bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell cycle parameters of Chinese hamster ovary cells during exponential, polyamine-limited growth.
Authors: Harada J J , Morris D R
Published: September 2015

We have previously shown that Chinese hamster ovary cells made polyamine deficient by treatment with alpha-methylornithine, an inhibitor of ornithine decarboxylase, grow exponentially in culture at low densities at one-half the rate observed in untreated (control) cultures. In this study, the cell cycle of polyamine-limited cells was examined by using thymidine autoradiography, mitotic index analysis, and fraction labeled mitoses analysis. We found that the longer doubling time of inhibitor-treated cultures was a consequence of increases in the lengths of the G1 and S phases. The expansion of the S phase was proportional to the increase in doubling time (twofold), whereas the G1 phase was lengthened by slightly more than a factor of 2. The lengths of the G2 and M phases were essentially unchanged. Putrescine stimulated the growth of inhibitor-treated cultures and restored the cell cycle parameters to those of untreated cells.
Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters.
Authors: Lee D C , Roeder R G
Published: September 2015

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.
Sensitivity of a mutator gene in Chinese hamster ovary cell to deoxynucleoside triphosphate pool alterations.
Authors: Meuth M
Published: September 2015

The Thy- mutants of Chinese hamster ovary cells have a 5- to 10-fold elevated pool of deoxycytidine 5'-triphosphate (dCTP) and are auxotrophic for thymidine as an apparent consequence of a single mutation. thy is also a mutator gene, elevating the spontaneous rate of mutation 5- to 200-fold for at least two genetic markers. Previous experiments suggested that this mutator activity was caused by the elevated pool of dCTP in Thy- cells. To test this, the dCTP and deoxythymidine 5'-triphosphate (dTTP) pools were manipulated by altering the external concentration of thymidine in the growth medium. The rate of mutation at one genetic locus, ouabain resistance, was directly related to cellular dCTP content. At the highest level of dCTP the rate in one Thy- strain was approximately 200 times that of wild-type cells. However, the relationship between dCTP content and the rate of mutation at the ouabain locus was different for two mutator strains and wild-type cells. The rate of mutation at a second locus, thioguanine resistance, was increased approximately 10-fold over wild type regardless of the dCTP-dTTP pools. These experiments suggest that the mutator activity of thy is clearly related to dCTP content, but the dCTP level alone does not appear to be the cause of the mutator.
Striking similarities are exhibited by two small Epstein-Barr virus-encoded ribonucleic acids and the adenovirus-associated ribonucleic acids VAI and VAII.
Authors: Rosa M D , Gottlieb E , Lerner M R , Steitz J A
Published: September 2015

The nucleotide sequence of the region of the Epstein-Barr virus genome that specifies two small ribonucleic acids (RNAs), EBER 1 and EBER 2, has been determined. Both of these RNAs are encoded by the right-hand 1,000 base pairs of the EcoRI J fragment of EBV deoxyribonucleic acid. EBER 1 is 166 (167) nucleotides long and EBER 2 is 172 +/- 1 nucleotides long; the heterogeneity resides at the 3' termini. The EBER genes are separated by 161 base pairs and are transcribed from the same deoxyribonucleic acid strand. In vitro, both EBER genes can be transcribed by RNA polymerase III; sequences homologous to previously identified RNA polymerase III intragenic transcription control regions are present. Striking similarities are therefore apparent both between the EBERs and the two adenovirus-associated RNAs, VAI and VAII, and between the regions of the two viral genomes that specify these small RNAs. We have shown that VAII RNA as well as VAI RNA and the EBERs exist in ribonucleoprotein complexes which are precipitable by anti-La antibodies associated with systemic lupus erythematosus. Finally, we have demonstrated that the binding of protein(s) from uninfected cells confers antigenicity on each of the four virus-encoded small RNAs.
Control of adenovirus early gene expression: posttranscriptional control mediated by both viral and cellular gene products.
Authors: Katze M G , Persson H , Philipson L
Published: September 2015

An adenovirus type 5 host range mutant (hr-1) located in region E1A and phenotypically defective in expressing viral messenger ribonucleic acid (RNA) from other early regions (Berk et al., Cell 17:935-944, 1979) was analyzed for accumulation of viral RNA in the presence of protein synthesis inhibitors. Nuclear RNA was transcribed from all early regions at the same rate, regardless of whether the drug was present or absent. As expected, low or undetectable levels of RNA were found in the cytoplasm of hr-1-infected cells compared with the wild-type adenovirus type 5 in the absence of drug. When anisomycin was added 30 min before hr-1 infection, cytoplasmic RNA was abundant from early regions E3 and E4 when assayed by filter hybridization. In accordance, early regions E3 and E4 viral messenger RNA species were detected by the S1 endonuclease mapping technique only in hr-1-infected cells that were treated with the drug. Similar results were obtained by in vitro translation studies. Together, these results suggest that this adenovirus type 5 mutant lacks a viral gene product necessary for accumulation of viral messenger RNA, but not for transcription. It is proposed that a cellular gene product serves as a negative regulator of viral messenger RNA accumulation at the posttranscriptional level.
Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.
Authors: Chan R K , Otte C A
Published: September 2015

