Mitochondrial DNA analysis of mouse-rat hybrid cells: Effect of chloramphenicol selection on the relative amounts of parental mitochondrial DNAs

Several mouse-rat somatic hybrid cell lines were isolated by fusing chloramphenicol-resistant (CAP^R) and CAP-sensitive (CAP^S) parent cells, and propagation of the parent mitochondrial DNA (mtDNA) species in the hybrid cells was studied. The restriction endonucleases EcoRI, HpaII, and HaeIII were used for identification of mtDNA species. Both mouse and rat mtDNAs were propagated in all the hybrid cells examined and maintained during long-term cultivation and repeated cell division. Moreover, in CAP^R mouse-rat hybrid cells, selection and successive cultivation in the presence of CAP did not increase the relative amount of mtDNA species of CAP^R parent cell origin, and when CAP was removed from the culture medium, mtDNA species of CAP^R parent cell origin did not decrease appreciably. The amount of mouse mtDNAs was consistently 1–4 times that of rat mtDNAs in the mouse-rat hybrid cells regardless of the species of parent cells from which the CAP resistance was derived. Thus mouse-rat hybrid cells have a stable mtDNA population in which the amount of mouse mtDNAs is larger than that of rat mtDNAs without any influence of CAP selection.     

Preferential amplification of mitochondrial DNA fragments in somatic hybrids of the Gramineae

Summary Somatic hybrid cell lines of Pennisetum americanum + Panicum maximum, and of Pennisetum americanum + Saccharum officinarum display unique mitochondrial DNA (mtDNA) restriction patterns suggestive of mitochondrial fusion and recombination. Apparent recombinant fragments of the hybrids were recovered, cloned, and hybridized to parental and somatic hybrid mtDNAs. In each somatic hybrid, novel fragments were found to be present at low copy number in one or both of the parental mtDNAs, and amplified 15–30 times in the hybrids. In pearl millet-sugarcane somatic hybrid cells, the amplification does not appear to involve enhanced recombination. The presumably amplified restriction fragment of the pearl millet-Guinea grass somatic hybrids is a junction fragment of a repeat, present in low copy number in both parents, and in high copy number in the hybrids. Thus protoplast and probable mitochondrial fusion results in a marked shift in the direction of mtDNA recombination events. We conclude that amplification of parental mtDNA fragments is a common event in somatic hybrid cells of these Gramineae.     

Production of transmitochondrial mouse cell lines by cybrid rescue of rhodamine-6G pre-treated L-cells

Treatment of mouse LMTK⁻ cells with the toxic mitochondrial dye rhodamine 6G (R-6G) at 2.5 μg/ml for 7 days prevented cell growth while maintaining viability, with less than 10⁻⁶ cells recovering to form colonies. Pre-treatment of LMTK⁻ cells with R-6G was followed by fusion with enucleated mouse 501–1 cells harboring a homoplasmic point mutation in the mitochondrial DNA (mtDNA) 16S rRNA gene conferring chloramphenicol resistance (CAP^R). Cybrids and any surviving unfused LMTK⁻ cells were selected in BrdU with or without CAP and their mtDNAs screened for the presence of the CAP^R marker. Approximately 1 colony per 2×10⁵ LMTK⁻ cells appeared in the fusion plates selected both with and without CAP. Most clones investigated were confirmed to be cybrids by showing the presence of the generally homoplasmic CAP^R mutation, whether or not CAP selection was used. Hence, R-6G pre-treatment permits construction of transmitochondrial cybrid cell lines carrying a variety of mtDNAs, without the need for ρ^o cell lines.     

Carcinoembryonic antigen (CEA) expression in somatic cell hybrids

Five hybrids (LSB) were formed between LS174T, a human CEA-producing colonic tumor cell line, and BU25.CAP^R, a HeLa derivative which does not produce CEA. All five hybrids produce CEA, but less per cell than LS174T. Approximately 10 % of the chromosomes have been lost from these hybrids. In an attempt to map the gene(s) coding for the protein moiety of CEA, 7 LSPG and 28 LSR hybrids were formed between LS174T and PG19, a mouse melanoma cell line, and LS174T and RAG, a mouse kidney adenocarcinoma cell line, respectively. These hybrids retain between 4 and 21 human chromosomes, and each human chromosome is represented in at least seven hybrids. Two hybrids appeared to produce trace amounts of CEA. These results might represent repression by the mouse genome of CEA production or the production of a structurally abnormal CEA molecule.Submitted by D.S. in partial fulfillment of the requirements for the D. Phil degree, Oxford University.     

