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Biomedical subjects

R E Fournier

Publications and source records attributed to R E Fournier.

At least 73 records · Page 4Linked to original sources
PubMed1986

Assignment of genes encoding dihydrofolate reductase and hexosaminidase B to Mus musculus chromosome 13.

The murine gene encoding dihydrofolate reductase (DHFR) has been localized to a particular mouse chromosome by complementation mapping. Microcells prepared from diploid mouse fibroblasts were fused with mutant hamster cells lacking the dihydrofolate reductase gene (Dhfr), and DHFR+ microcell hybrids were selected in medium lacking purines and pyrimidines. The complemented hybrids expressed wild-type levels of DHFR enzyme activity, and selectively retained a single mouse chromosome--chromosome 13. Genomic Southern blots of DNAs prepared from these hybrids and from an independently derived collection of clones isolated without selection on the DHFR phenotype confirmed the assignment of murine Dhfr gene sequences to chromosome 13. This assignment provides further evidence for the existence of genetic homology between regions of mouse chromosome 13 and human chromosome 5. In support of this view, we show that the gene encoding hexosamindase B, a chromosome 5 marker in man, also maps to mouse chromosome 13.

Animals↗
PubMed1986

Assignment of the human and mouse prion protein genes to homologous chromosomes.

Purified preparations of scrapie prions contain one major macromolecule, designated prion protein (PrP). Genes encoding PrP are found in normal animals and humans but not within the infectious particles. The PrP gene was assigned to human chromosome 20 and the corresponding mouse chromosome 2 using somatic cell hybrids. In situ hybridization studies mapped the human PrP gene to band 20p12----pter. Our results should lead to studies of genetic loci syntenic with the PrP gene, which may play a role in the pathogenesis of prion diseases or other degenerative neurologic disorders.

Animals↗
PubMed1986

Tissue-specific expression of hepatic functions. Genetic aspects.

Intertypic hybrids formed by fusing dissimilar cell types generally fail to express the tissue-specific products of either parent, a phenomenon termed extinction. To explore the genetic basis and phenotypic complexity of this phenomenon, 15 liver-specific cDNA clones have been used as probes to assay expression of the corresponding mRNAs in a series of hepatoma x fibroblast hybrids retaining different fibroblast chromosomes. Thirteen of the 15 liver-specific mRNAs were extinguished in karyotypically complete hepatoma x fibroblast hybrids, but were reexpressed in hybrid segregants that had eliminated fibroblast chromosomes. In three cases analyzed in detail, extinction of a particular hepatic marker required the presence of a single, specific fibroblast chromosome. These data define and localize a set of discrete genetic loci, the Tse loci, that negatively regulate liver-specific gene expression in trans.

Albumins↗
PubMed1986

Parasexual approaches to the study of human genetic disease.

We have used two different strategies to construct hybrid cells in which specific, individual human chromosomes or fragments thereof are maintained by direct selective pressure. Our first approach was to introduce a drug-resistance gene into human chromosomes using a retroviral vector, and to transfer the marked chromosomes via microcells into mouse cells. The second method was to fuse gamma-irradiated human cells with rodent cells to produce hybrids containing fragments of the human X chromosome. Such hybrid cell lines should greatly facilitate both human gene mapping and the isolation of human genes by molecular cloning. The gene-transfer technologies described here can also be used to construct cell lines in which the expression of genes involved in human diseases can be studied in vitro.

Chromosome Aberrations↗
PubMed1985

Assignment of the gene encoding cytosolic phosphoenolpyruvate carboxykinase (GTP) to Mus musculus chromosome 2.

The structural gene Pck-1, encoding cytosolic phosphoenolpyruvate carboxykinase (GTP) (PEPCK; EC 4.1.1.32), has been assigned to mouse chromosome 2. This assignment was made based on genomic Southern transfer of rat-mouse and hamster-mouse hybrid DNAs using a rat kidney cloned cDNA of the PEPCK gene as a probe. Conclusive evidence for the cosegregation of the PEPCK gene with mouse chromosome 2 was obtained using a monochromosomal microcell hybrid that selectively retained a Robertsonian translocation between mouse autosomes 2 and 8 and its back-selected hybrid clone.

Animals↗
PubMed1985

Chromosomal assignment and trans regulation of the tyrosine aminotransferase structural gene in hepatoma hybrid cells.