Eight independently isolated mutants which are supersensitive (Sst-) to the G1 arrest induced by the tridecapeptide pheromone alpha factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by alpha factor. These mutants carried lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to alpha factor, but MAT alpha sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on both MATa and MAT alpha cells. Even in the absence of added alpha pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology ("shmoo" shape) that normally develops only after MATa cells are exposed to alpha factor. This "self-shmooing" phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT alpha diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT alpha sst2-1/sst2-1) were still insensitive to alpha factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked to nor centromere distal to MAT on the right arm of chromosome III.
Selective linkage disruption in human-Chinese hamster cell hybrids: deletion mapping of the leuS, hexB, emtB, and chr genes on human chromosome 5.
Authors: Dana S , Wasmuth J J
Published: September 2015

Chinese hamster-human interspecific hybrid cells, which contain human chromosome 5 and express four genes linked on that chromosome, were subjected to selective conditions requiring them to retain one of the four linked genes, leuS (encoding leucyl-tRNA synthetase), but lose another, either emtB (encoding ribosomal protein S14) or chr. Cytogenetic and biochemical analyses of spontaneous segregants isolated by using these unique selective pressures have enabled us to determine the order and regional location of the leuS, hexB, emtB, and chr genes on human chromosome 5. These segregants arise primarily by terminal deletions of various portions of the long arm of chromosome 5. Our results indicate that the order of at least three of these genes is the same on human chromosome 5 and Chinese hamster chromosome 2. Thus, there appears to be extensive homology between Chinese hamster chromosome 2 and human chromosome 5, which represents an extreme example of the conservation of gene organization between very divergent mammalian species. In addition, these hybrids and selective conditions provide a very simple and quantitative means to assess the potency of various agents suspected of inducing gross chromosomal damage.
Actin from Saccharomyces cerevisiae.
Authors: Greer C , Schekman R
Published: September 2015

Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth.
Small RNA molecules related to the Alu family of repetitive DNA sequences.
Authors: Leinwand L A , Wydro R M , Nadal-Ginard B
Published: September 2015

A rodent 4.5S RNA molecule with extensive homology to the Alu family of interspersed repetitive DNA sequences has been found physically associated with polyadenylated nuclear and cytoplasmic RNAs (W. Jelinek and L. Leinwand, Cell 15:205-214, 1978; S. Haynes et al., Mol. Cell. Biol. 1:573-583, 1981). In this report, we describe a 4.5S RNA molecule in rat cells whose RNase fingerprints are identical to those of the equivalent mouse molecule. We show that the rat 4.5S RNA is part of a small family of RNA molecules, all sharing sequence homology to the Alu family of DNA sequences. These RNAs are synthesized by RNA polymerase III and are developmentally regulated and short-lived in the cytoplasm. Of this family of small RNAs, only the 4.5S RNA is found associated with polyadenylated RNA.
Mouse cells contain two distinct ras gene mRNA species that can be translated into a p21 onc protein.
Authors: Ellis R W , DeFeo D , Furth M E , Scolnick E M
Published: September 2015

The Kirsten (Ki) and Harvey (Ha) strains of murine sarcoma virus encode a 21,000-dalton protein (p21 ras) which is the product of the transforming gene of these viruses. Normal cells express low levels of p21 ras encoded by cellular genes (Ki-ras and Ha-ras) homologous to the Ki and Ha murine sarcoma virus transformation genes. A bone marrow-derived mouse cell line, 416B, has been shown to express unusually high levels of p21 ras. In this manuscript, we investigated the molecular biology of p21 ras gene expression in 416B and other normal mouse cells. We identified four distinct polyadenylated and polysome-associated RNAs, two related to Ki-ras and two to Ha-ras. The levels in 416B cells of the two Ki-ras RNAs, sized 5.2 and 2.0 kilobases, were both elevated approximately 25-fold over levels found in normal mouse cells; there was no corresponding change in 416B cells in the levels of the two Ha-ras RNAs. We partially purified the two Ki-ras mRNAs and separated them by velocity sedimentation in sucrose density gradients. Both the 5.2- and 2.0-kilobase mRNAs could be translated in vitro into p21 ras. These results show that a cellular onc protein can be translated from two distinct cellular mRNA species.
Identification of Proteins Whose Synthesis Is Modulated During the Cell Cycle ofSaccharomyces cerevisiae
Authors: Lörincz Attila T. , Miller Mark J. , Xuong Nguyen-Huu , Geiduschek E. Peter
Published: September 2015

