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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.

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.

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.

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.

The progress of mononucleate and cytochalasin-B-induced binucleate cells through the G1 period of the growth cycle has been studied. DNA synthesis was initiated in synchrony in both of the nuclei of cells rendered binucleate by a brief pulse of cytochalasin B. Furthermore, populations of these cells traversed the G1 period significantly faster than their mononucleate counterparts. That this effect was due to the presence of two nuclei in a common cytoplasm and not to the increased size of binucleate cells was suggested by studies of cells with altered nucleocytoplasmic ratios. No correlation was observed between protein content per cell and the rate of progress through G1. Evidence for the existence of nuclear-nuclear cooperation that results in a shortening of the G1 period is discussed.

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.

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.

Similarity of G-band patterns between the long arm of Chinese hamster chromosome 6 and mouse chromosome 2, combined with the assignments of AK1, ADA, and ITPA to hamster chromosome 6 and AK1 to mouse chromosome 2, suggested mouse chromosome 2 also might contain ADA and ITPA. Here, concordant segregation analysis of enzyme loci and chromosomes in mouse spleen X CHO as well as mouse microcell X CHO somatic cell hybrids established the assignments of ADA and ITPA onto mouse chromosome 2 in the region between the first G-band and the terminus (C1----ter). This assignment presents a demonstration of the conservation and evolution of enzyme and proto-oncogene loci linkage since two cellular homologs of viral oncogenes--c-src and c-abl--also map to mouse chromosome 2. In humans c-src, ADA, and ITPA remain conserved on chromosome 20, whereas AK1 and c-abl are together on chromosome 9. These observations and concepts are discussed with respect to the role of proto-oncogenes in chromosomal evolution and suggest the long arm of chromosome 6 as a fruitful place to look for c-src and c-abl in the Chinese hamster.

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.

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.

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.

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.

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.

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.

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.

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.

The site of association of the human transgenome and host murine chromosomes was determined in several subclones of a stable human/mouse transformed cell line. Chromosomes were transferred from each of three transformed subclones into Chinese hamster recipient cells, and selection was applied for the expression of human transgenome-encoded HPRT. A series of trispecific microcell hybrids was isolated and characterized for each subclone. Evidence is presented that, within a given transformed subclone, only a single host (murine) chromosome was associated with the human transgenome. This contrasts with previous results which utilized a newly stabilized transformed cell line as the microcell donor and in which a variety of chromosomal sites of association existed. The results presented here support the view that the heterogeneity of transgenome association (integration) sites in newly stabilized transformants was due to the fact that these populations were multiclonal mixtures resulting from independent stabilization events. The initial heterogeneity in the population was subsequently reduced upon prolonged cultivation, as a subset of the original population became predominant.

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.

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.

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.

In this report, we describe the production and characterization of proliferating hybrid cell populations generated by fusion of murine microcells with intact mouse, Chinese hamster, and human recipient cells. The microcell hybrids so produced contained one to five intact murine chromosomes derived from the microcell donor. these transferred chromosomes were maintained as functioning genetic elements in the hybrid cells. Our results firmly establish subnuclear particle-mediated chromosome transfer as a valid somatic cell genetic tool.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Evidence is presented for the assignment of the gene for adenosine kinase to Mus musculus chromosome 14 by synteny testing and karyotypic analysis of mouse X Chinese hamster somatic cell hybrid clones. ADOK and two enzymes previously mapped to mouse chromosome 14, NP and ES-10, were expressed concordantly in 29 hybrid clones. Chromosome analysis confirmed this assignment. Syntenic evidence is also presented using several clones of a gene transfer system in which the gene for human HPRT had integrated into modified mouse chromosome 14's.

Trispecific microcell hybrids were prepared by transferring limited numbers of chromosomes from a human/mouse gene-transfer cell line to a Chinese hamster recipient line. The donor cells employed were murine L-cells that stably expressed the human form of the enzyme hypoxanthine phosphoribosyltransferase. Karyotypic, zymographic, and back-selection tests of the resulting human/mouse/Chinese hamster microcell hybrids provided strong genetic evidence for a stable association of the human transgenome with host murine chromosomes in stable gene-transfer cell lines. This association, which may represent physical integration of the transgenome into the host cell genome, occurred at multiple chromosomal sites.

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.

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).