The structural gene encoding liver-specific tyrosine aminotransferase (TAT; EC 2.6.1.5) was assigned to mouse chromosome 8 by screening a series of hybrid cell lines for retention of murine Tat-1 gene sequences by genomic Southern blotting. This assignment demonstrated that the Tat-1 structural gene was not syntenic with Tse-1, a chromosome 11-linked locus that negatively regulates TAT expression in trans (A. M. Killary and R. E. K. Fournier, Cell 38:523-534, 1984). We also showed that the fibroblast Tat-1 gene was systematically activated in hepatoma X fibroblast hybrids retaining fibroblast chromosomes 8 in the absence of chromosome 11 but was extinguished in cells retaining both fibroblast chromosomes. Thus, the TAT structural genes of both parental cell types were coordinately regulated in the intertypic hybrids, and the TAT phenotype of the cells was determined by the presence or absence of fibroblast Tse-1.

Animals↗
PubMed1984

Identification of a cAMP regulatory region in the gene for rat cytosolic phosphoenolpyruvate carboxykinase (GTP). Use of chimeric genes transfected into hepatoma cells.

cAMP stimulates the transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) in rat liver. We have investigated the nucleotide sequences required for regulation of PEPCK gene expression by cAMP. A chimeric gene was constructed in which a 620-base pair fragment of the 5'-end of the PEPCK gene (including 547 base pairs of 5'-flanking sequence) was ligated to the herpes simplex virus thymidine kinase (TK) structural gene. The PEPCK promoter fragment was introduced either in the proper orientation for transcription of the TK gene or in the opposite orientation. These fusion genes and the parent vector, pOPF, which contains the intact TK gene, were transfected individually into TK-deficient FTO-2B rat hepatoma cells. FTO-2B cells contain an active endogenous PEPCK gene which is stimulated by cAMP. Cells were selected in HAT medium and grown either as mass cell cultures or as individual clones. Dibutyryl cyclic AMP (Bt2cAMP) plus theophylline (16 h) stimulated TK activity 1.6-6.1-fold in cell lines transfected with the PEPCK-TK fusion gene containing the PEPCK promoter fragment in the correct orientation. However, the intact TK gene was not induced by Bt2cAMP after transfection, nor was there any expression of the PEPCK-TK fusion gene in cells which contained the PEPCK promoter fragment in the wrong transcriptional orientation. Bt2cAMP also increased the levels of TK mRNA in cells transfected with the PEPCK-TK fusion gene, but not in cells transfected with the intact TK gene. The chimeric PEPCK-TK mRNA initiated at the PEPCK start site, as determined by S1 nuclease mapping. There was no relationship between the number of copies of the PEPCK-TK gene integrated in the various cell lines and either the basal level of TK activity or its inducibility of Bt2cAMP.

Animals↗
PubMed1984

Isolation of thymidine kinase-deficient rat hepatoma cells by selection with bromodeoxyuridine, Hoechst 33258, and visible light.

The photosensitivity of bromodeoxyuridine (BrdU)-substituted cells is known to be markedly enhanced by the fluorochrome Hoechst 33258. Since the incorporation of BrdU into nucleic acids depends upon its prior phosphorylation via thymidine kinase (TK; EC 2.7.1.21), cells deficient in TK activity are refractory to photoinduced killing. These observations suggested that combined treatment with BrdU, Hoechst 33258, and visible light would constitute an efficient selective strategy for the recovery of TK- mutant cells. In this report we describe a single-step selection protocol which reduced the survival of TK+ cells by a factor of 10(5) without affecting the viability of TK- mutants. This procedure was used to isolate H4IIEC3 -derived rat hepatoma cells deficient in TK activity. The properties of several TK- hepatoma clones are discussed.

Animals↗
PubMed1984

An isozyme-specific selective system for the recovery of mammalian cells deficient in hepatic alcohol dehydrogenase activity.

A selective system toxic towards mammalian cells expressing the liver-specific isozyme of alcohol dehydrogenase (L-ADH) has been developed. A number of alpha-unsaturated primary and secondary alcohols were assayed for their ability to serve as substrates for rat liver ADH and were screened for cytotoxicity towards L-ADH+ and L-ADH- cells. 1-Propen-3-ol and 1-penten-3-ol were identified as agents showing selective cytotoxicity. Reconstruction experiments demonstrated that 1-propen-3-ol at a concentration of 15 microM could be used to recover L-ADH- clones from mixed populations of L-ADH+ and L-ADH cells. Cells expressing the non-allelic S-ADH isozyme were not killed under these conditions. The selective system defined in this report is thus isozyme-specific.

1-Propanol↗
PubMed1984

A genetic analysis of extinction: trans-dominant loci regulate expression of liver-specific traits in hepatoma hybrid cells.

