Identification of a YAC from 16q24 carrying a senescence gene for breast cancer cells.

We have identified a 360 kb YAC that carries a cell senescence gene, SEN16. In our earlier studies, we localized SEN16 within a genetic interval of 3 - 7 cM at 16q24.3. Six overlapping YACs spanning the chromosomal region of senescence activity, were assembled in a contig. Candidate YACs, identified by the markers located in the vicinity of SEN16, were retrofitted to introduce a neo selectable marker. Retrofitted YACs were first transferred into mouse A9 cells to generate A9/YAC hybrids. YAC DNA present in A9/YAC hybrids was further transferred by microcell fusion into immortal cell lines derived from human and rat mammary tumors. YAC d792t2 restored senescence in both human and rat mammary tumor cell lines, while an unrelated YAC from chromosome 6q had no senescence activity.

Identification of a gene at 16q24.3 that restores cellular senescence in immortal mammary tumor cells.

We have mapped a cellular senescence gene, SEN16, within a genetic distance of 3 - 7 cM, at 16q24.3. Microcell mediated transfer of a normal human chromosome 16, 16q22-qter or 16q23-qter restored cellular senescence in four immortal cell lines, derived from human and rat mammary tumors. The resumption of indefinite cell proliferation, concordant with the segregation of the donor chromosome, confirmed the presence of a senescence gene at 16q23-qter. While microcell hybrids were maintained in selection medium to retain the donor chromosome, sporadic immortal revertant clones arose among senescent cells. Reversion to immortal growth could occur due to inactivation of the senescence gene either by a mutation or a deletion. The analysis for chromosome 16 specific DNA markers, in revertant clones of senescent microcell hybrids, revealed a consensus deletion, spanning a genetic interval of approximately 3 - 7 cM at 16q24.3.

Human sulfotransferase SULT1C1: cDNA cloning, tissue-specific expression, and chromosomal localization.

We have isolated and sequenced a cDNA that encodes an apparent human orthologue of a rat sulfotransferase (ST) cDNA that has been referred to as "ST1C1"-although it was recently recommended that sulfotransferase proteins and cDNAs be abbreviated "SULT." The new human cDNA was cloned from a fetal liver-spleen cDNA library and had an 888-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 62% identical with that encoded by the rat ST1C1 cDNA and included signature sequences that are conserved in all cytosolic SULT enzymes. Dot blot analysis of mRNA from 50 human tissues indicated that the cDNA was expressed in adult human stomach, kidney, and thyroid, as well as fetal kidney and liver. Northern blot analyses demonstrated that the major SULT1C1 mRNA in those same tissues was 1.4 kb in length. We next determined the partial human SULT1C1 gene sequence for a portion of the 5'-terminus of one intron. That sequence was used to design SULT1C1 gene-specific primers that were used to perform the PCR with DNA from human/rodent somatic cell hybrids to demonstrate that the gene was located on chromosome 2. PCR amplifications performed with human chromosome 2/rodent hybrid cell DNA as template sublocalized SULT1C1 to a region between bands 2q11.1 and 2q11.2.

A gene on 6q 14-21 restores senescence to immortal ovarian tumor cells.

We have identified a gene on 6q14-21 which restores senescence to immortal ovarian tumor cells. Single gpt tagged human chromosomes, present in mouse/human monochromosomal hybrids, were introduced into immortal human and rat ovarian tumor cells via microcell fusion. Analysis of chromosome transfer clones for cell morphology and growth properties revealed that chromosome 6 or 6q restored senescence to both human and rat ovarian tumor cells while chromosomes 10 or 14 did not affect the proliferative potential of these cells. Reversion to immortal growth concordant with loss of the donor chromosome confirmed the presence of a senescence gene on 6q. During continuous maintenance of microcell hybrids in MX medium, rare immortal revertant clones grew out of the human and rat senescent cell populations. Analysis of independent revertant clones of rat cells, for chromosome 6 markers, revealed a common deletion of chromosomal region 6q14-21 in all revertants. Restoration of senescence following introduction of a gpt tagged chromosome segment 6q13-21 into human and rat ovarian tumor cells confirmed the location of a senescence gene in this region. In contrast, introduction of a chromosome 6 lacking the region 6q14-21 did not impart senescence in these cells. Based on these results we assigned the senescence gene (SEN 6A) to region 6q14-21.

