Two related helix-loop-helix proteins participate in separate cell-specific complexes that bind the insulin enhancer.

Cell-specific expression of the insulin gene is dependent on a conserved 8-basepair sequence, GCCATCTG, present in two copies in the 5' flanking DNA of the rat insulin 1 gene (Nir and Far elements). A protein factor with well characterized binding affinities binds to this sequence and is unique to the nuclei of insulin-producing cells. Using the Nir element as a probe to screen a hamster insulinoma cDNA expression library, we cloned two cDNA inserts that encode two related helix-loop-helix DNA-binding proteins: Syrian hamster Pan-1 (shPan-1) and Syrian hamster Pan-2 (shPan-2). These clones have minimal differences from the previously reported human E47/E12 and rat PAN (rPan) DNA-binding proteins. In vitro translated protein products of both clones bound the insulin gene promoter Nir and far elements as well as the E2 elements of the mu heavy chain and kappa light chain immunoglobulin genes. Treating insulinoma cell nuclear extract with antiserum selectively directed to each of the two shPan proteins demonstrated the presence of each form of shPan in separate DNA-binding complexes, which together form the previously described, cell-specific, Nir element-binding complex. We conclude that shPan-1 and shPan-2 are the hamster homologs of the ubiquitous E47/E12 and rPan proteins, but form parts of distinct DNA-binding complexes apparently found only in the nuclei of insulin-producing cells.     

Mapping of the gene encoding the multifunctional protein carrying out the first three steps of pyrimidine biosynthesis to human chromosome 2

Summary The CAD gene encodes a trifunctional protein that carries the activities of the first three enzymes (carbamyl phosphate synthetase II, aspartate transcarbamylase, and dihydroorotase) of de novo pyrimidine biosynthesis. Genomic fragments of the human CAD gene have been obtained by screening a human genomic library in bacteriophage lambda using a Syrian hamster cDNA clone as a probe. These human genomic clones have been used to assign the CAD gene to human chromosome 2 using in situ hybridization to human metaphase chromosomes and Southern blot hybridization analysis of DNA isolated from a panel of Chinese hamster/human hybrid cells. In situ hybridization analysis has allowed further localization of this gene to the chromosomal region 2p21-p22.     

The human gene encoding insulin-like growth factor I is located on chromosome 12

Summary A cDNA probe corresponding to mRNA encoding human somatomedin-C/insulin-like growth factor I (IGF-I) was used for the chromosomal assignment of the IGF-I gene. Southern-blot hybridization analysis of DNA from human-Chinese hamster somatic cell hybrids showed that the IGF-I gene is located on chromosome 12. Comparison of the chromosomal assignments of the IGF-I gene and two other members of the insulin gene family, with three c-ras oncogenes, reveals a remarkable association of the two gene families.     

Chromosomal distribution of the hamster intracisternal A-particle (IAP) retrotransposons

The retrotransposon-like elements of the intracisternal A-particle (IAP) sequences occur in about 900 copies per haploid hamster cell genome. By applying the fluorescent in situ hybridization (FISH) technique and four different, cloned segments of the IAP element as hybridization probes, these elements were found to be distributed in specific patterns over many of the 44 hamster chromosomes. The hybridization patterns were very similar regardless of whether all four probes or only the IAPI probe carrying the long terminal repeat (LTR) region were used. The IAP elements were found most abundantly, though not exclusively, on the short arms of at least 12 of the autosomes. Of the sex chromosomes, the shorter Y chromosome was stained on both arms, and the X chromosome on one arm by the IAP probes. Primary Syrian hamster cells, the established Syrian hamster cell line BHK21, and the adenovirus type 12 (Ad12)-transformed BHK21 cell line T637 yielded very similar results. In Chinese hamster ovary (CHO) or 3T3 mouse cells, signals could not be elicited by FISH using the Syrian hamster IAP probes. On Southern blots, the DNAs from these cell lines hybridized very weakly, if at all, to the IAP sequences. Thus, IAP sequences were retroposed after Syrian hamster and mouse or Syrian and Chinese hamsters had diverged in evolution.     

