Suppressor genes for malignant and anchorage-independent phenotypes located on human chromosome 9 have no dosage effects

We have previously shown that microcell-mediated transfer of a der(9)t(X;9) human chromosome (HSA), derived from human fibroblast strain GM0705, into the Syrian hamster cell line BHK-191-5C produced only near-tetraploid hybrids, although the recipient cell line contained a 1:1 ratio of near-diploid and near-tetraploid cells. However, the tumorigenicity and the anchorage independence could be suppressed in the near-tetraploid hybrids with one copy of the der(9)t(X;9) chromosome. The introduction of an HSA X chromosome did not suppress either of these phenotypes. We concluded that in addition to two suppressor genes, one for tumorigenicity and another for anchorage independence, HSA 9 might carry a third gene capable of inhibiting cellular growth in vitro, which had dosage effects. In the present study, keeping one copy of the der(9)t(X;9) chromosome, we have increased the hamster background chromosome number beyond hexaploid level by fusing two microcell-generated hybrid cell lines, where both malignant and anchorage-independent phenotypes were suppressed, with the parental malignant BHK-191-5C cell line. Tests with nude mice showed that hybrids containing one copy of the der(9)t(X;9) chromosome against the increased background of chromosomes of malignant parental origin were still suppressed for both phenotypes. These results suggest that the suppressor genes for malignancy and for anchorage independence have no dosage effects, in contrast to the suppressor gene(s) for cellular growth.     

Comparative mapping of mouse and rat chromosomes by fluorescence in situ hybridization

Comparative fluorescence in situ hybridization mapping using DNA libraries from flow-sorted mouse chromosomes and region-specific mouse BAC clones on rat chromosomes reveals chromosomal homologies between mouse (Mus musculus, MMU) and rat (Rattus norvegicus, RNO). Each of the MMU 2, 3, 4, 6, 7, 9, 12, 14, 15, 16, 18, 19, and X chromosomes paints only a single rat chromosome or chromosome segment and, thus, the chromosomes are largely conserved between the two species. In contrast, the painting probes for MMU chromosomes 1, 5, 8, 10, 11, 13, and 17 produce split hybridization signals in the rat, disclosing evolutionary chromosome rearrangements. Comparative mapping data delineate several large linkage groups on RNO 1, 2, 4, 7, and 14 that are conserved in human but diverged in the mouse. On the other hand, there are linkage groups in the mouse, i.e., on MMU 1, 8, 10, and 11, that are disrupted in both rat and human. In addition, we have hybridized probes for Nap2, p57, Igf2, H19, and Sh3d2c from MMU 7 to RNO 1q and found the orientation of the imprinting gene cluster and Sh3d2c to be the same in mouse and rat. Hybridization of rat genomic DNA shows blocks of (rat-specific) repetitive sequences in the pericentromeric region of RNO chromosomes 3-5, 7-13, and 20; on the short arms of RNO chromosomes 3, 12, and 13; and on the entire Y chromosome.     

Conservation of the syntenies between porcine chromosome 7 and human chromosomes 6, 14 and 15 demonstrated by radiation hybrid mapping and linkage analysis

Comparative mapping studies facilitate the identification of genes located in quantitative trait locus (QTL) regions in domestic animals by utilizing information from the human genome. Radiation hybrid (RH) mapping is effective for this purpose because of its high resolution in ordered gene mapping on chromosomes. We constructed an RH map of pig chromosome 7, by adding 23 markers associated with genes. This RH map clearly demonstrated the mosaic of homology between pig chromosome 7 (SSC7) and human chromosomes 6, 14 and 15 at a 'gene' level, and was confirmed by linkage analysis. Clarification of the homology of SSC7 to human chromosomes will contribute to the elucidation of the gene(s) responsible for QTL detected on this chromosome.     

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

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 alpha 1-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.     

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.     

Analysis of chromosomal aberrations involving chromosome 1q31-->q53 in a DMBA-induced rat fibrosarcoma cell line: amplification and overexpression of Jak2

