be obtained. Thus, the method serves to test the essentiality of the target gene: when applied to essential genes, only cells with the wild-type allele on the chromosome are obtained.

E. Other techniques for making disruption mutations in E. coli have also been described. For example, Link et al. Also describes the insertion of in-frame sequence tags containing in-frame deletions to simplify analysis of the resulting recombinants. Further, Link et al. Describe gene disruption with a drug resistance marker such as the kanamycin resistance gene. Argonnie
t al. (Aragoni, F. et al, A Genomi-
based Approach for the identityificatio
no of Essential Bacterial Genes, Natur
eBiotechnology, 16, 851-856, the entire disclosure of which is incorporated herein by reference, describes a second copy of a test gene whose gene expression is to be controlled, and the essentiality of the gene. The use of gene disruption in combination with operation with an inducible promoter such as the arabinose promoter to test has been described. Many of these techniques insert large DNA fragments into the gene of interest (such as a drug selectable marker). Link et al. The advantage of the technique described by is that the coding region is accurately removed rather than relying on insertion into the gene, which causes loss of function. This ensures loss of polar effects on the expression of genes downstream from the target gene.

[0083] Recombinant DNA technology can be used to express the entire coding sequence, or a portion thereof, of a gene identified as required for growth. The overexpressed protein can be used as a reagent for further study. Using methods well known in the art,
The identified exogenous sequence is isolated, purified, and cloned into an appropriate expression vector. If desired, the nucleic acid can include a sequence encoding a single peptide to facilitate secretion of the expressed protein.

The expression of a fragment of a bacterial gene identified as necessary for growth is also within the scope of the invention. The fragment of the identified gene is one of the sequences identified in the present invention.
At least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40 of
, At least 45, at least 50, at least 55, at least 60, at least 65, (at least 70), at least 75, or a polypeptide comprising more than 75 contiguous amino acids. The nucleic acid inserted into the expression vector can also include sequences upstream and downstream of the coding sequence.

When expressing the coding sequence of a gene or a fragment thereof identified as necessary for bacterial growth, the nucleic acid sequence to be expressed is operably linked to a promoter in an expression vector using conventional cloning techniques. . The expression vector can be any bacterial, insect, yeast, or mammalian expression system known in the art. Various suppliers (Genetics Institute (Cambridge, MA)
), Slatagene (La Jolla, California), Pro
mega (Madison, Wisconsin), and Invitroge
n (including San Diego, California) can use commercially available vectors and expression systems. If desired, to improve expression and facilitate protein folding, see Hatfield et al. As described in U.S. Patent No. 5,082,767, incorporated herein by reference, the sequence codon usage and codon bias for the particular expression organism into which the expression vector is introduced is described. Can be optimized. Fusion protein expression systems are also within the scope of the present invention.

After expression of the protein encoded by the identified exogenous nucleic acid sequence, the protein is purified. Techniques for purifying proteins are well known in the art. The protein encoded and expressed from the identified exogenous nucleic acid sequence can be partially purified using precipitation techniques (such as precipitation with polyethylene glycol). Chromatographic methods that can be used in the present invention include ion exchange chromatography, gel filtration, the use of hydroxyapatite columns, immobilized reactive dyes, chromatofocusing, and the use of high-performance liquid chromatography. May be included. Electrophoresis (one-dimensional gel electrophoresis, high-resolution two-dimensional polyacrylamide electrophoresis, isoelectric focusing, etc.) and other purification methods are also contemplated as purification methods. Affinity separation methods, including antibody columns, columns with ligands, and other affinity chromatographic substrates are also contemplated as purification methods of the invention.

[0087] Purified proteins produced from the gene coding sequences identified as necessary for growth can be used in various production protocols for useful antimicrobial agents. In one embodiment of the invention, antibodies are raised against a protein expressed from the identified exogenous nucleic acid sequence. Both monoclonal and polyclonal antibodies can be raised against the expressed protein. Methods for making monoclonal and polyclonal antibodies are well-known in the art. Also contemplated are antibody fragment preparations prepared from the antibodies produced as described above.

The purified protein of the present invention can also be applied to screening a small molecule library of candidate compounds active against various target proteins of the present invention. The advantages in the art of binding chemistry, well known in the art, provide a way to generate a large number of candidate compounds that can have a binding or inhibitory effect on a target protein. Thus, screening of small molecule libraries for compounds having binding affinity or inhibitory activity against a target protein produced from the identified gene sequence is also contemplated by the present invention.

The present invention further provides Use against various other pathogenic organisms in addition to E. coli is also contemplated. For example, the invention finds use in identifying genes required for the growth of prokaryotes and eukaryotes. For example, the present invention relates to Plasmodium.
um), protists such as plants, plants of the genus Entamoeba and C
animals such as C. ontracaecum, fungi including Candida species such as Candida albicans, Saccharomyc
es cerevisiae, Cryptococcus neoforman
s, and Aspergillus fumigatus. In one embodiment of the invention, the organisms of the Monera kingdom, particularly bacteria, are searched for new genes required for growth. This embodiment is particularly important when drug resistant bacteria develop.

The number of bacterial species that are resistant to existing antibiotics is increasing. Some lists of these organisms include the genus Staphylococcus (such as S. aureus), the genus Enterococcus (such as E. faecalis), the Pseudo
genus (such as P. aeruginosa), Clostridium (
C. botulinum), the genus Haemophilus (H. influu
nzae), Enterobacter (genus E. cloacae), V
ibrio (such as V. cholera), Moraxala (such as M. cata)
rrhalis, etc.), the genus Streptococcus (S. pneumoni)
ae), Neisseria (N. gonorrhoeae, etc.), My
genus coplasma (such as Mycoplasma pneumoniae), S
almonella typhimurium, Helicobacter p
ylori, Escherichia coli, and Mycobacter
ium tuberculosis. These and other organisms can be searched using the sequences identified as required for growth of the present invention to identify homologous genes required for growth.

In one embodiment of the present invention, the nucleic acid sequences disclosed herein are used to transform E. coli.
A genomic library prepared from a target bacterial species other than E. coli can be screened. For example, a genomic library is Staphylococc
us aureus, Pseudomonas aeruginosa, Ent
erobacter cloacae, Helicobacter pylor
i, Neisseria gonorrhoeae, Enterococcus
faecalis, Streptococcus pneumoniae, H
aemophilus influenzae, Salmonella type
himurium, Saccharomyces cerevisiae, Ca
ndida albicans, Cryptococcus neoforma
ns, Aspergillus fumigatus, Klebsiella
pneumoniae, Salmonella typhi, Salmonel
la paratyphi, Salmonella cholerasuis,
Staphylococcus epidermidis, Mycobacterte
rium tuberculosis, Mycobacterium lepr
ae, Treponema pallidum, Bacillus anthr
acis, Yersinia pestis, Clostridium bot
ulinum, Campylobacter jejuni, Chlamydi
a trachomatus, Chlamydia pneumoniae, or any species within the genus to which any of the above species belongs. Genomic libraries from various microorganisms can be generated using standard molecular biology techniques. In one embodiment, a library is made and conjugated to nitrocellulose paper. The identified exogenous nucleic acid sequences of the present invention can be used as probes to screen libraries of homologous sequences. The identified homologous sequences can then be used as targets for novel antimicrobial compounds with higher activity than certain organisms.

For example, using the methods described above, SEQ ID NOs: 1-81, 405-485, 82-
88, 90 to 242, at least 97%, at least 95%, at least 90%, at least 85%, at least 80% or at least 70% identical to a nucleic acid sequence selected from the group consisting of one of the sequences: At least 10
, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200
, 300, 400, or 500 contiguous bases, and nucleic acids having sequences complementary thereto can be isolated. Identity can be measured using BLASTN version 2.0 with default parameters. (Altschul, SF et al., Gapped BLA.
ST and PSI-BLAST: A New Generation of
Protein Database Search Programs, Nu
cleic Acid Res. , 25, 3389-3402 (1997), the entire disclosure of which is incorporated herein by reference. For example, a homologous polynucleotide may have a coding sequence that is a naturally occurring allelic variant of one of the coding sequences described herein. SEQ ID NOs: 1-81, 405-485, 82
Such allelic variants can have one or more nucleic acid substitutions, deletions, or additions when compared to the nucleic acid of ~ 88, 90-242, or a sequence complementary thereto.

Further, using the above procedure, FASTA with default parameters
When identified using the version 3.0t78 algorithm, SEQ ID NO: 24
At least 9 for a polypeptide having one sequence of 3-357, 359-398;
9%, at least 95%, at least 90%, at least 85%, at least 8
A polypeptide having 0%, at least 70%, at least 60%, at least 50% or at least 40% identity or similarity, or at least 5,
10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 15
Nucleic acids encoding fragments containing 0 contiguous amino acids can be isolated. Alternatively, protein identity or similarity can be identified using BLASTP using default parameters, BLASTX using default parameters, or TBLASTN using default parameters. (Alschul, SF et al., Gapped BLAST a)
nd PSI-BLAST: A New Generation of Pro
tein Database Search Programs, Nuclei
c Acid Res. , 25, 3389-3402 (1997), the entire disclosure of which is incorporated herein by reference.

Alternatively, homologous nucleic acids or polypeptides may be searched through databases to identify sequences having a desired level of homology to nucleic acids or polypeptides associated with growth, or antisense nucleic acids to nucleic acids associated with microbial growth. Peptides can be identified. Various databases are available, including GenBank and GenSeq. In some embodiments, the database is screened for at least 97%, at least 95%, at least 90%, at least 85%, at least 80% of proliferation-associated nucleic acids or polypeptides, or proliferation-associated antisense nucleic acids. , Nucleic acids or polypeptides having at least 70%, at least 60%, at least 50% or at least 40% identity or similarity. For example, screening a database to identify nucleic acids or polypeptides homologous to one of SEQ ID NOs: 1-81, 405-485, 82-88, 90-242 or SEQ ID NOs: 243-357, 359-398. be able to. In some embodiments, the database is screened for Staphylococcus aureus, Pseudomonas a.
eruginosa, Enterobacter cloacae, Helic
observer pylori, Neisseria gonorrhoeae
, Enterococcus faecalis, Streptococcus
pneumoniae, Haemophilus influenzae, S
almonella typhimurium, Saccharomyces
cerevisiae, Candida albicans, Cryptoco
ccus neoformans, Aspergillus fumigatu
s, Klebsiella pneumoniae, Salmonella t
yphi, Salmonella paratyphi, Salmonella
cholerasuis, Staphylococcus epidermi
dis, Mycobacterium tuberculosis, Mycob
acterium leprae, Treponema pallidum, B
acillus anthracis, Yersinia pestis, Cl
Ostrium botulinum, Campylobacter je
juni, Chlamydia trachomatus, Chlamydia
E. pneumoniae, or an organism such as any species within the genus to which any of the above species belongs. Homologous nucleic acids or polypeptides from organisms other than E. coli can be identified.

[0095] In another embodiment, gene expression arrays and microarrays can be used. The gene expression array has a high-density array of DNA samples attached to a specific position on a glass chip, nylon membrane, or the like. Researchers can use such arrays to quantify relative gene expression under different conditions. Researchers can use gene expression arrays to help identify optimal drug targets, profile new compounds, and identify disease pathways.
An example of this technique can be found in US Pat. No. 5,807,522, which is incorporated herein by reference.

It is possible to study the expression of all genes in a particular microbial genome using one array. For example, Genosys arrays are available from E. coli. coli 4290
Consisted of a 12 × 24 cm nylon filter containing PCR products corresponding to one ORF. Each 10 ng is spotted on the filter at 1.5 mm intervals. Single-stranded labeled cDNA is prepared for hybridization to the array (no second strand synthesis or amplification step is performed) and placed in contact with the filter. Thus, the labeled cDNA is in the "antisense" orientation. Phosphor image (phosphorimag
er) performs a quantitative analysis.

After hybridization of a cDNA prepared from a sample of whole cellular mRNA to such an array, binding is detected by one or more of a variety of techniques known to those of skill in the art to determine if binding on the array hybridized to the cDNA has occurred. A signal is observed at each position. Thus, the intensity of the hybridization signal obtained from each location on the array reflects the amount of mRNA for a specific gene present in the sample. Thus, a comparison of the results obtained from mRNA isolated from cells grown under different conditions allows a comparison of the relative expression levels of each gene during growth under different conditions.

Gene expression arrays can be used to analyze total mRNA patterns at various measurement points after induction of antisense nucleic acids against genes required for growth. Analysis of expression patterns revealed by hybridization to the array indicates whether the target gene of the antisense nucleic acid is affected by antisense induction, how fast antisense affects the target gene, or slower Information about what other genes are affected by antisense expression at the measurement point is obtained. For example, the antisense ribosomal protein L7 / in the 50S subunit
When directed to the L12 gene, its targeted mRNA may first disappear, and then the other mRNA may increase, decrease, or remain the same. Similarly, when the antisense is directed to a different 50S subunit ribosomal protein mRNA (eg, L25), the mRNA is first abolished, and then an mRNA expression similar to that observed with L7 / L12 antisense expression. Change. Thus, based on the mRNA expression pattern observed for an antisense nucleic acid against a gene required for growth, a nucleic acid required for another growth in the same pathway as the target of the antisense nucleic acid can be identified. In addition, the mRNA found in the candidate drug compound
Expression patterns can be compared to those found with antisense nucleic acids to nucleic acids required for growth. If the mRNA expression pattern observed with the candidate drug compound is similar to that seen with the antisense nucleic acid, the drug compound may be a promising therapeutic candidate. Thus, this assay would be useful in assisting in selecting candidate drug compounds for use in screening methods, such as those described below.

If the source of the nucleic acid stuck on the array and the source of the nucleic acid hybridized to the array are from two different organisms, a gene expression array can identify homologous genes of the two organisms .

The present invention also contemplates additional methods of screening other microorganisms for genes required for growth. In this embodiment, conserved portions of sequences identified as required for growth can be used to make degenerate primers for use in the polymerase chain reaction (PCR). PCR technology is well known in the art. Successful production of a PCR product using a denatured probe made from the sequences identified herein indicates the presence of a homologous gene sequence for the species being screened. Then
This homologous gene is isolated, expressed and used as a target for candidate antimicrobial compounds. In another aspect of this embodiment, the homologous gene is expressed in the organism or heterologous organism in a manner that alters the level or activity of the homologous gene required for growth of the organism or heterologous organism. In yet another aspect of this embodiment, the homologous gene, or a portion thereof, is expressed in an antisense orientation in a manner that alters the level or activity of a nucleic acid required for the growth of an autologous or heterologous organism.

Using homologous sequences to genes required for growth identified using the techniques described herein, as described below, E. coli. Genes required for the growth of organisms other than E. coli are identified, or as described below. E. coli by inhibiting or reducing the activity of an identified homologous nucleic acid or polypeptide of an organism other than E. coli. inhibiting the growth of organisms other than E. coli; Compounds that inhibit the growth of organisms other than E. coli can be identified.

[0102] In another aspect of the invention, E. coli identified as required for growth. E. coli sequence,
Non-E. Transfer to an expression vector that can function in the E. coli species. As will be appreciated by those in the art, the expression vector should include certain elements that are specific species. These elements can include promoter sequences, operator sequences, repressor genes, origins of replication, ribosome binding sequences, termination sequences, and the like. To use the identified exogenous sequences of the present invention, one of skill in the art would isolate a vector containing the sequence of interest from cultured bacterial cells, isolate and purify the sequence, and use it for the bacterial species to be screened. Recognize the use of standard molecular biology techniques for subcloning these sequences into expression vectors as applied to US Pat.

[0103] Various other types of expression vectors are known in the art. For example, Cao et
al. Report the expression of a steroid receptor fragment of Staphylococcus aureus. (J. Steroid Biochem
Mol. Biol. 44 (1), 1-11, 1993. ) Also, Pla
et al. Is Salmonella typhimurium, Pseud
omonas putida and Pseudomonas aerugin
Expression vectors that are functional in a number of related hosts, including osa, have been reported. (J. Bacteriol., 172 (8), 4448-55, 1990.
These examples demonstrate the existence of molecular biology techniques that can construct expression vectors of the bacterial species of interest for the present invention.

After subcloning the identified nucleic acid sequence into an expression vector that is functional in the microorganism of interest, the identified nucleic acid sequence is conditionally transcribed and analyzed for bacterial growth inhibition. These expression vectors that, when transcribed, are found to contain sequences that inhibit bacterial growth may be similar to, or homologous to, the known genomic sequence of the screened pathogenic microorganism. If the sequence is unknown, as described above, the E. coli required for the desired growth is obtained. E. coli required for hybridization to E. coli sequences or desired growth. It can be identified and isolated by amplification using primers based on the E. coli sequence.

[0105] The antisense sequence from the second organism identified above can then be operably linked to a promoter, such as an inducible promoter, or introduced into the second organism. Thus, the E. coli required for growth described herein. col
The i-gene identification technique can be used to determine whether the identified sequence from the second organism inhibits the growth of the second organism.

