Regional asssignments of the loci AK3, ACONS, and ASS on human chromosome 9.

Experiments are described in which human cells carrying balanced reciprocal translocations involving four different regions of chromosome 9 were fused with a Chinese hamster cell line and the resulting hybrids used to obtain subchromosomal assignments of the loci ASS, AK3, and ACONS. ASS was localized on the distal portion of the long arm of chromosome 9, in the region 9q34 leads to 9qter, and AK3 and ACONS on the short arm, in the region 9pter leads to 9p13.     

Localization of the structural genes for hexokinase-1 and inorganic pyrophosphatase on region (pter-->q24) of human chromosome 10

Concordant expression of human hexokinase-1 and inorganic pyrophosphatase was established in somatic cell hybrids between thymidine kinase-deficient Chinese hamster cells and human fibroblasts carrying a translocation of the distal third of the long arm of chromosome 10 to chromosome 17. Neither human hexokinase-1 nor human inorganic pyrophosphatase expression segregated concordantly with human cytoplasmic glutamic-oxaloacetic transaminase expression.     

Mapping AK1, ACONs, and AK3 to chromosome 9 in man employing and X/9 translocation and somatic cell hybrids.

The adenylate kinase 1 (AK1), adenylate kinase3 (AK3), and aconitaseS (ACONS) genes have been assigned to chromosome 9 in man by employing an X/9 translocation segregating in man-mouse somatic cell hybrids. Segregation was controlled by taking advantage of the HAT/8-azaguanine selection-counterselection strategy directed at the X-linked HPRT locus. Assignment of AK1 to chromosome 9 has suggested the assignment of the ABO blood-group locus and the nail-patella (Np) locus to 9, since both loci are linked to AK1 by family studies.     

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.     

Mapping of human chromosome 22 with a panel of somatic cell hybrids

The adenylosuccinate lyase (ADSL) which is essential for generating adenylate, maps to the long arm of chromosome 22. By using a Chinese hamster ovary cell line deficient in ADSL activity, we have constructed a set of 17 somatic cell hybrids containing defined regions of human chromosome 22. This panel was extended with six additional hybrids, obtained in other laboratories using various methods of selection. Southern analysis of the hybrids with 38 chromosome 22 probes defined 14 different subregions which could be linearly organized on the long arm of chromosome 22. The order of the probes thus deduced is fully compatible with their previous localization and with the genetic map. The ADSL gene was further sublocalized between the MB and D22S22. This panel, which enables the rapid assignment of chromosome 22 single copy probes to small subregions, will be an important tool in the construction of a detailed physical map of this part of the genome.     

Requirement of the human chromosome 11 long arm for replication of herpes simplex virus type 1 in nonpermissive Chinese hamster x human diploid fibroblast hybrids

Somatic cell hybrids between Chinese hamster (CH) lung cells (V79/380-6), nonpermissive for productive infection by herpes simplex type 1 (HSV-1), and permissive human diploid cells support productive HSV-1 infection as long as they retain human chromosome 11. Human chromosome 3 has been reported to complement nonpermissivity in (CH) Don cells (1). Intraspecies hybrids between Don/a3 and V79/380-6 cells, however, did not support HSV-1 replication, indicating lack of complementation. The block in both nonpermissive CH cell lines was determined to involve a step beyond replication of the parental viral DNA. In cell hybrids between nonpermissive Don/a23 cells and human fibroblasts containing a t(11;15) (p11;p12) translocation, HSV-1 production was dependent solely on the presence of either human chromosome 11 or the der(11) (p11 leads to qter) translocation product containing the long arm of chromosome 11. Chromosome 3 was excluded by a discordancy rate of 59%. We conclude that the long arm of human chromosome 11 carries one or more genes coding for host functions necessary for the production of progeny HSV-1 DNA.     

Assignment of first random restriction fragment length polymorphism (RFLP) locus ((D14S1) to a region of human chromosome 14.

A locus responsible for a restriction fragment length polymorphism (RFLP) has been identified by hybridization of Eco RI fragments to the random human DNA sequence in recombinant plasmid pAW101. We have examined DNA extracted from 20 human X Chinese hamster somatic cell hybrids for the presence of sequences homologous to the human insert in pAW101. The hybrids were derived from six different human donors, five of whom were heterozygous, producing two bands on Southern transfers. The presence of homologous sequences in the hybrids correlated exclusively with the presence of human chromosome 14. Three hybrids contained chromosome 14 in a frequency of greater than one per cell and were positive for two alleles. Two hybrids contained only the distal half of the long arm of 14 as part of a translocation and were still positive. These results assign the first highly polymorphic random RFPL locus (D14S1) to region q21 leads to qter of chromosome 14.     

