Claims
- 1. A method for inhibiting the activity or expression of a gene in an operon required for proliferation wherein the activity or expression of at least one gene in said operon is inhibited by an antisense nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 1-127, said method comprising contacting a cell in a cell population with an antisense nucleic acid complementary to at least a portion of said operon.
- 2. The method of claim 1, wherein said antisense nucleic acid complementary to at least a portion of said operon comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-127 or a proliferation-inhibiting portion thereof.
- 3. The method of claim 2, wherein said proliferation-inhibiting portion of one of SEQ ID NO: 1-127 is a fragment comprising at least 10, at least 20, at least 25, at least 30, at least 50 or more than 51 consecutive nucleotides of one of SEQ ID NOs: 1-127.
- 4. The method of claim 1, wherein said gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 128-298.
- 5. The method of claim 1, wherein said gene encodes a polypeptide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 299-469.
- 6. The method of claim 1, wherein said cell is contacted with said antisense nucleic acid by expressing said antisense nucleic acid from an inducible promoter.
- 7. The method of claim 1, wherein said cell is contacted with said antisense nucleic acid by expressing said antisense nucleic acid from a plasmid.
- 8. The method of claim 1, wherein said cell is contacted with said antisense nucleic acid by expressing said antisense nucleic acid from a phage.
- 9. The method of claim 1, wherein said cell is contacted with said antisense nucleic acid by expressing said antisense nucleic acid from the chromosome of a cell in said cell population.
- 10. The method of claim 1, wherein said cell is contacted with said antisense nucleic acid by introducing a promoter adjacent to a chromosomal copy of said antisense nucleic acid such that said promoter directs the synthesis of said antisense nucleic acid.
- 11. The method of claim 1, wherein said cell is contacted with said antisense nucleic acid by expressing said antisense nucleic acid from a retron.
- 12. The method of claim 1, wherein said cell is contacted with said antisense nucleic acid by introducing a ribozyme into said cell, wherein a binding portion of said ribozyme is complementary to said antisense oligonucleotide.
- 13. The method of claim 1, wherein said cell is contacted with said antisense nucleic acid by introducing a liposome comprising said antisense oligonucleotide into said cell.
- 14. The method of claim 1, wherein said cell is contacted with said antisense nucleic acid by electroporation of said antisense nucleic acid into said cell.
- 15. The method of claim 1, wherein said cell is selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells.
- 16. The method of claim 1, wherein said cell is a Gram negative bacterium.
- 17. The method of claim 1, wherein said cell is an E. coli cell.
- 18. The method of claim 1, wherein said cell is from an organism selected from the group consisting of Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, Enterobacter cloacae, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Saccharomyces cerevisae, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Treponema pallidum, and Yersinia pestis or any species falling within the genera of any of the above species.
- 19. A method for inhibiting the activity or expression of a gene in an operon which encodes a gene product required for proliferation comprising contacting a cell in a cell population with an antisense nucleic acid comprising at least a proliferation-inhibiting portion of said operon in an antisense orientation, wherein said gene product is selected from the group consisting of a gene product having at least 70% nucleotide sequence identity as determined using BLASTN version 2.0 with the default parameters to a gene product whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs.: 1-127, a gene product encoded by a nucleic acid having at least 70% nucleotide sequence identity as determined using BLASTN version 2.0 with the default parameters to a nucleic acid encoding a gene product whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-127, and a gene product having at least 25% amino acid identity as determined using FASTA version 3.0t78 with the default parameters to a gene product whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs.: 1-127.
- 20. The method of claim 19, wherein said antisense nucleic acid comprises a nucleotide sequence having at least 70% nucleotide sequence identity as determined using BLASTN version 2.0 with the default parameters to a nucleotide sequence selected from the group consisting of SEQ ID NOs.: 1-127, and a proliferation inhibiting portion thereof.
- 21. The method of claim 20, wherein said antisense nucleic acid has at least 70% nucleotide sequence identity as determined using BLASTN version 2.0 with the default parameters to a nucleotide sequence comprising at least 10, at least 20, at least 25, at least 30, at least 50 or more than 51 consecutive nucleotides of one of SEQ ID NOs.: 1-127.
- 22. The method of claim 19, wherein said gene comprises a nucleic acid selected from the group consisting of a nucleic acid comprising a nucleic acid having at least 70% nucleotide sequence identity as determined using BLASTN version 2.0 with the default parameters to a nucleotide sequence selected from the group consisting of SEQ ID NOS.: 128-298.
