Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-04-25
2003-08-05
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S252300, C435S252330, C536S023100, C536S024100
Reexamination Certificate
active
06602666
ABSTRACT:
There are at present various screening methods for genotoxic and/or toxic compounds using microorganisms. The Ames test (Maron and Ames, 1983) appears to be the most widely used test for toxic compounds and is as such recommended worldwide. It however takes about three days for completion of the test with the obvious concomitant disadvantages.
Some short term methods have been introduced wherein the SOS response caused by DNA damage is measured as an amount of &bgr;-galactosidase expressed by the lacZ gene positioned downstream of the umuD,C or sfiA stress induced promoters that are SOS regulated. These tests are known respectively as the umu test (Oda et al. 1985) using
Salmonella typhimurium
as host microorganism and SOS chromotest (Quillardat et al., 1982) using
E. coli
as host microorganism. In the umu test and SOS chromotest the host microorganism is cultured in the presence of the sample to be tested and subsequently the host microorganism is disrupted. A substrate for &bgr;-galactosidase is then added and the ensuing reaction is terminated after some 10 minutes by addition of an inhibitor followed by OD measurement at two wave lengths and calculation or &bgr;-galactosidase activity. These tests overcome the long timespan problem of the Ames test, however, have their own disadvantages. The sensitivity is low and detection times are still a lengthy 7-8 hours. In particular the detection sensitivity of nitroarenes and polycyclic aromatic hydrocarbons is low. In addition the detection method requires a large number of actions and additions of various reagents thereby rendering the method complicated and expensive. Due to the fact that the cell has to be disrupted in order to carry out detection of any induction it is only possible to carry out one measurement on the cell.
EP-A-0.649.905 discloses that some of the disadvantages of the aforementioned tests can be overcome by placing an SOS gene upstream of a gene expressing luciferase activity such that luciferase expression occurs simultaneously with expression of the SOS gene. Subsequently a mutagenic substance can be detected or measured in a short time by measuring the luminescence. Any SOS gene is stated as being useful and the umuD,C gene (as used in the umu test) is illustrated in the examples. The sensitivity of this test is stated to be increased in comparison to that of the conventional tests i.e. the SOS chromotest and the umu test because of the smaller sample volume required for detection measurement. No relevance is attached to the promoter to be used other than the fact it must be SOS inducible. As luminescence production is immediate the measurement can occur earlier than with the lac system thereby shortening the detection time.
In WO 94/13831 DuPont also disclose the use of stress inducible promoters in combination with a luminescence gene complex to provide a genetically engineered microorganism. They state “although stress responses have been demonstrated to be useful in detecting the presence of various environmental insults it has yet to be linked to a sensitive easily detected reporter”. DuPont provides an extensive list of stress inducible promoters known in the state of the art that could be useful according to them but to which the invention is not restricted. This list comprises promoters from the following regulatory circuits: heat shock, SOS, hydrogen peroxide, superoxide, fatty acid starvation, universal stress, resting state, stringent, catabolite activation, P utilisation and N utilisation. In the Examples they use the heat shock regulated protein promoters dnaK and grpE, the SOS regulated promoters recA and uvrA, the oxidative damage regulated promoters katG and micF, the universal stress promoter uspA, the stationary phase promoter xthA, the his promoter from the amino acid starvation circuit, the lac promoter involved with the carbon starvation circuit, the phoA promoter from the phosphate limitation circuit and the glnA promoter from the nitrogen limitation circuit. This large number of examples from such a broad range of differently regulated promoters presumably serves to illustrate the broad applicability of their system. No preference is expressed or deducible for any particular group of promoters or any individual promoter. The only explicit reference to a specific SOS regulated promoter occurs in one of the examples (example 12 of the patent application) in which results with the SOS regulated promoter recA are presented. After exposing the microorganism to samples with the mutagen ethidium bromide at a concentration of 0.25 mg/ml an induction ratio of 1.9 was measured in one measurement after 180 minutes of the addition of mutagen. After addition of 0.5 &mgr;g/ml mitomycin C, the induction ratio's were measured after 100 minutes. Depending on the test strain used they varied between 4.7 to 20. This is apparent from Example 12 of the DuPont application.
Unexpectedly we have found a subgroup of stress induced promoters, in fact a subgroup of SOS regulated promoters which in combination with a luminescence reporter can be used in a microorganism system for assessing mutagenicity with improved results. This subgroup does not include the SOS regulated RecA promoter. This subgroup offers a number of advantages over the DuPont RecA-luciferase system and the other microbial toxicity testing sytems of the state of the art. In further testing and developing these new systems by mutating the promoters in a specific manner we were able to improve performance even more. In addition novel methods of testing were carried out. These novel methods of testing result in obtaining more and better data hitherto not described for any of the existing microbial toxicity tests.
The subject invention is directed at a recombinant nucleic acid sequence comprising an SOS regulated promoter with an induction ratio higher than 40, said promoter being operatively linked to a reporter encoding nucleic acid sequence encoding a reporter resulting in a signal that can be assayed as light production. Preferably a promoter with an even higher induction ratio, preferably higher than 50 is present in the recombinant nucleic acid sequence according to the invention. The induction ratio can e.g. be determined as disclosed by M. Schnurr et al. in Biochimie (1991) 73, 423-431. Alternatively the method disclosed by Peterson K. R. and Mount D. W. (1987), J. Mol. Biol. 193, 27-40 can be used. The disclosures are hereby incorporated by reference. The recA promoter disclosed in the state of the art in combination with luciferase does not fall within this category as the induction ratio of recA is appreciably lower. It is 11× at 30° C. which is the incubation temperature of the test. The induction ratio is an art recognised term and can be ascertained in a manner known per se for a person skilled in the art. In the literature Examples are given of how the induction ratio can be and has been ascertained together with numerical values for a number of promoters. The low induction ratio of recA is probably the reason why it is not particularly suited for a sensitive detection system based on promoter induction. The RecA promoter is the earliest promoter to be induced in the SOS regulated system. The signal(s) generated by metabolic defect(s) is (are) sensed by RecA protein. It also purportedly has a second function in mutagenesis in assisting DNA polymerase to bypass lesions. During the first twenty minutes after DNA damage the uvrA,B,C,D genes are activated to commence with excision repair. Then the recombinatorial repair pathway known as the RecF recombination pathway is very active during approximately 40 minutes. Finally, the SOS mutagenesis pathway involving umu D,C is induced.
The lowest level of mytomycin C that was used in the dupont system as illustrated in the above cited patent application was 500 ng/ml. No indication or suggestion of detection of lower levels is given. In the subject systems 7 ng/ml of mytomycin can be detected. This system is thus approximately 80 times more sensitive than the DuPont system
Borremans Brigitte Marie Francoise
Provoost Ann Irma Alice
Regniers Luc Agnes Louis Jean Bosco
Van Der Lelie Daniel
Verschaeve Luc Philipinne Edouard
Ketter James
Merchant & Gould P.C.
Vlaamse Instelling Technologisch Onderzoek (V.I.T.O.)
LandOfFree
Recombinant nucleic acid sequences and methods for... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Recombinant nucleic acid sequences and methods for..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Recombinant nucleic acid sequences and methods for... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3085183