bluuebunny/crossref_metadata_2025 huggingface dataset:
Sporulation and rna2 lower ribosomal protein mRNA levels by different mechanisms in Saccharomyces cerevisiae.
Authors: Kraig E , Haber J E , Rosbash M
Published: September 2015

In Saccharomyces cerevisiae, the levels of ribosomal protein mRNAs are regulated coordinately. Vegetative strains carrying the temperature-sensitive rna2 mutation exhibit a dramatic decrease in the levels of most ribosomal protein mRNAs at the restrictive temperature. Similarly, in wild-type cells induced to sporulate by nitrogen starvation, there is a fivefold reduction in the relative synthesis rate of ribosomal proteins. Using Northern gel analysis and cloned ribosomal protein genes, we compared the way in which ribosomal protein mRNA is affected under these two conditions. In vegetative rna2 cells, incubation at 34 degrees C led to the disappearance of ribosomal protein mRNAs and the accumulation of higher-molecular-weight precursor RNAs. A different phenotype was observed during sporulation. Although sporulating conditions led to a significant reduction in the relative abundance of ribosomal protein mRNA, there was no detectable accumulation of precursor RNAs even in rna2/rna2 diploids at 34 degrees C. A suppressor of rna2 and of other rna mutations, SRN1, at least partially relieved the block in the splicing of the ribosomal protein 51 intron in vegetative rna2 cells but did not detectably affect the level of ribosomal protein mRNA in sporulating cells. We concluded that the rna2 mutation and sporulation conditions affected ribosomal protein mRNA metabolism in two quite different ways. In vegetative cells the mutant rna2 effected a block which occurred primarily in post-transcriptional processing, whereas in sporulating cells the ribosomal protein mRNA levels were decreased by some other mechanism, presumably a change in the relative rate of transcription or mRNA turnover. Furthermore, the data suggest that the mutation rna2 has no additional effect on ribosomal protein mRNA metabolism in sporulating cells.
Revertants of an S49 cell mutant that expresses altered cyclic AMP-dependent protein kinase.
Authors: van Daalen Wetters T , Coffino P
Published: September 2015

Dibutyryl adenosine 3',5'-phosphate (Bt2cAMP)-sensitive (Bt2cAMPS) revertants were isolated from a resistant S49 cell mutant carrying a structural gene lesion in the regulatory subunit of cAMP-dependent protein kinase (cA-PK). This was accomplished with a counter-selection in which, first, Bt2cAMP was used to reversibly arrest revertants, and then a sequence of treatments with bromodeoxyuridine, 33258 Hoechst dye, and white light was used to kill cycling mutant cells. Reversion rates in nonmutagenized cultures could not be accurately measured, but spontaneous revertants do occur and with frequencies of less than 10(-7) to 10(-5). The mutagens ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitro-soguanidine (MNNG), and ICR191 increased the reversion frequency. In all cases, reversion to Bt2cAMP sensitivity was associated with restoration of wild-type levels and apparent activation constant for cAMP of cA-PK. MNNG induced revertants whose cell extracts contained cA-PK activity distinguishable from that of wild type by thermal liability. EMS did not. The counter-selection effectively isolates rare phenotypes and is therefore a useful tool in further somatic genetic experiments. The association of reversion with alterations in cA-PK function supports all previous data from this and other laboratories implicating cA-PK as the intracellular mediator of cAMP effects. Reversion is probably the result of a mutational event. Induction of reversion by ICR191 suggests the existence of a novel mechanism for generating revertants in somatic cells.
Polyacrylamide gel mapping of chicken tRNA: comparison of polysome-bound and whole-cell tRNA from normal and avian sarcoma virus-infected chicken embryo fibroblasts.
Authors: Reinisch F , Heyman T
Published: September 2015

