Search PubMed⌕ Search

Biomedical subjects

R E Fournier

Publications and source records attributed to R E Fournier.

At least 37 records · Page 2Linked to original sources
PubMed1995

The gene for the serpin thrombin inhibitor (PI7), protease nexin I, is located on human chromosome 2q33-q35 and on syntenic regions in the mouse and sheep genomes.

Protease nexin I (PNI) is the most important physiologic regulator of alpha-thrombin in tissues. PNI is highly expressed and developmentally regulated in the nervous system where it is concentrated at neuromuscular junctions and also central synapses in the hippocampus and striatum. Approximately 10% of identified proteins at mammalian neuromuscular junctions are serine protease inhibitors, consistent with their central role in balancing serine protease activity to develop, maintain, and remodel synapses. Southern blot hybridization of PNI cDNA to somatic cell hybrids placed the structural gene for PNI (locus PI7) on human chromosome 2q33-q35 and to syntenic chromosomes in the mouse (chromosome 1) and sheep (chromosome 2).

Amyloid beta-Protein Precursor↗
PubMed1995

Cloning mammary cell cDNAs from 17q12-q23 using interspecific somatic cell hybrids and subtractive hybridization.

We have cloned human genes that are encoded in the region 17q12-q23 and expressed in breast tissue using interspecific somatic cell hybrids and subtractive hybridization. Two mouse microcell hybrids containing fragments of human chromosome 17 with a nonoverlap region at 17q12-q23 were generated by microcell transfer. Radiolabeled cDNA was synthesized from the hybrid cell containing the 17q12-q23 interval and was subtracted with an excess of RNA from the hybrid cell lacking the interval. Resulting cDNA probes enriched for sequences from 17q12-q23 were used to screen a human premenopausal breast cDNA library, and 60 cDNAs were identified. Three of these cDNAs mapped to the hybrid cell nonoverlap region. These cDNAs were expressed in mammary epithelial cell hybrids, although none appeared to be breast-specific. Sequence analysis of the cDNAs revealed that clone 93A represents a previously unidentified gene, clone 98C has homology to an expressed sequence tag from goat mammary tissue, and clone 200A is identical to the human homologue of the Drosophila melanogaster flightless-I gene. These genes map outside a 1-cM region linked to early onset familial breast cancer but may be useful genetic markers in the 17q12-q23 region.

Animals↗
PubMed1995

Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain.

The human apolipoprotein B (apoB) gene resides within a 47.5-kb chromatin domain that is flanked by sequences that bind to the nuclear matrix. These matrix attachment regions (MARs) are boundaries between nuclease-sensitive and -resistant chromatin. As domain boundaries are thought to function as insulator elements, shielding sequences between them from effects of neighboring chromatin, this raised the possibility that the apoB MARs have functions that could be assayed by transfection. To test this possibility, we examined effects of the apoB MARs on transgene expression in transiently and stably transfected rat and human hepatoma cells. The apoB MARs had no effects on expression of transiently transfected reporters, but they altered expression of stably integrated transgenes in dramatic and reproducible ways. Single integrated copies of transgenes that contained the apoB promoter and second intron enhancer, which are sufficient for high-level expression in transient assays, were expressed at low and variable levels in stable transfectant clones. In contrast, transgenes containing the apoB 5' and 3' MARs were expressed at levels nearly 200-fold higher than levels of the minimal reporters in stable transfectants, and expression was position independent. Transgenes that contained the apoB MARs and an additional 3.3 kb of apoB 5' flanking sequence were also expressed in an elevated, position-independent manner. Surprisingly, tandem transgene arrays in multicopy transfectants were transcriptionally inactive. These observations suggest that the apoB MARs function as insulator elements, shielding transgene expression from effects of neighboring chromatin domains.

Animals↗
PubMed1994

Tissue-specific extinguisher loci in the murine genome: a screening study based on a rat/mouse microcell hybrid panel.

