A gene that regulates DNA replication in response to DNA damage is located on human chromosome 4q.

Inhibition of replicative DNA synthesis following gamma-irradiation is observed in eukaryotic cells but is defective in cells derived from patients with the cancer-prone inherited disorder ataxia-telangiectasia (A-T) and in A-T-like Chinese hamster cell mutants. Chinese hamster cells show a less pronounced inhibition of DNA synthesis after gamma-irradiation when compared to irradiated human HeLa or mouse A9 cells. Therefore, to identify new human genes involved in the regulation of DNA replication in response to ionizing radiation in mammalian cells, single human chromosomes were introduced into Chinese hamster cells by microcell-mediated chromosome transfer. It is found that a new gene on human chromosome 4q inhibits DNA synthesis following gamma- and UV irradiation in hamster cells. However, this delay of DNA replication did not improve cell survival or the level of chromosomal aberrations induced by X-rays, indicating that the lack of the inhibition of DNA synthesis after X-irradiation is not a prerequisite for the X-ray sensitivity and chromosomal instability, which is observed in A-T and A-T-like hamster cells.     

Amplified inverted duplications within and adjacent to heterologous selectable DNA.

Plasmids containing a dihydrofolate reductase (DHFR) expression unit were transfected into DHFR-deficient Chinese hamster ovary (CHO) cells. Methotrexate exposure was used to select cells with amplified DHFR sequences. Three cell lines were isolated containing amplified copies of transfected DNA that had integrated into the Chinese hamster genome. Plasmid DNA was found to co-amplify with flanking hamster sequences that were repetitive (2 cell lines) and unique (1 cell line). Fragments comprising the junctions of amplified plasmid and CHO DNA were found to exist as inverted duplications in all three cell lines. These observations provide evidence that inverted duplication occurred prior to DNA amplification, thus underscoring the importance of inverted duplication in the DNA amplification process.     

The binding of the mucoprotein from gastric mucus to cells in tissue culture and the inhibition of cell adhesion.

The mucoprotein, which is responsible for the formation of gastric mucous gel in pig, has been shown to bind equally well to suspensions of baby hamster kidney cells, polyoma-virus-transformed baby hamster kidney cells and HeLa cells. The binding of the mucoprotein to the cells is dependent on Ca 2     

Resistance of Chinese hamster ovary cell chromatin to endonuclease digestion. I. Reversal by cAMP

A portion of the DNA within intact nuclei of a spontaneously transformed Chinese hamster ovary cell line (CHO-Kl) is relatively resistant to digestion by pancreatic deoxyribonuclease, as compared to nuclei from primary cultures of Chinese hamster ovary fibroblasts. Treatment of CHO-Kl cells with derivatives of 3',5' cyclic AMP (cAMP) under conditions which effect the reverse transformation (RT) of these cells, results in restoration of the increased sensitivity of their DNA to hydrolysis by pancreatic deoxyribonuclease, to the level characteristic of an untransformed, morphologically normal Chinese hamster fibroblast cell line. Dibutyryl (db-)cAMP and 8-bromo-cAMP (Br-cAMP) yielded similar results. The cAMP derivatives employed had no effect on the normal fibroblasts.     

Unique features of Chinese hamster S13 gene relative to its human and Xenopus analogs.

We have cloned and sequenced the ribosomal protein S13 gene from the Chinese hamster fibroblast HA-1 cells. The predicted protein encoded by this gene is identical to the human ribosomal protein S13, except for one amino acid substitution at residue 29, which is an alanine in the hamster protein and a threonine in that of humans. The physical organization of the six exons and five introns in the hamster S13 gene is also identical to that found in the human and Xenopus genes with respect to the amino acid codes, even though there are small differences in the lengths of the introns. The striking feature is that unlike its human and Xenopus counterparts, which encode two U14 snoRNAs in two separate introns, the hamster S13 gene encodes no U14 snoRNA. Instead, the hamster gene has a pseudo-U14 coding sequence in its third intron. Our data support the idea that the single copy of the hsc70/U14 gene, which we had previously characterized, is the only source for the production of both U14 snoRNA and hsc70 mRNA species in hamster HA-1 cells.     