An extended panel of hamster-human hybrids for chromosome 2q.

A hamster-human hybrid containing only the q arm of chromosome 2 has been used to construct a panel of hybrids bearing reduced regions of chromosome 2 using the technique of irradiation fusion gene transfer. The human chromosome 2 carried the Ecogpt gene and all hybrids were selected using this marker. The integrated Ecogpt gene was localized to the region 2q33-34, resulting in the selective retention of this region in the hybrids. These data were combined with another previously constructed panel of hybrids containing regions of 2q, which were enriched for the region 2q36-37. The combined hybrid panel is useful for the mapping of new markers to defined regions of chromosome 2 and for the cloning of genes located on 2q by a positional strategy.

Senescence of immortal human fibroblasts by the introduction of normal human chromosome 6.

In these studies we show that introduction of a normal human chromosome 6 or 6q can suppress the immortal phenotype of simian virus 40-transformed human fibroblasts (SV/HF). Normal human fibroblasts have a limited life span in culture. Immortal clones of SV/HF displayed nonrandom rearrangements in chromosome 6. Single human chromosomes present in mouse/human monochromosomal hybrids were introduced into SV/HF via microcell fusion and maintained by selection for a dominant selectable marker gpt, previously integrated into the human chromosome. Clones of SV/HF cells bearing chromosome 6 displayed limited potential for cell division and morphological characteristics of senescent cells. The loss of chromosome 6 from the suppressed clones correlated with the reappearance of immortal clones. Introduced chromosome 6 in the senescing cells was distinguished from those of parental cells by the analysis for DNA sequences specific for the donor chromosome. Our results further show that suppression of immortal phenotype in SV/HF is specific to chromosome 6. Introduction of individual human chromosomes 2, 8, or 19 did not impart cellular senescence in SV/HF. In addition, introduction of chromosome 6 into human glioblastoma cells did not lead to senescence. Based upon these results we propose that at least one of the genes (SEN6) for cellular senescence in human fibroblasts is present on the long arm of chromosome 6.

The human myeloid cell nuclear differentiation antigen gene is one of at least two related interferon-inducible genes located on chromosome 1q that are expressed specifically in hematopoietic cells.

We have previously shown that the human myeloid cell nuclear differentiation antigen (MNDA) is expressed at both the antigen and mRNA levels specifically in human monocytes and granulocytes and earlier stage cells in the myeloid lineage. A 200 amino acid region of the MNDA is strikingly similar to a region in the proteins encoded by a family of interferon-inducible mouse genes, designated Ifi-201, Ifi-202, Ifi-203, etc, that are not regulated in a cell- or tissue-specific fashion. However, a new member of the Ifi-200 gene family, D3, is induced in mouse mononuclear phagocytes but not in fibroblasts by interferon. The same 200 amino acid region, duplicated in the mouse Ifi-200 gene family, is also repeated in the recently characterized human IFI 16 gene that is constitutively expressed specifically in lymphoid cells and is induced in myeloid cells by interferon gamma. The 1.8-kb MNDA mRNA, which contains an interferon-stimulated response element in the 5' untranslated region, was significantly upregulated in human monocytes exposed to interferon alpha. Characterization of the MNDA gene showed that it is a single-copy gene and localized to human chromosome 1q 21-22 within the large linkage group conserved between mouse and human that contains the Ifi-200 gene family. The IFI 16 gene is also located on human chromosome 1q. Our observations are consistent with the proposal that the MNDA is a member of a cluster of related human interferon-regulated genes, similar to the mouse Ifi-200 gene family. In addition, one mouse gene in the Ifi-200 gene family and the human MNDA and IFI 16 genes show expression and/or regulation restricted to cells of the hematopoietic system, suggesting that these genes participate in blood cell-specific responses to interferons.

