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Studies of hprt mutation, hprt mRNA levels and karyotypes in human T-cell clones
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Tokyo Women's Medical College, Box 116, NS BLD., 2-4-1, Hishi-Shinjuku, Shinjuku-ku, Tokyo, 163 (Japan), In vivo somatic cell mutations at the hprt locus in atomic bomb survivors Gennett, I.N., and W.G. Thilly, Center for Environmental Health Sciences, MIT, Cambridge, MA 02139 (U.S.A.), Mutational spectra in the human hprt gene Gibbs, R.A., P. Nga Nguyen and C.T. Caskey, Institute for Molecular Genetics and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77098 (U.S.A.), Direct DNA sequence analysis of in vitro amplified hprt cDNA from Lesch-Nyhan patients Cole, J., and A. Lehmann, MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, Sussex BN1 9RR (U.K.), Hprt mutations in the DNA of cells from patients with repair-deficient disorders Melton, D.W., Department of Molecular Biology, Edinburgh University, Mayfield Road, Edinburgh EH 3JR (U.K.), Expression of the hprt gene in cultured cells and mice Vrieling, H., M.L. van Rooijen, M.Z. Zdzienicka, J.W.I.M. Simons, P.H.M. Lohman and A.A. van Zeeland, Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, 2333 AL Leiden (The Netherlands), Development of the PCR method for sequencing single base pair changes in the hprt gene of man, mouse and hamster: different mutation spectra in normal and repair-deficient UV-sensitive V79 Chinese hamster cells Tates, A.D., A.M. Rossi and A.T. Natarajan, Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, 2333 AL Leiden (The Netherlands), Detection of hprt- mutants in human T-lymphocytes and mouse splenocytes Jones, I., Lawrence Livermore National Laboratory, Livermore, CA 94550 (U.S.A.), Analyses of in vivo mutations of the mufine hprt locus Borresen, A.-L., Department of Genetics, Institute for Cancer Research, The Norwegian Radiumhospital, Montebello, 0310 Oslo 3 (Norway), Denaturing gradient gel electrophoresis: a tool in mutation analysis Eriksson, L., H. Amn~us and J. Grawfi, Department of Genetics, University of Uppsala and Department of Pathology, Faculty of Veterinary Medicine, Swedish Agricultural University, Uppsala (Sweden), Flow sorting and cloning of 6-thioguanine-resistant cells Johnson, P., and T. Friedmann, Department of Pediatrics and Center for Molecular Genetics, School of Medicine UCSD, La Jolla, CA 92093 (U.S.A.), Bidirectional function of the human hprt promoter
Andersson, B., S.-H. He, D. Hellgren, K. Holmberg, S. Marcus, A.-M. Steen and B. Lambert, Department of Clinical Genetics, Karolinska In-
stitute, Karolinska Hospital, 10401 Stockholm (Sweden) Studies of hprt mutation, hprt mRNA levels and karyotypes in human T-cell clones In vivo mutation at the hprt locus was studied in thioguanine-resistant T-cell clones from healthy male and female subjects and melphalan-treated ovarian carcinoma patients (He et al., 1989). Southern blot analysis of PstI-digested DNA from a total of 108 hprt- clones hybridized with a full-length hprt cDNA probe (pHPT 30) showed alterations in 4 / 2 9 male clones, 2 / 4 7 healthy female clones and 1 / 3 2 clones from patients. No alteration was detected in any of 19 unselected clones studied. The low proportion of alterations in the hprt female clones is probably due to the presence of the inactive X chromosome, making alterations not associated with novel bands difficult to detect. Two alterations (both in male clones) were total deletions of the hprt gene, the others were partial deletions. One of the female clones showed loss of one of the X chromosomes, which facilitated the analysis in this case. Four clones (2 male and 2 female) showed novel bands, which were further identified using probes specific for exons 1-2, 3-6 and 7 - 9 respectively. Karyotype analysis of hprt- clones (He et al., 1989; Lambert et al., 1988) revealed one clonal abnormality in 29 male clones (3.6%). Loss or structural aberration of one X chromosome occurred in 6% of the clones from healthy females. The frequency of other karyotypic abnormalities was significantly increased in clones from the patients (37%) as compared to healthy females (5.6%). No aberration was found to affect the hprt locus at Xq27 in any of the 82 hprt- clones studied, which indicates that mutation in the hprt locus in human T-lymphocytes in vivo is very seldom (less than 1%) associated with microscopically detectable chromosome deletion or rearrangement. A quantitative R N A / R N A hybridization assay was used to determine the levels of hprt m R N A in human cells (Steen et al., 1989). Unstimulated peripheral lymphocytes were found to contain low levels, which increased 10-fold after PHA stimulation to a mean of 0.93 pg hprt mRNA//~g DNA, corresponding to approximately 7 hprt mRNA
72 molecules per cell. Unselected T-cell clones showed a mean level of 0.6 pg/btg DNA. Hprt clones with a normal D N A fragment pattern in Southern blots fell in either of 2 groups with regard to their levels of hprt mRNA. One group was similar to the unselected clones, whereas hprt m R N A in the other group was not detectable at all. These groups are likely to represent different classes of hprt mutation. The sensitivity of this method for quantitation of small and variable amounts of hprt m R N A in normal and mutant human cells makes it useful for further studies of hprt gene expression and mutation.