Assignment of human pepsinogen A locus to the q12-pter region of chromosome 11

Summary A 0.9 kb cDNA fragment, corresponding to a large part of Rhesus monkey pepsinogen A mRNA, was used as probe for the chromosomal localization of the human pepsinogen A gene(s) using human-rodent somatic cell hybrids. Southern blot analysis of 14 human-Chinese hamster and three human-mouse cell hybrids, strongly indicates that the human PGA locus is on chromosome 11. The human-mouse hybrids, containing a translocation involving chromosome 11, allow sublocalization to the region q12-pter.     

Regional assignment of seven genes on chromosome 1 of man by use of man-Chinese hamster somatic cell hybrids. I. Results obtained after hybridization of human cells carrying reciprocal translocations involving chromosome 1.

Regional localization studies of genes coding for human PGD, PPH1, PGM1, UGPP, GuK1, Pep-C, and FH, which have been assigned to chromosome 1, were performed with man-Chinese hamster somatic cell hybrids, Informative hybrids that retained fragments of the human chromosome 1 were produced by fusion of hamster cells with human cells carrying reciprocal translocations involving chromosome 1. Analysis of the hybrids that retained one of the translocation chromosomes or de novo rearrangements involving the human 1 revealed the following gene positions: PGD and PPH1 in 1pter leads to 1p32, PGM1 in 1p32 leads to 1p22, UGPP and GuK1 in 1q21 leads to 1q42, FH in 1qter leads to 1q42, and Pep-C probably in 1q42.     

Assignment of PGM3 to the long arm of human chromosome 6. Studies using Chinese hamster X human cell hybrids containing a human 6/15 translocation

Hybrids derived from the fusion of thymidine kinase deficient Chinese hamster cells and human cells carrying a 6/15 translocation, 46,XX,t(6;15)(cen;p13), were analyzed for the expression of human PGM3, GLO and ME1. The results show that PGM3 and ME1 are on the long arm and GLO is on the short arm of human chromosomes 6.     

Regional localization of human phosphoglucomutase-2 locus on chromosome 4

Analysis of somatic cell hybrids derived from fusion of human lymphocytes with a karyotype of 46,XX,t(3;4) (q27;q25) to a pseudo-tetraploid HPRT deficient Chinese hamster line, CH 1103, has permitted assignment of the human phosphoglucomutase-2 locus (PGM₂) to the pter→q25 region of chromosome 4. This is the first confirmation of the assignment of this locus to chromosome 4 and, combined with earlier mapping studies of MeAlpine et al., permits localization of the PGM₂ to the 4p14→q25 region.     

Regional assignment of ADA and ITPA to mouse chromosome 2 (C1----ter). A demonstration of the conserved linkage of enzyme and proto-oncogene loci

Similarity of G-band patterns between the long arm of Chinese hamster chromosome 6 and mouse chromosome 2, combined with the assignments of AK1, ADA, and ITPA to hamster chromosome 6 and AK1 to mouse chromosome 2, suggested mouse chromosome 2 also might contain ADA and ITPA. Here, concordant segregation analysis of enzyme loci and chromosomes in mouse spleen X CHO as well as mouse microcell X CHO somatic cell hybrids established the assignments of ADA and ITPA onto mouse chromosome 2 in the region between the first G-band and the terminus (C1----ter). This assignment presents a demonstration of the conservation and evolution of enzyme and proto-oncogene loci linkage since two cellular homologs of viral oncogenes--c-src and c-abl--also map to mouse chromosome 2. In humans c-src, ADA, and ITPA remain conserved on chromosome 20, whereas AK1 and c-abl are together on chromosome 9. These observations and concepts are discussed with respect to the role of proto-oncogenes in chromosomal evolution and suggest the long arm of chromosome 6 as a fruitful place to look for c-src and c-abl in the Chinese hamster.     

Genetic and biochemical studies with the adenosine analogs toyocamycin and tubercidin: mutation at the adenosine kinase locus in Chinese hamster cells.

The pyrrolopyrimidine nucleosides toyocamycin and tubercidin show several unique features of growth inhibition in Chinese hamster ovary (CHO) cells. Stable mutants which are more than 600-fold resistant to these drugs are obtained in CHO cells at a strikingly high frequency of approximately 10(-3), in the absence of mutagenesis. The mutants resistant to toyocamycin (Toyr) and tubercidin (Tubr) exhibit similar cross-resistance patterns to the two selective drugs as well as to adenosine and 6-methyl mercaptopurine riboside, indicating that the same lesion is probably involved in all cases. The mutants examined were found to be deficient in the enzyme adenosine kinase (AK), indicating that the phosphorylation of these analogs is an essential first step in their toxic action. The above mutants (AK-) behaved recessively in cell hybrids, and segregation studies indicate that the AK locus is not linked to the X chromosome. The frequencies of similar Toyr mutants in other Chinese hamster lines, e.g., V79, CHW, M3-1, GM7, and CHO-K1, varied from similar to more than three logs less than that observed for CHO cells, indicating that various cell lines probably differ in the number of functional gene copies for this locus.     

