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Biomedical subjects

M Meuth

Publications and source records attributed to M Meuth.

At least 55 records · Page 3Linked to original sources
PubMed1989

The molecular basis of mutations induced by deoxyribonucleoside triphosphate pool imbalances in mammalian cells.

Alterations of the balanced supply of the precursors of DNA synthesis, the deoxyribonucleoside triphosphates, have dramatic genetic consequences for mammalian cells including the induction of mutations, the sensitization to DNA damaging agents, and the production of gross chromosomal abnormalities. The use of recombinant DNA techniques has allowed the analysis of some of these effects and has revealed further mechanisms by which mammalian cells control the accuracy of DNA replication.

Alkylating Agents↗
PubMed1989

The genetic consequences of DNA precursor pool imbalance: sequence analysis of mutations induced by excess thymidine at the hamster aprt locus.

To determine the effect of deoxyribonucleoside triphosphate pool imbalances on the accuracy of DNA replication within the cell, we examined the base pair alterations induced by excess intracellular dTTP at the adenine phosphoribosyl transferase (aprt) locus of CHO cells. The mutations were predominantly simple (C----T) transitions (38/44) and transversions (G----T, 5/44) explicable by the misincorporation of the DNA precursor supplied in excess (dTTP). Only one small deletion was observed. The context of the mutations is notable as the nucleotide incorporated after the error was usually the nucleotide in excess for the great majority of the transitions but not the transversions. As next nucleotide effects are characteristic of replication complexes having proofreading exonuclease activity, our data indicate that this mechanism functions within the cell to control the occurrence of some types of replicational errors.

Adenine Phosphoribosyltransferase↗
PubMed1989

DNA sequence determination of gamma-radiation-induced mutations of the hamster aprt locus.

From a collection of 85 independent gamma-radiation hamster aprt- mutants, 27 having no major structural alterations were analysed at the nucleotide level by using the polymerase chain reaction to amplify mutant exons and then directly sequencing the double-stranded products. The majority of these mutations were simple base substitutions of all types, particularly transversions (11/27). Frameshifts and small deletions were also induced. The 'spectrum' of mutations produced by gamma-radiation was not significantly different from that occurring spontaneously at this locus. Differences with respect to the target and structure of frameshifts and small deletions occurring in the two collections were apparent.

Adenine Phosphoribosyltransferase↗
PubMed1989

Deletion formation in mammalian cells: molecular analysis of breakpoints and junctions in the hamster aprt locus.

To examine the mechanisms governing deletion formation in mammalian cells, we have analyzed the breakpoints and junction fragments produced by seven such mutations at the aprt locus of Chinese hamster ovary cells at the base sequence level. The deletions were heterogeneous both in size, varying from 38 bp to 170 kb, and in sequence in that no recurring sequence or structural motifs were evident. Most were simple exchanges at overlapping di- or trinucleotides, but one was the result of a complex rearrangement in which breakpoints approximately 34 kb apart were joined by 16- and 398-bp inserted fragments originating some distance from the target. Unlike many human germ line deletions, few of the breakpoints fell within hamster repetitive elements. The directionality of the deletions at aprt indicates that an essential gene or structure may determine the pattern of such mutations.

Adenine Phosphoribosyltransferase↗
PubMed1988

Insertion of unique and repetitive DNA fragments into the aprt locus of hamster cells.

Two insertion mutations occurring at the aprt locus of Chinese hamster ovary cells were analyzed at the nucleotide level by cloning and sequencing the mutant genes. The insertions are similar with respect to events at the target site in that both are accompanied by small deletions. The nature of the DNA introduced, on the other hand, is very different, a unique fragment in a spontaneously occurring mutant and a highly dispersed, repetitive fragment in a gamma radiation-induced strain. The inserts are small (285 and 58 base-pairs) and have none of the structural features or sequences related to putative mobile sequences in mammalian cells. The mechanism of transposition was further examined by cloning the unique donor fragment of the spontaneous mutant. These analyses revealed that the insert in the mutant gene was a precise duplicate of the donor DNA fragment.

