bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasminogen activator: analysis of enzyme induction by ultraviolet irradiation mapping.
Authors: Miskin R , Reich E , Dixon K
Published: September 2015

Ultraviolet irradiation mapping techniques have previously been used to study the organization of eucaryotic gene classes and transcription units. We used the same method to probe some regulatory phenomena observed in the induction of plasminogen activator (PA) biosynthesis: PA synthesis in chicken embryo fibroblasts is induced by tumor-promoting phorbol esters and by retinoic acid; furthermore, PA induction by phorbol esters is synergistic with transformation, being 10- to 20-fold greater in virus-transformed cells than in normal cells. We found that the ultraviolet irradiation inactivation cross sections for PA induction by phorbol esters and by retinoate differed significantly, suggesting that these agents induce PA biosynthesis by different mechanisms. On the other hand, the ultraviolet irradiation sensitivity of phorbol ester induction in normal chicken embryo fibroblasts was the same as in transformed cells, indicating that the synergism of transformation and phorbol esters is probably not due to different pathways of PA induction.
Genetic regulation: yeast mutants constitutive for beta-galactosidase activity have an increased level of beta-galactosidase messenger ribonucleic acid.
Authors: Dickson R C , Sheetz R M , Lacy L R
Published: September 2015

Mutants of Kluyveromyces lactis with elevated uninduced levels of beta-galactosidase (EC 32.1.2.3) activity, constitutive mutants (lac10c), were isolated and characterized to determine the basis for their constitutiveness. These lesions are not operator-type regulatory mutants because they are not closely linked to the beta-galactosidase structural gene. In a constitutive strain having a 7-fold increase in beta-galactosidase activity, the concentration of beta-galactosidase messenger ribonucleic acid (mRNA) was 8- to 10-fold higher than uninduced wild type. The half-life of beta-galactosidase mRNA was the same in the mutant strain (t1/2 = 4.5 +/- 0.2 min) as in uninduced wild-type cells (t1/2 = 4.8 +/- 0.1 min), indicating that the elevated mRNA level in the mutant was not due to a decreased rate of mRNA degradation. Consequently, we hypothesize that the LAC10 product regulates transcription of the beta-galactosidase gene; it probably affects the rate of transcription initiation. Parallel increases in enzyme protein, in constitutive levels of beta-galactosidase activity, and in mRNA further support this position, making translational or posttranslational control by LAC10 unlikely. Several types of data suggest that the LAC10 product functions as a negative regulatory element to prevent transcription. Other data demonstrate that lac10c mutations have pleiotrophic effects, there being constitutive levels not only of beta-galactosidase activity, but also the other lactose-inducible activities of galactokinase (EC 2.7.5.1), galactose-1-phosphate uridyl transferase (EC 2.7.7.10), and lactose transport. It would appear that LAC10 regulates lactose-inducible proteins.
Control of dihydrofolate reductase messenger ribonucleic acid production.
Authors: Leys E J , Kellems R E
Published: September 2015

We used methotrexate-resistant mouse cells in which dihydrofolate reductase levels are approximately 500 times normal to study the effect of growth stimulation on dihydrofolate reductase gene expression. As a result of growth stimulation, the relative rate of dihydrofolate reductase protein synthesis increased threefold, reaching a maximum between 25 and 30 h after stimulation. The relative rate of dihydrofolate reductase messenger ribonucleic acid production (i.e., the appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm) increased threefold after growth stimulation and was accompanied by a corresponding increase in the relative steady-state level of dihydrofolate reductase ribonucleic acid in the nucleus. However, the increase in the nuclear level of dihydrofolate reductase ribonucleic acid was not accompanied by a significant increase in the relative rate of transcription of the dihydrofolate reductase genes. These data indicated that the relative rate of appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm depends on the relative stability of the dihydrofolate reductase ribonucleic acid sequences in the nucleus and is not dependent on the relative rate of transcription of the dihydrofolate reductase genes.
Three segments from the monkey genome that hybridize to simian virus 40 have common structural elements.
Authors: Queen C , Lord S T , McCutchan T F , Singer M F
Published: September 2015

Three cloned segments that hybridize to a region of simian virus 40 (SV40) deoxyribonucleic acid including the origin of replication have been isolated from a monkey genomic library. The primary structure of one segment was previously reported (T. McCutchan and M. Singer, Proc. Natl. Acad. Sci. U.S.A. 78:95-99, 1981). We report here the sequences of the other two segments and a comparison of all three. The SV 40-hybridizing region in each segment is limited to several hundred base pairs. All of the segments contain multiple and disconnected sequences homologous to the region of SV40 directly surrounding the viral replication origin. The number and arrangement of the homologous sequences is different in the three segments. However, the segments have the following features in common: (i) each contains multiple copies of the sequence GGGCGGPuPu, which also appears six times near the origin of SV40; (ii) each contains several strong homologies to the central dyad symmetry of SV40; (iii) each contains a long internal repeat, as does the origin region of SV40. The three SV40-hybridizing segments are members of a larger family of genomic sequences that hybridize well to each other, but not necessarily to SV40.
Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line.
Authors: Kaufman R J , Schimke R T
Published: September 2015

