Showing papers by "Stephen J. O'Brien published in 1983"

The cheetah is depauperate in genetic variation.

Abstract: A sample of 55 South African cheetahs (Acinonyx jubatus jubatus) from two geographically isolated populations in South Africa were found to be genetically monomorphic at each of 47 allozyme (allelic isozyme) loci. Two-dimensional gel electrophoresis of 155 abundant soluble proteins from cheetah fibroblasts also revealed a low frequency of polymorphism (average heterozygosity, 0.013). Both estimates are dramatically lower than levels of variation reported in other cats and mammals in general. The extreme monomorphism may be a consequence of a demographic contraction of the cheetah (a population bottleneck) in association with a reduced rate of increase in the recent natural history of this endangered species.

Unique seminal quality in the South African cheetah and a comparative evaluation in the domestic cat.

Abstract: Analysis of 40 semen samples collected by electroejaculation from 18 cheetahs revealed no major differences in seminal traits among Transvaal, South West (Namibia) or hybrid (Transvaal X South West) males. However, mean spermatozoal concentration (14.5 X 10(6) spermatozoa/ml of ejaculate) and percent motility (54.0%) were less in cheetahs than in domestic cats (147.0 X 10(6) spermatozoa/ml of ejaculate, 77.0% motility) subjected to the same electroejaculation regimen. On the average, cheetah ejaculates contained 71.0% morphologically abnormal spermatozoa compared to 29.1% aberrant spermatozoal forms in the domestic cat. These results indicate that seminal characteristics in the cheetah are markedly inferior compared to the domestic cat, particularly with respect to the incidence of pleiomorphic spermatozoa. Because a recent parallel study demonstrates that the cheetah lacks genetic variation, it appears likely that spermatozoal abnormalities are a genetic consequence of genomic homozygosity characteristic of this endangered species.

Clonal Variation of MCF-7 Breast Cancer Cells In Vitro and in Athymic Nude Mice

Abstract: The MCF-7 human breast cancer cell line and four derived variant sublines (R27, R3, R3–12, and R3–98), which were all estrogen receptor positive, as well as the receptor-deficient line, MDA-MB-231, were compared both in vitro and as heterotransplants into athymic nude mice. The cell lines and heterotransplanted tumors were evaluated in terms of growth, receptor status, morphology by light and electron microscopy, karyotype, and allozyme phenotype analyses. The R3 and R3–12 variant lines exhibited markedly retarded growth rates in vitro as compared with those of the MCF-7 parent line. The R3-12 line, which had the slowest growth rate in vitro as compared to those of the other cell lines, failed to produce tumors in nude mice after many attempts using various concentrations of tumor cell inocula. The MCF-7 and derived lines would not grow in oophorectomized animals without 17β-estradiol replacement, with one rare exception, while the MDA-MB-231 receptor-deficient line was able to grow in oophorectomized mice with and without 17β-estradiol replacement. Compared to the parent MCF-7 and R27 line in vivo and in vitro , the R3 line and its subclones had reduced progesterone receptor markedly. By light microscopy, all of the cell lines in vivo and in vitro could be identified as adenocarcinoma. Using electron microscopy, the MCF and variant lines showed better tissue organization and more squamous features in vivo than in vitro where glandular features were more prominent. The R27 line in vivo and in vitro showed the most cellular polarity and differentiation as compared to the MCF or R3 lines (R3, R3–12, and R3–98), while the R3 lines were the least differentiated of all the lines. The MDA-MB-231 line in vivo and in vitro had the most pronounced glandular features of all the lines and was not affected by changes in media additions of fetal calf serum, 17β-estradiol, or charcoal-treated calf serum as were the MCF-7- and MCF-7-derived lines. All the tumors in vivo and in vitro showed a human karyotype, and the MCF-7 and each of the derived lines showed both common and unique chromosome markers. The original cell line (MCF-7), the derived sublines (R27 and R3), and tumors derived in nude mice from these lines were typed for species identity using standard isozyme procedures and were found to be human. The allozyme phenotype at seven polymorphic human loci (allozyme genetic signature) demonstrated that the MCF-7 line and its derivatives were derived from the same individual and were distinct from the signatures of HeLa and a variety of other human breast cancer lines. This multifaceted study, using both in vivo and in vitro systems, may provide a model for better understanding the nature of tumor heterogeneity and its implications in therapeutic designs.