Animals↗
PubMed1988

Direct selection of Chinese hamster ovary strains deficient in CTP synthetase activity.

Mutant CHO cell strains dependent upon cytidine for growth and survival were isolated by a direct selection protocol. The mutants obtained are deficient in CTP synthetase activity (less than 2% residual activity) and have low reversion frequencies (less than 10(-7)). Cytidine deprivation of these stains leads to rapid depletion of intracellular CTP pools, but not dCTP pools, and a surprisingly rapid loss of cell viability. These properties should make the cytidine auxotrophs useful for a number of biochemical and genetic studies.

Animals↗
PubMed1987

[2 cases of death following cell therapy].

Fresh-cell therapy is a paramedical procedure whose claimed therapeutic success has not been proven by customary clinical tests (randomized, double-blind trials). In addition, the qualitatively and quantitatively non-standardized parenteral application of heterologous antigens presents considerable danger for the recipient in the form of fatal immune reactions. Two cases are reported in which history, clinical findings and autopsy provided evidence of a causal relationship between cell therapy and death. In one instance, a 75-year-old woman died 30 days after an intramuscular injection of quick-frozen fresh cells from the effects of an immune-complex vasculitis; in the other, a 60-year-old woman died 14 days after "original fresh-cell treatment after Prof. Niehans" from perivenous leucoencephalitis.

Adult↗
PubMed1987

Next-nucleotide effects in mutations driven by DNA precursor pool imbalances at the aprt locus of Chinese hamster ovary cells.

Imbalances of the intracellular pools of the precursors of DNA synthesis, the deoxyribonucleoside triphosphates, produce marked shifts in the spectrum of mutations at the aprt locus of Chinese hamster ovary cells. Mutations induced by excess dTTP or dCTP are dominated by misincorporation of the nucleotide in excess, as determined by sequence analysis of cloned mutant genes. The shift in spectrum is also apparently influenced by the nucleotides surrounding the one altered--those 3' to the nucleotide misincorporated being present in excess in most of the mutant genes characterized. Since next-nucleotide effects are a property of DNA polymerases with "proofreading" activities, our data suggest that this function is part of the mammalian DNA replication complex.

Adenine Phosphoribosyltransferase↗
PubMed1987

DNA sequence analysis of spontaneous mutations at the aprt locus of hamster cells.

To determine the nature of spontaneous mutational events in cellular genes in hamster cells, mutant adenine phosphoribosyltransferase (aprt) genes were cloned and the regions to which we mapped alterations were sequenced. A variety of nucleotide changes were found to occur in the 12 mutant genes analyzed. Most mutations were simple base-pair substitutions-transitions (both G X C----A X T and A X T----G X C) and transversions. The only multiple mutation was a simple transition next to a single-base-pair insertion. Of the 12 mutations, 4 were more complex, involving small deletions or duplications. Two of these were similar to previously described deletions in that they occurred between short direct sequence repeats. No hot spots were detected. Three independent mutations were characterized at one restriction endonuclease site, although no other mutations were detected in the nucleotides surrounding this site in other mutant strains. At a functional level, sequence changes were either in exons (resulting in missense and, in one instance, nonsense mutations) or at splicing sites.

Adenine Phosphoribosyltransferase↗
PubMed1986

Structure and sequence of mutations induced by ionizing radiation at selectable loci in Chinese hamster ovary cells.

The spectrum of mutations induced by ionizing radiation at two non-essential genetic loci varies markedly. Those at the adenine phosphoribosyl transferase (aprt) locus predominantly have no detectable alterations of gene structure on Southern blots, while those at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus are largely massive deletions eliminating all coding sequence. Insertion mutations were detected at both loci. To characterize the sequence alterations producing the minor changes at the aprt locus, two mutant genes were cloned from lambda genomic libraries and sequenced. One of these mutants proved to be a 20 base-pair deletion formed between two short (3 base-pair) direct repeat sequences, while the second was the result of a 58 base-pair insertion accompanied by a 13 base-pair deletion.