Extinction of tissue-specific traits in intertypic somatic cell hybrids is a well-known phenomenon. In the past few years, microcell hybrids have been used in attempts to dissect this phenotype genetically, and tissue-specific extinguisher loci have been mapped to two different mouse chromosomes. When transferred from fibroblast into hepatoma cells by microcell fusion, these loci down-regulate expression of specific liver genes in trans. However, other liver genes that are extinguished in genotypically complete hybrids seem not to be extinguished in monochromosomal hybrids. To assess the generality of monochromosomal extinction phenotypes, we assembled a collection of rat hepatoma/mouse fibroblast microcell hybrids that represent most of the mouse chromosome complement, and we screened them for expression of a large number of liver-specific genes. Phosphoenolpyruvate carboxykinase gene expression was down-regulated in hybrids containing mouse chromosome 7 or mouse chromosome 11, but other extinction phenotypes were not readily apparent. These results indicate that extinction of many liver genes may be a polygenic trait.

Animals↗
PubMed1994

Tissue-specific extinguisher loci in the human genome: a screening study based on random marking and transfer of human chromosomes.

Expression of many liver-specific genes is extinguished when cultured hepatoma cells are fused with fibroblasts, but liver genes can be reexpressed in hybrid segregants that have lost fibroblast chromosomes. To map extinguisher loci involved in this process, hepatoma microcell hybrids retaining single fibroblast chromosomes have been employed. Two different, transdominant loci that affect liver gene expression have been defined in this way. To determine whether other monochromosomal extinction phenotypes could be observed, we inserted a selectable marker into many human chromosomal sites and transferred the marked human chromosomes into rat hepatoma recipient cells by microcell fusion. Nearly 200 microcell hybrid clones were isolated and screened for expression of liver-specific mRNAs. Most liver transcripts continued to be expressed. However, PEPCK mRNA was extinguished in 12 hybrid clones. Some of these hybrids contained human TSE1, the previously characterized extinguisher locus on chromosome 17, but others contained a novel extinguishing function that mapped to human chromosome 14. The implications of these findings are discussed.

Animals↗
PubMed1994

Genetic analysis of a transcriptional activation pathway by using hepatoma cell variants.

A hierarchy of liver-enriched transcription factors plays an important role in activating expression of many hepatic genes. In particular, hepatocyte nuclear factor 4 (HNF-4) is a major activator of the gene encoding HNF-1, and HNF-1 itself activates expression of more than 20 liver genes. To dissect this activation pathway genetically, we prepared somatic cell variants that were deficient in expression of the liver-specific alpha 1-antitrypsin (alpha 1AT) gene, which requires both HNF-1 and HNF-4 for high-level gene activity. This was accomplished in two steps. First, hepatoma transfectants that stably expressed two selectable markers under alpha 1AT promoter control were prepared; second, variant sublines that could no longer express either transgene were isolated by direct selection. In this report, we demonstrate that the variants contain defects in the HNF-4/HNF-1 activation pathway. These defects functioned in trans, as expression of many liver genes was affected, but the variant phenotypes were recessive to wild type in somatic cell hybrids. Three different variant classes could be discriminated by their phenotypic responses to ectopic expression of either HNF-4 or HNF-1. Two variant clones appeared specifically deficient in HNF-4 expression, as transfection with an HNF-4 expression cassette fully restored their hepatic phenotypes. Another line activated HNF-1 in response to forced HNF-4 expression, but activation of downstream genes failed to occur. One clone was unresponsive to either HNF-1 or HNF-4. Using the variants, we demonstrate further that the chromosomal genes encoding alpha 1AT, aldolase B, and alpha-fibrinogen display strict requirements for HNF-1 activation in vivo, while other liver genes were unaffected by the presence or absence of HNF-1 or HNF-4. We also provide evidence for the existence of an autoregulatory loop in which HNF-1 regulates its own expression through activation of HNF-4.

Animals↗
PubMed1992

Multiple elements regulate phosphoenolpyruvate carboxykinase gene expression in hepatoma hybrid cells.