Extinction is an operational term that refers to the lack of expression of tissue-specific traits that is generally observed in hybrid cells formed by fusing dissimilar cell types. To define the genetic basis of this phenomenon, a series of rat hepatoma x mouse fibroblast hybrids has been isolated and characterized. We report here that the extinction of hepatic marker traits in these clones was strictly correlated with the retention of five particular fibroblast chromosomes (autosomes 8, 9, 10, 11, and 13). In order to dissect this correlation into its component parts, hepatoma microcell hybrids containing single, specific fibroblast chromosomes were constructed. Hepatoma clones retaining only fibroblast chromosome 11 were specifically extinguished for liver-specific tyrosine aminotransferase (TAT) expression, while expression of four other hepatic traits and of numerous constitutive markers was unaffected. Furthermore, removal of fibroblast chromosome 11 from the populations by back-selection resulted in reexpression of TAT activity to full parental levels. These data define and localize a genetic locus, tissue-specific extinguisher-1 (Tse-1), which regulates hepatic TAT expression in trans. We also provide evidence that human Tse-1 resides on the homologous chromosome (human chromosome 17), and that hybrids retaining active Tse-1 loci lack TAT-specific mRNA.

Animals↗
PubMed1984

Cellular homologs of the avian erythroblastosis virus erb-A and erb-B genes are syntenic in mouse but asyntenic in man.

Avian erythroblastosis virus, a retrovirus that causes erythroblastosis and sarcomas in infected birds, possesses two host cell-derived genes [viral (v) erb-A and erb-B]. Although v-erb-B seems to be responsible for oncogenic transformation, v-erb-A might have an enhancing effect on transformation. In chickens, the natural host for avian erythroblastosis virus, cellular (c) erb-A and erb-B genes appear to be unlinked, but their chromosomal locations in other species are unknown. To ascertain the chromosomal location of c-erb genes in man and mouse, we analyzed interspecies somatic cell and microcell hybrids by Southern filter hybridization techniques using specific v-erb-A and v-erb-B probes. We found c-erb-A sequences on human chromosome 17 (17p11----qter) and located c-erb-B on human chromosome 7 (7pter----q22). In contrast, both c-erb-A and c-erb-B reside on mouse chromosome 11.

Alpharetrovirus↗
PubMed1983

Multigene family for sarcomeric myosin heavy chain in mouse and human DNA: localization on a single chromosome.

Cloned myosin heavy chain DNA probes from rat and human were hybridized to restriction endonuclease digests of genomic DNA from somatic cell hybrids and their parental cells. The mouse myosin heavy chain genes detectable by this assay were located on chromosome 11, and three different human sarcomeric myosin heavy chain genes were mapped to the short arm of chromosome 17. A synteny between myosin heavy chain and two unrelated markers, thymidine kinase and galactokinase, was found to be preserved in the rodent and human genomes.

Animals↗
PubMed1983

Complementation mapping in microcell hybrids: localization of Fpgs and Ak-1 on Mus musculus chromosome 2.

The gene encoding folylpolyglutamyl synthetase (FPGS) was assigned to mouse chromosome 2 by complementation mapping. Chinese hamster ovary cells (AuxBl) deficient in FPGS, and consequently auxotrophic for glycine, adenosine, and thymidine (gat-), were employed as recipients in microcell-mediated chromosome transfer experiments. Mouse chromosomes derived from diploid embryo fibroblasts were introduced into hamster AuxBl cells, and gat+ microcell hybrids were selected in medium lacking adenosine and thymidine. Mouse chromosome 2 was the only donor chromosome whose presence correlated with expression of FPGS activity. Furthermore, every gat+ hybrid clone expressed murine AK-1, a marker previously assigned to chromosome 2. Eight of 20 clones analyzed retained deletion chromosomes derived from mouse chromosome 2. These clones were used to localize murine Fpgs and Ak-1 to a region of this chromosome, namely 2 (cen leads to Cl).

Adenosine↗
PubMed1983

Microcell-mediated transfer of carcinogen-induced ouabain resistance from C3H/10T1/2 Cl 8 mouse fibroblasts to human cells.