- 23. The method of claim 19, wherein said gene encodes a polypeptide comprising a polypeptide selected from the group consisting of a polypeptide having at least 25% amino acid identity as determined using FASTA version 3.0t78 to a polypeptide selected from the group consisting of SEQ ID NOs.: 299-469.
- 24. The method of claim 19, wherein said cell is contacted with said antisense nucleic acid by expressing said antisense nucleic acid from an inducible promoter.
- 25. The method of claim 19, wherein said cell is contacted with said antisense nucleic acid by expressing said antisense nucleic acid from a plasmid.
- 26. The method of claim 19, wherein said cell is contacted with said antisense nucleic acid by expressing said antisense nucleic acid from a phage.
- 27. The method of claim 19, wherein said cell is contacted with said antisense nucleic acid by expressing said antisense nucleic acid from the chromosome of a cell in said cell population.
- 28. The method of claim 19, wherein said cell is contacted with said antisense nucleic acid by introducing a promoter adjacent to a chromosomal copy of said antisense nucleic acid such that said promoter directs the synthesis of said antisense nucleic acid.
- 29. The method of claim 19, wherein said cell is contacted with said antisense nucleic acid by expressing said antisense nucleic acid from a retron.
- 30. The method of claim 19, wherein said cell is contacted with said antisense nucleic acid by introducing a ribozyme into said cell, wherein a binding portion of said ribozyme is complementary to said antisense oligonucleotide.
- 31. The method of claim 19, wherein said cell is contacted with said antisense nucleic acid by introducing a liposome comprising said antisense oligonucleotide into said cell.
- 32. The method of claim 19, wherein said cell is contacted with said antisense nucleic acid by electroporation of said antisense nucleic acid into said cell.
- 33. The method of claim 19, wherein said cell is selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells.
- 34. The method of claim 19, wherein said cell is a Gram negative bacterium.
- 35. The method of claim 19, wherein said cell is an E. coli cell.
- 36. The method of claim 19, wherein said cell is from an organism selected from the group consisting of Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, Enterobacter cloacae, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Saccharomyces cerevisae, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Treponema pallidum, and Yersinia pestis or any species falling within the genera of any of the above species.
- 37. A method for screening a candidate compound for the ability to reduce cellular proliferation, said method comprising the steps of:
(a) providing a sublethal level of an antisense nucleic acid complementary to at least a portion of an operon comprising a gene which encodes a gene product required for proliferation of a cell to reduce the activity or amount of said gene product in said cell, thereby producing a sensitized cell, wherein said gene product is a gene product whose activity or amount is reduced by an antisense nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs.: 1-127; (b) contacting said sensitized cell with a compound; and (c) determining the degree to which said compound inhibits proliferation of said sensitized cell relative to a nonsensitized cell.
- 38. The method of claim 37, wherein said antisense nucleic acid complementary to at least a portion of said operon comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-127 or a proliferation-inhibiting portion thereof.
- 39. The method of claim 38, wherein said proliferation-inhibiting portion of one of SEQ ID NO: 1-127 is a fragment comprising at least 10, at least 20, at least 25, at least 30, at least 50 or more than 51 consecutive nucleotides of one of SEQ ID NOs: 1-127.
- 40. The method of claim 37, wherein said gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 128-298.
- 41. The method of claim 37, wherein said gene encodes a polypeptide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 299-469.
- 42. The method of claim 37, wherein said determining step comprises determining whether said compound inhibits the growth of said sensitized cell to a greater extent than said compound inhibits the growth of a nonsensitized cell.
- 43. The method of claim 37, further comprising the step of contacting said cell with a concentration of inducer which induces said antisense nucleic acid to a sublethal level.
- 44. The method of claim 37, wherein said inhibition of proliferation is measured by monitoring optical density of a liquid culture.
- 45. The method of claim 37, wherein said cell is selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells.
- 46. The method of claim 37, wherein said cell is a Gram negative bacterium.
- 47. The method of claim 37, wherein said cell is an E. coli cell.
- 48. The method of claim 37, wherein said cell is from an organism selected from the group consisting of Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, Enterobacter cloacae, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Saccharomyces cerevisae, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Treponema pallidum, and Yersinia pestis or any species falling within the genera of any of the above species.
- 49. The method of claim 37, wherein said cell is provided with said antisense nucleic acid by expressing said antisense nucleic acid from an inducible promoter.
- 50. The method of claim 37, wherein said cell is provided with said antisense nucleic acid by introducing an oligonucleotide into said cell..
- 51. The method of claim 37, wherein said compound is a natural product.
- 52. A compound identified using the method of claim 37.