In a study of DMBA-induced rat fibrosarcomas we repeatedly found deletions and/or amplifications in the long arm of rat chromosome 1 (RNO1). Comparative genome hybridization showed that there was amplification involving RNO1q31-->q53 in one of the DMBA-induced rat fibrosarcoma tumors (LB31) and a cell culture derived from it. To identify the amplified genes we physically mapped rat genes implicated in cancer and analyzed them for signs of amplification. The genes were selected based on their locations in comparative maps between rat and man. The rat proto-oncogenes Ccnd1, Fgf4, and Fgf3 (HSA11q13.3), were mapped to RNO1q43 by fluorescence in situ hybridization (FISH). The Ems1 gene was mapped by radiation hybrid (RH) mapping to the same rat chromosome region and shown to be situated centromeric to Ccnd1 and Fgf4. In addition, the proto-oncogenes Hras (HSA11p15.5) and Igf1r (HSA15q25-->q26) were mapped to RNO1q43 and RNO1q32 by FISH and Omp (HSA11q13.5) was assigned to RNO1q34. PCR probes for the above genes together with PCR probes for the previously mapped rat genes Bax (RNO1q31) and Jak2 (RNO1q51-->q53) were analyzed for signs of amplification by Southern blot hybridization. Low copy number increases of the Omp and Jak2 genes were detected in the LB31 cell culture. Dual color FISH analysis of tumor cells confirmed that chromosome regions containing Omp and Jak2 were amplified and were situated in long marker chromosomes showing an aberrant banding pattern. The configuration of the signals in the marker chromosomes suggested that they had arisen by a break-fusion-bridge (BFB) mechanism.     

Chromosome ideograms of the laboratory rat (Rattus norvegicus) based on high-resolution banding, and anchoring of the cytogenetic map to the DNA sequence by FISH in sample chromosomes

A detailed banded ideogram representation of the rat chromosomes was constructed based on actual G-banded prometaphase chromosomes. The approach yielded 535 individual bands, a significant increase compared to previously presented ideograms. The new ideogram was adapted to the existing band nomenclature. The gene locus positions in the rat draft DNA sequence were compared to the chromosomal positions as determined by dual-color FISH, using rat (RNO) chromosomes 6 and 15 and a segment of RNO4 as sample regions. It was found that there was generally an excellent correlation in the chromosome regions tested between the relative gene position in the DNA molecules and the sub-chromosomal localization by FISH and subsequent information transfer on ideograms from measurements of chromosomal images. However, in the metacentric chromosome (RNO15), the correlation was much better in the short arm than in the long arm, suggesting that the centromeric region may distort the linear relationship between the chromosomal image and the corresponding DNA molecule.     

Comparative study of pig-rodent somatic cell hybrids

The pig chromosome complement of six different types of pig-rodent hybrid cell lines was examined by means of fluorescence in situ hybridization with a porcine SINE probe. The cell lines were obtained by fusing pig lymphocytes with cells of the Chinese hamster cell lines wg3h, BK14-150 and E36, and of the mouse cell lines NSO, PU and LMTK^-. The hybrids were analysed with respect to: (1) the number of pig chromosomes, (2) the type of pig chromosomes, (3) the occurrence of pig-rodent chromosome trans-locations, and (4) the presence of pig chromsome fragments. The results show that the number of pig chromosomes varied within and among hybrid cell lines. The pig-hamster hybrids mainly retained nontelocentric pig chromosomes, whereas the pig-mouse hybrids also retained telocentric pig chromosomes. Pig-rodent chromosome translocations were found in all types of hybrids, but the incidence was in general low. Chromosome fragments were abundant in BK14-150 hybrids, and rare in most other hybrid cell lines. It is concluded that the SINE probe is a useful tool to make a preliminary characterization of the porcine chromosome complement of pig-rodent somatic cell hybrids. The results of this characterization can be used to select hybrids for further cytogenetic analysis. Furthermore, our data show that different rodent cell lines will have to be used as fusion partners for the production of hybrids when constructing a panel informative for all pig chromosomes.     

Comparative mapping of genes flanking the human chromosome 12 evolutionary breakpoint in the pig

Genes located on human chromosome 12 (HSA12) are conserved on pig chromosomes 5 and 14 (SSC5 and SSC14), with HSA12q23.3-->q24.11 harboring the evolutionary breakpoint between these chromosomes. For this study, pig sequence-tagged sites (STS) were developed for nine HSA12 genes flanking this breakpoint. Radiation hybrid (RH) mapping using the IMpRH panel revealed that COL2A1, DUSP6, KITLG, PAH and STAB2 map to SSC5, while PXN, PLA2G1B, SART3 and TCF1 map to SSC14. Polymorphisms identified in COL2A1, DUSP6, PAH, PLA2G1B and TCF1 were used for genetic linkage mapping and confirmed the map locations for these genes. Our results indicate that the HSA12 evolutionary breakpoint occurs between STAB2 and SART3 in a region spanning less than five million basepairs. These results refine the comparative map of the HSA12 evolutionary breakpoint region and help to further elucidate the extensive gene order rearrangements between HSA12 and SSC5 and 14.     