E. To evaluate homology between E. coli sequences and nucleic acids required for growth from other organisms, An antisense nucleic acid or a gene corresponding thereto required for the growth of an organism other than E. coli is It can also hybridize with microarrays containing the E. coli ORF. For example, nucleic acids required for growth are Staphyloc
occus aureus, Pseudomonas aeruginosa,
Enterobacter cloacae, Helicobacter py
lori, Neisseria gonorrhoeae, Enterococ
cus faecalis, Streptococcus pneumonia
e, Haemophilus influenzae, Salmonella
typhimurium, Saccharomyces cerevisiae
, Candida albicans, Cryptococcus neofo
rmans, Aspergillus fumigatus, Klebsiel
la pneumoniae, Salmonella typhi, Salmo
nella paratyphi, Salmonella cholerasu
is, Staphylococcus epidermidis, Mycoba
cterium tuberculosis, Mycobacterium l
eprae, Treponema pallidum, Bacillus an
thracis, Yersinia pestis, Clostridium
botulinum, Campylobacter jejuni, Chlam
ydia trachomatus, Chlamydia pneumonia
e, or from any species within the genus to which any of the above species belongs. If the probes have different levels of homology to sequences on the microarray, coli
Nucleic acids required for growth from other organisms can hybridize to the array under various conditions under which hybridization occurs. This will provide a key to homology across organisms as well as other possible essential genes in these organisms.

In yet another embodiment of the invention, an exogenous nucleic acid sequence of the invention identified as being required for bacterial growth or growth can be used as an antisense therapeutic for bacterial killing. An antisense sequence can be used for the gene required for growth whose sequence corresponds to the exogenous nucleic acid probe identified herein (
That is, an antisense nucleic acid can hybridize to a gene or a portion thereof). Alternatively, the antisense therapeutic can be directed to the operon where the gene required for growth is present (ie, the antisense nucleic acid can hybridize to any gene in the operon where the gene required for growth is present). ). In addition, antisense therapeutics may be used to identify the actual sequence required for bacterial growth or the adjacent non-coding intragenic sequence upstream or downstream from the operon containing the gene required for growth (ie, Sequence), intergenic sequence (
That is, a sequence that spans at least a portion of two or more genes, a 5 ′ non-coding sequence, or a gene or portion thereof that is required for growth with or without a 3 ′ non-coding region. can do.

In addition to therapeutic applications, the present invention involves the use of nucleic acids complementary to sequences required for growth as diagnostic tools. For example, a nucleic acid complementary to a sequence required for specific growth of a particular microorganism can be used as a probe to identify a particular microorganism species in a clinical specimen. This application provides a quick and reliable way to identify the causative agent of a bacterial infection. This use will allow physicians to formulate species-specific antimicrobial compounds to treat such infections. This expanded use allows screening of specific microorganisms that produce such proteins in a species-specific manner using antibodies raised against proteins translated from mRNA transcribed from sequences required for growth. You can also.

The following examples illustrate E. coli identified as required for growth. The genes and subsets of the invention used for the E. coli gene are taught. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Examples The following examples illustrate the E. coli required for growth. E. coli gene identification and development. Methods for identifying genes and various uses of the identified sequences are discussed.

E. Gene exogenous nucleic acid sequences identified as required for E. coli growth were cloned into an inducible expression vector and analyzed for growth inhibitory activity. Example 1 describes the testing of a library of exogenous nucleic acid sequences cloned into an IPTG-derived expression vector. Upon activation or induction, the expression vector is:
An RNA molecule corresponding to the subcloned exogenous nucleic acid sequence was produced. The RNA product was originally derived from E. coli. It was in the antisense orientation with respect to the E. coli gene. This antisense RNA can then be used for various E. coli. interacts with the sense mRNA produced from the E. coli gene and generates a sense messenger RNA (mRN
Buffering or inhibiting the translation of A) prevented protein production from these sense mRNA molecules. When the sense mRNA encoded a protein required for growth, bacterial cells containing the activated expression vector could not grow or grew at a substantially reduced rate.

Example 1 Inhibition of Bacterial Growth After IPTG Induction To study the effect of transcription induction in liquid media, should 1 mM IPTG be included?
Without dilution, 1: 200 medium back dilution into fresh medium and OD every 30 minutes ₄₅₀ A growth curve was prepared by measuring. Study the effect of inducing transcription on solid media
For an overnight culture of 10^Two, 10^Three, 10^Four, 10^Five, 10⁶, 10⁷, 10⁸Double
An exudate was prepared. Aliquots of 0.5-3 μl of these dilutions were made with 1 mM I
Spotted on selective agar plates with or without PTG. Overnight incubation
After the application, the plates were compared to evaluate the sensitivity of the clone to IPTG.

[0113] Of the large number of clones tested, some clones were E. coli after IPTG induction.
It was identified as containing sequences that inhibited E. coli growth. Thus, the gene corresponding to the inserted nucleic acid, or the gene in the operon containing the inserted nucleic acid, is E. coli. coli
May be required for growth.

E. Characterization of isolated clones that negatively affect E. coli growth After identification of expression vectors that negatively affect E. coli growth or growth, the inserts or nucleic acid fragments contained in these expression vectors were isolated for subsequent characterization. The desired expression vector was subjected to nucleic acid sequencing.

Example 2 Determination of the Nucleic Acid Sequence of Identified Clones Expressing Nucleic Acid Fragments that Detrimentally Affect E. coli Propagation
ia, CA) and the method supplied by the manufacturer, and was determined using the isolated plasmid DNA. The primer used for sequencing the insert was 5'-TGT
TTATCGACCGCTT-3 ′ (SEQ ID NO: 403) and 5′-ACAA
TTTCACACACGCCTC-3 ′ (SEQ ID NO: 404). These sequences are adjacent to the polylinker in pLEX5BA. The SEQ ID NOs of the identified inserts are listed in Table 1 and discussed below.

Example 3 Comparison of Isolated Sequence with Known Sequences The nucleic acid sequence of the subcloned fragment obtained from the above expression vector was converted to BLAST version 1.4 or version 2.10 using the following default parameters: 0.6 using GenBank's known E.C. compared to E. coli sequences: filtering off, cost to open a gap -5, cost to extend a gap -2, penalty for mismatch in the running part of blast-3, Reward for a match in the blast portion of run-1, expected value (e) -10.0, word size-11, online display count-100, alignment display count (B ) -100. BLAST is described in Altschul, J .; Mo
l. Biol. 215, 403-10, 1990, the entire disclosure of which is incorporated herein by reference. The expression vector was found to contain the nucleic acid sequence in both sense and antisense orientation. A known gene,
The open reading frame and the presence of the ribosome binding site were identified by comparing the genetic information held by public databases and various computer programs such as the Genetics Computer Group programs FRAMES and CODONPREFERENCE. If the cloned fragment was oriented to the promoter such that the RNA transcript produced was complementary to the mRNA expressed from the chromosomal locus, the clone was called "antisense". A clone was called "sense" if it encoded an RNA fragment that was identical to a portion of the wild-type mRNA from the chromosomal locus.

Examples 1 to 4 Inhibiting Bacterial Growth and Containing Gene Fragments in Antisense Orientation
The sequences described in Table 2 are listed in Table 1. This table shows the National Center
r for Biotechnology Information (NCBI
) Blattner (Science 277: 1453-1474 (1997)
Year) E. coli K-12 genomic sequence) or Rudd (Micro
. and Mol. Rev .. 62: 985-1019 (1998). Each sequence identified by the gene corresponding to the sequence identification number, molecular number, and the identified sequence was listed, respectively. (Both articles are incorporated herein by reference). CONTIG number in each identified sequence; coli
Indicates the position of the first and last base pairs present on the chromosome. Molecular numbers with “ ^** ” indicate clones corresponding to the intergenic sequence.

SEQ ID NOs: 1 from US Provisional Patent Application No. 60 / 117,405 which inhibits growth
The 81 nucleic acid insert sequences were further analyzed. US Provisional Patent Application No. 60/117,
The reanalyzed sequence corresponding to SEQ ID NOs: 1-81 of SEQ ID NO: 405 is SEQ ID NO: 4
05 to 485.

SEQ ID NOs: 82-242 of US Provisional Patent Application No. 60 / 117,405 are identical to SEQ ID NOs: 82-242 of the present application with the following exceptions. SEQ ID NO: 1 of the present application
48 is the complementary strand of SEQ ID NO: 148 of US Provisional Patent Application No. 60 / 117,405. Accordingly, the protein of SEQ ID NO: 308, encoded by SEQ ID NO: 148, has also been modified. SEQ ID NO: 163 of the present application corresponds to US Provisional Patent Application No. 60
/ 117,405 is the complementary strand of SEQ ID NO: 163. Therefore, SEQ ID NO: 1
The protein of SEQ ID NO: 323 encoded by 63 has also been modified.

[0120] The target genes of SEQ ID NOs: 18 and 19 of US Provisional Patent Application No. 60 / 117,405 (SEQ ID NOs: 18, 19, 422, 423 of the present application) are those whose SEQ ID NOs: Reflecting the fact that antisense molecules contain dic
It has been modified from F to ftsZ.

[0121] SEQ ID NOs: 198 and 239-2 of US Provisional Patent Application No. 60 / 117,405.
42 and the gene products of the nucleic acids of the present application (SEQ ID NOs: 358 and 399-402 of the present application) have been modified to reflect the fact that these nucleic acids encode untranslated tRNA and rRNA. Accordingly, Tables I and II have been modified. SEQ ID NOs: 89 and 402 in Table II also correspond to US Provisional Patent Application No. 6
Revised to reflect the facts identified in 0 / 117,405.

[0122]

[Table 1]

Example 4 Identification of genes affected by antisense inhibition and their corresponding operons The sequencing of the E. coli genome is described in Blattner et al. , Sc
gene, 277, 1453-1474, 1997, which is incorporated herein by reference in its entirety, and the genomic sequence is GenBank Accession No. U00096, the entire disclosure of which is incorporated herein by reference. , Incorporated by reference). An operon corresponding to the growth-inhibiting nucleic acid was obtained from Regulon D
Identification was performed using B and information in the literature. E. FIG. The coordinates of the boundaries of these operons on the E. coli genome are listed in Table III. Table II shows the molecular number of the insert containing the growth-inhibiting nucleic acid fragment, the gene in the operon corresponding to the insert, the SEQ ID NO of the gene containing the insert, the SEQ ID NO of the protein encoded by the gene,
E. FIG. The start and end points of the gene on the E. coli genome, the orientation of the gene on the genome, whether the operon is estimated or described in the literature, and the putative function of the gene are listed. The identified operons, their putative functions, and whether the genes are currently considered necessary for growth are discussed below.

The function of the identified genes was identified by Blattner's functional class nomenclature or by comparison of the identified sequences to known sequences in various databases. Various biological functions have been described for the genes to which the clones of the present invention correspond. The function of the gene of interest is shown in Table II.

The proteins listed in Table II are associated with a wide range of biological functions.

[0126]

[Table 2]

[0127] Some expression vectors contain fragments that correspond to or have a known function, when the gene is essential or unknown. For example, EcXA
001, 003, 007, 008, 013, 015, 016, 017, 018,
019, 020, 021, 022, 023, 024, 025, 026, 027,
028, 029, 030, 032, 033, 034, 035, 036, 037,
038, 039, 040, 041, 047, 048, 049, and 050 all do not indicate whether the function is unknown or whether the function is required or not. col
An exogenous nucleic acid sequence corresponding to the i protein.

The present invention provides a number of novel E. coli required for growth. The E. coli gene and operon have been reported. From the list, the clone sequences identified herein are each E. coli.
It was identified as part of the operon required for E. coli growth. E. FIG. Cloned sequences corresponding to genes already known to be necessary for the growth of E. coli include EcX
A002, 004, 005, 010, 012, 014, 031, 02, 043,
045, 051, 052, 054, and 055. The remaining identified sequences have been previously identified in the art as not required for growth. E. coli gene.

An interesting observation of the present invention is that E. coli not considered necessary for E. coli growth
Some sequence fragments corresponding to the E. coli gene are also present.
Nevertheless, under the conditions described above, antisense expression of these gene fragments reduces cell proliferation. This result indicates that the gene corresponding to the identified sequence is actually required for growth. The molecular numbers corresponding to these genes are EcXA006, 044, and 053.

After identifying the sequences of interest, these sequences were localized to the operon. Since bacterial genes are expressed in a polycistronic fashion, antisense inhibition of one gene in the operon can affect the expression of all other genes on the operon or genes downstream from one identified gene. To identify which gene products in the operon are required for growth, each gene contained within the operon can be analyzed for its effect on viability as described below.

[0131]

[Table 3] Example 5 Identification of Each Gene in the Operon Required for Growth The following example illustrates a method for identifying which genes in the operon are required for growth. The clone insert corresponding to molecule number EcXA004 was obtained from E. coli.
It carries nucleic acid sequences homologous to the E. coli genes rspG and rspL. This molecule corresponds to an operon containing two additional genes, fusA and tufA.
The rpsL gene is the first gene in the operon. To identify which genes in this operon are required for growth, each gene is selectively inactivated using homologous recombination. Gene rpsL is the inactivated first gene.

E. Deletion inactivation of a chromosomal copy of a gene in E. coli can be performed by integrated gene replacement. This method (Hamilton, CM et al.
. 1989; Bacteriol. 171, 4617-4622) is to construct a mutant allele of the target gene, transfer it to this allele using a conditional suicide vector, and remove the wild-type allele and vector sequence. This replaces the native gene with the desired mutation, but with the promoter,
Operators and the like remain intact. The essentiality of the gene is identified either by speculation by genetic analysis or by conditional expression of a wild-type copy of the target gene (trans homology).

The first step is the generation of a mutant rpsL allele using PCR amplification. Two sets of PCR primers are selected to produce a copy of rpsL with a large central deletion to inactivate the gene. To eliminate the polarity effect, rpsL
It is desirable to construct a mutant allele in which most or all of the coding region of the gene has been deleted in frame. Each set of PCR primers is chosen such that the region adjacent to the gene to be amplified is long enough to recombine (typically,
At least 500 nucleotides on each deletion site). Target deletions or mutations may be included within this fragment. To facilitate cloning of PCR products,
PCR primers may also include a restriction endonuclease site found in the cloning region of a conditional knockout vector such as pK03 (Link
et al. 1997; Bacteriol. , 179, (20), 6
228-6237). Suitable sites include NotL, SalI, BamHI, and SmaI. The rpsL gene fragment was prepared using standard PCR conditions (
Hot Start taq PCR kit (Qiagen, Inc., Vale)
ncia, CA), including but not limited to the conditions outlined in the product insert. The PCR reaction can produce two fragments that can be fused together. Alternatively, the desired deletion can be made in one step using crossover PCR (Ho, SN, et a).
l. 1989, Gene, 77, 51-59; Horton, R .; M. , Et
al. , 1989, Gene, 77, 61-68). Therefore, the mutated allele produced is referred to as "null" because it cannot produce a functional gene product.
Called alleles.

The mutated allele from the PCR amplification is cloned into the multiple cloning site of pK03. No directional cloning of the rpsL "null" allele is required. p
The K03 vector has a temperature-sensitive origin of replication from pSC101. Therefore, clones are grown at the permissive temperature (30 ° C.). The vector also provides two selectable marker genes (such as Bacillus subtilis s) that confer resistance to chloramphenicol and the like and are capable of counterselecting against sucrose containing growth medium.
acB gene)). Vector DN with "null" allele inserted
Clones containing A are confirmed by restriction endonuclease analysis and DNA sequence analysis of the isolated plasmid DNA. Plasmids containing the rpsL "null" allele insertion are known as knockout plasmids.

[0135] Once the knockout plasmid has been constructed and its sequence confirmed, it can be transferred to Rec ⁺ E. coli. E. coli host cells. Transformation can be performed by any standard method, such as electroporation. In some fractions of the transformed cells, the plasmid is transformed into E. coli by homologous recombination between the rpsL null allele in the plasmid and the rpsL gene in the chromosome. coli
Integrated into the chromosome. Transformant colonies in which such an event occurs are readily selected for by growth in the non-permissive temperature (43 ° C) and in the presence of chloramphenicol. At this temperature, the plasmid will not replicate as an episome and will be lost from the cell as it grows and divides. These cells are no longer resistant to chloramphenicol and will not proliferate in the presence of chloramphenicol. However, the knockout plasmid was E. coli. Cells integrated into the E. coli chromosome maintain resistance to chloramphenicol and proliferate.

