Transfer of the human X chromosome to human--Chinese hamster cell hybrids via isolated HeLa metaphase chromosomes.

Evidence is presented for the uptake of the human X chromosome by human-Chinese hamster cell hybrids which lack H P R T activity, following incubation with isolated human HeLa S3 chromosomes. Sixteen independent clonal cell lines were isolated in H A T medium, all of which contained a human X chromosome as determined by trypsin-Giemsa staining. The frequency of H A T-resistant clones was 32 x 10(-6) when 10(7) cells were incubated with 10(8) HeLa chromosomes. Potential reversion of the hybrid cells in H A T medium was less than 5 x 10(-7). The 16 isolated cell lines all contained activity of the human X-linked marker enzymes H P R T, P G K,alpha-Gal A, and G6PD, as determined by electrophoresis. The phenotype of G6PD was G6PD A, corresponding to G6PD A in HeLa cells. The human parental cells used in the fusion to form the hybrids had the G6PD B phenotype. The recipient cells gave no evidence of containing human X chromosomes. These results indicate that incorporation and expression of HeLa X chromosomes is accomplished in human-Chinese hamster hybrids which lack a human X chromosome.     

Suppression of replication of SV40 and polyoma virus in mouse-human hybrids.

Mouse-human heterokaryons are permissive for the replication of both SV40 virus and polyoma virus. If the hybrids which develop from these heterokaryons segregate human chromosomes (mouse greater than human hybrids), the hybrids are permissive for replication of polyoma virus but not for replication of SV40 virus. If the subsequent hybrids segregate mouse chromosomes (human greater than mouse hybrids), such hybrids support the replication of SV40 virus but not the replication of polyoma virus, even when the hybrids contain at least one copy of each mouse chromosome. This indicates that during the transition from heterokaryon to hybrid cell, suppression of expression of species-specific function(s) required for the replication of these species-specific viruses occurs in parallel with the direction of chromosome loss and suppression of nucleolus organizer activity.     

Maintenance of replication patterns in human-mouse hybrids retaining only one human chromosome.

The time of termination of DNA replication of human chromosomes in human-mouse hybrids retaining only one human chromosome was analyzed. Hybrids between SV40-transformed human skin fibroblasts and mouse peritoneal macrophages were used for these studies. Data obtained from hybrids containing only human chromosome 7 or 17 were compared with data from related hybrids containing additional human chromosomes. When either human chromosome 7 or 17 was present alone, it terminated replication at the same stage of the S phase as in hybrids in which other human chromosomes were present (relative to the time of termination of replication of the mouse chromosomes). In comparing the hybrids containing single human chromosomes, it was found that chromosome 17 terminated replication much earlier than chromosome 7. Therefore, the relationship between the replication times of these chromosomes normally observed in human cells was maintained in the hybrids in the absence of all other human chromosomes. The results also indicate that the presence of SV40 gene sequences in chromosomes 7 and 17 did not alter the relative times of termination of replication of those chromosomes.     

Mouse A9 cells containing single human chromosomes for analysis of genomic imprinting.

To develop an systematic in vitro approach for the study of genomic imprinting, we generated a new library of human/mouse A9 monochromosomal hybrids. We used whole cell fusion and microcell-mediated chromosome transfer to generate A9 hybrids containing a single, intact, bsr-tagged human chromosome derived from primary fibroblasts. A9 hybrids were identified that contained either human chromosome 1, 2, 4, 5, 7, 8, 10, 11, 15, 18, 20, or X. The parental origin of these chromosomes was determined by polymorphic analysis using microsatellite markers, and matched hybrids containing maternal and paternal chromosomes were identified for chromosomes 5, 10, 11 and 15. The imprinted gene KVLQT1 on human chromosome 11p15.5 was expressed exclusively from the maternal chromosome in A9 hybrids, and the parental-origin-specific expression patterns of several other imprinted genes were also maintained. This library of human monochromosomal hybrids is a valuable resource for the mapping and cloning of human genes and is a novel in vitro system for the screening of imprinted genes and for their functional analysis.     

Association of human chromosome 14 with a ts defect in G1 of Chinese hamster K12 cells.