We examined the synthesis and turnover of individual proteins in theSaccharomyces cerevisiaecell cycle. Proteins were pulse-labeled with radioactive isotope (35S or14C) in cells at discrete cycle stages and then resolved on two-dimensional gels and analyzed by a semiautomatic procedure for quantitating gel electropherogram-autoradiographs. The cells were obtained by one of three methods: (i) isolation of synchronous subpopulations of growing cells by zonal centrifugation; (ii) fractionation of pulse-labeled steady-state cultures according to cell age; and (iii) synchronization of cells with the mating pheromone, α-factor. In confirmation of previous studies, we found that the histones H4, H2A, and H2B were synthesized almost exclusively in the late G1 and early S phases. In addition, we identified eight proteins whose rates of synthesis were modulated in the cell cycle, and nine proteins (of which five, which may well be related, were unstable, with half-lives of 10 to 15 min) that might be regulated in the cell cycle by periodic synthesis, modification, or degradation. Based on the time of maximal labeling in the cell cycle and on experiments with α-factor and hydroxyurea, we assigned the cell cycle proteins to two classes: proteins in class I were labeled principally in early G1 phase and at a late stage of the cycle, whereas those in class II were primarily synthesized at times ranging from late G1 to mid S phase. At least one major control point for the cell cycle proteins occurred between “start” and early S phase. A set of stress-responsive proteins was also identified and analyzed. The rates of synthesis of these proteins were affected by certain perturbations that resulted during selection of synchronous cell populations and by heat shock.
Heterogeneous nuclear RNA promotes synthesis of (2',5')oligoadenylate and is cleaved by the (2',5')oligoadenylate-activated endoribonuclease.
Authors: Nilsen T W , Maroney P A , Robertson H D , Baglioni C
Published: September 2015

Heterogeneous nuclear RNA contains double-stranded regions that are not found in mRNA and that may serve as recognition elements for processing enzymes. The double-stranded regions of heterogeneous nuclear RNA prepared from HeLa cells promoted the synthesis of (2',5')oligoadenylate [(2',5')oligo(A) or (2'5')An] when incubated with (2',5')An polymerase. This enzyme is present in elevated levels in interferon-treated cells, and labeled heterogeneous nuclear RNA incubated with extracts of these cells is preferentially cleaved, since mRNA included in the same incubations is not appreciably degraded. The cleavage of heterogenous nuclear RNA is caused by the synthesis of (2'5')An and by a "localized" activation of the (2',5')An-dependent endonuclease, since it was enhanced by ATP, the substrate of the (2',5')An polymerase, and inhibited by 2'-dATP and ethidium bromide. Both of these compounds suppress the synthesis of (2',5')An, the first by competitive inhibition and the latter by intercalating into double-stranded RNA. The possible role of double-stranded regions and of the (2',5')An polymerase-endonuclease system in the processing of heterogeneous nuclear RNA is discussed.
Prolactin-deficient variants of GH3 rat pituitary tumor cells: linked expression of prolactin and another hormonally responsive protein in GH3 cells.
Authors: Ivarie R D , Morris J A , Martial J A
Published: September 2015

GH3 cells normally synthesize and secrete two pituitary polypeptide hormones, prolactin and growth hormone. From an ethyl methane sulfonate-mutagenized population, prolactin low-producing variants have been isolated at a frequency near 20%. Intracellular prolactin synthesis in the variants was reduced 40- to 100-fold compared to wild-type cells while growth hormone synthesis varied less than 2-fold. This decrease was paralleled by a decrease in intracellular preprolactin mRNA. Although reduced, prolactin synthesis was still repressible by glucocorticoids. There was a coordinate loss of expression of p21, a thyroid and glucocorticoid hormone-regulated protein, in GH3 cells, whereas the synthesis and regulation of other hormonally responsive proteins were unimpaired in the variants. Since p21 expression was coordinately regained in a high-producing prolactin revertant cell, expression of the two proteins is tightly coupled in GH3 cells. The stability of the low-producing phenotype differed among variants. One (B2) gave rise to revertants at about 20% frequency even after two rounds of subcloning, whereas another (B3) was more stable in that only 1 weak revertant was found in 47 subclones. The reversion frequency of B3 cells was also measured at less than 0.5%. Unmutagenized GH3 cells were phenotypically stable in that no prolactin-deficient variant was found among 57 subclones. Since variants were ony found after ethyl methane sulfonate mutagenesis, the DNA alkylating agent appears to have promoted an epigenetic change in pituitary gene expression.
Nuclear ligation of RNA 5'-OH kinase products in tRNA.
Authors: Winicov I , Button J D
Published: September 2015

