Age-related accumulation of mutations in human T-lymphocytes.
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Aging↗
Biomedical subjects
H Vrieling
Publications and source records attributed to H Vrieling.
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.
Animals↗
Molecular analysis of mutations affecting hprt mRNA splicing in human T-lymphocytes in vivo.
Molecular analysis of hypoxanthine-guanine phosphoribosyltransferase (hprt) cDNA from 6-thioguanine-resistant T-lymphocytes cloned from smoking and non-smoking adult donors showed that 35% of these mutants were defective in splicing of hprt mRNA. Among a set of 42 hprt splice mutants, we observed i) complete loss of one or more exons, ii) partial loss of one exon, or iii) inclusion of part of an intron sequence between adjacent exons. Loss of exon 4 was significantly more frequent than of the other exons, suggesting that the sequences that regulate splicing of this exon are either larger than those of the other exons or especially prone to mutation. In order to identify the molecular nature of DNA alterations causing aberrant splicing of hprt mRNA, 17 splice mutants were analyzed in more detail by sequencing the genomic regions flanking the mis-spliced exon. Base pair substitutions or small deletions causing defective splicing were either detected in exon sequences or in splice site consensus sequences of introns. Furthermore, genomic deletions encompassing entire exons were found. In some mutants, the alteration responsible for incorrect splicing could not be identified, suggesting that the target sequence for splice mutations is larger than merely the splice junctions. Molecular characterization of hprt splice mutations will lead to the identification of specific sequences regulating splicing of hprt mRNA and will reveal whether the mutational spectrum in splice mutants is similar to that found in the hprt coding region.
Adult↗
Human HPRT mutant database: software for data entry and retrieval.
We have developed a computer database containing information on over 1,000 human hypoxanthine guanine phosphoribosyl transferase (HPRT) mutants. Both published and unpublished data are present. The database itself is maintained in a dBASE format (.DBF) and we provide a set of programs to examine and extract information from the database. A program to input information into the database is also supplied. The database and programs are available directly from us or via remote FTP (file transfer protocol) using BITNET/INTERNET. All programs require an IBM-compatible computer, the MS-DOS operating system (version 3.3 or greater), and a hard disk with about 5 megabytes of free disk space. The purpose of the database is 1) to allow investigators to contribute their HPRT mutants directly to the database in a standardized fashion, and 2) to allow access to the entire database with a set of programs that allows manipulation and extraction of data. For example, using our programs it is possible to i) order the database by base pair position, ii) examine only information regarding mutagenesis by a particular agent, iii) search for a particular author, iv) create a report which contains selected portions of the database, the report can be printed or saved as a file. The database will be updated every several months and distributed.
Databases, Bibliographic↗
The gene encoding hypoxanthine-guanine phosphoribosyltransferase as target for mutational analysis: PCR cloning and sequencing of the cDNA from the rat.
In this paper, the cloning and nucleotide sequence of the cDNA of the rat gene coding for hypoxanthine-guanine phosphoribosyltransferase (hprt) is reported. Knowledge of the cDNA sequence is needed, among other reasons, for the molecular analysis of hprt mutations occurring in rat cells, such as skin fibroblasts isolated according to the granuloma pouch assay. The rat hprt cDNA was synthesized and used as a template for in vitro amplification by PCR. For this purpose, oligonucleotide primers were used, the nucleotide sequences of which were based on mouse and hamster hprt cDNA sequences. Sequence analysis of 1146 bp of the amplified rat hprt cDNA showed a single open reading frame of 654 bp, encoding a protein of 218 amino acids. In the predicted rat hprt amino acid sequence, the proposed functional domains for 5'-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide binding in phosphoribosylating enzymes as well as a region near the carboxyl terminal part were highly conserved when compared with amino acid sequences of other mammalian hprt proteins. Analysis of hprt amino acid sequences of 727 independent hprt mutants from human, mouse, hamster and rat cells bearing single amino acid substitutions revealed that a large variety of amino acid changes were located in these highly conserved regions, suggesting that all 3 domains are important for proper catalytic activity. The suitability of the hprt gene as target for mutational analysis is demonstrated by the fact that amino acid changes in at least 151 of the 218 amino acid residues of the hprt protein result in a 6-thioguanine-resistant phenotype.
Amino Acid Sequence↗
(6-4) photoproducts and not cyclobutane pyrimidine dimers are the main UV-induced mutagenic lesions in Chinese hamster cells.
