Hamster Somatic Cell Hybrids Research Articles

The gene and the pseudogene for mouse p53 cellular tumor antigen are located on different chromosomes.

The chromosomal assignments of the two genes encoding the murine p53 cellular tumor antigen were determined by using a panel of mouse-Chinese hamster somatic cell hybrid clones and a mouse p53-specific cDNA clone. One gene, probably the functional member of the family, was found to be on chromosome 11. The other gene, which is probably a processed pseudogene, was assigned to chromosome 14. The potential relevance of these findings to documented cases of chromosome 11 trisomy are also discussed.

The gene and the pseudogene for mouse p53 cellular tumor antigen are located on different chromosomes.

The chromosomal assignments of the two genes encoding the murine p53 cellular tumor antigen were determined by using a panel of mouse-Chinese hamster somatic cell hybrid clones and a mouse p53-specific cDNA clone. One gene, probably the functional member of the family, was found to be on chromosome 11. The other gene, which is probably a processed pseudogene, was assigned to chromosome 14. The potential relevance of these findings to documented cases of chromosome 11 trisomy are also discussed.

Localization of a gene which complements branched-chain amino acid transaminase deficiency to the short arm of human chromosome 12.

A human X Chinese hamster somatic cell hybrid (J1) containing only one human chromosome (number 11) does not express branched-chain amino acid transaminase and, therefore, cannot grow if the branched-alpha-keto acids are substituted for the branched-chain amino acids in the growth medium. J1 cells, which are glycine auxotrophs (Gly-A), were fused with normal human lymphocytes and the resulting hybrids selectively isolated in glycine-free medium. A total of 16 primary and 46 secondary clones were analyzed for isozymic and nutritional markers. Cytogenetic analysis with chromosome banding was also performed on selected hybrid clones. The results provide evidence that branched-chain amino acid transaminase is syntenic with lactic dehydrogenase B and is located on the short arm of human chromosome 12.

The identification of a DNA polymorphism of the alpha fibrinogen gene, and the regional assignment of the human fibrinogen genes to 4q26-qter.

We have identified a common restriction fragment length polymorphism of the alpha fibrinogen gene with the enzyme TaqI. This polymorphism is probably due to a single base change that creates or destroys a TaqI recognition site about 1000 basepairs from the 3' end of the alpha fibrinogen géne. The frequency of the rare allele in 83 unrelated healthy individuals is 0.33. We have used in situ hybridisation of the alpha fibrinogen cDNA to localise the gene on chromosome 4q29-31. We have confirmed this regional localisation by restriction fragment detection in a human X Chinese hamster somatic cell hybrid which contains a translocated human chromosome 4 with a breakpoint at 4q26. The alpha, beta, and gamma fibrinogen genes are all present on human chromosome 4q26-qter.

Chromosome assignments of four mouse cellular homologs of sarcoma and leukemia virus oncogenes.

Molecular probes for the oncogenes of Rous sarcoma virus (v-src), avian myeloblastosis virus (v-myb), Kirsten murine sarcoma virus (v-Ki-ras), and Harvey murine sarcoma virus (v-Ha-ras) were hybridized to the DNA from mouse-Chinese hamster somatic cell hybrids. The v-src, v-myb, v-Ki-ras, and v-Ha-ras genes each detected one or a few homologous mouse DNA fragments whose segregation was analyzed in cell hybrids. Mouse cellular homologs c-src, c-Ki-ras, c-Ha-ras, and c-myb segregated concordantly with chromosomes 2, 6, 7, and 10, respectively. Comparison with the known locations of human c-src (chromosome 20) and human c-Ha-ras1 (chromosome 11 short arm) suggests that the human and mouse homologs of these two viral oncogenes reside in conserved linkage groups. The c-Ki-ras gene on mouse chromosome 6 might reside also in a conserved linkage group, along with glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase. However, direct confirmation of this suggestion must await a demonstration that c-Ki-ras on mouse chromosome 6 is homologous to c-Ki-ras2 on the short arm of human chromosome 12.

Glutathione S-transferase Ya subunit is coded by a multigene family located on a single mouse chromosome.

A cloned DNA probe of Ya, the major glutathione S-transferase subunit in rat liver, was used to study the organization of Ya genes in the mouse genome. Southern blot analysis of mouse genomic DNA indicates that the Ya subunit is encoded by a multigene family. The chromosomal distribution of Ya genes was determined by analysis of DNA from a panel of mouse-Chinese hamster somatic cell hybrids. All detectable Ya genes were found to be located on chromosome 9. At least some of the Ya-specific DNA sequences are clustered since, by screening a mouse genomic library, two recombinant phages, each containing two different Ya DNA sequences in the same insert, have been isolated. The finding that Ya is encoded by a cluster of different genes raises the question of the specificity of the different Ya DNA sequences.