During stepwise increases in the methotrexate concentration in culture medium, we selected Chinese hamster ovary cells that contained elevated dihydrofolate reductase levels which were proportional to the number of dihydrofolate reductase gene copies (i.e., gene amplification). We studied the dihydrofolate reductase levels in individual cells that underwent the initial steps of methotrexate resistance by using the fluorescence-activated cell sorter technique. Such cells constituted a heterogeneous population with differing dihydrofolate reductase levels, and they characteristically lost the elevated enzyme levels when they were grown in the absence of methotrexate. The progeny of individual cells with high enzyme levels behaved differently and could lose all or variable numbers of the amplified genes.
Loss and stabilization of amplified dihydrofolate reductase genes in mouse sarcoma S-180 cell lines.
Authors: Kaufman R J , Brown P C , Schimke R T
Published: September 2015

We studied the loss and stabilization of dihydrofolate reductase genes in clones of a methotrexate-resistant murine S-180 cell line. These cells contained multiple copies of the dihydrofolate reductase gene which were associated with double minute chromosomes. The growth rate of these cells in the absence of methotrexate was inversely related to the degree of gene amplification (number of double minute chromosomes). Cells could both gain and lose genes as a result of an unequal distribution of double minute chromosomes into daughter cells at mitosis. The loss of amplified dihydrofolate reductase genes during growth in the absence of methotrexate resulted from the continual generation of cells containing lower numbers of double minute chromosomes. Because of the growth advantage of these cells, they became dominant in the population. We also studied an unstably resistant S-180 cell line (clone) that, after 3 years of continuous growth in methotrexate, generated cells containing stably amplified dihydrofolate reductase genes. These genes were present on one or more chromosomes, and they were retained in a stable state.
Post-translational regulation of the 54K cellular tumor antigen in normal and transformed cells.
Authors: Oren M , Maltzman W , Levine A J
Published: September 2015

The 54K cellular tumor antigen has been translated in vitro, using messenger ribonucleic acids from simian virus 40 (SV40)-transformed cells or 3T3 cells. The in vitro 54K product could be immunoprecipitated with SV40 tumor serum and had a peptide map that was similar, but not identical, to the in vivo product. The levels of this 54K protein in SV3T3 cells were significantly higher than those detected in 3T3 cells (D. I. H. Linzer, W. Maltzman, and A. J. Levine, Virology 98:308-318, 1979). In spite of this, the levels of translatable 54K messenger ribonucleic acid from 3T3 and SV3T3 cells were roughly equivalent or often greater in 3T3 cells. Pulse-chase experiments with the 54K protein from 3T3 or SV3T3 cells demonstrated that this protein, once synthesized, was rapidly degraded in 3T3 cells but was extremely stable in SV3T3 cells. Similarly, in an SV40 tsA-transformed cell line, temperature sensitive for the SV40 T-antigen, the 54K protein was rapidly turned over at the nonpermissive temperature and stable at the permissive temperature, whereas the levels of translatable 54K messenger ribonucleic acid at each temperature were roughly equal. These results demonstrate a post-translational regulation of the 54K cellular tumor antigen and suggest that this control is mediated by the SV40 large T-antigen.
RAD6+ gene of Saccharomyces cerevisiae codes for two mutationally separable deoxyribonucleic acid repair functions.
Authors: Tuite M F , Cox B S
Published: September 2015

The response of two mutant alleles of the RAD6+ gene of Saccharomyces cerevisiae to the ochre translational suppressor SUQ5 was determined. Both the ultraviolet sensitivity phenotype and the deficiency in ultraviolet-induced mutagenesis phenotype of the rad6-1 allele were suppressed in a [psi+] background. For the rad6-3 allele, only the ultraviolet-sensitivity phenotype was suppressible in a [psi+] background. An SUQ5 rad6-3 [psi+] strain that was examined showed the normal rad6-3 deficiency in ultraviolet-induced mutagenesis. We propose that the RAD6+ gene is divided into two cistrons, RAD6A and RAD6B. RAD6A codes for an activity responsible for the error-prone repair of ultraviolet-induced lesions in deoxyribonucleic acid but is not involved in a cell's resistance to the lethal effects of ultraviolet light. RAD6B codes for an activity essential for error-free repair of potentially lethal mutagenic damage.
Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells.
Authors: Cooper J A , Hunter T
Published: September 2015

Rous sarcoma virus encodes a tyrosine-specific protein kinase (p60src) which is necessary for cell transformation. To identify substrates for this kinase, we set out to detect phosphotyrosine-containing proteins in Rous sarcoma virus-transformed chicken embryo cells, making use of the known alkali stability of phosphotyrosine. 32P-labeled phosphoproteins were separated by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were then incubated in alkali. Using this procedure with normal cells, we detected a total of about 190 alkali-resistant phosphoproteins. In Rous sarcoma virus-transformed cells, five phosphoproteins were found which were not detectable in normal cells. Two of these are probably structural proteins of the virus. The other three transformation-dependent phosphoproteins, and four other phosphoproteins which were elevated by transformation, all contained phosphotyrosine. Increased phosphorylation of these proteins did not occur with cells infected with a mutant Rous sarcoma virus, temperature sensitive for transformation, grown at the restrictive temperature. We conclude that these seven proteins are probably substrates of p60src, although they may be substrates for other tyrosine-specific protein kinases activated by p60src.
Large heterogeneous nuclear ribonucleic acid has three times as many 5' caps as polyadenylic acid segments, and most caps do not enter polyribosomes.
Authors: Salditt-Georgieff M , Harpold M M , Wilson M C , Darnell J E
Published: September 2015