Coding sequence and growth regulation of the human vimentin gene.

We have established the complete coding sequence of the human vimentin gene. It had 91% homology to the coding sequence of the Syrian hamster vimentin gene (Quax et al., Cell 35:215-223, 1983) and partial homology to several other sequences coding for intermediate filament proteins. The most striking difference between the Syrian hamster and human vimentin genes was in the 3' untranslated region, which was considerably longer in the Syrian hamster. Using RNA blots and a human vimentin cDNA clone from an Okayama-Berg library, we have established that expression of the vimentin gene was growth regulated. The steady-state levels of cytoplasmic vimentin mRNA in 3T3 cells were increased by serum and platelet-derived growth factor, but not by epidermal growth factor, insulin, or platelet-poor plasma. The increase in expression of the vimentin gene that occurred when G0-phase cells were stimulated to proliferate was detected in six different cell types from four different species. The expression of the vimentin gene was also increased when HL60 cells were induced to differentiate by phorbol esters; it decreased when differentiation was induced by retinoic acid.     

Distribution of DNA replication origins between matrix-attached and loop DNA in mammalian cells

Using a previously developed procedure (Gencheva et al. [1996] J Biol Chem 271:2608-2614), we isolated a DNA fraction consisting of short fragments originating from the regions of initiation of DNA synthesis from exponentially growing Chinese hamster ovary cells. This fraction arbitrarily designated as "collective origin fraction" was labeled in vitro and used to probe the abundance of origin containing sequences in preparations of matrix-attached and loop DNA isolated by two different procedures from Chinese hamster ovary cells. Alternatively, an individual DNA replication origin sequence - a 478-bp long DNA fragment located at about 17-kb downstream of the dihydrofolate reductase gene - was used to probe the same matrix-attached and loop DNA fractions. The results with both the collective and individual DNA replication origins showed that there was random distribution of the origin sequences between DNA attached to the matrix and DNA from the loops.     

Co-amplification of rRNA genes with CAD genes in N-(phosphonacetyl)-L-aspartate-resistant Syrian hamster cells.

The amplified CAD genes in N-(phosphonacetyl)-L-aspartate (PALA)-resistant Syrian hamster cells are located in an expanded chromosomal region emanating from the site of the wild-type gene at the tip of the short arm of chromosome B-9. The terminus of B-9 in PALA-sensitive cells contains a cluster of rRNA genes (i.e., a nucleolus organizer, rDNA). We have used a molecular clone containing sequences complementary to Syrian hamster 28S rRNA to investigate whether rDNA is coamplified with CAD genes in the PALA-resistant mutants. In situ hybridization of this probe to metaphase chromosomes demonstrates that rDNA and CAD genes do coamplify in two independently isolated PALA-resistant mutants. The tight linkage of CAD and rDNA genes was demonstrated by their coordinate translocation from B-9 to the end of the long arm of chromosome C-11 in one mutant. Blot hybridization studies substantiate the in situ hybridization results. Both types of analysis indicate that only one or two rDNA genes, on the average, are coamplified per CAD gene. The data are consistent with the model that unequal exchanges between rDNA genes mediate the amplification of CAD genes in the Syrian hamster mutants that were analyzed.     

Assignment of a human DNA double-strand break repair gene (XRCC5) to chromosome 2.

