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Biomedical subjects

A R Lehmann

Publications and source records attributed to A R Lehmann.

At least 19 recordsLinked to original sources
PubMed1996

Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse.

DNA-dependent protein kinase (DNA-PK) consists of a heterodimeric protein (Ku) and a large catalytic subunit (DNA-PKcs). The Ku protein has double-stranded DNA end-binding activity that serves to recruit the complex to DNA ends. Despite having serine/threonine protein kinase activity, DNA-PKcs falls into the phosphatidylinositol 3-kinase superfamily. DNA-PK functions in DNA double-strand break repair and V(D)J recombination, and recent evidence has shown that mouse scid cells are defective in DNA-PKcs. In this study we have cloned the cDNA for the carboxyl-terminal region of DNA-PKcs in rodent cells and identified the existence of two differently spliced products in human cells. We show that DNA-PKcs maps to the same chromosomal region as the mouse scid gene. scid cells contain approximately wild-type levels of DNA-PKcs transcripts, whereas the V-3 cell line, which is also defective in DNA-PKcs, contains very reduced transcript levels. Sequence comparison of the carboxyl-terminal region of scid and wild-type mouse cells enabled us to identify a nonsense mutation within a highly conserved region of the gene in mouse scid cells. This represents a strong candidate for the inactivating mutation in DNA-PKcs in the scid mouse.

3T3 Cells↗
PubMed1996

Development of new molecular procedures for the detection of genetic alterations in man.

The Restriction Site Mutation (RSM) procedure is a DNA-based method for detecting mutations at any unselected locus. Mutations are identified as alterations of the DNA sequence at a chosen restriction site. DNA from cells exposed to mutagenic treatment is exhaustively digested with the restriction enzyme (RE). Sequences containing the mutated target site are specifically amplified using the polymerase chain reaction (PCR), whereas DNA without mutations at this site will have been cleaved and can not therefore provide a substrate for PCR. We have developed this procedure using both bacterial and mammalian cells. With bacteria, in plasmid reconstruction experiments we were able to detect mutations at a frequency of 10(-6) at an EcoRI site in the AraA locus of Salmonella typhimurium. The detection limit with an RsaI site in the lacI gene of Escherichia coli was 10(-5), and we were able to detect DNA damage and repair after treatment with N-methyl-N-nitrosourea (MNU). With mammalian cells, we have detected mutations induced by ethyl methanesulphonate (EMS) at a TaqI site in the aprt gene of Chinese hamster cells. In extensive studies with normal and repair-deficient human cells, we have detected and sequenced mutations induced by UV-C or UV-B in fibroblasts and lymphoblastoid cells from repair-deficient xeroderma pigmentosum (XP) donors. Similar results were obtained at TaqI sites in three genes, hprt, c-Ha-rasI and p53. These results demonstrate that the system is able to detect and analyse mutations induced at high frequencies. In our extensive attempts to extend the work to conditions of lower mutation frequencies, we have encountered several obstacles, the most serious being false-positive mutant DNA in totally untreated cells. This appeared to be a cell-line specific phenomenon, which we have not been able to eliminate by altering conditions. We propose therefore that, at present, RSM is a suitable method for studying high mutation frequencies at different loci and could be used for mutagen testing with repair-deficient cells. As yet, however, its sensitivity and specificity is not sufficient for population monitoring.

Animals↗
PubMed1996

DNA repair and ultraviolet mutagenesis in cells from a new patient with xeroderma pigmentosum group G and cockayne syndrome resemble xeroderma pigmentosum cells.

Xeroderma pigmentosum (XP)/Cockayne syndrome (CS) complex is a combination of clinical features of two rare genetic disorders in one individual. A sun-sensitive boy (XP20BE) who had severe symptoms of CS, with dwarfism, microcephaly, retinal degeneration, and mental impairment, had XP-type pigmentation and died at 6 y with marked cachexia (weight 14.5 lb) without skin cancers. We evaluated his cultured cells for characteristic CS or XP DNA-repair abnormalities. The level of ultraviolet (UV)-induced unscheduled DNA synthesis was less than 5% of normal, characteristic of the excision-repair defect of XP. Cell fusion studies indicated that his cells were in XP complementation group G. His cells were hypersensitive to killing by UV, and their post-UV recovery of RNA synthesis was abnormally low, features of both CS and XP. Post-UV survival of plasmid pSP189 in his cells was markedly reduced, and post-UV plasmid mutation frequency was higher than with normal cells, as in both CS and XP. Sequence analysis of the mutated plasmid marker gene showed normal frequency of plasmids with multiple base substitutions, as in CS, and an abnormally increased frequency of G:C-->A:T mutations, a feature of XP. Transfection of UV-treated pRSVcat with or without photoreactivation revealed that his cells, like XP cells, could not repair either cyclobutane pyrimidine dimers or non-dimer photoproducts. These results indicate that the DNA-repair features of the XP20BE (XP-G/CS) cells are phenotypically more like XP cells than CS cells, whereas clinically the CS phenotype is more prominent than XP.

