Search PubMed⌕ Search

Biomedical subjects

H Vrieling

Publications and source records attributed to H Vrieling.

64 records · Page 4Linked to original sources
PubMed1990

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1988

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↗
PubMed1988

Nucleotide sequence determination of point mutations at the mouse HPRT locus using in vitro amplification of HPRT mRNA sequences.

Cloning of genomic and cDNA sequences of mammalian genes has made it possible to analyze at the molecular level mutations induced by radiation and chemical mutagens. The X-linked HPRT gene is very suitable for these investigations because in addition to the availability of cell culture systems, HPRT mutants can also be obtained directly from the lymphocytes of mouse and man. Recently a new technique has been introduced by Saiki and co-workers which allows the cloning and sequencing of small specific DNA segments from total genomic DNA after in vitro amplification of those segments up to 200,000-fold (Saiki et al., 1985). We have adapted this so-called polymerase chain reaction (PCR) procedure in such a way that the entire mouse HPRT-coding region could be amplified, cloned and sequenced. Instead of genomic DNA, we have used RNA as template in the PCR reactions. This allows us to detect point mutations in HPRT exon sequences in a very efficient way, since the DNA sequence of all 9 exons, which are scattered over 34 kb of DNA, can be obtained from only one amplification experiment. We studied the nature of 3 N-ethyl-N-nitrosourea (ENU)-induced HPRT mutants from cultured mouse lymphoma cells. One contains an A:T----G:C transition, the second an A:T----T:A transversion, whereas the third mutant is the result of abnormal splicing events, probably due to a mutation in the 3' splice site of the first intron.

Animals↗
PubMed1985

Mutations induced by X-rays at the HPRT locus in cultured Chinese hamster cells are mostly large deletions.

We investigated the molecular basis of 19 X-ray-induced HPRT-deficient mutants of V79 Chinese hamster cells with Southern hybridisation techniques. 12 of those mutants suffer from a big deletion (greater than 10 kb) of HPRT DNA sequences. Cytological studies of chromosome preparations of those 12 deletion mutants showed that in at least 3 of these mutants part of the long arm of the X-chromosome was lost. After correction for spontaneous arising mutations we estimate that at least 70-80% of X-ray-induced mutations are caused by large deletions.

Animals↗
PubMed1983

Transcription initiation of Mu mom depends on methylation of the promoter region and a phage-coded transactivator.

The product of the bacteriophage Mu gene mom modifies adenine residues of DNA within the consensus sequence CGAGCNPy, providing protection against various restriction endonucleases (ref. 1 and D. Kamp, personal communication cited in ref. 2). The mom gene is only expressed during lytic development of the phage. It is known that mom is nonfunctional in Escherichia coli host mutants in a gene (dam) which itself encodes an adenine methylation system. We show here that the E. coli dam gene is essential for transcription initiation of the mom gene, and that this dependence on dam seems to lie in a short segment preceding the mom coding region, which also contains the mom promoter. The sequence of this segment reveals the presence of dam methylation sites (GATC), and suggests a model for the regulation of mom gene expression based on DNA secondary structure, which may explain why mom is only expressed during phage lytic development. We also show that expression of phage-coded proteins (A, B and C) is needed for transactivation of mom transcription.

Bacteriophage mu↗
PubMedDate not supplied

Hierarchies of DNA repair in mammalian cells: biological consequences.

Mammalian cells exposed to genotoxic agents exhibit heterogeneous levels of repair of certain types of DNA damage in various genomic regions. For UV-induced cyclobutane pyrimidine dimers we propose that at least three levels of repair exist: (1) slow repair of inactive (X-chromosomal) genes, (2) fast repair of active housekeeping genes, and (3) accelerated repair of the transcribed strand of active genes. These hierarchies of repair may be related to chromosomal banding patterns as obtained by Giemsa staining. The possible consequences of defective DNA repair in one or more of these levels may be manifested in different clinical features associated with UV-sensitive human syndromes. Moreover, molecular analysis of hprt mutations reveals that mutations are primarily generated by DNA damage in the poorly repaired non-transcribed strand of the gene.

Animals↗