Somatic cell hybrids between human lymphoblastoid cells (Raji) and temperature-sensitive Chinese hamster cells (K12) were selected from monolayer cultures in MEM at 40 degrees C. A total of 21 hybrid clones were isolated and karyotyped. All clones contained a near complete set of Chinese hamster chromosomes and 1 to 5 human chromosomes. Human chromosome 14 present in the hybrid cells of all clones; and was the only human chromosome retained in 10 clones. The presence of human chromosome 14 in hybrids was further confirmed by the demonstration of human nucleoside phosphorylase activity in the hybrid cells. Only one hybrid clone was positive for EBNA, the Epstein-Barr virus antigen present in Raji cells. These findings indicate that human chromosome 14 contains the necessary information for the K12 cells to overcome their G1 defect in the cell cycle and grow at non-permissive temperature. The present study lends strong support to the possibility that different steps in the G1 phase of the cell cycle are controlled by genes located on different chromosomes.     

Localization of the human alpha-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

We have used 16 human × mouse somatic cell hybrids containing a variable number of human chromosomes to demonstrate that the human α-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human α-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which have been characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the α-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human α-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human α-globin sequences, one contained chromosome 2 (JFA 14a 5), three contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and two contained chromosome 5 (AHA 16E and JFA14a 13 5) in >10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which had been suggested by other investigators to contain human globin genes. Only chromosome 16 was present in each one of the three hybrid cell clones found to be strongly positive for the human α-globin gene. Two clones (WAIV A and WAV) positive for the human α-globin gene and chromosome 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked both human chromosome 16 and the α-globin gene. These studies establish the localization of the human α-globin gene to chromosome 16 and represent the first assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.     

Assignment of the structural gene encoding human aspartylglucosaminidase to the long arm of chromosome 4 (4q21----4qter)

The structural gene for the human lysosomal enzyme aspartylglucosaminidase (AGA) has been assigned to chromosome 4 using somatic cell hybridization techniques. The human monomeric enzyme was detected in Chinese hamster-human cell hybrids by a thermal denaturation assay that selectively inactivated the Chinese hamster isozyme, while the thermostable human enzyme retained activity. Twenty informative hybrid clones, derived from seven independent fusions, were analyzed for the presence of human AGA activity and their human chromosomal constitutions. Without exception, the presence of human AGA in these hybrids was correlated with the presence of human chromosome 4. All other human chromosomes were excluded by discordant segregation of the human enzyme and other chromosomes. Two hybrid clones, with interspecific Chinese hamster-human chromosome translocations involving the long arm of human chromosome 4, permitted the assignment of human AGA to the region 4q21----4qter.     

Chromosome mapping of human cell surface molecules: monoclonal anti-human lymphocyte antibodies 4F2, A3D8, and A1G3 define antigens controlled by different regions of chromosome 11

Monoclonal antibodies 4F2, A3D8, and A1G3, directed against cell surface antigens present on subsets of human cells, were used to identify the human chromosome regions that code for the antigenic determinants. Human fibroblasts expressed all three antigens, and no cross-reactivity with Chinese hamster or mouse cells was found. Fourteen rodent X human somatic cell hybrids, derived from six different human donors and from two different Chinese hamster and one mouse cell line, were studied simultaneously for human chromosome content and for antibody binding as detected by indirect immunofluorescence. Concordancy with binding of all three antibodies was observed only for human chromosome 11. All other chromosomes were excluded by three or more discordant hybrid clones. Data from six hybrids containing three different regions of chromosome 11 indicate that it is the long arm of chromosome 11 which is both necessary and sufficient for expression of the human antigen defined by 4F2 while the antigen(s) defined by A3D8 and A1G3 map to short arm.     

The human gene for beta glucuronidase is on chromosome 7.

Inconsistent assignments of the human gene for beta glucuronidase (GUS) to chromosomes 7 and 9 have previously been reported. In this study, we have correlated the expression of human GUS in 22 primary Chinese hamster/human hybrid lines with quantitative cytogenetic analysis. Eight hybrids were positive for human GUS as manifested by a five-band pattern on electrophoresis. All of them contained a human chromosome 7 in 34% or more of cells, and seven of them had not retained chromosome 9. One hybrid with only 6% of metaphases possessing a chromosome 7 had no detectable human GUS activity. Human GUS expression was absent in 10 hybrid clones containing chromosome 9 but not 7 and in control fibroblasts from a patient with GUS deficiency. These results support the assignment of presumably the structural gene for beta glucuronidase to chromosome 7.     