Mouse L-cell nuclei incorporate gamma-32P from ATP in vitro predominantly in 5'-monophosphoryl termini and internal phosphodiester bonds with a nonrandom nearest-neighbor distribution. In the presence of 1 microgram of alpha-amanitin per ml the gamma-32P showed a time-dependent appearance in RNA bands which migrated with mature tRNA species but not with pre-tRNA and 5S RNA. The gamma-32P was found in internal phosphodiester bonds as shown by alkaline phosphatase resistance and was identified in 3'-monophosphates after RNase T2, T1, and A digestion. The specificity of this incorporation was indicated by a limited number of labeled oligonucleotides from a T1 digest and identification of 70 to 80% of the 32P label as Cp on complete digestion of the eluted tRNA band. We also observed transiently [gamma-32P]ATP-labeled RNA bands (in 5'-monophosphate positions) that were 32 to 45 nucleotides long. The results presented suggest splicing of several mouse L-cell tRNA species in isolated nuclei which involve the RNA 5'-OH kinase products as intermediates.
Properties of dispersed, highly repeated DNA within and near the hamster CAD gene.
Authors: Padgett R A , Wahl G M , Stark G R
Published: September 2015

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.
Cells of pea (Pisum sativum) that differentiate from G2 phase have extrachromosomal DNA.
Authors: Van't Hof J , Bjerknes C A
Published: September 2015

Velocity sedimentation in an alkaline sucrose gradient of newly replicated chromosomal DNA revealed the presence of extrachromosomal DNA that was not replicated by differentiating cells in the elongation zone. The extrachromosomal DNA had a number average molecular weight of 12 X 10(6) to 15 X 10(6) and a weight average molecular weight of 25 X 10(6), corresponding to about 26 X 10(6) and 50 X 10(6) daltons, respectively, of double-stranded DNA. The molecules were stable, lasting at least 72 h after being formed. Concurrent measurements by velocity sedimentation, autoradiography, and cytophotometry of isolated nuclei indicated that the extrachromosomal molecules were associated with root-tip cells that stopped dividing and differentiated from G2 phase but not with those that stopped dividing and differentiated from G1 phase.
Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.
Authors: Reed S I , Ferguson J , Groppe J C
Published: September 2015

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.
Influence of the nuclear gene tmp3 on the loss of mitochondrial genes in Saccharomyces cerevisiae.
Authors: Zelikson R , Luzzati M
Published: September 2015

The Saccharomyces cerevisiae tmp3 mutant is deficient in the mitochondrial enzyme complex that participates in the formation of one-carbon-group-tetrahydrofolate coenzymes, serine transhydroxymethylase, dihydrofolate reductase, and thymidylate synthetase, thus leading to multiple nutritional requirements of dTMP, adenine, histidine, and methionine. The tmp3 mutant quickly loses its mitochondrial genome even when grown on fully supplemented medium or on a high concentration of 5-formyl tetrahydrofolate, which replaces all the four requirements. A study of the loss of the mitochondrial genome by following several mitochondrial genetic markers showed that there was a preferential specific loss of a large region of the mitochondrial genome, covering mit ts983, Er, Cr, and mit ts982 up to OrI, and retention of the region of Pr and mit tscs1297. A kinetic study showed that there was a preferentially rapid loss of the region covering the mit+ alleles ts983 to tscs902 at the rate of 10% per generation.
Newly formed mRNA lacking polyadenylic acid enters the cytoplasm and the polyribosomes but has a shorter half-life in the absence of polyadenylic acid.
Authors: Zeevi M , Nevins J R , Darnell J E
Published: September 2015

Labeled adenovirus type 2 nuclear RNA molecules from cells treated with 3'-deoxyadenosine (3'dA) were earlier reported to lack polyadenylic acid [poly(A)], but to be correctly spliced in the nucleus (M. Zeevi et al., Cell 26:39-46, 1981). We have now found that the shortened mRNA molecules, lacking poly(A), can also be found in the cytoplasm of 3'dA-treated cells in association with the polyribosomes. In addition, the accumulation of labeled, nuclear adenovirus-specific RNA complementary to early regions 1a, 1b, and 2 of the adenovirus genome was approximately equal in 3'dA-treated and control cells. At the initial appearance of newly labeled adenovirus type 2 RNA (10 min) in the cytoplasm, there was one-half as much labeled RNA in 3'dA-treated cells as in the control. However, control cells accumulated additional mRNA in the cytoplasm very rapidly in the first 40 min of labeling, whereas the 3'dA-treated cells did not. Therefore, it appears that the correctly spliced, poly(A)- mRNA molecules that are labeled in the presence of 3'dA can be transported from the nucleus with nearly the same frequency and the same exit time as in control cells and can be translated in the cytoplasm but have a much shorter half-life than the poly(A)+ mRNA molecules from control infected cells. From these results it is suggested that the role of poly(A) may be entirely to increase the longevity of cytoplasmic mRNA.
Construction of microcell hybrid clones containing specific mouse chromosomes: application to autosomes 8 and 17.
Authors: Fournier R E , Frelinger J A
Published: September 2015