Isolation of genomic clones homologous to transcribed sequences from human X chromosome

Several X chromosome DNA clones homologous to transcribed sequences were isolated from a human X chromosome library. The clones were selected for their ability to hybridize either with³²P -labeled human cDNA in the presence of an excess of unlabeled human repetitive DNA or with mouse fibroblast cDNA. The X chromosome specificity of these sequences was demonstrated by two criteria: A dosage effect was seen when the clones were hybridized to Southern blots of DNA from 1X and 5X cells, and they hybridized to DNA from mouse-human hybrid cells containing only the human X chromosome. The presence of transcribed sequences in these X clones was detected by hybridization with mouse cDNA or with human cDNA in the presence of unlabeled human repetitive sequences, by identifying restriction fragments which hybridize with cDNA but not with human repetitive DNA, and by hybridization with poly A⁺ RNA on Northern blots. These clones were mapped on the human X chromosomes using a panel of mouse-human somatic cell hybrids carrying various translocated human X chromosomes.     

Mouse immune interferon (IFN-γ) gene is on chromosome 10

A cDNA clone for mouse immune interferon has been used to map the mouse interferon gene (Ifg)to a specific chromosome. This clone, which contains a 638-bp insert, detects an 18-kb HindIII fragment of mouse DNA. The presence of the mouse Ifgtgene in cell hybrids and its chromosomal location were determined by assaying cell hybrid DNA for the presence of the 18-kb HindIII fragment by Southern filter hybridization. Under the hybridization conditions used, Chinese hamster DNA did not hybridize to the cDNA probe. The segregation of mouse chromosomes in cell hybrids indicated that Ifgis located on chromosome 10. Previously, we have mapped immune interferon to the p12.05 qter region of chromosome 12 in humans (1). This region of chromosome 12 also contains the genes for peptidase B and citrate synthase. The homologous genes in mouse are also located on chromosome 10, suggesting that these genes comprise a conserved linkage group.     

Human and mouse cellularmyc protooncogenes reside on chromosomes involved in numerical and structural aberrations in cancer 1

A molecular clone of viralmyc (v-myc, the oncogene of avian myelocytomatosis virus, MC29, detected homologous human, mouse, and Chinese hamster cellularmyc (c-myc sequences by Southern filter hybridization. A v-myc probe, containing sequences from the 3′ domain of the gene, hybridized to single human HindIII and mouse EcoRI genomic DNA fragments of the cellular myc genes whose segregation could be followed in interspecies somatic cell hybrids. Human c-myc segregated concordantly with the enzyme marker glutathione reductase and with a karyotypically normal chromosome 8. A rearrangement of human c-myc was observed in Burkitt's lymphoma cells possessing the t(8;14) translocation. These results suggest that human c-myc is located close to the breakpoint on chromosome 8 (q24) involved in the t(8;14) translocation. The mouse c-myc gene segregated concordantly with chromosome 15 in mouse-Chinese hamster cell hybrids. These gene assignments are noteworthy, as structural and numerical abnormalities of human chromosome 8 and mouse chromosome 15 are associated frequently with B-cell neoplasms.     

Genetic counterselective procedure to isolate interspecific cell hybrids containing single human chromosomes: Construction of cell hybrids and recombinant DNA libraries specific for human chromosomes 3 and 4

Counterselection against genes on human chromosome 5 was applied to interspecific human-Chinese hamster cell hybrids which retained this and one additional human chromosome in order to generate cell hybrids retaining single, nonselected human chromosomes. Using this procedure, stable cell hybrids which retain human chromosome 3 exclusively or human chromosome 4 exclusively were isolated. Complete recombinant genomic DNA libraries were prepared from each hybrid using the cloning vector EMBL-4. These libraries represent sources of human DNA fragments derived specifically from chromosomes 3 and 4, respectively. Low-copy or unique human DNA fragments isolated from both libraries were analyzed to confirm their chromosomal origin and to determine the complexity of their hybridization patterns to total human DNA. These single human chromosome libraries represent a means to efficiently saturate chromosomes 3 and 4 with informative, polymorphic genetic markers. DNA fragments from the chromosome 4 library will be particularly useful in identifying additional genetic markers close to the Huntington's disease gene. The same genetic counterselective procedure can be utilized to derive several additional cell hybrids with single human chromosomes.     