The rate of synthesis in Chinese hamster cells of 5' cap structures, m7 GpppNmp, in large (greater than 700 bases) heterogeneous nuclear ribonucleic acid (RNA) molecules is two to three times faster than the synthesis of 3'-terminal polyadenylic acid segments. As judged by presence of caps, newly synthesized polysomal messenger RNA, exclusive of messenger RNA the size of histone messenger RNA, is more than 90% in the polyadenylated category. It appears, therefore, that between half and two-thirds of the long capped heterogeneous nuclear RNA molecules do not contribute a capped polysomal derivative to the cytoplasm. There are capped, nonpolysomal, non-polyadenylated molecules with a rapid turnover rate that fractionate with the cytoplasm. These metabolically unstable molecules either could represent leakage into the cytoplasm during fractionation or could truly spend a brief time in the cytoplasm before decay.
Regulation of tropomyosin gene expression during myogenesis.
Authors: Moss M , Schwartz R
Published: September 2015

In skeletal muscle, tropomyosin has a critical role in transduction of calcium-induced contraction. Presently, little is known about the regulation of tropomyosin gene expression during myogenesis. In the present study, qualitative and quantitative changes in the nucleic acid populations of differentiating chicken embryo muscle cells in culture have been examined. Total nucleic acid content per nucleus increased about fivefold in fully developed myotubes as compared to mononucleated myoblasts. The contribution of deoxyribonucleic acid to the total nucleic acid population decreased from 24% in myoblasts to 5% of total nucleic acid in myotubes. Concomitant with the decrement in deoxyribonucleic acid contribution to total nucleic acid was an increase in polyadenylated ribonucleic acid (RNA) content per cell which reached levels in myotubes that were 17-fold higher than those of myoblasts. Specific changes in the RNA population during myogenesis were further investigated by quantitation of the synthetic capacity (messenger RNA levels) per cell for alpha- and beta-tropomyosin. Cell-free translation and immunoprecipitation demonstrated an approximately 40-fold increase in messenger RNA levels per nucleus for alpha- and beta-tropomyosin after fusion in the terminally differentiated myotubes. Indirect immunofluorescence with affinity-purified tropomyosin antibodies demonstrated the presence of tropomyosin-containing filaments in cells throughout myogenesis. Thus, the tropomyosin genes are constitutively expressed during muscle differentiation through the production of tropomyosin messenger RNA and translation into tropomyosin protein.
Microcell-mediated cotransfer of genes specifying methotrexate resistance, emetine sensitivity, and chromate sensitivity with Chinese hamster chromosome 2.
Authors: Worton R , Duff C , Flintoff W
Published: September 2015

Many selectable mutants of somatic Chinese hamster cells have been described, but very few of the mutations have been mapped to specific chromosomes. We have utilized the microcell-mediated gene transfer technique to establish the location of three selectable genetic markers on chromosome 2 of Chinese hamster. Microcells were prepared from the methotrexate-resistant MtxRIII line of Flintoff et al. (Somatic Cell Genet. 2:245-261, 1976) and fused to wild-type CHO cells, and microcell hybrids (transferants) were selected in medium containing methotrexate. All transferants were karyotyped and found to contain a marker chromosome from the donor MtxRIII line. This marker chromosome, called 2p-, consisted of a chromosome 2 with a reduced short arm resulting from a reciprocal translocation between 2p and 5q. In experiments utilizing emetine-resistant (Emtr) or chromate-resistant (Chrr) recipient cells it was found that the emt+ and chr+ wild-type genes were cotransferred with the 2p- chromosomes. Karyotype analysis of several transferants with rearranged or broken 2p- markers allowed regional localization of the emt and chr loci to the proximal third of the long arm and localization of the gene or genes conferring methotrexate resistance to the short arm. These results confirm our earlier assignment of the emt and chr loci to chromosome 2 in Chinese hamster.
Segregation of recessive phenotypes in somatic cell hybrids: role of mitotic recombination, gene inactivation, and chromosome nondisjunction.
Authors: Campbell C E , Worton R G
Published: September 2015