Fibroblast cultures prepared from mice homozygous for a Robertsonian translocation (centric fusion) between autosomes 8 and 17 [Rb(8.17)] were used as donors in microcell-mediated chromosome transfer experiments. By using hamster recipient cells deficient in adenine phosphoribosyltransferase (APRT-) and selecting for expression of murine APRT (a chromosome 8 marker), microcell hybrids were isolated which retained only the mouse Rb(8.17) translocation in addition to the hamster chromosome complement. The translocation was stable in cells maintained under APRT+ selective pressure, and mouse marker traits encoded by genes on both chromosomes 8 and 17 segregated concordantly. A second family of hybrid clones was constructed by fusing microcells derived from wild-type mouse fibroblasts with APRT- hamster cells. Four of six clones analyzed retained only mouse chromosome 8. These studies demonstrated that microcell hybrids containing specific Robertsonian translocations as the only donor-derived genetic material can be obtained. Furthermore, a number of Robertsonian translocations between chromosomes which carry selectable markers (chromosomes 3, 8, and 11) and other autosomes have been described. By using fibroblast cultures prepared from mice containing these translocations as donors in microcell fusions, 18 of the 20 mouse chromosomes could be selectively fixed in different hybrid clones. Thus, a collection of 20 hybrid clones, each containing a single, specific mouse chromosome, can be constructed by using the strategy described in this report. The potential utility of such a monochromosomal hybrid panel is discussed.
A suppressor of temperature-sensitive rna mutations that affect mRNA metabolism in Saccharomyces cerevisiae.
Authors: Pearson N J , Thorburn P C , Haber J E
Published: September 2015

We have isolated a dominant suppressor of rna mutation (SRN1) that relieves the temperature-sensitive inhibition of mRNA synthesis of ribosomal protein genes in the yeast Saccharomyces cerevisiae. The suppressor was selected for its ability to alleviate simultaneously the temperature-sensitive growth phenotypes of rna2 and rna6. Several independently isolated suppressors appeared to be recessive lethal mutations. One suppressor, SRN1, was recovered as viable in haploid strains. SRN1 can suppress rna2, rna3, rna4, rna5, rna6, and rna8 singly or in pairs, although some combinations of rna mutations are less well suppressed than others. The suppressor allows strains with rna mutations to grow at 34 degrees C but is unable to suppress at 37 degrees C; however, SRN1 does not, by itself, prevent growth at 37 degrees C. In addition, SRN1 suppresses the rna1 mutation which affects general mRNA levels and also leads to the accumulation of precursor tRNA for those tRNAs that have intervening sequences. SRN1 can suppress the rna1 mutation as well as the rna1 rna2 double mutation at 34 degrees C. The suppressor does not affect the temperature-sensitive growth of two unrelated temperature-sensitive mutations, cdc4 and cdc7.
Comparison of phenotypic expression with genotypic transformation by using cloned, selectable markers.
Authors: Linsley P S , Siminovitch L
Published: September 2015

The frequency of phenotypic expression of the herpes simplex virus type 1 tk and Escherichia coli gpt genes was compared with the frequency of genotypic transformation after calcium phosphate-mediated DNA transfection of a number of tk- and hprt- cell lines. In three of the five lines tested, the frequency of phenotypic expression was at most 10-fold higher than that of genotypic transformation as indicated by frequency of HAT resistance. The remaining two lines showed phenotypic responses which were 50- to 100-fold greater than the genotypic responses. The data indicate that the efficiency of DNA-mediated transformation with some cell lines can be limited by events after the uptake and expression of transfected DNA.
Topographical distribution of 5-methylcytosine in animal and plant DNA.
Authors: Naveh-Many T , Cedar H
Published: September 2015