Assignment of the gene for human arylsulfatase B, ARSB, to chromosome region 5p11----5qter

The structural gene coding for human arylsulfatase B, ARSB, is assigned to 5p11----5qter by analysis of somatic cell hybrids isolated from two separate fusions of human fibroblasts carrying a translocation involving chromosome 5 with the Chinese hamster cell line a3.     

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.     

Mapping of a locus correcting lack of phosphoribosylaminoimidazole carboxylase activity in Chinese hamster ovary cell Ade-D mutants to human chromosome 4

The human phosphoribosylaminoimidazole (AIR) carboxylase locus has been until this report one of the genes encoding purine biosynthetic enzymes that had not been assigned to an individual human chromosome. Characterization of Chinese hamster ovary (CHO) cell mutant Ade-D showed that the cell line was unable to produce IMP and accumulated AIR. CHO Ade-D cells were fused with normal human lymphocytes utilizing inactivated Sendai virus and the resulting hybrid cell lines were selected for purine prototrophy. Cytogenetic analysis showed a 100% concordance value for chromosome 4. Two of the isolated subclones contained only the long arm of chromosome 4 translocated onto a CHO chromosome, providing evidence for a regional assignment of the Ade-D gene to the long arm of chromosome 4. Two of the subclones containing chromosome 4 were subjected to the BrdU visible light segregation. All of the isolated purine auxotrophic cell lines showed a loss of the q arm of chromosome 4. The localization of the Ade-D locus to the long arm of chromosome 4 may reveal further clustering of the mammalian purine genes since the Ade-A locus has previously been regionally assigned to 4pter-q21.     

Lack of tumor suppression but induced loss of copies of indigenous chromosome 10 in vitro following microcell-mediated transfer of a deleted human der(9)t(X;9) chromosome to Syrian hamster BHK-191-5C cells

We have previously shown that microcell-mediated transfer of a der(9)t(X;9) chromosome, containing an almost complete human chromosome (HSA) 9 derived from the human fibroblast strain GM0705, into the Syrian hamster (Mesocricetus auratus) cell line BHK-191-5C suppressed the anchorage independence and tumorigenicity of the hybrids. Transfer of a normal HSA X did not have any effect on these phenotypes. Although the recipient cell line contained a 1:1 ratio of near-diploid and near-tetraploid cells, all hybrids retaining the der(9) chromosome were near-tetraploid, in contrast to hybrids retaining a normal X chromosome. In the present study, we have generated microcell hybrids by transferring another der(9)t(X;9) chromosome derived from the human fibroblast strain GM01429. This derivative chromosome contained a deletion on the short arm of HSA 9 and was also missing the distal part of the long arm of HSA 9 due to the involvement in a reciprocal (constitutive) translocation of this chromosome with HSA X. Cytogenetic analysis showed that all hybrid clones were near-tetraploid, confirming our previous finding. We also observed that the introduction of the deleted der(9) chromosome forced the hybrids to lose Syrian hamster chromosome 10. A soft agar test and nude mice assay indicated that none of the hybrids was suppressed for either anchorage independent growth or tumor formation. These data suggest that there is an antagonistic relationship between growth-promoting genes and antiproliferative genes. The observed dosage effects of both growth-promoting and growth-suppressing genes indicate that cellular growth may be a quantitative trait.     

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.     

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.     

Gene mapping in the Chinese hamster and conservation of syntenic groups and Q-band homologies between Chinese hamster and mouse chromosomes

Using Chinese hamster/mouse somatic cell hybrids segregating hamster chromosomes, we assigned 15 enzyme genes to six different Chinese hamster autosomes. Of the 15 loci, three genes, HK1, PEPC, and SORD, were newly assigned to chromosomes 1, 5, and 6, respectively, while ENO1, PGD, and PGM1 were assigned to the long arm of chromosome 2, in the segment 2q113----qter. The locations of the following loci were confirmed: ESD, NP, and PEPB on chromosome 1, ME1 and MPI on chromosome 4, AK1 on chromosome 6, and GPI and PEPD on chromosome 9. Comparative mapping of Chinese hamster and laboratory mouse chromosomes revealed conservation of syntenic groups and extensive banding homology between the Chinese hamster and mouse chromosomes on which homologous enzyme markers have been mapped.