Somatic cell hybrids heterozygous at the emetine resistance locus (emtr/emt+) or the chromate resistance locus (chrr/chr+) are known to segregate the recessive drug resistance phenotype at high frequency. We have examined mechanisms of segregation in Chinese hamster cell hybrids heterozygous at these two loci, both of which map to the long arm of Chinese hamster chromosome 2. To follow the fate of chromosomal arms through the segregation process, our hybrids were also heterozygous at the mtx (methotrexate resistance) locus on the short arm of chromosome 2 and carried cytogenetically marked chromosomes with either a short-arm deletion (2p-) or a long-arm addition (2q+). Karyotype and phenotype analysis of emetine- or chromate-resistant segregants from such hybrids allowed us to distinguish four potential segregation mechanisms: (i) loss of the emt+- or chr+-bearing chromosome; (ii) mitotic recombination between the centromere and the emt or chr loci, giving rise to homozygous resistant segregants; (iii) inactivation of the emt+ or chr+ alleles; and (iv) loss of the emt+- or chr+-bearing chromosome with duplication of the homologous chromosome carrying the emtr or chrr allele. Of 48 independent segregants examined, only 9 (20%) arose by simple chromosome loss. Two segregants (4%) were consistent with a gene inactivation mechanism, but because of their rarity, other mechanisms such as mutation or submicroscopic deletion could not be excluded. Twenty-one segregants (44%) arose by either mitotic recombination or chromosome loss and duplication; the two mechanisms were not distinguishable in that experiment. Finally, in hybrids allowing these two mechanisms to be distinguished, 15 segregants (31%) arose by chromosome loss and duplication, and none arose by mitotic recombination.
Localization of lipophosphonoglycan in membranes of Acanthamoeba by using specific antibodies.
Authors: Bailey C F , Bowers B
Published: September 2015

Antibodies against two electrophoretically distinct forms of lipophosphonoglycan (LPG) were produced in rabbits. Antibody specificity was demonstrated by the coupled antibody 125I-protein A assay (Adair et al., J. Cell Biol. 79:281-285, 1978). Indirect immunofluorescent labeling of intact Acanthamoeba showed that antibodies to both LPG components had the same uniform distribution on the cell surface. Both antibodies also bound to the cytoplasmic surface of isolated phagosomes. The location of LPG in other membranes of the amoeba was demonstrated on sections by the unlabeled antibody method. Although LPG was absent from the nuclear membrane, virtually all of the internal vacuole membranes were labeled, including the contractile vacuole. Antibodies directed against LPG were utilized to label lipophosphonoglycan in the plasma membrane of living amoebae. Labeled membrane was internalized and then localized by immunofluorescence in cytoplasmic vacuoles within 10 min of incubation. Although these results are evidence for exchange between plasma and cytoplasmic vacuolar membranes, the contractile vacuole remained unlabeled and can be considered, therefore, a separate membrane compartment. Concanavalin A also was bound and internalized by the amoeba, but electron microscopy showed that this label caused pronounced membrane perturbation, limiting its usefulness as a membrane marker in this system.
Synthesis and turnover of mitochondrial ribonucleic acid in HeLa cells: the mature ribosomal and messenger ribonucleic acid species are metabolically unstable.
Authors: Gelfand R , Attardi G
Published: September 2015

The synthesis rates and half-lives of the individual mitochondrial ribosomal ribonucleic acid (RNA) and polyadenylic acid-containing RNA species in HeLa cells have been determined by analyzing their kinetics of labeling with [5-3H]-uridine and the changes in specific activity of the mitochondrial nucleotide precursor pools. In one experiment, a novel method for determining the nucleotide precursor pool specific activities, using nascent RNA chains, has been utilized. All mitochondrial RNA species analyzed were found to be metabolically unstable, with half-lives of 2.5 to 3.5 h for the two ribosomal RNA components and between 25 and 90 min for the various putative messenger RNAs. A cordycepin "chase" experiment yielded half-life values for the messenger RNA species which were, in general, larger by a factor of 1.5 to 2.5 than those estimated in the labeling kinetics experiments. On the basis of previous observations, a model is proposed whereby the rate of mitochondrial RNA decay is under feedback control by some mechanism linked to RNA synthesis or processing. A short half-life was determined for five large polyadenylated RNAs, which are probably precursors of mature species. A rate of synthesis of one to two molecules per minute per cell was estimated for the various H-strand-coded messenger RNA species, and a rate of synthesis 50 to 100 times higher was estimated for the ribosomal RNA species. These data indicate that the major portion of the H-strand in each mitochondrial deoxyribonucleic acid molecule is transcribed very infrequently, possibly as rarely as once or twice per cell generation. Furthermore, these results are consistent with a previously proposed model of H-strand transcription in the form of a single polycistronic molecule.
Mutator genes of baby hamster kidney cells.
Authors: Chan V L , Guttman S , Juranka P
Published: September 2015

Two mutator genes of mammalian cells were demonstrated. One was associated with the ribonucleoside diphosphate reductase, and the other was associated with an extreme adenosine sensitivity.
Biological role of the general control of amino acid biosynthesis in Saccharomyces cerevisiae.
Authors: Niederberger P , Miozzari G , Hütter R
Published: September 2015

The biological role of the "general control of amino acid biosynthesis" has been investigated by analyzing growth and enzyme levels in wild-type, bradytrophic, and nonderepressing mutant strains of Saccharomyces cerevisiae. Amino acid limitation was achieved by using either bradytrophic mutations or external amino acid imbalance. In the wild-type strain noncoordinate derepression of enzymes subject to the general control has been found. Derepressing factors were in the order of 2 to 4 in bradytrophic mutant strains grown under limiting conditions and only in the order of 1.5 to 2 under the influence of external amino acid imbalance. Nonderepressing mutations led to slower growth rates under conditions of amino acid limitation, and no derepression of enzymes under the general control was observed. The amino acid pools were found to be very similar in the wild type and in nonderepressing mutant strains under all conditions tested. Our results indicate that the general control affects all branched amino acid biosynthetic pathways, namely, those of the aromatic amino acids and the aspartate family, the pathways for the basic amino acids lysine, histidine, and arginine, and also the pathways of serine and valine biosyntheses.
Transcriptional regulation of the Kluyveromyces lactis beta-galactosidase gene.
Authors: Lacy L R , Dickson R C
Published: September 2015