Cells containing the integrated knockout plasmid are usually the result of a mutation in rpsL separated by the vector sequence and a single crossover creating the tandem sequence of the native wild-type allele. The result is that rpsL will be expressed in these cells as well. To determine if a gene is essential for growth, the wild-type copy must be removed. This is done by choosing plasmid removal by a process in which homologous recombination between the two alleles circularizes away from the plasmid sequence. Cells that have undergone such removal and lack the plasmid sequence containing the sacB gene are selected by adding sucrose to the medium. s
The acB gene product converts sucrose to a toxic molecule. Thus, counter-selection on sucrose confirms that the plasmid sequence is no longer present in the cell. In addition, the loss of plasmid sequence is confirmed by a test for susceptibility to chloramphenicol (loss of chloramphenicol resistance gene). The latter test is important because sometimes mutations in the sacB gene occur as a result of loss of sacB function without affecting plasmid replication (L
ink, et al. 1997; Bacteriol. , 179, (2
0), 6228-6237). These artificial clones retain the plasmid sequence and are therefore resistant to chloramphenicol.

In the plasmid removal step, one of the two rpsL alleles has been deleted from the chromosome along with the plasmid DNA. Generally, null or wild-type alleles appear to be deleted. Thus, if the rpsL gene is not essential, half of the clones obtained in this experiment will have the wild-type allele on the chromosome, and half will be the null allele. However, when the rpsL gene is essential,
Since one copy of the null allele is lethal, cells containing the null allele will not be obtained.

To identify the indispensability of rpsL, the resulting statistically significant number of clones (at least 20) is analyzed by PCR amplification of the rpsL gene. Because the null allele lacks a significant portion of the rpsL gene, its PCR product is significantly shorter than that of the wild-type gene, and both are easily distinguished by gel base analysis. For further confirmation, the PCR product can be subjected to sequencing by methods well known in the art.

The above experiments are generally suitable for identifying the essentiality of genes such as rpsL. However, it may be necessary or desirable to more directly confirm the essentiality of the gene. There are several ways that this can be achieved. In general, these include the following three steps: 1) construction of an episome containing the wild-type allele, 2) one of the mutant null alleles as described above, except in the presence of the episome wild-type allele. Isolation of clones containing two chromosomal copies, and 3) identification of whether cells survive if expression of episomal alleles is blocked.
In this case, the transcopy of wild-type rpsL was performed by PCR of the entire coding region of rpsL.
Cloning and E. coli. It is made by insertion in the sense direction downstream of an inducible promoter, such as the E. coli lac promoter. Transcription of this rpsL allele is induced in the presence of IPTG, which inactivates the lac repressor. IP
Under TG induction, as long as the recombinant gene also has a ribosome binding site, rps
The L protein is expressed and is known as the "Shine-Dalgarno sequence". The rpsL transcopy is cloned into a plasmid compatible with pSC101. Compatible vectors include p15A, pBR322, and pUC plasmids. Replication of compatible plasmids is not temperature sensitive. To confirm that the expression of the functional rpsL protein was fully maintained, rpsL
The entire process of integration of the null allele and subsequent plasmid removal is
Perform in the presence of After confirming that the null rpsL allele is integrated into the chromosome instead of the wild-type rpsL allele, the IPTG is recovered to block expression of the functional rpsL protein. If the rpsL gene is essential, the cells will not grow under these conditions. However, if the rpsL gene is not essential, cells will continue to grow under these conditions. In this experiment, indispensability is identified by the conditional expression of a wild-type gene copy rather than the inability of the intended chromosome disruption.

An advantage of this method over some other gene disruption techniques is that the target gene can be deleted and mutated without the transfer of large segments of foreign DNA. Thus, polar effects on downstream genes are eliminated or minimized. Methods exist that describe inducible promoter transfer upstream of potentially essential bacterial genes. However, in such cases, polarity from multiple transcription start points can be a problem.
One way to avoid this is to insert a gene disruption cassette that contains a strong transcription terminator upstream of the integrated inducible promoter (Zhang,
Y and Cronan, J.M. E. FIG. 1996; Bacteriol.
178, 12, 3614-3620). The techniques described are well known to those skilled in the art.

Following analysis of the rpsL gene, other genes in the operon are examined to identify if they are required for growth.

Example 6 Expression of a Protein Encoded by a Gene Identified as Required for E. coli Propagation An exemplary method for expressing a protein required for growth encoded by the above identified sequences is provided below. First, the start and stop codons of the gene are identified. If necessary, methods for improving the transcription or expression of proteins are well known in the art. For example, if the nucleic acid encoding the expressed polypeptide lacks a start site, a strong Shine-Dalgarno sequence, or a methionine codon that acts as a stop codon, these sequences can be added. Similarly, if the identified nucleic acid sequence lacks a transcription termination signal, this sequence can be added to the construct, for example, by splicing such a sequence from a suitable donor sequence. In addition, if desired, the coding sequence can be operably linked to a strong or inducible promoter. The identified nucleic acid sequence encoding the polypeptide to be expressed, or a part thereof, corresponds to the NcoI incorporated into the 5 ′ primer and the 3 ′ primer using an oligonucleotide primer complementary to the identified nucleic acid sequence or a part thereof. The 5'-terminal BglII restriction endonuclease sequence is obtained by PCR from a bacterial expression vector or genome, taking into account the evidence that the identified nucleic acid sequence is present in frame with a termination signal. The purified fragment obtained from the resulting PCR reaction is digested and purified with NcoI and BglII and ligated into an expression vector.

[0143] The ligated product can be combined with DH5α or some other E. coli suitable for overexpression of potential proteins. E. coli strain. Transformation protocols are well-known in the art. For example, the transformation protocol is a Current Protocol.
ols in Molecular Biology, Volume 1, Unit 1.
8, Ausubel, et al. Hen, John Wiley & Sons,
Inc. , (1997). 50-100 μg of transformed cells
After growth on plates containing / ml ampicillin (Sigma, St. Louis, Missouri), positive transformants are selected. In one embodiment, the expressed protein is retained in the cytoplasm of the host organism. In another embodiment, the expressed protein is released into the culture medium. In yet another embodiment, the expressed protein can be sequestered in the periplasmic space or released therefrom using any one of a number of cell lysis techniques known in the art. For example, Curren
t Protocols in Molecular Biology, Volume 2, Ausubel, et al. Hen, John Wiley & Sons, I
nc. Osmotic shock cell lysis as described in Chapter 16, Vol. Each of these methods can be used to express proteins required for growth.

The expressed proteins released in the culture medium or from the periplasmic space or cytoplasm are then analyzed by ammonium sulfate precipitation, standard chromatography, immunoprecipitation, immunochromatography, size exclusion chromatography, ion exchange chromatography, HPLC, etc. Purify or enrich from the supernatant using conventional techniques. Alternatively, the secreted protein can be fully enriched or present in a host supernatant or growth medium that can be used for the intended purpose without further enrichment. The purity of the resulting protein product can be assessed by using techniques such as Coomassie or silver staining, or by using antibodies to control proteins. Coomassie or silver staining methods
It is well known to those skilled in the art.

An antibody capable of specifically recognizing a protein of interest can be produced by using a synthetic peptide and a method well known in the art. Antibody
s: A Laboratory Manual, Harlow and Lan
e, Cold Spring Harbor Laboratory, (19
1988). For example, a 15-mer peptide having the sequence encoded by the appropriate identified gene sequence of interest or a portion thereof can be chemically synthesized. Synthetic peptides are injected into mice to generate antibodies against the polypeptide encoded by the identified nucleic acid sequence of interest or a portion thereof. Alternatively, a protein sample expressed from the above expression vector is purified and subjected to amino acid sequencing,
The identity of the recombinantly expressed protein can be confirmed and then used to raise antibodies. An example describing in detail the production of monoclonal and polyclonal antibodies is given in Example 7.

[0146] The protein encoded by the identifying nucleic acid sequence of interest or a portion thereof can be purified by standard immunochromatographic techniques. In such a procedure, a solution containing a secreted protein (such as a culture medium or cell extract) is passed through a column having antibodies to the secreted protein bound to a chromatography substrate. The secreted protein is bound to an immunochromatographic column. Thereafter, the column is washed to remove non-specifically bound proteins. The specifically bound secreted protein is then released from the column and recovered using standard techniques. These procedures are well-known in the art.

In another protein purification scheme, the identified nucleic acid sequence of interest or a portion thereof can be incorporated into an expression vector designed for use in a purification scheme using a chimeric polypeptide. In such a strategy, the coding sequence for the identifying nucleic acid sequence of interest, or a portion thereof, is inserted in frame with the gene encoding the other half chimera. The other half chimera may be a maltose binding protein (MBP) or a nickel binding polypeptide coding sequence. The chimeric protein can then be purified using a chromatographic substrate with antibodies to MBP or nickel. The protease cleavage site can be engineered between the MBP gene or nickel binding polypeptide and the identified gene of interest or a portion thereof. Thus, the two polypeptides of the chimera can be separated from each other by protease digestion.

One useful expression vector for making maltose binding protein fusion proteins is pMAL (New England Biol) encoding the malE gene.
abs). In the pMal protein fusion system, the cloned gene is
Insert into the pMal vector downstream of the gene. Thereby, the MBP fusion protein is expressed. Purify by fusion protein affinity chromatography. These techniques described are well known to those skilled in the field of molecular biology.

Example 7 Isolation Production of Antibodies Against E. coli Proteins Substantially pure proteins or polypeptides are isolated from transformed cells as described in Example 6. The protein concentration in the final preparation is, for example,
AMICON filter device (Millipore)
, Bedford, MA) by concentration to a level of a few μg / ml. A monoclonal or polyclonal antibody to this protein can then be prepared as follows.

Monoclonal Antibody Production by Hybridoma Fusion Monoclonal antibodies against any peptide epitope identified and isolated as described were prepared by Kohler, G .; and Milstein, C.I. , N
It can be prepared from mouse hybridomas according to the classical method of atature, 256, 495, 1975 or a known method derived from this method. Briefly, mice are repeatedly inoculated with several μg of the selected protein or peptides derived therefrom over several weeks. The mice are then sacrificed and the antibody-producing cells of the spleen are isolated. The spleen cells are fused with polyethylene glycol with mouse myeloma cells and excess non-fused cells are destroyed by growing the system on a selective medium containing aminopterin (HAT medium). Dilute the successfully fused cells and place aliquots of the dilutions into the wells of a microtiter plate to continue growing the culture. Immunoassay (Engvall, E. "Enzyme immunoas
say ELISA and EMIT ", Meth. Enzymol. , 70
Known production cells are identified by detection of antibodies in the supernatant of wells by the ELISA method described in US Pat. The selected positive clone was expanded and its monoclonal antibody product was recovered. Detailed methods for producing monoclonal antibodies are described in Davis, L .; et al. , Basic Methods
in Molecular Biology Eliseviewer, New
York, Chapter 21-2.

Production of Polyclonal Antibodies by Immunization Polyclonal antibodies, including antibodies against heterologous epitopes of one protein or polypeptide, are expressed or derived from the above-described proteins that can be unmodified or modified to enhance immunogenicity It can be prepared by immunizing a suitable animal with the peptide. Effective polyclonal antibody production is affected by a number of factors related to both the antigen and the species of the host. For example, small molecules are less immunogenic than larger molecules, requiring the use of carriers and adjuvants. Also, host animals change in response to both the site of inoculation and the dose, and inappropriate or excessive amounts of antigen result in low titers of antisera. Administration of small amounts (ng level) of antigen to multiple intradermal sites appears to be most reliable. An effective rabbit immunization protocol is described in Vaitukaitis, J .; et al. J. Clin
. Endocrinol. Metab. , 33, 988-991, 1971.

Booster injections are administered at regular intervals and are identified semiquantitatively by double immunodiffusion in agar against known concentrations of antigen, for example, when their antibody titers begin to decrease. Antiserum can be recovered. See, for example, Ouchterlony, O .;
et al. , Handbook of Experimental Immu
nology, D.E. See Chapter 19 of Wier, Backwell, 1973. Antibody plateau concentrations typically range from 0.1 to 0.2 mg / ml serum (approximately 12M). The affinity of the antigen for antisera can be determined, for example, by Fisher, D.
. Manual of Clinical Immunology, 2nd edition 4
Chapter 2, Rose and Friedman, Amer. Soc. For M
microbiol. Washington, D .; C. , 1980 by generating a competitive binding curve.

[0153] Antibody preparations prepared by either protocol are useful in quantitative immunoassays to identify antigen-carrying substrate concentrations in biological samples. Using the preparation
A semi-quantitative or qualitative identification of the presence of the antigen in the biological sample. Antibodies,
It can also be used in therapeutic compositions for killing bacterial cells that express the protein.

Example 8 Screening of Chemical Libraries A. Protein-Based Assays If proteins identified as required for growth are isolated and expressed,
The present invention further contemplates the use of these expressed proteins in screening assays of libraries of compounds as potential drug candidates. Generation of chemical libraries is well known in the art. For example, using combinatorial chemistry,
Libraries of compounds screened by the assays described herein can be made. Combinatorial chemical libraries are collections of various compounds made by either chemical or biological synthesis by the combination of a number of chemical "bond blocking" reagents. For example, a linear combinatorial chemical library, such as a polypeptide library, is formed by combining amino acids in any possible combination to obtain a peptide of a given length. Theoretically, countless compounds can be synthesized by combinatorial mixing of such chemical bond blockers. For example, one skilled in the art has recognized that the synthetic combinatorial mixing of 100 exchangeable binding blockers theoretically synthesizes 100 million tetramers or 10 billion pentamer compounds. . (Gallop et al., "A
applications of Combinatorial Technology
ogies to Drug Discovery, Background a
nd Peptide Combinatorial Libraries ",
Journal of Medicinal Chemistry, Vol. 37,
9, No. 1233-1250, 1994). Other chemical libraries known to those of skill in the art, including natural product libraries, can also be used.

[0155] Once created, combinatorial libraries can be screened for compounds that possess the desired biological properties. For example, a compound that may be useful as a drug or for developing a drug will likely have the ability to bind to the target protein isolated, expressed, and purified above. Furthermore, if the identified target protein is an enzyme, the candidate compound will likely interfere with the enzymatic properties of the target protein. Any enzyme can be the target protein. For example, the enzymatic function of the target protein can act as a protease, phosphatase, dehydrogenase, transporter protein, transcriptase, and any other type of enzyme known or unknown. Thus, the present invention also contemplates the use of the above protein products to screen combinatorial chemical libraries.

Those of skill in the art will recognize that there are a number of techniques for characterizing target proteins to identify molecules useful for the discovery and development of therapeutics. For example, some techniques include the production and use of small molecule peptides to biologically and genetically search and analyze target proteins to identify and develop drugs. Such techniques include, but are not limited to, PCT Publication Nos. WO 99/35494, WO 98/191.
62, WO 99/54728, the entire disclosure of which is incorporated herein by reference.

In another example, the target protein is a serine protease, and the substrate for this enzyme is known. This example relates to the analysis of a library of compounds to identify compounds that function as inhibitors of the target enzyme. First, a library of small molecules is generated using combinatorial library formation methods well known in the art. Ag
rafiotis, et al. U.S. Patent Nos. 5,463,564 and 5,574,656 (titles "Systems and Met") issued to
hod of Automatically Generating Chem
ical Compound with Desired Property
s ") are the teachings of the above method. The compounds of the library are then screened to identify library compounds that possess the desired structural and functional properties. U.S. Patent No. 5,684,711 also describes a method for screening a library.

To illustrate the screening step, the combined targets and compounds of the library are exposed to and interact with purified enzymes. A labeled substrate is added to this incubation. The label on the substrate is one in which the detectable signal originates from the metabolized substrate molecule. The generation of this signal makes it possible to determine the effect of the compounds of the combination library on the enzymatic activity of the target enzyme. The characteristics of the compounds in each library are coded so that compounds that are active against the enzyme can be analyzed to isolate characteristics common to the various compounds identified and incorporated into further interactions of the library. Become

Once a library of compounds has been screened, the library is then made using chemical bond blockers that possess the features identified in the first screen to have activity against the target enzyme. Using this method, the subsequent interaction of the candidate compound will reduce the structure required for the inhibition of one or more functions of the target enzyme until a group of enzyme inhibitors with high specificity for the enzyme can be discovered. Possesses strategic and functional characteristics. These compounds can then be further tested for safety and efficacy as antibiotics for use in animals.

It will be readily recognized that this particular screening method is for illustrative purposes only. Other methods are well known to those skilled in the art. For example, where the biochemical function of the target protein is known, a wide variety of screening techniques for a large number of naturally occurring targets are known.

B. Cell-Based AssaysCurrent cell-based assays used to identify or characterize compounds for drug discovery and development often involve the inhibition of the activity of target molecules that are present intracellularly or on the surface of cells. Depends on the detection of the ability of the test compound. The most commonly used target molecules are proteins such as enzymes, receptors and the like. However, target molecules can also include other molecules such as DNA, lipids, carbohydrates, and RNA (including messenger RNA, ribosomal RNA, tRNA, etc.). Those skilled in the art
A number of sensitive cell-based assays are available for detecting binding and interaction of a test compound with a specific target molecule. However, these methods are generally not very effective when the test compound binds or interacts with its target molecule with moderate or low affinity. In addition, the target molecule cannot be readily accessible to the test compound in solution, such as when the target molecule is present in a cell or in a cellular compartment such as the periplasm of a bacterial cell. Thus, current cell-based assays cannot effectively identify or characterize compounds that interact with a target with moderate to low affinity or that cannot be easily accessed. Limited.