Assignment of human gamma crystallin multigene family to chromosome 2

The multigene family for human -crystallin has been assigned to chromosome 2 using rodent-human somatic cell hybrids and filter hybridization analysis of cell hybrid DNA. Two genomic DNA probes containing human -crystallin gene sequences hybridize to five fragments in human DNA digested with the restriction enzyme EcoRI. By correlating the presence of these fragments in somatic cell hybrid DNA with the human chromosome content of the hybrids, at least six human -crystallin genes can be mapped to chromosome 2. Data obtained with a hybrid clone containing a mouse-human interspecies translocation suggest that these genes may be clustered together on the long arm of human chromosome 2.     

Human Luteinizing hormone-releasing hormone gene (LHRH) is located on short arm of chromosome 8 (region 8p11.2 → p21)

Luteinizing hormone-releasing hormone (LHRH) is synthesized by hypothalamic neurons and affects the release of gonadotropic hormones from the anterior pituitary gland. A cDNA clone encoding the human LHRH precursor molecule was used to assign theLHRH gene to a human chromosome by in situ hybridization and Southern blot analysis. Metaphase spreads from two normal individuals were hybridized with³H -labeled LHRH-specific sequence. Of 120 cells analyzed, 33 had silver grains over the p11.2 p21 bands of chromosome 8. No other chromosomal site was labeled above background, indicating the presence of a single site for LHRH sequences in the human genome. Independent confirmation for this location of the humanLHRH gene on chromosome 8p was provided by analysis of DNA from human × Chinese hamster somatic cell hybrids. DNA samples were digested with EcoRI, blotted, and hybridized with the³²P-labeled human LHRH precursor cDNA probe. The single 11.5-kb human-specific band was detected only in hybrids containing human chromosome 8. Also, hybridization was observed in DNA from hybrids in which a portion of human chromosome 8 (region 8pter 8q21) had been spontaneously translocated onto a Chinese hamster chromosome.Presented in part at the 8th Human Gene Mapping Conference, Helsinki, August 1985.     

The chloroplast and mitochondrial DNA type are correlated with the nuclear composition of somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginfolia

This paper describes the analysis of chloroplast (cp) DNA and mitochondrial (mt) DNA in 21 somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia by means of Southern-blot hybridization. Each of these calli contained only one type of cpDNA; 14 had the N. plumbaginifolia (Np) type and seven the S. tuberosum (St) type. N. plumbaginifolia cpDNA was present in hybrids previously shown to contain predominantly N. plumbaginifolia chromosomes whereas hybrids in which S. tuberosum chromosomes predominated possessed cpDNA from potato. We have analyzed the mtDNA of these 21 somatic hybrid calli using four restriction enzyme/probe combinations. Most fusion products had only, or mostly, mtDNA fragments from the parent that predominated in the nucleus. The hybrids containing mtDNA fragments from only one parent (and new fragments) also possessed chloroplasts from the same species. The results suggest the existence of a strong nucleo-cytoplasmic incongruity which affects the genome composition of somatic hybrids between distantly related species.     

Chromosome mapping of biological pathways by fluorescence-activated cell sorting and cell fusion: Human interferon gamma receptor as a model system

Human chromosome 6 encodes both the interferon gamma receptor as well as the class I major histocompatability complex antigens, HLA-A, -B, and -C. However, the presence of chromosome 6 in somatic cell hybrids is insufficient to confer sensitivity to human interferon gamma (Hu-IFN-) as assayed by class I HLA induction; it is necessary for both human chromosomes 6 and 21 to reside in the hybrid to generate a response to Hu-IFN-. Treatment of such a hamster-human hybrid, Q72-18, with Hu-IFN- induces the class I HLA antigens. Q72-18 cells selected by fluorescence-activated cell sorting for the loss of class I HLA induction also lost human chromosome 21. Fusions of such cells to a hybrid that contains only human chromosome 21 reconstitutes HLA antigen induction by Hu-IFN-. Furthermore, fusions of hybrids containing a translocated human chromosome 6q and the HLA-B7 gene to a line containing only human chromosome 21 or a translocated 21q also reconstitutes HLA-B7 mRNA and antigen induction by Hu-IFN-. Thus the segregation of cells on the basis of a biological effect by fluorescence-activated cell sorting and reconstitution by hybrid fusion provides a strategy by which some biological pathways can be mapped at a chromosomal level.     