Isolation of cDNA clones coding for the alpha-subunit of human beta-hexosaminidase. Extensive homology between the alpha- and beta-subunits and studies on Tay-Sachs disease

The lysosomal beta-hexosaminidases (N-acetyl-beta-glucosaminidase, EC 3.2.1.30) occur as two major isozymes, hexosaminidase A (alpha beta a beta b) and hexosaminidase B (2(beta a beta b)). To facilitate the investigations of the biosynthesis and structure of the enzymes and the nature of mutation in Tay-Sachs disease, we have isolated cDNA clones coding for the alpha-subunit. The polypeptide chains of hexosaminidase A (30 mg) were digested with trypsin, and peptides were isolated by reverse phase high pressure liquid chromatography and their amino acid sequences determined. One of alpha-chain peptides contained a string of seven amino acids from which two sets of oligonucleotides were specified. They were used to screen the SV40-transformed human fibroblast cDNA library of Okayama and Berg. Three cDNA clones, designated pHexA, identified from among 5 X 10(5) clones screened, contained the deduced amino-acid sequences of five alpha-chain peptides. Genomic DNA homologous to pHexA cDNA mapped to human chromosome 15 in somatic cell hybrids, as expected for the pre-alpha-polypeptide. Two of the clones contained identical polyadenylation sites, while the third was polyadenylated about 450 base pairs downstream. The two types of clones were found to correspond to a major 2.0-kilobase pair and a minor 2.3-kilobase pair mRNA species. Blot hybridizations of mRNA and DNA from Tay-Sachs variant fibroblasts revealed absence or reduction of levels of both mRNA species among infantile and juvenile variants, but no observable DNA alterations. Alignment of the pre-alpha- and pre-beta-polypeptides revealed 55% nucleotide and 57% amino acid homology. These data suggest a common origin of the HEXA and HEXB genes and account for the similar substrate specificities of the alpha-dimer subunit, hexosaminidase S, and hexosaminidase B.     

Assignment of the structural genes for the alpha subunit of hexosaminidase A, mannosephosphate isomerase, and pyruvate kinase to the region q22-qter of human chromosome 15.

Concordant segregation of the expression of the alpha subunit of human hexosaminidase A, human mannosephosphate isomerase, and pyruvate kinase was observed in somatic cell hybrids between either thymidine kinase-deficient mouse cells or thymidine kinase-deficient Chinese hamster cells and human white blood cells carrying a translocation of the distal half (q 22-qter) of the long arm of chromosome 15 to chromosome 17. A positive correlation was established between the expression of these human phenotypes and the presence of the distal half of the long arm of human chromosome 15.     

Gene for human CD59 (likely Ly-6 homologue) is located on the short arm of chromosome 11

The CD59 (MEM-43) antigen, which probably is a human homologue of mouse Ly-6 antigens, is a broadly expressedM _r 18000–25000 human leucocyte surface glycoprotein recognized by monoclonal antibody MEM-43. Ten mouse-human T-lymphocyte hybrids, carrying all mouse chromosomes and a limited number of human chromosomes, were analyzed for expression of CD59 by indirect immunofluorescence and immunoblotting with MEM-43 antibody. Karyotypic analysis of the tested clones showed that the presence of human chromosome 11 correlated with the expression of CD59 in all clones tested. Three other human chromosome 11-encoded antigens, 4F2 (Trop-4), Leu 7 (HNK-1, CD57), and lymphocyte homing receptor, were expressed concordantly with CD59. A more exact localization of the gene for CD59 was obtained by the study of Chinese hamster-human cell hybrids containing short or long arm deletions of human chromosome 11. CD59 segregated with hybrids containing part of the short arm of human chromosome 11, but not with the hybrids containing the long arm. Based on these studies we assign the gene for CD59 to regionP14–p13 of the short arm of chromosome 11.     

Requirement of human chromosomes 19, 6 and possibly 3 for infection of hamster x human hybrid cells with baboon M7 type C virus.

