Hamster Gene Research Articles

Partial cDNA sequence to a hamster gene corrects defect in Escherichia coli pyrB mutant.

The first three enzymes of pyrimidine biosynthesis (carbamoyl-phosphate synthetase, aspartate carbamoyl-transferase, and dihydro-orotase) are carried on a multifunctional protein in mammalian cells and are on separate proteins in bacteria. A plasmid containing a cDNA sequence corresponding to 80% of a hamster mRNA for this protein was transformed into Escherichia coli mutants lacking aspartate carbamoyltransferase (pyrB) or dihydro-orotase (pyrC). Only pyrB transformants were able to grow in the absence of uracil. Plasmid recovered from primary transformants was similar in size to the original plasmid and could yield prototrophs after secondary transformation of E. coli pyrB mutants. When cell extracts were prepared from pyrB transformants, high levels of aspartate carbamoyltransferase activity were found, and the enzyme had properties identical to the mammalian enzyme, including lack of allosteric regulation, precipitation by antiserum specific to the hamster multifunctional protein, and presence of a strong aggregation center. These results demonstrate that (i) a partial hamster protein can complement E. coli defective in pyrimidine biosynthesis, (ii) the order of the enzyme domains of the multifunctional protein is likely to be NH2-dihydro-orotase-carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-COOH, and (iii) the enzyme domains appear to be self-contained at the DNA and protein levels. The protocol described here may be a general means for studying the domains of multifunctional proteins and for isolating other mammalian genes for which bacterial mutants have been prepared. It also permits study of the structure and function of the same gene in both prokaryotic and eukaryotic cells and may provide new insight into the evolution of complex genes.

Organization of a Chinese hamster ovary dihydrofolate reductase gene identified by phenotypic rescue.

We have constructed a genomic DNA library from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) in the cosmid vector pHC79. By utilizing a murine dihydrofolate reductase (DHFR) cDNA clone, we have identified 66 DHFR+ clones among the 11,000 colonies screened by colony hybridization. To isolate a recombinant cosmid containing the entire DHFR gene, we have tested these colonies for their ability to rescue a DHFR- Chinese hamster ovary cell line, using the spheroplast fusion method of gene transfer developed by W. Schaffner (Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980). One clone (cH1) was able to transform DHFR- cells to the DHFR+ phenotype and was shown in hybridization studies to contain all of the gene except a small portion of the 3' untranslated region. We have mapped cosmid cH1 and several overlapping cosmids with a variety of restriction enzymes and have determined the approximate positions of the five (and possibly six) exons within the DHFR gene. Differences between the sizes of homologous genes in hamster cells (24.5 kilobases [kb]) and in mouse cells (31.5 kb) are shown to reside primarily in the length of the 3' intron, which is 8 kb in the hamster gene and 16 kb in length in the mouse gene. Our studies confirm the utility of cosmid libraries for the isolation of large genes, as previously shown by R. de Saint Vincent et al. (Cell 27:267-277, 1981). In addition, a cosmid that contains a functional DHFR gene will be a useful vector for the co-amplification and subsequent overexpression of other cloned genes.

Organization of a Chinese hamster ovary dihydrofolate reductase gene identified by phenotypic rescue.

We have constructed a genomic DNA library from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) in the cosmid vector pHC79. By utilizing a murine dihydrofolate reductase (DHFR) cDNA clone, we have identified 66 DHFR+ clones among the 11,000 colonies screened by colony hybridization. To isolate a recombinant cosmid containing the entire DHFR gene, we have tested these colonies for their ability to rescue a DHFR- Chinese hamster ovary cell line, using the spheroplast fusion method of gene transfer developed by W. Schaffner (Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980). One clone (cH1) was able to transform DHFR- cells to the DHFR+ phenotype and was shown in hybridization studies to contain all of the gene except a small portion of the 3' untranslated region. We have mapped cosmid cH1 and several overlapping cosmids with a variety of restriction enzymes and have determined the approximate positions of the five (and possibly six) exons within the DHFR gene. Differences between the sizes of homologous genes in hamster cells (24.5 kilobases [kb]) and in mouse cells (31.5 kb) are shown to reside primarily in the length of the 3' intron, which is 8 kb in the hamster gene and 16 kb in length in the mouse gene. Our studies confirm the utility of cosmid libraries for the isolation of large genes, as previously shown by R. de Saint Vincent et al. (Cell 27:267-277, 1981). In addition, a cosmid that contains a functional DHFR gene will be a useful vector for the co-amplification and subsequent overexpression of other cloned genes.

Properties of dispersed, highly repeated DNA within and near the hamster CAD gene.