Factors affecting the binding of polycyclic aromatic hydrocarbons to human embryo cells, and transformable and non-transformable hamster embryo cells.

The effects of various factors, including population doubling number, percent of confluence, serum concentration and storage in liquid nitrogen on the binding of several polycyclic aromatic hydrocarbons to human and hamster embryo cells were studied. The binding of 7,12-dimethylbenz[a]-anthracene (DMBA) to hamster embryo cells DNA, RNA and protein was maximal after 22 h of treatment. In contrast, binding to human embryo cell macromolecules increased for at least 55 h. Treatment of hamster embryo cells at 100% confluence resulted in much less binding than treatment at 70% confluence, whereas with human embryo cells the binding increased, or remained constant, following treatment at the greater confluence. The transforming frequency of hamster embryo cells decreases with increasing population doubling number. Accordingly, we found that the binding of DMBA to hamster embryo DNA, RNA and protein decreased approximately 100-fold between population doubling numbers 8 and 20. In transformable cell cultures, DMBA was bound to hamster embryo cell DNA to a greater extent than to RNA or protein. The binding of DMBA to nucleic acids was much greater than binding by either dibenz[a,h]anthracene (DB[a,h]A) or dibenz-[a,c]anthracene (DB[a,c]A), both of which had low binding values at all population doubling numbers tested. Therefore, the best correlation of binding with carcinogenicity and transforming activity was observed with DMBA. Storage of hamster embryo cells in liquid nitrogen did not alter their binding characteristics. Binding of all three hydrocarbons to human embryo cell nucleic acids was low during all population doubling numbers studied, while binding to cellular protein increased until population doubling number 70 and then decreased sharply.     

Materials released into culture medium by normal and oncogenic virus-transformed mammalian cells.

Materials released into culture medium by transformed and untransformed baby hamster kidney cells labelled with glucosamine, sulfate, fucose or leucine were characterized. Some of the components could also be labelled by iodination of intact cells, indicating their surface origin. Analysis on gradient polyacrylamide sodium lauryl sulfate gels demonstrated that a group of high apparent molecular weight glucosamine-labelled components were more abundant in materials released from Rous sarcoma virus-transformed baby hamster kidney cells than from baby hamster kidney cells or polyoma virus-transformed baby hamster kidney cells. The relative rates of release of glucosamine-labelled components from transformed and untransformed cells were similar except that the transformed baby hamster kidney cells released some large molecular weight components slightly more rapidly than baby hamster kidney cells. Treatment of labelled medium materials with testicular hyaluronidase removed much glucosamine label from the materials but did not affect the amounts of other labels. After treatment with hyaluronidase, the patterns of labelled conditioned media from both transformed and untransformed baby hamster kidney cells were qualitatively and quantitatively very similar, suggesting that the differences seen in untreated labelled conditioned media were due to the presence of hyaluronidase-sensitive materials associated with medium materials rather than to actual differences in glycoproteins.     

Incorporation of isolated chromosomes and induction of hypoxanthine phosphoribosyltransferase in Chinese hamster cells.

Evidence is presented for the uptake of radioactive-labeled isolated Chinese hamster chromosomes following incubation with Chinese hamster cells. Metaphases were found which contained radioactive labeled chromosomes in a very low frequency, and in some of the labeled chromosomes only one chromatid was labeled. Incubation of hypoxanthine phosphoribosyltransferas (HPRT)-deficient Chinese hamster cells with chromosomes isolated from HPRT+ Chinese hamster or human cells resulted in the appearance of HPRT+ cells. Clones derived from these cells were isolated in HAT medium. Cells in mitosis during incubation with the chromosomes yielded thr-e times more HPRT+ clones than did cells in interphase. The intraspecies combination involving recipient cells and chromosomes from Chinese hamster origin yielded significantly higher numbers of HPRT+ clones than did the interspecies system using human chromsomes and Chinese hamster recipient cells (5 X 10(-5) and 6 X 10(-6) respectively). Electrophoresis of HPRT from Chinese hamster cells treated with human chromosomes revealed the pattern of the human enzyme.     