Analysis of the tRNA population from chicken cells was performed by means of polyacrylamide gel mapping. About 60 species were detected; most of these were positively identified by their acceptor specificity. The comparison of polysome-bound and overall cellular tRNA gel patterns from normal and Rous sarcoma virus-infected chicken embryo fibroblasts led us to the following observations: some tRNA species were present in the same relative proportions in all the preparations, and within isoaccepting groups the same species was preponderant; however, although about 8% of whole-cell tRNA was recovered in polysomal preparations, amounts ranging from 3 to 30% were found for individual tRNA species. This points to the absence of a direct correlation between the amount of each mature tRNA species produced and the frequency with which it is used in this case of embryonic cells. No significant difference was observed between the whole-cell tRNA patterns from normal and infected cells. Thus, tRNA transcription appears unaltered when cells are transformed and virus producing. No change was observed in the extent of a post-transcriptional modification of tRNAPhe (the base Y). However, viral infection led to some changes in the relative proportions of individual species from polysomal preparations.
Somatic cells efficiently join unrelated DNA segments end-to-end.
Authors: Wilson J H , Berget P B , Pipas J M
Published: September 2015

Molecular substrates for probing nonhomologous recombination in somatic cells were constructed by inserting pBR322 sequences at selected sites on the simian virus 40 (SV40) genome. The chimeric products are too large to be packaged into an SV40 capsid. Therefore, production of viable progeny requires that most of the pBR322 sequences be deleted without altering any SV40 sequences that are essential for lytic infection. As judged by plaque assay, these recombination events occur at readily detectable frequencies after transfection into CV1 monkey kidney cells. Depending on the site of pBR322 insertion, the infectivities of the full-length circular or linear chimeras ranged from 0.02 to 2% of the infectivity of linear wild-type SV40 DNA. Nucleotide sequence analysis of several recombinant progeny revealed three distinct classes of recombination junction and indicated that the causative recombination events were minimally dependent on sequence homology. Potential mechanisms involving recombination at internal sites or at ends were distinguished by measuring the infectivity of chimeric molecules from which various lengths of pBR322 had been removed. These data support end-to-end joining as the primary mechanism by which DNA segments recombine nonhomologously in somatic cells. This end joining appears to be very efficient, since SV40 genomes with complementary single-stranded tails or with short non-complementary pBR322 tails were comparably infectious. Overall, this study indicates that mammalian somatic cells are quite efficient at the willy-nilly end-to-end joining of unrelated DNA segments.
Calcium control of Saccharomyces cerevisiae actin assembly.
Authors: Greer C , Schekman R
Published: September 2015

Low levels of Ca2+ dramatically influence the polymerization of Saccharomyces cerevisiae actin in KCl. The apparent critical concentration for polymerization (C infinity) increases eightfold in the presence of 0.1 mM Ca2+. This effect is rapidly reversed by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or of 0.1 mM Mg2+. Furthermore, the addition of Ca2+ to polymerized actin causes a reversible increase in the apparent C infinity. In the presence of Ca2+, at actin concentrations below the apparent C infinity, particles of 15 to 50 nm in diameter are seen instead of filaments. These particles are separated from soluble actin when Ca2+-treated filamentous actin is sedimented at high speed; both the soluble and particulate fractions retain Ca2+-sensitive polymerization. The Ca2+ effect is S. cerevisiae actin-specific: the C infinity for rabbit muscle actin is not affected by the presence of Ca2+ and S. cerevisiae actin. Ca2+ may act directly on S. cerevisiae actin to control the assembly state in vivo.
Adenovirus type 5 induces progression of quiescent rat cells into S phase without polyamine accumulation.
Authors: Cheetham B F , Shaw D C , Bellett A J
Published: September 2015

