Showing papers in "Somatic Cell and Molecular Genetics in 1983"

Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule

Abstract: Hybridomas have been prepared that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells by fusion of mouse myeloma cells with spleen cells from mice and rats immunized with delipidated human milk fat globules. Using a novel method for molecular weight determination, the three different monoclonal antibodies, BLMRL-HMFG-Mc3, BLMRL-HMFG-McR2, and BLMRLHMFG-Mc5, were found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The latter is a mucin-like glycoprotein with a high sugar content and has not previously been described as a component of the human milk fat globule or of human mammary epithelial cell membranes. Single-cell quantitation of binding of monoclonal BLMRL-HMFG-Mc5 to three breast tumor cell lines using a Microscope Spectrum Analyzer and indirect immunofluorescence revealed a heterogeneous expression. Further, using a competitive radioimmunoassay, it was found that breast tumor cell lines differed by at least 10-fold in the 400,000-molecular-weight antigen content. None of the three antigens are detectable on several nonbreast cell lines, including normal breast fibroblasts.

Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell

Abstract: A technique for the isolation of gamma ray-sensitive Chinese hamster ovary (CHO) cell mutants is described, which uses nylon cloth replica plating and photography with dark-field illumination to directly monitor colonies for growth after gamma irradiation. Two gamma raysensitive mutants were isolated using this method. One of these cells (XR-1) had a two-slope survival curve: an initial steep slope and then a flattening of the curve at about 10% survival. Subsequently, it was found that this cell is sensitive to gamma irradiation in G1,early S, and late G2phases of the cell cycle, whereas in the resistant phase (late S phase) its survival approaches that of the parental cells. The D37in the sensitive G1period is approximately 30 rads, compared with 300 rads of the parental cell. This mutant cell is also sensitive to killing by the DNA breaking agent, bleomycin, but is relatively insensitive to UV light and ethyl methane sulfonate, suggesting that the defect is specific for agents that produce DNA strand breakage.

A simple method for premature chromosome condensation induction in primary human and rodent cells using polyethylene glycol

Abstract: Even though polyethylene glycol (PEG) has been shown to be a potent fusogen, it has not been widely exploited as an alternative to the Sendai virus for premature chromosome condensation (PCC) induction. A simple, rapid, and reproducible PEG protocol for primary cells in suspension is presented which allows satisfactory cell fusion and PCC indices, giving at the same time high cell viability and low giant, multinucleated cell formation. Technical details for PEG-mediated fusion and premature chromosome condensation induction in human and rat lymphocytes, rodent spleen cells, and spleen and whole body cells of newborn mice are provided. Further applications of the method are suggested.

Chromosome assignment of genes encoding the alpha and beta subunits of glycoprotein hormones in man and mouse.

Abstract: The chromosomal locations of the genes for the common alpha subunit of the glycoprotein hormones and the beta subunit of chorionic gonadotropin in humans and mice have been determined by restriction enzyme analysis of DNA isolated from somatic cell hybrids. The CG alpha gene (CGA), detected as a 15-kb BamHI fragment in human DNA by hybridization to CG alpha 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 leads to q21 region. The greater than 30- and 6.5-kb BamHI CGB fragments hybridizing to human CG beta 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 CG beta gene are all located on human chromosome 19. In the mouse, the alpha subunit gene, detected by a mouse thyrotropin (TSH) alpha subunit probe, and the CG beta-like sequences (CG beta-LH beta), detected by the human CG beta cDNA probe, are on chromosomes 4 and 7, respectively.

Dominant and recessive aryl hydrocarbon hydroxylase-deficient mutants of mouse hepatoma line, Hepa-1, and assignment of recessive mutants to three complementation groups.

Abstract: Fifty-four benzo[a]pyrene (BP)-resistant, aryl hydrocarbon hydroxylase (AHH)-deficient mutants were found to be recessive, while five were dominant. Hybrids between the former mutants and the wild-type were killed by BP, and possessed AHH activities of at least 0.5 (relative to the wild-type). Dominant-mutant-wild-type hybrids were resistant to BP and had activities of about 0.05. Additional experiments assigned the recessive mutants to three complementation groups, designated A through C. Group-B-group-C hybrids were exceptional in possessing a mean AHH activity (0.36), less than the value (0.5) expected from gene dosage. This deficiency was probably due, in part, to instability of AHH activity in these hybrids. However, all hybrids tested retained stable DNA complements, equal to the sum of those of their parents, for 140 days in culture. Previous studies have shown that group B and group C mutations both affect the functioning of a cytosolic receptor required for AHH induction (1).