[0162] The cell-based assays of the present invention have substantial advantages over current cell-based assays practiced in the art. This advantage stems from the use of sensitized cells in which the level or activity of the gene product (target molecule) required for growth is specifically reduced to the point where the rate of cell growth becomes the rate-limiting step due to the presence or absence of that function. . Bacteria, fungi, plant or animal cells can all be used in the method of the invention. Such sensitized cells are even more sensitive to compounds that are active on the affected target molecule. Thus, the cell-based assays of the present invention detect compounds that exhibit low or moderate potency against the target molecule of interest, as the compounds are substantially more potent on sensitized cells than unsensitized cells can do. This effect is indicated when the test compound is 2- to several-fold more potent, at least 10-fold more potent, or even at least 10-fold more potent when compared to unsensitized cells when tested against sensitized cells.
It can be 0 times stronger.

Due to the increased signs of antibiotic resistance of some pathogenic microorganisms and the significant side effects associated with some currently used antibiotics, new antibiotics that act on new targets in the art are becoming increasingly sophisticated. Has been pursued. However, another current limitation of cell-based assays is the problem of identifying targets for some species of target molecules in some restricted sets of biological pathways. This is because compounds acting on `` old '' targets are more frequently encountered and are more potent than compounds acting on new targets, so compounds acting on such new targets may be truncated and ignored. Or if not detected. As a result, most of the currently used antibiotics interact relatively with a small number of target molecules within a more restricted set of biological pathways.

The use of the sensitized cells of the present invention solves the above problem in two ways. First, when tested against the sensitized cells of the present invention, the desired compound that will interact with the target of interest (either a new target or a target that is already known but less utilized) will be identified as such Because of the specific and substantial increase in potency of any desired compound, the "noise" of the compound acting on the "old" target can be detected. Second,
The methods used to sensitize cells to compounds that act on the target of interest can also sensitize these molecules to compounds that interact with other target molecules in the same first biological pathway. For example, expression of an antisense molecule against a gene encoding a ribosomal protein predicts sensitization of the cell to a compound that acts on the ribosomal protein, and any ribosome component (protein or rRNA
) Can be sensitized to a compound that interacts with a compound that further acts with any target that is part of the protein synthesis pathway. Thus, a key advantage of the present invention is the ability to identify novel targets and pathways that were not previously readily accessible to drug discovery methods.

The sensitized cells of the present invention are prepared by reducing the activity or level of the target molecule.
The target molecule can be a gene product, such as an RNA or polypeptide produced from the nucleic acids required for growth described herein. Alternatively, the target may be a gene product such as an RNA or polypeptide produced from the same sequence in the operon as the nucleic acids required for growth described herein. In addition, the target can be an RNA or polypeptide in the same biological pathway as the nucleic acids required for growth described herein. Such biological pathways include, but are not limited to, enzymatic, biochemical, and metabolic pathways, as well as pathways associated with the production of cellular structures such as cell walls.

Current methods used in the fields of medicine and combinatorial chemistry provide structure-activity relationship information derived from testing compounds in a variety of biological assays, including direct binding assays and cell-based assays. Can be used. Occasionally, compounds are identified in assays that are sufficiently effective for drug development. The more frequently used initial hit compounds show moderate or low potency. Once a compound of interest has been identified as low or moderate potency, a directed library of compounds (directed library
ries) is synthesized and tested. In general, these directed libraries are combinatorial chemical libraries consisting of compounds having a structure related to the compound of interest, but including systematic variations that include the addition, reduction, and substitution of various structural features. When tested for activity on a target molecule, structural features that alone or in combination with other features increase or decrease activity are identified. This information is used to design additional directed libraries containing compounds with increased activity on the target molecule. After one or more of the interactions in this step, compounds with substantially increased activity on the target molecule can be identified and further developed as drugs. Compounds that act on the selected target increase their potency in this cell-based assay and can be characterized as more compounds that provide more useful information than otherwise obtained. The process is facilitated by the use of the sensitized cells of the present invention.

Accordingly, in order to identify or characterize compounds that were not readily identified or characterized previously, including compounds that act on targets that were not readily exploited previously using cell-based assays, The cell-based assays of the invention can be used. Because more structure-function relationship information is likely to be revealed, perhaps for the same number of test compounds, the process of developing a potent drug derived from the first compound of interest is also substantially improved by the cell-based assay of the present invention. Be improved.

[0168] Methods of sensitizing cells require the selection of appropriate genes or operons. Suitable genes or operons are those whose expression is necessary for the growth of cells sensitized. The next step is the transfer of the appropriate gene or operon, or an antisense RNA capable of hybridizing to the RNA encoded by the appropriate gene or operon, to the cells to be sensitized. The transfer of the antisense RNA can be in the form of an expression vector in which the antisense RNA is produced under the control of an inducible promoter. The amount of antisense RNA produced is limited to changes in the inducer concentration at which exposure of the cells changes the promoter-driven transcriptional activity of the antisense RNA. Thus, cells are sensitized by exposing the cells to an inducer concentration at which antisense RNA expression is at a sublethal level.

In one embodiment of the cell-based assay, the identified exogenous E. coli of the present invention is
The E. coli nucleotide sequence is used to inhibit the production of proteins required for growth. Expression vectors that produce antisense RNA against the identified gene required for growth are used to limit the protein concentration required for growth without severely disrupting growth. To this end, a growth inhibitory dose curve of the inducer is calculated by plotting different doses of the inducer against the corresponding growth inhibition caused by antisense expression. From this curve, the various percentages (1-100%) of the antisense at which growth inhibition is induced can be identified. When the promoter contained in the expression vector contains a lac operator, transcription is controlled by the lac repressor, and expression from the promoter is induced by IPTG. For example, the highest concentration of the inducer IPTG that does not reduce the growth rate (0% growth inhibition) can be predicted from this curve. Cell growth can be monitored by turbidity of the growth medium by OD measurement. In another example, the concentration of inducer that reduces proliferation by 25% can be predicted from the curve. In yet another example, the concentration of an inducer that reduces proliferation by 50% can be calculated. Additional parameters such as colony forming units (cfu) can be used to measure cell viability.

The cells to be assayed are exposed to the inducer concentrations determined above. The presence of the inducer at this sublethal concentration reduces the amount of the gene product required for growth to the smallest amount in cells that support growth. Thus, due to the presence of this inducer concentration, an inhibitor of a protein or RNA required for the desired growth, or of a protein or RNA in the same biological pathway as the protein or RNA required for the desired growth, It is more sensitive specifically to inhibitors that are not inhibitors of unrelated proteins or RNA.

Cells that are pre-treated with subinhibitory concentrations of an inducer, thereby reducing the amount of target gene product required for growth, are used to screen for compounds that reduce cell growth. The sublethal concentration of the inducer can be any concentration consistent with the intended use in the identification assay for candidate compounds that render the cells more sensitive. For example, a sublethal concentration of an inducer may increase proliferation by at least about 5%, at least about 8%, at least about 10%, at least about 20%, at least about 30%,
The concentration can be at least about 40%, at least about 50%, at least about 60%, at least about 75% or more. Cells pre-sensitized using the above method contain the target protein with less inhibition than wild-type cells and are more sensitive to inhibitors of the target protein.

In another embodiment of the cell-based assay of the invention, the level or activity of a gene product required for growth is determined using temperature sensitive mutations in sequences required for growth and antisense nucleic acids to sequences required for growth. To reduce. Growth of cells at temperatures intermediate between the permissive and limiting temperatures of the temperature-sensitive mutant when the mutation is present in the gene product required for growth reduces the activity of the gene product required for cell growth I do. Antisense RNA directed to sequences required for growth will further reduce the activity of the gene product required for growth. Drugs that cannot be found using either the temperature-sensitive mutation or the antisense nucleic acid alone induce the expression of the antisense nucleic acid, and cells grown at a temperature between the permissive temperature and the limiting temperature will cause Cells that do not induce expression and are substantially more sensitive to the test compound than cells growing at the permissive temperature can be identified. Also, drugs previously found from either the antisense nucleic acid alone or the temperature sensitive mutant alone have different sensitivity profiles when used in cells combining the two approaches, and the sensitivity profile is It may exhibit a more specific effect of the drug that inhibits one or more activities of the product.

[0173] Temperature sensitive mutations are present at different sites in the gene and may correspond to different domains of the protein. For example, the Escherichia coli dnaB gene encodes a replication fork DNA helicase. DnaB has several domains, including domains for oligomerization, ATP hydrolysis, DNA binding, interaction with primase, interaction with DnaC, and interaction with DnaA. [(B
iwas, E .; E. FIG. and Biswas, S.M. B. , 1999. Escherichia coli mechanism and DnaB helicase: structural domains involved in ATP hydrolysis, DNA binding, and oligomerization. Biochem. , 38, 1091
9-10928, Hiasa, H .; and Marians, K.M. J. , 199
Nine years, the initiation of bidirectional replication from chromosomal origin is directed to the interaction between helicase and primase. J. Biol. Chem. , 274, 27244-2724
8, San Martin, C.I. , Randermacher, M .; , Wolp
ensinger, B.S. Engel, A .; Miles, C .; S. , Dixo
n, N. E. FIG. , And Carazo, J .; M. , 1998). Three-dimensional reconstruction from cryo-EM images reveals a detailed complex between helicase DnaB and its loading partner DnaC. Structure, 6
501-9, Sutton, M .; D. , Carr, K .; M. , Victorent
, M .; , And Kaguni, J .; M. , 1998. Esrichia Kori
DnaA protein. E. FIG. Loading of the N-terminal domain and DnaB helicase on the E. coli chromosome. J. Biol. Chem. , 273, 34255-6
2), the entire disclosure of which is incorporated herein by reference)]. Temperature-sensitive mutations in different domains of DnaB confer different phenotypes at the limiting temperature, which can be abrupt or gradual termination of DNA replication with or without DNA disruption (Wechsler, JA and Gross, JD, 19
71. An Escherichia coli mutant whose DNA synthesis is temperature-sensitive. Mol. Gen.
Genetics, 113, 273-284, the entire disclosure of which is incorporated herein by reference and including either termination of proliferation or cell death. Antisense to dnaB Gene Causes Cell Death at Limited Temperature
By a combination of the use of temperature-sensitive mutations in the B gene, very specific and effective inhibitors of one or a subset of the activities exhibited by DnaB can be found.

When screening for antibiotics for a gene product required for growth, inhibition of growth of cells containing the gene product required for a limited amount of growth can be assayed.
Growth inhibition can be measured by a direct comparison of the amount of growth between the experimental and control samples, as measured by the optical density of the growth medium. Alternative cell proliferation assays include measurements of green fluorescent protein (GFP) reporter construct publication, various enzyme activity assays, and others well known in the art.

It will be appreciated that the above method can be performed in a solid phase, a liquid phase, or a combination of the two. For example, cells grown on nutrient agar containing the inducer of the antisense construct can be exposed to compounds spotted on the agar surface. The effect of the compound can be determined from the diameter of the resulting killed area (the area around the compound application point where no cells proliferate). Transfer a number of compounds to an agar plate and use a multichannel pipettor (eg, Beckman Multi).
imek) and multi-channel spotters (eg, Genomic Sol)
tests can be performed simultaneously using automated and semi-automated equipment, including, but not limited to, U.s. Flexions. In this way, large numbers of plates and countless compounds can be tested in one day.

Compounds can also be tested in their entirety in the liquid phase using the microtiter plates described below. To screen large numbers of plates and thousands to millions of compounds per day, 96,384,
Liquid phase screening can be performed on microtiter plates containing 1536 or more wells. Automated and semi-automated devices can be used to identify additional reagents (eg, cells and compounds) and cell density.

Example 9 The efficacy of the cell-based assay described above was tested for ribosomal protein L7 / L12
, L10, and L23, respectively. coli gene rpIL, r
Confirmation was made with constructs expressing antisense RNA against pIJ and rpIW. These proteins are part of the cell's protein synthesizer and as such are required for growth. These constructs were used to test the effect of antisense expression on cell sensitivity to antibodies known to bind ribosomes and inhibit protein synthesis. Several other genes (elaD, v
Constructs expressing antisense RNA against isC, yohH, and aptE / B) (products not involved in protein synthesis) were used for comparison.

The first expression vector containing the antisense construct for rpIW or elaD was replaced with another E. coli. E. coli cell population. Vector transfer is a technique well known to those skilled in the art. The expression vector of this example contains an IPTG-inducible promoter that drives the expression of antisense RNA in the presence of an inducer. However, one skilled in the art will recognize that other inducible promoters can also be used. Suitable expression vectors are also well known in the art. Ribosomal proteins L7 / L12,
E. L. encoding L10 and L23. E. coli antisense clones were used to test the effect of antisense expression on cell susceptibility to antibiotics known to bind to these proteins. First, an expression vector containing an antisense to any of the genes encoding L7 / L12, L10, or L23 was added to another E. coli. E. coli cell population.

The cell population was exposed to a range of IPTG concentrations in liquid medium to obtain an inhibitory dose curve for each clone (FIG. 1). First, the seed culture was grown at the optical density (OD
) To a specific turbidity determined by The OD of a solution is directly related to the number of bacterial cells contained in the solution. Sixteen 200 μl liquid medium cultures were then incubated at 37 ° C. in a 96-well microtiter plate at a range of IPTG concentrations for 1 hour.
Grow in duplicate 2-fold serial dilutions from 600 μM to 12.5 μM (final concentration).
In addition, duplicate control cells without IPTG were grown. These cultures were started from equal amounts of cells from the same seed culture of the clone of interest. Cells were grown for up to 15 hours and the growth range was identified by measuring the optical density of the culture at 600 nm. When control cultures reached mid-log, each IP
Growth inhibition dose-response curves for IPTG were generated by plotting the control% growth of the cultures containing TG against the log concentration of IPTG. Then, 0 mM
The IPTG concentration that inhibits cell growth by 50% compared to the IPTG control (0% growth inhibition) was calculated from the curve (IC ₅₀ ). Under these conditions, growth is 50%
The amount of antisense RNA that reduced the level of rpIW and elaD expression that was inhibited was obtained.

[0180] Other measures of proliferation are also contemplated. An example of this method involves measuring the protein (the expression of the protein can be easily measured by engineering it into the cells to be tested). Examples of such proteins include green fluorescent protein (GFP) and various enzymes.

The cells were pretreated with a selected concentration of IPTG and used to test the sensitivity of the cell population to tetracycline, erythromycin, and other protein synthesis inhibitors. Examples of tetracycline dose response curves for the rpIW and elaD genes are shown in FIGS. 2A and 2B, respectively. After growing to log phase, cells were diluted in medium alone or medium containing IPTG at growth inhibitory concentrations of 20% and 50% as identified by IPTG volume response curves. After 2.5 h, the cells were diluted to 96 OD ₆₀₀ well plates in 0.002 including the following: (1) 2.5 hour pre-incubation with the same concentrations of +/- IPTG and (2) tetracycline Two-fold dilutions of tetracycline such that the final concentration of is in the range of 1 μg / ml to 15.6 ng / ml and 0 μg / ml. 37 96-well plates
℃ incubated at and read on a plate reader up to 15 hours OD ₆₀₀ every 5 minutes. When the control (without tetracycline) reaches OD _{60 0} 0.1 were identified tetracycline dose-response curves for control without the IPTG concentration and IPTG. To compare tetracycline sensitivity with and without IPTG, the tetracycline IC was
50 were identified (FIGS. 2A-2B). Cells with reduced L23 (rpIW) levels showed increased sensitivity to tetracycline (FIG. 2A) compared to cells with reduced elaD levels (FIG. 2B). FIG. 3 shows IP showing 50% growth inhibition.
The ratio of the tetracycline IC50 identified in the presence of TG to the tetracycline IC50 identified without IPTG (fold increase in tetracycline sensitivity) was plotted. Cells with reduced levels of either L7 / L12 (genes rpIL, rpIJ) or L23 (rpIW) showed increased sensitivity to tetracycline (FIG. 3). Genes with unknown involvement in protein synthesis (atpB / E,
Cells expressing antisense to visC, elaD, yohH) did not show the same increase in sensitivity to tetracycline, confirming the specificity of the assay (FIG. 3).

[0182] In addition to the above, genes involved in protein synthesis (ribosome protein L7
/ L12 and L10, clones expressing antisense RNA against L7 / L12 alone, genes encoding L22 and L18, and genes encoding rRNA and elongation factor G) have increased sensitivity to macrolide, erythromycin Is the non-protein synthetic gene elaD, atpB / E,
Initial experiments showed that clones expressing antisense to and visC did not increase sensitivity. In addition, clones expressing antisense to rpIL and rpIJ do not have increased sensitivity to nalidixic acid and ofloxacin, an antibiotic that does not inhibit protein synthesis.