The phosphoenolpyruvate carboxykinase (PEPCK) gene is highly expressed in cultured rat hepatoma cells, but extinguished in hepatoma x fibroblast hybrids. Extinction of PEPCK gene expression in hybrids is a polygenic process that involves several fibroblast loci, only one of which (tissue-specific extinguisher-1, TSE1) has been characterized to date. To identify sequence elements of the PEPCK gene that are involved both in TSE1-mediated extinction and in TSE1-independent processes, we assayed expression of chimeric PEPCK transgenes in transiently and stably transfected hybrid cells. We report that TSE1 responsiveness mapped to the PEPCK CRE (cAMP response element), as shown previously for the tyrosine aminotransferase gene. This was expected from the recent identification of the TSE1 gene product as a regulatory subunit of protein kinase A. However, none of the transgenes we assayed were responsive to TSE1-independent extinction mechanisms, suggesting that these controls require DNA sequences and/or chromatin structures that were not present in the transfected reporters. The implications of these findings are discussed.

Animals↗
PubMed1992

Direct selection of hepatoma cell variants deficient in alpha 1-antitrypsin gene expression.

Expression plasmids containing the human alpha 1-antitrypsin (alpha 1 AT) promoter fused to either adenine phosphoribosyltransferase (aprt) or xanthine-guanine phosphoribosyltransferase (gpt) coding sequences were sequentially introduced into APRT- HPRT- rat hepatoma cells. Stable transfectants expressing both transgenes were isolated and characterized. Nonexpressing variants were subsequently obtained by selecting against expression of one or both transgenes. Variants isolated by selecting against expression of either transgene alone generally displayed deficiency phenotypes in cis, as only three of 20 clones tested were affected for expression of alpha 1AT mRNA. In contrast, double selection yielded predominantly trans effects: 12 of 14 lines tested showed impaired ability to express their chromosomal alpha 1AT genes. Furthermore, expression of several other liver genes, including the gene encoding the HNF-1 trans-activator, was repressed in many of the variant lines. Thus, double selection using chimeric transgenes is a useful approach for generating variant cell lines deficient in expression of specific mammalian genes.

Adenine Phosphoribosyltransferase↗
PubMed1992

Generation of region- and species-specific expressed gene probes from somatic cell hybrids.

Human genes expressed in interspecific somatic cell hybrids can be cloned specifically by subtractive cDNA hybridization. This approach is based on the observation that cDNA fragments from noncoding segments of mature human transcripts do not form stable heteroduplexes with their rodent homologues under high-stringency hybridization conditions. Thus, small, oligo-dT primed cDNAs from a rat/human hybrid retaining a fragment of human chromosome 17 were enriched for human sequences by hybridization with RNA from a sister clone containing a smaller human chromosome fragment. The enriched probe was used to screen a human cDNA library, and nine expressed genes from within the non-overlap region were obtained. This method should be useful for cloning active human genes from defined chromosome segments.

Animals↗
PubMed1992

Extinction of alpha 1-antitrypsin gene expression in somatic cell hybrids: evidence for multiple controls.

Expression of the liver-specific alpha 1-antitrypsin (alpha 1AT) gene is extinguished in hepatoma/fibroblast hybrids. To define the mechanism of extinction, we identified DNA sequences involved in this process by transiently transfecting mutant alpha 1AT promoters into parental and hybrid cells. The wild-type alpha 1AT promoter (-554 to +44 bp) was highly expressed in rat hepatoma cells, but activity was 100-fold less in fibroblasts or cell hybrids. Mutations in this region failed to activate alpha 1AT expression in nonhepatic cells, but mutations in the binding site for liver factor B1 (LF-B1) reduced hepatic-specific expression greater than 100-fold. Furthermore, the hybrid cells failed to express LF-B1-binding activity and mRNA. This suggested that alpha 1AT extinction in hybrids might be an indirect, lack-of-activation phenotype mediated primarily through repression of LF-B1. To test this possibility, we stably transfected an LF-B1 expression cassette into parental and hybrid cells and monitored expression of transfected and endogenous alpha 1AT genes. Surprisingly, although constitutive LF-B1 expression could activate alpha 1AT-CAT transgenes in these cells, it neither prevented nor reversed extinction of the chromosomal alpha 1AT genes. We conclude that although extinction of the LF-B1 trans-activator accompanies alpha 1AT extinction in cell hybrids, it does not play a causal role in this process.