Adenovirus type 5 induces cellular DNA synthesis and thymidine kinase in quiescent rat cells but does not induce ornithine decarboxylase. We now show that unlike serum, adenovirus type 5 fails to induce S-adenosylmethionine decarboxylase or polyamine accumulation. The inhibition by methylglyoxal bis(guanylhydrazone) of the induction of thymidine kinase by adenovirus type 5 is probably unrelated to its effects on polyamine biosynthesis. Thus, induction of cellular thymidine kinase and DNA replication by adenovirus type 5 is uncoupled from polyamine accumulation.
Chinese hamster cell mutant resistant to ML236B (Compactin) is defective in endocytosis of low-density lipo-protein.
Authors: Masuda A , Akiyama S , Kuwano M
Published: September 2015

A fungal metabolite, ML236B (Compactin), isolated from Penicillium citrinum, is a specific inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase (EC 1.1.1.34). Three ML236B-resistant (ML236Br) mutants, MF-1, MF-2, and MF-3, were isolated from V79 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The fluctuation test showed 2.2 X 10(-6) mutants per cell per generation of a spontaneous mutation frequency of ML236Br clones. These ML236Br clones showed a four- to fivefold-higher resistance to the drug than did their parental V79. Radioactive acetate, but not mevalonate, incorporation into the sterol fraction increased about 10-fold in ML236Br clones in comparison with that in V79. The cellular level of HMG-coenzyme A reductase in three ML236Br mutants was found to be a few-fold higher than that of V79 when cultured in the presence of lipoproteins. The 125I-labeled low-density lipoprotein-binding assay showed binding activity in three ML236Br clones comparable to that of the parental V79 cells. By contrast, an internalization assay of 125I-labeled low-density lipoprotein into the cells showed significantly reduced activity in three ML236Br clones in comparison with V79.
DNA sequence analysis of a mouse pro alpha 1 (I) procollagen gene: evidence for a mouse B1 element within the gene.
Authors: Monson J M , Friedman J , McCarthy B J
Published: September 2015

In a 3.8-kilobase mouse DNA sequence encoding amino acid sequences for the pro alpha 1(I) chain of type I procollagen, 14 coding sequences were identified which specify a sequence 95% homologous to amino acid residues 568 to 963 of the bovine alpha 1(I) chain. All of these coding sequences were flanked by appropriate splice junctions following the GT/AG rule. These observations suggest, but do not prove, that this pro alpha 1(I) gene is transcriptionally active. Of the 14 coding sequences, 7 were 54 base pairs in length, whereas the remainder were higher multiples of 54 base pairs. Nonrandom utilization of codons pertained throughout all of the coding sequences showing a preference (56%) for U in the wobble position. Two of the intervening sequences encoded imperfect vestiges of coding sequences which exhibited a codon preference different from that of the pro alpha 1(I) gene proper and were not flanked by splice junctions. One intervening sequence encoded a member of the mouse B1 family of middle repetitive sequences. It was flanked by 8-base-pair direct repeats and had a truncated A-rich region, suggesting that it may be a mobile element. Within this element were sequences which could function as a RNA polymerase III split promoter.
Chromosomes XIV and XVII of Saccharomyces cerevisiae constitute a single linkage group.
Authors: Klapholz S , Esposito R E
Published: September 2015

We present several lines of evidence that chromosomes XIV and XVII of Saccharomyces cerevisiae are not independent chromosomes, but rather constitute a single linkage group. Studies which made use of a new mapping method based on the haploidization-without-recombination meiotic phenotype of the spoll mutant initially indicated that markers on chromosomes XIV and XVII were linked. Tetrad analysis was used to establish gene-gene distances, and a new chromosome XIV map incorporating markers originally assigned to chromosome XVII was derived. During the course of trisomic segregation studies, we discovered that a 2n + 2 homothallic diploid, originally believed to be tetrasomic for chromosome XVII (now XIV), carries two normal chromosome XIV homologs and two aberrant homologs which appear to be deficient for a large portion of the right arm of XIV. The previous evidence that established chromosome XVII as an independent linkage group is discussed in the light of these findings.
Properties and Possible Functions of the Adenylate Cyclase in Plasma Membranes ofSaccharomyces cerevisiae
Authors: Jaynes Patrick K. , McDonough James P. , Mahler Henry R.
Published: September 2015