The results with the ribosomal protein genes rpIL, rpIJ, and rpIW, as well as the initial results with various other antisense clones and antibiotics, indicate that cells are specific for the protein due to the limited concentration of the antibiotic target. Shows greater sensitivity to interacting antimicrobials. The results also indicate that these cells are susceptible to antimicrobial agents that inhibit all functions that involve protein targets but are not sensitive to antimicrobial agents that inhibit other functions.

The cell-based assays described above can also be used to identify biological pathways in which nucleic acids or their gene products required for growth are present. In such a method, cells expressing sublethal levels of antisense and cells that do not induce antisense expression against the target nucleic acid required for proliferation are treated with various antibiotics by known antibiotics. Contact the panel. Cells that induce antisense expression are more susceptible to antibiotics than cells that do not induce antisense expression when the antibiotic acts in a pathway where the target nucleic acid or its gene product is required for growth. .

As a control, by contacting a panel of cells expressing an antisense nucleic acid with a number of different genes required for growth, including those targeted for growth,
The result of the assay can be confirmed. When the antibiotics specifically act, cells expressing antisense to the target gene required for growth (or antisense to other genes required for growth in the same pathway as the gene required for target growth). Increased sensitivity to antibiotics is observed only in cells expressing sense, but generally not in cells expressing antisense to a gene required for growth.

Similarly, the methods described above can be used to identify the pathway by which a test antibiotic acts. A panel of cells, each expressing antisense to a nucleic acid required for growth in a known pathway, is contacted with a compound desired to identify the pathway in which the panel acts. A panel of cells is identified for sensitivity to a test compound in cells in which antisense expression is induced and in control cells in which antisense expression is not induced. When the test antibiotic acts on the pathway in which the antisense nucleic acid acts, cells in which antisense expression is induced are more sensitive to the antibiotic than cells in which antisense expression is not induced. Furthermore, control cells in which antisense expression is induced for genes required for proliferation in other pathways do not show high sensitivity to antibiotics. In this way, the pathway by which the test antibiotic acts can be identified.

The following example illustrates one method for performing such an assay.

Example 10 Identification of pathways where genes required for growth are present or where antibiotics act. A.
Preparation of Bacterial Stock for Assay To obtain a consistent source of cells for screening, a frozen stock of host bacteria containing the desired antisense construct is prepared using standard microbiological techniques. For example, a single clone of the organism can be isolated by streaking a sample of the original stock on an agar plate containing antibiotics that contain genes for which cell growth nutrients and antisense constructs confer resistance. . After overnight growth,
The isolated clone is picked from the plate with a sterile needle and transferred to a suitable liquid medium containing the antibiotics necessary for plasmid maintenance. Cells were incubated for 4-6 hours with vigorous shaking at 30 ° C.-37 ° C. to allow the culture to grow exponentially. Add sterile glycerol to 15% (v / v), 100 μL-500 μL
Aliquots are dispensed into sterile cryotubes, snap frozen with liquid nitrogen injection and stored at -80 <0> C for further assays.

B. Growth of bacteria used in the assay One day prior to the assay, the stock virus is removed from the freezer and rapidly thawed.
Thaw (37 ° C water bath) and scrape medium with nutrients and antisense structure for cell growth.
The streak is streaked on agar plates containing antibiotics that confer resistance. Overnight at 37 ° C
After growth, randomly selected isolated colonies were removed from the plate using the antisense vector
Transfer to a sterile tube containing 5 mL of LB medium containing antibiotics that confer resistance (
In a sterile inoculation loop). After vigorous mixing to form a uniform cell suspension, 60
0 nm (OD₆₀₀Measure the optical density of the suspension in) and, if necessary,
The OD to 0.02 or less in a second sterile tube containing LB medium and antibiotics. ₆₀₀ Dilute to absorbance units in. The culture is then placed on the OD₆₀₀Is OD 0.2
Incubate with agitation at 37 ° C. for 1-2 hours until ０．３0.3 is reached.
At this point, the cells are ready for use in the assay.

C. Selection of Medium Used in Assay For maintenance of the antisense construct, make a two-fold serial dilution of the inducer in culture medium containing the appropriate antibiotic. Several media are tested in parallel and three out of four wells are used to assess the effect of the inducer at each concentration of each media. For example, M9 minimal media, LB broth, TBD broth, and Muller
-Hinton medium can be tested with the following concentrations of inducer IPTG: 50 μM, 100 μM, 200 μM, 400 μM, 600 μM,
800 μM, and 1000 μM. Equal volumes of test media-inducer and cells are added to the wells of a 384-well microtiter plate and mixed. Cells are prepared as described above and diluted 1: 100 in a suitable medium containing the test antibiotic just prior to addition to the wells of the microtiter plate. As a control,
Cells are also added to some wells of each medium without inducer (eg, 0M IPTG). Cell growth is continuously monitored by incubating at 37 ° C. in a microtiter plate reader and monitoring the OD ₆₀₀ of the wells for 18 hours. % Cell inhibition obtained with each concentration of inducer
Is calculated by comparing the log growth rate with the cell growth rate in media without inducer. The medium showing the highest sensitivity to the inducer is selected for the assay described below.

D. Determination of sensitivity of test antibiotics in the absence of induction of antisense constructs In selected culture media for the development of additional assays supplemented with the antibiotics used to maintain the constructs, select antibiotics with known mechanisms of action. Make two-fold serial dilutions. A panel of test antibiotics known to act by different routes is tested in parallel with three of the four wells used to evaluate the effect of the test antibiotic on cells grown at each concentration. Equal volumes of test antibiotic and cells are added to the wells of a 384-well microtiter plate and mixed. Cells are prepared using media selected for the development of assays supplemented with the antibiotics necessary for maintenance of the antisense construct as described above, and in the same media immediately prior to addition to the wells of the microtiter plate: Dilute to 100. As a control, cells are added to some wells containing the solvent used for lysis of the antibiotic but without the antibiotic.
Cell growth is continuously monitored by incubating at 37 ° C. in a microtiter plate reader and monitoring the OD ₆₀₀ of the wells for 18 hours. The% cell inhibition obtained with each concentration of antibiotic is calculated by comparing the log growth rate with the cell growth rate in media without antibiotic. A plot of% inhibition versus log [antibiotic concentration] allows one to estimate the IC ₅₀ for each antibiotic.

E. Determination of the sensitivity of the test antibiotic in the presence of the antisense construct inducer In the culture medium selected for the assay, the concentration of the cells shown to inhibit cell growth by 50% and 80% as described above Supplement the inducer. In each of these media, make two-fold serial dilutions of the test panel used above. Some antibiotics are tested in parallel with three of the four wells used to assess the effect of the antibiotic on cells grown at each concentration in each medium. Equal volumes of test antibiotic and cells are added to the wells of a 384-well microtiter plate and mixed. Cells are prepared using media selected for assays supplemented with the antibiotics necessary for maintenance of the antisense construct as described above. The cells are diluted 1: 100 with two 50 mL aliquots of the same medium containing the inducers at concentrations that have been found to inhibit cell growth by 50% and 80%, respectively. Immediately prior to addition to the wells of the microtiter plate, the culture is diluted to the appropriate OD ₆₀₀ (by dilution in warm (37 ° C.) sterile medium supplemented with the same concentration of inducer and antibiotic used to maintain the antisense construct. Typically 0.0
02). As a control, cells are also added to some wells containing a solvent used for lysis of the antibiotic but no antibiotic. Cell growth is continuously monitored by incubating at 37 ° C. in a microtiter plate reader and monitoring the OD ₆₀₀ of the wells for 18 hours. The% cell inhibition obtained with each concentration of antibiotic is calculated by comparing the log growth rate with the cell growth rate in media without antibiotic. A plot of% inhibition versus log [antibiotic concentration] allows one to estimate the IC ₅₀ for each antibiotic.

F. Comparison an IC ₅₀ obtained by antibiotics known mechanism of action under the conditions identified antisense specificity of the test antibiotics is not induced or induced, to identify a path exists nucleic necessary for growth be able to. If cells expressing an antisense nucleic acid against a gene required for growth are selectively susceptible to the action of an antibiotic via a particular pathway, then the antisense-acting gene is associated with the pathway for the antibiotic to act .

G. Third Embodiment Identifying the Pathway for the Test Compound to Act As discussed above, cell-based assays can also be used to identify the pathway for the test compound to act. In such an analysis, the pathway in which each panel of antisense nucleic acids acts is identified as described above. A panel of cells, each containing an induced antisense vector for a gene in a pathway required for known growth, is contacted with a test antibiotic desired to identify a pathway that operates under non-induced conditions. If induced cells expressing antisense to a gene in a particular pathway show increased sensitivity but not induced cells expressing antisense to a gene in another pathway, the test antibiotic will be sensitive. Acts on pathways with elevated levels.

The skilled artisan will appreciate that the inducer concentrations used to induce antisense expression and / or
Or that further optimization of assay conditions, such as the growth conditions (eg, incubation temperature and media components) used in the assay, can further increase the selectivity and / or magnitude of the sensitivity of the indicated antibiotic. recognize.

The following example confirms the effectiveness of the above method.

Example 11 Identification of Pathways Where Genes Required for Proliferation Are Present Antibiotics of various chemical classes and modes of action are available from Sigma Chemica
ls (St. Louis, MO). Based on the information obtained from the manufacturer, stock solutions were prepared by dissolving each antibiotic in the appropriate aqueous solution.
The final calibration curve solution for each antibiotic contained only 0.2% (w / v) of any organic solvent. To identify its potency against bacterial strains that have engineered expression of antisense to the 50S ribosomal protein required for growth, each antibiotic was treated in growth medium supplemented with an appropriate antibiotic for maintenance of the antisense construct. Serially diluted 3-fold. At least 10 dilutions were prepared for each antibiotic. A 25 μL aliquot of each dilution was transferred to a separate well of a 384-well microplate (assay plate) using a multichannel pipette. Quadruplicate wells were used for each dilution of antibiotic under each treatment condition (+ and-inducer).
Each assay plate contained 20 wells for cell growth control (replacing antibiotics with growth medium) and for each treatment (+ and − inducers, IPTG in this example).
) Contains 10 wells. Typically, the assay plates were divided into two treatment groups: one half containing the induced cells and an appropriate concentration of an inducer (IPTG in this example) to maintain the induced state, and the other half containing non-IPTG. Plate containing non-induced cells in the presence.

Cells for the assay were prepared as follows. Bacterial cells containing a construct capable of inducing the expression of antisense nucleic acids against rpIL and rpIJ in the presence of IPTG, encoding 50S ribosomal subunit proteins required for growth,
Grow exponentially (OD ₆₀₀ 0.2-0.3), 400 μM or 0 μM
1: 100 in fresh medium containing any of the above inducers (IPTG). These media were incubated at 37 ° C. for 2.5 hours. After 2.5 hours of incubation, the induced and uninduced cells were diluted to a final OD ₆₀₀ values in each assay medium becomes 0.0004. The medium contained the appropriate concentration of antibiotic for maintenance of the antisense construct. Further, the medium used for dilution of the induced cells was supplemented with 800 μM IPTG so that the final IPTG concentration was 400 μM by adding to the assay plate. Induced and uninduced cell suspensions were dispensed (25 μl / well) into wells of the appropriate assay plate described above. The plates were then loaded on a plate reader, incubated at a constant temperature, and cell growth was monitored for each well by measuring 595 nm light scatter. Growth was monitored every 5 minutes until the cell medium reached a stationary growth phase. Control wells associated with% growth inhibition for each concentration of antibiotic (no antibiotic, + or -IPTG)
Were calculated at the measurement points corresponding to the mid-logarithmic growth phase. For each antibiotic and condition (+ or -IPTG), plot% inhibition versus log antibiotic concentration,
The IC ₅₀ was identified. I for each antibiotic in the presence and absence of IPTG
Comparison of C _50, antisense constructs to cells, whether sensitized revealed to the action mechanism of the antibiotic showed. Cells showed a significant reduction in the presence of inducer (standard statistical analysis) IC ₅₀ values were considered sensitive is increased to the test antibiotics.

The results are shown in the table below, which shows the class and name of the antibiotic used in the analysis.
, An antibiotic target, IC in the absence of IPTG₅₀, IC in the presence of IPTG ₅₀ , IC₅₀Unit in the presence of IPTG₅₀Fold increase, and sensitivity
Are listed in the presence of IPTG.

[0200]

[Table 4]

The above results indicate that cells are selectively and very significantly sensitized to antibiotics that inhibit ribosome function and protein synthesis by inducing antisense RNA against the gene encoding the 50S ribosomal subunit protein. Indicates that The above results further indicate that the organism is sensitized to a compound that interferes with the biological role of that gene product by induction of an antisense construct against the essential gene. Sensitization is limited to compounds that interfere with pathways associated with the target gene and its products.

Assays using antisense constructs against essential genes can be used to identify compounds that specifically inhibit a number of target activities in the pathway. Using such a construct, a sample can be simultaneously screened against multiple targets in one pathway in one reaction (combination HTS).

In addition, as described above, antisense constructs, including cells, are used to characterize the point of entry of any compound that affects essential biological pathways, including antibiotics of unknown mechanism of action. be able to.

Another embodiment of the present invention is directed to a method wherein the activity of a target protein or nucleic acid associated with a pathway required for growth is determined by the interaction of a cell with a sublethal concentration of a known antibiotic acting on the target protein or nucleic acid. A method of identifying pathways in which a test antimicrobial is active, which is reduced by contact. In one embodiment, the target protein or nucleic acid is a target protein or nucleic acid corresponding to a nucleic acid required for growth identified using the method described above. This method does not use a sublethal level of antisense to the nucleic acid required for growth to reduce the activity or level of the gene product required for growth, but rather to reduce the sublethal level acting on the gene product required for growth. Similar to the above-described method of identifying the pathway in which a test antibiotic acts, except that the known antibiotics used to reduce the activity or level of the gene product required for growth.

Interactions between drugs affecting the same biological pathway have been described in the literature. For example, mesilinam (amdinocillin) is a penicillin binding protein 2 (P
BP2, E.C. (product of mrdA in E. coli). This antibiotic interacts with other antibiotics that inhibit PBP2 as well as other penicillin binding proteins such as PBP3. [Gutmann, L .; ,
Vincent, S.M. , Billot-Klein, D .; Acar, J .; F.
, Mrena, E .; , And Williamson, R .; 1986. β-
Relationship between penicillin binding protein 2 and other penicillin binding proteins in lysates of Escherichia coli by a synergistic dissolution effect with lactam antibiotics alone or with amdinocillin (mesilinam). Antimicrobial
Agents & Chemotherapy, 30, 906-912, the entire disclosure of which is incorporated herein by reference. Thus, the interaction between drugs may involve two drugs that inhibit the same target protein or nucleic acid in the same pathway or different proteins or nucleic acids [Fukuoka, T. et al.
, Domon, H .; , Kakuta, M .; , Ishii, C.I. , Hirasaw
a, A. Utsui, Y .; , Ohya, S .; , And Yasuda, H .; ,
1997. Staphylococcus aureu with high methicillin resistance
Effect of combined use of panipenem and vancomycin on s. Japan. J. Ant
ibio. , 50, 411-419; Smith, C.I. E. FIG. Foleno, B .;
E. FIG. Barrett, J .; F. , And Frosc, M .; B. , 1997. E by checkboard agar dilution and time-kill method
Evaluation of the synergistic effect of levofloxacin and ampicillin on N. terococcus faecium. Diagnostics. Microbiol. Infect
. Disease, 27, 85-92; den Hollander, J. et al. G. FIG.
, Horreports, A .; M. , Van Goor, M .; L. , Verbr
Ugh, H .; A. And Mouton, J .; W. , 1997. In vitro (i
n vitro) resistant Pseudomona tested in a pharmacokinetic model
Synergy between tobramycin and ceftazidime on S. aeruginosa strains. Antimicrobial Agents & Chemother
apy, 41, 95-110, the entire disclosures of which are hereby incorporated by reference.

The two drugs can interact even if they inhibit different targets. For example, the proton pump inhibitor omeprazole and the antibiotic amoxicillin are synergistic compounds that work together; these are Helicobacter
pylori infection can be treated [Gabrylewicz, A. et al. ,
Laszewicz, W.C. , Dzieniszewski, J .; , Cook, J
. , Marlicz, K .; Bielecki, D .; , Popiela, T .; ,
Legutko, J .; , Knapik, Z .; , Poniewierka, E .; ,
1997. Multicenter evaluation (two-year observation) of dual treatment (omeprazole and amoxicillin) for Helicobacter pylori-related duodenal and gastric ulcers. J. Physiol. Pharmacol. , 48, Suppl.
4, 93-105, the entire disclosure of which is incorporated herein by reference.

Sublethal concentrations of growth inhibition from known antibiotics are at least about 5%, at least about 8%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%. %, At least about 60, or at least about 75% or more.

Alternatively, sublethal concentrations of known antibiotics can be identified by measuring the activity of the target gene product required for growth rather than by measuring growth inhibition.