Animals↗
PubMed1986

DNA amplification--deletion in a spontaneous mutation of the hamster aprt locus: structure and sequence of the novel joint.

In a collection of spontaneous mutants of Chinese hamster ovary cells selected for deficiency in adenine phosphoribosyl transferase (aprt) activity, one was detected having not only a deletion of aprt coding sequences but also an apparent amplification of remaining sequences. The HindIII fragment bearing the novel joint was cloned and sequenced revealing a complex gene rearrangement. A deletion of at least 9 kb extending upstream from the aprt locus is accompanied by an inverted duplication of flanking sequences 672 bp downstream from the novel joint. This unit is amplified three to four times with the net result of some sequences being increased as much as eight fold in copy number because of the duplication. The fidelity of the sequences involved is preserved. We propose a model which could account for this inverted duplication.

Adenine Phosphoribosyltransferase↗
PubMed1986

Spontaneous deletion formation at the aprt locus of hamster cells: the presence of short sequence homologies and dyad symmetries at deletion termini.

To examine the factors governing the generation of DNA sequence rearrangements in mammalian somatic cells, we have cloned and sequenced novel junctions produced by six spontaneous deletion mutations at the aprt locus of Chinese hamster ovary cells. Our analyses indicate that these rearrangements were produced by non-homologous recombinational events occurring between short (2-7 bp) sequence repeats at the two termini of the deletion which leave one copy of the repeat in the mutant gene. Certain tri- and tetranucleotides recur at the deletion termini, suggesting that these may possibly be a recognition sequence for an enzyme involved in the event. No other gene structural alterations were found at the novel junctions or in neighbouring sequences. The deletions are not randomly distributed over the aprt gene; four termini clustered in a 40-bp sequence. This region of aprt is unusual as it contains both significant stretches of dyad symmetry which could potentially form stable DNA secondary structures and short direct repeats. Regions of dyad symmetry were also found at at least one terminus of all the deletions. In view of the similar properties of this set of deletions, possible mechanisms for the formation of this type of gene rearrangement are considered.

Adenine Phosphoribosyltransferase↗
PubMed1986

Fresh cell therapy followed by fatal coma.

A 60-year-old woman received a 3-day course of nine injections of "fresh" cells from fetal lamb ovary, placenta, brain (hypothalamus) and liver. There were no immediate complications, but a few days later she developed headache, fever and hemiparesis. She subsequently fell into a coma and died 3 weeks after her fresh cell therapy and 2 weeks after the onset of her clinical symptoms. Autopsy revealed perivenous leucoencephalopathy with a probably steroid-treatment-induced paucity of perivascular inflammation. Fresh cell therapy, clinical symptomatology and morphological findings suggest, though do not prove, that this patient's monophasic and probably immune-mediated disease is a rare fatal complication of fresh cell therapy.

Blood-Brain Barrier↗
PubMed1984

Biochemical characterization of the hamster thy mutator gene and its revertants.

The thy- mutator phenotype of Chinese hamster ovary cells is distinguished by increased intracellular levels of dCTP, auxotrophy for thymidine, and elevated spontaneous mutational rates. To determine the biochemical lesion responsible for this complex phenotype, enzymes responsible for the synthesis of dCTP and dTTP were investigated. Levels of ribonucleotide reductase and dCMP deaminase were identical in mutant and wild type strains. In contrast, CTP synthetase activity in extracts from thy- strains was consistently altered in that 50% of enzyme activity was resistant to feedback inhibition by CTP. Additionally, thy- strains obtained by DNA transfection also had CTP-resistant CTP synthetase. Thy+ revertants lost the resistant enzyme, and total activity was reduced. CTP-resistant CTP synthetase was regained in thy- mutants reselected from thy+ revertants, but in these strains all activity was resistant. These experiments demonstrate that the thy- mutator phenotype is a consequence of a mutation of CTP synthetase and suggest that one pathway of reversion to the wild type state is by loss or inactivation of the mutant allele rendering the revertants hemizygous for the gene.

Animals↗