Animals↗
PubMed1991

The relaxin gene is located on chromosome 19 in the mouse.

Relaxin is a polypeptide hormone that exerts a variety of physiological effects during pregnancy. To investigate the possibility that known genetic mutations affecting aspects of reproductive physiology result in alterations in the structure or production of relaxin, we have determined the chromosomal location of the mouse gene. The finding of a BamHI restriction fragment length polymorphism in AKR mice enabled us to use recombinant inbred strains to make an assignment to chromosome 19. This was confirmed by Southern analysis of DNA from microcell hybrids.

Animals↗
PubMed1991

Subtractive hybridization cloning of a tissue-specific extinguisher: TSE1 encodes a regulatory subunit of protein kinase A.

Tissue-specific extinguisher 1 (TSE1) is a trans-acting locus on human chromosome 17 that down-regulates expression of seven liver genes in hepatoma x fibroblast hybrids. To study the mechanism by which TSE1 functions, we used subtractive cDNA hybridization to clone transcripts encoded within a 2-4 Mb segment of chromosome 17 that includes TSE1. High resolution mapping within this region indicated that 8 of 9 different human cDNAs so obtained were distinct from TSE1. The remaining cDNA clone mapped concordantly with TSE1 in a panel of fragment-containing hybrids. DNA sequencing indicated that this cDNA encoded regulatory subunit RI alpha of cAMP-dependent protein kinase, and RI alpha mRNA levels correlated with TSE1 activity in various hybrid lines. Stable transfection of wild-type or cAMP-binding mutant RI alpha alleles into hepatoma recipients produced an extinction phenotype indistinguishable from that encoded by human TSE1. We conclude that TSE1 encodes a regulatory subunit of protein kinase A whose activity differs in different cell types.

Base Sequence↗
PubMed1990

Two genetically defined trans-acting loci coordinately regulate overlapping sets of liver-specific genes.

Mice homozygous for deletions around the albino locus fail to activate expression of a set of neonatal liver functions and die shortly after birth. This phenotype is thought to result from the loss of a positive transacting factor, denoted alf, in deletion homozygotes. Using differential cDNA screening, we isolated and characterized genes whose cell type-specific transcription is affected by alf and found as a common feature that expression of these genes is induced by glucocorticoids and cAMP. Surprisingly, a subset of these alf-responsive genes is negatively controlled by the tissue-specific extinguisher locus Tse-1. Administration of glucocorticoids and cAMP leads to reversal of Tse-1-mediated extinction of these genes. These results show that two trans-acting factors coordinately regulate expression of overlapping sets of liver-specific genes. We suggest that both the lethal phenotype and the extinguished state result from interference with hormone signal transduction.

Animals↗
PubMed1990

A cyclic AMP response element mediates repression of tyrosine aminotransferase gene transcription by the tissue-specific extinguisher locus Tse-1.

Tyrosine aminotransferase (TAT) gene expression is liver specific and inducible by glucocorticoids and via the cAMP signaling pathway. In fibroblasts and other nonliver cells the gene is subject to negative control by the trans-dominant tissue-specific extinguisher locus Tse-1. We identified a hepatocyte-specific enhancer that is repressed by Tse-1. Two distinct sequence motifs are absolutely essential for function of this enhancer: a cAMP response element (CRE), which is the target for repression by Tse-1, and a hepatocyte-specific element. The specificity of the enhancer is generated by the combination of these two essential elements, which are fully interdependent. In vivo footprinting indicates that Tse-1 acts by affecting protein binding at the CRE. A direct antagonism between Tse-1 and the cAMP signaling pathway suggests that Tse-1 plays a role in control of developmental activation of the TAT gene.

Animals↗
PubMed1990

Clustering of cytokine genes on mouse chromosome 11.