Differential reaction of condensed and diffuse chromatin to polyamines. I. Reaction of interphase nuclei chromatin to putrescine]

Condensation of the interphase nuclei chromatin under putrescine treatment was studied in cultured human fibroblasts 46, XX: 47, XXX: 49, XXXXY, and aneuploid cells of the Chinese hamster. The effect was tested separately for diffuse and condensed chromatin. Putrescine treatment did not affect the percentage of cell nuclei with X-chromatin bodies in the human cell strains while significantly increasing the percentage of nuclei with coarse chromatin network and chromocenters. In cultured Chinese hamster cells, putrescene did not change the percentage of nuclei with identified chromocenters and no significant condensation of diffuse chromatin was observed either.     

Cutting edge: cells that carry A null allele for toll-like receptor 2 are capable of responding to endotoxin.

Toll-like receptor (TLR) 2 and TLR4 have been implicated in the responses of cells to LPS (endotoxin). CD14-transfected Chinese hamster ovary (CHO)-K1 fibroblasts (CHO/CD14) are exquisitely sensitive to endotoxin. Sequence analysis of CHO-TLR2, compared with human and mouse TLR2, revealed a single base pair deletion. This frameshift mutation resulted in an alternative stop codon, encoding a protein devoid of transmembrane and intracellular domains. CHO-TLR2 cDNA failed to enable LPS signaling upon transient transfection into human epithelial kidney 293 cells. Site-directed mutagenesis of CHO-TLR2 enabled expression of a presumed full-length hamster TLR2 that conferred LPS responsiveness in human epithelial kidney 293 cells. Genomic TLR2 DNA from primary hamster macrophages also contained the frameshift mutation found in CHO fibroblasts. Nevertheless, hamster peritoneal macrophages were found to respond normally to LPS, as evidenced by the induction of cytokines. These results imply that expression of TLR2 is sufficient but not essential for mammalian responses to endotoxin.     

Identification and characterization of a third complementation group of emetine-resistant Chinese hamster cell mutants.

We have isolated emetine-resistant cell lines from Chinese hamster peritoneal fibroblasts and have shown that they represent a third distinct class or complementation group of emetine-resistant mutants, as determined by three different criteria. These mutants, like those belonging to the two other complementation groups we have previously defined, which were isolated from Chinese hamster lung and Chinese hamster ovary cells, have alterations that directly affect the protein biosynthetic machinery. So far, there is absolute cell line specificity with respect to the three complementation groups, in that all the emetine-resistant mutants we have isolated from Chinese hamster lung cells belong to one complementation group, all those we have isolated from Chinese hamster ovary cells belong to a second complementation group, and all those isolated from Chinese hamster peritoneal cells belong to a third complementation group. Thus, in cultured Chinese hamster cells, mutations in at least three different loci, designated emtA, emtB, and emtC, encoding for different components of the protein biosynthetic machinery, can give rise to the emetine-resistant phenotype.     

Low molecular weight RNAs with homologies to cellular DNA at sites of adenovirus DNA insertion in hamster or mouse cells.

The adenovirus type 2 (Ad2)-transformed hamster cell line HE5 contains one or very few integrated copies of Ad2 DNA. At the site of insertion of Ad2 DNA, the cellular DNA sequence has been completely preserved and has homologies to small unpolyadenylated, cytoplasmic RNAs of 300 nucleotides in length and to minority populations of smaller RNAs present in HE5 cells and in normal hamster cells. The 300-nucleotide RNA is present on average in approximately 20 copies per cell. This RNA, and shorter RNAs, reveal homologies to the hamster DNA sequence of approximately 400 nucleotides to the right of the site of insertion of Ad2 DNA, which is present in one or very few copies per genome. The nucleotide sequence of the DNA segment homologous to this RNA does not contain open reading frames in excess of a sequence encoding 18 amino acids. Thus, it is unlikely that the small RNAs are actually translated and their function is unknown. The nucleotide sequence does not exhibit similarities to known low mol. wt. RNAs of eukaryotic origin. The low mol. wt. cellular RNA has been found in HE5 cells, in other hamster cell lines and organs, and also in mouse cells. There are differences with respect to size and abundance in the RNAs smaller than 300 nucleotides between HE5 cells and LSH hamster embryo cells. The adenovirus type 12 (Ad12)-induced mouse tumor CBA-12-1-T carries greater than 30 copies of integrated Ad12 DNA. The cellular DNA sequence at the site of Ad12 DNA insertion exhibits homologies to small RNAs (approximately 300 nucleotides long) from mouse cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Viral and cellular factors governing hamster cell infection by murine and gibbon ape leukemia viruses.

