Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-12
2002-12-03
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S029000, C435S243000, C435S320100, C435S325000, C435S410000, C435S254200, C435S254210, C536S023100, C536S023500, C536S024100
Reexamination Certificate
active
06489099
ABSTRACT:
The present invention relates to methods for detecting agents that cause or potentiate DNA damage and to molecules and transformed cells that may be usefully employed in such methods.
DNA damage is induced by a variety of agents such as ultraviolet light, X rays, free radicals, methylating agents and other mutagenic compounds. These agents may cause damage to the DNA that comprises the genetic code of an organism and cause mutations in genes. In microorganisms such mutations may lead to the evolution of new undesirable strains of the microorganism. For instance, antibiotic or herbicide resistant bacteria may arise. In animals these mutations can lead to carcinogenesis or may damage the gametes to give rise to congenital defects in offspring.
These DNA damaging agents may chemically modify the nucleotides that comprise DNA and may also break the phosphodiester bonds that link the nucleotides or disrupt association between bases (T-A or C-G). To counter the effect of these DNA damaging agents cells have evolved a number of mechanisms. For instance the SOS response in
E. coli
is a well characterised cellular response induced by DNA damage in which a series of proteins are expressed, including DNA repair enzymes, which repair the damaged DNA.
There are numerous circumstances when it is important to identify what agents may cause or potentiate DNA damage. It is particularly important to detect agents that cause DNA damage when assessing whether it is safe to expose a person to these agents. For instance a method of detecting these agents may be used as a mutagenesis assay for screening compounds that are candidate food additives, medicaments or cosmetics to assess whether or not the compound of interest induces DNA damage. Alternatively methods of detecting DNA damaging agents may be used to monitor for contamination of water supplies with pollutants that contain mutagenic compounds.
Various methods, such as the Ames Test, for determining the toxicity of an agent are known. More recent developments are disclosed in WO 95/00834 which relates to the use of a light emitting organism (particularly the bacterium
Photobacterium phosphoreum
) for measuring the toxicity of industrial effluents. WO 95/07463 discloses a gene construct formed from DNA encoding for Green Fluorescent Protein and DNA encoding for a regulatory element (such as a promoter induced by heavy metals) which may be used to detect pollution. However these developments do not disclose means of specifically monitoring for the presence of agents that may cause or potentiate DNA damage. Furthermore these known methods are not sensitive enough to detect agents that cause or potentiate DNA damage at low concentrations.
According to a first aspect of the present invention, there is provided a recombinant DNA molecule comprising a regulatory element that activates gene expression in response to DNA damage operatively linked to a DNA sequence that encodes a light emitting reporter protein.
According to a second aspect of the invention, there is provided a recombinant vector comprising a DNA molecule in accordance with the first aspect of the present invention and a DNA vector.
According to a third aspect of the invention, there is provided a cell containing a DNA molecule in accordance with the first aspect of the present invention or a recombinant vector in accordance with the second aspect of the present invention.
According to a fourth aspect of the present invention, there is provided a method of detecting for the presence of an agent that causes or potentiates DNA damage comprising subjecting a cell in accordance with the third aspect of the present invention to an agent and monitoring the expression of the light emitting reporter protein from the cell.
By “regulatory element” we mean a DNA sequence which regulates the transcription of a gene with which it is associated.
By “operatively linked” we mean that the regulatory element is able to induce the expression of the reporter protein.
By “reporter protein” we mean a protein which when expressed in response to the regulatory element of the DNA molecule of the invention is detectable by means of a suitable assay procedure.
The method of the fourth aspect of the invention is suitable for assessing whether or not an agent may cause DNA damage. It is particularly useful for detecting agents that cause DNA damage when assessing whether it is safe to expose a person to DNA damaging agents. For instance, the method may be used as a mutagenesis assay for screening whether or not known agents, such as candidate foodstuffs, medicarnents or cosmetics, induce DNA damage. Alternatively the method of the invention may be used to monitor for contamination of water supplies with pollutants containing DNA damaging agents.
The method of the fourth aspect of the invention may equally be used for assessing whether an agent may potentiate DNA damage. For example, certain agents can cause DNA damage by inhibiting DNA repair (for instance by preventing expression of a repair protein) without directly inflicting DNA damage. These agents are often known as co-mutagens and include agents such as lead.
The regulatory element of the DNA molecule of the first aspect of the invention activates expression of the reporter protein when DNA damage occurs. Such regulatory elements ideally comprise a promoter sequence which induces RNA polymerase to bind to the DNA molecule and start transcribing the DNA encoding for the reporter protein. The regulatory element may also comprise other functional DNA sequences such as translation initiation sequences for ribosome binding or DNA sequences that bind transcription factors which promote gene expression following DNA damage. Regulatory elements may even code for proteins which act to dislodge inhibitors of transcription from the regulated gene and thereby increase transcription of that gene.
Preferred regulatory elements are DNA sequences that are associated in nature with the regulation of the expression of DNA repair proteins. For instance, the regulatory elements from genes such as RAD2, RAD6, RAD7, RAD18, RAD23, RAD51, RAD54, CDC7, CDC8, CDC9, MAG1, PHR1, DIN1, DDR48, RNR1, RNR2, RNR3 and UB14 from yeast may be used to make molecules according to the first aspect of the invention. There are also regulatory elements associated with inducible excision repair genes in Neurospora, inducible recombinational repair genes in Ustilago and UV inducible irradiation damage recovery pathway genes in mammalian cells which may be used.
A preferred regulatory element comprises the promoter and 5′ regulatory sequences of the RAD54 repair gene. Such a regulatory element may be derived from yeast and particularly
Saccharomyces cerevisiae
. It is most preferred that the regulatory element comprises the promoter and 5′ regulatory sequences of the RAD54 repair gene which correspond to the DNA sequence identified as SEQ ID NO 1 or a functional analogue or fragment thereof.
Another preferred regulatory element comprises the promoter and 5′ regulatory sequences of the RNR2 gene. The RNR2 gene may be found on chromosome X of
Saccharomyces cerevisiae.
A preferred regulatory element may be derived from between co-ordinates 387100 and 398299 associated with the RNR2 gene on chromosome X as identified in the
Saccharomyces cerevisiae
genome database. The database may be accessed by the World Wide Web at many sites. For example at genome-www.stanford.edu.
The DNA sequences that encode a light emitting reporter protein may code for any light emitting protein, however it is preferred that the DNA sequences code for a protein that is fluorescent.
Preferred DNA sequences that encode a light emitting reporter protein code for Green Fluorescent Protein (GFP) and light emitting derivatives thereof. GFP is from the jelly fish
Aquorea Victoria
and is able to absorb blue light and re-emits an easily detectable green light and is thus suitable as a reporter protein. GFP may be advantageously used as a reporter protein because its measurement is simple and reagent free and t
Heyer Wolf Dietrich
Walmsley Richard Maurice
Gentronix Limited
Hogan & Hartson L.L.P.
McKelvey Terry
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