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R A Schultz

Publications and source records attributed to R A Schultz.

At least 55 records · Page 3Linked to original sources
PubMed1994

Structural and mutational analysis of the xeroderma pigmentosum group D (XPD) gene.

Individuals affected by the autosomal recessive disease xeroderma pigmentosum (XP) are acutely sensitive to sunlight and predisposed to skin cancer on exposed areas. Cells cultured from XP patients are both UV sensitive and defective in the nucleotide excision repair of damaged DNA. These cellular phenotypes are amenable to experimental strategies employing complementation, an approach previously used to demonstrate the correction of XP-D phenotypes following the introduction of the XPD (ERCC2) gene. In the present study, we have characterized the genomic organization of the XPD (ERCC2) gene and found it to be comprised of 23 exons. These data were helpful in evaluating the functional integrity of alleles in two XP-D cell lines. In cell line GM436 a C-->G transversion was found at nucleotide position 1411 in the XPD (ERCC2) cDNA, a change expected to result in a Leu461Val substitution. Cell line XP67MA carries a C-->T transition in genomic DNA at nucleotide position 2176 in exon 22, introducing the termination codon TAG at amino acid 726. The latter would be expected to produce a protein truncated by 34 amino acids. Although expression of the normal XPD cDNA could be shown to correct the UV sensitivity phenotype in XP-D cells, cDNA constructs bearing either of the two mutations failed to yield complementation. These results confirm the role of ERCC2 in XP-D and illustrate the power of utilizing cellular phenotypes to evaluate the significance of single nucleotide substitutions.

Alleles↗
PubMed1994

Perinatal mortality in lambs of ewes exposed to cultures of Diplodia maydis (= Stenocarpella maydis) during gestation. A study of the central-nervous-system lesions.

Previously published results of dosing trials, involving 82 ewes, revealed that 66% of the offspring of dams exposed to cultures of Diplodia maydis in the second trimester of pregnancy and 87% of lambs of ewes exposed in the third trimester, were born dead or died soon afterwards. Spongiform degeneration of myelin of varying degree was present in the brains of all the stillborn and non-viable lambs in these trials. Myelin lamellae were separated at the intraperiod line. The spongiform degeneration occurred throughout the central nervous system in severely affected animals, whereas in mildly affected lambs the lesions had a predilection for the white matter, particularly of the cerebellum and cerebrum. In 28 lambs the spongiform degeneration progressed to lytic necrosis, and hydrocephalus was recorded in two animals.

Animal Feed↗
PubMed1993

DNA ligase III is the major high molecular weight DNA joining activity in SV40-transformed human fibroblasts: normal levels of DNA ligase III activity in Bloom syndrome cells.

The phenotypes of cultured cell lines established from individuals with Bloom syndrome (BLM), including an elevated spontaneous frequency of sister chromatid exchanges (SCEs), are consistent with a defect in DNA joining. We have investigated the levels of DNA ligase I and DNA ligase III in an SV40-transformed control and BLM fibroblast cell line, as well as clonal derivatives of the BLM cell line complemented or not for the elevated SCE phenotype. No differences in either DNA ligase I or DNA ligase III were detected in extracts from these cell lines. Furthermore, the data indicate that in dividing cultures of SV40-transformed fibroblasts, DNA ligase III contributes > 85% of high molecular weight DNA joining activity. This observation contrasts with previous studies in which DNA ligase I was reported to be the major DNA joining activity in extracts from proliferating mammalian cells.

Bloom Syndrome↗
PubMed1993

DNA content measurements and an improved idiogram for the Indian muntjac.

The Indian muntjac, an asiatic deer, has the lowest diploid chromosome number among mammals (female 2N = 6; male 2N = 7). Using flow cytometric quantification of propidium iodide-stained cells, we determined the DNA content of muntjac cells to be 94% that of human. This suggests that the muntjac may serve as a model for investigation of karyotypic evolution and rearrangement. In order to facilitate future comparative gene mapping studies, computer-aided analysis of digitized metaphase chromosomes allowed development of a detailed Indian muntjac G-banded idiogram incorporating both ISCN-type nomenclature and quantitative estimates of the size of each band and position.

Animals↗
PubMed1993

Elevation of sister chromatid exchange frequency in transformed human fibroblasts following exposure to widely used aminoglycosides.