Subchromosomal localization of a gene (XRCC5) involved in double strand break repair to the region 2q34-36.

We have previously shown that human chromosome 2 can complement both the radiation sensitivity and the defect in double strand break rejoining characteristic of ionizing radiation (IR) group 5 mutants. A number of human-hamster hybrids containing segments of human chromosome 2 were obtained by microcell transfer into two group 5 mutants. In most, but not all, of these hybrids, the repair defect was complemented by the human chromosomal DNA. Two complementing microcell hybrids were irradiated and fused to XR-V15B, an IR group 5 mutant, to generate further hybrids bearing smaller regions of chromosome 2. All hybrids were examined for complementation of the repair defect. The region of chromosome 2 present was determined using PCR with primers specific for various human genes located on chromosome 2. A complementing hybrid bearing only a small region of chromosome 2 was finally generated. From this analysis we deduced that the XRCC5 gene was tightly linked to the marker, TNP1, which is located in the region 2q35.

A hamster-human subchromosomal hybrid cell panel for chromosome 2.

We have constructed hamster-human hybrid cell lines containing fragments of human chromosome 2 as their only source of human DNA. Microcell-mediated chromosome transfer was used to transfer human chromosome 2 from a monochromosomal mouse-human hybrid line to a radiation-sensitive hamster mutant (XR-V15B) defective in double-strand break rejoining. The human chromosome 2 carried the Ecogpt gene and hybrids were selected using this marker. The transferred human chromosome was frequently broken, and the resulting microcell hybrids contained different sized segments of the q arm of chromosome 2. Two microcell hybrids were irradiated and fused to XR-V15B to generate additional hybrids bearing reduced amounts of human DNA. All hybrids were analyzed by PCR using primers specific for 27 human genes located on chromosome 2. From these data we have localized the integrated gpt gene on the human chromosome 2 to the region q36-37 and present a gene order for chromosome 2 markers.

Complementation of DNA repair defect in xeroderma pigmentosum cells of group C by the transfer of human chromosome 5.

Complementation of DNA excision repair defect in xeroderma pigmentosum cells of group C (XP-C) has been achieved by the transfer of human chromosome 5. Individual human chromosomes tagged with a selectable marker were transferred to XP-C cells by microcell fusion from mouse-human hybrid cell lines each bearing a single different human chromosome. Analysis of the chromosome transfer clones revealed that introduction of chromosome 5 into XP-C cells corrected the DNA repair defect as well as UV-sensitive phenotypes, while chromosomes 2, 6, 7, 9, 13, 15, 17, and 21 failed to complement. The introduced chromosome 5 in complemented UVr clones was distinguished from the parental XP-C chromosomes by polymorphism for dinucleotide (CA)n repeats at two loci, D5S117 and D5S209. In addition, an intact marked chromosome 5 was rescued into mouse cells from a complemented UVr clone by microcell fusion. Five subclones of a complemented clone that had lost the marked chromosome 5 exhibited UV-sensitive and repair-deficient phenotypes identical to parental XP-C cells. Concordant loss of the transferred chromosome and reappearance of XP-C phenotype further confirmed the presence of a DNA repair gene on human chromosome 5.

A xeroderma pigmentosum complementation group A related gene: confirmation using monoclonal antibodies against the cyclobutane dimer and (6-4) photoproduct.

