Identification of genes

Abstract

A method for identifying a microorganism having a reduced adaptation to a particular environment comprising the steps of: (1) providing a plurality of microorganisms each of which is independently mutated by the insertional inactivation of a gene with a nucleic acid comprising a unique marker sequence so that each mutant contains a different marker sequence, or clones of the said microorganism; (2) providing individually a stored sample of each mutant produced by step (1) and providing individually stored nucleic acid comprising the unique marker sequence from each individual mutant; (3) introducing a plurality of mutants produced by step (1) into the said particular environment and allowing those microorganisms which are able to do so to grow in the said environment; (4) retrieving microorganisms from the said environment or a selected part thereof and isolating the nucleic acid from the retrieved microorganisms; (5) comparing any marker sequences in the nucleic acid isolated in step (4) to the unique marker sequence of each individual mutant stored as in step (2); and (6) selecting an individual mutant which does not contain any of the marker sequences as isolated in step (4).

1 . A method for identifying a microorganism having a reduced adaptation to a particular environment comprising the steps of: 
 (1) providing a plurality of microorganisms each of which is independently mutated by the insertional inactivation of a gene with a nucleic acid comprising a unique marker sequence so that each mutant contains a different marker sequence, or clones of the said microorganism;    (2) providing individually a stored sample of each mutant produced by step (1) and providing individually stored nucleic acid comprising the unique marker sequence from each individual mutant;    (3) introducing a plurality of mutants produced by step (1) into the said particular environment and allowing those microorganisms which are able to do so to grow in the said environment;    (4) retrieving microorganisms from the said environment or a selected part thereof and isolating the nucleic acid from the retrieved microorganisms;    (5) comparing any marker sequences in the nucleic acid isolated in step (4) to the unique marker sequence of each individual mutant stored as in step (2); and    (6) selecting an individual mutant which does not contain any of the marker sequences as isolated in step (4),    
     
     
         2 . A method according to  claim 1  wherein the plurality of microorganisms as defined in step (1) is produced from a plurality of microorganisms, each of which comprises a nucleic acid comprising a unique marker sequence, by changing their condition from a first given condition to a second given condition wherein (a) in the first given condition the said nucleic acid comprising a unique marker is maintained episomally and (b) in the second given condition the said nucleic acid comprising a unique marker sequence insertionally inactivates a gene.  
     
     
         3 . (canceled)  
     
     
         4 . A method according to  claim 1  followed by the step: 
 (7) isolating the insertionally-inactivated gene from the individual mutant selected in step (6).    
     
     
         5 . A method according to  claim 4  further comprising the step: 
 (8) isolating from a wild-type microorganism the corresponding wild-type gene using the insertionally-inactivated gene isolated in step (7) as a probe.    
     
     
         6 . A method according to  claim 1  wherein the particular environment is a differentiated multicellular organism.  
     
     
         7 . A method according to  claim 6  wherein the multicellular organism is a plant.  
     
     
         8 . A method according to  claim 6  wherein the multicellular organism is a non-human animal.  
     
     
         9 . A method according to  claim 8  wherein the animal is a mouse, rat, rabbit, dog or monkey.  
     
     
         10 . A method according to  claim 9  wherein the animal is a mouse.  
     
     
         11 . A method according to  claim 6  wherein in step (4) the microorganisms are retrieved from the said environment at a site remote from the site of introduction in step (3).  
     
     
         12 . A method according to  claim 8  wherein in step (3) the microorganism is introduced orally or intraperitoneally.  
     
     
         13 . The method of  claim 12  wherein in step (4) the microorganisms are retrieved from the spleen.  
     
     
         14 . A method according to  claim 1  wherein the microorganism is a bacterium.  
     
     
         15 . A method according to  claim 1  wherein the microorganism is a fungus.  
     
     
         16 . A method according to  claim 7  wherein the microorganism is a bacterium pathogenic to plants.  
     
     
         17 . A method according to  claim 7  wherein the microorganism is a fungus pathogenic to plants.  
     