Dispersed, highly repeated DNA sequences were found within and near the Syrian hamster gene coding for the multifunctional protein CAD. Most of the repeated sequences were homologous to each other and had similar properties. They hybridized to many cytoplasmic polyadenylated RNAs and to 7S and 4.5S cytoplasmic non-polyadenylated RNAs. Cloned DNA fragments containing repeated sequences were transcribed in vitro by RNA polymerase III. The repeated sequences from Syrian hamsters share many properties with the Alu family of repetitive DNA from humans. The hamster sequences were homologous to total repetitive human DNA but only very weakly homologous to two cloned members of the human Alu family.

Properties of dispersed, highly repeated DNA within and near the hamster CAD gene.

Dispersed, highly repeated DNA sequences were found within and near the Syrian hamster gene coding for the multifunctional protein CAD. Most of the repeated sequences were homologous to each other and had similar properties. They hybridized to many cytoplasmic polyadenylated RNAs and to 7S and 4.5S cytoplasmic non-polyadenylated RNAs. Cloned DNA fragments containing repeated sequences were transcribed in vitro by RNA polymerase III. The repeated sequences from Syrian hamsters share many properties with the Alu family of repetitive DNA from humans. The hamster sequences were homologous to total repetitive human DNA but only very weakly homologous to two cloned members of the human Alu family.

Molecular cloning of retrovirus-like genes present in multiple copies in the Syrian hamster genome.

Endogenous retrovirus-like sequences homologous to intracisternal type-A particle (IAP) genes, which are present in the inbred mouse (Mus musculus) genome, were cloned from a Syrian hamster gene library. A typical hamster IAP gene was 7 kb long and segments homologous to long terminal repeat (IAP) sequences present in Mus musculus IAP genes were located at both ends of the gene. Contrary to the pattern found in the Mus musculus IAP genes, the organization of the cloned hamster IAP genes was not markedly polymorphic and deletion was not observed among these cloned genes. A sequence about 0.8 kb long and located close to the 3' end of the hamster IAP gene was well conserved in both IAP gene families, although they showed less overall homology with one another. The reiteration frequency of the hamster IAP genes was calculated to be 950 copies per haploid genome. Since such IAP genes with the above properties were not found in the genome of the Chinese hamster, whose progenitors diverged from those of the Syrian hamster about 7.5 Myr ago, the integration of a huge number of Syrian hamster IAP genes must have occurred subsequent to such divergence.

Isolation of transforming DNA: Cloning the hamster aprt gene

We have isolated the hamster gene coding for the enzyme adenine phosphoribosyl transferase (aprt) using gene transfer and molecular cloning of transforming DNA. Mouse aprt − cells were transformed to the aprt + phenotype with the product of ligation of Hind III-cleaved hamster genomic DNA and pBR322 DNA. In this manner, the aprt gene was linked to a marker plasmid sequence and segregated from other hamster sequences. A λ-recombinant phage containing pBR322 DNA sequences was isolated from a library of aprt + transformed cell DNA. The phage DNA transfers hamster aprt + activity at a frequency expected of a pure gene. Furthermore, sequences homologous to this clone are present in all hamster aprt + transformants examined. This experimental design should in theory permit the isolation of any gene coding for selectable or identifiable functions for which DNA-mediated gene transfer can be effected.

Transformation of mammalian cells with an amplifiable dominant-acting gene.

We have transferred a mutant hamster gene coding for an altered dihydrofolate reductase to wild-type cultured mouse cells by using total genomic DNA from methotrexate-resistant Chinese hamster ovary A29 cells as donor. By demonstrating the presence of hamster gene sequences in transformants we have provided direct evidence for gene transfer. Transformants selected for increased resistance to methotrexate contain increased amounts of the newly transferred gene. We have used this mutant dhfr gene to introduce the Escherichia coli antibiotic resistance plasmid pBR322 into animal cells. Amplification of the dhfr sequences results in amplification of the pBR322 sequences as well. The use of this gene may allow the introduction and amplification of virtually any genetic element in various new cellular environments.

The Chinese hamster gene map: Assignment of four genes (DTS, PGM2, 6PGD, EnoI) to chromosome 2

Chinese hamster X mouse somatic cell hybrids constructed according to our methods, tend to retain a full complement of both sets of chromosomes, with eventual segregation of Chinese hamster chromosomes. Diphtheria toxin is an effective selective agent against Chinese hamster cells and their somatic cell hybrids. In Chinese hamster X mouse hybrids, sensitivity to diphtheria toxin (DTS) is associated with the presence of Chinese hamster chromosome 2. In addition, three Chinese hamster isozymes, PGM2, 6PGD, and EnoI, segregate with diphtheria toxin sensitivity, and are linked to Chinese hamster chromosome 2. Present data allow one to draw evolutionary comparisons with regard to genome organization among three mammalian species, human, mouse, and Chinese hamster.

Genetic and biochemical analysis of the a1 cell-surface antigen associated with human chromosome 11.