Hamster cells are resistant to infection by most retroviruses, including Moloney murine leukemia virus (MoMLV) and gibbon ape leukemia viruses (GaLVs). We have constructed MoMLV-GaLV hybrid virions to identify viral and cellular determinants responsible for the inability of GaLV and MoMLV to infect hamster cells. The substitution of MoMLV core components for GaLV core components circumvents the resistance of hamster cells to infection by GaLV, demonstrating that hamster cells have receptors for GaLV but are not efficiently infected by this primate retrovirus because of a postpenetration block. In contrast, hamster cells are apparently resistant to MoMLV infection because although they bear a receptor for MoMLV, the receptor is nonfunctional. Treatment of CHO K1 or BHK 21 hamster cells with the glycosylation inhibitor tunicamycin allows the cells to be infected by MoMLV. The construction of MoMLV-GaLV hybrid virions that can efficiently infect resistant cells has allowed the identification of viral and cellular factors responsible for restricting infection of hamster cells by MoMLV and GaLV.     

Amplification of portions of the genome in the somatic cells of mammals resistant to colchicine. IV. Genetic transformation using amplified genes from Djungarian hamster cells highly resistant to colchicine

DNA-mediated transfer of colchicine-resistance from Djungarian hamster DM5/7 cell line, 750-fold resistant to the drug, was studied. The resistance to colchicine of DM5/7 cells is due to amplification of the genes, possibly coding for the polypeptide p22. Both high-molecular weight DNA (presumably, chromosomal DNA) and low-molecular weight DNA (presumably, extrachromosomal DNA) effectively transferred the colchicine-resistance to Djungarian hamster and mouse cells. DNA of sensitive to colchicine but resistant to ouabain mouse cells CAK-143OuaR was not capable to transfer colchicine-resistance, but effectively transferred ouabain-resistance connected with a mutation in Na+/K+-dependent ATP-ase locus. The differences in genetic transformation with amplified p22 genes and mutant Na+/K+-dependent ATP-ase genes were revealed. All cells of 3 colchicine-resistant transformants of DM-15 cells and all 10 spontaneously derived resistant clones contain the additional chromosome 4. The role of trisomy 4 in the development of colchicine-resistance in DM-15 cells is discussed.     

The induction by X-rays of chromosome aberrations in germ cells of male guinea-pigs, golden hamsters and rabbits. I. Dose response in post-meiotic stages.

The induction by X-rays of translocations in post-meiotic germ cells of the guinea-pig, golden hamster and rabbit was studied by cytological analysis of male offspring of the irradiated animals. As reported previously for the mouse, the pattern of sensitivity to dominant lethal induction, as indicated by litter-size, was similar to that for translocation induction in both the guinea-pig and golden hamster. In both speciesspermatids were more sensitive than spermatozoa, and in the golden hamster spermatocytes gave a lower yield than spermatids. The translocation frequency among post-meiotic germ cells treated with 600 rad was higher in the rabbit than the guinea-pig, and both were above that for the golden hamster. However, for spermatozoa, species differences with respect to the recovered translocation yield appeared to depend on dose. In the hamster, the translocation frequency after 600 rad, as measured in the female offspring, was similar to that obtained in the male offspring. A small amount of data on the induction of sex-chromosome aneuploidy by 200 rad in golden hamsters suggested that the hamster might be as sensitive as the mouse.     

Transfer of human genes conferring resistance to methylating mutagens, but not to UV irradiation and cross-linking agents, into Chinese hamster ovary cells.