Dispersion of the ras family of transforming genes to four different chromosomes in man.

Abstract: Cellular transforming genes (c-onc) are evolutionarily conserved vertebrate DNA segments which have been identified by two different approaches. One group of these cellular genes has been defined by their close homology to the transforming genes of the acute transforming retroviruses (v-onc)1–3. The second group, which represent activated forms of normal cellular genes1,4–9, has been detected by the ability of certain genes from animal and human tumours to induce focal transformation of tissue culture cells. Investigation of the possibility that the same cellular gene might have given rise to both a retroviral and a tumour transforming gene revealed that two of the c-onc genes identified by transfecting genomic DNA from human tumours to murine 3T3 fibroblasts were related to the transforming genes of two closely related acute transforming retroviruses, Harvey murine sarcoma virus (HaMuSV) and Kirsten murine sarcoma virus (KiMuSV)10–12. The transforming genes of HaMuSV and KiMuSV are derived from two members of a cellular onc gene family called ras, which is a rather divergent group of normal vertebrate genes originally found by analysis of the cellular homologues of the v-onc genes of HaMuSV and KiMuSV13. Four distinct human cellular homologues of v-Ha-ras and v-Ki-ras (designated c-Ha-ras and c-Ki-ras, respectively) have been characterized14; two (c-Ha-ras-1 and c-Ha-ras-2) are more closely related to v-Ha-ras, while the others (c-Ki-ras-1 and c-Ki-ras-2) are more closely related to v-Ki-ras. On ligation with a retroviral long terminal repeat, the c-Ha-ras-1 gene of both rat and human have been shown to induce in vitro transformation of mouse NIH 3T3 cells by DNA transfection15,16. This gene and c-Ki-ras-2 have also been isolated as activated transforming genes in human tumours10–12. An understanding of the genetic relationship of the c-ras genes and additional genetic loci possibly involved in neoplastic transformation would be greatly facilitated by placement of the ras genes on the human chromosome map. Using DNA analysis of rodent×human somatic cell hybrids, we have now assigned each of the human genes to a different chromosome.

Mapping of an endogenous retroviral sequence to human chromosome 18.

Abstract: The application of recombinant DNA technologies has allowed the detection of at least three families of moderately repetitive DNA segments in the human genome that are homologous to retroviruses previously isolated from mice and primates. One of these DNA segments has been shown by nucleotide sequence comparisons to be distantly related to both Moloney murine leukaemia virus (MoMuLV) and the endogenous baboon retrovirus and to have the sequence organization characteristic of an integrated retrovirus. Isolation of the homologous locus from chimpanzee DNA indicated that the integration event preceded the evolutionary divergence of chimpanzees and man. Here we have used a panel of rodent x human somatic cell hybrids to assign the chromosomal localization of this segment, called ERV1 (endogenous retrovirus-1), to human chromosome 18 (HSA 18).

Murine retroviral restriction genes Fv-4 and Akvr-1 are alleles of a single locus.

Abstract: The two murine retroviral restriction genes, Fv-4 and Akvr-1, are very similar in their effects, distributions, ranges of action, and phenotypes. Akvr-1 has been shown to segregate independently in backcrosses with a variety of retroviral restriction loci, including Fv-1, Fv-2, Ril-1, and Ril-2. An allelism test cross of FRG (Fv-4R) X LCRR (Akvr-1R) hybrids mated to AKR mice failed to produce any viremic offspring. These results suggested that Akvr-1R and Fv-4R are alleles of a single locus, Fv-4, on mouse chromosome 12.

Mycoplasma hominis- tissue cell interactions: a review with new observations on phenotypic and genotypic properties.