A highly versatile system for genetic, biochemical, and immunological analysis of human cell surface components has been developed using a human-Chinese hamster somatic cell hybrid containing chromosome 11 as its only human chromosome. This system lends itself to studies such as identification of human cell surface antigens and other genetic markers, regional gene mapping of these markers on chromosome 11, mutational analysis of these markers, and exploration of distribution of these antigens in normal and pathological human tissues. Genetic analysis of a1, one of the human antigens expressed by this hybrid, has been accomplished by subjecting a series of a1- variants to complementation analysis. These experiments have shown that the a1- variants behave in a recessive manner and that at least four genes, including three Chinese hamster genes, are needed for a1 antigen expression. Biochemical analysis has shown that a macroglycolipid isolated from human erythrocytes contains the a1 antigenic activity, so that genes coding for glycosyltransferases are required for its biosynthesis and may correspond to the complementation groups identified. The power of combined genetic, biochemical, and immunological approaches to understanding cell membrane molecules is demonstrated.

Alloimmune reactions among recently wild syrian hamsters and classical inbred strains include alloantibody production

Partially inbred lines of Syrian hamsters, the descendants of animals caught wild in Syria within the past decade, have been studied to determine their immunogenetic relationships to the long established domestic inbred strains. These new sources of hamster genes display strong alloimmune reactions when confronted with tissues from the domestic inbred strains and vice versa: acute skin graft rejection, strong mixed lymphocyte reactions, and intense graft-versus-host reactions. While these forms of reactivity have also characterized the disparities among the domestic inbred strains, no evidence of alloantibodies specific for putative transplantation antigens has ever been found in hamsters. We report, for the first time, that immunization of recently wild hamsters with tissues from domestic inbred strain donors elicits high titer cytotoxic antibodies directed at alloantigens. Similarly, animals of the domestic inbred strains produce comparably strong alloantibody responses when immunized with tissues from the recently wild hamsters. Availability of these alloantisera will make the possibility of defining the major histocompatibility complex equivalent in this species much more likely.

Regulation of endogenous virus production by bromodeoxyuridine and dibutyryl cyclic AMP in Chinese hamster ovary cells

Either dibutyryl cyclic AMP or bromodeoxyuridine can enhance RNA type-C virus production in Chinese hamster ovary cells. The resultant effect of simultaneous treatment of cells with 1 mM cyclic AMP and 65 μM bromodeoxyuridine is greater than their additive effects when used separately. The increase in virus production does not seem to involve viral gene amplification, but correlates with an increase in viral RNA transcription. The virus is also shown immunologically to be unique to the Chinese hamster cells and does not appear to be a result of external infection by other common laboratory type-C viruses. The control of this endogenous viral genomic expression may therefore be proposed to be under the normal Chinese hamster gene regulatory mechanism.

Expression of ?early? and ?late? viral functions in a somatic cell hybrid between a mouse cell and a spontaneous yielder SV 40-transformed Chinese hamster cell

A somatic cell hybrid (Cl. 6d) was originated from the fusion of mouse 3T3-4E) and spontaneous yielder SV 40-transformed Chinese hamster (CHK/SVLP AG) cells. During the early stages of its history, the C1. 6d hybrid underwent a rapid chromosome loss, preferentially loosing hamster chromosomes. This was not a constant tendency of the hybrid cells. As the parental CHK)SVLP AG cells, the hybrid cells were always found 100 per cent SV40 T-antigen positive. While CHK/SVLP AG cells infectious SV 40 DNA, V-antigen and virus were regularly detected, in the hybrid cells only infectious DNA was occasionally detected. This was not due either to the loww of an essential Chinese hamster gene(s) or to the presence of an inhibiting mouse cell component(s); it was apparently the consequence of inability of the cells to properly activate the resident SV 40 genome(s). After superinfection with SV 40 DNA, the hybrid cells-though capable of synthesizing SV 40 V-antigen--were unable to ensure virus assembly. Experimental evidence was obtained suggesting that SV 40 maturation is dependent of a cellular function(s).

Cotransfer of thymidine kinase and galactokinase genes by chromosome-mediated gene transfer.

The Chinese hamster genes for thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75) and galactokinase (ATP:D-galactose 1-phosphotransferase, EC 2.7.1.6) have been cotransferred to mouse cells by chromosome-mediated gene transfer. Hamster metaphase chromosomes were incubated with mouse B82 cells and 22 independent colonies were isolated in a selective medium. All of the 12 colonies analyzed expressed the donor form of thymidine kinase; the hamster form of galactokinase was also expressed in 2 of these colonies, indicating cotransfer with a frequency of about 20%. There was coordinate loss of both transferred genes from each colony when selection was applied for the loss of thymidine kinase alone. Comparison of the regional localization of these two linked genes with the frequency of cotransfer suggests that the transgenome is probably not larger than about 0.25% of the donor genome.