Mapping of 443 porcine EST improves the comparative maps for SSC1 and SSC7 with the human genome

Numerous mapping studies of complex traits in the pig have resulted in quantitative trait loci (QTL) intervals of 10-20 cM. To improve the chances to identify the genes located in such intervals, increased expressed sequence tags (EST)-based marker density, coupled with comparative mapping with species whose genomes have been sequenced such as human and mouse, is the most efficient tool. In this study, we mapped 443 porcine EST with a radiation hybrid (RH) panel (384 had LOD > 6.0) and a somatic cell hybrid panel. Requiring no discrepancy between two-point and multipoint RH data allowed robust assignment of 309 EST, of which most were located on porcine chromosomes (SSC) 1, 4, 7, 8 and X. Moreover, we built framework maps for two chromosomes, SSC1 and SSC7, with mapped QTL in regions with known rearrangement between pig and human genomes. Using the Blast tool, we found orthologies between 407 of the 443 pig cDNA sequences and human genes, or to existing pig genes. Our porcine/human comparative mapping results reveal possible new homologies for SSC1, SSC3, SSC5, SSC6, SSC12 and SSC14 and add markers in synteny breakpoints for chromosome 7.     

The Ha-ras-induced transformed phenotype of rat-1 cells can be suppressed in hybrids with rat embryonic fibroblasts

Somatic cell hybrids were isolated from fusions of diploid embryonic rat fibroblasts with transformed Rat-1 cells which contained 4 to 5 copies of the transforming human Ha-ras 1 gene. In contrast to their transformed parental cells four hybrid clones showed normal morphology, long latency periods of tumorigenicity in newborn rats, anchorage requirement of proliferation, and an eightfold-reduced amount of secreted transforming growth factor activity. Thus these hybrids are called suppressed with regard to expression of the Ha-ras-induced transformed phenotype. Tumorigenic derivatives of the suppressed hybrids that had segregated chromosomes were isolated. Since two of the tumorigenic hybrid clones showed the similar low level of secreted transforming growth factors as the suppressed hybrids, decreased production of transforming growth factor activity is unlikely to be a sufficient criterion for suppression of malignancy. Whereas one of the suppressed hybrids expressed the transforming gene product p21 at a level similar to that of the transformed parental cells, other suppressed hybrids expressed less p21. This suggests that the suppressed phenotype can be regulated at the posttranslational level of p21 but that additional controls of expression of p21 are likely to exist. DNA of the suppressed hybrids transformed Rat-1 cells to proliferation in the presence of semisolid agar. Thus the activated human Ha-ras gene in the suppressed hybrids retained its biological activity even though it did not transform these cells to tumorigenicity.     

Chromosomal assignment of retinoic acid receptor (RAR) genes in the human, mouse, and rat genomes.

The human genes encoding the alpha and beta forms of the retinoic acid receptor are known to be located on chromosomes 17 (band q21.1:RARA) and 3 (band p24:RARB). By in situ hybridization, we have now localized the gene for retinoic acid receptor gamma, RARG, on chromosome 12, band q13. We also mapped the three retinoic acid receptor genes in the mouse, by in situ hybridization, on chromosomes 11, band D (Rar-a); 14, band A (Rar-b); and 15, band F (Rar-g), respectively, and in the rat, using a panel of somatic cell hybrids that segregate rat chromosomes, on chromosomes 10 (RARA), 15 (RARB), and 7 (RARG), respectively. These assignments reveal a retention of tight linkage between RAR and HOX gene clusters. They also establish or confirm and extend the following homologies: (i) between human chromosome 17, mouse chromosome 11, and rat chromosome 10 (RARA); (ii) between human chromosome 3, mouse chromosome 14, and rat chromosome 15 (RARB); and (iii) between human chromosome 12, mouse chromosome 15, and rat chromosome 7 (RARG).     