The Chinese hamster ovary (CHO-K1) cell mutant XRS-6 is defective in rejoining of DNA double-strand breaks and is hypersensitive to X-rays, gamma-rays, and bleomycin. Radiation resistance or sensitivity of somatic cell hybrids constructed from the fusion of XRS-6 cells with primary human fibroblasts strongly correlated with the retention of human chromosome 2 isozyme and molecular markers. Discordancies between some chromosome 2 markers and the radiation resistance phenotype in some of the hybrid cells suggested the location of the X-ray repair cross complementing 5 (XRCC5) gene on the p arm of chromosome 2. Introduction of human chromosome 2 by microcell-mediated chromosome transfer into the radiation-sensitive XRS-6 cells resulted in hybrid cells in which the radiation sensitivity was complemented. The chromosome 2p origin of the complementing human DNA in the microcell hybrids was supported by fluorescent in situ hybridization analysis of human metaphases using human DNA amplified from the hybrids by inter-Alu-PCR as chromosome-painting probes. XRCC5 is therefore provisionally assigned to human chromosome 2p.     

Expression and chromosome location of hamster Ku70 and Ku80

Ku proteins play an important role in DNA double-strand break (DSB) repair, chromosome maintenance, and growth regulation. To understand the fundamental characteristics of Ku proteins, we examined the electrophoretic mobility and expression of hamster Ku70 and Ku80 and determined the chromosome locations of their genes. The electrophoretic mobility of hamster Ku proteins are different from that of human Ku proteins. No significant changes in the quantity of Ku proteins were observed in CHO-K1 cells treated with 10 Gy of ionizing radiation, suggesting that both proteins are expressed constitutively in amounts adequate to repair DNA DSBs. The chromosome locations of the Ku genes were determined by direct R-banding fluorescence in situ hybridization. The Ku70 gene was localized to Syrian hamster chromosome 4qa4.1--> qa4.2 and Chinese hamster chromosome 2p3.1, and the Ku80 gene was localized to Syrian hamster chromosome 4qb5--> qb6.1 and Chinese hamster chromosome 2p3.5-->p3.6. These results provide clues to the biological functions of Ku, as well as useful information for constructing comparative chromosome maps between hamsters and other mammalian species, including human, mouse, and rat.     

Characterization of the hamster DDT-1 cell aFGF/HGBF-I gene and cDNA and its modulation by steroids.

Syrian hamster DDT-1 cells are derived from smooth muscle of the ductus deferens. DDT-1 cell growth is increased by the addition of testosterone (T). Acidic fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF) also known as heparin binding growth factor I and II (HBGF-I and HBGF-II) can replace T in the stimulation of growth in these cells. This phenomenon is correlated with testosterone's ability to elevate aFGF/HBGF-I mRNA. The increase steady-state levels of aFGF/HBGF-I mRNA were documented by northern blots and by in situ hybridization. Using a 520 bp human aFGF/HBGF-I cDNA probe, a genomic clone with a 38 kb DNA insert was isolated from a cosmid library. By restriction enzyme analysis and southern hybridization, it was determined that there are three coding exons. DNA sequence analysis showed all of the coding region and 3' noncoding sequences were on this clone. A 5' noncoding exon not in the 38 kb insert is indicated, based on the cDNA sequences and genomic sequences of aFGF/HBGF-I's from hamster DDT-1 cells and several other species. The cDNA for hamster aFGF/HBGF-I was isolated from a DDT-1 lambda gt11 library and sequenced. Comparison of the coding region of aFGF/HBGF-I from four species shows a greater than 90% conservation of amino acid sequence.     

Chromosomal localization and preliminary characterization of the human gene encoding insulin-like growth factor II

Summary A cDNA probe corresponding to mRNA encoding a closely related variant of human insulin-like growth factor II (IGF-II) was used for the chromosomal assignment of the IGF-II gene. Southern blot hybridization analysis of DNA from human-Chinese hamster somatic cell hybrids showed that the human IGF-II gene is located on chromosome 11. Using the same IGF-II variant probe, a cosmid was isolated which contains the human IGF-II gene. Restriction enzyme analysis revealed that the gene encoding IGF-II has a discontinuous structure and contains at least four exons.     

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.     