Xeroderma pigmentosum complementation group A was partially complemented by a cosmid genomic clone containing a 42-kb human DNA insert selected with a cDNA clone that we obtained through cDNA competition between the repair-proficient and repair-deficient cell line. The relationship between these two clones was confirmed using PCR amplifications. The enhancement in DNA-repair capacity of the transformants was assessed with the monoclonal antibodies specific for cyclobutane dimers and (6-4) photoproducts and partially correct the xeroderma pigmentosum complementation group A defect. Furthermore, the level of the photoproduct-repair capacity is in agreement with the survival enhancement calculated from the D37 values. This gene was mapped to chromosome 8, suggesting that this may represent one of the defective gene(s) in xeroderma pigmentosum complementation group A.

A gene that partially complements xeroderma pigmentosum group A cells maps to human chromosome 8.

A gene that partially complements sensitivity of xeroderma pigmentosum cells of group A to UV irradiation has been mapped to human chromosome 8. Isolation of this gene has previously been described. A cDNA clone pEMKR that represents part of this gene was used for mapping. Based upon the nucleotide sequence of pEMKR, a set of oligonucleotide primers were designed for PCR amplification of DNAs from hybrid cell lines. A panel of rodent-human hybrid cell lines representing the total human genome was screened by PCR and Southern blot analysis for chromosomal assignment of this gene. PCR amplification and hybridization occurred only in the case of human and hybrid cell lines that contained human chromosome 8. The pEMKR thus represents a different gene than a DNA repair gene XPAC that has been mapped to human chromosome 9.

Localization of a DNA repair gene (XRCC5) involved in double-strand-break rejoining to human chromosome 2.

Complementation of the repair defect in hamster xrs mutants has been achieved by transfer of human chromosome 2 using the method of microcell-mediated chromosome transfer. The xrs mutants belong to ionizing radiation complementation group 5, are highly sensitive to ionizing radiation, and have an impaired ability to rejoin radiation-induced DNA double-strand breaks. Both phenotypes were corrected by chromosome 2, although the correction of radiation sensitivity was only partial. Complementation was achieved in two members of this complementation group, xrs6 and XR-V15B, derived independently from the CHO and V79 cell lines, respectively. The presence of human chromosome 2 in complemented clones was examined cytogenetically and by PCR analysis with primers directed at a human-specific long interspersed repetitive sequence or chromosome 2-specific genes. Complementation was observed in 25/27 hybrids, one of which contained only the q arm of chromosome 2. The two noncomplementing hybrids were missing segments of chromosome 2. The use of a back-selection system enabled the isolation of clones that had lost the human chromosome and these regained radiation sensitivity. Transfer of several other human chromosomes did not result in complementation of the repair defect in XR-V15B. These data show that the gene defective in xrs cells, XRCC5, which is involved in double-strand break rejoining, is located on human chromosome 2q.

Complementation of a DNA repair defect in xeroderma pigmentosum cells by transfer of human chromosome 9.

Complementation of the repair defect in xeroderma pigmentosum cells of complementation group A was achieved by the transfer of human chromosome 9. A set of mouse-human hybrid cell lines, each containing a single Ecogpt-marked human chromosome, was used as a source of donor chromosomes. Chromosome transfer to XPTG-1 cells, a hypoxanthine/guanine phosphoribosyltransferase-deficient mutant of simian virus 40-transformed complementation group A cells, was achieved by microcell fusion and selection for Ecogpt. Chromosome-transfer clones of XPTG-1 cells, each containing a different human donor chromosome, were analyzed for complementation of sensitivity to UV irradiation. Among all the clones, increased levels of resistance to UV was observed only in clones containing chromosome 9. Since our recipient cell line XPTG-1 is hypoxanthine/guanine phosphoribosyltransferase deficient, cultivation of Ecogpt+ clones in medium containing 6-thioguanine permits selection of cells for loss of the marker and, by inference, transferred chromosome 9. Clones isolated for growth in 6-thioguanine, which have lost the Ecogpt-marked chromosome, exhibited a UV-sensitive phenotype, confirming the presence of the repair gene(s) for complementation group A on chromosome 9.