A partial revertant (RH1-26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1-26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1-26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1-26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1-26 cells but different from that of V-H1 cells. Since in RH1-26 as well as in V79 cells most hprt mutations were induced by lesions in the non-transcribed DNA strand, in contrast to the transcribed DNA strand in V-H1, the observed mutation-strand bias suggests that normally (6-4) photoproducts are preferentially repaired in the transcribed DNA strand. The dramatic influence of the impaired (6-4) photoproduct repair in V-H1 on UV-induced mutability and the molecular nature of hprt mutations indicate that the (6-4) photoproduct is the main UV-induced mutagenic lesion.
Animals↗
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.
Animals↗
N-acetylated and deacetylated 4'-fluoro-4-aminobiphenyl and 4-aminobiphenyl adducts differ in their ability to inhibit DNA replication of single-stranded M13 in vitro and of single-stranded phi X174 in Escherichia coli.
Calf thymus single-stranded (ss) DNA was modified with the N-sulfate conjugate of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP) or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) to yield predominantly N-acetylated adducts of 2-aminofluorene, 4-aminobiphenyl and 4'-fluoro-4-amino-biphenyl respectively to C8 of deoxyguanosine (dG-C8-AAF, dG-C8-AABP and dG-C8-FAABP). The modified DNAs were used as templates for in vitro DNA synthesis. DNA replication on the randomly primed template was inhibited as compared to control (unmodified) DNA to the same extent by all three types of adducts, irrespective of whether polymerization was performed by Escherichia coli DNA polymerase I, modified T7 DNA polymerase or Thermus aquaticus (Taq) DNA polymerase. In addition, all three types of adducts completely blocked replication of ss phi X174 in an E. coli host: on average one adduct per DNA molecule was sufficient to inactivate the bacteriophage. Polyacrylamide gel electrophoresis of DNA fragments synthesized by E. coli DNA polymerase I on FAABP- and AABP-modified ss M13mp9 DNA templates, showed that termination occurred predominantly one nucleotide before (and occasionally opposite) a modified deoxyguanosine in the template. However, the deacetylated adducts, dG-C8-AF, dG-C8-ABP and dG-C8-FABP (obtained by reacting DNA with their N-trifluoroacetyl-N-acetoxy esters) were frequently bypassed during replication of ss phi X174 in E. coli, though with different efficiencies: 1 out 7, 1 out of 2 and 1 out of 3 adducts on average respectively caused bacteriophage inactivation. Polyacrylamide gel electrophoresis showed that termination of DNA synthesis occurred at least as frequently opposite as 3' to a modified deoxyguanosine in the template.
Aminobiphenyl Compounds↗
Enhanced hprt mutant frequency but no significant difference in mutation spectrum between a smoking and a non-smoking human population.
Recently, we have observed a small (36%), but significant, enhancement of the frequency of 6-thioguanine (6-TG)-resistant T-lymphocytes in blood from smokers. The molecular nature of 43 hypoxanthine-guanine phosphoribosyltransferase (hprt) mutant T-lymphocyte clones from nine smoking individuals was determined to investigate whether the increase in hprt mutant frequency would lead to a changed mutation spectrum. The types and distribution of hprt mutations in smokers was compared with those found in 55 6-TGr T-lymphocyte clones from 12 members of a control group of non-smokers. From this control group 25 hprt mutants were novel, whereas 31 have been described previously. Among smokers and non-smokers, a similar proportion of base substitutions (approximately 35%), mutations causing aberrant splicing (approximately 37%), frameshifts (approximately 16%) and deletions (approximately 9%) was found. In both groups, GC----AT base pair changes were found to be predominant among transitions. However, whereas all types of transversions were about equally represented in non-smokers, GC----TA transversions were not recovered among smokers. Investigation of the distribution of base substitutions over the hprt coding region showed no differences between the two groups. These data provide no clues on the nature of DNA adducts induced by smoking, which are thought to be responsible for the increased mutation frequency at the hprt locus in T-lymphocytes from smokers.
Adult↗
Contributions from molecular/biochemical approaches in epidemiology to cancer risk assessment and prevention.
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Animals↗
Strand specificity for UV-induced DNA repair and mutations in the Chinese hamster HPRT gene.
DNA excision repair modulates the mutagenic effect of many genotoxic agents. The recently observed strand specificity for removal of UV-induced cyclobutane dimers from actively transcribed genes in mammalian cells could influence the nature and distribution of mutations in a particular gene. To investigate this, we have analyzed UV-induced DNA repair and mutagenesis in the same gene, i.e. the hypoxanthine phosphoribosyl-transferase (hprt) gene. In 23 hprt mutants from V79 Chinese hamster cells induced by 2 J/m2 UV we found a strong strand bias for mutation induction: assuming that pre-mutagenic lesions occur at dipyrimidine sequences, 85% of the mutations could be attributed to lesions in the nontranscribed strand. Analysis of DNA repair in the hprt gene revealed that more than 90% of the cyclobutane dimers were removed from the transcribed strand within 8 hours after irradiation with 10 J/m2 UV, whereas virtually no dimer removal could be detected from the nontranscribed strand even up to 24 hr after UV. These data present the first proof that strand specific repair of DNA lesions in an expressed mammalian gene is associated with a strand specificity for mutation induction.