We examined the molecular basis for beta-D-galactosidase (EC 3.2.1.23) induction in the yeast Kluyveromyces lactis. The protein synthesis inhibitor anisomycin effectively blocked both protein synthesis and enzyme induction by lactose. Further, hybridization analysis with the cloned beta-galactosidase gene indicated coordinate increases in the concentration of beta-galactosidase messenger ribonucleic acid and enzyme activity. The half-life of beta-galactosidase messenger ribonucleic acid was the same (4.8 +/- 0.4 min) when measured both before and at succeeding times during enzyme induction. These results strongly support the hypothesis that expression of the yeast beta-galactosidase gene is subject to transcriptional regulation.
Selection of specific wheat germ agglutinin-resistant (WgaR) phenotypes from Chinese hamster ovary cell populations containing numerous lecR genotypes.
Authors: Stanley P
Published: September 2015

Three distinct Chinese hamster ovary mutants selected for resistance to wheat germ agglutinin were previously described by this laboratory. In this paper, evidence is provided that each phenotype occurs at a similar frequency in an unmutagenized population of Chinese hamster ovary cells. Two novel wheat germ agglutinin resistance phenotypes (WgaR), which also appear to occur at similar frequencies were uncovered in the course of these studies. One mutant type belongs to a new, recessive complementation group (VIII), and the second belongs to a previously defined complementation group (VI). Mutants from each of the four WgaR complementation groups (I, II, III, and VIII) exhibited characteristic and unique patterns of resistance to the toxicity of a variety of plant lectins. These properties were used in developing independent selection protocols which were highly specific for the isolation of each of the mutant types.
Characterization of mouse cellular deoxyribonucleic acid homologous to Abelson murine leukemia virus-specific sequences.
Authors: Dale B , Ozanne B
Published: September 2015

The genome of Abelson murine leukemia virus (A-MuLV) consists of sequences derived from both BALB/c mouse deoxyribonucleic acid and the genome of Moloney murine leukemia virus. Using deoxyribonucleic acid linear intermediates as a source of retroviral deoxyribonucleic acid, we isolated a recombinant plasmid which contained 1.9 kilobases of the 3.5-kilobase mouse-derived sequences found in A-MuLV (A-MuLV-specific sequences). We used this clone, designated pSA-17, as a probe restriction enzyme and Southern blot analyses to examine the arrangement of homologous sequences in BALB/c deoxyribonucleic acid (endogenous Abelson sequences). The endogenous Abelson sequences within the mouse genome were interrupted by noncoding regions, suggesting that a rearrangement of the cell sequences was required to produce the sequence found in the virus. Endogenous Abelson sequences were arranged similarly in mice that were susceptible to A-MuLV tumors and in mice that were resistant to A-MuLV tumors. An examination of three BALB/c plasmacytomas and a BALB/c early B-cell tumor likewise revealed no alteration in the arrangement of the endogenous Abelson sequences. Homology to pSA-17 was also observed in deoxyribonucleic acids prepared from rat, hamster, chicken, and human cells. An isolate of A-MuLV which encoded a 160,000-dalton transforming protein (P160) contained 700 more base pairs of mouse sequences than the standard A-MuLV isolate, which encoded a 120,000-dalton transforming protein (P120).
Initiation points for cellular deoxyribonucleic acid replication in human lymphoid cells converted by Epstein-Barr virus.
Authors: Oppenheim A , Shlomai Z , Ben-Bassat H
Published: September 2015

Replicon size was estimated in two Epstein-Barr virus (EBV)-negative human lymphoma lines, BJAB and Ramos, and four EBV-positive lines derived from the former ones by infection (conversion) with two viral strains, B95-8 and P3HR-1. Logarithmic cultures were pulse-labeled with [3H]thymidine, and the deoxyribonucleic acid was spread on microscopic slides and autoradiographed by the method of Huberman and Riggs. After developing, replication forks were visualized as silver grain tracks on the autoradiograms. Average replicon size was estimated by scoring the number of replication forks per constant length of deoxyribonucleic acid and by measuring distances between centers of adjacent tracks, followed by detailed statistical analyses. Three of the four EBV-converted cell lines, BJAB/B95-8, Ra/B95-8, and Ra/HRIK, were found to have significantly shorter replicons (41, 21, 54% shorter, respectively), i.e., more initiation points, than their EBV-negative parents. BJAB/HRIK had replicons which were only slightly shorter (11%) than those of BJAB. However, analysis of track length demonstrated that extensive track fusion occurred during the labeling of BJAB/ HRIK, implying that its true average replicon size is shorter than the observed value. The results indicate that in analogy to simian virus 40, EBV activates new initiation points for cellular DNA replication in EBV-transformed cells.
Intracellular monosaccharide and amino acid concentrations and activities and the mechanisms of insulin action.
Authors: Horowitz S B , Pearson T W
Published: September 2015

