Li-Hua Zhang

Spectrum of spontaneously occurring mutations in the hprt gene of V79 Chinese hamster cells.

A total of 76 independent spontaneous mutants in the hprt gene of V79 Chinese hamster cells have been analyzed. These mutants were obtained in two different laboratories, 17 and 59 mutants in sets 1 and 2, respectively, under different cell culture conditions. Mutation analysis was performed by amplification of hprt cDNA with the polymerase chain reaction and direct sequencing of the products. The data obtained showed similar spectra of spontaneous mutations in both sets of mutants, suggesting that culture does not play a major role in spontaneous mutagenesis. The majority of the mutations were base substitutions (greater than 60%), with twice as many transversions as transitions. Base changes were evenly distributed throughout the structural gene, including the splice junctions. All types of base substitutions appeared in comparable frequencies, except for A.T to T.A transversions, which were almost absent. The fraction of deletion mutations was low (13%). A striking feature of the observed mutation spectra is that one third of the spontaneous mutations analyzed involved aberrant splicing of the hprt primary transcript, with exon 4 being affected most frequently, indicating that splice mutations are a common mechanism of mutation in the hprt gene.

UV-induced hprt mutations in a UV-sensitive hamster cell line from complementation group 3 are biased towards the transcribed strand

The molecular nature of 254 nm ultraviolet light (UV)-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in UV24 Chinese hamster ovary (CHO) cells, which are defective in nucleotide excision repair, was determined. Sequence analysis of 19 hprt mutants showed that single base substitutions (9 mutants) and tandem base changes (7 mutants) dominated the UV mutation spectrum in this cell line. Sixty-five percent of the base substitutions were GC greater than AT transitions, whereas the rest consisted of transitions and transversions at AT base pairs. Analysis of the distribution of dipyrimidine sites over the two DNA strands, where the photoproducts causing these mutations presumably were formed, showed that 12 out of 14 mutations were located in the transcribed strand of the hprt gene. A similar strand distribution of mutagenic photoproducts as in UV24 has previously been found in two other UV-sensitive Chinese hamster cell lines (V-H1 and UV5), indicating that under defective nucleotide excision repair conditions the induction of mutations is strongly biased towards lesions in the transcribed strand of the hprt gene. A plausible explanation for this phenomenon is that during DNA replication large differences exist in the error rate with which DNA polymerase(s) bypass lesions in the templates for the leading and lagging strand, respectively.

CHARACTERIZATION OF MUTANTS INVOLVING PARTIAL EXON DUPLICATIONS IN THE HPRT GENE OF CHINESE HAMSTER V79 CELLS

Sequencing ofhprt cDNA revealed that three spontaneous mutants in V79 Chinese hamster cells exhibit tandem duplications of exon(s), i.e., either exons 2 and 3 or exon 7. Sequences of different sizes (4.5—8 Kb) were found to be duplicated and inserted in tandem into thehprt gene. These mutants demonstrated spontaneous reversion frequencies which were about 40-fold higher than those observed with other types of spontaneous mutants, but on the same order of magnitude as spontaneous reversions in Sp5, a mutant with a duplication insertion involving exon 2 in this gene. These data suggest that all of the duplications found have the same genetic instability, regardless of the type, size or position of the duplictaed fragment. The coding sequence of thehprt cDNA and the restriction pattern of the revertants were virtually identical to the wild-type, indicating restoration of a functionalhprt gene by precise deletion of the duplicated fragment.

Isolation and characterization of spontaneously occurring mutations at the HPRT locus in V79 Chinese hamster cells

The aim of the present investigation was to screen for rare types of spontaneously occurring mutational events in order to provide information on the organization of the mammalian genome. For this purpose a hierarchical sequence of analyses is used with a first step utilizing a forward reverse mutation approach. The present paper deals with the characterization of 22 isolated mutants from 2 groups, 11 spontaneously appearing mutants and, in comparison, 11 ethyl methanesulfonate-induced mutants at the HPRT locus in V79 Chinese hamster cells, by means of reverse mutation analyses using selection with medium containing L-azaserine. Nine out of the 11 mutant clones of each group could be reverted either spontaneously or induced by treatments with ethyl nitrosourea (ENU), ICR191 or 5-azacytidine (5AC), which indicates that they were caused by point mutations. Two of the revertible mutant clones of spontaneous origin were found to be resistant to HAT but not HAsT medium. These 2 6TGrHATr mutants were the only mutants isolated which could be affected by 5AC with a significant increase in reversion frequency. Chromosome aberration analysis did not indicate any enhancement in aberration frequency in the X-chromosome by 5AC treatment. Studies on the mutagenicity at the OUA locus indicated that the 5AC- and ENU-induced mutation frequencies in these 2 mutants were comparable to the effects in the parent wild-type cell line. Their cellular incorporation of 3H-hypoxanthine was enhanced in the presence of aminopterin, but decreased with L-azaserine indicating that they were phosphoribosyl pyrophosphate (PRPP) mutants. On the basis of these results, it is hypothesized that reversion of these 2 6TGrHATr mutants may occur by a gene amplification mechanism and that this process may be facilitated by 5AC treatment.

CHARACTERIZATION OF HAT- AND HAST-RESISTANT HPRT MUTANT CLONES OF V79 CHINESE HAMSTER CELLS

HPRT mutant clones of V79 Chinese hamster cells, isolated after 6-thioguanine (6TG) selection, normally exhibit sensitivity to growth in medium containing the folic acid inhibitor aminopterin or the glutamine analogue L-azaserine (e.g., HAT or HAsT medium). However, it has been shown that some HPRT- clones are resistant to both HAT and HAsT medium. The present study was undertaken to investigate whether any common structural gene alteration exists for such 6TGr-HATr-HAsTr clones. Four clones were studied, 1 of spontaneous origin, 2 induced by a low dose of MNU and 1 EMS-induced. In contrast to wild-type cells and a mutant clone carrying a complete deletion of the HPRT gene, these 4 investigated 6TGr-HATr-HAsTr clones all showed an enhanced incorporation of exogenous 3H-hypoxanthine in the presence of aminopterin and L-azaserine suggesting that these clones carry mutations in the structural part of the HPRT gene. Sequence analysis of PCR-amplified HPRT cDNA from these mutants showed that the spontaneous and the 2 MNU-induced mutant clones lacked exon 4, while the EMS-induced mutant had a GC to AT transition in exon 6. Southern blot analysis of genomic DNA after digestion with BglII, EcoRI and PstI showed no changes in fragment patterns as compared to the wild type. Further sequence analysis of PCR-amplified genomic DNA using exon 4-specific primers showed that all these 3 mutants had an AT to GC or GC to AT transition in exon 4, but had no alterations in the splice sites of exon 4. Based on their characteristics of hypoxanthine incorporation, the present mutant clones fit the model for the proposed functional domains of the HPRT protein.