Search

Irradiation of cells with UVC light induces two types of mutagenic DNA photoproducts, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). To investigate the relationship between the frequency of UV-induced photolesions at specific sites and their ability to induce mutations, we quantified CPD formation at the nucleotide level along exons 3 and 8 of the hprt gene using ligation-mediated PCR, and determined the mutational spectrum of 132 UV-induced hprt mutants in the AA8 hamster cell line and of 165 mutants in its nucleotide excision repair-defective derivative UV5. In AA8 cells, transversions predominated with a strong strand bias towards thymine-containing photolesions in the non-transcribed strand. As hamster AA8 cells are proficient in global genome repair of 6-4PP but selectively repair CPD from the transcribed strand of active genes, most mutations probably resulted from erroneous bypass of CPD in the non-transcribed strand. However, the relative incidence of CPD and the positions where mutations most frequently arose do not correlate. In fact some major damage sites hardly gave rise to the formation of mutations. In the repair-defective UV5 cells, mutations were almost exclusively C > T transitions caused by photoproducts at PyC sites in the transcribed strand. Even though CPD were formed at high frequencies at some TT sites in UV5, these photoproducts did not contribute to mutation induction at all. We conclude that, even in the absence of repair, large variations in the level of induction of CPD at different sites throughout the two exons do not correspond to frequencies of mutation induction.

Accumulation of damage in undifferentiated cells may threaten homeostasis and regenerative capacity. Remarkably, p53 has been suggested to be transcriptionally inactive in these cells. To gain insight in the kinetics and interplay of the predominant transcriptional responses of DNA damage signalling pathways in undifferentiated cells, mouse embryonic stem cells were exposed to cisplatin at four different time points (2, 4, 8 and 24h) and concentrations (1, 2, 5 and 10 microM). RNA was isolated and subjected to genome-wide expression profiling. Up to one fourth of the tested genes could be identified as being differentially expressed (false discovery rate=10%) after the cisplatin treatment. Clustering of the expression changes showed a strong time dependency. To investigate the relationship between affected genes, a gene set analysis method was used. Functionally related gene sets were defined using gene ontologies or transcription factor binding sites and were tested for overrepresentation within the differentially expressed genes. A variety of gene sets were clearly enriched among which 'apoptosis' and 'cell cycle' were the most pronounced. Furthermore, there was a strong enrichment of genes with a p53-binding motif. The involvement of the 'cell cycle' and 'apoptosis' gene sets in the cisplatin response was detected at concentrations and time points where the respective biological assays were still negative. The results reveal novel insights into the mechanisms which maintain the genomic integrity in undifferentiated cells. Additionally the results illustrate that gene set analysis of genome-wide expression changes provides a sensitive instrument to detect cellular stress responses to DNA damage.

Loss of heterozygosity (LOH) contributes significantly to the inactivation of tumor suppressor genes and may involve a variety of mechanisms. Studying loss of HLA-A2 alleles in human lymphoblastoid cell lines, we previously showed that mitotic recombination and chromosome loss with concomitant duplication of the non-selected chromosome were the most frequent mechanisms of LOH. In the present study we used the HLA system to determine the rate and spectrum of LOH mutations in the EBV transformed lymphoblastoid cell line R83-4915. Spontaneous loss of HLA-A2 in R83-4915 occurred with a rate of 7.9×10−7 which was 5 to 10-times lower compared to the previously observed rate of loss of HLA-A2 in other lymphoblastoid cell lines. Among the HLA-A2 mutants, 27% did not show LOH of additional chromosome 6 markers. Molecular analysis showed that neither large deletion nor gene conversion was the cause for their mutant phenotype. The remaining mutants showed LOH, which was caused by mitotic recombination (40%) and chromosome loss (33%). However, the chromosome loss observed in mutants of R83-4915 was not accompanied by the duplication of the remaining chromosome. Instead 3 out of 5 mutants became polyploid suggesting that different mechanisms exist to compensate for chromosome loss. In conclusion, the rate and types of LOH that can be observed in cell lines obtained from various donors may depend on the genetic make-up or the transformation status of these cells

Mutation spectra at the nucleotide sequence level of five hprt cDNA genes integrated in different genomic positions of a HPRT(-) derivative of the human lymphoblastoid TK6 cell line were compared with each other and with the spectrum of mutations confined to the 657 bp coding region of the endogenous hprt gene in the parental TK6 cells. The mutation rates in these genomic positions vary significantly and also the mutation spectra are different. In each genomic position the majority of mutations are basepair substitutions and deletions. the ratios of which vary among the genomic positions. Although it is likely that the different rates of deletion are to a large extent the net result of different rates of misalignment and repair of these errors in the various genomic positions, for the basepair substitutions it is not possible to deduce which mechanisms have caused these mutations and what causes the differences among the genomic positions. Taken together, the differences in mutation rates and spectra cannot be explained by a single mutagenic process.

