Genetic analysis of mammalian cell differentiation.
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Animals↗
Biomedical subjects
R E Fournier
Publications and source records attributed to R E Fournier.
The angiotensinogen gene is located on mouse chromosome 8.
We have recently identified a cis-acting genetic lesion affecting angiotensinogen gene expression in testis and salivary gland. Accordingly, the angiotensinogen gene was assigned to mouse chromosome 8 by screening a series of hybrid cell lines for retention of mouse angiotensinogen sequences by genomic Southern analysis. In AKXD recombinant inbred mice, the angiotensinogen gene is 2.4 +/- 1.8 centiMorgan from Rn7S-8,a 7S RNA gene located on chromosome 8 (Taylor, B.A., personal communication). However, the segregation of salivary and testicular angiotensinogen expression phenotypes into inbred mouse strains was not concordant with the known chromosome 8 proviruses Emv-2, Mtv-21, Xmv-12 or Xmv-26.
Angiotensinogen↗
Two NF1 translocations map within a 600-kilobase segment of 17q11.2.
Balanced translocations, each involving chromosome 17q11.2, have been described in two patients with von Recklinghausen neurofibromatosis (NF1). To better localize the end points of these translocation events, and the NF1 gene (NF1) itself, human cosmids were isolated and mapped in the immediate vicinity of NF1. One cosmid probe, c11-1F10, demonstrated that both translocation breakpoints, and presumably NF1, are contained within a 600-kilobase Nru I fragment.
Animals↗
Cytokeratin gene expression in hepatoma hybrid cells: evidence for regulation in cis.
The genes encoding intermediate filament (IF) proteins are expressed in a cell-lineage restricted fashion. To analyze the regulation of such genes, we studied cytokeratin and vimentin expression in hepatoma x fibroblast hybrids. These hybrids continued to express both hepatoma cell-derived cytokeratins and fibroblast-specific vimentin. Furthermore, the cytokeratin subunits that were produced were exclusively of rat hepatoma origin. Thus, IF protein genes were neither extinguished nor activated in cell hybrids, providing evidence for regulation in cis. This behavior contrasts sharply with that of most tissue-specific genes, which tend to be regulated in trans in hybrid cells.
Animals↗
Physical mapping of human chromosome 17 using fragment-containing microcell hybrids.
Hybrid cell lines were generated by microcell-mediated transfer of human chromosome 17 into rat recipient cells. The genotypes of 36 such lines were analyzed using a set of human chromosome 17-derived sequences to probe the structural integrity of the chromosome. Four classes of hybrids were obtained: clones with an apparently intact chromosome 17, clones containing large fragments of the chromosome including both the centromere and the selected marker, clones containing only the selected marker and flanking sequences, and clones containing two 17-derived fragments--the pericentric region plus the region of the selected marker. Data from these hybrids were used in conjunction with published regional localization information to obtain a provisional linear map of the chromosome. Results of this analysis are compared to the gene maps predicted from recent linkage studies and from other somatic cell hybrid experiments.
Blotting, Southern↗
Characterization of cDNAs encoding human leukosialin and localization of the leukosialin gene to chromosome 16.
We describe the isolation and characterization of cDNA clones encoding human leukosialin, a major sialoglycoprotein of human leukocytes. Leukosialin is very closely related or identical to the sialophorin molecule, which is involved in T-cell proliferation and whose expression is altered in Wiskott-Aldrich syndrome (WAS), an X chromosome-linked immunodeficiency disease. Using a rabbit anti-serum to leukosialin, a cDNA clone was isolated from a lambda gt11 cDNA library constructed from human peripheral blood cells. This lambda gt11 clone was used to isolate longer cDNA clones that correspond to the entire coding sequence of leukosialin. DNA sequence analysis reveals three domains in the predicted mature protein. The extracellular domain is enriched for Ser, Thr, and Pro and contains four contiguous 18-amino acid repeats. The transmembrane and intracellular domains of the human leukosialin molecule are highly homologous to the rat W3/13 molecule. RNA gel blot analysis reveals two polyadenylylated species of 2.3 and 8 kilobases. Southern blot analysis suggests that human leukosialin is a single-copy gene. Analysis of monochromosomal cell hybrids indicates that the leukosialin gene is not X chromosome linked and in situ hybridization shows leukosialin is located on chromosome 16. These findings demonstrate that the primary mutation in WAS is not a defect in the structural gene for leukosialin.
