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.     

Human chromosome 9 can complement UV sensitivity of xeroderma pigmentosum group A cells

A single human chromosome derived from normal human fibroblasts and tagged with the G418 resistance gene was transferred into SV40-transformed xeroderma pigmentosum group A (XP-A) cells via microcell fusion. When chromosome 1 or 12 was transferred, UV sensitivity of microcell hybrid cells was not changed. By contrast, after transferring chromosome 9, 7 of 11 recipient clones were as UV-resistant as normal human cells. Four other clones were still as UV-sensitive as the parental XP-A cells. Southern hybridization analysis using a polymorphic probe, pEKZ19.3, which is homologous to a sequence of the D9S17 locus on chromosome 9, has confirmed that at least a part of normal human chromosome 9 was transferred into the recipient clones. However, amounts of UV-induced unscheduled DNA synthesis in the UV-resistant clones were only one-third of those in normal human cells. These results indicate that a gene on chromosome 9 can confer complementation of high UV sensitivity of XP-A cells although it is still possible that 2 or more genes might be involved in the defective-repair phenotypes of XP-A.     

Complementation of repair gene mutations on the hemizygous chromosome 9 in CHO: a third repair gene on human chromosome 19

A human DNA repair gene, ERCC2 (Excision Repair Cross Complementing 2), was assigned to human chromosome 19 using hybrid clone panels in two different procedures. One set of cell hybrids was constructed by selecting for functional complementation of the DNA repair defect in mutant CHO UV5 after fusion with human lymphocytes. In the second analysis, DNAs from an independent hybrid panel were digested with restriction enzymes and analyzed by Southern blot hybridization using DNA probes for the three DNA repair genes that are located on human chromosome 19: ERCC1, ERCC2, and X-Ray Repair Cross Complementing 1 (XRCC1). The results from hybrids retaining different portions of this chromosome showed that ERCC2 is distal to XRCC1 and in the same region of the chromosome 19 long arm (q13.2-q13.3) as ERCC1, but on different MluI macrorestriction fragments. Similar experiments using a hybrid clone panel containing segregating Chinese hamster chromosomes revealed the hamster homologs of the three repair genes to be part of a highly conserved linkage group on Chinese hamster chromosome number 9. The known hemizygosity of hamster chromosome 9 in CHO cells can account for the high frequency at which genetically recessive mutations are recovered in these three genes in CHO cells. Thus, the conservation of linkage of the repair genes explains the seemingly disproportionate number of repair genes identified on human chromosome 19.     

Phenotypic correction of the defect in xeroderma pigmentosum cells after fusion with isolated cytoplasts

The cybridization technique was used to study the role of cytoplasmic and nuclear factors in complementation of the repair defects in xeroderma pigmentosum (XP) cells. Cybrids were prepared by fusion of UV-exposed XP cells with cytoplasts derived from normal human or complementing XP cells. Phenotypic correction of the DNA repair defect measured by unscheduled DNA synthesis (UDS) occurred in these cybrids. The results show that the correcting factors are present in the cytoplasts and can move into the nucleus of the UV-exposed XP cell almost immediately after fusion. The defective repair in the nuclei of XP complementation group A cell strains is corrected with fast kinetics reaching normal UDS levels within 2 h after fusion. In the A-group cybrids the correcting activity decreased with a half-time of about 12 h. Correction of the XP group C defect occurred at a much slower rate, indicating that different factors are involved in the correction of the XP-A and XP-C defects.     

Regional localization of a beta-galactosidase locus on human chromosome 22.

Human white blood cells with an X/22 translocation [46, XX, t(X;22)(q23;q13)] were fused with Chinese hamster cells. The isolated hybrids were analyzed for human chromosomes and 21 enzyme markers. An electrophoretic technique for studying the beta-galactosidase isoenzymes in man-Chinese hamster hybrid cells was developed. Immunological studies showed that the beta-galactosidase marker studied in these hybrids did contain immunological determinants of human origin. Furthermore the results provided evidence that a locus for beta-galactosidase is situated on chromosome 22 distal to the breakpoint in q13.     

Interspecies complementation analysis of xeroderma pigmentosum and UV-sensitive chinese hamster cells

Complementation analysis was performed 24 h after fusion of UV-sensitive CHO cells (CHO 12 RO) with XP cells of complementation groups A, B, C, D, F and G. The parental cells are characterized by low levels of unscheduled DNA synthesis (UDS). In all combinations, the UDS levels observed in heterokaryons were higher than those in parental mutant cells, clearly indicating cooperation of human and Chinese hamster repair functions. In heterokaryons of CHO 12 RO with XP-A and XP-C cells, the UDS values reached about the normal human level, whereas in heterokaryons with XP-B, XP-D and XP-F, UDS was restored at a level approaching that in wild-type CHO cells. The results obtained after fusion of CHO cells with two representative cell strains from the XP-G group, XP 2 BI and XP 3 BR, were inconsistent. Fusion with XP 3 BR cells yielded UDS levels ranging from wild-type Chinese hamster to normal human, whereas fusion with XP 2 BI cells resulted in a slight increase in UDS which even after 48 h remained below the level found in wild-type CHO cells. The occurrence of complementation in these interspecies heterokaryons indicates that the genetic defect in the CHO 12 RO cells is different from the defects in the XP complementation groups tested.     