Chinese hamster ovary cells were transfected by human DNA ligated to the bacterial gpt (xanthine-guanine-phosphoribosyltransferase) gene which was used either in its native form or after partial inactivation with methylnitrosourea. The gpt+ transfectants were screened for resistance to high doses of N-methyl-N'-nitro-N-nitrosoguanidine. Using this approach, we showed that Chinese hamster ovary cells can acquire N-methyl-N'-nitro-N-nitrosoguanidine resistance upon transfection with DNA from diploid human fibroblasts, that this resistance is transferable by secondary transfection and is specific for methylating mutagens, and that it is not caused by increased removal of O6-methylguanine, 3-methyladenine, and 7-methylguanine from DNA.     

Bodians Protargol Method Applied to Other than Neurological Preparations

Some applications of the Bodian technic for other than neurological purposes are described. After fixation for 48 hours, in formalin, 5 ml., acetic acid, glacial, 5 ml., and 80% ethyl alcohol, 90 ml., the routine procedures are recommended, with the exception that the exposure to protargol for 24 hours and subsequent reduction should be repeated once. Gold toning may be o-mitted. With this method the argentaffin (chromaffin) cells of the digestive tract (rat, hamster), the alpha cells of the pancreatic islands (hamster) and the medullary cells of the suprarenal gland (hamster) are selectively impregnated. In the mammalian pituitary gland (rat, hamster) certain of the granulated chromophile cells are impregnated. In the rat only the basophiles appear to react with the silver.     

High resistance of cultured Mongolian gerbil cells to X-ray-induced killing and chromosome aberrations.

The Mongolian gerbil (Meriones unguiculatus) is known to be one of the most radioresistant animals. We have examined the X-ray sensitivity of normal diploid fibroblasts from Mongolian gerbil embryos compared with those of cultured embryo cells obtained from various laboratory animals and a normal human. There was a wide difference in X-ray sensitivity for cell killing among different mammalian species. The D0 values for Mongolian gerbil cells ranged from 2.08 to 2.28 Gy, values which are twice as high as those for human cells. The mean D0 value for human cells was 1.06 Gy. Mouse, rat, Chinese hamster, and Syrian/golden hamster cells showed similar D0 values ranging from 1.30 to 1.56 Gy. When cells were irradiated with X rays, ten times more chromosome aberrations were detected in human cells than in Mongolian gerbil cells. The frequencies of chromosome aberrations in other rodent cells were between the values for cells from humans and those from gerbils. These data indicate that the Mongolian gerbil cells are resistant to X-ray-induced cell killing and chromosome aberrations, and that the radiation sensitivity of mammalian cells in primary culture may be reflected by their radioresistance in vivo.     

Metabolism of 25-hydroxycholesterol in mammalian cell cultures. Side-chain scission to pregnenolone in mouse L929 fibroblasts

Three mammalian cell lines were examined for their ability to metabolize the regulatory oxysterol, 25-hydroxycholesterol, and derepress 3-hydroxy-3-methylglutaryl CoA reductase. In mouse L cell fibroblasts reductase activity was restored with the concomitant metabolism of 25-hydroxycholesterol via side-chain hydroxylation and scission of the C20-C22 bond. Chinese hamster lung cells did not appear to derepress the reductase and these cells and Chinese hamster ovary cells did not metabolize 25-hydroxycholesterol to a significant extent. Only 5-10% of the oxysterol became esterified with a fatty acid in any of the cell lines when grown in the described culture conditions.     

Regulation of melanin synthesis in mammalian cells, as studied by somatic hybridization. I. Evidence for negative control

Somatic hybrids between pigmented Syrian hamster cells and unpigmented mouse cells were isolated and propagated in vitro. These hybrids are unpigmented and lack dopa oxidase (and tyrosinase) activity, which is correlated with the pigmentation of the Syrian hamster cells. In contrast, the presence of three other enzymes (LDH, MDH, and thymidine kinase) also specified by the hamster genome but unrelated to pigment synthesis is observed in the hybrid cells. This suggests that the repression of dopa oxidase in these cells is a specific effect on the enzyme associated with the differentiated state of pigment cells. It is concluded that the genetic control of differentiation in this case involves a diffusible regulator substance which functions negatively.