The topographical distribution of 5-methylcytosine on animal and plant cell DNA has been examined with methyl-sensitive restriction enzymes and gel electrophoresis analysis. These DNAs digested with the enzyme HpaII have a partially bimodal size distribution, indicating the existence of clusters of methylated and unmethylated CCGG sites in the DNA. By analyzing the methylation state of all CG moieties in restricted DNA fractions, it was possible to show that these genomes are, in general, arranged as clusters of relatively highly methylated and undermethylated regions. Plant DNA also contains 5-methylcytosine in the prototype sequence C-X-G. Restriction of this DNA with EcoRII revealed that these methyl groups are also distributed in clusters, suggesting that this is a general phenomenon. The undermethylated areas may correspond to the active fraction of the genome.
BK virus-transformed inbred hamster brain cells: status of viral DNA in subclones.
Authors: Pater M M , Pater A , di Mayorca G , Beth E , Giraldo G
Published: September 2015

We have recently reported that viral DNA sequences in inbred LSH hamster brain cells transformed by the GS variant of BK virus (LSH-BR-BK) are present predominantly in a free form (Beth et al., J. Virol. 40:276-284, 1981). In this report, we confirm that the presence of viral DNA sequences in these cells is not due to virus production, since viral capsid proteins were not detected by immunoprecipitation. Furthermore, we examined the status of viral DNA in 15 subclones of this cell line and detected free and integrated viral DNA sequences in only 5 of the subclones. The other 10 subclones contained exclusively integrated viral DNA sequences, as shown by the blot hybridization of high-molecular-weight cell DNA which was uncleaved or digested with HincII, for which there are no sites in viral DNA. The arrangement of viral DNA in these clones was further analyzed by cleavage of cellular DNA with HpaII and HindIII. Mitomycin (0.03 microgram/ml) treatment of subclones containing only integrated sequences resulted in the appearance of free viral DNA sequences in some of these cells. This result supports the postulation that free viral DNA in LSH-BR-BK cells is made up of excision products of observed tandemly repeated integrated sequences. In addition to the large T- and small t-antigens, LSH-BR-BK and all of its 15 subclones contained two antigen species which were larger than large T and one species which was smaller than small t. The number of tumor antigens in the LSH- BR-BK cell line and its subclones with a large copy number in a free form was not more than in the subclones with low copy number and integrated DNA. This suggests that free viral DNA is not a template for tumor antigen production in transformed cells.
Association of the transforming proteins of Rous, Fujinami, and Y73 avian sarcoma viruses with the same two cellular proteins.
Authors: Lipsich L A , Cutt J R , Brugge J S
Published: September 2015

Two forms of the transforming proteins of Fujinami (pp140fps) and Yamaguchi 73 (pp94yes) sarcoma viruses were detected in lysates of chicken cells transformed by these viruses; the majority of pp140fps and pp94yes molecules were present as monomers; however, a small percentage of these proteins was associated in a complex with two cellular proteins of Mr 90,000 and 50,000. These cellular proteins were shown to be identical to those previously found to be complexed with the transforming protein of Rous sarcoma virus, pp60src. These results suggest a common role for the interaction of pp90 and pp50 with viral transforming proteins encoding tyrosyl-protein kinases.
Small nuclear ribonucleoproteins of Drosophila: identification of U1 RNA-associated proteins and their behavior during heat shock.
Authors: Wieben E D , Pederson T
Published: September 2015

In Drosophila, two nuclear proteins of approximately 26,000 and 14,000 molecular weight are recognized by a human autoimmune antibody for mammalian ribonucleoprotein (RNP) particles that contain U1 small nuclear RNA. The antibody-selected Drosophila RNP contains, in addition to these two proteins, a single RNA species that has been identified as U1 by hybridization with a cloned Drosophila U1 DNA probe. Small nuclear RNP isolated from human cells under the same conditions as used for Drosophila and selected by the anti-U1 RNP-specific antibody contains eight proteins, two of which are similar in molecular weight to the two Drosophila U1 RNP proteins. Thus, even though the nucleotide sequences of Drosophila and human U1 RNA are about 72% homologous, and the corresponding RNPs are both recognized by the same human autoantibody, Drosophila U1 RNP appears to have a simpler protein complement than its mammalian counterpart. The two Drosophila U1 RNA-associated proteins are synthesized at normal or slightly increased rates during the heat shock response and are incorporated into antibody-recognizable RNP complexes. This raises the possibility that U1 RNP is an indispensable nuclear element for cell survival during heat shock.
Saccharomyces carlsbergensis fdp mutant and futile cycling of fructose 6-phosphate.
Authors: Bañuelos M , Fraenkel D G
Published: September 2015