The cells are contacted with a combination of sublethal levels of each member of the known antibiotic community with various concentrations of the test antibiotic. As a control, cells are contacted only with various concentrations of the test antibiotic. The IC ₅₀ of the test antibiotic in the presence or absence of the known antibiotic is identified. If the IC ₅₀ in the presence or absence of the known antibiotic is substantially similar, the test drug and the known drug act by different routes. If the IC ₅₀ of substantially different, the test drug and the known drugs which act in the same pathway.

Another embodiment of the present invention relates to a method for reducing a target protein or a pathway involved in a pathway required for growth, which is reduced by contacting a cell with a sublethal concentration of a known antibiotic acting on the target protein or nucleic acid. This is a method for identifying a candidate compound to be used as an antibiotic for the activity of a nucleic acid. In one embodiment, the target protein or nucleic acid is
A target protein or nucleic acid corresponding to a nucleic acid required for growth, identified using the above method. This method does not use a lethal level of antisense to the nucleic acid required for growth to reduce the activity or level of the gene product required for growth, but rather to reduce the sublethal level acting on the gene product required for growth. Similar to the above-described method for identifying candidate compounds to be used as antibiotics, except that known antibiotics are used to reduce the activity or level of the gene product required for growth.

[0211] Sublethal concentrations of growth inhibition from known antibiotics are at least about 5%, at least about 8%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%. %, At least about 60%, or at least 75% or more.

Alternatively, sublethal concentrations of known antibiotics can be identified by measuring the activity of the target gene product required for growth rather than by measuring growth inhibition.

To characterize the test compound of interest, the cells are contacted with sublethal levels of a known antibiotic group and one or more test compound concentrates. As a control, cells are contacted with the same concentration of test compound only. The IC ₅₀ of the test compound in the presence or absence of a known antibiotic is identified. If the IC ₅₀ of the test compound in the presence or absence of the known drug is substantially different, the test compound is a good candidate for use as an antibiotic. As described above, once a candidate compound has been identified using the method described above, its structure can be optimized using standard techniques such as combinatorial chemistry.

[0214] The following table lists representative known antibiotics that can be used in each of the above methods. However, it will be appreciated that other antibiotics can be used.

[0215]

[Table 5]

Example 12 coli expression vector or E. coli. Introduction of Exogenous Nucleic Acid Sequences into Other Bacterial Species Using Expression Vectors Functional in Bacterial Species Other than E. coli The above method was evaluated using antisense nucleic acids that inhibit E. coli growth. Induction of antisense RNA expression with IPTG An expression vector that inhibits E. coli growth is
ter cloacae, Klebsiella pneumonia or S
almonella typhimurium was directly transformed. The transformed cells were then assayed for growth inhibition according to the method of Example 1. After growth in liquid culture, cells are plated in various serial dilutions,
Induced versus uninduced antisense RN identified at up to 10-fold dilution where colonies were observed
Scores were determined by calculating the log difference in proliferation for A expression. The results of these experiments are listed in Table 6 below. If there is no effect of antisense RNA expression on the organism, the clone is negative in Table 6. In contrast, positive in Table 6 means that at least 10-fold cells are required to find colonies on induced rather than uninduced plates under the conditions used and in the organism.

Twenty-six of the constructs were found to inhibit growth in all organisms tested upon induction of antisense RNA expression with IPTG. One skilled in the art will recognize that a negative result in a heterologous organism does not mean that the organism has lost its gene or that the gene is not essential. However, a positive result means that the heterologous organism contains the homologous genes necessary for the growth of that organism. Homologous genes can be obtained using the methods described herein. These cells, which are inhibited by antisense, can be used in the cell-based assays described herein for identifying and characterizing compounds to develop effective antibiotics for these organisms. One skilled in the art will recognize that an antisense molecule that acts in that organism does not always act in a heterologous organism.

[0218]

[Table 6]

Example 13 Use of the Identified Exogenous Nucleic Acid Sequence as a Probe The identified sequence of the present invention can be used as a probe to obtain a further gene sequence of interest from a second organism. For example, probes for potential bacterial target proteins can be hybridized with nucleic acids from other organisms, including other bacteria and higher organisms, to identify homologous sequences. Such hybridization will indicate that the protein encoded by the gene to which the probe corresponds is found in humans and is not necessarily a good drug target. Alternatively, the gene can be stored only in bacteria and would be a good drug target for a wide range of antibiotics or antibacterials.

[0220] A probe derived from the identified nucleic acid sequence of interest or a portion thereof can be labeled with a detectable label (including radioisotopes and non-radioactive labels) well known to those of skill in the art to provide a detectable probe. . Detectable probes can be single-stranded or double-stranded and can be made using techniques known in the art, including in vitro transcription, nick translation, or a kinase reaction. A nucleic acid sample containing a sequence capable of hybridizing to a labeled probe is contacted with the labeled probe. If the nucleic acid in the sample is double-stranded, it can be denatured before contacting the probe. For some applications, the nucleic acid sample can be immobilized on a surface such as nitrocellulose or a nylon membrane. Nucleic acid samples can be obtained from various sources (
(Including genomic DNA, cDNA libraries, RNA, or tissue samples).

The methods used to detect the presence of a nucleic acid capable of hybridizing to a detectable probe include well known techniques such as Southern blotting, Northern blotting, dot blotting, colony hybridization, and plaque hybridization. It is. For some applications, to facilitate characterization and expression of the hybridizing nucleic acid in the sample, the nucleic acid capable of hybridizing to the labeled probe is converted to a vector, such as an expression vector, a sequencing vector, or an in vitro transcription vector. Can be cloned. For example, using such a technique, sequences capable of hybridizing to probes generated from the sequences identified in Examples 5 and 6 were isolated from genomic libraries generated from various bacterial species. , Purified, and cloned.

Example 14 Preparation of PCR Primers and Amplification of DNA Identification of E. coli directly corresponding to or located within an operon or a portion thereof of a nucleic acid sequence required for growth. Using the E. coli gene, other species containing some or all of the homologous genes (eg, Salmonella typhimurium, Enter)
obactor cloacae and Klebsiella pneumo
PCR primers can be prepared for various uses, including the identification or isolation of homologous sequences from nia). Since homologous genes are related but not identical in sequence, those skilled in the art often use degenerate sequence PCR primers. Such a degenerate sequence primer may be used as a conserved or known conserved sequence region (eg, a conserved coding region).
Design based on Successful production of a PCR product using a denatured probe made from the sequences identified herein indicates the presence of a homologous gene sequence for the species being screened. PCR primers are at least 10 bases in length, preferably at least 20 bases in length. More preferably, PCR primers are at least 20 to 30 bases in length. In some embodiments, the PCR primer is 3
It can exceed 0 bases in length. Primer pairs should have similar melting temperatures,
Preferably, the G / C ratios are approximately the same. Various PCR techniques are well known to those skilled in the art. For an overview of PCR technology, see Molecular Cloning.
to Genetic Engineering, White, B .; A. Hen, M
methods in Molecular Biology, 67, Human
a Press, Totowa, 1997. If the entire coding sequence of the target gene is known, the 5 'and 3' regions of the target gene can be used as a sequence source for making PCR probes. In each of these PCR methods, PCR primers on either end of the nucleic acid sequence to be amplified are added to an appropriately prepared nucleic acid sample with dNTPs and a thermostable polymerase (such as Taq polymerase, Pfu polymerase, or Vent polymerase). I do. The nucleic acid in the sample is denatured and the PCR primer specifically hybridizes to the complementary nucleic acid sequence in the sample. The hybridized primer is extended. afterwards,
Initiate the next cycle of denaturation, hybridization, and extension. Cycle
Repeat multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites.

Example 15 Inverse PCR Inverse polymerase chain reaction technology can be used to extend the known nucleic acid sequence identified in Examples 5 and 6. Inverse PCR reactions generally involve Ochm
an et al. , PCR Technology: Principles
and Applications for DNA Amplificati
on, Chapter 10, Henry A. Ed. Erlich, W.M. H. Freeman
and Co. , 1992. Conventional PCR requires two primers to initiate the synthesis of the complementary strand of DNA. In reverse PCR, only the core sequence needs to be known.

Using sequences taught in Examples 5 and 6 and identified as relevant by techniques applied to other species of bacteria, an exogenous gene corresponding to a gene or operon required for bacterial growth is used. Identify a subset of the nucleic acid sequences. In species where the genomic sequence is unknown, the inverse PCR technique provides a method for obtaining genes to determine the sequence or place a probe sequence in the entire range of target sequences to which the identified exogenous nucleic acid sequence binds.

To perform this technique, the genome of the target organism is digested with appropriate restriction enzymes to produce fragments of the nucleic acid containing the identified sequence as well as unknown sequences that flank the identified sequence. These fragments are then circularized and used as templates for PCR reactions. PCR primers were designed according to the teachings of Example 15 and PCR primers directed to the ends of the identified sequences were synthesized. The primer synthesizes nucleic acid in a direction away from the known sequence contained in the circular template and toward the unknown sequence. After completion of the PCR reaction, the resulting PCR product can be sequenced to extend the identified gene sequence past the core sequence of the identified exogenous nucleic acid sequence. In this manner, each new entire gene sequence can be identified. In addition, flanking coding and non-coding region sequences can be identified.

Example 16 Identification of Genes Required for Staphylococcus aureus Proliferation Genes required for Staphylococcus aureus proliferation are identified according to the method described above.

Example 17 Identification of Genes Required for Neisseria gonorrhoeae Propagation Genes required for Neisseria gonorrhoeae propagation are identified according to the method described above.

Example 18 Identification of Genes Required for Pseudomonas aeruginosa Proliferation Genes required for Pseudomonas aeruginosa growth are identified according to the method described above.

Example 19 Identification of Genes Required for Growth of Enterococcus faecalis Genes required for growth of Enterococcus faecalis are identified according to the method described above.

Example 20 Identification of Genes Required for Haemophilus influenzae Growth Genes required for Haemophilus influenzae growth are identified according to the method described above.

Example 21 Identification of Genes Required for Salmonella typhimurium Growth Genes required for Salmonella typhimurium growth are identified according to the methods described above.

Example 22 Identification of Genes Required for Propagation of Helicobacter pylori Genes required for propagation of Helicobacter pylori are identified according to the method described above.

Example 23 Identification of Genes Required for Mycoplasma pneumoniae Proliferation Genes required for Mycoplasma pneumoniae proliferation are identified according to the method described above.

Example 24 Identification of Genes Required for Propagation of Plasmodium ovale Genes required for propagation of Plasmodium ovale are identified according to the method described above.

Example 25 Identification of Genes Required for Saccharomyces cerevisiae Growth Genes required for Saccharomyces cerevisiae growth are identified according to the method described above.

Example 26 Identification of Genes Required for Entamoeba histolytica Proliferation Genes required for Entamoeba histolytica growth are identified according to the method described above.

Example 27 Identification of Genes Required for Growth of Candida albicans Genes required for growth of Candida albicans are identified according to the method described above.

Example 28 Identification of Genes Required for Growth of Klebsiella pneumoniae Genes required for growth of Klebsiella pneumoniae are identified according to the method described above.

Example 29 Identification of Genes Required for Salmonella typhi Propagation Genes required for Salmonella typhi growth are identified according to the method described above.

Example 30 Identification of Genes Required for Salmonella paratyphi Growth Genes required for Salmonella paratyphi growth are identified according to the method described above.

Example 31 Identification of Genes Required for Salmonella cholerasuis Propagation Genes required for Salmonella cholerasuis growth are identified according to the method described above.

Example 32 Identification of Genes Required for Staphylococcus epidermis Propagation Genes required for Staphylococcus epidermis propagation are identified according to the method described above.

Example 33 Identification of Genes Required for Mycobacterium tuberculosis Growth Genes required for Mycobacterium tuberculosis growth are identified according to the method described above.

Example 34 Identification of Genes Required for Propagation of Mycobacterium leprae Genes required for growth of Mycobacterium leprae are identified according to the method described above.

Example 35 Identification of Genes Required for Growth of Treponema pallidum Genes required for growth of Treponema pallidum are identified according to the method described above.

Example 36 Identification of Genes Required for Bacillus anthracis Growth Genes required for Bacillus anthracis growth are identified according to the methods described above.

Example 37 Identification of Genes Required for Growth of Yersinia pestis Genes required for growth of Yersinia pestis are identified according to the method described above.

Example 38 Identification of Genes Required for Growth of Clostridium botulinum Genes required for growth of Clostridium botulinum are identified according to the method described above.

Example 39 Identification of Genes Required for Growth of Campylobacter jejuni Genes required for growth of Campylobacter jejuni are identified according to the method described above.

Example 40 Identification of Genes Required for Growth of Chlamydia trachomatis Genes required for growth of Chlamydia trachomatis are identified according to the method described above.

Use of Isolated Exogenous Nucleic Acid Fragments as Antisense Antibiotics In addition to the use of identifying sequences that allow screening of molecular libraries to identify compounds useful for antibiotic identification, Can be used as a therapeutic. In particular, an individual can be provided with an identifying exogenous sequence in the antisense orientation so as to inhibit translation of a bacterial target gene.

Generation of Antisense Therapeutics from Identified Exogenous Sequences The sequences of the invention are used as antisense therapeutics for treating bacterial infections in vitro or in vivo, or simply for inhibiting bacterial growth. be able to. This treatment utilizes a biological process in cells where genes are transcribed into messenger RNA (mRNA) and translated into proteins. Antisense R
NA technology contemplates the use of antisense oligonucleotides directed to a target gene that binds to the target and reduces or inhibits translation of the target mRNA. In one embodiment, antisense oligonucleotides can be used to treat and regulate bacterial infection of cell cultures containing the desired bacterially contaminated cell population. In another embodiment, an antisense oligonucleotide can be used to treat a bacterially infected organism.

Antisense oligonucleotides can be synthesized from any of the sequences of the present invention using methods well known in the art. In a preferred embodiment, the antisense oligonucleotide is synthesized using artificial means. Uhlmann &
Peymann, Chemical Rev. , 90, 543-584, 199
Year 0 reviews the antisense oligonucleotide technology in detail. Modified or unmodified antisense oligonucleotides can be used as therapeutics. Since antisense oligonucleotides are known to be very unstable, modified antisense oligonucleotides are preferred. Modifications of the phosphate backbone of antisense oligonucleotides can be made by replacement of phosphate residues between nucleotides with methylphosphonates, phosphorothioates, phosphoramidates, and phosphate esters. Siloxane crosslinking, carbonic acid crosslinking, thioester crosslinking,
And non-phosphate internucleotide analogs, such as many other bridges known in the art. The preparation of certain antisense oligonucleotides using modified intranucleotide linkages is described in US Pat. No. 5,142,047, which is incorporated herein by reference.

Modifications to the nucleotide units of the antisense oligonucleotide are also contemplated. This modification can increase the half-life of the oligonucleotide in vivo and increase the rate of cellular uptake. For example, α-anomeric nucleotide units and modified bases (such as 1,2-dideoxy-d-ribofuranose, 1,2-dideoxy-1-phenylribofuranose, and N ⁴ , N ⁴ -ethano-5-methyl-cytosine). Is intended for use in the present invention.

Further forms of modified antisense molecules are found in peptide nucleic acids. Peptide nucleic acids (PNA) have been developed that hybridize to standard single- and double-stranded nucleic acids. PNA is a nucleic acid analog in which the total deoxyribose phosphate skeleton is exchanged for a polyamide (peptide) skeleton containing 2-aminoethylglycine units that are chemically distinct but structurally homologous. Unlike highly negatively charged DNA,
The PNA backbone is neutral. Thus, it is more stable because the repulsive energy between complementary strands in a PNA-DNA hybrid is much lower than a similar DNA-DNA hybrid. PNA is Watson / Crick or Hoogste
en format (Demidov et al., Proc. Natl. Acad. S.
ci. U. S. A. , 92, 2637-2641, 1995, Egholm,
Nature, 365, 566-568, 1993; Nielsen et.
al. , Science, 254, 1497-1500, 1991; Dueh.
olm et al. New J .; Chem. , 21, 19-31, 1997
Year) can hybridize with DNA.

A molecule called a PNA "clamp" having two identical PNA sequences joined by a flexible hairpin linker containing three 8-amino-3,6-dioxaoctanoic acid units has been synthesized. When the PNA clamp is mixed with a complementary homopurine or homopyrimidine DNA target sequence, a PNA-DNA-PNA triple hybrid that has been shown to be very stable can be formed (Bent
in et al. , Biochemistry, 35, 8863-8869,
1996; Egholm et al. , Nucleic Acids Re
s. Griffith et al., 23, 217-222, 1995; , J
. Am. Chem. Soc. 117, 831-832, 1995).