Cell Survival↗
PubMed1996

Defects in the DNA repair and transcription gene ERCC2(XPD) in trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterized by brittle hair with reduced sulfur content, ichthyosis, peculiar face, and mental and growth retardation. Clinical photosensitivity is present in approximately 50% of TTD patients but is not associated with an elevated frequency of cancers. Previous complementation studies show that the photosensitivity in nearly all of the studied patients is due to a defect in the same genetic locus that underlies the cancer-prone genetic disorder xeroderma pigmentosum group D (XP-D). Nucleotide-sequence analysis of the ERCC2 cDNA from three TTD cell strains (TTD1V1, TTD3VI, and TTD1RO) revealed mutations within the region from amino acid 713-730 and within previously identified helicase functional domains. The various clinical presentations and DNA repair characteristics of the cell strains can be correlated with the particular mutations found in the ERCC2 locus. Mutations of Arg658 to either His or Cys correlate with TTD cell strains with intermediate UV-sensitivity, mutation of Arg722 to Trp correlates with highly UV-sensitive TTD cell strains, and mutation of Arg683 to Trp correlates with XP-D. Alleles with mutation of Arg616 to Pro or with the combined mutation of Leu461 to Val and deletion of 716-730 are found in both XP-D and TTD cell strains.

Alleles↗
PubMed1995

The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH.

The hereditary disease Cockayne syndrome (CS) is characterized by a complex clinical phenotype. CS cells are abnormally sensitive to ultraviolet radiation and are defective in the repair of transcriptionally active genes. The cloned CSB gene encodes a member of a protein family that includes the yeast Snf2 protein, a component of the transcriptional regulator Swi/Snf. We report the cloning of the CSA cDNA, which can encode a WD repeat protein. Mutations in the cDNA have been identified in CS-A cell lines. CSA protein interacts with CSB protein and with p44 protein, a subunit of the human RNA polymerase II transcription factor IIH. These observations suggest that the products of the CSA and CSB genes are involved in transcription.

Amino Acid Sequence↗
PubMed1995

U.v.-hypermutability of xeroderma pigmentosum cells demonstrated with a DNA-based mutation system.

We have developed a DNA-based system, to detect mutations at restriction sites without any selection in culture. DNA is exhaustively digested with a restriction enzyme. Primers flanking a chosen site for this enzyme are used in the polymerase chain reaction (PCR). Only DNA molecules mutated at the chosen site are resistant to digestion and can serve as templates for the PCR. We have initially used this system to demonstrate the generation of mutations by ethyl methanesulphonate (EMS) at a TaqI site in the aprt gene of Chinese hamster cells, and by u.v.-C irradiation at a TaqI site in the hprt gene of human fibroblasts. In repair-deficient xeroderma pigmentosum (XP) cells the u.v.-induced mutant frequency was greatly enhanced. We have been able to detect and analyse mutations in XP cells at TaqI sites in three different genes, hprt, p53 and c-Ha-ras1. Both u.v.-C and u.v.-B irradiation have been used as mutagenic agents with both lymphoblastoid and fibroblast cells from XP patients from complementation group G. The mutant DNA molecules have been sequenced. Following u.v.-C-irradiation, the majority of mutations analysed were GC-->AT transitions, but several double and tandem mutations were also found.

Animals↗
PubMed1995

G2 phase repair of X-ray-induced chromosomal DNA damage in trichothiodystrophy cells.