Retention of both parental mitochondrial DNA species in mouse-Chinese hamster somatic cell hybrids.

Interspecific somatic cell hybrids were isolated following the fusion of an oligomycin-resistant derivative of LM (TK-) mouse cells to a chloramphenicol-resistant derivative of AK412 Chinese hamsters cells. Hybrids were selected in either HAT medium, HAT plus chloramphenicol (CAP), HAT plus oligomycin (OLI), or HAT plus chloramphenicol and oligomycin. Cytogenetic analysis of the hybrids indicated that their karyotype reflected the sum of the parents. Hybrids selected in HAT medium alone or HAT plus OLI retained primarily mouse mitochondrial DNA while those selected in HAT plus CAP, or HAT plus CAP plus OLI retained both species of mitochondrial DNA. There was no evidence for mitochondrial DNA recombination, despite the continued growth of these hybrids in CAP plus OLI. Hybrids that were removed from dual antibiotic selection for over three months retained both species of mitochondrial DNA in approximately equal amounts with no detectable loss or rearrangement.

Chromosomal location of mouse gene 202 which is induced by interferons and specifies a 56.5 kD protein.

The 202 gene which specifies a 56.5 kD protein can be induced in Ehrlich ascites tumor cells by treatment with mouse beta interferon. This treatment increases the level of the gene 202-specific mRNA at least 12-fold. For determining the chromosomal location of this gene a 1.5 kb fragment of the gene was hybridized to EcoR1 digested DNA samples from a set of mouse-hamster somatic cell hybrids. Each of the cell hybrids used contained a complete array of hamster chromosomes and one or more mouse chromosomes. The 202 gene fragment hybridized to every DNA sample from cell hybrids containing mouse chromosome 1 (8 hybrids in total) and to none of the DNA samples from hybrids lacking this chromosome (7 hybrids in total). These and other data indicate that the 202 gene is located on mouse chromosome 1.

Chromosome localization of the genes for ENO1, HK1, ADK, ACP2, MPI, ITPA, ACON1 and ?-GAL in the American mink (Mustela vison)

Twenty-eight American mink × Chinese hamster somatic cell hybrids were analysed for the expression of mink enzymes and the segregation of mink chromosomes. The results demonstrated that the gene for enolase-1 is located on the long arm of mink chromosome 2, and those for hexokinase-1 and adenosine kinase, on its short arm. Segregation analysis of mink chromosomes and mink acid phosphatase-2, mannose phosphate isomerase, inosine triphosphatase and aconitase-1 provided data allowing us to assign the genes for these markers to mink chromosomes 7, 10, 11 and 12, respectively. The expression of mink α-galactosidase was highly coincidental with mink × chromosome as well as with its markers: hypoxanthine-phosphoribosyltransferase, glucose-6-phosphate dehydrogenase and phosphoglycerate kinase-1. This result confirms the assignment of the gene for α-galactosidase to the mink × chromosome.

Assignment of murine cellular Harvey ras gene to chromosome 7.

Mouse-Chinese hamster somatic cell hybrids containing various combinations of mouse chromosomes were analyzed for the presence of the mouse c-Ha-ras (1) sequences after restriction endonuclease digestion and hybridization with a 32P-labeled Ha-ras specific probe according to the procedure of Southern (2). The presence of the mouse c-Ha-ras containing fragment was correlated with the presence of mouse chromosome 7 in the hybrids.

Normal X chromosome induced reversion in the direction of chromosome segregation in mouse-Chinese hamster somatic cell hybrids

The effect of a normal mouse X chromosome on the chromosome segregation of mouse-Chinese hamster somatic cell hybrids was determined by ( i) producing hybrids between the mouse sarcoma line CMS4 and a microcell hybrid (mfe4) of the hamster line E36, containing a mouse X chromosome from a normal cell; ( ii) isolating hybrids between CMS4 and a 6-thioguanine selected (X minus) mfe4 subpopulation; ( iii) comparing the direction of segregation in the two sets of hybrids. It was found that the normal X chromosome, like the X chromosomes from two MCA-transformed sarcoma lines reported previously [9], has the ability to switch the chromosome segregation of mouse-Chinese hamster somatic cell hybrids. We conclude that the reversal in chromosome segregation is mediated by factors located on the X chromosome. We designate these genetic elements as segregation reversal genes or sr genes.