Isolation and characterization of irradiation fusion hybrids from mouse Chromosome 1 for mapping Rmc-1, a gene encoding a cellular receptor for MCF class murine retroviruses

An irradiation-reduced somatic cell hybrid mapping panel was constructed of BALB/c mouse Chromosome 1. Nineteen hybrids were selected from a pool of 292 clones to generate a fine structure physical map of the distal 40 cM of the chromosome. The hybrids contain mouse DNA fragments only from Chromosome 1, ranging from 5 cM to 20 cM. Utilizing a viral infectibility assay, a cellular receptor gene,Rmc-1, for the MCF class of murine retroviruses was found to be linked toLamb2, in the region between theLamb2 andBxv-1 loci. In addition, analysis of the hybrid mapping panel resulted in the remapping of three loci,Atpb, Ly-5, andPmv-24, as compared to the mouse linkage map. Two previously unmapped endogenous proviruses are also putatively assigned positions on the chromosome.     

The proopiocortin (adrenocorticotropin/β-lipotropin) gene is located on chromosome 2 in humans

The proopiocortin gene is located on chromosome 2 in humans. A 13-kb DNA fragment containing proopiocortin gene sequences was identified in human cells while proopiocortin-related gene sequences of 9.8 and 6.2 kb were present in mouse cells. In human-mouse cell hybrids which contained reduced numbers of human chromosomes and a complete set of mouse chromosomes, the 9.8- and 6.2-kb fragments were always present while the 13-kb fragment segregated with human chromosome 2 and the chromosome 2 enzyme markers acid phosphatase-1 (ACP1), malate dehydrogenase-1 (MDH1), and isocitrate dehydrogenase-1 (IDH1). Analysis of a single cell hybrid with a broken chromosome 2 indicates that the proopiocortin andACP1 genes are closely linked and in the distal region of the short arm of chromosome 2.     

GM1-gangliosidosis: Chromosome 3 assignment of theβ-galactosidase-A gene (β GAL A )

The structural gene (GAL_A) coding for lysosomal -galactosidase- A (EC 3.2.1.23) has been assigned to human chromosome 3 using man-mouse somatic cell hybrids. Human -galactosidase-A was identified in cell hybrids with a species-specific antiserum to human liver -galactosidase-A. The antiserum precipitates -galactosidase-A from human tissues, cultured cells, and cell hybrids, and recognizes cross-reacting material from a patient with G_M1 gangliosidosis. We have analyzed 90 primary man-mouse hybrids derived from 12 separate fusion experiments utilizing cells from 9 individuals. Enzyme segregation analysis excluded all chromosomes for GAL_A assignment except chromosome 3. Concordant segregation of chromosomes and enzymes in 16 cell hybrids demonstrated assignment of GAL_A to chromosome 3; all other chromosomes were excluded. The evidence suggests that G_M1 gangliosidosis is a consequence of mutation at this GAL_A locus on chromosome 3.     

Chromosomal assignments of genes coding for human leukocyte common antigen,T-200, and lymphocyte function-associated antigen 1, LFA-1β subunit

Five hybrids reactive with monoclonal antibodies against human leukocyte common antigen (T-200) and/or lymphocyte function-associated antigen 1 (LFA-1) subunit were obtained from the fusion of human blood lymphocytes or T-cell chronic lymphocytic leukemia cells with BW5147 mouse T-cell leukemia cells. Chromosome analyses of 20 clones showed concordance between the presence of human chromosomes 1 and 21 and the expression of T-200 and LFA-1 subunit, respectively. Confirmation of human chromosomes in the hybrids was made by the electrophoretic analyses of phosphoglucomutase 1 for chromosome1and Superoxide dismutase _A for chromosome 21. The results suggested that the presence of human chromosomes 1 and 21 was essential for the expression of T-200 and LFA-1 subunit, respectively.     

Assignment of the gene for uridine diphosphate galactose-4-epimerase to human chromosome 1 by human-mouse somatic cell hybridization

Hybrid cell clones between mouse cells deficient in hypoxanthine phosphoribosyltransferase and human diploid cells were analyzed for the expression of human uridine diphosphate galactose-4-epimerase (UDPGal-4-epimerase, EC 5.1.3.2) and for the segregation of human chromosomes. Ten of the 18 independent hybrid clones analyzed were found to express human UDPGal-4-epimerase. Karyotype analysis of these hybrids showed concordant segregation of human UDPGal-4-epimerase with chromosome 1. Human UDPGal-4-epimerase is expressed as a dimeric structure demonstrated by the formation of an intermediate heterodimer migrating between the human and mouse forms in the cell hybrids. The gene specifying this enzyme was also observed to be syntenic with enolase-1. These results indicate that the structural gene for human UDPGal-4-epimerase is located on chromosome 1.     

