Molecular analysis of X-ray-induced mutants at the HPRT locus in V79 Chinese hamster cells

Spontaneous and X-ray-induced mutants at the hypoxanthine phosphoribosyl transferase (HPRT) locus have been isolated from V79 Chinese hamster cells and characterized at the biochemical and cytogenetic levels. Fourteen spontaneous and 24 X-ray-induced clones were azaguanine and thioguanine resistant, did not grow in HAT medium (AZRTGRHATS) and failed to incorporate significant levels of [14C]hypoxyanthine. Cytogenetic analysis of two spontaneous and eight X-ray-induced mutants revealed no major X chromosome rearrangements. In two induced mutants, one of which was hypotetraploid (mode 35-39) with 2 X chromosomes, the short arm of the chromosome (Xp) was slightly shorter than normal. A third mutant was hyperdiploid (mode 22-23) compared with the parental clone (mode 21). When compared with wild-type clones, no other cytogenetic changes were evident in the remaining mutants. Analysis at the DNA level using a Chinese hamster HPRT cDNA probe showed major deletion of HPRT sequences in two and partial deletion in another two induced mutants. In two of the mutants with deletions of HPRT sequences there was a visible shortening of the Xp arm. In the other six mutants two spontaneous and four induced) no karyotypic changes or alterations in restriction fragment patterns were detected suggesting that they carry small deletions or point mutations at the HPRT locus.     

Chromosome loss is responsible for segregation at the HPRT locus in Chinese hamster cell hybrids

The phenomenon of segregation of gene expression has been examined in intraspecific somatic cell hybrids. Specifically, segregation at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus has been studied in hybrids of Chinese hamster cell lines. The role of chromosome segregation, or other chromosomal events has been assessed by detailed comparison of karyotypes in the 6-thioguanine resistant segregants with those of the parental hybrid lines. The results clearly demonstrate that loss of an entire X chromosome is the primary event responsible for segregation at the HPRT locus, while deletion of a portion of the short arm of an X chromosome was also a frequent event. The results provide the first direct evidence for the assignment of the mapping of this locus to the distal region of the short arm. Analysis of chromosome number distributions in the hybrids and segregants suggests that in selecting chromosomal segregants one may also select for hybrid lines with reduced chromosome stability.     

DNase I sensitivity of Microtus agrestis active,inactive and reactivated X chromosomes in mouse-Microtus cell hybrids

We isolated Microtus agrestis-mouse somatic cell hybrid clones which had retained either the active or the inactive M. agrestis X chromosome. In both hybrid clones the X chromosomes retained their original chromatin conformation as studied by the in situ nick translation technique — the active X chromosome retained its high sensitivity to DNase I while the inactive one remained insensitive. A clone in which the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene had been spontaneously reactivated was isolated from the hybrid containing the inactive X chromosome. The in situ nick translation technique was used to study possible DNA conformation changes in the euchromatin of the inactive X chromosome with special reference to the reactivated HPRT locus. We found that the euchromatin in this X chromosome exhibited the same low sensitivity to DNase I as is characteristic of the inactive X chromosome.Professor Marcus passed away on 2 January 1987     

Regional localization of human gene loci on chromosome 9: studies of somatic cell hybrids containing human translocations.

Somatic cell hybrids were derived from the fusion of (1) Chinese hamster cells deficient in hypoxanthine guanine phosphoribosyltransferase (HPRT) and human cells carrying an X/9 translocation and (2) Chinese hamster cells deficient in thymidine kinase (TK) and human cells carrying a 17/9 translocation. Several independent primary hybrid clones from these two series of cell hybrids were analyzed cytogenitically for human chromosome content and electrophoretically for the expression of human markers known to be on human chromosome 9. The results allow the assignment of the loci for the enzymes galactose-1-phosphate uridyltransferase (GALT), soluble aconitase (ACONs), and adenylate kinase-3 (AK3) to the short arm of chromosome 9 (p11 to pter) and the locus for the enzyme adenylate kinase-1 (AK1) to the distal end of the long arm of human chromosome 9 (hand q34). Earlier family studies have shown that the locus for AK1 is closely linked to the ABO blood group locus and to the locus of the nail-patella (Np) syndrome. Thus the regional localization of AK1 locus permits the localization of the AK1-Np-ABO linkage group.     

Assignment of the gene for adenosine kinase to chromosome 14 in Mus musculus by somatic cell hybridization.