Regional localization of the gene for thyroid peroxidase to human chromosome 2pter----p12

A 2.0-kb thyroid peroxidase cDNA of human origin was used as probe for Southern blot hybridization of genomic DNA from human somatic cells and human-rodent somatic cell hybrids. The results showed that the gene coding for human thyroid peroxidase is located on chromosome. 2. Further analysis of hybrids derived from Burkitt lymphoma cells carrying a (2;8)(p12;q24) translocation revealed that the gene maps to the region 2pter----p12.     

Evolutionary relationships of the Chinese hamster X chromosome and autosomes: a comparison using solution hybridization techniques

The evolutionary relationships of Chinese hamster X chromosome and autosome DNA sequences were compared by solution hybridization techniques. Chinese hamster X chromosome tracer was prepared by radiolabeling DNA from chromosomes isolated by fluorescence-activated sorting. Radiolabeled Chinese hamster total genomic DNA, approximately 90% of which is of autosome origin, was used as autosome tracer. Each tracer was mixed with excess driver DNA of Chinese hamster, Syrian hamster, rat, rabbit, cat, cow, or human origin. Reaction mixtures were melted and allowed to reassociate to an equivalent CoT of 12000, under conditions which permitted 35% mismatch in DNA duplexes. Both the extent of duplex formation (the normalized percentage hybridization or NPH) and the average thermal stability of the duplexes formed (melting temperature or Tm) were measured; these values were used to compare the evolutionary relatedness of tracer and driver DNAs. The pattern of evolutionary relatedness revealed by comparing either the Tm or NPH values obtained with different drivers was the same for X chromosome and autosome DNA and was consistent with the phylogeny of the species examined. Although NPH and Tm values for X chromosome and autosome tracers differed, differences fell within the range of experimental error. The results of these studies provide no evidence for differential conservation of Chinese hamster X chromosome sequences, suggesting that the constraints on the mammalian X chromosome which act to maintain its gene linkage group intact do not markedly reduce the extent to which its sequences diverge during evolution.     

Assignment of a human DNA double-strand break repair gene (XRCC5) to chromosome 2

The Chinese hamster ovary (CHO-K1) cell mutant XRS-6 is defective in rejoining of DNA double-strand breaks and is hypersensitive to X-rays, γ-rays, and bleomycin. Radiation resistance or sensitivity of somatic cell hybrids constructed from the fusion of XRS-6 cells with primary human fibroblasts strongly correlated with the retention of human chromosome 2 isozyme and molecular markers. Discordancies between some chromosome 2 markers and the radiation resistance phenotype in some of the hybrid cells suggested the location of the X-ray repair cross complementing 5 (XRCC5) gene on the p arm of chromosome 2. Introduction of human chromosome 2 by microcell-mediated chromosome transfer into the radiation-sensitive XRS-6 cells resulted in hybrid cells in which the radiation sensitivity was complemented. The chromosome 2p origin of the complementing human DNA in the microcell hybrids was supported by fluorescent in situ hybridization analysis of human metaphases using human DNA amplified from the hybrids by inter-Alu-PCR as chromosome-painting probes. XRCC5 is therefore provisionally assigned to human chromosome 2p.     

Uncoupled expression of mRNAs for alpha 1(I) and alpha 2(I) procollagen chains in chemically transformed Syrian hamster fibroblasts

Syrian hamster embryo fibroblasts transformed by 4-nitroquinoline-1-oxide (NQT-SHE cells) failed to synthesize the pro-alpha 1(I) subunit of type I procollagen but continued to synthesize altered forms of the other subunit, pro-alpha 2(I) (Peterkofsky, B., and Prather, W. (1986) J. Biol. Chem. 261, 16818-16826). This was unusual, since synthesis of the two subunits generally is coordinately regulated. Present experiments using cell-free translation and hybridization of RNA from normal and transformed Syrian hamster fibroblasts with labeled pro-alpha 1(I) DNA probes show that mRNA for pro-alpha 1(I) is absent from the transformant. In contrast, dot-blot and Southern blot hybridizations of cellular DNAs with pro-alpha 1(I) DNA probes demonstrated that the transformed cells contained pro-alpha 1(I) gene sequences and that the gross structure of the gene was unchanged by transformation. mRNA for the other type I procollagen subunit, pro-alpha 2(I), was present in transformed cells and the major collagenous polypeptide translated from this RNA migrated like the normal pro-alpha 2 subunit during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The translated procollagen chain was cleaved to an alpha 2(I)-sized collagen chain by pepsin at 4 degrees C. These studies provide a molecular basis for the observed collagen phenotype of NQT-SHE cells.     

