Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-24
2001-02-27
Elliott, George C. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S252800, C435S320100, C536S023100, C536S024100
Reexamination Certificate
active
06194159
ABSTRACT:
FIELD OF INVENTION
The invention relates to the discovery and characterization of regulated promoters aided by use of reporter gene fusions. More specifically the invention creates gene fusion comprising bioluminescent reporter genes in transformed hosts grown in liquid media to identify regulated promoters.
BACKGROUND OF THE INVENTION
Technological advances within the chemical arts supporting the agrochemical, pharmaceutical and environmental industries have made possible the synthesis of vast arrays of chemical compounds. The utility of these compounds is modeled on various structure-function relationships. Compound utility of often confirmed through screening methods designed to associate these compounds with desired known activities. Currently, methods of synthesis are capable of producing far more compounds than can be reasonably screened. A need exists for the development of rapid high-throughput screens that are able to analyze vast numbers of compounds for putative agrochemical, environmental, or pharmaceutical activities.
Current screening methods are often costly, time intensive, and lacking in specificity where the screens must rely on studies in whole plants and animals. A variety of methods have been developed using engineered microorganisms to detect and characterize compound activity. One such test is the Ames test (McCann et al.
Mutat. Res.
(1984), 134(1), 1-47) where Salmonella sp. are used to characterize compounds as mutagenic or pro-mutagenic. The Ames test relies on the unique enzymatic properties of
S. typhimurium
to characterize xenobiotics as mutagens or pro-mutagens. Other microorganism-based systems for the characterization of compounds have relied on the specificity of gene promoters or regulatory regions to identify potential compound activity. For example, Orser et al. (
In Vitro Toxicol.
(1995), 8(1):71-85) utilizes a stress promoter fused to a promoterless lacZ structural gene to screen compounds for environmental toxicity, Molders et al. (WO 9008836) teaches the use of recombinant bacteria to detect the presence of mercury using a gene complex consisting of a mer regulatory region that is hypersensitive to induction by mercury and Burlage et al. (
J. Bacteriol,
172 (9):4749-4757 (1990)) recites a method using a naphthalene sensitive regulatory region from plasmid NAH7 to detect naphthalene-like compounds.
Microbiological methods such as these hold great promise for the screening of compounds for specific activities; however they are impeded by the lack of a facile reporting system and by the difficulty in identifying regulatory regions specific for the activities to be screened for. A rapid method for the identification of new, compound sensitive regulatory regions combined with a facile reporting gene would represent a significance advance in the art of compound screens. Genes responsible for bacterial bioluminescence are gaining increasing interest as facile reporters and offer a partial solution to the development of rapid high-throughput screens for new compound activities.
Bioluminescent bacteria are found in marine and terrestrial environments. The lux gene products from marine organisms often exhibit thermolability such that they do not function well at typical growth temperatures for other bacteria (Szittner and Meighen,
J. Biol. Chem.,
265:16581-16587 (1990); Rupani et al.,
Biotechnol Prog,
12:387-392 (1996); Hill et al.,
Biotechnol Appl Biochem,
17:3-14 (1993)). In contrast, the lux gene products from the terrestrial microorganism
Photorhabdus luminescens
(formerly called
Xenorhabdus luminescens
) are stable at temperatures up to 45° C. (Szittner and Meighen,
J. Biol. Chem.,
265:16581-16587 (1990)). Therefore, the advantage of the thermostable lux genes is that a larger range of assay temperatures are available for use.
Recent advances in recombinant DNA technology have made it possible to express the luciferase (lux) gene complex as heterologous gene products. This is generally accomplished by placing the lux structural gene complex under the control of a host promoter. So, for example, cDNA encoding firefly luciferase has been expressed in
E. coli
under the control of the lacZ promoter. (Tatsumi et al.,
Biochem. Biophys Acta.,
1131, 2:161-165, (1992)), and the luxAB fusion gene has been expressed in Bacillus at levels comparable to those achievable in
E. coli
by placing it under the control of the powerful Pxyn promoter (Jacobs et al.,
Mol. Gen. Genet.,
230(1-2):251-256, (1991)).
Stress genes are found in all cells and are defined as those genes activated as a result of any type of insults that might alter the normal cellular metabolism. Environmental insults often induce synthesis of an overlapping set of proteins. The most well recognized class of stress genes are the heat shock genes encoding a set of cellular proteins thought to have roles in refolding, recycling and resynthesis of proteins. The heat shock phenomenon was first described as a response to an increased temperature. Subsequent work has shown that exposure to a variety of insults including phage infection, macrophage envelopment, as well as the presence of organic molecules and heavy metals can also trigger the heat shock response. The common theme of the inducing agents may be unfolding of some proteins within the cell. (LaRossa et al.,
Mol. Micriobiol.,
5(3):529-523, (1991)). Thus the response may integrate and report a wide range of environmental insults. VanBogelen et al., (
J. Bacteriol.,
169(1):26-32, (1987)) have demonstrated that a variety of chemicals are able to induce the heat shock genes in
E. coli
, including CdCl
2
, H
2
O
2
, ethanol, puromycin and nalidixic acid. Blom et al., (
Appl. Environ. Microbiol.,
58(1):331-334, (1992)) teach that the exposure of
E. coli
cultures to benzene, CdCl
2
, chlorpyrivos, 2,4-dichloraniline, dioctylphtalate, hexachlorobenzene, pentachlorophenol, trichlorethylene, and tetrapropylbenzosulfonate leads to the induction of up to 39 different stress proteins, as analyzed by two dimensional gel electrophoresis. LaRossa et al. (PCT International Application WO 94/13831) have transformed
E. coli
with a construct comprised of luxCDABE operably linked to a variety of stress promoters. They have used the microorganisms to detect a variety of environmental insults such as ethanol, CdCl
2
, and toluene. The presence of a sublethal concentration of the insult is indicated by an increase in bioluminescence. The detector organism described in WO 94/13831 was also used in a lyophilized form to detect similar environmental stresses by an increase in bioluminescence (Van Dyk, T. and Wagner, W., International Application No. PCT/US 95/15224.
Since the cell attempts to maintain a steady state, stress responses are activated well below the minimal inhibitory concentration for any condition that serves as a triggering factor. It would be useful to identify complete sets of promoters induced by any particular stress.
Thus, genes responsible for bacterial bioluminescence offer a partial solution to the need for a microorganism-based screening method for compound activity. Still needed, however, is a rapid method for the identification of useful regulatory regions.
Various methods of screening for bacterial promoter activity and for regulatory regions affected by various conditions are known. It is very common to use reporter genes for such a task. For example, transposons which can be inserted throughout the genome can be engineered to have reporter genes that require an external promoter sequence to be expressed and hence activity of the reporter gene is indicative of transcription initiated at the upstream chromosomal promoter sequences. A classic example of this approach is the work of Kenyon and Walker in discovering genes induced by DNA damage (Kenyon and Walker,
Proc. Natl. Acad. Sci. U.S.A.,
77:2819-2823 (1980)). Many such transposons are available and have been recently reviewed (Berg and Berg,
Transposable element tools for microbial genetics, in Escherichia coli and Salmonella Cellular and Molecula
LaRossa Robert Alan
Van Dyk Tina Kangas
E. I. Du Pont de Nemours and Company
Elliott George C.
Sandals William
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