The presence of positionally conserved amino acid residues suggests that the mouse proteins TCA3, P500, MIP1-alpha, MIP1-beta, and JE are members of a single gene family. These proteins are activation specific and can be expressed by both myeloid and lymphoid cells. MIP1-alpha/MIP1-beta and MCAF (the putative human homologue of JE) act as chemotactic and activating agents for neutrophils and macrophages, respectively. The functions of TCA3 and P500 are unknown. We have used interspecies somatic cell hybrids and recombinant inbred mouse strains to show that the genes encoding TCA3, MIP1-alpha, MIP1-beta, and JE (provisionally termed Tca3, Mip-1a, Mip-1b, and Sigje, respectively) map as a cluster on the distal portion of mouse chromosome 11 near the Hox-2 gene complex. DNA sequence analysis indicates that the P500 and TCA3 proteins are encoded by alternative splicing products of one genomic gene. Additionally, the genes encoding TCA3 and JE are found to be strikingly similar with respect to the positions of intron-exon boundaries. Together, these data support the model that the cytokines TCA3, P500, MIP1-alpha, MIP1-beta, and JE are encoded by a single cluster of related genes. The gene encoding IL-5 (Il-5), which acts as a T cell-replacing factor, a B cell growth factor, and an eosinophil differentiation factor, is also mapped to mouse chromosome 11.Il-5 maps approximately 25 cM proximal to the Tca-3 gene and appears tightly linked to a previously described gene cluster that includes Il-3, Il-4, and Csfgm. We discuss the potential relevance of the two cytokine gene clusters described here with particular attention to specific human hematologic malignancies associated with chromosomal aberrations at corresponding locations on human chromosomes 5 and 17.

Amino Acid Sequence↗
PubMed1990

Regulation of chimeric phosphoenolpyruvate carboxykinase genes by the trans-dominant locus TSE1.

Extinction of phosphoenolpyruvate carboxykinase (PCK) gene expression in hepatoma x fibroblast hybrids is mediated by a trans-acting genetic locus designated tissue-specific extinguisher 1 (TSE1). To identify PCK gene sequences required for extinction, hepatoma transfectants expressing PCK-thymidine kinase (TK) chimeric genes were fused with TK- fibroblasts and PCK-TK expression in the resulting hybrids was monitored. Expression of a PCK-TK chimera containing PCK sequences between base pairs -548 and +73 was extinguished in four of five hepatoma transfectants tested, although hybrids derived from one transfectant clone failed to extinguish PCK-TK expression. In contrast, crosses between hepatoma transfectants expressing the herpesvirus TK gene from its own promoter and TK- fibroblasts produced TK+ hybrids; extinction of the transfected TK gene was not observed. Thus, rat PCK gene sequences between base pairs -548 and +73 are sufficient for tissue-specific extinction in hybrid cells. Extinction of PCK-TK gene expression in transfectant microcell hybrids mapped specifically to human chromosome 17, the site of human TSE1.

Animals↗
PubMed1990

Extinction of phosphoenolpyruvate carboxykinase gene expression is associated with loss of a specific chromatin-binding protein from a far upstream domain.

We have analyzed the chromatin structure of the phosphoenolpyruvate carboxykinase (PEPCK) gene in hepatoma x fibroblast hybrids with different extinction phenotypes. These hybrids included a karyotypically complete hybrid in which all liver gene activity was extinguished, a microcell hybrid that contained a single mouse chromosome 11 and in which PEPCK gene activity was decreased but inducible by cyclic AMP, and a segregant line that had lost all mouse chromosomes and in which the PEPCK gene was reexpressed. We found that only in the completely extinguished hybrid was PEPCK chromatin structure radically different from that in the parental hepatoma cells. In this hybrid, there was no evidence of any factors binding to the promoter or to the upstream hypersensitive site at -4800 base pairs. In the other cell lines, even when PEPCK gene transcription was low, the PEPCK chromatin showed characteristic structures typical of a transcriptionally competent gene, with hypersensitive sites at positions previously described. Loss of the upstream hypersensitive site was also shown to be correlated with the absence of a liver-specific protein factor that binds specifically to the upstream region.

Animals↗