The sequence-specific, high-affinity duplex and triplex PNA binding has been extensively described (Nielsen et al., Science, 254, 1497-1).
500, 1991; Egholm et al. J. Am. Chem. Soc
. Egholm et al., 114, 9677-9678, 1992; ,
Nature 365, 566-568, 1993; Almarssone
t al. Proc. Natl. Acad. Sci. U. S. A. , 90, 9
542-9546, 1993; Demidov et al. Proc. N
atl. Acad. Sci. U. S. A. , 92, 2637-2641, 199
5 years). They have also been shown to be resistant to nucleotide and protease digestion (Demidov et al., Biochem. Pharm., 48).
1010-1313, 1994). PNA is used to inhibit gene expression (Hanvey et al., Science, 258, 1481-1485).
Nielsen et al., 1992. , Nucl. Acids. Re
s. , 21, 197-200, 1993; Nielsen et al. , G
ene, 149, 139-145, 1994; Good & Nielsen
Science, 95, 2073-2076, 1998, all of which are incorporated herein by reference) and block restriction enzyme activity (Nielsen).
et al. , Supra, 1993), acting as an artificial transcription promoter (
Mollegaard, Proc. Natl. Acad. Sci. U. S. A,
91, 3892-3895, 1994), acting as a pseudorestriction endonuclease (Demidov et al., Nucl. Acids. Res.
, 21, 2103-2107, 1993). Recently, PNAs have also been shown to have antiviral and antitumor activities mediated by an antisense mechanism (Norton, Nature Biotechnol., 14).
Hirschman et al., 615-619, 1996; J. In
vestig. Med. 44, 347-351 (1996)). PNAs have been linked to various peptides to facilitate PNA entry into cells (Basue
t al. , Bioconj. Chem. 8, 481-488, 1997;
Pardridge et al. Proc. Natl. Acad. Sci.
U. S. A, 92, 5592-5596, 1995).

The antisense oligonucleotides contemplated by the present invention can be administered by direct application of the oligonucleotide to the target using standard techniques well known in the art. Plasmids or phage can be used to produce antisense oligonucleotides in the target. Alternatively, antisense nucleic acids can be expressed from sequences in the chromosome of the target cell. It is further contemplated that the intended antisense oligonucleotide can be incorporated into a ribozyme sequence to specifically bind the antisense and cleave its target mRNA. For technical applications of ribozymes and antisense oligonucleotides, see Rossi
et al. Pharmacol. Ther. , 50 (2), 245-25
4, 1991, incorporated herein by reference. The invention also contemplates the use of retrons to direct antisense oligonucleotides to cells. Retron technology is exemplified in US Pat. No. 5,405,775, which is incorporated herein by reference. Antisense oligonucleotides can also be delivered using liposomes or by electroporation techniques well known in the art.

The antisense nucleic acids of the present invention can also be used to design antimicrobial compounds containing nucleic acids that function in an intracellular triple helix form. Triple helix oligonucleotides are used to inhibit transcription from the genome. Sequences or portions thereof identified as required for growth according to the invention can be used as templates to inhibit microbial gene expression in individuals infected with the microorganism. Heretofore, homopurine sequences were considered to be most useful for triple helix strategies. However, homopyrimidine sequences can also inhibit gene expression. Such homopyrimidine oligonucleotides bind to the major groove of the homopurine: homopyrimidine sequence. Accordingly, both types of sequences required for propagation based on the sequences of the present invention are contemplated for use as templates for antimicrobial compounds.

The antisense oligonucleotides of this example use the identifying sequences of the present invention to induce bacterial cell death or at least bacterial quiescence by inhibiting translation of the target gene. Antisense oligonucleotides comprising a sequence of the invention of about 8-40 bases are sufficiently complementary to form a duplex with the target sequence under physiological conditions.

To kill bacterial cells or inhibit their growth, antisense oligonucleotides are applied to the bacteria or target cells under conditions that promote their uptake.
These conditions include sufficient incubation time of the cells and oligonucleotide so that the antisense oligonucleotide is taken up by the cells. In one embodiment, a 7-10 day incubation period is sufficient to kill bacteria in the sample. For use, select the optimal concentration of the antisense oligonucleotide.

The antisense oligonucleotide concentration used will depend on the type of bacteria that are believed to be modulated, the nature of the antisense oligonucleotide used, and the relative antisense oligonucleotide to the desired cells in the treated medium. Can vary with toxicity. Antisense oligonucleotides can be introduced into a cell sample at a number of different concentrations, preferably between 1 × 10 ⁻¹⁰ M and 1 × 10 ⁻⁴ M. Once the minimum concentration that can adequately regulate gene expression is obtained, the optimal dosage is converted into a dosage suitable for in vivo use. For example, an inhibitory concentration of 1 × 10 ⁻⁷ in culture is converted to a dose of about 0.6 mg / kg body weight. Following toxicity testing of oligonucleotides in experimental animals, oligonucleotide levels reaching or exceeding 100 mg / kg body weight may be possible. It is further contemplated that cells from the subject are removed, treated with an antisense oligonucleotide, and reintroduced into the subject. This range is merely exemplary, and one skilled in the art can determine the optimal concentration to use in a given case.

After killing or adjusting bacterial cells in the desired medium, the desired cell population can be used for other purposes.

Example 41 The following example illustrates the use of E. coli. Figure 4 shows the ability of E. coli antisense oligonucleotides to act as antibacterial or bacteriostatic agents for treating contaminated cell culture systems. It is believed that the application of the antisense oligonucleotides of the invention inhibits translation of bacterial gene products required for growth.

The antisense oligonucleotide of this example corresponds to a 30-base phosphorothioate-modified oligodeoxynucleotide (eg, molecule number EcXA001) complementary to a nucleic acid involved in growth. A sense oligodeoxynucleotide complementary to the antisense sequence is synthesized and used as a control. Oligonucleotides were purchased from Matsukura, et al. Gene, 72, 343, 198
Synthesize and purify according to the 8-year procedure. The test oligonucleotide is dissolved in a small amount of autoclaved water and added to the culture medium to make a 100 micromolar stock solution.

Human bone marrow cells are obtained from the peripheral blood of two patients and cultured according to standard procedures well known in the art. Cultures contain E. coli. coli K-12 strain,
Incubate at 37 ° C. overnight until bacteria are infected.

Control and antisense oligonucleotides containing solutions are added to the contaminated culture to monitor bacterial growth. Ten hours after incubation of the cultures and oligonucleotides, samples are removed from control and experimental cultures and analyzed for translation of the target bacterial gene using standard bacterial techniques well known in the art. Target E. coli in control medium treated with control oligonucleotides. Although translation of the E. coli gene was observed, translation of the target gene was not detected or reduced in experimental cultures treated with the antisense oligonucleotides of the invention.

Example 42 A subject infected with E. coli is treated with the antisense oligonucleotide preparation of Example 39. The antisense oligonucleotide is included in a pharmaceutically acceptable carrier at a concentration effective to inhibit translation of the target gene. The subject is treated with an antisense oligonucleotide concentration sufficient to achieve a blood concentration of about 100 micromolar. To maintain this concentration, patients are given an antisense oligonucleotide injection daily for one week. At the end of the week a blood sample is taken
E. coli using standard techniques well known in the art. Analyze for the presence of E. coli. E. FIG. coli is not detected and the treatment is terminated.

Example 43 Preparation and Use of a Triple Helix Probe The gene sequence of a microorganism required for growth according to the present invention can be scanned and used as a triple helix-based strategy for inhibiting gene expression in a 10-mer. ~ 20
Identify dimeric homopyrimidine or homopurine stretches.
After identification of a candidate homopyrimidine or homopurine stretch, its efficacy in inhibiting gene expression is assessed by introducing various amounts of oligonucleotides containing the candidate sequence into bacterial cell populations that normally express the target gene. Oligonucleotides can be prepared on an oligonucleotide synthesizer or purchased from specialized oligonucleotide synthesis companies (GENSET, Paris, France, etc.).

The oligonucleotides can be prepared by various methods known to those skilled in the art (calcium phosphate precipitation, D
Cells can be introduced using EAE-dextran, electroporation, liposome-mediated transfection, or including, but not limited to, native uptake.

Treated cells are monitored for reduced proliferation compared to untreated cells using techniques such as monitoring the level of proliferation using optical densitometry. The oligonucleotide can then be introduced in vivo at an effective dosage level effective to inhibit gene expression in cultured cells using techniques well known in the art.

In some embodiments, the natural (β) anomer of the oligonucleotide unit is converted to α to provide the oligonucleotide with higher resistance to nucleases.
It can be replaced by an anomer. Further, an intercalating agent such as ethidium bromide is added to α
The triple helix can be stabilized by binding to the three termini of the oligonucleotide. For information on making oligonucleotides suitable for triple helix formation, see Griffin et al. , Science, 245, 967-971,
See 1989, incorporated herein by reference.

Example 44 Identification of Bacterial Strains from Isolated Specimens by PCR Conventional methods for the biological detection of various bacterial species are time consuming and expensive. These methods include the growth of bacteria isolated from the subject in a particular medium, culturing on selective agar medium, followed by a set of confirmatory assays that take 8-10 days or more to complete. Use of the identification sequences of the present invention provides a method that dramatically reduces the time to detect and identify a particular bacterial species present in a sample.

In one exemplary method, the bacteria are grown on an enrichment medium,
DNA samples are isolated by conventional methods, for example, from a sample of blood, urine, feces, saliva, or central nervous system fluid. Then, using a PCR primer group based on the identification sequences unique to various microorganism species as in Example 12, about 100 to 100
Amplify a 200 base long DNA. A separate PCR reaction is set up for each PCR primer pair, and after completion of the PCR reaction, the reaction mixture is assayed for the presence of the PCR product. Various tests were performed to determine the presence of bacteria from the species in which the PCR primer pair was present.
Identify by the presence of the PCR product in the CR reaction tube.

Although PCR reactions are used to assay isolated samples for the presence of various bacterial species, other assays such as Southern blot hybridization are also contemplated.

[Sequence list]

[Brief description of the drawings]

FIG. 1. E. coli required for protein synthesis and essential cell growth. E. coli ribosomal protein rplW antisense clone (AS-rplW) or IPTG inducible containing any of the antisense clone against elaD (AS-elaD) genes that are not known to be involved in protein synthesis and are also essential for growth E. coli transformed with the plasmid FIG. 2 shows an IPTG dose response curve in E. coli.

FIG. 2A shows E. coli transformed with an IPTG-inducible plasmid containing antisense to rplW (AS-rplW) in the presence of 0, 20, or 50 μM IPTG. FIG. 4 shows a tetracycline dose response curve in E. coli. FIG. 2B shows E. coli transformed with an IPTG-inducible plasmid containing antisense to elaD (AS-elaD) in the presence of 0, 20, or 50 μM IPTG. FIG. 4 shows a tetracycline dose response curve in E. coli.

FIG. 3. E. coli transfected with antisense clones against the essential ribosomal proteins L23 (AS-rplW) and L7 / L12 and L10 (AS-rplLrpIJ). 5 is a graph showing a fold increase in E. coli sensitivity to tetracycline. Antisense clones against known genes not involved in protein synthesis (atpB / E (AS-atpB / E), visC (AS-visC, ela)
D (AS-elaD), yohH (AS-yohH)) have very low sensitivity to tetracycline.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 16/12 C12N 1/15 4C084 C12N 1/15 1/19 4C086 1/19 1/21 4H045 1/21 C12P 21/02 C 5/10 C12Q 1/02 C12P 21/02 1/68 A C12Q 1/02 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 33/53 M 33/50 33/566 33/53 C12N 15/00 ZNAA 33/566 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU , MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K) E, LS, MW, SD, L, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Olsen, Carrier. United States California 92117 San Diego Vista de la Oli La 3560 (72) Inventor Trawick, John United States of America 91942 La Mesa Baldrich Street 7210 (72) Inventor Forsys, Earl. Allin United States California 92109 San Diego Beryl Street 1135 (72) Inventors Froerich, Jamie M. M. United States California 92116 San Diego 35th Street 5057 (72) Inventor Carl, Grant Jay. United States California 92029 Escondido Sonrize Glen 2210 (72) Inventor Yamamoto, Robert Tee. United States California 92131 San Diego Noto Deme Avenue 3725 (72) Inventor Shui, H. et al. Howard USA California 92131 San Diego Ivy Hill Drive 11142 F-term (reference) 2G045 AA34 AA35 BB20 BB46 BB50 CB21 DA13 DA36 FB02 FB12 4B024 AA01 AA11 AA13 BA48 CA01 CA12 FA02 GA11 GA27 HA06 HA12 4B06QQQ QQ1 QR58 QR75 QS22 QS24 QS28 QX01 4B064 AG01 CA02 CA19 CC24 DA01 DA13 4B065 AA26X AA26Y AB01 AC14 BA02 BA25 BD50 CA23 CA44 CA46 4C084 AA13 NA14 ZB35 4C086 AA01 AA02 AA03 EA16 MA01 MA04 NA14 AB AA AA AA AA AA AA A4 AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA

Claims (110)