We previously developed a quantitative assay for measuring the induction of ouabain-resistant (Ouar) variants in transformable C3H/20T1/2 Cl 8 mouse fibroblasts following treatment of the cells with chemical carcinogens. To further define the nature of the Ouar phenotype, we conducted microcell-mediated chromosome transfer studies using Ouar cell lines induced by chemical carcinogens in C3H/10T1/2 Cl 8 cells as donors and 8-azaguanine-resistant (Azgr) derivatives of the human cell lines, D98/AH2 and HT 1080, as recipients. Microcells prepared from one spontaneous and two carcinogen-induced Ouar mouse cell lines were able to transfer resistance to 0.01 and 1 mM Oua to ouabain-sensitive D98 and HT 1080 cells. The frequency of microcell hybrid formation ranged from 10(-6) to 10(-5). Karyotypic analysis of the microcell hybrids indicated that the Ouar phenotype of C3H/10T1/2 Cl 8 derivatives mapped to mouse chromosome 3, the chromosome to which the wild-type murine Oua-1 allele had previously been assigned. These studies show that both spontaneous and chemically induced high level Ouar phenotypes of C3H/10T1/2 Cl 8 mouse fibroblasts can be transferred via microcell-mediated chromosome transfer, and provide strong genetic evidence that chemically induced Ouar phenotypes of C3H/10T1/2 Cl 8 cells arise from mutations at Oua-1. In addition, this study sufficiently standardizes microcell-mediated chromosome transfer in the C3H/10T1/2 Cl 8 cell line so that this technique can be used to investigate the nature of other phenotypic changes in these cells, such as the chemically transformed phenotype.

Animals↗
PubMed1982

Construction of microcell hybrid clones containing specific mouse chromosomes: application to autosomes 8 and 17.

Fibroblast cultures prepared from mice homozygous for a Robertsonian translocation (centric fusion) between autosomes 8 and 17 [Rb(8.17)] were used as donors in microcell-mediated chromosome transfer experiments. By using hamster recipient cells deficient in adenine phosphoribosyltransferase (APRT-) and selecting for expression of murine APRT (a chromosome 8 marker), microcell hybrids were isolated which retained only the mouse Rb(8.17) translocation in addition to the hamster chromosome complement. The translocation was stable in cells maintained under APRT+ selective pressure, and mouse marker traits encoded by genes on both chromosomes 8 and 17 segregated concordantly. A second family of hybrid clones was constructed by fusing microcells derived from wild-type mouse fibroblasts with APRT- hamster cells. Four of six clones analyzed retained only mouse chromosome 8. These studies demonstrated that microcell hybrids containing specific Robertsonian translocations as the only donor-derived genetic material can be obtained. Furthermore, a number of Robertsonian translocations between chromosomes which carry selectable markers (chromosomes 3, 8, and 11) and other autosomes have been described. By using fibroblast cultures prepared from mice containing these translocations as donors in microcell fusions, 18 of the 20 mouse chromosomes could be selectively fixed in different hybrid clones. Thus, a collection of 20 hybrid clones, each containing a single, specific mouse chromosome, can be constructed by using the strategy described in this report. The potential utility of such a monochromosomal hybrid panel is discussed.

Adenine Phosphoribosyltransferase↗
PubMed1981

A general high-efficiency procedure for production of microcell hybrids.

The relative efficiency of microcell-mediated chromosome transfer vs. somatic cell hybridization has been determined. The prolonged mitotic arrest generally used to micronucleate donor cells also reduced the fusion efficiency to 1/10th-1/5th of that in whole cell hybridizations. Here we report an alternative micronucleation protocol, involving sequential treatment of the donor cells with Colcemid and cytochalasin B, which yielded micronucleated cells that hybridized with the same efficiency as whole cells. The enucleation, purification, and fusion steps of the microcell procedure have also been refined. By using these modifications the practical yield of microcell hybrid clones can be increased 50- to 100-fold.

Adenine Phosphoribosyltransferase↗
PubMed1979

Assignment of the gene governing cellular ouabain resistance to Mus musculus chromosome 3 using human/mouse microcell hybrids.

Human/mouse microcell hybrids were used to establish the assignment of the gene governing resistance to the cardiac glycoside ouabain (Oua-1) to Mus musculus chromosome 3. Microcells were prepared from primary mouse embryo fibroblasts and fused with HeLa S3 cells, and microcell hybrids were isolated and maintained in medium containing 10(-6) M ouabain. Resistance to ouabain was not expressed concordantly with any of 26 murine isozyme markers. Karyotypic analysis of five primary clones showed that one to five murine chromosomes had been transferred from donor to recipient in these experiments. Only mouse chromosome 3 was common to all ouabain-resistant primary clones. Both ouabain-resistant and -sensitive subclones were isolated from hybrids grown in the absence of selective pressure, and karyotyping showed that loss of resistance to ouabain was concordant with the loss of murine chromosome 3.

Animals↗