Human parathyroid hormone gene (PTH) is on short arm of chromosome 11.

Abstract: The human gene for parathyroid hormone (PTH) was chromosomally mapped using human-rodent hybrids and Southern filter hybridization of cell hybrid DNA. A recombinant DNA probe containing human PTHcDNA insert (pPTHm122) hybridized to a 3.7-kb fragment in human DNA cleaved with the restriction enzyme EcoRI. By correlating the presence of this fragment in somatic cell hybrid DNA with the human chromosomal content of the hybrid cells, the PTHgene was mapped to the short arm of the chromosome 11.

Lectin-resistant CHO cells: Selection of new mutant phenotypes

Abstract: Cytotoxic plant lectins select for mutants which exhibit unique structural changes in surface carbohydrates reflecting specific defects in glycosylation reactions. However, lectins are not highly specific selective agents and, as a result, only the most frequently occurring mutants are obtained from single lectin selections. We have previously shown that the specificity of lectin selections may be improved by utilizing a combination of lectins added together or sequentially. This strategy has now been further exploited in the search for novel lectin-resistant mutants of Chinese hamster ovary cells. Five new LeeR phenotypes have been uncovered. One belongs to a new, recessive complementation group, two behave dominantly in somatic cell hybrids, and the remaining two appear to represent new phenotypes which fall into previously described complementation groups.

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

Abstract: 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.

Structural gene coding for multifunctional protein carrying orotate phosphoribosyltransferase and OMP decarboxylase activity is located on long arm of human chromosome 3.

Abstract: In humans, deficiency in the last two enzymes of UMP biosynthesis, orotate phosphoribosyltransferase (OPRT) and OMP decarboxylase results in the inborn error of metabolism hereditary orotic aciduria, type 1. In this manuscript, we present immunologic, molecular, biochemical, and genetic evidence that the gene coding for this set of enzymatic activities is located on the long arm of human chromosome 3. The evidence presented here is consistent with both these activities being carried on the same multifunctional protein in mammalian cells. These studies allow further genetic analysis of human chromosome 3, confirming that human markers ACY-1, previously assigned to 3p21, and β-gal, previously assigned by others to the region 3(p21-q21), must be in the region 3 (cen-p21) and confirming the regional assignment of a human DNA segment, D3S1, to 3q12. The significance of these studies to genetic analysis of genes on human chromosome 3, some of which appear to play a role in some forms of malignancy, is discussed.

Coreactivation of four inactive X genes in a hamster x human hybrid and persistence of late replication of reactivated X chromosome.

Abstract: Hamster-human hybrids which contained an inactive human X chromosome were treated by 5-azacytidine. Hypoxanthine guanine phosphoribosyltransferase derepressed hybrids were selected and derepression of three other loci, phosphoglycerate kinase, alpha-galactosidase, and glucose-6-phosphate dehydrogenase were studied. Among 32 hybrids selected for hypoxanthine guanine phosphoribosyltransferase, two were found to be reactivated at four X loci. The independence or nonindependence of the reactivation events will be discussed. No correlation was found between the time of replication and the expression or nonexpression of the X chromosome genes: X chromosomes reactivated at four loci remained late replicating; conversely early replication can exist without the expression of some X genes.

Origin, cellular expression, and cybrid transmission of mitochondrial CAP-R, PYR-IND, and OLI-R mutant phenotypes.