[Claims] 1. A purified or isolated nucleic acid sequence consisting essentially of one of SEQ ID NOs: 405-485, wherein the nucleic acid inhibits growth of a microorganism. 2. The nucleic acid sequence of claim 1, wherein said nucleic acid sequence is complementary to at least a portion of a coding sequence of a gene whose expression is required for growth of the microorganism. 3. The nucleic acid sequence comprises one fragment of SEQ ID NOs: 405-485, wherein the fragment comprises at least 10 of one of SEQ ID NOs: 405-485.
At least 20, at least 25, at least 30, at least 50, or 5
The nucleic acid sequence according to claim 1 or 2, wherein the nucleic acid sequence is selected from the group consisting of fragments containing more than 0 consecutive bases. 4. The nucleic acid sequence of claim 3, wherein said nucleic acid sequence is complementary to the coding sequence of a gene whose expression is required for microbial growth. 5. A vector comprising a promoter operably linked to a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 405-485. 6. The promoter according to claim 6, wherein the promoter is Escherichia coli.
li), Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae, Helicobacter pyl
ori), Neisseria gonorrhoeae, Enterococcus faecalis, Streptococcus pneumoniae, Haemophilus influenzae
influenzae), Salmonella typhimurium, Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans (Crypto)
coccus neoformans), Aspergillus fumigatus
, Klebsiella pneumoniae, Salmonella typhi, Salmonella paratyphi,
Salmonella cholerasuis (Salmonella cholerasuis), Staphylococcus
Epidermidis (Staphylococcus epidermidis), Mycobacterium tuberculosis, Mycobacterium leprae (Mycobacterium leprae), Treponema pallidum (Treponema pallidum),
Bacillus anthracis, Yersinia pestis (
Yersinia pestis), Clostridium botulinum (Clostridium botulinum)
, Campylobacter jejuni, Chlamydia trachomatus, Chlamydum pneumonia
ia pneumoniae) or an organism selected from the group consisting of any species within the genus to which any of the species belongs. The vector according to claim 5, which is active in an organism selected from the group consisting of: 7. A host cell comprising the vector according to claim 5 or 6. 8. A purified or isolated nucleic acid consisting essentially of the coding sequence of one of SEQ ID NOs: 82-88, 90-242. 9. The fragment comprises one of SEQ ID NOs: 82 to 88, 90 to 242.
9. A fragment of a nucleic acid according to claim 8, comprising at least 10, at least 20, at least 25, at least 30, at least 50, or more than 50 contiguous bases. 10. A vector comprising a promoter operably linked to the nucleic acid according to claim 8 or 9. 11. An intragenic sequence, an intergenic sequence, and at least one of two or more genes in an operon encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 243 to 357, 359 to 398. Sequence across the part, 5 '
A purified or isolated nucleic acid comprising a nucleic acid sequence complementary to a non-coding region, or at least a portion of a 3 'non-coding region. 12. SEQ ID NOs: 405-485, 82-88, 90-, as determined using BLASTN version 2.0 with default parameters.
242 or a purified or isolated nucleic acid comprising a nucleic acid having at least 70% homology to a sequence selected from the group consisting of sequences complementary thereto. 13. The nucleic acid according to claim 1, wherein the nucleic acid is Staphylococcus aureus.
coccus aureus), Pseudomonas aeruginosa
), Enterobacter cloacae, Helicobacter pylori, Neisseria gonorhea
rhoeae), Enterococcus faecalis, Streptococcus pneumoniae, Haemophilus influenzae, Salmonella typhimurium (Salm)
onella typhimurium, Saccharomyces cerevi
siae), Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Klebsiella pneumonium
moniae), Salmonella typhi, Salmonella paratyphi, Salmonella cholera Switzerland (Salmonella cholerasu)
is), Staphylococcus epidermidis
, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Bacillus anthracis,
Yersinia pestis, Clostridium botulinum, Campylobacter yejunyi
er jejuni), Chlamydia trachomatus, Chlamydia pneumoniae, or an organism selected from the group consisting of any species within the genus to which any of the species belongs. Nucleic acids. 14. A purified or isolated nucleic acid consisting essentially of a nucleic acid encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 243-357, 359-398. 15. A vector comprising a promoter operably linked to a nucleic acid encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 243-357, 359-398. 16. A host cell comprising the vector according to claim 15. 17. A purified or isolated polypeptide comprising the sequence of one of SEQ ID NOs: 243-357, 359-398. 18. A fragment comprising one of the polypeptides of SEQ ID NOs: 243-357, 359-398, wherein the fragments comprise SEQ ID NOs: 243-357,3.
At least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, one of the 59 to 398 polypeptides.
Or a purified or isolated polypeptide selected from the group consisting of fragments comprising more than 60 contiguous amino acids. (19) an antibody capable of specifically binding to the polypeptide according to (17) or (18); 20. introducing a vector comprising a promoter operably linked to a nucleic acid encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 243-357, 359-398, into a cell; A method for producing a polypeptide. 21. The method of claim 20, further comprising isolating the protein. 22. A step of inhibiting or reducing the activity of a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 243-357, 359-398, or inhibiting the activity of a nucleic acid encoding said polypeptide. Or a method of inhibiting the growth thereof, comprising the steps of: 23. A method for identifying a compound that affects the activity of a polypeptide required for growth, comprising: a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 243-357, 359-398; and a candidate compound. And identifying whether the compound affects the activity of the polypeptide. 24. The method of claim 23, wherein said activity is an enzymatic activity. 25. The method of claim 23, wherein said activity is a carbon compound catabolism activity. 26. The method of claim 23, wherein said activity is a biosynthetic activity. 27. The method of claim 23, wherein said activity is a transporter activity. 28. The method of claim 23, wherein said activity is a transcription activity. 29. The method of claim 23, wherein said activity is a DNA replication activity. 30. The method of claim 23, wherein said activity is a cell division activity. 31. A method of analyzing a compound for the ability to reduce activity or the level of a polypeptide required for growth, comprising: encoding a coding sequence of a sequence selected from the group consisting of SEQ ID NOs: 82-88, 90-242. A method comprising: obtaining a target comprising: contacting the target with a candidate compound; and measuring the activity of the target. 32. The method of claim 31, wherein said target is a messenger RNA molecule transcribed from one of the coding regions of SEQ ID NOs: 82-88, 90-242, and wherein said activity is transcription of said messenger RNA. 33. The target is a coding region of one of SEQ ID NOs: 82-88, 90-242, and the activity is transcription of the messenger RNA.
2. The method according to 2. 34. A compound identified using the method of claim 31. 35. A method of identifying a compound that decreases the activity or level of a gene product required for cell growth, comprising: reducing the gene product in a cell to reduce the activity or amount of the gene product in the cell. Expressing an antisense nucleic acid against the encoding nucleic acid, thereby producing a sensitized cell, contacting the sensitized cell with a compound, and preventing the compound from inhibiting the growth of non-sensitized cells. Identifying whether the compound inhibits the growth of the sensitized cells to a greater extent. 36. The method of claim 35, wherein said cells are selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells. 37. The method of claim 36, wherein said cells are Escherichia coli cells. 38. The cell, wherein the cell is Staphylococcus aureus.
coccus aureus), Pseudomonas aeruginosa
), Enterobacter cloacae, Helicobacter pylori, Neisseria gonorhea
rhoeae), Enterococcus faecalis, Streptococcus pneumoniae, Haemophilus influenzae, Salmonella typhimurium (Salm)
onella typhimurium, Saccharomyces cerevi
siae), Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Klebsiella pneumonium
moniae), Salmonella typhi, Salmonella paratyphi, Salmonella cholera Switzerland (Salmonella cholerasu)
is), Staphylococcus epidermidis
, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Bacillus anthracis,
Yersinia pestis, Clostridium botulinum, Campylobacter yejunyi
38. The method according to claim 36, wherein the organism is derived from an organism selected from the group consisting of Chlamydia trachomatus, Chlamydia pneumoniae, or any species within the genus to which any of the species belongs. Method. 39. The method of claim 35, wherein said antisense nucleic acid is transcribed from an inducible promoter. 40. The method of claim 39, further comprising the step of contacting said cell with an inducer at a concentration that induces said antisense nucleic acid to a sublethal level. 41. A growth inhibition of 8% with the sublethal concentration of the inducer
41. The method of claim 40, or more. 42. The inducer is isopropyl-1-thio-β-D-
41. The method of claim 40, wherein the method is galactoside. 43. The method of claim 35, wherein said growth inhibition is measured by monitoring the optical density of a culture growth solution. 44. The method of claim 35, wherein said gene product is a polypeptide. 45. The method of claim 35, wherein said gene product is RNA. 46. The gene product according to any one of SEQ ID NOs: 243 to 357, 359 to 3
45. The method of claim 44, comprising a polypeptide having a sequence selected from the group consisting of 98. 47. A compound identified using the method of claim 35. 48. A method for cell proliferation comprising the step of introducing a compound having activity against a gene corresponding to one of SEQ ID NOs: 82 to 88, 90 to 242 or an activity against said gene product into a cell population expressing the gene. Inhibition method. 49. The method of claim 48, wherein said compound is an antisense oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 405-485 or a growth inhibiting portion thereof. 50. The growth inhibiting moiety of one of SEQ ID NOs: 405-485 is at least 10, at least 20, at least 25 of one of SEQ ID NOs: 405-485.
50. The method of claim 49, wherein the fragment comprises at least 30, at least 50, or more than 50 contiguous bases. 51. The method of claim 48, wherein said compound is a triple helix oligonucleotide. 52. SEQ ID NOS: 405-485 in a pharmaceutically acceptable carrier
A preparation comprising an effective concentration of an antisense oligonucleotide or a growth inhibiting portion thereof, comprising a sequence selected from the group consisting of: 53. The growth-inhibiting moiety of one of SEQ ID NOs: 405-485 comprises at least 10, at least 20, at least 25, one of SEQ ID NOs: 405-485.
53. The preparation of claim 52, comprising at least 30, at least 50, or more than 50 contiguous bases. 54. A method of inhibiting gene expression in an operon required for growth,
Contacting a cell in a cell population with an antisense nucleic acid, wherein said cell expressing a gene corresponds to one of SEQ ID NOs: 82-88, 90-242, wherein said antisense nucleic acid comprises at least one of said operon. A method comprising a growth inhibiting moiety in an antisense orientation that effectively inhibits expression of said gene. 55. The antisense nucleic acid according to any one of SEQ ID NOs: 82 to 88, 90 to 24.
55. The method of claim 54, which is complementary to a sequence of a gene comprising one or more of the two. 56. The method of claim 54, wherein said antisense nucleic acid is one of SEQ ID NOs: 405-485 or a portion thereof. 57. The method of claim 54, wherein said cells are contacted with said antisense nucleic acid by introducing a plasmid expressing said antisense nucleic acid into said cell population. 58. The method of claim 54, wherein said cells are contacted with said antisense nucleic acid by introducing phage expressing said antisense nucleic acid into said cell population. 59. The method of claim 54, wherein said cell is contacted with said antisense nucleic acid by introducing into said cell population a sequence encoding said antisense nucleic acid in the chromosome of said cell. 60. The method of claim 54, wherein said cells are contacted with said antisense nucleic acid by introducing a retron expressing said antisense nucleic acid into said cell population. 61. The method of claim 54, wherein said cell is contacted with said antisense nucleic acid by introducing a ribozyme into said cell population, and wherein said ribozyme binding portion is complementary to said antisense oligonucleotide. 62. The method of claim 54, wherein said cell is contacted with said antisense nucleic acid by introducing liposomes comprising said antisense oligonucleotide into said cell. 63. The method of claim 54, wherein said cells are contacted with said antisense nucleic acid by electroporation. 64. The antisense nucleic acid comprises SEQ ID NOs: 82 to 88, 90 to 24.
At least 10, at least 20, at least 25, at least 30 of one of the two
55. The method of claim 54, wherein the fragment comprises at least 35, at least 50, or more than 50 contiguous bases. 65. The method of claim 54, wherein said antisense nucleic acid is an oligonucleotide. 66. A step of obtaining a sample containing the bacterial species, and identifying the bacterial species using a species-specific probe having a sequence selected from the group consisting of SEQ ID NOs: 405-485, 82-88, 90-242. And the step of
Identification of bacterial strains. 67. A method for identifying a gene in a microorganism required for growth, comprising:
(A) a step of identifying an inhibitory nucleic acid that inhibits the activity of a gene or a gene product required for the growth of the first microorganism; (b) a step of contacting a second microorganism with the inhibitory nucleic acid; (c) Determining whether the inhibitory nucleic acid from the first microorganism inhibits the growth of the second microorganism; and (d) identifying a gene in the second microorganism inhibited by the inhibitory nucleic acid. And a step. 68. A method of analyzing a compound for its ability to inhibit the growth of a microorganism, comprising: (a) identifying a gene or gene product required for growth of the first microorganism;
(B) identifying a homolog of the gene or gene product in the second microorganism; (c) identifying an inhibitory nucleic acid sequence that inhibits the activity of the homolog in the second microorganism; (d) ) Contacting the second microorganism with a growth-inhibiting amount of the inhibitory nucleic acid to sensitize the second microorganism; and (e) contacting the sensitized microorganism of step (d) with a compound. And (f) determining whether the compound inhibits the growth of the sensitized microorganism more than the compound inhibits the growth of the non-sensitized microorganism. 69. The step of identifying a gene associated with the growth of the first microorganism, comprising introducing a nucleic acid comprising a random genomic fragment from the first microorganism operably linked to a promoter, Wherein said random genomic fragment is present in an antisense orientation; and said growing said first microorganism to transcribe said random genomic fragment at a first level and said first transcript to a lower level of said random genomic fragment.
Comparing the growth of said microorganism with said microorganism, wherein said difference in growth indicates that said random genomic fragment contains a gene involved in growth. 70. The step of identifying a homologue of the gene in the second microorganism is selected from the group consisting of BLASTN version 2.0 using default parameters and FASTA version 3.0t78 algorithm using default parameters. 70. The method of claim 69, comprising identifying a homologous nucleic acid or a nucleic acid encoding a homologous polypeptide in a database using an algorithm. 71. The step of identifying a homologue of a gene in the second microorganism comprises the step of identifying a nucleic acid encoding a homologous nucleic acid or a homologous polypeptide by identifying a nucleic acid that hybridizes to the first gene. 70. The method of claim 69, comprising:
The described method. 72. The step of identifying a homologue of the gene of the second microorganism,
70. The method of claim 69, comprising expressing a nucleic acid that inhibits growth of said first microorganism in said second microorganism. 73. The method of claim 69, wherein said inhibitory nucleic acid is an antisense nucleic acid. 74. The method of claim 69, wherein said inhibitory nucleic acid comprises an antisense nucleic acid to a portion of said homolog. 75. The method of claim 69, wherein said inhibitory nucleic acid comprises an antisense nucleic acid to a portion of an operon encoding said homolog. 76. The method of claim 69, wherein contacting the second microorganism with a growth-inhibiting amount of the nucleic acid sequence comprises directly contacting the second microorganism with the nucleic acid. 77. The method of claim 69, wherein contacting the second microorganism with a growth-inhibiting amount of the nucleic acid sequence comprises expressing an antisense nucleic acid in the homolog of the second microorganism. . 78. A compound identified using the method of claim 68. 79. (a) identifying an inhibitory nucleic acid sequence that inhibits the activity of a gene or gene product required for growth of the first microorganism; and (b) a second microorganism and a growth inhibitory amount of the inhibitory nucleic acid. (C) contacting the compound with the growth-inhibiting microorganism of step (b); and (d) contacting the compound with a non-sensitizing second microorganism. Identifying whether the compound inhibits the growth of the second microorganism in the sensitization to a greater extent than the inhibition of the growth of the second microorganism. Analysis method. 80. The method of claim 79, wherein said inhibitory nucleic acid is an antisense nucleic acid that inhibits growth of said first microorganism. 81. The method of claim 79, wherein said inhibitory nucleic acid comprises a portion of an antisense nucleic acid that inhibits growth of said first microorganism. 82. The method of claim 79, wherein said inhibitory nucleic acid comprises an antisense molecule to the entire coding region of a gene associated with growth of said first microorganism. 83. The method of claim 79, wherein said inhibitory nucleic acid comprises an antisense nucleic acid to a portion of an operon encoding a gene associated with growth of said first microorganism. 84. A compound identified using the method of claim 79. 85. A method of sensitizing a cell by expressing an antisense nucleic acid against a nucleic acid encoding a gene product required for growth in the cell to reduce the activity or amount of the gene product, And contacting the compound with, and identifying whether the compound inhibits the growth of the sensitized cells to a greater extent than the compound inhibits the growth of unsensitized cells, Methods for analyzing compounds for activity on a biological pathway required for growth. 86. The method of claim 85, wherein said cells are selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells. 87. The cell may be E. coli. 89. The method of claim 86, wherein the method is an E. coli cell. 88. The cell may be a Staphylococcus aureus.
coccus aureus), Pseudomonas aeruginosa
), Enterobacter cloacae, Helicobacter pylori, Neisseria gonorhea
rhoeae), Enterococcus faecalis, Streptococcus pneumoniae, Haemophilus influenzae, Salmonella typhimurium (Salm)
onella typhimurium, Saccharomyces cerevi
siae), Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Klebsiella pneumonium
moniae), Salmonella typhi, Salmonella paratyphi, Salmonella cholera Switzerland (Salmonella cholerasu)
is), Staphylococcus epidermidis
, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Bacillus anthracis,
Yersinia pestis, Clostridium botulinum, Campylobacter yejunyi
86. The method of claim 85, wherein the organism is derived from an organism selected from the group consisting of Chlamydia trachomatus, Chlamydia pneumoniae, or any species within the genus to which any of the species belongs. Method. 89. The method of claim 85, wherein said antisense nucleic acid is transcribed from an inducible promoter. 90. The method of claim 89, further comprising contacting the cell with an agent that induces expression of the antisense nucleic acid from the inducible promoter, wherein the antisense nucleic acid is expressed at sublethal levels. the method of. 91. The method of claim 90, wherein said sublethal level of said antisense nucleic acid inhibits proliferation by 8% or more. 92. The method of claim 90, wherein said agent is isopropyl-1-thio-β-D-galactoside (IPTG). 93. The method of claim 91, wherein said growth inhibition is measured by monitoring the optical density of a liquid culture. 94. The gene product is SEQ ID NOS: 243-357, 359-3
86. The method of claim 85, comprising a polypeptide having a sequence selected from the group consisting of 98. 95. A compound identified using the method of claim 85. 96. A method for analyzing the ability of a compound to inhibit cell proliferation, comprising the steps of: contacting a cell with an agent that reduces the activity or level of a gene product required for the proliferation of the cell; And determining whether the compound reduces the growth of cells that have not been contacted with the agent more than the compound does. 97. The method of claim 96, wherein said agent that decreases the activity or level of said gene product required for growth of said cell comprises an antisense nucleic acid against a gene or operon required for growth. 98. The method of claim 96, wherein said agent that decreases the activity or level of said gene product required for cell growth comprises an antibiotic. 99. The method of claim 96, wherein said cell comprises a temperature sensitive mutation that reduces the activity or level of said gene product required for growth of said cell. 100. The method of claim 99, wherein said antisense nucleic acid is directed to a nucleic acid that encodes the same functional domain as said gene product required for growth of said cell to which said antisense nucleic acid is directed. 101. The antisense nucleic acid of claim 99, wherein said antisense nucleic acid is directed to a nucleic acid that encodes a functional domain of said gene product that is different from the functional domain to which said antisense nucleic acid is directed for growth of said cell. Method. 102. A compound identified using the method of claim 96. 103. A method for identifying a pathway in which a nucleic acid required for growth or a gene product thereof is present, comprising: expressing a sublethal level of an antisense nucleic acid directed to the nucleic acid required for growth in a cell; Contacting the cell with an antibiotic, wherein the biological pathway through which the antibiotic acts is known, and the cell is more resistant to the antibiotic than a cell that does not express the sublethal level of the antisense nucleic acid. Determining whether the substance is substantially sensitive to the substance. 104. A method for determining a pathway in which a test compound acts, comprising: (a) expressing a sublethal level of an antisense nucleic acid directed to a nucleic acid required for proliferation in a cell; (B) contacting the cells with the test compound; and (c) cells that do not express the sublethal level of the antisense nucleic acid. Determining whether said cells are substantially more sensitive to said test compound. (D) expressing a sublethal level of a second antisense nucleic acid directed to a nucleic acid required for a second growth in the cell, wherein the nucleic acid is required for the second growth. Is present in a different biological pathway than the nucleic acid required for the growth of step (a), and (e) said cell is less than the cell that does not express said sublethal level of a second antisense nucleic acid. Identifying whether said test compound is substantially sensitive to said test compound. 106. A purified or isolated nucleic acid consisting essentially of one of SEQ ID NOs: 358, 399-402. 107. A compound identified using the method of claim 23. 108. SEQ ID NOs: 82-88, 90-2 to inhibit growth
A compound that interacts with a gene or gene product of a nucleic acid comprising one of the 42 sequences. 109. SEQ ID NOS: 243-357,359 to inhibit growth
-398, a compound that interacts with a polypeptide comprising one of: 110. SEQ ID NOs: 358, 399-402 for inhibiting growth
A compound that interacts with a nucleic acid comprising one of the following.