References He, S.-M., K. Holmberg, B. Lambert and N. Einhorn (1989) Hprt mutations and karyotypeabnormalities in T-cell clones from healthy subjects and melphalan-treated ovarian carcinoma patients, Mutation Res., 210, 353-358. Lambert, B., K, Holmberg, S.-M. He and N. Einhorn (1988) Karyotypes of human T-lymphocyteclones, IARC Scientific Publ. No. 89, pp. 469-476. Steen, A.-M., H. Luthman, D. Hellgren and B. Lambert (1989) Levels of hypoxanthine phosphoribosyl transferase mRNA in human cells, submitted.
Morley, A., Department of Haematology, Flinders Medical Centre, Bedford Park, Adelaide, SA 5042 (Australia)
Comparison of hprt and HLA-A mutations The molecular basis for somatic mutations has been studied in human lymphocytes mutated at the X-linked hprt locus or at the autosomal HLAA locus on chromosome 6. Genomic D N A from hprt mutants was probed using the c D N A probes pHPT30 or pHPT31. Partial or complete deletions were found in 6 of 33 mutants present spontaneously in vivo, in 17 of 43 mutants generated spontaneously in vitro, in 15 of 33 mutants generated by X-radiation in vitro and in 0 of 23 mutants generated by methylnitrosourea in vitro. Mutants in which a deletion has not been detected presumably result from deletions too small to be
detected or from point mutations involving the gene or controlling regions. The hprt locus, being X-linked, cannot detect all mutational mechanisms and a mutation assay has therefore been developed using the autosomal H L A - A locus. Wild-type cells have been eliminated by antibody-complement cytotoxicity and the presumptively mutant cells expanded for phenotypic and genotypic study. The mean frequency of HLA-A mutants is 3 x 10-s in young adults and 7 X 10 s in individuals aged over 70. This mutation frequency is significantly greater than that measured at the hprt locus. Genomic D N A probed with a genomic probe for the 3' untranslated portion of the gene has shown that 40% of mutants show loss of the index H L A - A allele. For 8 of 17 mutants produced by X-radiation the loss was due to a simple gene deletion the magnitude of which could be mapped using markers separate from HLA-A. For the remaining mutants produced by X-irradiation, and in all 8 spontaneous mutants and 11 mutants produced by mitomycin, loss of the index H L A - A allele was associated with doubling of gene dose for the homologous HLA-A allele, indicating acquired homozygosity due to either chromosome duplication or mitotic recombination (including gene conversion). Preliminary results using polymorphic gene loci on both arms of chromosome 6 have shown that mitotic recombination is the underlying mechanism in most of the mutants so far studied. Mitotic recombination is an important aetiological mechanism in a number of forms of cancer and the present results provide a clear link between mutagenesis and carcinogenesis.
Albertini, R.J., J.A. Nicklas and J.P. O'Neill, Genetics Laboratory, The University of Vermont, Burlington, VT 05401 (U.S.A.)