In Saccharomyces, the addition of glucose to cells grown in media lacking sugars causes irreversible inactivation of fructose bisphosphatase. One function of this process might be to prevent a futile cycle of formation and hydrolysis of fructose 1,6-bisphosphate. We tested such cycling by assessing the labeling of the 1-position of glucose in polysaccharides from [6-14C]glucose (J.P. Chambost and D. G. Fraenkel, J. Biol. Chem. 225:2867-2869, 1980) by using mutants impaired in glucose growth and known not to inactivate the phosphatase normally (i.e., the fdp mutant of Saccharomyces carlsbergensis [van de Poll et al., J. Bacteriol. 117:965-970, 1974] and the similar cif mutant of Saccharomyces cerevisiae [Navon et al., Biochemistry 18:4487-4499, 1979] ), as well as in the wild-type strain tested in the 1-h period before inactivation is complete. There was marginal, if any, cycling in any situation, and we conclude that the phosphatase activity is controlled by means other than inactivation or that the extent of cycling is too low to be significant, or both. For the fdp mutant data are also presented on growth, rate of glucose metabolism, metabolite accumulations, enzyme levels, and glucose transport, but the primary lesion is unknown.
Isolation and characterization of human DNA fragments with nucleotide sequence homologies with the simian virus 40 regulatory region.
Authors: Conrad S E , Botchan M R
Published: September 2015

A recombinant library of human DNA sequences was screened with a segment of simian virus 40 (SV40) DNA that spans the viral origin of replication. One hundred and fifty phage were isolated that hybridized to this probe. Restriction enzyme and hybridization analyses indicated that these sequences were partially homologous to one another. Direct DNA sequencing of two such SV40-hybridizing segments indicated that this was not a highly conserved family of sequences, but rather a set of DNA fragments that contained repetitive regions of high guanine plus cytosine content. These sequences were not members of the previously described Alu family of repeats and hybridized to SV40 DNA more strongly than do Alu family members. Computer analyses showed that the human DNA segments contained multiple homologies with sequences throughout the SV40 origin region, although sequences on the late side of the viral origin contained the strongest cross-hybridizing sequences. Because of the number and complexity of the matches detected, we could not determine unambiguously which of the many possible heteroduplexes between these DNAs was thermodynamically most favored. No hybridization of these human DNA sequences to any other segment of the SV40 genome was detected. In contrast, the human DNA segments isolated cross-hybridized with many sequences within the human genome. We tested for the presence of several functional domains on two of these human DNA fragments. One SV40-hybridizing fragment, SVCR29, contained a sequence which enhanced the efficiency of thymidine kinase transformation in human cells by approximately 20-fold. This effect was seen in an orientation-independent manner when the sequence was present at the 3' end of the chicken thymidine kinase gene. We propose that this segment of DNA contains a sequence analogous to the 72-base-pair repeats of SV40. The existence of such an "activator" element in cellular DNA raises the possibility that families of these sequences may exist in the mammalian genome.
Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells.
Authors: Breitman M L , Tsui L C , Buchwald M , Siminovitch L
Published: September 2015

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells.
Covalent guanylyl intermediate formed by HeLa cell mRNA capping enzyme.
Authors: Wang D , Furuichi Y , Shatkin A J
Published: September 2015

Guanylyltransferase, an enzyme that catalyzes formation of mRNA 5'-terminal caps, was isolated from HeLa cell nuclei. The partially purified preparation, after incubation with [alpha-32P]GTP, yielded a single radiolabeled polypeptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The guanylylated product was stable at neutral and alkaline pHs and had a pI of 4 by isoelectric focusing. An apparent molecular weight of approximately 68,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The formation of a covalently linked, radiolabeled GMP-protein complex and the associated release of PPi required the presence of [alpha-32P]GTP and divalent cations and incubation between pH 7 and 9. Reaction with [beta-32P]GTP, [alpha-32P]CTP, [alpha-32P]UTP, or [alpha-32P]ATP did not label the approximately 68,000-dalton polypeptide. Phosphoamide linkage of the GMP-enzyme complex was indicated by its sensitivity to cleavage by acidic hydroxylamine or HCl and not by NaOH or alkaline phosphatase. Both formation of the GMP-enzyme intermediate and synthesis of cap structures of type GpppApG from GTP and ppApG were remarkably temperature independent; the rates of enzyme activity at 0 to 4 degrees C were 30% or more of those obtained at 37 degrees C. Radiolabeled GMP-enzyme complex, isolated by heparin-Sepharose chromatography from reaction mixtures, functioned effectively as a GMP donor for cap synthesis with 5'-diphosphorylated oligo- and polynucleotide acceptors. Alternatively, protein-bound GMP could be transferred to PPi to form GTP. The formation of a guanylylated enzyme intermediate appears to be characteristic of viral and cellular guanylyltransferases that modify eucaryotic mRNA 5' termini.
Genetic selection for reciprocal translocation at chosen chromosomal sites in Saccharomyces cerevisiae.
Authors: Potier S , Winsor B , Lacroute F
Published: September 2015