Opal suppressor phosphoserine tRNA gene and pseudogene are located on human chromosomes 19 and 22, respectively

An opal suppressor phosphoserine tRNA gene and pseudogene have been isolated from a human DNA library and sequenced (O'Neill, V., Eden, F., Pratt, K., and Hatfield, D. (1985) J. Biol. Chem. 260, 2501-2508). Southern hybridization of human genomic DNA with an opal suppressor tRNA probe suggested that the gene and pseudogene are present in single copy. In this study, we have determined the chromosome location of the human gene and pseudogene by utilizing a 193-base pair fragment encoding the opal suppressor phosphoserine tRNA gene as probe to examine DNAs isolated from human-rodent somatic cell hybrids that have segregated human chromosomes. These studies show that the probe hybridized with two regions in the human genome; one is located on chromosome 19 and the second on chromosome 22. By comparing the restriction sites within these two regions to those previously determined for the human opal suppressor phosphoserine tRNA gene and pseudogene, we tentatively assigned the gene to chromosome 19 and the pseudogene to chromosome 22. These assignments were confirmed by utilizing a 350-base pair fragment which was isolated from the 5'-flanking region of the human gene as probe. This fragment hybridized only to chromosome 19, demonstrating unequivocally that the opal suppressor phosphoserine tRNA gene is located on chromosome 19. The flanking probe hybridized to a single homologous band in hamster and in mouse DNA to which the gene probe also hybridized, demonstrating that the 5'-flanking region of the opal suppressor tRNA gene is conserved in mammals. Restriction analysis of DNAs obtained from the white blood cells of 10 separate individuals demonstrates that the gene is polymorphic. This study provides two additional markers for the human genome and constitutes only the second set of two tRNA genes assigned to human chromosomes.     

Physical assignments of human chromosome 13 genes on pig chromosome 11 demonstrate extensive synteny and gene order conservation between pig and human

Previous mapping between the human and pig genomes suggested extensive conservation of human chromosome 13 (HSA13) to pig chromosome 11 (SSC11). The objectives of this study were comparative gene mapping of pig homologs of HSA13 genes and examining gene order within this conserved synteny group by physical assignment of each locus. A detailed HSA13 to SSC11 comparison was chosen since the comparative gene map is not well developed for these chromosomes and a rearranged gene order within conserved synteny groups was observed from the comparison between HSA13 and bovine chromosome 12 (BTA12). Heterologous primers for PCR were designed and used to amplify pig homologous fragments. The pig fragments were sequenced to confirm the homology. Six pig STSs (FLT1, ESD, RB1, HTR2A, EDNRB, and F10) were physically mapped using a somatic cell hybrid panel to SSC11, and fluorescent in situ hybridization (FISH) mapping was also applied to improve map resolution and determine gene order. Results from this study increase the comparative information available on SSC11 and suggest a conserved gene order on SSC11 and HSA13, in contrast to human:bovine comparisons of this syntenic group.     

Suppression of tumorigenicity in somatic cell hybrids. IV. Chromosomes of normal human cells associated with suppression of tumorigenicity in hybrids with D98AH2 carcinoma cells

An analysis for cosegregation of chromosomes and tumorigenicity in 52 hybrids of human diploid X D98AH2 human carcinoma-derived cells reveals the consistent presence of four copies of chromosome 11 in all nontumorigenic hybrids (two from each of the parental cells) and a consistent loss of one or two copies of the 11 in all tumor cells derived from tumorigenic hybrids that grow in nude mice. In our earlier study, assays with restriction fragment length polymorphic (RFLP) markers for the cell parent origin of the chromosomes 11 in the hybrids indicated that at least one of the Nos. 11 lost in the tumor cells is from the diploid. Thus both Nos. 11 of the diploid seem to be required for complete and stable suppression of the tumorigenic phenotype. The results of the present study suggest that chromosome 2 may also carry suppressor information, but this causes only partial suppression of the tumorigenic phenotype in the absence of both Nos. 11. On the other hand, when the hybrids contain full complements of the 2 and the 11, suppression is very stable. All other chromosomes except for Nos. 1, 16, 17, 19, and 21 are clearly discordant with suppression. The latter chromosomes are not discordant often enough to allow their exclusion as possible carriers of suppressor information, particularly in the absence of RFLP evaluations. It is clear, however, that if they do carry such information it is not adequate for maintaining a stably suppressed phenotype in the absence of both Nos. 11 of the diploid.     

Rescue of the HNF4 → HNF1α Pathway in Hepatoma Variant Cells Containing Human Chromosome 12

Expression of liver-enrichedtrans-acting hepatocyte nuclear factors 1α (HNF1α) and 4 (HNF4) is correlated with the hepatic phenotype in cultured rat hepatoma cells. We have used a hepatoma variant cell line, H11, that specifically lacks the HNF4 → HNF1α pathway as a model to understand mechanisms controlling hepatic gene expression. We have introduced randomly marked human chromosomes into H11 cells and have isolated a number of microcell hybrids that have rescued hepatic gene expression, including HNF4, HNF1α, and α1-antitrypsin. Chromosomal analysis of cell hybrids showed that the rescued hepatic phenotype correlated closely with the presence of human chromosome 12p sequences. Although the gene encoding HNF1α is located on chromosome 12q24, its retention was not required to rescue the hepatic phenotype. Thus, we suggest that a locus on human chromosome 12p plays an important role in maintenance of hepatic gene expression through activation of the HNF4 → HNF1α pathway.     