Optimization strategies for production of mammalian embryos by nuclear transfer

In order to optimize each of the individual steps in the nuclear transfer procedure, we report alternative protocols useful for producing recipient cytoplasts and for improving the success rate of nuclear transfer embryos in cattle, rhesus monkey, and hamster. Vital labeling of maternal chromatin/spindle is accomplished by long wavelength fluorochromes Sybr14 and rhodamine labeled tubulin allowing constant monitoring and verification during enucleation. The use of Chinese hamster ovary (CHO) donor cells expressing the viral influenza hemagglutinin fusion protein (HA-300a+), to adhere and induce fusion between the donor cells and enucleated cow, rhesus and hamster oocytes was examined. Cell surface hemagglutinin was activated with trypsin prior to nuclear transfer and fusion was induced by a short incubation of a newly created nuclear transfer couplet at pH 5.2 at room temperature. Donor cell cytoplasm was dynamically labeled with CMFDA, or further transfected with the green fluorescence protein (GFP) gene, so that fusion could be directly monitored using live imaging. High rates of fusion were observed between CHO donor cells and hamster (100%), rhesus (100%), and cow recipient cytoplasts (81.6%). Live imaging during fusion revealed rapid intermixing of cytoplasmic components between a recipient and a donor cell. Prelabeled donor cytoplasmic components were uniformly distributed throughout the recipient cytoplast, within minutes of fusion, while the newly introduced nucleus remained at the periphery. The fusion process did not induce activation as evidenced by unchanged distribution and density of cortical granules in the recipient cytoplasts. After artificial activation, the nuclear transfer embryos created in this manner were capable of completing several embryonic cell divisions. These procedures hold promise for enhancing the efficiency of nuclear transfer in mammals of importance for biomedical research, agriculture, biotechnology, and preserving unique, rare, and endangered species.     

Evidence for postmeiotic expression of ribosomal RNA genes during male gametogenesis

Summary In the progeny of somatic cell hybrids formed by fusion of human lymphocytes and Chinese hamster mutant cells, a single human chromosome A2 was selectively retained when grown in appropriate medium.Spontaneous breakage of this chromosome in different hybrid subclones led to the assignment of the gene for galactose-1-phosphate uridyltransferase to the centromeric region of this chromosome (2q11-2q14). This gene is shown to be syntenic to the previously mapped genes for acid phosphatase 1 and malate dehydrogenase 1.     

Purification of cybrids by fluorescence-activated cell sorting

A general method to isolate and purify substantial numbers of viable cybrids from cultured mammalian cells immediately following cytoplast-cell fusion is described. This method uses cytoplasts whose mitochondria are selectively stained in vivo by the cationic fluorescent rhodamine dye, rhodamine 123. Large numbers of highly purified, rhodamine-stained cytoplasts are fused to appropriate recipient cell lines and then the fusion mixture is sorted based on forward angle scatter and fluorescence parameters. Plating the positively sorted population in culture for as short as 12 h eliminates contaminating cytoplasts which, lacking a nucleus, are unable to adhere or survive. The resultant population, based on an analysis of genetic markers, is 75-100% cybrids, an enrichment of 1000- to 10,000-fold over the initial fusion mixture. Cybrids purified by cell sorting may be useful for detailed molecular studies of mitochondrial DNA gene expression and in the specific induction of new mitochondrial DNA mutants.     

Fibroblast interferon in man is coded by two loci on separate chromosomes.

We have examined viral and poly(rl):poly(rC) induction of interferon synthesis in several human, mouse and Chinese hamster cell lines, and in hybrids derived from the fusion of such cells. We observed species and cell-type differences in inducer effectiveness and in the kinetics of interferon production. In some cases, parental characteristics are preserved in somatic cell hybrids, and in other cases, the expression of the donor phenotype is modulated by the epigenetic state of the recipient cell. Mapping studies in human/mouse and human/Chinese hamster hybrids indicate that there are at least two structural genes for human fibroblast interferon. Chromosomes 2 and 5 each contain genetic information for the synthesis of fibroblast interferon. Gene dosage experiments indicate that one gene is on the long arm of chromosome 2 and another is on the short arm of chromosome 5. Leukocyte interferon genes could not be mapped to these chromosomes, but this negative result could be influenced by the epigenetic state of the hybrid cells.     