Abstract: Chloramphenicol-resistant (CAP-R) mouse and Chinese hamster lines were isolated in a single selection step in drug medium containing pyruvate. Cellular expression of the CAP-R phenotype required pyruvate— or an appropriate substitute—as a nutritional supplement. Subclone lines which were pyruvate independent (PYR-IND) arose in second-step selections at a high frequency. CAP-R PYR-IND Chinese hamster mutants could be directly isolated in single-step selections but at a very low frequency. Subclone lines (OLI-R) which were cross-resistant to oligomycin were isolated in a third selection cycle. The PYR-IND and OLI-R phenotypes were cotransmitted with the CAP-R mtDNA mutation but were expressed at the cellular level only if the number of mutant mitochondrial genomes exceeded a minimum threshold value. Analysis of a mtDNA restriction fragment alteration in one series of mutants supported this model. Threshold limits for cellular expression of mitochondrial mutant phenotypes are likely to be a general phenomenon and will constrain models of the origin and segregation of mtDNA mutations.

Complementation mapping in microcell hybrids: Localization ofFpgs andAk-1 onmus musculus chromosome 2

Abstract: 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→C1).

Expression of recessive Aprt- mutations in mouse CAK cells resulting from chromosome loss and duplication

Abstract: Karyotypes of recessive mutants at the autosomal adenine phosphoribosyltransferase (Aprt) locus in a clone of the neardiploid mouse CAK cell line have been analyzed. The Aprt gene is located on chromosome 8. One copy of chromosome 8 was morphologically abnormal in the parental clone (CAK-B3-Toyr13) from whichAprt − mutants were isolated. Among 22 mutants, there were ten in which one copy of chromosome 8 had been lost. Four of these were monosomic, and in the others duplication of the remaining homolog had occurred. These findings indicate that newly induced recessive mutations in cultured mammalian cells can be expressed as the result of loss of one chromosome carrying a wild-type allele, with or without duplication of the homolog carrying the mutant allele. Loss and duplication would not be detcted in cell lines lacking morphologically marked chromosomes.

Activation of human alpha 1-antitrypsin gene in rat hepatoma x human fetal liver cell hybrids depends on presence of human chromosome 14.

Abstract: In order to study the involvement of human chromosomes in the expression of liver-specific functions, we have produced somatic cell hybrids between a rat hepatoma (7777) cell line and human diploid skin fibroblasts (series XIX) or human fetal liver cells (series XXII). Production of human serum proteins was detected by immunoelectrophoretic analyses of concentrated serum-free hybrid culture supernatants. Human α1-antitrypsin (AAT) was secreted by a subset of hybrids but not by the parental cells. The activated human AAT phenotype segregated concordantly with human chromosome 14 in 18 primarily HAT-selected and five azaguanine back-selected series XXII hybrids. All other chromosomes were excluded as playing a role in AAT expression. Therefore, the AAT gene PI is assigned to chromosome 14. This quasi-constitutive expression of a liver-specific function was not observed for the other serum proteins studied, nor was it seen in the skin fibroblast-derived hybrids (series XIX) although AAT was produced by some of them.

LPS-nonresponsive variants of mouse B cell lymphoma, 70Z/3: isolation and characterization.

Abstract: We have used a genetic approach to study the differentiation of B lymphocytes. The cultured murine cell line 70Z/3 resembles pre-B cells in containing the heavy chain of the immunoglobulin IgM, Μ, as an internal protein in the absence of light chain, L. However, overnight incubation with the B cell mitogen lipopolysaccharide (LPS) induces the cells to mature to a B lymphocyte-like state by the induction of L chain synthesis and the appearance of IgM on the cell surface. We have used immunoselection against surface-bound IgM to isolate LPS uninducible variants of 70Z/3. These fall into two complementation groups, LPS A and LPS B. LPS A variants predominated and were found at a frequency of 1/1200. These cells were completely unresponsive to LPS. LPS B was represented by a single variant in which a subset of cells was induced to display wild-type levels of membranebound IgM, and the proportion of induced cells increased with prolonged incubation with LPS. We detected no structural defects in either variant group, but LPS B may represent a defect in the decision to differentiate in response to LPS.

Human and mouse cellular myc protooncogenes reside on chromosomes involved in numerical and structural aberrations in cancer.

Abstract: A molecular clone of viral myc (v-myc), the oncogene of avian myelocytomatosis virus, MC29, detected homologous human, mouse, and Chinese hamster cellular myc (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.