Aminoglycosides are a class of antibiotics that interfere with protein translation. Geneticin and hygromycin are two such agents, which have been shown to exhibit highly toxic effects in mammalian cells. Cloned bacterial genes, which inactivate these antibiotics, have facilitated the establishment of dominant selection systems, which are widely used in eukaryotic molecular genetics. We have examined the effect of aminoglycosides on the sister chromatid exchange (SCE) frequency in transformed human fibroblast cell lines. Geneticin and hygromycin were both found to increase SCE frequency in all cell lines examined, including a cell line derived from a patient with Bloom syndrome, a disorder exhibiting an elevated spontaneous SCE frequency. Induction was seen to occur in a dose-responsive manner and was also observed in cells expressing the resistance genes that inactivate the cellular toxicity of these antibiotics. The implications of these findings for somatic cell genetics and for human gene therapy protocols are discussed.

Cell Line, Transformed↗
PubMed1993

Mapping of the human cardiac Na+/Ca2+ exchanger gene (NCX1) by fluorescent in situ hybridization to chromosome region 2p22-->p23.

The cDNA that encodes the human Na+/Ca2+ exchanger (NCX1) involved in regulation of intracellular calcium levels has been isolated from a cardiac cDNA library. Using fluorescent in situ hybridization, the human cDNA was mapped to chromosome region 2p23-->p22 by co-hybridization with fluorescinated alu517-PCR amplified total human DNA to obtain an R-banding pattern.

Carrier Proteins↗
PubMed1992

Elevated sister chromatid exchange phenotype of Bloom syndrome cells is complemented by human chromosome 15.

Bloom syndrome (BSx) is a rare autosomal-recessive chromosome-instability disorder manifested by a constellation of clinical features including a significant predisposition to early onset of neoplasia. BSx cells display cytogenetic abnormalities, the pathognomonic feature being an increased rate of spontaneous sister chromatid exchanges (SCEs), 10- to 15-fold more frequent than SCEs seen in control cells. Identification of the primary biochemical defect in BSx and its relationship to SCE frequency and neoplasia have been complicated by reports that BSx cell lines exhibit defects in the structure and/or activity of a number of different enzymes. The rare occurrence of the disorder and lack of informative families have precluded mapping of the primary defect by standard linkage analysis. We have utilized BSx cells as recipients for microcell-mediated chromosome transfer to map a locus that renders complementation of the elevated SCE phenotype. Studies with the BSx cell line GM08505 demonstrated a stable frequency of SCEs 10-fold higher than control values, offering a phenotype suitable for complementation studies. Transfer of different independent human chromosomes from somatic cell hybrids into BSx cells permitted identification of a single chromosome that dramatically reduced the SCE frequency to a level near that seen in control cells. Detailed characterization revealed this complementing element to be human chromosome 15.

Base Sequence↗
PubMed1992

Correction of xeroderma pigmentosum complementation group D mutant cell phenotypes by chromosome and gene transfer: involvement of the human ERCC2 DNA repair gene.

Cultured cells from individuals afflicted with the genetically heterogeneous autosomal recessive disorder xeroderma pigmentosum (XP) exhibit sensitivity to UV radiation and defective nucleotide excision repair. Complementation of these mutant phenotypes after the introduction of single human chromosomes from repair-proficient cells into XP cells has provided a means of mapping the genes involved in this disease. We now report the phenotypic correction of XP cells from genetic complementation group D (XP-D) by a single human chromosome designated Tneo. Detailed molecular characterization of Tneo revealed a rearranged structure involving human chromosomes 16 and 19, including the excision repair cross-complementing 2 (ERCC2) gene from the previously described human DNA repair gene cluster at 19q13.2-q13.3. Direct transfer of a cosmid bearing the ERCC2 gene conferred UV resistance to XP-D cells.

Blotting, Southern↗
PubMed1992

Characterization of a complex chromosomal rearrangement maps the locus for in vitro complementation of xeroderma pigmentosum group D to human chromosome band 19q13.

Microcell-mediated chromosome transfer (MMCT) is a powerful genetic technique that permits the transfer of a single chromosome from one mammalian cell to another. The utility of MMCT for gene mapping strategies is critically dependent on the careful characterization of the chromosomes being transferred. We have recently reported the identification of a single rearranged human chromosome, designated Tneo, which corrects the UV sensitivity and excision repair defect of cells of xeroderma pigmentosum genetic complementation group D (XP-D) in culture (Flejter WL et al., Proc Natl Acad Sci USA 89:261-265, 1992). Additionally, those studies demonstrated a role for the excision repair cross-complementing 2 (ERCC2) gene in the observed phenotypic correction. We now report the results of detailed conventional and molecular cytogenetic characterization of the complementing Tneo chromosome. This analysis revealed a complex rearrangement involving material from human chromosomes 16, 17, and 19. Characterization of deletions of Tneo which retained or lost XP-D complementing ability mapped the gene responsible for phenotypic correction to a small region of the terminal q-arm of this chromosome. This region includes the previously described human DNA repair gene cluster located in the region 19q13.2-q13.3, a result consistent with the notion that the in vitro correction of XP-D cells by the Tneo chromosome is rendered by the ERCC2 locus. The data illustrate the potential value of detailed cytogenetic characterization of a human chromosome present in a somatic cell hybrid, even when that material involves complex rearrangements.