Current amino acid and monosaccharide transport models are based on an assumption which equates the intracellular chemical activity of a solute with its concentration. This assumption was tested for alpha-aminoisobutyric acid and 3-O-methylglucose in a giant cell, the amphibian oocyte, by using recently developed cryomicrodissection and internal reference phase techniques. We found the following. (i) alpha-Aminoisobutyric acid and 3-O-methylglucose activities were much greater in cytoplasm than was suggested by concentration data; i.e., activity coefficients were higher than in ordinary water solutions. This is attributable to the inaccessibility of considerable water as solvent (solute exclusion). (ii) Solute concentrations varied regionally as follows: nucleus > > animal cytoplasm > vegetal cytoplasm. Insulin increased the nucleus/cytoplasm concentration asymmetry, apparently by increasing cytoplasmic solute exclusion. (iii) Nuclear activity coefficients more closely resembled those of ordinary saline solutions so that nucleus/ extracellular concentration ratios reflected transmembrane activity gradients better than did cytoplasm (or whole cell)/extracellular ratios. (iv) Mediated passive alpha-aminoisobutyric acid and 3-O-methylglucose transport were constituent oocyte membrane properties. Membrane active transport was initiated with time (in the presence of substrate) and by insulin. (v) Increased temperature mimicked insulin in enhancing transmembrane alpha-aminoisobutyric acid activity gradients and increasing the nucleus/cytoplasm concentration asymmetry. These results indicated that concentration data are a misleading measure of cellular amino acid and monosaccharide activity; some consequences of this observation were explored. A model is proposed in which cell water has reduced solvent capacity or is compartmentalized (considered less likely) and is susceptible to physiological modulation. The model accounts for many observations in small cells, suggesting generality of the exclusion phenomenon and a previously unrecognized metabolic control mechanism.
Genetic complementation of the Saccharomyces cerevisiae leu2 gene by the Escherichia coli leuB gene.
Authors: Storms R K , Holowachuck E W , Friesen J D
Published: September 2015

The leucine operon of Escherichia coli was cloned on a plasmid possessing both E. coli and Saccharomyces cerevisiae replication origins. This plasmid, pEH25, transformed leuA, leuB, and leuD auxotrophs of E. coli to prototrophy; it also transformed leu2 auxotrophs of S. cerevisiae to prototrophy. beta-Isopropylmalate dehydrogenase was encoded by the leuB gene of E. coli and the leu2 gene of yeast. Verification that the leuB gene present on pEH26 was responsible for complementing yeast leu2 was obtained by isolating in E. coli several leuB mutations that resided on the plasmid. These mutant leuB- plasmids were no longer capable of complementing leu2 in S. cerevisiae. We conclude that S. cerevisiae is capable of transcribing at least a portion of the polycistronic leu operon of E. coli and can translate a functional protein from at least the second gene of this operon. The yeast Leu+ transformants obtained with pEH25, when cultured in minimal medium lacking leucine, grew with a doubling time three to four times longer than when cultured in medium supplemented with leucine.
Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors.
Authors: Subramani S , Mulligan R , Berg P
Published: September 2015

A mouse complementary deoxyribonucleic acid segment coding for the enzyme dihydrofolate reductase has been cloned in two general classes of vectors containing simian virus 40 deoxyribonucleic acid: (i) those that can be propagated as virions in permissive cells and (ii) those that can be introduced into and maintained stably in various mammalian cells. Both types of vectors express the mouse dihydrofolate reductase by using signals supplied by simian virus 40 deoxyribonucleic acid sequences. Moreover, plasmid vectors carrying the complementary deoxyribonucleic acid segment can complement Chinese hamster ovary cells lacking dihydrofolate reductase.
Conditions necessary for inhibition of protein synthesis and production of cytopathic effect in Aedes albopictus cells infected with vesicular stomatitis virus.
Authors: Gillies S , Stollar V
Published: September 2015

The relationship between the development of cytopathic effect (CPE) and the inhibition of host macromolecular synthesis was examined in a CPE-susceptible cloned line of Aedes albopictus cells after infection with vesicular stomatitis virus. To induce rapid and maximal CPE, two conditions were required: (i) presence of serum in the medium and (ii) incubation at 34 degrees C rather than at 28 degrees C. In the absence of serum, incubation of infected cultures at 34 degrees C resulted in a significant increase in viral protein and RNA synthesis compared with that observed at 28 degrees C. However, when serum was present in the medium, by 6 h after infection protein synthesis (both host and viral) was markedly inhibited when infected cells were maintained at 34 degrees C. RNA synthesis (host and viral) was also inhibited in vesicular stomatitis virus-infected cells maintained at 34 degrees C with serum, but somewhat more slowly than protein synthesis. Examination of polysome patterns indicated that when infected cultures were maintained under conditions which predispose to CPE, more than half of the ribosomes existed as monosomes, suggesting that protein synthesis was being inhibited at the level of initiation. In addition, the phosphorylation of one (or two) polysome-associated proteins was reduced when protein synthesis was inhibited. Our findings indicate a strong correlation between virus-induced CPE in the LT-C7 clone of A. albopictus cells and the inhibition of protein synthesis. Although the mechanism of the serum effect is not understood, incubation at 34 degrees C probably predisposes to CPE and inhibition of protein synthesis by increasing the amount of viral gene products made.
Direct demonstration of genetic alterations at the dihydrofolate reductase locus after gamma irradiation.
Authors: Graf L H , Chasin L A
Published: September 2015