Evidence is presented for the assignment of the gene for adenosine kinase to Mus musculus chromosome 14 by synteny testing and karyotypic analysis of mouse X Chinese hamster somatic cell hybrid clones. ADOK and two enzymes previously mapped to mouse chromosome 14, NP and ES-10, were expressed concordantly in 29 hybrid clones. Chromosome analysis confirmed this assignment. Syntenic evidence is also presented using several clones of a gene transfer system in which the gene for human HPRT had integrated into modified mouse chromosome 14's.     

Molecular characterization of thymidine kinase mutants of human cells induced by densely ionizing radiation

In order to characterize the nature of mutants induced by densely ionizing radiations at an autosomal locus, we have isolated a series of 99 thymidine kinase (tk) mutants of human TK6 lymphoblastoid cells irradiated with either fast neutrons or accelerated argon ions. Individual mutant clones were examined for alterations in their restriction fragment pattern after hybridization with a human cDNA probe for tk. A restriction fragment length polymorphism (RFLP) allowed identification of the active tk allele. Among the neutron-induced mutants, 34/52 exhibited loss of the previously active allele while 6/52 exhibited intragenic rearrangements. Among the argon-induced mutants 27/46 exhibited allele loss and 10/46 showed rearrangements within the tk locus. The remaining mutants had restriction patterns indistinguishable from the TK6 parent. Each of the mutant clones was further examined for structural alterations within the c-erbA1 locus which has been localized to chromosome 17q11-q22, at some unknown distance from the human tk locus at chromosome 17q21-q22. A substantial proportion (54%) of tk mutants induced by densely ionizing radiation showed loss of the c-erb locus on the homologous chromosome, suggesting that the mutations involve large-scale genetic changes.     

Retinoblastoma, chromosome 13, and in vitro cellular radiosensitivity.

Five diploid fibroblast strains from patients with deletions mapping in the long arm of chromosome 13 and three strains bearing partial trisomies of chromosome 13 were assayed for clonogenic survival following X-irradiation. Results of these experiments suggest the existence of a region on 13q14 which is related to increased sensitivity to cell killing in vitro by X-rays. This locus seems to be distinct from but close to the retinoblastoma locus, which has been associated with the same band.     

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.     

A mouse-human hybrid cell panel for mapping human chromosome 16

A mouse-human hybrid cell panel for human chromosome 16 was constructed from human cell lines with breakpoints on chromosome 16 at p13.11, q13, q22 and q24. Fusions with the human fibroblast line GM3884, t(X;16)(q26;q24) allowed the isolation of clones with either the derivative X or the derivative 16 as the only human chromosome. This was a consequence of both the genes APRT and HPRT being involved in the translocation. The breakpoints of the line GM3884 were confirmed by aphidicolin induction of the common fragile site at 16q23. The results of the fusions with this line suggest a localisation of the APRT gene at 16q24 and confirm the localisation of HPRT to Xq26 to Xq27.3. These hybrid cell lines enable the localisation of genes and DNA fragments to six clearly defined regions. Further localisation within three of these regions is possible by use of the three fragile sites on chromosome 16. In situ hybridisation with the probe pBLUR confirmed that of three lines tested all contained a single human chromosome.     

Exclusion of human chromosome 13q34 as the site of the cystic fibrosis mutation.

We have studied a family in which both cystic fibrosis (CF) and an unbalanced translocation between chromosomes 6 and 13 are found. As CF occurs in the child who is effectively monosomic for the translocated part of the long arm of chromosome 13, it was suggested that the locus of the gene mutation causing CF is on chromosome 13q34. The gene for human coagulation factor X is located at 13q34, and we have found a restriction fragment length polymorphism (RFLP) that is revealed by a cloned cDNA coding for this protein. Linkage analysis in eight CF families shows no evidence of cosegregation between CF and the gene for factor X, strongly suggesting that the locus for the defect causing cystic fibrosis is not at 13q34.     

Pericentric inversion of the X chromosome: presentation of a case and review of the literature.

A large pericentric inversion of the X chromosome [inv(X)(p22.31q26.3)] was found to be transmitted in four generations through phenotypically normal males and females. In one female carrier, the inv(X) was late replicating in 70% of lymphocytes and 46% of skin fibroblasts. Steroid sulfatase (STS), an enzyme which normally escapes inactivation has been located to Xp22.32 and, in our case, has been moved to an aberrant position. We have assayed its activity in clones with the inv(X) inactive or the normal X inactive and found no significant differences. Thus, the STS locus escaped X inactivation in both the normal and the inverted X chromosomes. A review of the literature shows that almost half of the breakpoints on the short arm are found at region p22 and we propose that low-copy repetitive DNA segments along the X chromosome are responsible for non-homologous pairing and production of inversions.     

