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Identification of genes

a technology of identification and gene, applied in the field of identification of genes, can solve the problems of not being able to identify virulence genes, not being able to complete screening of a bacterial genome for virulence genes, and not being able to provide information on whether the gene(s) which have been identified are genuine or no

Inactive Publication Date: 2006-09-28
IMPERIAL INNOVATIONS LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This method significantly reduces the number of animals needed for screening and effectively identifies genes essential for virulence, enabling the development of vaccines and drug screening for reducing virulence.

Problems solved by technology

Because of the extremely large numbers of animals required, comprehensive screening of a bacterial genome for virulence genes has not been feasible.
However, it will not identify virulence genes that are regulated posttranscriptionally, and more importantly, will not provide information on whether the gene(s) which have been identified are actually required for, or contribute to, the infection process.
The Walsh and Cepko method uses a tag that contains a unique nucleic acid sequence and the lacZ gene but there is no indication that useful mutants or genes could be detected by their method.

Method used

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  • Identification of genes
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Examples

Experimental program
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Effect test

example 1

Identification of Virulence Genes in Salmonella typhimurium

Materials and Methods

Bacterial Strains and Plasmids

[0201]Salmonella typhimurium strain 12023 (equivalent to American Type Collection of Type Cultures (NCTC), Public Health Laboratory Service, Colindale, London, UK. A spontaneous nalidixic acid resistant mutant of this strain (12023 Nalr) was selected in our laboratory. Another derivative of strain 12023, CL1509 (aroA::Tn10) was a gift from Fred Heffron. Escherichia coli strains CC118 lambdapir (δ[ara-leu], araD, .DELTA.lacX74, galE, galK, phoA20, thi-1, rpsE, rpoB, argE(Am), recA1, lambdapir phage lysogen) and S17-1 lambdapir(Tpr, Smr, recA, thi, pro, hsdR−, M+, RP4:2-Tc:Mu:KmTn7, lambdapir) were gifts from Kenneth Timmis. E. coli DH5α was used for propagating pUC18 (Gibco-BRL) and Bluescript (Stratagene) plasmids containing S. typhimurium DNA. Plasmid pUTmini-Tn5Km2 (de Lorenzo et al, 1990) was a gift from Kenneth Timmis.

Construction of Semi-random Sequence Tags and Lig...

example 2

Cloning and Partial Characterisation of Sequences Flanking the Transposon

[0216] DNA was extracted from one of the mutants described in Example 1 (Pool 1, F10), digested with SstI, and subcloned on the basis of kanamycin resistance. The sequence of 450 bp flanking one end of the transposon was determined using primer P7. This sequence shows 80% identity to the E. coli clp (Ion) gene, which encodes a heat-regulated protease (FIG. 5; SEQ ID NOS 39 and 40). To our knowledge, this gene has not previously been implicated as a virulence determinant.

[0217] Partial sequences of thirteen further Salmonella typhimurium virulence genes are shown in FIG. 6 (sequences A2 to A9 and B1 to B5; SEQ ID NOS 8-36). Deduced amino acid sequences of P2D6, S4C3, P3F4, P7G2 and P9B7 bear similarities to a family of secretion-associated proteins that have been conserved throughout bacterial pathogens of animals and plants, and which are known in Salmonella as the inv family. In S. typhimurium the inv genes ...

example 3

LD50 Determinations and Mouse Vaccination Study

[0219] Mutations identified by the method of the invention attenuate virulence.

[0220] Five of the mutations in genes not previously implicated in virulence were transferred by P22-mediated transduction to the nalidixic acid-sensitive parent strain of S. typhimurium 12028. Transductants were checked by restriction mapping then injected by the intraperitoneal route into groups of BALB / c mice to determine their 50% lethal dose (LD50). The LD50 values for mutants S4C3, P7G2, P3F4 and P9B7 were all several orders of magnitude higher than that of the wild-type strain. No difference in the LD50 was detected for mutant P1F10; however, there was a statistically significant decrease in the proportion of P1F10 cells recovered from the spleens of mice injected with an inoculum consisting of an equal proportion of this strain and the wild-type strain. This implies that this mutation does attenuate virulence, but to a degree that is not detectable ...

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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).

Description

[0001] This application is a divisional of pending application U.S. Ser. No. 09 / 714,602 filed Nov. 16, 2000 entitled “Identification of Genes” by David William Holden; which is a continuation of U.S. Ser. No. 09 / 201,945, filed Dec. 1, 1998, now U.S. Pat. No. 6,342,215, which is a continuation of Ser. No. 08 / 637,759, filed Jul. 19, 1997, now U.S. Pat. No. 5,876,931, which is a 371 of PCT / GB95 / 02875 filed Dec. 11, 1995. application Ser. No. 09 / 201,945, filed Dec. 1, 1998, and application Ser. No. 08 / 637,759, filed Jul. 19, 1997, are hereby incorporated herein by reference.[0002] The present invention relates to methods for the identification of genes involved in the adaptation of a microorganism to its environment, particularly the identification of genes responsible for the virulence of a pathogenic microorganism. BACKGROUND OF THE INVENTION [0003] Antibiotic resistance in bacterial and other pathogens is becoming increasingly important. It is therefore important to find new therapeu...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/00A61K39/02C12Q1/68C07H21/04C12Q1/04C07K14/255C07K14/39A61K31/7088C12N15/09A61K38/00A61K39/112A61K48/00A61P31/04A61P31/10C07H21/02C07K14/21C07K14/315C07K14/38C12N1/10C12N1/15C12N1/21C12N15/00C12N15/10C12N15/11C12N15/31G01N33/53
CPCA61K38/00A61K39/00A61K2039/522A61K2039/523C07K14/21C07K14/255C07K14/315C07K14/3156C07K14/38C12N15/102C12N15/1065C12N15/1082C12N15/1086C12Q1/68C12R1/42A61P31/00A61P31/04A61P31/10A61P37/04Y02A50/30C12R2001/42C12N1/205C12N15/10C12Q1/04C12Q1/6888C12Q2600/136
Inventor HOLDEN, DAVID
Owner IMPERIAL INNOVATIONS LTD