Cell Line↗
PubMed1992

Comparative gene mapping in the species Muntiacus muntjac.

An extreme case of chromosomal evolution is presented by the two muntjac species Muntiacus muntjac (Indian muntjac, 2n = 6 [females], 7 [males]) and M. reevesi (Chinese muntjac, 2n = 46). Despite disparate karyotypes, these phenotypically similar species produce viable hybrid offspring, indicating a high degree of DNA-level conservation and genetic relatedness. As a first step toward development of a comparative gene map, several Indian muntjac homologs of known human type I anchor loci were mapped. Using flow-sorted, chromosome-specific Southern hybridization techniques, homologs of the protein kinase C beta polypeptide (PRKCB1) and the DNA repair genes ERCC2 and XRCC1 have been assigned to Indian muntjac chromosome 2. The male-specific ZFY gene was presumptively mapped to Indian muntjac chromosome Y2. Ultimate generation of a comparative physical map of both Indian and Chinese muntjac chromosomes will prove invaluable in the study of mammalian karyotype evolution.

Animals↗
PubMed1992

Galenia africana L. poisoning in sheep and goats: hepatic and cardiac changes.

Lesions in 4 field cases (3 sheep and 1 goat) of 'waterpens' or water belly, caused by the plant Galenia africana, are described. The clinical pathological and pathological findings in 7 sheep which were drenched with toxic plant material are also reported. Inappetence, ruminal stasis and apathy as well as tachycardia were noticed in some of the sheep towards the end of the dosing period. The most prominent clinical pathological change in the experimental animals was an increase in the activity of gamma-glutamyltransferase which in some animals occurred within days after commencement of dosing. This indicates liver involvement in the early stages of the intoxication, and at this stage no heart abnormalities were detected clinically, clinical pathologically or with cardiac function tests. Decrease in cardiac function were recorded in 2 sheep towards the end of the dosing period. Liver and heart lesions were present in all the animals. In some cases hepatic changes were mild and characterized by dilation of central veins and sinusoids and, less commonly, centrilobular fibrosis. More advanced lesions included centrilobular fibrosis and bridging between neighbouring lobules with adjacent areas of coagulative necrosis, lysis and ballooning degeneration of hepatocytes. Myocardial changes occurred in the free ventricular walls and interventricular septum and comprised hypertrophy of myocytes with consequent degeneration and necrosis and fibrosis. In cases of longer duration myocytes were diffusely atrophic with scattered groups of remaining hypertrophic fibres. The clinical pathological and pathological features suggest that G. africana is primarily hepatotoxic with myocardial involvement occurring only in the terminal stages of the intoxication.

Animals↗
PubMed1991

Functional complementation of ataxia-telangiectasia group D (AT-D) cells by microcell-mediated chromosome transfer and mapping of the AT-D locus to the region 11q22-23.

The hereditary human disease ataxia-telangiectasia (AT) is characterized by phenotypic complexity at the cellular level. We show that multiple mutant phenotypes of immortalized AT cells from genetic complementation group D (AT-D) are corrected after the introduction of a single human chromosome from a human-mouse hybrid line by microcell-mediated chromosome transfer. This chromosome is cytogenetically abnormal. It consists primarily of human chromosome 18, but it carries translocated material from the region 11q22-23, where one or more AT genes have been previously mapped by linkage analysis. A cytogenetically normal human chromosome 18 does not complement AT-D cells after microcell-mediated transfer, whereas a normal human chromosome 11 does. We conclude that the AT-D gene is located on chromosome 11q22-23.

Ataxia Telangiectasia↗
PubMed1991

Anti-kinetochore staining for single laser, bivariate flow sorting of Indian muntjac chromosomes.

Flow cytometric chromosome sorting typically relies upon dual-laser, bivariate analysis after staining with two different base pair-specific dyes for resolution of chromosomes with similar DNA content. The availability of FITC-conjugated antibodies offers the possibility of single-laser bivariate analysis when combined with propidium iodide (PI) DNA staining, but requires exploitable antigenic differences between chromosomes of interest. A technique was developed for indirect immunofluorescent anti-kinetochore staining of Indian muntjac chromosomes in suspension. Primary antibody binding within permeabilized whole cells minimized centrifugation-induced loss of chromosomal integrity. Subsequent FITC-conjugated second antibody binding was not affected by concurrent PI-counterstaining. Anti-kinetochore staining facilitated resolution of chromosomes No. 2 and X, which formed a doublet peak upon univariate DNA content analysis, as well as recognition of the Y2 peak which was indistinguishable from debris by univariate analysis. The technique allowed greater than 90% purification of each Indian muntjac chromosome.