Distribution of genes for gap junction membrane channel proteins on human and mouse chromosomes

Gap junctions are widely distributed structures that mediate communication between cells. The channels that allow passage of small molecules between adjacent cells are made up of oligomeric proteins (connexins) that are encoded by a family of related genes. By probing somatic cell hybrid DNA on Southern filters with rat or human cDNAs or human genomic fragments, we have mapped four functioning gap junction genes, (₁, ₁, ₂, and ₃), to different sites on human chromosomes:GJA1 (connexin43) to 6p21.1–q24.1;GJB1 (connexin32) to Xcen-q22;GJB2 (connexin26) to 13; andGJA3 (connexin46) also to 13, probably nearGJB2. The GJA3 probe also hybridized to a restriction fragment that was mapped to chromosome 1. A GJA1-related pseudogeneGJA1P was assigned to chromosome 5. The homologous loci in mouse were assigned to regions of known conserved syntenic groups:Gja-1 to chromosome 10;Gjb-1 to XD-F4 andGjb-2 to 14. Of two sites of hybridization with the GJA3 probe, on mouse 14 and 5, we assume that the site on 14 corresponds to theGJA3 locus on human 13. Based on these data, additional members of this family of related genes can be isolated and characterized, and possible human and mouse mutations can be identified.     

Chromosome assignment of genes encoding theα andΒ subunits of glycoprotein hormones in man and mouse

The chromosomal locations of the genes for the common α subunit of the glycoprotein hormones and the Β subunit of chorionic gonadotropin in humans and mice have been determined by restriction enzyme analysis of DNA isolated from somatic cell hybrids. The CGα gene (CGA), detected as a 15-kb BamHI fragment in human DNA by hybridization to CGα cDNA, segregated with the chromosome 6 enzyme markers ME1 (malic enzyme, soluble) and SOD2 (superoxide dismutase, mitchondrial) and an intact chromosome 6 in human-rodent hybrids. Cell hybrids containing portions of chromosome 6 allowed the localization of CGA to the q12 → q21 region. The >30- and 6.5-kb BamHI CGB fragments hybridizing to human CGΒ cDNA segregated concordantly with the human chromosome 19 marker enzymes PEPD (peptidase D) and GPI (glucose phosphate isomerase) and a normal chromosome 19 in karyotyped hybrids. A KpnI-HindIII digest of cell hybrid DNAs indicated that the multiple copies of the CGΒ gene are all located on human chromosome 19. In the mouse, the α subunit gene, detected by a mouse thyrotropin (TSH) α subunit probe, and the CGΒ-like sequences (CGΒ-LHΒ), detected by the human CGΒ cDNA probe, are on chromosomes 4 and 7, respectively.     

Cell-surface antigens determined by human chromosomes 1 and 12: Comparative serological and somatic cell genetic analysis of eight antigenic systems

We have previously assigned several genes controlling expression of cell-surface antigens to human chromosomes 1 and 12. In the present study, we characterize three additional cell-surface antigens determined by these chromosomes. Two monoclonal antibodies, AbSR75 and AbMG6, define antigens expressed on a wide range of cultured human cells. In contrast, AbK66 defines an antigen with a more restricted distribution which is expressed on normal and malignant human epithelial cells but not on neuroectoderm-derived cells or hematopoietic cells. Normal adult fibroblasts are K66^–, whereas fetal fibroblasts are K66⁺. Serological analysis of rodent-human hybrid cells permitted the assignment of MSK31,controlling SR75, and MSK32,controlling K66, to chromosome 1, whereas MSK27, controlling MG6, maps to chromosome 12. Analysis of hybrids containing only deleted copies of chromosome 1 or chromosome 12 established regional assignments forMSK31, MSK32, and MSK27 and also for the previously defined genesMSK1, MSK4, andMSK7. TheMSK1 gene, coding for a 140,000 mol wt cell surface glycoprotein, was found to map to the same band (1p22) as the NRAS protooncogene and the gene encoding the -subunit of nerve growth factor (NGFB).