Hprt mutation in human T-lymphocytes: cumulative results of quantitative and molecular studies Our clonal method for measuring hprt mutant frequencies in human T-lymphocytes has been described. We use either crude T C G F or super-
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14
15
Tokyo Women's Medical College, Box 116, NS BLD., 2-4-1, Hishi-Shinjuku, Shinjuku-ku, Tokyo, 163 (Japan), In vivo somatic cell mutations at the hprt locus in atomic bomb survivors Gennett, I.N., and W.G. Thilly, Center for Environmental Health Sciences, MIT, Cambridge, MA 02139 (U.S.A.), Mutational spectra in the human hprt gene Gibbs, R.A., P. Nga Nguyen and C.T. Caskey, Institute for Molecular Genetics and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77098 (U.S.A.), Direct DNA sequence analysis of in vitro amplified hprt cDNA from Lesch-Nyhan patients Cole, J., and A. Lehmann, MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, Sussex BN1 9RR (U.K.), Hprt mutations in the DNA of cells from patients with repair-deficient disorders Melton, D.W., Department of Molecular Biology, Edinburgh University, Mayfield Road, Edinburgh EH 3JR (U.K.), Expression of the hprt gene in cultured cells and mice Vrieling, H., M.L. van Rooijen, M.Z. Zdzienicka, J.W.I.M. Simons, P.H.M. Lohman and A.A. van Zeeland, Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, 2333 AL Leiden (The Netherlands), Development of the PCR method for sequencing single base pair changes in the hprt gene of man, mouse and hamster: different mutation spectra in normal and repair-deficient UV-sensitive V79 Chinese hamster cells Tates, A.D., A.M. Rossi and A.T. Natarajan, Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, 2333 AL Leiden (The Netherlands), Detection of hprt- mutants in human T-lymphocytes and mouse splenocytes Jones, I., Lawrence Livermore National Laboratory, Livermore, CA 94550 (U.S.A.), Analyses of in vivo mutations of the mufine hprt locus Borresen, A.-L., Department of Genetics, Institute for Cancer Research, The Norwegian Radiumhospital, Montebello, 0310 Oslo 3 (Norway), Denaturing gradient gel electrophoresis: a tool in mutation analysis Eriksson, L., H. Amn~us and J. Grawfi, Department of Genetics, University of Uppsala and Department of Pathology, Faculty of Veterinary Medicine, Swedish Agricultural University, Uppsala (Sweden), Flow sorting and cloning of 6-thioguanine-resistant cells Johnson, P., and T. Friedmann, Department of Pediatrics and Center for Molecular Genetics, School of Medicine UCSD, La Jolla, CA 92093 (U.S.A.), Bidirectional function of the human hprt promoter
Andersson, B., S.-H. He, D. Hellgren, K. Holmberg, S. Marcus, A.-M. Steen and B. Lambert, Department of Clinical Genetics, Karolinska In-
stitute, Karolinska Hospital, 10401 Stockholm (Sweden) Studies of hprt mutation, hprt mRNA levels and karyotypes in human T-cell clones In vivo mutation at the hprt locus was studied in thioguanine-resistant T-cell clones from healthy male and female subjects and melphalan-treated ovarian carcinoma patients (He et al., 1989). Southern blot analysis of PstI-digested DNA from a total of 108 hprt- clones hybridized with a full-length hprt cDNA probe (pHPT 30) showed alterations in 4 / 2 9 male clones, 2 / 4 7 healthy female clones and 1 / 3 2 clones from patients. No alteration was detected in any of 19 unselected clones studied. The low proportion of alterations in the hprt female clones is probably due to the presence of the inactive X chromosome, making alterations not associated with novel bands difficult to detect. Two alterations (both in male clones) were total deletions of the hprt gene, the others were partial deletions. One of the female clones showed loss of one of the X chromosomes, which facilitated the analysis in this case. Four clones (2 male and 2 female) showed novel bands, which were further identified using probes specific for exons 1-2, 3-6 and 7 - 9 respectively. Karyotype analysis of hprt- clones (He et al., 1989; Lambert et al., 1988) revealed one clonal abnormality in 29 male clones (3.6%). Loss or structural aberration of one X chromosome occurred in 6% of the clones from healthy females. The frequency of other karyotypic abnormalities was significantly increased in clones from the patients (37%) as compared to healthy females (5.6%). No aberration was found to affect the hprt locus at Xq27 in any of the 82 hprt- clones studied, which indicates that mutation in the hprt locus in human T-lymphocytes in vivo is very seldom (less than 1%) associated with microscopically detectable chromosome deletion or rearrangement. A quantitative R N A / R N A hybridization assay was used to determine the levels of hprt m R N A in human cells (Steen et al., 1989). Unstimulated peripheral lymphocytes were found to contain low levels, which increased 10-fold after PHA stimulation to a mean of 0.93 pg hprt mRNA//~g DNA, corresponding to approximately 7 hprt mRNA
72 molecules per cell. Unselected T-cell clones showed a mean level of 0.6 pg/btg DNA. Hprt clones with a normal D N A fragment pattern in Southern blots fell in either of 2 groups with regard to their levels of hprt mRNA. One group was similar to the unselected clones, whereas hprt m R N A in the other group was not detectable at all. These groups are likely to represent different classes of hprt mutation. The sensitivity of this method for quantitation of small and variable amounts of hprt m R N A in normal and mutant human cells makes it useful for further studies of hprt gene expression and mutation.