Animals↗
PubMed1991

Perinatal mortality in lambs of ewes exposed to cultures of Diplodia maydis (= Stenocarpella maydis) during gestation.

During 1987 when cob rot was rife, perinatal losses were reported in flocks and herds that had been exposed to diplodiosis. The affected lambs or calves were either stillborn or died soon after birth. Dosing trials at the Onderstepoort Veterinary Research Institute involving 82 ewes revealed that 66% of the offspring of dams exposed to cultures of Diplodia maydis (= Stenocarpella maydis) in the second trimester of pregnancy and 87% of lambs of ewes exposed in the third trimester were born dead or died soon afterwards. A status spongiosus similar to that of the field cases was present in the white matter of the brains of all the affected lambs. The lambs of ewes that had shown nervous signs and those of ewes that had not shown such signs were equally affected. The experiments also clearly demonstrated that foetuses were much more susceptible to diplodiosis than the adults, e.g. in the third trimester 87% of the lambs were affected compared to only 44% of the dams. No perinatal mortalities were recorded in the group exposed to the culture in the first trimester. Ewes developed significant resistance to intoxication after initial exposure to the culture and both the length of gestation and birth mass of the affected lambs were less than those of their untreated counterparts.

Animal Feed↗
PubMed1990

Gene complementing xeroderma pigmentosum group A cells maps to distal human chromosome 9q.

Phenotypic complementation of xeroderma pigmentosum group A (XP-A) cells by microcell-mediated transfer of a single rearranged neo-tagged human chromosome from a human-mouse somatic cell hybrid designated K3SUB1A9-3 was reported previously. Extended growth of this human-mouse hybrid in culture led to deletion of the small arm of the human chromosome, with concomitant loss of complementing ability when introduced into XP-A cells by microcell-mediated chromosome transfer. Cytogenetic analysis of both hybrids suggests that the complementing locus is on chromosome 9q22.2-q34.3, and Southern blot analysis confirms the presence of distal chromosome 9q sequences.

Animals↗
PubMed1989

Clinical pathological changes in gousiekte, a plant-induced cardiotoxicosis of ruminants.

Twenty sheep were dosed with either Pachystigma pygmaeum or Fadogia homblei belonging to the Rubiaceae. The experimentally-induced cardiotoxicoses were monitored by various clinical pathological parameters and heart function tests. Elevated AST (aspartate transaminase) activity in the serum proved to be a more reliable indicator of cardiac damage in gousiekte than either LD (lactate dehydrogenase) or CK (creatine kinase). Persistent increases of AST activity were recorded from c. 14 days after commencement of dosing, and this activity sometimes peaked as late as 30 days after the dosing had ceased. Tachycardia and diminished heart function were registered only terminally. Lesions of gousiekte were present in all the sheep that were exposed to the plants. In a field outbreak of P. pygmaeum, where 60 out of 90 sheep died, 14 out of the 15 animals examined had increased AST levels compared with none of the 15 controls. These results indicated that increased enzyme levels can be of use to identify affected animals during latency in a natural outbreak of gousiekte.

Animals↗
PubMed1989

Human chromosome 15 confers partial complementation of phenotypes to xeroderma pigmentosum group F cells.

Microcell-mediated transfer of a single human chromosome from repair-proficient human cells to genetic complementation group F cells from the hereditary disease xeroderma pigmentosum (XP) results in partial complementation of repair-defective phenotypes. The complementing chromosome was identified by cytogenetic and molecular analysis as human chromosome 15. Transfer of this chromosome to XP-F cells restores approximately 20% of the resistance of wild-type cells to killing by UV radiation or by the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), as well as partial repair synthesis of DNA measured as unscheduled DNA synthesis. Additionally, complemented XP-F cells have an enhanced capacity for reactivation of the plasmid-borne E. coli cat gene following its inactivation by UV radiation. Phenotypic complementation of XP cells by chromosome 15 is specific to genetic complementation group F; no effect on the UV sensitivity of XP-A, XP-C, or XP-D cells was detected. The observation that phenotypic complementation is partial is open to several interpretations and does not allow the definitive conclusion that the XP-F locus is carried on chromosome 15.

Cell Division↗