ABSTRACT
A recently derived in vitro chemiluminescence assay (Salles et al. [1995] Anal. Biochem., 232, 37-42) has been used to investigate the effects of a panel of twenty-two anticancer drugs and certain antibiotics on the excision repair activity of cell-free extracts from the human cell line, HeLa. This methodology, termed the 3D (Damaged DNA Detection) assay, based on the in vitro excision repair assay previously developed (Wood et al. [1988] Cell, 53, 97-106) has provided data indicating definite in vitro inhibition of DNA repair by actinomycin D, aphidicolin, doxorubicin, distamycin A and mithramycin A. This assay therefore offers the potential for identifying agents with the ability to inhibit DNA repair.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/pharmacology , DNA Repair/drug effects , DNA, Bacterial/drug effects , DNA, Bacterial/radiation effects , Dose-Response Relationship, Drug , HeLa Cells , Humans , Luminescent Measurements , Methods , Plasmids , Ultraviolet RaysABSTRACT
A damaged DNA detection assay (3D assay) using plasmid DNA adsorbed on sensitized microplates as the substrate for an in vitro repair reaction is presented. DNA lesions are repaired by the excision repair pathway which implies an incision-excision reaction followed by DNA repair synthesis. In the 3D assay, we took advantage of (i) plasmid DNA adsorption on polylysine-coated microplates that allowed various DNA-damaging treatments; (ii) a protein extract that reproduced the repair reaction in vitro; (iii) incorporation of digoxigenylated deoxynucleotide monophosphate during the DNA polymerization step which was quantified by a chemiluminescent reaction. Under experimental conditions for quantitative DNA adsorption, a dose-response relationship between the extent of DNA modification and the repair synthesis activity was found. Optimization of the biochemical parameters with UVC light-induced DNA lesions allowed the detection of about one photoproduct per plasmid circle. This new assay that permits a quick and easy assessment of DNA damage is applicable to the screening of genotoxic compounds and to the testing of DNA-damaging treatments.
Subject(s)
DNA Damage , DNA/analysis , Luminescent Measurements , Cell Extracts , DNA/drug effects , DNA/metabolism , DNA/radiation effects , DNA Repair , Deoxyribonucleotides/metabolism , HeLa Cells , Humans , Kinetics , Methyl Methanesulfonate/toxicity , Methylnitrosourea/toxicity , Mutagens/toxicity , Plasmids , Polylysine/metabolism , Ultraviolet RaysABSTRACT
The 9149-bp transcription unit encoding ovine beta-casein (Cas) and 4636 bp of 5' flanking region were completely sequenced. The gene is composed of nine exons and its overall organization is similar to that of its counterparts from other species. Intron 4, the largest, shares three similar stretches (sizes ranging from 0.1 to 0.3 kb) with the region upstream from the transcription unit. These common sequences are part of short interspersed nuclear elements (SINE) specific to Bovidae (Bov). Intron 4 contains two 274-bp Bov-A2 SINE in opposite orientation, as well as a full-length 569-bp Bov-B SINE. This latter SINE, also present in caprine intron 4, is missing in cattle. This suggests that the amplification of Bov-SINE has continued after the divergence of cattle from sheep and goats, assuming that the presently known sequences are representative of these species.
Subject(s)
Caseins/genetics , Sheep/genetics , Amino Acid Sequence , Animals , Base Sequence , Cattle , Conserved Sequence , DNA/genetics , DNA, Satellite , Exons , Goats , Introns , Molecular Sequence Data , Phylogeny , Repetitive Sequences, Nucleic Acid , Sequence Analysis, DNA , Species Specificity , Transcription, GeneticABSTRACT
Great progress is being made in understanding the process of nucleotide excision repair (NER) in eukaryotes. Different lines of research have been developed, among them an in vitro assay with cell-free extracts has played a major role. This in vitro repair assay takes advantage of a cell-free system that can mediate DNA excision-repair by transcriptionally active protein extracts from mammalian cells incubated in the presence of two plasmids of different sizes, one damaged and the other undamaged as internal control. The extent of repair activity is determined by following the level of radiolabeled incorporation during the repair synthesis step consecutive to the excision of DNA lesions. We discuss the interest and drawbacks of this biochemical assay in light of the main results obtained. We report the modifications that we have undertaken in order to determine repair synthesis activity in a chemiluminescent-directed reaction as well as to assess incision activity in protein extracts.
Subject(s)
DNA Repair/genetics , DNA/genetics , Eukaryotic Cells/physiology , Animals , Genetic Techniques , Humans , Isotope Labeling , Luminescent MeasurementsABSTRACT
Restriction fragment length polymorphism (RFLP) of ovine casein genes was investigated. Genomic DNA from 56 rams was digested with 10 restriction endonucleases and Southern blots probed with the four ovine casein cDNAs (alpha s1-, beta-, alpha s2- and kappa-Cn). Five enzymes, namely, BglI, PvuII, RsaI, TaqI and HindIII revealed nine different RFLPs. The inheritance of six of these polymorphisms was studied by segregation analysis of gametes in nine rams' families, and each of them could be related to the existence of alleles at the relevant casein locus. A close linkage between the four ovine casein genes was demonstrated since no recombination within the four pairs of loci examined, alpha s1-beta-Cn, alpha s1-kappa-Cn, beta-kappa-Cn and alpha s2-kappa-Cn, was observed in the progeny of double heterozygous rams. The casein genes are thus clustered in the ovine species as in the case of other mammals.
Subject(s)
Caseins/genetics , Genetic Linkage/genetics , Polymorphism, Restriction Fragment Length , Sheep/genetics , Alleles , Animals , Blotting, Southern , Crosses, Genetic , DNA Probes , Endonucleases , Heterozygote , Male , Multigene Family , Pedigree , Recombination, Genetic/geneticsABSTRACT
Bovine and ovine (pseudo)genes homologous to the alpha-lactalbumin-encoding gene are described. In both cases, sequence analysis reveals homology extending downstream from exon 2. Southern analysis indicates the presence of a family of alpha-lactalbumin-related sequences in the bovine genome.
Subject(s)
Genes , Genomic Library , Lactalbumin/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Cattle , Cloning, Molecular/methods , Molecular Sequence Data , Restriction Mapping , Sequence Homology, Nucleic AcidABSTRACT
The complete nucleotide sequence of ovine beta-casein mRNA has been determined by sequencing, according to Sanger-Messing, both a recombinant clone isolated from a mammary cDNA pUC 18 library and a single-stranded cDNA generated by reverse transcription from a synthetic 17-mer primer complementary to the 5' part of the mRNA coding frame. The 1088 nucleotide long beta-casein mRNA, excluding the poly(A) tail, contains a coding frame of 669 nucleotides including the stop codon, flanked by 60 and 359 nucleotides in the 5' and 3' untranslated regions, respectively. It arises from the splicing of 9 exons as deduced from gene sequence data. The deduced amino acid sequence differs at 3 positions from that previously determined by direct sequencing of mature beta-casein. Comparison of the ovine, bovine, rat, mouse, and rabbit beta-casein mRNA sequences shows a higher homology in the 3' and 5' untranslated regions. The most conserved regions in the open reading frame are essentially those encoding the signal peptide and the major phosphorylation site.