Animals↗
Strand-specific mutation spectra in repair-proficient and repair-deficient hamster cells.
The mutation spectrum induced by UV light has been determined at the hprt locus for both cultured normal (AA8) and UV-sensitive (UV-5) Chinese hamster ovary cells to investigate the effect of DNA repair on the nature of induced mutations. DNA base-pair changes of 23 hprt mutants of AA8 and of 28 hprt mutants of UV-5 were determined by sequence analysis of in vitro amplified hprt cDNA. Almost all mutants in AA8 carried single-base substitutions, transitions and transversions accounting for 38% and 62% of the base changes, respectively. In contrast, in repair-deficient cells (UV-5) tandem and nontandem double mutations represented a considerable portion of the mutations observed (30%), whereas the vast majority of base-pair substitutions were GC greater than AT transitions (87%). Moreover, 5 splice mutants and 2 frameshift mutations were found in the UV-5 collection. In almost all mutants analyzed base changes were located at dipyrimidine sites where UV photoproducts could have been formed. In AA8 the photolesions causing mutations were predominantly located in the nontranscribed strand whereas a strong bias for mutation induction towards photolesions in the transcribed strand was found in UV-5. We hypothesize that preferential removal of lesions from the transcribed strand of the hprt gene accounts for the observed DNA strand specificity of mutations in repair-proficient cells. Furthermore, differences in the degree of misincorporation opposite a lesion for lagging and leading strand DNA synthesis may dictate the pattern of UV-induced mutations in the absence of DNA repair.
Animals↗
Mutations affecting RNA splicing in man are detected more frequently in somatic than in germ cells.
The spectrum of DNA sequence alterations in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of HPRTase-deficient T-lymphocytes isolated from the blood of healthy male donors was determined and compared with the spectrum found in patients suffering from genetic diseases (Lesch-Nyhan syndrome or gouty arthritis) associated with a mutation in the same gene. Most of the T-cell mutants still produced hprt mRNA which was converted into cDNA and used for DNA sequence analysis after amplification using the polymerase chain reaction (PCR). In 39% of the 31 analyzed T-cell mutants of normal donors 1 or 2 exons were completely or partially deleted from hprt mRNA, probably because of a mutation in a splice acceptor site. Among patients suffering from the Lesch-Nyhan syndrome or gouty arthritis, the class of splice mutations amounts only to 7%. These data suggest that carriers of splice mutations often do not show the characteristics of HPRTase deficiency associated with these genetic diseases, because correctly spliced hprt mRNA is still produced at a low level.
Arthritis, Gouty↗
Influence of DNA excision repair on UV-induced mutation spectra in Chinese hamster cells.
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Animals↗
Screening for base mutations in the PAH and HPRT loci using the polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE).
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Animals↗
DNA strand specificity for UV-induced mutations in mammalian cells.
The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.
Amino Acid Sequence↗
Relationship between DNA-adduct formation, DNA repair, mutation frequency and mutation spectra.
DNA-adduct formation by a series of ethylating agents was determined and correlated with induction of gene mutations. This approach gave information concerning the DNA-adduct(s) likely to be responsible for the observed mutations. A methodology has been developed which is used for the DNA sequence analysis of point mutations in the HPRT gene of mammalian cells. This method can be used to obtain mutation spectra and to determine whether the base-pair changes do occur at those sites where DNA-adducts are likely to occur. Measurements of DNA repair in specific DNA sequences show that actively transcribed genes are repaired faster than the genome overall. This stresses the importance of studying removal DNA-adducts in the gene that is used for the analysis of mutation induction.
Animals↗
Molecular analysis of mutations induced by N-ethyl-N-nitrosourea at the HPRT locus in mouse lymphoma cells.
The molecular basis of 29 N-ethyl-N-nitrosourea (ENU)-induced HPRT-deficient mutants of mouse lymphoma cells (GRSL 13-2) was investigated using nucleic acid blot hybridization techniques. DNA from all 29 mutants showed normal restriction patterns on Southern blots when probed with HPRT cDNA, but 10 mutants differed from wild-type cells in their cytoplasmic HPRT mRNA level. In 5 mutants we found 10-25% of the normal amount of HPRT mRNA, whereas in another 5 mutants no HPRT mRNA could be detected at all. These mutants do not seem to be induced by hypermethylation of regulatory sequences of the HPRT gene, since they could not be reverted to an HPRT-proficient phenotype by treatment of the cells with 5-azacytidine.
Animals↗