The polypeptides encoded by the early region of the Mu genome (comprising approximately 8000 base-pairs of DNA extending from the immunity encoding end of the linear Mu genome) have been determined. Minicells were infected by wildtype, suppressor-sensitive-mutation-carrying Mu phages and by λpMu hybrid phages. Comparison of the polypeptides synthesized in these infected minicells with polypeptides synthesized in minicells containing recombinant plasmids, which carry the first 5100 base-pairs of the early region of Mu DNA, have permitted a definitive correlation of the region of DNA with the encoded gene product. The Mu represser protein, gpc (Mr, 26,000), is encoded by approximately 900 base-pairs of DNA extending from the c-end of the genome † . The region 1300 to 4300 base-pairs encodes gpA (Mr, 70,000) and gpB (Mr, 33,000) and the region 4300 to 5100 basepairs encodes gpcim (Mr, 7000) and gpkil (Mr, 8000). A 14,000 Mr polypeptide, which is probably gpgam, is located in the region 4800 to 5400 base-pairs, and three polypeptides of molecular weights 20,500, 19,000 to 22,000 and 11,500 are located in the region 5200 to 6600 base-pairs. One of these polypeptides is gpsot. The basis for the apparent range of molecular weights (19,000 to 22,000) exhibited by one of the polypeptides encoded in this region is unknown. The region 6200 to 8600 base-pairs encodes three polypeptides (Mr = 10,000, 10,000 and 8000) of unknown function. The polypeptides identified as early polypeptides from their location on the Mu genome are also those polypeptides detected earliest after infection of minicells and are the major translation products of Mu RNA synthesized in minicells infected in the presence of chloramphenicol. These early polypeptides, assuming no overlapping genes, require the expression of approximately 80% of the coding capacity of the early region of the Mu genome.

This paper describes the identification and functional role of late gene products of bacteriophage Mu, including an analysis of the structural proteins of the Mu virion. In vitro reconstitution of infectious phage particles has shown that four genes ( E , D , I , J ) control the formation of phage heads and that a cluster of eight genes ( K , L , M , N , P , Q , R , S ) controls the formation of phage tails. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of Mu polypeptides synthesized in Escherichia coli minicells infected by Mu phages carrying amber mutations in various late genes has resulted in the identification of the products of gene C (15.5 × 10 3 M r ); H (64 × 10 3 M r ); F (54 × 10 3 M r ); G (16 × 10 3 M r ); L (55 × 10 3 M r ); N (60 × 10 3 M r ); P (43 × 10 3 M r ) and S (56 × 10 3 M r ). Minicells infected with λpMu hybrid phages and deletion mutants of Mu were used to identify polypeptides encoded by the V-β region of the Mu genome. These are the products of genes V , W or R (41.5 × 10 3 M r , and 45 × 10 3 M r ); U (20.5 × 10 3 M r ) and of genes located in the β region (24 × 10 3 M r (gp gin ) and 37 × 10 3 M r (possibly gp mom )). Analytical separation of the proteins of the Mu virion revealed that it consists of a major head polypeptide with a molecular weight of 33 × 10 3 , a second head polypeptide of 54 × 10 3 (gp F ) and two major tail polypeptides with molecular weights of 55 × 10 3 and 12.5 × 10 3 (gp L and gp Y , respectively). In addition, there are five minor components of the tail (including gp N , gp S and gp U ) and approximately seven minor components of the head structure of the virion (including gp H ).

A method is described, which allows identification of early gene products of bacteriophage Mu. This method is based on ultraviolet irradiation of host cells prior to bacteriophage infection. It appears that only early genes of Mu are expressed in u. v. -irradiated cells. Two polypeptides with molecular weights of 23,000 and 35,000, as measured on sodium dodecylsulphate/polyacrylamide gels, were identified as Mu products, both after infection with Mu and after infection with a λdMu transducing phage. From the results obtained with various amber mutants we conclude that the polypeptide with a molecular weight of 35,000 is the product of gene B . The possibility that the Mu protein with a molecular weight of 23,000 is the product of a gene downstream of gene B will be discussed.