A chromosome study of 6-thioguanine-resistant mutants in T lymphocytes of Hiroshima atomic bomb survivors

Cytogenetic characterizations were made of lymphocyte colonies established from somatic mutation assays for 6-thioguanine (TG) resistance in Hiroshima atomic bomb survivors. G-banded chromosomes were analyzed in both TG-resistant (TGr) and wild-type colonies. Included were 45 TGr and 19 wild-type colonies derived from proximally exposed A-bomb survivors, as well as colonies from distally exposed control individuals who did not receive a significant amount of A-bomb radiation (18 TGr and 9-wild type colonies). Various structural and numerical chromosome abnormalities were observed in both TGr and wild-type colonies. Aberrations of the X chromosome, on which the hypoxanthine guanine phosphoribosyl transferase (HPRT) locus is present, were found in 6 colonies: 2 resistant colonies from controls (45,X/46,XX; 46,X,ins(X)), 3 resistant colonies (45,X/46,XX/46,X, + mar; 46,X,t(Xq +;14q-); 46,Y,t(Xq-;5q +)), and 1 wild-type colony (45,X/47,XXX) from proximally exposed persons. In cases with exchange aberrations, each of the break points on the X chromosome was situated proximally to band q26 where the HPRT locus is known to be assigned. DNA-replicating patterns were also studied, and it was found that abnormal X chromosomes showed early replicating patterns, while normal X chromosomes showed late replicating patterns.     

Silver fox gene mapping: conserved chromosome regions in the order Carnivora

Twenty-three silver fox x hamster somatic cell hybrid clones were used to assign 15 fox genes: GPI to chromosome 1; PGD to chromosome 2; MDH2 to chromosome 3; ESD to chromosome 6; LDHB to chromosome 8; NP to chromosome 10; LDHA to chromosome 11; APRT, ENO1, and PGM1 to chromosome 12; IDH1 and MDH1 to chromosome 16; and GLA, G6PD, and HPRT to the X chromosome. High-resolution G-banding of human, cat, mink, and fox chromosomes containing homologous regions (according to genetic maps) revealed regions of putative homology. The results lend support to the suggestion that the most considerable karyotypic reorganization of the ancestral genome in the order Carnivora occurred during Canidae formation. The details of karyotypic evolution in mammals are discussed.     

Regional mapping of the human No. 1 and X chromosome in interspecific cell hybrids using an X/1 translocation.

Fibroblasts from a carrier of an X/1 translocation, 46,XY,t(X;1)(q28;q31), were fused with Chinese hamster cells. The resulting hybrids were analyzed for human No. 1 and X-chromosome markers. The data indicate that the loci for PGM1, PGD, PPH, and GuK1 are situated either in the long arm proximal to a break point in band 1q31 or in the short arm. The loci for Pep-C, FH, and GuK1 are located distal to the break point. HPRT and G6PD are probably situated distal to a break point in band q28 of the X chromosome; alpha-Gal A is situated proximal to the break point, either on the long or short arm of the chromosome.     

Frequency of reactivation and variability in expression of X-linked enzyme loci.

A mouse-human cell hybrid clone retaining an inactive human X chromosome was treated with 5-azacytidine. Following treatment, expression of the X-linked enzyme markers, hypoxanthine-guanine phosphoribosyltransferase (HPRT), glucose-6-phosphate dehydrogenase (G6PD), phosphoglycerate kinase (PGK), and alpha-galactosidase A (GLA) was examined. Results presented here show that 45 of the 62 clones positive for human HPRT expressed human GLA, while only four of 68 clones negative for human HPRT expressed human GLA. These results strongly suggest that there is coordinate reactivation of GLA and HPRT. Reactivated expression of G6PD was studied in detail. The studies show that 5-azacytidine can induce heritable changes in the inactive human X chromosome resulting in the expression of G6PD activity at a level lower than that from an active human X chromosome.     

In vivo hprt mutant frequencies in T-cells of normal human newborns

Mutation at the hypoxanthine-guanine phosphoribosyl transferase locus (hprt; HPRT enzyme) in the human fetus was studied by clonal assay of placental cord blood samples from full-term newborns. Conditions for determining hprt mutant frequencies, as defined for adults, were also optimal for studies in newborns. The mean mutant frequency for 45 normal human newborns (37 male, 8 female) was 0.64 X 10(-6) (SD = 0.41 X 10(-6); median value = 0.58 X 10(-6). These values are approx. 10-fold lower than corresponding adult hprt mutant frequency values. Factors such as limiting-dilution cloning efficiencies, delay prior to study of sample, sex, cryopreservation or technician performing the assay did not significantly affect assay results. Maternal smoking did not result in elevated mutant frequency values. Most wild-type and mutant clones studied were CD4 surface antigen positive (helper/inducer). All hprt mutants analyzed lacked HPRT activity.     