Gamma ray-induced mutants of Chinese hamster ovary cells lacking dihydrofolate reductase activity were screened for DNA sequence changes at the locus specifying this activity by using a cloned cDNA probe. Two of nine mutants screened displayed an altered restriction fragment pattern suggesting the occurrence of DNA deletions or rearrangements.
Polyoma virus middle T antigen: relationship to cell membranes and apparent lack of ATP-binding activity.
Authors: Schaffhausen B S , Dorai H , Arakere G , Benjamin T L
Published: September 2015

Middle T antigen of polyoma virus is associated principally with the plasma membrane. Comparison of the trypsin sensitivity of middle T in intact cells and "inside out" membrane preparations showed that middle T is oriented towards the inside of the cell. This was confirmed by labeling of middle T in permeabilized cells, but not in intact cells, using [gamma-32P]ATP. Middle T molecules active in the in vitro kinase reaction could be differentiated from the bulk (metabolically labeled) middle T based on resistance to trypsin treatment. The active fraction also behaved differently from the bulk when cell frameworks were prepared with Triton-containing buffers; whereas the bulk middle T was evenly distributed in the soluble and cell framework fractions, the kinase-active forms were largely associated with the framework. Middle T molecules labeled in vivo with 32PO4 were found largely in the framework fraction, like the molecules that show kinase activity in vitro. Experiments with ATP affinity reagents 8-azido-ATP and 2,3-dialdehyde ATP have failed to label the middle T antigen. However, 2,3-dialdehyde ATP could be used to inhibit the kinase reaction. This raises the question of whether middle T antigen possesses intrinsic kinase activity or, rather, associates with a cellular tyrosine kinase.
Further evidence that the rna2 mutation of Saccharomyces cerevisiae affects mRNA processing.
Authors: Bromley S , Hereford L , Rosbash M
Published: September 2015

The relative rate at which ribosomal protein 51 (rp51) mRNA is synthesized was measured by pulse-labeling cells in vivo with [3H]adenine. Two strains of Saccharomyces cerevisiae were compared: A364A (wild type) and ts368 (rna2), a temperature-sensitive strain in which the level of rp51 mRNA decreases and an intron-containing rp51 precursor RNA increases. When cells were shifted up to the nonpermissive temperature (36 degrees C), the rate of rp51 RNA synthesis was only marginally affected (75% of wild type) by the presence of the rna2 mutation. The precursor RNA was the predominant transcription product at 36 degrees C. This precursor could be converted into RNA equal in size to mature mRNA by further incubation at either 36 or 23 degrees C in the presence of unlabeled adenine. The relative half-life of the rp51 transcripts at 36 degrees C also decreased approximately twofold in ts368 as compared with A364A. All of these data imply that the precursor (intron-containing) RNA is processed inefficiently to mature mRNA and that the rp51 precursor RNA is continuously synthesized and degraded in the mutant strain at 36 degrees C.
Deletions of N-terminal sequences of polyoma virus T-antigens reduce but do not abolish transformation of rat fibroblasts.
Authors: Katinka M , Yaniv M
Published: September 2015

Polyoma virus transforms, upon infection or DNA transfection, nonpermissive Fisher rat fibroblasts. Cloned viral DNA was deleted of sequences around the Bg/I site at nucleotide 86 by Bal31 nuclease treatment and then recloned in Escherichia coli. The extent of deletion for each mutant was then determined by DNA sequencing. Deletions included the early transcription control signals; others stretched into the N-terminal coding sequences of the viral tumor antigens. The transformation efficiency of 16 mutants was tested by transfecting rat fibroblasts. Expression of the T antigens was analyzed by immunofluorescence detection after transfection of rat fibroblasts, mouse secondary embryo cells, and HeLa cells. We found that the absence of the early transcription control sequences (TATA and CAAT boxes) did not significantly alter the transformation capacity of the virus. On the other hand, deletion of the initiator methionine ATG codon or further into the coding sequences did abolish the transformation capacity in some mutants, whereas others maintained a reduced transforming activity, possibly by initiation of translation in a penultimate methionine.
Construction of a modular dihydrofolate reductase cDNA gene: analysis of signals utilized for efficient expression.
Authors: Kaufman R J , Sharp P A
Published: September 2015