Abstract: Strains of Mycoplasma hominis isolated from different tissues of patients with a variety of disease processes and from cell culture substrates show marked phenotypic and genotypic heterogeneity, as determined by their antigenic and isozyme properties and by [3H]DNA-DNA hybridization and DNA cleavage pattern analyses. Strains isolated from the same tissues (blood of postpartum patients or human urogenital tract or cell culture substrates) have very high genomic homology and form clusters of similar strains. Clusters of strains that colonize similar specialized urogenital tissues may initiate diseases that reflect damage to the particular tissue colonized. Antigenic markers such as those for attachment components for different strain clusters may help determine the role, if any, played by strain differences in the etiology of a family of urogenital diseases.

On the distribution and characteristics of isozyme expression in Mycoplasma, Acholeplasma, and Ureaplasma species.

Abstract: A summary of a survey of three genera of mycoplasmatales (Mycoplasma, Acholeplasma, and Ureaplasma) for isozyme expression is presented. Isozyme analysis of mycoplasmas has been employed in at least three distinct areas: (1) as genetic markers for identification, individualization, and taxonomic classification; (2) as markers for cell culture contamination; and (3) as a qualitative measure of the operative metabolic pathways in the diverse species. We have found five ubiquitous enzymes: purine nucleoside phosphorylase, adenylate kinase, inorganic pyrophosphatase, dipeptidase, and esterase. Three enzymes, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, and superoxide dismutase, were restricted to Acholeplasma species and were not detected in Mycoplasma or Ureaplasma. Four glycolytic enzymes, glucose phosphate isomerase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase, were restricted to those species of Mycoplasma and Acholeplasma capable of glucose fermentation. Two of these glycolytic enzymes, glucose phosphate isomerase and lactate dehydrogenase, were detected in serovars I and II of U. urealyticum, which is inconsistent with the non-glycolytic activity in this genus.

Parallels of Genomic Organization and of Endogenous Retrovirus Organization in Cat and Man

Abstract: A combination of technical advances (most notably heterologous cell fusion, high resolution G-banding, and molecular cloning) has contributed to an accelerated advance in genetic analysis in mammals. The present human genetic map contains over 400 gene assignments and the map is growing rapidly as each new molecular clone or immunological reagent is developed. In our laboratory, we have developed a panel of rodent X human somatic cell hybrids that have been utilized in chromosome assignment of several classes of genes including oncogenes (ras, raf) and endogenous human retroviral sequences (ERVL, 2, etc). Using similar techniques, a biochemical genetic map of the domestic cat has been derived. The cat has 19 chromosome pairs and, to date, 40 genes have been mapped to 16 linkage or syntenic groups. Comparison of linkage relationships between homologous enzymes has revealed a striking conversation of chromosomal linkage association between cat and man. A comparison of syntenically homologous, highly extended high resoultion G-banded chromosomes between the two mammalian families revealed that 20–25%, by length, of the human karyotype can be precisely aligned (chromomere to chromomere) between cats and man despite the evolutionary divergence of the species nearly 80 million years ago. Moderately repetitive families of retrovirus-related DNAs exist within the feline and the human genomes. We have isolated molecular clones of several members of the feline RD-114 retrovirus family from a genomic library of normal cat cellular DNA. The endogenous sequences analyzed were similar to each other in that they were colinear with RD-114 proviral DNA, were bounded by long terminal redundancies, and conserved many restriction sites in the gag and pol regions. Several sequences were apparently deleted, relative to the previously characterized inducible RD-114 genome. The env regions of a number of endogenous RD-114 sequences examined were substantially deleted or diverged; a subset of these sequences contained information at the position of the env region that was not homologous to inducible RD-114. The RD-114 virogenes were dispersed to several cat chrosomes that were localized using a panel of rodent x cat somatic cell hybrids. A comparison of the genetic properties of endogenous human retroviral sequences revealed several similarities between the human and feline status of endogenous retroviruses.