- 53. A method for screening a candidate compound for the ability to reduce cellular proliferation, said method comprising the steps of:
(a) providing a sublethal level of an antisense nucleic acid complementary to at least a portion of an operon comprising a gene which encodes a gene product required for proliferation of a cell to reduce the activity or amount of said gene product in said cell, thereby producing a sensitized cell, wherein said gene product is selected from the group consisting of a gene product having at least 70% nucleotide sequence identity as determined using BLASTN version 2.0 with the default parameters to a gene product whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs.: 1-127, a gene product encoded by a nucleic acid having at least 70% nucleotide sequence identity as determined using BLASTN version 2.0 with the default parameters to a nucleic acid encoding a gene product whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-127, and a gene product having at least 25% amino acid identity as determined using FASTA version 3.0t78 with the default parameters to a gene product whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs.: 1-127; (b) contacting said sensitized cell with a compound; and (c) determining the degree to which said compound inhibits proliferation of said sensitized cell relative to a nonsensitized cell.
- 54. The method of claim 53, wherein said antisense nucleic acid comprises a nucleotide sequence having at least 70% nucleotide sequence identity as determined using BLASTN version 2.0 with the default parameters to a nucleotide sequence selected from the group consisting of SEQ ID NOs.: 1-127, and a proliferation inhibiting portion thereof.
- 55. The method of claim 54, wherein said antisense nucleic acid has at least 70% nucleotide sequence identity as determined using BLASTN version 2.0 with the default parameters to a nucleotide sequence comprising at least 10, at least 20, at least 25, at least 30, at least 50 or more than 51 consecutive nucleotides of one of SEQ ID NOs.: 1-127.
- 56. The method of claim 53, wherein said gene comprises a nucleic acid selected from the group consisting of a nucleic acid comprising a nucleic acid having at least 70% nucleotide sequence identity as determined using BLASTN version 2.0 with the default parameters to a nucleotide sequence selected from the group consisting of SEQ ID NOS.: 128-298.
- 57. The method of claim 53, wherein said gene encodes a polypeptide comprising a polypeptide selected from the group consisting of a polypeptide having at least 25% amino acid identity as determined using FASTA version 3.0t78 to a polypeptide selected from the group consisting of SEQ ID NOs.: 299-469.
- 58. The method of claim 53, wherein said determining step comprises determining whether said compound inhibits the growth of said sensitized cell to a greater extent than said compound inhibits the growth of a nonsensitized cell.
- 59. The method of claim 53, further comprising the step of contacting said cell with a concentration of inducer which induces said antisense nucleic acid to a sublethal level.
- 60. The method of claim 53, wherein said inhibition of proliferation is measured by monitoring optical density of a liquid culture.
- 61. The method of claim 53, wherein said cell is selected from the group consisting of bacterial cells, fungal cells, plant cells, and animal cells.
- 62. The method of claim 53, wherein said cell is a Gram negative bacterium.
- 63. The method of claim 53, wherein said cell is an E. coli cell.
- 64. The method of claim 53, wherein said cell is from an organism selected from the group consisting of Aspergillus fumigatus, Bacillus anthracis, Campylobacter jejuni, Candida albicans, Chlamydia pneumoniae, Chlamydia trachomatus, Clostridium botulinum, Cryptococcus neoformans, Enterobacter cloacae, Enterococcus faecalis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Saccharomyces cerevisae, Salmonella cholerasuis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Treponema pallidum, and Yersinia pestis or any species falling within the genera of any of the above species.
- 65. The method of claim 53, wherein said cell is provided with said antisense nucleic acid by expressing said antisense nucleic acid from an inducible promoter.
- 66. The method of claim 53, wherein said cell is provided with said antisense nucleic acid by introducing an oligonucleotide into said cell..
- 67. The method of claim 53, wherein said compound is a natural product.
- 68. A compound identified using the method of claim 53.
RELATED APPLICATIONS
[0001] This application is a division of copending U.S. patent application Ser. No. 09/711,164, entitled GENES ESSENTIAL FOR MICROBIAL PROLIFERATION AND ANTISENSE THERETO, filed Nov. 9, 2002, which claims priority from U.S. Provisional Patent Application No. 60/164,415, entitled GENES ESSENTIAL FOR MICROBIAL PROLIFERATION AND ANTISENSE THERETO, filed Nov. 9, 1999, the disclosures of which are incorporated herein by reference in there entireties.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60164415 |
Nov 1999 |
US |
Divisions (1)
|
Number |
Date |
Country |
Parent |
09711164 |
Nov 2000 |
US |
Child |
10287274 |
Oct 2002 |
US |