Identification of DNA-damaging substances by means of cell lines

Details Identification of DNA-damaging substances by means of cell lines Identification of DNA-damaging substances by means of cell lines

1996-07-24

1999-09-14

Guzo, David

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 71, 435 29, 435 405, 4353201, 435325, 435352, 435455, 536 235, C12Q 168, C12Q 102, C12N 510, C07H 2104

Patent

active

059521694

DESCRIPTION:

BRIEF SUMMARY
TABLE OF CONTENTS



I. FIELD OF THE INVENTION

The present invention relates to a DNA construct containing a cDNA sequence for human poly(ADP-ribose)-polymerase, cell lines containing the DNA construct and a process for identifying DNA damaging substances by means of these cell lines which overexpress poly(ADP-ribose)-polymerase.


II. BACKGROUND OF THE INVENTION

So far, DNA-damaging substances (physical and chemical carcinogens) which cause the breakage of DNA strands have been identified by measuring the strand breakage either in cell lysates by alkaline DNA denaturation methods (e.g., "alkaline elution") or in situ by means of individual cells (what is called "comet assay"). However, the lysate tests included the drawback that they were technically relatively complicated and required the experimenter's great manual skill. In "comet assays" the reading is also carried out by means of a microscope, which calls for the employment of a complicated apparatus.
Therefore, the object underlying the present invention is to provide a process for rapidly and reliably identifying chemical carcinogens.


III. SUMMARY OF THE INVENTION

The present invention relates to a DNA construct containing a cDNA sequence for human poly(ADP-ribose)-polymerase, cell lines containing the DNA construct, and a process for identifying DNA-damaging substances by means of these cell lines which overexpress poly(ADP-ribose)-polymerase.


IV. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the eukaryotic expression vector pL15TK.
FIG. 2 depicts the cloning diagram for pPARP31.
FIG. 3 depicts the genetic map of the plasmid pPARP31.


V. BRIEF DESCRIPTION OF THE INVENTION

The principle of the present invention is based on the detection of the cellular poly(ADP-ribose) production as indicator for breakages of DNA strands occurring in living cells. Poly(ADP-ribose) is synthesized by poly(ADP-ribose)-polymerase (PARP) in the presence of DNA strand breakages and by consuming NAD.sup.+. This happens under the influence of carcinogenic substances in almost every nucleated cell. However, in such a cell very much poly(ADP-ribose) is formed by manipulation, i.e., by inserting a PARP-DNA sequence in a predetermined combination with a promoter, and can be detected by suitable methods, e.g., immunofluorescence.
An example of a construct containing the PARP-DNA sequence is the plasmid pPARP31, deposited with the Deutsche Sammlung von Microorganismen und Zellkulturen (German Type Collection of Microorganisms and Cell Cultures) (DSMZ) at Mascheroder Weg 1b, D-38124 Braunschweig, Germany, under DSMZ Accession Number DSM 12290 on Jun. 29, 1998.
pPARP31 can be obtained by the following steps: poly(ADP-ribose)-polymerase from human embryonal fibroblasts (HEF cells) into pBluescript, thereby obtaining the plasmid pPARP25 (Kupper, doctoral thesis, University of Heidelberg, 1990). The full open reading frame of one of the known PARP-cDNAs is published in the colony bank/EMBL data bank, access number J 03473 (Kurosaki), for example. bp HincII/AvaII fragment of the human cytomegalovirus-promoter/enhancer (HCMV Prom) which has blunt ends, is ligated into the blunt EcoRI site of pUC19 so as to obtain the "intermediate vector" pL15. Then, the 629 bp SmaI/HindIII fragment of the poly-adenylation signal of the herpes simplex virus thymidine kinase gene (TK poly A) is ligated into the blunt SphI site and the HindIII site of pL15 so as to obtain the vector pL15TK. The vector pL15TK contains the resistance gene for ampicillin (Amp), a bacterial replication origin (ori), as well as portions of the Lac operon (Z and I). The polylinker cassette of pUC19 is intact except for the EcoRI and SphI sites. shown in FIG. 2), thereby obtaining the plasmid pPARP31 (see FIG. 3), the XhoI site of the insert and the BamHI site of the vector being destroyed. The XbaI sites of vector and insert remain intact.
For the production of cell lines, the embryonal hamster cell line CO60 (Lavi, 1981 PNAS 78:6144) is stably transfected with a plasmid expressing a poly(ADP-ribose)-polymerase, p

REFERENCES:
Alkhatib et al., 1987 " Cloning and Expression of cDNA For Human Poly(ADP-Ribose) Polymerase,"Proc. Natl. Acad. Sci. U.S.A. 84:1224-1228.
Bhatia et al., 1990, "Expression of The Poly(ADP-Ribose) Polymerase Gene Following Natural And Induced DNA Strand Breakage And Effect of Hyperexpression On DNA Repair," Carcinogenesis 1:123-128.
Burkle et al., 1992, "Poly(ADP-Ribosyl)atiobin: Its Role In Inducible DNA Amplification, And Its Correlation With The Longevity of Mammalian Species," Exp. Clin. Immunogenet. 9:230-240.
Cherney et al., 1987, "cDNA Sequence, Protein Structure And Chromosonal Location Of The Human Gene For Poly(ADP-Ribose)Polymerase," Proc. Natl. Acad. Sci. U.S.A. 84:8370-8374.
Fritz et al., 1994, "Effects of Transfection Of Human Poly(ADP-Ribose) Polymerase In Chinses Hamster Cells On Mutagen Resistance," Mutation Research 308:127-133.
Kawamitsu et al., 1984, "Monoclonal Antibodies to Poly(adenosine diphosphate ribose) Recognize Different Structures," Biochemistry 23:3771.
Lavi, 1981, "Carcinogen-mediated Amplification of Viral DNA Sequences in Simian Virus 40-Transformed Chinese Hamster Embryo Cells," PNAS 78:6144.
Suzuki et al., 1987, "Molecular Cloning of cDNA For Human Poly(ADP-Ribose) Polymerase and Expression Of Its Gene During HL-60 Cell Differentiation," Biochem. Biophys. Res. Commun. 146:403-409.
Uchida et al., 1987, "Nucleotide Sequence Of A Full-Length cDNA For Human Fibroblast Poly(ADP-Ribose) Polymerase," Biochem. Biophys. Res. Commun. 148:617-622.

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