Localization of G6PD and HPRT to different arms of the X chromosome of the North American marsupial (Didelphis virginiana) by in situ hybridization and deletion mapping: evolutionary significance

We have mapped HPRT and G6PD loci on the X chromosome in the American opossum, Didelphis virginiana, by in situ hybridization to cells derived from two females by using genomic opossum DNA as probes. The localizations (G6PD to Xp13 and HPRT to Xq21), indicating that the two genes are separated by the centromere, were confirmed by results of hybridization to X chromosomes with deletions that include the HPRT locus and opossum-mouse cell hybrids containing the relevant fragment of the opossum X chromosome.     

The sockeye salmon neo-Y chromosome is a fusion between linkage groups orthologous to the coho Y chromosome and the long arm of rainbow trout chromosome 2

Unlike other Pacific salmon, sockeye salmon (Oncorhynchus nerka) have an X(1)X(2)Y sex chromosome system, with females having a diploid chromosome number of 2n = 58 and males 2n = 57 in all populations examined. To determine the origin of the sockeye Y chromosome, we mapped microsatellite loci from the rainbow trout (O. mykiss; OMY) genetic map, including those found on the Y chromosomes of related species, in kokanee (i.e. non-anadromous sockeye) crosses. Results showed that 3 microsatellite loci from the long arm of rainbow trout chromosome 8 (OMY8q), linked to SEX (the sex-determining locus) in coho salmon (O. kisutch), are also closely linked to SEX in the kokanee crosses. We also found that 3 microsatellite loci from OMY2q are linked to those markers from OMY8q and SEX in kokanee, with both linkage groups fused to form the neo-Y. These results were confirmed by physical mapping of BAC clones containing microsatellite loci from OMY8q and OMY2q to kokanee chromosomes using fluorescence in situ hybridization. The fusion of OMY2q to the ancestral Y may have resolved sexual conflict and, in turn, may have played a large role in the divergence of sockeye from a shared ancestor with coho.     

Spontaneous reactivation of the inactive X chromosome in mouse embryonal carcinoma cells

The mouse embryonal carcinoma cell line MC12 carries two X chromosomes, one of which replicates late in S phase and shares properties with the normal inactive X chromosome and, therefore, is considered to be inactivated. Since the hypoxanthine phosphoribosyl transferase (HPRT) gene on the active X chromosome is mutated (HPRT(NDASH;)), MC12 cells lack HPRT activity. After subjecting MC12 cells to selection in HAT medium, however, a number of HAT-resistant clones (HAT(R)) appeared. The high frequency of HAT resistance (3.18 x 10(-4)) suggested reactivation of HPRT(PLUS;) on the inactive X chromosome rather than reversion of HPRT(NDASH;). Consistent with this view, cytological analyses showed that the reactivation occurred over the length of the inactive X chromosome in 11 of 20 HAT(R) clones isolated. The remaining nine clones retained a normal heterochromatic inactive X chromosome. The spontaneous reactivation rate of the HPRT(PLUS;) on the inactive X chromosome was relatively high (1.34 x 10(-6)) and comparable to that observed for XIST-deleted somatic cells (Csankovszki et al., 2001), suggesting that the inactivated state is poorly maintained in MC12 cells.     

A t(X;15)(q23;q25) with Xq reactivation in a lymphoblastoid cell line from Fanconi anemia.

A t(X:15)(q23;q25) was detected during cytogenetic investigation of a lymphoblastoid cell line established from a female patient with Fanconi anemia. The translocation was apparently balanced at passage 300 and unbalanced at passage 13. A chromatid exchange between both the normal and the der(15), between the centromere and band 15q25, may explain these results. Replication studies, following BrdU incorporation, indicate that the segment Xq23----qter from the der(15) is early replicating whereas segment Xpter----q23 from the der(X) is late replicating. Since the normal X was early replicating, it is concluded that the segment of the long arm of chromosome X, separated from its inactivation center by the translocation, was reactivated. This interpretation is confirmed by the methylation patterns of the hypoxanthine phosphoribosyltransferase gene (HPRT), mapped on Xq26, which corresponds to that of an active gene, whereas that of phosphoglycerate kinase (PGK1), which remained on the der(X), corresponds to that of an inactive gene. This is the first example of reactivation of a segment of the X chromosome following a structural rearrangement in somatic cells.