Assignment of structural gene for asparagine synthetase to human chromosome 7

Abstract: Somatic cell hybrids obtained from the fusion of human B lymphocytes and an asparagine synthetase-deficient Chinese hamster ovary cell line were isolated after growth in asparagine-free medium. The human and hamster forms of asparagine synthetase differ significantly in their rate of inactivation at 47.5 degrees C. The asparagine synthetase activity expressed in the hybrids was inactivated at 47.5 degrees C at the same rate as the human form of the enzyme. Karyotypic analysis and analysis for chromosome-specific enzyme markers showed that the structural gene for asparagine synthetase is located on chromosome 7 in humans. The heat-inactivation profile for asparagine synthetase in extracts of hybrids formed between human peripheral leukocytes and a hamster cell line expressing asparagine synthetase activity was intermediate between the two parental types when human chromosome 7 was present, but was identical to the hamster parent when chromosome 7 was absent.

Isolation and characterization of interspecific heat-resistant hybrids between a temperature-sensitive chinese hamster cell asparaginyl-tRNA synthetase mutant and normal human leukocytes: assignment of human asnS gene to chromosome 18.

Abstract: We isolated interspecific somatic cell hybrids between human peripheral leukocytes and a temperature-sensitive CHO cell line with a thermolabile asparaginyl-tRNA synthetase. The hybrids were selected at 39 degrees C so as to require the expression of the human gene complementing the deficient CHO enzyme. In vitro heat-inactivation profiles of cell-free extracts from temperature-resistant hybrid cells indicate the presence of two forms of asparaginyl-tRNA synthetase. One form is very resistant to thermal inactivation, like the normal human enzyme, while the other form is very thermolabile, like the altered enzyme from the CHO parent. Hybrids and temperature-sensitive segregants derived from them were analyzed for the expression of known human chromosomal marker enzymes. The strong correlation between the expression of the human form of asparaginyl-tRNA synthetase and the presence of human chromosome 18 in hybrids suggests that the human gene, asnS, which corrects the heat-sensitive phenotype of the CHO asparaginyl-tRNA synthetase mutant, is located on chromosome 18.

Gene mapping and linkage analysis in Chinese hamster: assignment of the genes for APRT, LDHA, IDH2, and GAA to chromosome 3.

Abstract: Interspecific somatic cell hybrids were generated by fusion of Chinese hamster spleen cells or primary fibroblasts with cells from an adenine phosphoribosyltransferase (APRT)-deficient mouse subline derived from LMTK− Cl1D Subclones which had been selected for either retention or loss ofAPRT were subjected to combined isozyme and chromosome segregation analysis Concordant expression or segregation of Chinese hamster APRT, lactate dehydrogenase A (LDHA), isocitrate dehydrogenase 2 (IDH2), and α-glucosidase (GAA) with Chinese hamster chromosome 3 allowed provisional assignment of all four loci to that chromosome Exceptional subclones, in which coordinate segregation of these syntenic markers was disrupted by chromosome breakage or deletions, allowed further localization of these genes to specific regions of the 3 chromosome

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

Abstract: 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.

Mitochondrial genetics of mammalian cells: A mouse antimycin-resistant mutant with a probable alteration of cytochromeb

Abstract: Mouse LA9 antimycin-resistant mutants (ANT-R) were isolated and characterized. Genetic analyses established that this phenotype is encoded within the mtDNA: (1) the ANT-R phenotype showed frequent mitotic segregation and reassortment in hybrid clonal lines; (2) it was transmitted directly in cybrid crosses; and (3) it was cotransmitted in cybrid crosses with the mitochondrial CAP-R marker. Furthermore, the genetic studies suggested that the LA9 CAP-R ANT-R cells were heteroplasmic and contained at least two mtDNA genotypes, cap-r ant-s and cap-s ant-r. Cellular respiration of the ANT-R mutant was markedly more resistant to inhibition by antimycin than that of the parental ANT-S cells. The increased resistance of cellular respiration was entirely accounted for by an increase in the resistance of mitochondrial succinate-cytochrome coxidoreductase to antimycin inhibition. There was no detectable change in the specific activity of the oxidoreductase in mitochondria of resistant ANT-R cells nor in the sensitivity of the complex to three other specific inhibitors of the complex: TTFA, myxothiazol, and HQNO. Taken together, these studies indicate that the ANT-R phenotype is most likely encoded within the mitochondrial cytochrome bgene and, more specifically, within an antimycin binding domain.