Genetic effects of chromosomal rearrangements in Chinese hamster ovary cells: expression and chromosomal assignment of TK, GALK, ACP1, ADA, and ITPA loci.

Polyethylene glycol-mediated fusion of Chinese hamster ovary (CHO) cells with mouse Cl1D cells produced interspecific somatic cell hybrids which slowly segregated CHO chromosomes. Cytogenetic and isozyme analysis of HAT- and bromodeoxyuridine-selected hybrid subclones and of members of a hybrid clone panel retaining different combinations of CHO chromosomes enabled provisional assignments of the following enzyme loci to CHO chromosomes: TK, GALK, and ACP1 to chromosome 7; TK and GALK to chromosome Z13; ACP1, ADA, and ITPA to chromosome Z8; and ADA and ITPA to chromosome Z9. These genetic markers reflect the origin of each of these Z group chromosomes and indicate the functional activity of alleles located on rearranged chromosomes. Identification of diploid electrophoretic shift mutations for ADA and ITPA was consistent with those observations. Assignment of the functional TK locus in TK+/- CHO-AT3-2 cells indicated that gene deletion may be responsible for TK hemizygosity in this subline.     

Human chromosome 11 complements ataxia-telangiectasia cells but does not complement the defect in AT-like Chinese hamster cell mutants

It has been shown that the X-ray-sensitive Chinese hamster V79 mutants (V-E5, V-C4 and V-G8) are similar to ataxia-telangiectasia (A-T) cells. To determine whether the AT-like rodent cell mutants are defective in the gene homologous to A-T (group A, C or D), human chromosome 11 was introduced to the V-E5 and V-G8 mutant cells by microcell-mediated chromosome transfer. Forty independent hybrid clones were obtained in which the presence of chromosome 11 was determined by in situ hybridization. The presence of the region of chromosome 11q22–23 was shown by molecular analysis using polymorphic DNA markers specific for the ATA, ATC and ATD loci. Seventeen of the obtained monochromosomal Chinese hamster hybrids contained a cytogenetically normal human chromosome 11, but only twelve hybrid cell lines were shown to contain an intact 11q22–23 region. Despite the complementation of the X-ray sensitivity by a normal chromosome 11 introduced to A-T cells (complementation group D), these twelve Chinese hamster hybrid clones showed lack of complementation of X-ray and streptonigrin hypersensitivity. The observed lack of complementation does not seem to be attributable to hypermethylation of the human chromosome 11 in the rodent cell background, since 5-azacytidine treatment had no effect on the streptonigrin hypersensitivity of the hybrid cell lines. These results indicate that the gene defective in the AT-like rodent cell mutants is not homologous to the ATA, ATC or ATD genes and that the human gene complementing the defect in the AT-like mutants seems not to be located on human chromosome 11.     

The gene coding for a sphingolipid activator protein,SAP-1, is on human chromosome 10

Summary SAP-1 is a sphingolipid activator protein found in human tissues required for the enzymatic hydrolysis of GM1 ganglioside and sulfatide. It appears to be missing in patients who have a genetic lipidosis resembling juvenile metachromatic leukodystrophy. Using rabbit antibodies against human SAP-1 it could be visualized in extracts from cultured human skin fibroblasts after sodium dodecylsulfate-polyacrylamide gel electrophoresis, followed by electroblotting to nitrocellulose membrane and immunochemical staining (Western blotting). A series of 23 human-Chinese hamster ovary cell hybrids containing different human chromosomes were examined. The parent Chinese hamster ovary cells did not have a reacting protein in the region of human SAP-1. Only in the eight hybrid clones containing human chromosome 10 was a reacting protein identified. Other chromosomes were excluded by this method. Therefore the gene for SAP-1 and the genetic mutation resulting in a fatal lipidosis are located on human chromosome 10. Present address: Department of Pediatrics, Osaka University Medical School, Fukushima-Ku, Osaka, Japan     

Characteristics of the ultrastructural organization of the nuclei of somatic cell hybrids

A study was made of the ultrastructure of cell nuclei of two types of hybrid clones obtained from the fusion of Chinese hamster with human skin fibroblasts, and from that of mouse hepatoma cells with mink fibroblasts. In cell nuclei of the eight hybrid clones deep invaginations of the inner membrane, not characteristic of the parent cells, were revealed. Analysis of serial sections, and application of electron microscopic radioautography and histochemistry have suggested that these structures are associated with the nuclear envelope which is necessary for regulating the superfluous chromosome localization in the hybrid nucleus.     

Human alpha-globin gene expression following chromosomal dependent gene transfer into mouse erythroleukemia cells.