Assignment of the gene coding for human beta-glucocerebrosidase to the region q21-q31 of chromosome 1 using monoclonal antibodies.

A series of man-Chinese hamster somatic cell hybrids with a variable content of human chromosomes was used to study the localization of the human gene coding for the lysosomal enzyme beta-glucocerebrosidase (EC 3.2.1.45). In lysates made from hybrid cells, the human enzyme was specifically recognized by a mouse monoclonal antibody raised against human placental beta-glucocerebrosidase. This monoclonal antibody did not cross-react with Chinese hamster beta-glucocerebrosidase. After reaction of the antibody with the enzyme, beta-glucocerebrosidase was precipitated by addition of Protein A-Sepharose beads, and was detected on the beads by its enzymatic activity. From the analysis of a series of man-Chinese hamster hybrids, among which were hybrids with specific segments of chromosome 1, we conclude that the gene coding for human beta-glucocerebrosidase is localized in the region q21-q31 of chromosome 1.

Regulation of glucose 6-phosphate dehydrogenase expression in CHO-human fibroblast somatic cell hybrids

Human--hamster somatic cell hybrids have been obtained by fusion of a CHO line (NA31) doubly deficient in hypoxanthine guanine phosphoribosyltransferase and glucose 6-phosphate dehydrogenase (G6PD) with normal G6PD(+) human fibroblasts. Analysis of NA31 extracts has revealed that, although G6PD activity is nearly absent, significant activity can be detected with 2-deoxyglucose 6-phosphate as substrate, so that the mutant and normal forms of the enzyme can both be easily detected. The cell hybrids obtained express human G6PD. The human G6PD subunits are distributed in homodimeric molecules as well as in human--hamster heterodimeric molecules. However, whereas the amount of hamster G6PD subunits present in the hybrid is similar to that in the hamster parental cells, the amount of human G6PD subunits is decreased by 3- to 10-fold when compared to the human parental cell. These results indicate that either the expression of the G6PD gene or the stability of the gene product is altered in the hybrid. By mutagenesis and selection in diamide (a substance that oxidizes intracellular glutathione), we have isolated a clone with a 3- to 5-fold increase in human G6PD activity. This derivative may have an increased rate of expression of the human G6PD structural gene.

X chromosome-induced reversion of chromosome segregation in mouse/Chinese hamster somatic cell hybrids: Cellular recognition of native and foreign X chromosomes

The direction of chromosome loss in two sets of mouse-Chinese hamster hybrids was compared with the direction of segregation of the same hybrids, to which an additional X chromosome derived from either of the mouse sarcoma lines MethAa, MethAs, or CMS4, was introduced at the time of the fusion. The addition of the X chromosome was carried out by substituting in place of the Chinese hamster parent a mouse X containing microcell hybrid of the latter. It was found that the addition of an X chromosome reverses the direction of chromosome segregation, but it can do so only if the mouse parent in the hybridization is different from the line from which the X originated. The possible reasons for recognition by the cells of a native and a foreign X are discussed. The existence of a multigene family on the X chromosome, involved in this recognition, is proposed.

Assignment of the genes for mouse type I procollagen to chromosome 16 using mouse fibroblast-Chinese hamster somatic cell hybrids.

Somatic cell hybrids between mouse and Chinese hamster fibroblasts have been used to identify the chromosome responsible for the synthesis of both mouse type I procollagen subunit chains (MCOLA1 and MCOLA2). Thirty-one separate hybrid clones and subclones from ten separate hybridization events were isolated in hypoxanthine-aminopterin-thymidine (HAT) selection medium and were used for detailed gene-mapping studies. ELISA and "Western blotting" immunochemical analysis were used to detect the production of mouse type I procollagen in each hybrid clone. Mouse and Chinese hamster chromosomes were identified in each hybrid clone by trypsin-Giemsa banding of metaphase chromosome spreads and by isozyme analysis. We have found that mouse type I procollagen production segregates concordantly with mouse superoxide dismutase-1, previously mapped to mouse chromosome 16, and with the presence of mouse chromosome 16 karyotypically. Western blotting immunochemical analysis of the separated mouse procollagen chains produced by each hybrid line demonstrated that apparently the genes for both subunit chains are located on the same chromosome. These studies, therefore, assign the structural genes for mouse type I procollagen pro alpha 1 (MCOLA1) and pro alpha 2 (MCOLA2) chains to mouse chromosome 16.