The replication pattern of the endogenous baboon type C virus M7 was studied in 29 primary Chinese hamster × human hybrid clones generated with leukemic cells from two different patients with acute lymphoblastic or myeloblastic leukemia. There was no evidence of viral particulate RDDP or M7 antigen before viral infection. M7 virus replicated in human and some hybrid cells but not in Chinese hamster cells, indicating that M7 requires dominantly expressed human gene(s) for replication. Enzyme and cytogenetic analyses show that a gene(s) coded for by human chromosome 19 is necessary for M7 infection of these hybrids. Detailed cytogenetic correlations revealed, however, that the chromosome 19+/M7 + hybrid clones with intact chromosomes also had copies of chromosomes 3 and 6. Previously, Bevi, the putative integration site for M7 virus, has been assigned to human chromosome 6. Many clones with various combinations of chromosomes 3 and 6 lacked chromosome 19, however, and failed to replicate exogenously applied M7 virus, while tests of 27 secondary clones showed that M7 markers co-segregated with chromosome 19 markers. These findings all confirm the need for a chromosome 19-coded function in Chinese hamster × human hybrids. In addition, the yield of viral particulate RDDP produced into the culture fluid was 50–100 fold less per viral antigen-positive cell in the hybrids compared with human cells. Thus some form of regulation of viral components exists in the hybrid cells. When the virus replicating in hybrid cells was transferred back to human cells, this regulation was relaxed and the yield of RDDP per FA(+) cell greatly increased. We conclude that human chromosomes 6 and 19 code for functions involved in M7 virus metabolism, and we cannot exclude a function coded for by chromosome 3.     

A genetic analysis of extinction: trans-dominant loci regulate expression of liver-specific traits in hepatoma hybrid cells

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.     

Necessity for two human chromosomes for human chorionic gonadotropin production in human-mouse hybrids

Through a series of human-mouse hybrids we have identified that two human chromosomes, 10 and 18, must be present for production of the pregnancy protein hormone human chorionic gonadotropin (hCG). Human choriocarcinoma cells producing hCG were hybridized to mouse cells. From 49 independent clones three hybrid clones continued to produce whole hCG. Chromosomal analysis was done on the 3 producer clones and 5 nonproducer clones. The additional 41 nonproducer clones were genetically characterized by isozymes. Only when chromosomes 10 and 18 were present in a clone would the whole hCG molecule be produced. Clones with only 10 or only 18 did not produce hormone. Nine subclones of a producer clone confirmed this observation. Three subclones retaining both 10 and 18 continued to produce hCG. This study demonstrated the need to use cellular chromosome data and population enzyme data to identify two chromosomes necessary for hCG production in heterogeneous human-mouse hybrids.     

Assignment of the gene for lactic dehydrogenase A to mouse chromosome 7 using mouse-human hybrids

We have studied somatic cell hybrids between either mouse peritoneal macrophages or spleen cells and HT-1080-6TG human fibrosarcoma cells for the expression of mouse lactic dehydrogenase A (LDH-A). The hybrids were also studied for the expression of mouse glucose phosphate isomerase (GPI-1), the gene for which has been assigned to chromosome 7. Concordant segregation of the expression of mouse GPI-1 and LDH-1 was observed in 61 independent hybrid clones. These results indicate that the gene coding for LDH-A is located on mouse chromosome 7.     

Establishment of hybridomas secreting human monoclonal antibodies against tetanus toxin and hepatitis B virus surface antigen

Mouse-human heterohybrids (M X H) were constructed and compared with other cell lines (human or mouse) as parental cells to obtain hybrids secreting human monoclonal antibody (MoAb). One of the M X H lines, HM-5, was far superior to the others and useful for establishing hybrids secreting human MoAb. Using HM-5 as a parental cell line, we have obtained 2 hybrids secreting human anti-tetanus toxoid MoAb with neutralizing activity and a hybrid secreting human anti-hepatitis B virus surface antigen (HBsAg) MoAb which recognizes the a-determinant of HBsAg.     

Quantitation of the viral DNA present in somatic cell hybrids between mouse and SV40-transformed human cells

Recent studies of somatic cell hybrids between mouse cells and SV40-transformed human cells have demonstrated a correlation between the expression of SV40 T-antigen and the presence of human chromosome 7. We have used two types of nucleic acid hybridization procedures to detect and quantitate the presence of viral DNA sequences in the DNA of the hybrid cell clones. Results of reassociation kinetics as well as hybridization with a single-strand probe indicate that SV40 DNA is present only in those hybrid clones which both contain human chromosome 7 and express the SV40 T-antigen. SV40 DNA was not detectable either in the clones which had lost human chromosome 7, or in the rare clones which retain human chromosome 7 but which do not express T-antigen. We have thus extended the correlation between human chromosome 7 and the SV40 T-antigen to the presence of integrated SV40 DNA in somatic cell hybrid clones.