References He, S.-M., K. Holmberg, B. Lambert and N. Einhorn (1989) Hprt mutations and karyotypeabnormalities in T-cell clones from healthy subjects and melphalan-treated ovarian carcinoma patients, Mutation Res., 210, 353-358. Lambert, B., K, Holmberg, S.-M. He and N. Einhorn (1988) Karyotypes of human T-lymphocyteclones, IARC Scientific Publ. No. 89, pp. 469-476. Steen, A.-M., H. Luthman, D. Hellgren and B. Lambert (1989) Levels of hypoxanthine phosphoribosyl transferase mRNA in human cells, submitted.
Morley, A., Department of Haematology, Flinders Medical Centre, Bedford Park, Adelaide, SA 5042 (Australia)
Comparison of hprt and HLA-A mutations The molecular basis for somatic mutations has been studied in human lymphocytes mutated at the X-linked hprt locus or at the autosomal HLAA locus on chromosome 6. Genomic D N A from hprt mutants was probed using the c D N A probes pHPT30 or pHPT31. Partial or complete deletions were found in 6 of 33 mutants present spontaneously in vivo, in 17 of 43 mutants generated spontaneously in vitro, in 15 of 33 mutants generated by X-radiation in vitro and in 0 of 23 mutants generated by methylnitrosourea in vitro. Mutants in which a deletion has not been detected presumably result from deletions too small to be
detected or from point mutations involving the gene or controlling regions. The hprt locus, being X-linked, cannot detect all mutational mechanisms and a mutation assay has therefore been developed using the autosomal H L A - A locus. Wild-type cells have been eliminated by antibody-complement cytotoxicity and the presumptively mutant cells expanded for phenotypic and genotypic study. The mean frequency of HLA-A mutants is 3 x 10-s in young adults and 7 X 10 s in individuals aged over 70. This mutation frequency is significantly greater than that measured at the hprt locus. Genomic D N A probed with a genomic probe for the 3' untranslated portion of the gene has shown that 40% of mutants show loss of the index H L A - A allele. For 8 of 17 mutants produced by X-radiation the loss was due to a simple gene deletion the magnitude of which could be mapped using markers separate from HLA-A. For the remaining mutants produced by X-irradiation, and in all 8 spontaneous mutants and 11 mutants produced by mitomycin, loss of the index H L A - A allele was associated with doubling of gene dose for the homologous HLA-A allele, indicating acquired homozygosity due to either chromosome duplication or mitotic recombination (including gene conversion). Preliminary results using polymorphic gene loci on both arms of chromosome 6 have shown that mitotic recombination is the underlying mechanism in most of the mutants so far studied. Mitotic recombination is an important aetiological mechanism in a number of forms of cancer and the present results provide a clear link between mutagenesis and carcinogenesis.
Albertini, R.J., J.A. Nicklas and J.P. O'Neill, Genetics Laboratory, The University of Vermont, Burlington, VT 05401 (U.S.A.)
Hprt mutation in human T-lymphocytes: cumulative results of quantitative and molecular studies Our clonal method for measuring hprt mutant frequencies in human T-lymphocytes has been described. We use either crude T C G F or super-