Dihydrofolate reductase (DHFR) modular genes have been constructed with segments containing the adenovirus major late promoter, a 3' splice site from a variable region immunoglobulin gene, a DHFR cDNA, and portions of the simian virus 40 (SV40) genome. DNA-mediated transfer of these genes transformed Chinese hamster ovary DHFR- cells to the DHFR+ phenotype. Transformants contained one to several copies of the transfected DNA integrated into the host genome. Clones subjected to growth in increasing concentrations of methotrexate eventually gave rise to lines containing several hundred copies of the transforming DNA. Analysis of the DHFR mRNA produced in amplified lines indicated the following. (i) All clones utilize the adenovirus major late promoter for transcription initiation. (ii) A hybrid intron formed by the 5' splice site of the adenovirus major late leader and a 3' splice site from a variable-region immunoglobulin gene is properly excised. (iii) The mRNA is not efficiently polyadenylated at sequences in the 3' end of the DHFR cDNA but rather uses polyadenylation signals downstream from the DHFR cDNA. Three independent clones produce a DHFR mRNA containing SV40 or pBR322 and SV40 sequences, and the RNA is polyadenylated at the SV40 late polyadenylation site. Another clone has recombined into cellular DNA and apparently uses a cellular sequence for polyadenylation. Introduction of a segment containing the SV40 early polyadenylation signal into the 3' end of the DHFR cDNA gene generated a recombinant capable of transforming cells to the DHFR+ phenotype with at least a 10-fold increase in efficiency, demonstrating the necessity for an efficient polyadenylation signal. Attachment of a DNA segment containing the transcription enhancer (72-base pair repeat) of SV40 further increased the biological activity of the modular DHFR gene 50- to 100-fold.
Sequence comparison in the crossover region of an oncogenic avian retrovirus recombinant and its nononcogenic parent: genetic regions that control growth rate and oncogenic potential.
Authors: Tsichlis P N , Donehower L , Hager G , Zeller N , Malavarca R , Astrin S , Skalka A M
Published: September 2015

NTRE 7 is an avian retrovirus recombinant of the endogenous nononcogenic Rous-associated virus-0 (RAV-0) and the oncogenic, exogenous, transformation-defective (td) Prague strain of Rous sarcoma virus B (td-PrRSV-B). Oligonucleotide mapping had shown that the recombinant virus is indistinguishable from its RAV-0 parent except for the 3'-end sequences, which were derived from td-PrRSV-B. However, the virus exhibits properties which are typical of an exogenous virus: it grows to high titers in tissue culture, and it is oncogenic in vivo. To accurately define the genetic region responsible for these properties, we determined the nucleotide sequences of the recombinant and its RAV-0 parent by using molecular clones of their DNA. These were compared with sequences already available for PrRSV-C, a virus closely related to the exogenous parent td-PrRSV-B. The results suggested that the crossover event which generated NTRE 7 took place in a region -501 to -401 nucleotides from the 3' end of the td-PrRSV parental genome and that sequences to the right of the recombination region were responsible for its growth properties and oncogenic potential. These sequences included a 148-base-pair exogenous-virus-specific region that was absent from the RAV-0 genome and the U3 region of the long terminal repeat. Since the exogenous-virus-specific sequences are expected to be missing from transformation-defective mutants of the Schmidt-Ruppin strain of RSV, which, like other exogenous viruses, grow to high titers in tissue culture and are oncogenic in vivo, we concluded that the growth properties and oncogenic potential of the exogenous viruses are determined by sequences in the U3 region of the long terminal repeat. However, we propose that the exogenous-virus-specific region may play a role in determining the oncogenic spectrum of a given oncogenic virus.
Stepwise isolation and properties of unstable Chinese hamster cell variants that overproduce adenylate deaminase.
Authors: Debatisse M , Berry M , Buttin G
Published: September 2015

Addition of coformycin (0.5 microgram/ml) to a culture medium containing adenine causes in Chinese hamster fibroblasts a lethal depletion of IMP. Resistant variants have been recovered, some of which exhibit increased adenylate deaminase activity. (Debatisse et al., J. Cell. Physiol., 106:1-11, 1981). The selective medium was made more specific for the isolation of this class of variants by supplementation with azaserine. The hyperactive variants remained sensitive to coformycin concentrations above that used for their selection and were unstable. Their frequency was not increased by ethyl methane sulfonate mutagenesis. The resistant phenotype and the increased activity of adenylate deaminase behaved as semidominant traits in hybrids. No change was detected in the Km for AMP, the cofactor requirement, or the chromatographic properties of adenylate deaminase in the variants. Through stepwise selection in media supplemented with increasing coformycin concentrations, unstable clones with adenylate deaminase activity up to 150-fold the wild-type level were isolated; from an unstable clone, a stable subclone with reduced resistance and enzyme activity was recovered. Evidence that increased adenylate deaminase activity is the manifestation of overaccumulation of the enzyme protein was supplied by the correlation of enzyme activity with the intensity of a protein band comigrating with purified adenylate deaminase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. Several unidentified additional bands showed comparable quantitative changes. The striking similarity between the adenylate deaminase-overproducing lines and unstable dihydrofolate reductase-overproducing lines generated by gene amplification strongly suggests that the coformycin-resistant variants also resulted from amplification of an adenylate deaminase gene.
Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.
Authors: Folger K R , Wong E A , Wahl G , Capecchi M R
Published: September 2015

We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene. (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk- and RAT-2tk- cells to the TK+ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by homologous recombination.