The repair of X-ray-induced DNA damage during G2 cell-cycle phase has been examined in lines of skin fibroblasts from three patients with trichothiodystrophy (TTD), one with apparently normal and two with defective nucleotide excision repair (NER). These responses are compared with those of five lines from clinically normal controls, lines from xeroderma pigmentosum (XP), Cockayne syndrome (CS), Down syndrome (DS), and ataxia telangiectasia (AT) patients. Chromosomal DNA repair was measured as the chromatid aberration frequency (CAF) or total number of chromatid breaks and long gaps per 100 metaphase cells, determined 0.5-1.5 h after X-irradiation (53 rad). Chromatid breaks and gaps (as defined herein) represent unrepaired DNA strand breaks. Only one of the TTD lines, TTD 1BR, showed an abnormally high CAF. This line was shown subsequently to be of a different complementation group, representing a new nucleotide excision repair gene. An abnormally high CAF was also observed, as reported previously, in XP-C, AT and DS but not in CS skin fibroblasts. In addition, cell lines were examined for DNA incision activity by an indirect method in which chromatid aberrations were enumerated with or without ara-C, an inhibitor of repair synthesis, added after X-irradiation. All TTD lines had abnormally low incision activity.

Abnormalities, Multiple↗
PubMed1995

Nucleotide excision repair and the link with transcription.

Nucleotide excision repair (NER) uses the products of about 30 genes to remove a damage-containing oligonucleotide from cellular DNA. The transcription factor TFIIH is an essential component of NER. In man, defects in NER can result in three distinct genetic disorders, whose features can be ascribed to abnormalities in DNA repair or transcription.

Cockayne Syndrome↗
PubMed1995

The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair.

The rad18 mutant of Schizosaccharomyces pombe is very sensitive to killing by both UV and gamma radiation. We have cloned and sequenced the rad18 gene and isolated and sequenced its homolog from Saccharomyces cerevisiae, designated RHC18. The predicted Rad18 protein has all the structural properties characteristic of the SMC family of proteins, suggesting a motor function--the first implicated in DNA repair. Gene deletion shows that both rad18 and RHC18 are essential for proliferation. Genetic and biochemical analyses suggest that the product of the rad18 gene acts in a DNA repair pathway for removal of UV-induced DNA damage that is distinct from classical nucleotide excision repair. This second repair pathway involves the products of the rhp51 gene (the homolog of the RAD51 gene of S. cerevisiae) and the rad2 gene.

Amino Acid Sequence↗
PubMed1995

Trichothiodystrophy with sideroblastic anaemia and developmental delay.

A patient with sideroblastic anaemia, development delay, and trichothiodystrophy is presented. Trichothiodystrophy is a feature of several autosomal recessive diseases. Photosensitivity, failure to thrive, and developmental delay are commonly observed in affected cases. X linked inheritance accounts for the bulk of cases with sideroblastic anaemia. This case highlights the importance of routine hair microscopy in cases of atypical ectodermal dysplasia.

Anemia, Sideroblastic↗
PubMed1994

Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination.

The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cells, is defective in DNA double-strand break repair and in ability to undergo V(D)J recombination. The human XRCC5 DNA repair gene, which complements this mutant, is shown here through genetic and biochemical evidence to be the 80-kilodalton subunit of the Ku protein. Ku binds to free double-stranded DNA ends and is the DNA-binding component of the DNA-dependent protein kinase. Thus, the Ku protein is involved in DNA repair and in V(D)J recombination, and these results may also indicate a role for the Ku-DNA-dependent protein kinase complex in those same processes.

Animals↗
PubMed1994

Molecular analysis of the XP-D gene in Italian families with patients affected by trichothiodystrophy and xeroderma pigmentosum group D.

In several patients with the rare hereditary disorder trichothiodystrophy (TTD), a DNA repair defect has been shown to be in the same gene as in xeroderma pigmentosum complementation group D (XP-D). The ERCC-2 gene (excision repair cross-complementing rodent repair deficiency of group 2) has recently been identified as a strong candidate gene for XP-D, since it restores normal UV sensitivity to XP-D cells after transfection. Using Southern blotting, we have analysed the ERCC-2 gene in DNA samples from 28 members of nine Italian families with individuals affected by XP-D (three patients) or by TTD with photosensitivity due to the XP-D defect (eight patients). No major modifications of the ERCC-2 gene were detected with two cDNA probes in either XP-D or TTD patients indicating that the association between TTD and XP-D is not likely to result from a large deletion or rearrangement involving this gene. We found two RFLPs after digestion of the DNA samples with TaqI or MspI, but neither of them could be related to the molecular alteration determining the pathological phenotype. We also analysed a human homologue detected with the hamster sequence isolated by Arrand et al. (1989), which specifically, but partially, complements the DNA repair deficiency in XP-D cells. Our analysis demonstrated that this gene is not the primary gene defective in XP-D. In fact two RFLPs detected with a genomic probe do not co-segregate with the disease in an XP-D family.

DNA↗