We have constructed viable Saccharomyces cerevisiae strains containing a reciprocal translocation between the URA2 site of chromosome X and the HIS3 site of chromosome XV. Our methodology is an extension of the method originally developed to introduce an altered cloned sequence at the chromosomal location from which the parent sequence was derived (S. Scherer and R.W. Davis, Proc. Natl. Acad. Sci. U.S.A. 76:4951-4955, 1979). It comprises three essential steps. First, a nonreverting ura2- strain was constructed by deleting a 3.7-kilobase fragment from the coding sequence of the wild-type URA2 gene. Second, part of the coding sequence of the wild-type URA2 gene (without promotor) was inserted at the HIS3 locus of the ura2- strain. Third, after several generations of growth on uracil-supplemented medium, ura2+ colonies were selected which resulted from mitotic recombination between the nonoverlapping deletions of URA2 located on chromosomes X and XV.
Mutations affecting the structure and function of immunoglobulin M.
Authors: Shulman M J , Heusser C , Filkin C , Köhler G
Published: September 2015

Using a hybridoma cell line which secretes hapten-specific immunoglobulin M (IgM), we have isolated a variety of mutants which produce abnormal immunoglobulin. Immunoglobulin was tested for the size and composition of the component heavy and light chains and for variable and constant region related functional and serological activities. Some mutants secrete IgM which seems to be defective in hapten binding; others make IgM which appears not to activate complement. Many of the mutants secrete monomeric as opposed to pentameric IgM. In some cases, the defect apparently correlates with structural alterations in the mu heavy chain: partial deletion, polypeptide addition, and abnormal glycosylation have been observed. These mutant cell lines provide a means of identifying the structural basis of IgM function and of studying the biochemistry of IgM synthesis and processing.
Mating-defective ste mutations are suppressed by cell division cycle start mutations in Saccharomyces cerevisiae.
Authors: Shuster J R
Published: September 2015

Temperature-sensitive mutants which arrest in the G1 phase of the cell cycle have been described for the yeast Saccharomyces cerevisiae. One class of these mutants (carrying cdc28, cdc36, cdc37, or cdc39) forms a shmoo morphology at restrictive temperature, characteristic of mating pheromone-arrested wild-type cells. Therefore, one hypothesis to explain the control of cell division by mating factors states that mating pheromones arrest wild-type cells by inactivating one or more of these CDC gene products. A class of mutants (carrying ste4, ste5, ste7, ste11, or ste12) which is insensitive to mating pheromone and sterile has also been described. One possible function of the STE gene products is the inactivation of the CDC gene products in the presence of a mating pheromone. A model incorporating these two hypotheses predicts that such STE gene products will not be required for mating in strains carrying an appropriate cdc lesion. This prediction was tested by assaying the mating abilities of double mutants for all of the pairwise combinations of cdc and ste mutations. Lesions in either cdc36 or cdc39 suppressed the mating defect due to ste4 and ste5. Allele specificity was observed in the suppression of both ste4 and ste5. The results indicate that the CDC36, CDC39, STE4, and STE5 gene products interact functionally or physically or both in the regulation of cell division mediated by the presence or absence of mating pheromones. The cdc36 and cdc39 mutations did not suppress ste7, ste11, or ste12. Lesions in cdc28 or cdc37 did not suppress any of the ste mutations. Other models of CDC and STE gene action which predicted that some of the cdc and ste mutations would be alleles of the same locus were tested. None of the cdc mutations was allelic to the ste mutations and, therefore, these models were eliminated.
Regulation by vitamin A of envelope cross-linking in cultured keratinocytes derived from different human epithelia.
Authors: Green H , Watt F M
Published: September 2015

When keratinocytes derived from different squamous epithelia are cultured in the absence of vitamin A, they form cross-linked envelopes during the last stage of terminal differentiation. Addition of the vitamin inhibits envelope formation, but the degree of inhibition is not the same for different keratinocyte subtypes. In the presence of low concentrations of retinyl acetate, conjunctival keratinocytes form virtually no cross-linked envelopes; esophageal and vaginal keratinocytes are less sensitive to the vitamin, and epidermal keratinocytes are the least sensitive. The suppression of cross-linked envelope formation is not associated with a proportional decrease in the concentration of involucrin, a precursor of the envelope, but occurs at the level of cross-linking itself, a process dependent on an increase in the intracellular concentration of calcium ions. Keratinocytes in which spontaneous envelope cross-linking has been prevented by retinyl acetate promptly form cross-linked envelopes if Ca2+ is introduced into the cytoplasm.