Mammalian mitochondrial mutants selected for resistance to the cytochrome b inhibitors HQNO or myxothiazol.

Abstract: Mouse LA9 cell lines were selected for increased resistance to either HQNO or myxothiazol, inhibitors of electron transport which bind to the mitochondrial cytochrome b protein. Two phenotypically distinguishable HQNO-resistant mutants were recovered while the myxothiazol-resistant isolates had a common phenotype. All three mutant phenotypes were transmitted cytoplasmically in cybrid crosses. Biochemical studies further established that for all three mutant types, resistance at the cellular level was paralleled by an increase in inhibitor resistance of mitochondrial succinate-cytochrome c oxidoreductase, the respiratory complex containing cytochrome b. As with the previously described mitochondrial antimycin-resistant mutant, the initial biochemical and genetic studies indicated that these mutations occur within the mitochondrial cytochrome b gene. This conclusion was strongly supported by the results of mtDNA restriction fragment analyses in which it was found that one HQNO-resistant mutant had undergone a small insertion or duplication in the apocytochrome b gene. Finally, all four mitochondrial cytochrome b mutants have been analyzed in both cell plating studies and succinate-cytochrome c oxidoreductase assays to determine the pattern of cross-resistance to inhibitors of cytochrome b other than the one used for selection.

Further studies on a hybrid cell-surface antigen associated with human chromosome 11 using a monoclonal antibody

Abstract: A monoclonal antibody has been obtained that recognizes an antigen encoded by human chromosome 11. We present evidence that this monoclonal antibody recognizes the same or a similar antigenic activity as that previously called a1. Genetic information necessary for a1 expression and recognition by the monoclonal antibody both map to 11p13 → 11pter. Mutants that have lost a1 are no longer recognized by the monoclonal antibody. The macroglycolipid fraction of human erythrocyte membranes which contains the a1 antigenic activity is able to convert antigen-negative Chinese hamster ovary cells into cells which are killed by the monoclonal antibody plus complement.

Alanine-resistant mutants of Chinese hamster ovary cells, CHO-K1, producing increases in velocity of proline transport through the A, ASC, and P systems.

Abstract: We have developed a method for the isolation of transport mutants with increases in velocity of transport through the A and ASC systems and through a newly discovered P system utilizing the amino acid antagonism between A system amino acids and proline in CH0-K1 pro − cells. Mutants ala r 2and ala r 3,isolated in a single-step procedure, resistant to 25 mM alanine in MEM-10 plus 0.05 mM proline are pro −,stable, cross resistant to α-(methylamino)isobutyric acid (MeAIB) and show an approximately twofold increase in the initial velocity of proline uptake. Ethyl methane sulfonate (EMS) increases the frequency of pro − ala r clones in the population by at least 50 times the spontaneous frequency. The increased velocity of proline transport by ala r 2and ala r 3can be attributable to the 1.5 to 3 times increase in velocity of transport of proline through systems A, ASC, and P. The V max for proline transport through the A system has increased two times for ala r 2while the K m and V max for ala r 3has increased by 1.4 and 2.3 times that of CH0-K1. There is a corresponding increase in V max of proline transport by ala r 2through the P system. The P system is defined operationally as that portion of the Na +-dependent velocity that remains when the A, ASC, and glutamine-inhibitable fraction are eliminated. The system is concentrative. Proline appears to be the preferred substrate. Li + cannot be substituted for Na +.The system is moderately dependent upon pH. It obeys Michaelis-Menten kinetics and is not derepressible by starvation. There is no evidence for an N system in CHO-K1.

Events preceding stable integration of SV40 genomes in a human cell line.