Amino Acid Sequence↗
Differential activity of a tissue-specific extinguisher locus in hepatic and nonhepatic cells.
Tissue-specific extinguisher 1 (Tse-1) is a genetic locus on mouse chromosome 11 that can repress expression of several liver genes in trans. This locus is clearly active in fibroblasts, as hepatoma cells retaining fibroblast chromosome 11 are extinguished for both tyrosine aminotransferase and phosphoenolpyruvate carboxykinase gene expression. To assess the activity of Tse-1 in other tissues, we transferred mouse chromosome 11 from several different cell types into rat hepatoma recipients. Tse-1 was active in nonhepatic cell lines derived from each primary germ layer, but Tse-1 activity was not apparent in hybrids between hepatoma cells and primary mouse hepatocytes. These differences in the genetic activity of murine Tse-1 were apparently heritable in cis.
Animals↗
Hormonal regulation of TSE1-repressed genes: evidence for multiple genetic controls in extinction.
Somatic cell hybrids formed by fusing hepatoma cells with fibroblasts generally fail to express liver functions, a phenomenon termed extinction. Previous studies demonstrated that extinction of the genes encoding tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, and argininosuccinate synthetase is mediated by a specific genetic locus (TSE1) that maps to mouse chromosome 11 and human chromosome 17. In this report, we show that full repression of these genes requires a genetic factor in addition to TSE1. This conclusion is based on the observation that residual gene activity was apparent in monochromosomal hybrids retaining human TSE1 but not in complex hybrids retaining many fibroblast chromosomes. Furthermore, TSE1-repressed genes were hormone inducible, whereas fully extinguished genes were not. Analysis of hybrid segregants indicated that genetic loci required for the complete repression phenotype were distinct from TSE1.
Animals↗
Tse-2: a trans-dominant extinguisher of albumin gene expression in hepatoma hybrid cells.
Serum albumin gene expression is generally extinguished in hepatoma x fibroblast hybrids. To define the genetic basis of this phenomenon, we screened a panel of hepatoma hybrids retaining different fibroblast chromosomes for albumin production by immunofluorescence. We report that albumin extinction in these clones was strictly correlated with the retention of mouse chromosome 1. Furthermore, albumin was systematically reexpressed in chromosome 1 segregants. These data define a tissue-specific extinguisher locus (Tse-2) that affects albumin gene expression in trans. Two other liver genes, those encoding liver alcohol dehydrogenase and liv-10, were coordinately extinguished with albumin in monochromosomal hybrids that specifically retained mouse chromosome 1.
Animals↗
Physical mapping of the von Recklinghausen neurofibromatosis region on chromosome 17.
The von Recklinghausen neurofibromatosis (NF1) locus has been linked to chromosome 17, and recent linkage analyses place the gene on the proximal long arm. NF1 probably resides in 17q11.2, since two unrelated NF1 patients have been identified who possess constitutional reciprocal translocations involving 17q11.2 with chromosomes 1 and 22. We have used a somatic-cell hybrid from the t(17;22) individual, along with other hybrid cell lines, to order probes around the NF1 locus. An additional probe, 17L1, has been isolated from a NotI linking library made from flow-sorted chromosome 17 material and has been mapped to a region immediately proximal to the translocation breakpoint. While neither NF1 translocation breakpoint has yet been identified by pulse-field gel analysis, an overlap between two probes, EW206 and EW207, has been detected. Furthermore, we have identified the breakpoint in a non-NF1 translocation, SP-3, on the proximal side of the NF1 locus. This breakpoint has been helpful in creating a 1,000-kb pulsed-field map, which includes the closely linked NF1 probes HHH202 and TH17.19. The combined somatic-cell hybrid and pulsed-field gel analysis we report here favors the probe order D17Z1-HHH202-TH17.19-CRYB1-17L1-NF1- (EW206, EW207, EW203, L581, L946)-(ERBB2, ERBA1). The agreement in probe ordering between linkage analysis and physical mapping is excellent, and the availability of translocation breakpoints in NF1 should now greatly assist the cloning of this locus.