Assignment of two rat dihydrofolate (DHFR) genes to chromosomes 2 and 4

A panel of rat x mouse cell hybrids was used in the chromosomal mapping of the rat dihydrofolate reductase (DHFR) gene. It was determined that the probe hybridized to gene sequences on two different chromosomes (Nos. 2 and 4), possibly representing the active gene and a pseudogene. Hybridization of the DHFR probe to DNA from a methotrexate resistant rat cell line revealed that the gene on chromosome 2 was amplified, but not the gene on chromosome 4. This result was taken to suggest that the active DHFR gene is located on rat chromosome 2 and that the sequence on chromosome 4 is a pseudogene.     

Mapping of growth hormone releasing hormone receptor to swine chromosome 18

The growth hormone releasing hormone receptor (GHRHR) was mapped in the pig for study as a potential candidate gene in controlling pig quantitative growth and carcass characteristics. Primers were designed from the pig GHRHR sequence to amplify a 1·65-kb intronic fragment between exons 6 and 7. By using a pig–rodent somatic cell hybrid panel, GHRHR was mapped to pig chromosome 18 (SSC18) with 100% concordance, and the regional assignment was SSC18q24 with 89% concordance. The polymerase chain reaction–restriction fragment length polymorphisms (PCR–RFLPs) with Mse I and Taq I were developed to confirm this assignment with linkage analysis by using the European Pig Gene Mapping Project (PiGMaP) reference families. Pig GHRHR was mapped with strong linkage to SSC18 markers S0062 and S0120 (lod > 8). The GHRHR and IGFBP3 were found to map near to each other on human chromosome 7 (HSA7), and the pig IGFBP3 gene has been mapped to SSC18 by others. Our mapping of pig GHRHR increases the comparative information available on the SSC18 maps and further confirms the synteny conservation between HSA7 and SSC18.     

Improving the comparative map of SSC2p-q13 by sample sequencing of BAC clones

To improve the comparative map for pig chromosome 2 and increase the gene density on this chromosome, a porcine bacterial artificial chromosome (BAC) library was screened with 17 microsatellite markers and 18 genes previously assigned to pig chromosome 2. Fifty-one BAC clones located in the region of a maternally imprinted quantitative trait locus for backfat thickness (BFT) were identified. From these BACs 372 kb were sample sequenced. The average read length of a subclone was 442 basepair (bp). Contig assembly analysis showed that every bp was sequenced 1.28 times. Subsequently, sequences were compared with sequences in the nucleotide databases to identify homology with other mammalian sequences. Sequence identity was observed with sequences derived from 35 BACs. The average percentage identity with human sequences was 87.6%, with an average length of 143 bp. In total, sample sequencing of all BACs resulted in sequence identity with 29 human genes, 13 human expressed sequence tags (ESTs), 17 human genomic clones, one rat gene, one porcine gene and nine porcine ESTs. Eighteen genes located on human chromosome 11 and 19, and seven genes from other human locations, one rat gene and one porcine gene were assigned to pig chromosome 2 for the first time. The new genes were added to the radiation hybrid map at the same position as the locus from which the BAC that was sequenced was derived. In total 57 genes were placed on the radiation hybrid map of SSC2p-q13.     

The majority of independently transformed BHK cell clones share a single functional lesion which determines anchorage independence and influences tumorigenicity

Summary Expression of the anchorage-independent transformed phenotype in BHK 21/13 cells generally behaves as a recessive trait. When chemically induced and spontaneously arising transformants are fused to the nontransformed parent line, transformation is initially suppressed, reappearing after extended growth of the hybrids. In this paper, complementation for the expression of anchorage independence was sought among a large group of such transformants, all independently derived from BHK 21/13 cells. Tumorigenicity studies on selected hybrids and parental lines indicated that the in vitro trait of anchorage independence is an accurate indicator of in vivo neoplasia for these cells. Seventeen of the 18 clones tested did not complement one or more of three tester strains. This result indicates that anchorage independence arose in these clones as a result of lesions in the same genetic function and suggests that the final step in the progressive changes of carcinogenesis may frequently be restricted to lesions at a single locus. This investigation was supported by National Institutes of Health grant CA27306.