Abstract: We have examined the organization of integrated SV40 sequences in an uncloned population of a transformed human fibroblast cell line. Somatic cell hybrids between mouse B82 cells and human GM847 cells were examined for SV40 T-antigen expression and individual human chromosome presence. This analysis revealed that a functional SV40 genome is located on human chromosome 7. Restriction endonuclease digestion followed by blot hybridization of the parental human cell line revealed that it contains mutliple normal and defective SV40 copies integrated into the host genome in tandem. A similar analysis of several T-ag+ hybrid cell lines indicated that the integrated viral sequences in different hybrid cell lines (thus in different cells of the original population) are very closely related but not always identical. Analysis of subclones of GM847 also revealed such differences. Based upon these results, we postulate that following the initial integration event, viral as well as the flanking host DNA sequences become unstable and are subject to deletions and rearrangements. This short-lived structural instability is followed by highly stable integration of SV40 which is maintained in these cells or their hybrid derivatives for at least hundreds of cell generations.

Chromosome-mediated gene transfer of HPRT and APRT in an intraspecific human cell system

Abstract: Chromosome-mediated transfer of genes between human cell lines was accomplished using HeLa cells as chromosome donors and HT1080 fibrosarcoma lines as recipients. This report describes the intraspecific transfer of two genetic markers, hypoxanthine-guanine-phosphoribosyl-transferase (HPRT+)and adenine phosphoribosyltransferase (APRT+).The isolation and characterization of the necessary enzyme-deficient (HPRT−and APRT−)recipient HT1080 cell lines are also described. The chromosome-mediated gene transfer was carried out using a modification of the procedure of Miller and Ruddle, including treatment of the donor chromosomes with calcium phosphate and subsequent exposure of the recipient cells of dimethyl sulfoxide. In experiments to optimize this procedure for HT1080 cell recipients, we found that a brief (2-min) exposure to high DMSO concentration (20%) was effective for enhancing transfer efficiencies in this system. Transfer frequencies (transferents per recipient cells assayed) averaged approximately 1×10−6for HPRT+and were >2×10−6for APRT+.

Transformation of Hprt gene with sperm DNA.

Abstract: Inactivation of the X chromosome during mammalian spermatogenesis has been postulated to occur by the same mechanism that controls female somatic X chromosome inactivation. We have used DNA-mediated transformation of HPRT− cells to test this idea, because it has been shown previously that inactive X chromosome DNA from somatic cells will not transform HPRT− cells. Isolated DNA from the mature sperm of five mammals (human, mouse, horse, bull, rabbit) were all capable of HPRT transformation, and transformants were confirmed electrophoretically. Measures were taken to ensure that the transformation frequencies observed could not be due to somatic contamination. The positive HPRT transformation result indicates that mature sperm X chromosomal DNA is not modified in the same manner as that of female inactive X chromosomal DNA. Since there is evidence for methylation of the somatic inactive X chromosome, it is possible that methylation, at least for the genes studied, is not involved in sperm X chromosome inactivation.

Assignment of gene coding for cell surface glycoprotein with a molecular weight of 75,000 to human chromosome 11.

Abstract: The gene for a cell surface glycoprotein recognized by a mouse monoclonal antibody (Mab 4), has been assigned to human chromosome 11 by the study of mouse-human lymphocyte hybrids. The antigen is present on all human peripheral blood leukocytes, on human fibroblasts, and on human lymphoid and erythroid cell lines, but not on erythrocytes. Immunoprecipitation and polyacrylamide slab gel electrophoresis of both human cells and mouse-human hybrid clones carrying human chromosome 11 show that the apparent molecular weight of this glycoprotein is 75,000.

Assignment of murine cellular harvey ras gene to chromosome 7.

Abstract: 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 a32P-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.

Human chromosome 11 carries at least four genes controlling expression of cell-surface antigens.

Abstract: We have mapped two new genes to chromosome 11 which control the cell-surface expression of two distinct antigens defined by monoclonal antibodies. One of the antigens has a general tissue distribution and is associated with a molecular complex of two polypeptides of 80,000 dalton and 40,000 dalton molecular weight. The second antigen has a restricted tissue distribution and is carried on a polypeptide of 100,000 daltons. We have used a combination of genetic and biochemical techniques to demonstrate that these new markers are distinct from the antigens defined by the monoclonal antibodies F10.44.2 and W6/34 which are also encoded by genes on chromosome 11. It is concluded that human chromosome 11 carries at least four distinct genes controlling cell-surface antigen expression.