Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-27
2002-10-29
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S008000, C435S252300, C435S252330
Reexamination Certificate
active
06472152
ABSTRACT:
FIELD OF THE INVENTION
The present invention is related to a diagnostic system and to a method for determining the presence of a genotoxic compound in a sample.
BACKGROUND OF THE INVENTION AND STATE OF THE ART
The International Patent Application PCT/EP96/01745 describes a recombinant nucleic acid sequence, a host micro-organism comprising said nucleic acid sequence and the use of said host micro-organism for detecting the presence of genotoxic compounds in a sample. Said bacterial genotoxicity test is based on bioluminescence and allows an easy, very rapid and low cost detection of genotoxic compounds. The test was shown to be at least as sensitive as the Ames test and SOS-chromotest and to allow genotoxicity kinetics measurements as well as a simultaneous evaluation of the toxicity of the test compound or -material (van der Lelie et al., 1997). This new test, referred to as the VITOTOX® test was therefore considered to be a valuable short-term genotoxicity and toxicity test for many different purposes.
The test is based on bacteria that contain the lux operon of
Vibrio fischeri
under transcriptional control of the recN gene, that is part of the SOS-system. After incubation of the bacteria in the presence of a genotoxic compound, the recN promoter is derepressed, resulting in expression of the lux operon. This expression results in light production in function of genotoxicity. Originally, the test was performed with different modified
Escherichia coli
and
Salmonella typhimurium
strains.
Salmonella typhimurium
strains (TA98, TA100 and TA104) were further used, as the bacteria are well known for mutagenicity testing and because the same bacteria could also be used for a classical Ames test, should this be required. The construct using a recN promoter up mutation (recN 2-4) gave the best results in all strains. Furthermore, as all Salmonella strains gave very comparable results, it has been proposed to further use only the TA104 constructs [called TA104 (recN2-4)] as it was shown to be sometimes a little more sensitive than the other hybrid strains (van der Lelie et al., 1997).
However, some compounds act directly on the light production (e.g. aldehydes, organic solvents) or enhance the metabolism of the bacteria creating false-positive results.
AIMS OF THE INVENTION
The present invention aims to provide a new diagnostic system and method for the detection of environmental insults such as the presence of a genotoxic compound into a sample, which do not present the drawbacks of the state of the art.
A main aim of the present invention is to provide a new diagnostic system and method which will exclude false-positive and false-negative results.
A further aim of the present invention is to provide such a diagnostic system and method which could be used for the screening of genotoxic compounds obtained in the chemical, cosmetical or pharmaceutical industry field, as a prevalidation study of intermediate or active chemical, cosmetical and/or pharmaceutical compounds.
A last aim of the present invention is to provide such a diagnostic system and method which allow an automatic screening upon very small volume of sample.
DESCRIPTION OF THE INVENTION
The present invention is related to a diagnostic system made of
a transformed micro-organism capable of an increased reporter activity upon exposure to an environmental insult, preferably exposure to a genotoxic compound, said micro-organism having a stress inducible promoter sequence, preferably a promoter sequence which is inducible by a genotoxic compound, said promoter sequence being operatively linked to a reporter encoding nucleic acid sequence encoding a reporter molecule resulting in a signal that can be assayed, and of
a transformed micro-organism having a constitutive and non-stress inducible promoter sequence, preferably a constitutive promoter sequence which is not inducible by said genotoxic compound, said promoter sequence being operatively linked to a reporter encoding nucleic acid sequence encoding a reporter molecule resulting in a signal that can be assayed.
Preferably, both transformed micro-organisms are bioluminescent micro-organisms and the signal can be assayed as light production. Other possibilities are the peroxydase, alkaline phosphatase, &bgr;-gal and gus genetic sequence, where the signal will be assayed as a colorimetric modification or a chemiluminescent light production by using a colorimetry analyser or a photomultiplier device.
Advantageously, the diagnostic system according to the invention is made of two transformed bioluminescent micro-organisms, the first bioluminescent micro-organism is “activated” in the presence of a genotoxic compound and results in a signal that can be assayed as light production, while the light production of the second bioluminescent micro-organism is not influenced by the presence of a genotoxic compound in the sample.
Nucleic acid sequence encoding a reporter resulting in a signal that can be assayed as light production has been already described in the state of the art. Preferably, the transformed bioluminescent micro-organisms according to the invention comprise luciferase A and B genes, also identified as expressive lux genes complex, comprising the luxA and luxB genes or a translational luxAB fusion gene. The transformed bioluminescent micro-organisms may also comprise the luciferase C, D and E genes, required for production of limiting fatty acid substrate that is used in recycling.
According to a preferred embodiment of the present invention, the diagnostic system comprises a transformed bioluminescent micro-organism having a constitutive and non stress inducible promoter sequence with a Sigma 70 consensus sequence (TTGACA(−35) . . . 17/18 bp . . . TATAAT(−10)) and whose transcription is not regulated positively or negatively at the promoter level. Said promoter consensus is described in Hoopes B C and McClure W R (1987): Strategies in Regulation of Transcriptional Initiation; in “
Escherichia Coli
and
Salmonella typhimurium,
Cellular and Molecular Biology, FC Neidhart, J L Ingraham, K B Low, B Maganasik, M Schaechter, H E Umbaeger (eds.), American Society for Microbiology, Washington D.C., pp 1231-1240.
According to a preferred embodiment of the present invention, the transformed bioluminescent micro-organisms are selected from the group consisting of
E. coli
or
Salmonella typhimurium,
and are advantageously suitable Ames test micro-organism(s), preferably selected from the group consisting of TA98, TA100, TA102, TA104, TA1535, TA1538, TA7001 to TA7006, and TA7041 to TA7046, and/or having the micro-organism deposit number LMG P-18318. The micro-organism with deposit number LMG P-18318 will be identified hereafter as the “pr1” strain.
Advantageously, the stress inducible promoter sequence in the transformed bioluminescent micro-organism of the diagnostic system according to the invention is selected from the group consisting of groEL, dnaK, grpE, phoA, glnA, lon, lysU, rpoD, clpB, clpP, uspA, katG, uvrA, frda, micF, fabA, lac, his, sodA, sodB, soi-28, recA, xthA, narG, recF, recj, recN, recO, recQ, ruv, uvrD, ars, cad, mer, pco, and sfiA.
According to a preferred embodiment of the present invention, the stress inducible promoter sequence is recN, advantageously recN2-4. Said micro-organism will be identified hereafter as the “recN2-4” strain
In a preferred embodiment, the diagnostic system according to the invention comprises a transformed bioluminescent micro-organism having a stress inducible promoter sequence being a SOS regulator promoter sequence, having preferably an induction ration higher than 40, and comprising advantageously a mutation improving the promoter strength or regulation, said mutation not destroying the SOS regulation.
A specific example of mutated recN promoter sequence is described in the International Patent Application PCT/EP96/01745, incorporated hereafter by reference.
Said stress inducible promoter sequence comprises also a promoter up-mutation, preferably a promoter up-mutation in the −35 region of s
Corbisier Philippe Gilbert Ghislain
Regniers Luc Agnes Louis Jean Bosco
Taghavi Safiyh
Van Der Lelie Daniel
Verschaeve Luc Philippine Edouard
Katcheves Konstantina
Vlaamse Instelling Voor Technologish Onderzoek (VITO)
Yucel Remy
LandOfFree
Diagnostic systems and method for determining the presence... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Diagnostic systems and method for determining the presence..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Diagnostic systems and method for determining the presence... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2998221