     
         18 . A method according to  claim 8  wherein the microorganism is a bacterium pathogenic to animals.  
     
     
         19 . A method according to  claim 8  wherein the microorganism is a fungus pathogenic to animals.  
     
     
         20 . A method according to  claim 18  wherein the bacterium is any one of  Bordetella pertussis, Campylobacter jejuni, Clostridium botulinum, Escherichia coli, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Listeria  spp.,  Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas  spp.,  Salmonella  spp.,  Shigella  spp.,  Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Vibrio  spp., and  Yersinia pestis.    
     
     
         21 . A method according to  claim 19  wherein the fungus is any one of  Aspergillus  spp.,  Cryptococcus neoformans  and  Histoplasma capsulatum.    
     
     
         22 . A method according to  claim 1  wherein in step (1) the gene is insertionally inactivated using a transposon or transposon like element or other DNA sequence carrying a unique marker sequence.  
     
     
         23 . A method according to  claim 1  wherein in step (1) each different marker sequence is flanked on either side by sequences common to each said nucleic acid.  
     
     
         24 . A method according to  claim 23  wherein in step (2) the nucleic acid comprising the unique marker is isolated using DNA amplification techniques and oligonucleotide primers which hybridise to the said common sequences.  
     
     
         25 . A method according to  claim 23  wherein in step (4) the nucleic acid comprising a plurality of said marker sequences is isolated using DNA amplification techniques and oligonucleotide primers which hybridise to the said common sequences.  
     
     
         26 . (canceled)  
     
     
         27 . (canceled)  
     
     
         28 . (canceled)  
     
     
         29 . (canceled)  
     
     
         30 . (canceled)  
     
     
         31 . A gene which is isolated from the  Salmonella typhimurium  genome and hybridizes to the sequence shown in  FIG. 5  or  FIG. 6  under stringent conditions, or a polypeptide encoded thereby.  
     
     
         32 . (canceled)  
     
     
         33 . (canceled)  
     
     
         34 . (canceled)  
     
     
         35 . (canceled)  
     
     
         36 . (canceled)  
     
     
         37 . (canceled)  
     
     
         38 . (canceled)  
     
     
         39 . (canceled)  
     
     
         40 . A molecule which selectively interacts with, and substantially inhibits the function of, a gene hybridizing under stringent conditions to the sequence of  FIG. 5  or  FIG. 6  or a nucleic acid product thereof.  
     
     
         41 . A molecule according to  claim 40  which is an antisense nucleic acid or nucleic acid derivative.  
     
     
         42 . A molecule according to  claim 40  which is an antisense oligonucleotide.  
     
     
         43 . (canceled)  
     
     
         44 . (canceled)  
     
     
         45 . The molecule or compound according to  claim 40  further comprising a pharmaceutically acceptable carrier.  
     
     
         46 . The VGC2 DNA of  Salmonella typhimurium  or a part thereof, or a variant of said DNA or a variant of a part thereof, or a polypeptide encoded thereby.  
     
     
         47 . A mutant bacterium wherein if the bacterium normally contains a gene that is the same as or equivalent to a gene in VGC2, said gene is mutated or absent in said mutant bacterium.  
     
     
         48 . (canceled)  
     
     
         49 . (canceled)  
     
     
         50 . (canceled)  
     
     
         51 . (canceled)  
     
     
         52 . A method of identifying a compound which reduces the ability of a bacterium to infect or cause disease in a host comprising the step of selecting a compound which interferes with the function of a gene in the VGC2 DNA of  Salmonella typhimurium  or a part thereof, or a variant of said DNA or a variant of a part thereof or a polypeptide encoded thereby.  
     
     
         53 . (canceled)  
     
     
         54 . (canceled)  
     
     
         55 . (canceled)  
     
     
         56 . (canceled)  
     
     
         57 . A non-human animal or plant, or an animal or plant cell culture, containing a plurality of mutant microorganisms wherein each mutant contains a different marker sequence.