We have used the genetic marker, adenine phosphoribosyl transferase (APRT), an enzyme known to be on human chromosome 16, to establish a method for the transfer of human α-globin genes into mouse erythroleukemia cells. Mouse erythroleukemia cells devoid of detectable levels of APRT were fused with fractions of human marrow enriched in human erythroid cells. The hybrid cells arising from this fusion were isolated in medium supplemented with aminopterin and thymidine, and used adenine as the sole purine source. This population of hybrid cells was dominated by cells (80%) in which human chromosome 16 was present. Human chromosomes 4, 5 and 6 were also found in these cells. The hybrid cells were then placed in medium supplemented with diaminopurine (DAP), which is lethal for cells containing APRT. Greater than 95% of the DAP-selected hybrid cells lacked human chromosome 16. Cytoplasmic RNA was extracted from the two hybrid cell populations and assayed by molecular hybridization for sequences coding for human α-globin. Carboxymethyl cellulose chromatography was used to study the level of synthesis of human a-globin in the hybrids. The original hybrid cell, which contained a high frequency of human chromosome 16, also contained high levels of human a-globin mRNA and human α-globin chains. Hybrid cells counter-selected in DAP and thus lacking human chromosome 16 were devoid of detectable levels of human APRT, human α-globin mRNA and human α-globin chains. This work shows that transfer of human chromosome 16 into the MEL cell is possible using a chromosomedependent, APRT-mediated method of gene transfer. Using this system in which expression of the human α-globin gene occurs, we were also able to confirm our earlier assignment of the human α-globin gene to human chromosome 16. This system may be of further use in identifying genetic elements governing expression of the human α-globin gene which can be carried with human chromosome 16 as it is donated to the mouse erythroleukemia cell by donor cells of different epigenotypes.     

Human chromosome 12 is required for optimal interactions between Tat and TAR of human immunodeficiency virus type 1 in rodent cells.

Levels of trans activation of the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) by the virally encoded transactivator Tat show marked species-specific differences. For example, levels of transactivation observed in Chinese hamster ovary (CHO) rodent cells are 10-fold lower than those in human cells or in CHO cells that contain the human chromosome 12. Thus, the human chromosome 12 codes for a protein or proteins that are required for optimal Tat activity. Here, the function of these cellular proteins was analyzed by using a number of modified HIV-1 LTRs and Tats. Neither DNA-binding proteins that bind to the HIV-1 LTR nor proteins that interact with the activation domain of Tat could be implicated in this defect. However, since species-specific differences were no longer observed with hybrid proteins that contain the activation domain of Tat fused to heterologous RNA-binding proteins, optimal interactions between Tat and the trans-acting responsive RNA (TAR) must depend on this factor(s).     

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.     

Regional assignment of seven genes on chromosome 1 of man by use of man-Chinese hamster somatic cell hybrids. II. Results obtained after induction of breaks in chromosome 1 by X-irradiation.

The position of genes coding for PGD, PPH1, UGPP, GuK1, PGM1, Pep-C, and FH on human chromosome 1 was investigated by analysis of karyotype and enzyme phenotypes in man-Chinese hamster somatic cell hybrids carrying aberrations involving chromosome 1. Suitable hybrid cell lines were obtained by X-irradiation of hybrid cells carrying an intact chromosome 1 and by fusion of human cells from a clonal population carrying a translocation involving chromosome 1 with Chinese hamster cells. The latter human cell population had been isolated following X-irradiation of primary Lesch-Nyhan fibroblasts. In addition, products of de novo chromosome breakage in the investigated hybrid lines were utilized. By integrating the results of these analyses with earlier findings in our laboratory, the following positions of genes are deduced: PGD and PPH1 in 1p36 leads to 1p34; PGM1 in 1p32; UGPP in 1q21 leads to 1q23; GuK1 in 1q31 leads to 1q42; Pep-C in 1q42; and FH in 1qter leads to 1q42.     

Assignment of a human DNA double-strand break repair gene (XRCC5) to chromosome 2.

The Chinese hamster ovary (CHO-K1) cell mutant XRS-6 is defective in rejoining of DNA double-strand breaks and is hypersensitive to X-rays, gamma-rays, and bleomycin. Radiation resistance or sensitivity of somatic cell hybrids constructed from the fusion of XRS-6 cells with primary human fibroblasts strongly correlated with the retention of human chromosome 2 isozyme and molecular markers. Discordancies between some chromosome 2 markers and the radiation resistance phenotype in some of the hybrid cells suggested the location of the X-ray repair cross complementing 5 (XRCC5) gene on the p arm of chromosome 2. Introduction of human chromosome 2 by microcell-mediated chromosome transfer into the radiation-sensitive XRS-6 cells resulted in hybrid cells in which the radiation sensitivity was complemented. The chromosome 2p origin of the complementing human DNA in the microcell hybrids was supported by fluorescent in situ hybridization analysis of human metaphases using human DNA amplified from the hybrids by inter-Alu-PCR as chromosome-painting probes. XRCC5 is therefore provisionally assigned to human chromosome 2p.