用DNA分子杂交研究两种人体胸苷激酶基因间的同源性

本实验从人体细胞质胸苷激酶(TK-C)基因分离出不含Alu重复顺序的3个DNA片段,分别次级克隆在质粒pBR322上,定名为pRR0.92,pXR1.5和pHK1.25。用pXR1.5和pHK1.25为探针,作Southern印迹杂交分析,都不能与小鼠细胞Ltk、CD-1、A9和BALB/c的DNA杂交。但是pXR1.5能与中国仓鼠细胞E36 DNA 23kb Bam HI片段杂交,出现信号很弱的杂交带.这提示以TK-C基因来说,人同中国仓鼠间的同源程度似大于人同小鼠的同源程度。在降低杂交严紧度的条件下,pHK1.25能与含人体16号染色体而不含17号染色体的人鼠杂种细胞DNA杂交,只是杂交信号极弱。我们推测人体TK-C基因(位于17号染色体)与人体TK-M基因(位于16号染色体)可能有某种程度的同源性,pHK1.25对进一步克隆TK-M基因也许是有用的。     

Assignment of the alpha 1-antitrypsin gene and a sequence-related gene to human chromosome 14 by molecular hybridization

alpha 1-Antitrypsin is a major plasma protease inhibitor synthesized in the liver. Genetic deficiency of this protein predisposes the affected individuals to development of infantile liver cirrhosis or chronic obstructive pulmonary emphysema. The human chromosomal alpha 1-antitrypsin gene has been cloned and shown to contain three introns in the peptide-coding region. When the cloned alpha 1-antitrypsin gene was used as a hybridization probe to analyze Eco RI-digested genomic DNA from different individuals, two distinct bands of 9.6 kilobases (kb) and 8.5 kb in length were observed in every case. Further analysis using only labeled intronic DNA as the hybridization probe has indicated that the authentic alpha 1-antitrypsin gene resides within the 9.6-kb fragment. Thus the 8.5-kb fragment must contain another gene that is closely related in sequence to the alpha 1-antitrypsin gene. Using a series of human-Chinese hamster somatic cell hybrids containing unique combinations of human chromosomes, the alpha 1-antitrypsin gene as well as the sequence-related gene have been assigned to human chromosome 14 by Southern hybridization and synteny analysis.     

Isolation of probes specific to human chromosomal region 6p21 from immunoselected irradiation-fusion gene transfer hybrids

A hybrid cell line (R21/B1) containing a truncated human chromosome 6 (6pter-6q21) and a human Y chromosome on a hamster background was irradiated and fused to A23 (TK-) or W3GH (HPRT-) hamster cells. Clones containing expressed HLA class I genes (4/40) were selected using monoclonal antibodies. These clones were recloned and analyzed with a panel of probes from the HLA region. One hybrid (4G6) contained the entire HLA complex. Two other hybrids (4J4 and 4H2) contained only the HLA class I region, while the fourth hybrid (5P9) contained HLA class I and III genes in addition to other genes located in the 6p21 chromosomal region. In situ hybridization showed that the hybrid cells contained more than one fragment of human DNA. Alu and LINE PCR products were derived from these cells and compared to each other as well as to products from two somatic cell hybrids having the 6p21 region in common. The PCR fragments were then screened on conventional Southern blots of the somatic cell hybrids to select a panel of novel probes encompassing the 6p21 region. In addition, the origin of the human DNA fragments in hybrid 4J4 was determined by regional mapping of PCR products.