Basic defect in the expression of adenosine deaminase in ADA-SCID disease. II. Deficiency of ADA-CRM detected in heterozygote human-Chinese hamster cell hybrids.

A specific competitive radioimmunoassay (RIA) was employed to quantify human adenosine deaminase molecules produced in human-Chinese hamster somatic cell hybrids. Studies on a set of hybrids in which the normal and aberrant expressions of adenosine deaminase (assigned earlier to human chromosome 20) were segregating, have demonstrated that in the patient with ADA-SCID disease reported by Herbschleb-Voogt et al. (1981 a), the deficiency of ADA activity was associated with a comparable deficiency of adenosine deaminase specific immuno-crossreacting material (ADA-CRM).

Relationship between H1 histone phosphorylation and genome replication in a mouse-Chinese hamster somatic cell hybrid

The relationship between stable genome replication and the presence of species-specific H1 and H2b histones was studied in a mouse-Chinese hamster somatic cell hybrid (LMC-1). LMC-1 contained the complete chromosome complements of the mouse ( LM TK − , 46 chromosomes) and Chinese hamster (CHW-1102, 22 chromosomes) parents. No chromosome loss occurred in over a year in continuous culture, and both mouse and Chinese hamster enzymes were expressed. Chromatographic resolution of H1 histone subtypes showed that only mouse H1 genes were expressed. Resolution of chromatographically purified H2b histone variants using Triton X-100-containing gels also demonstrated that LMC-1 cells made mouse, not Chinese hamster, H2b histones. Species-specific tryptic phosphopeptides were resolved from purified H1 histones using high voltage paper electrophoresis. These studies demonstrated that characteristic mouse, but not Chinese hamster, phosphopeptides were formed in H1 histones in actively-growing LMC-1 cells. We conclude that the Chinese hamster genome may replicate in a stable manner in the presence of mouse H1 (and H2b) histones, and that there is no obligatory relationship between species-specific phosphorylation of H1 histones and stable genome replication.

Localization of a gene controlling the expression of the human transferrin receptor to the region q12 leads to qter of chromosome 3.

A monoclonal antiserum, 66-IG10, raised against human thymocytes was found to be directed against the human transferrin receptor. A panel of human X Chinese hamster somatic cell hybrids, in conjunction with the 66-IG10 reagent, was used to assign the gene(s) coding for the transferrin receptor to the q12 leads to qter region of human chromosome 3.

Genetic analysis of tumorigenesis: XII. Genetic control of the anchorage requirement in CHEF cells.

Chinese hamster somatic cell hybrids between diploid anchorage-independent CHEF/204Bu50 cells and diploid anchorage dependent CHEF/205-30 cells are anchorage dependent but can segregate subclones at low frequency which reexpress anchorage independence. Thus, anchorage independence, like other characteristics of the transformed phenotype, is suppressed in these hybrids. Anchorage-independent subclones were recovered from the anchorage-dependent hybrids under conditions which favored the retention of most chromosomes. Karyotype analysis of suppressed hybrids and their anchorage-independent subclones showed that segregation of anchorage dependence was correlated with the loss of one copy of chromosome 1 in CHEF Chinese hamster hybrids. Thus, suppression of anchorage independence has a chromosomal basis. Several genetic models are considered for the origin of anchorage-independent subclones from suppressed Chinese hamster hybrids.

DNA sequence associated with chromosome translocations in mouse plasmacytomas.

A DNA sequence that generates aberrantly rearranged immunoglobulin heavy chain constant region genes in murine plasmacytomas is shown to participate in a chromosome translocation. We have previously termed this DNA sequence NIARD for non-immunoglobulin-associated rearranging DNA. NIARD rearrangements were found frequently in murine plasmacytomas but were not detected in normal lymphocytes. These rearrangements occasionally involve the switch region of the C alpha gene. In this study, DNA samples obtained from mouse-Chinese hamster somatic cell hybrid lines were digested with various restriction endonucleases and analyzed by the Southern transfer technique with a NIARD hybridization probe. These experiments show that NIARD resides on chromosome 15 in the mouse germ line. Since NIARD is found adjacent to the C alpha gene (located on chromosome 12) in some plasmacytomas, it is apparent that a translocation involving these two chromosomes has occurred. We have proposed a rcpT(12;15) model to explain our data. The implications of NIARD rearrangements for malignant transformation are discussed.