Animals↗
Genetic activity of a trans-regulatory locus in hepatoma hybrid cells.
Tissue-specific extinguisher-1 (TSE1) is a genetic locus on mouse chromosome 11 that can repress expression of several liver genes in trans. The activity of this locus is apparent in rat hepatoma microcell hybrids that retain chromosome 11 from mouse fibroblasts: such hybrids fail to accumulate particular hepatic mRNAs, including those encoding tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK). In this report, we show that TSE1 from a TAT-, PEPCK- mouse hepatoma cell line expressing a fetal liver phenotype also induced TAT and PEPCK extinction when transferred into rat hepatoma recipients. Thus, TSE1 appears to be active in TAT-, PEPCK- cells of both hepatic and non-hepatic lineages. This suggests that TSE1 may play a role in the developmental regulation of hepatic gene expression in the liver.
Animals↗
Coordinate regulation of two genes encoding gluconeogenic enzymes by the trans-dominant locus Tse-1.
Tissue-specific extinguisher-1 (Tse-1) is a mouse genetic locus that can repress liver-specific tyrosine aminotransferase gene expression in trans. To search for other Tse-1-responsive genes, hepatoma microcell hybrids retaining mouse chromosome 11 or human chromosome 17, containing murine Tse-1 and human TSE1, respectively, were screened for expression of liver-specific mRNAs. While most liver gene activity was unaffected in such hybrids, phosphoenolpyruvate carboxykinase and tyrosine aminotransferase gene expression was coordinately repressed in these clones. Extinction of both genes was apparently mediated by a single genetic locus that resides on human chromosome 17.
Animals↗
Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.
A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins.
Animals↗
Assignment of the Ly-6--Ril-1--Sis--H-30--Pol-5/Xmmv-72--Ins-3--Krt-1--Int-1 --Gdc-1 region to mouse chromosome 15.
Previous work has demonstrated linkage between Ly-6, H-30, and a locus, Ril-1, that affects susceptibility to radiation-induced leukemia. Results or preliminary linkage analyses suggested further that the cluster might be linked to Ly-11 on the proximal portion of mouse chromosome 2. Using molecular probes to examine somatic cell lines and recombinant inbred and congenic strains of mice, we have re-evaluated these linkage relationships. A cloned genomic DNA fragment derived from a retroviral site has been used to define a novel locus, Pol-5, that is tightly linked to both H-30 and Ril-1 as shown by analysis of the B6.C-H-30c congenic mouse strain. Following the segregation of the Pol-5 mouse-specific DNA fragment in a series of somatic cell hybrids carrying various combinations of mouse chromosomes on a rat or Chinese hamster background mapped Pol-5 to mouse chromosome 15. During the course of these studies, restriction fragment length polymorphisms were defined associated with several loci, including Pol-5, Ly-6, Sis, Ins-3, Krt-1, Int-1, and Gdc-1. Three of these loci, Sis, Int-1, and Gdc-1, have been previously mapped to chromosome 15 by others using somatic cell hybrids or isoenzyme analyses. Following the inheritance of these eight loci in recombinant inbred strains of mice allowed the definition of a linkage group on the chromosome with the order Ly-6--Ril-1--Sis--H-30--Pol-5--Ins-3--Krt-1--Int-1--Gdc-1. Analyses of alleles inherited as passengers in B6.C-H-30c, C3H.B-Ly-6b, and C57BL/6By-Eh/+ congenic mouse strains and in situ hybridization experiments support the above gene order and indicate further that the cluster is located on distal chromosome 15, with Ly-6 and Sis near Eh.
