Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-26
2003-12-23
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06667153
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and compositions for detecting a mutagen. The compositions include a DNA construct, an expression vector, and a host cell including a mutagen sensitive gene operably linked to a fluorescent protein. The method includes exposing a host cell including a mutagen sensitive gene operably linked to a fluorescent protein and monitoring the fluorescent protein.
BACKGROUND OF THE INVENTION
The current increased awareness of environmental contamination by diverse classes of chemicals has led to increasing concern about the impact of this contamination on human health. Some of these environmental contaminants may be toxic and/or carcinogenic, which emphasizes the need for rapid and inexpensive screening methods for the hazards of environmental contaminants. Many existing tests for carcinogens are based on animal models or tissue culture techniques, which are both time-consuming and costly. These animal and tissue culture tests can be supplemented with simpler tests on bacterial cell cultures. However, the most widespread bacterial mutagenicity the test, the Ames test, requires repeated culturing of many cell samples, lengthy incubation, and tedious analysis. These consume time, money, and reagents. There exists a need for simpler and more robust bacterial or cell culture tests for mutagens.
SUMMARY OF THE INVENTION
The present invention relates to methods and compositions for detecting a mutagen. The compositions include a DNA construct, an expression vector, and a host cell each including a mutagen sensitive gene operably linked to a fluorescent protein. The method includes exposing a host cell including a mutagen sensitive gene operably linked to a fluorescent protein and monitoring the fluorescent protein. The present invention includes a DNA construct including a mutagen sensitive gene operably linked to the coding sequence for a fluorescent protein. In a preferred embodiment, the mutagen sensitive gene includes an SOS gene. The SOS gene can include one or more promoters or coding sequences whose expression is induced or up regulated in a cellular response to a mutagen. A preferred SOS gene is derived from
E. coli
or
S. typhimurium
and includes a umuC gene, a umuD gene, a control sequence for either of these genes, or a combination thereof. The DNA construct of the invention can include the coding sequence for any of a variety of fluorescent proteins. Preferably, the fluorescent protein is a green fluorescent protein, preferably from the jellyfish
Aequorea victoria
. The coding sequence for the fluorescent protein can encode a variant green fluorescent protein. The mutagen sensitive gene and/or the fluorescent protein can be a naturally occurring or variant gene or protein. The expression construct can encode a protein of the invention, a polypeptide including an amino acid sequence of a UmuD protein, a UmuC protein, or a combination thereof and an amino acid sequence of a fluorescent protein.
The invention also includes an expression vector including a mutagen sensitive gene operably linked to a coding sequence for a fluorescent protein. The expression vector can include the DNA construct of the invention. Preferably, the expression vector includes an SOS gene, or variant thereof, derived from plasmid pSE 117 and a coding sequence for a fluorescent protein. A preferred plasmid expression vector is pTJgfp.
In another embodiment, the invention includes a host cell including a mutagen sensitive gene operably linked to a coding sequence for a fluorescent protein. Preferred host cells include
S. typhimurium
and
E. coli
. The host cell can include a DNA construct or expression vector of the invention. Alternatively, the host cell can have its genomic DNA altered to include a heterologous mutagen sensitive gene operably linked to a homologous coding sequence for a fluorescent protein or to include a heterologous coding sequence for a fluorescent protein operably linked to a homologous mutation sensitive gene.
In one embodiment, the invention includes a method of determining or detecting a mutagen. The method includes contacting a test compound with a host cell of the invention, monitoring the host cell for the fluorescent protein, and, when an amount or distribution of the fluorescent protein meets or exceeds a predetermined threshold value, determining that the test compound is a mutagen. The method can employ a host cell either at stationary phase or at logarithmic phase. Contact with the test compound can deplete a nutrient, or even starve the host cell, and the method will still robustly detect a mutagen. Preferably, when contacting depletes a nutrient or starves a cell, the cells are in a logarithmic phase. Monitoring fluorescent protein typically includes detecting fluorescence, such as with a fluorescence detector that reads a microtiter plate. The mutagen can be detected through increases in the level or output of fluorescence compared to a control sample. Alternatively, the mutagen can be determined or detected by statistically analyzing the amount of green fluorescent protein in replicate wells. The statistical analysis can include analysis of the location, shape, or distribution of fluorescent output, cells, or other data.
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Merchant & Gould P.C.
Thomas Susan Margaret
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