Animals↗
A genetic analysis of extinction: trans-regulation of 16 liver-specific genes in hepatoma-fibroblast hybrid cells.
Hybrid cells formed by fusing different cell types generally fail to express the tissue-specific products of either parent, a phenomenon termed extinction. We have investigated the generality of this effect by assaying hepatoma-fibroblast hybrids for expression of 16 different liver-specific mRNAs. Fourteen of the mRNAs failed to accumulate in karyotypically complete hybrid clones, and quantitative measurements indicated that steady-state mRNA levels were depressed by a factor of at least 500-1000. However, all 14 liver-specific mRNAs were reexpressed in the hybrids following fibroblast chromosome loss. These data indicate that expression of whole sets of tissue-specific genes is affected in trans in intertypic hybrids and suggest that negative regulation of heterologous functions may be common form of gene control.
Alcohol Dehydrogenase↗
Chromosomal localization of the mouse homolog of the Huntington's disease linked G8 (D4S10) marker.
The human DNA probe G8 defines D4S10, a polymorphic locus tightly linked to the Huntington's disease gene on human chromosome 4. A subclone of G8, pSC33, showed significant cross-hybridization to discrete restriction fragments in total genomic mouse DNA. The probe detected restriction fragment length polymorphisms (RFLPs) with the enzymes Taq I and Msp I, permitting chromosomal localization of the mouse G8 homolog by linkage analysis using three sets of recombinant inbred mouse strains: BXH, BXD, and AKXD. The mouse locus was mapped to the central region of chromosome 11 at 1 centiMorgan from the SPARC gene, a locus whose human counterpart is on human chromosome 5.
Animals↗
Unstable and stable CAD gene amplification: importance of flanking sequences and nuclear environment in gene amplification.
We analyzed the amplification of the CAD gene in independently isolated N-(phosphonacetyl)-L-aspartate-resistant clones derived from single parental clones in two mouse cell lines. We report for the first time that the CAD gene is amplified unstably in mouse cells, that the degree of instability varies greatly between clones, and that minute chromosomes and highly unstable chromosomelike structures contain the amplified sequences. These data are most consistent with the idea that the amplified unit in each clone consists of different flanking DNA and that such differences engender amplified sequences with unequal stability. We also introduced the mouse chromosome containing the CAD gene into hamster cells by microcell-mediated chromosome transfer to determine whether the propensity for unstable extrachromosomal amplification of the mouse CAD gene would prevail in the hamster cell nuclear environment. We report that the mouse CAD gene was amplified stably in expanded chromosomal regions in each of seven hybrids that were analyzed. This observation is consistent with the idea that the nuclear environment influences whether mutants containing intra- or extrachromosomally amplified sequences will be isolated.
Animals↗
Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes.
We sought an efficient means to introduce specific human chromosomes into stable interspecific hybrid cells for applications in gene mapping and studies of gene regulation. A defective amphotropic retrovirus was used to insert the gene conferring G418 resistance (neo), a dominant selectable marker, into the chromosomes of diploid human fibroblasts, and the marked chromosomes were transferred to mouse recipient cells by microcell fusion. We recovered five microcell hybrid clones containing one or two intact human chromosomes which were identified by karyotype and marker analysis. Integration of the neo gene into a specific human chromosome in four hybrid clones was confirmed by segregation analysis or by in situ hybridization. We recovered four different human chromosomes into which the G418 resistance gene had integrated: human chromosomes 11, 14, 20, and 21. The high efficiency of retroviral vector transformation makes it possible to insert selectable markers into any mammalian chromosomes of interest.
Animals↗