We have examined the possible role of adenosine 3′,5′-phosphate (cAMP) in functions associated with the plasma membranes ofSaccharomyces cerevisiae.Purified membranes from this source contained an adenylate cyclase which was insensitive to activation by fluoride or guanine nucleotides, only weakly responsive to changes of carbon source in the growth medium, and strongly stimulated by vanadate. They also contained at least two classes of receptor proteins for guanine nucleotides (as measured by binding of labeled 5′-guanylyl methylene diphosphate) with apparent dissociation constants equal to 1.0 × 10−7and 3 × 10−6M, a protein kinase capable of phosphorylating added histones, the activity of which was stimulated by cAMP, and cAMP receptors that may function as regulatory subunits for this kinase. Membrane proteins were also susceptible to phosphorylation by endogenous kinase(s), with polypeptides of apparent molecular weights equal to 160 × 103, 135 × 103, 114 × 103, and 58 × 103as the major targets. Of these, the 114,000-molecular-weight polypeptide was probably identical to the proton-translocating ATPase of the membranes. However, the cAMP-dependent protein kinase did not appear to be involved in these reactions. Intact (rho+orrho0) cells responded to dissipation of the proton electrochemical gradient across their plasma membranes by rapid and transient changes in their intracellular level of cAMP, as suggested earlier (J. M. Trevillyan and M. L. Pall, J. Bacteriol.,138:397-403, 1979). Thus, although yeast plasma membranes contain all the essential components of a stimulus-responsive adenylate cyclase system, the precise nature of the coupling device and the targets involved remain to be established.
Identification of the genetic locus for the structural gene and a new regulatory gene for the synthesis of repressible alkaline phosphatase in Saccharomyces cerevisiae.
Authors: Kaneko Y , Toh-e A , Oshima Y
Published: September 2015

Two lines of evidence showed that the PHO8 gene encodes the structure of repressible, nonspecific alkaline phosphatase in Saccharomyces cerevisiae: (i) the enzyme produced by a temperature-sensitive pho8 mutant at the permissive temperature (25 degrees C) was more thermolabile than that of the wild-type strain, and (ii) the PHO8 gene showed a gene dosage effect on the enzyme activity. The pho8 locus has been mapped on chromosome IV, 8 centimorgans distal to rna3. A new mutant carrying the pho9 gene was isolated which lacks repressible alkaline phosphatase, but has the normal phenotype for the synthesis of repressible acid phosphatase. The pho9 gene segregated independently of all known pho-regulatory genes and did not show the gene dosage effect on repressible alkaline phosphatase activity. The pho9/pho9 diploid hardly sporulated and showed no commitment to intragenic recombination when it was inoculated on sporulation medium. Hence the pho9 mutant has a phenotype similar to the pep4 mutant, which was isolated as a pleiotropic mutant with reduced levels of proteinases A and B and carboxypeptidase Y. An allelism test indicated that pho9 and pep4 are allelic.
Developmental regulation of a sporulation-specific enzyme activity in Saccharomyces cerevisiae.
Authors: Clancy M J , Smith L M , Magee P T
Published: September 2015

An alpha-glucosidase activity (SAG) occurs in a/alpha Saccharomyces cerevisiae cells beginning at about 8 to 10 h after the initiation of sporulation. This enzyme is responsible for the rapid degradation of intracellular glycogen which follows the completion of meiosis in these cells. SAG differs from similar activities present in vegetative cells and appears to be a sporulation-specific enzyme. Cells arrested at various stages in sporulation (DNA replication, recombination, meiosis I, and meiosis II) were examined for SAG activity; the results show that SAG appearance depends on DNA synthesis and some recombination events but not on the meiotic divisions.
Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA.
Authors: Zakian V A , Scott J F
Published: September 2015

Transformation studies with Saccharomyces cerevisiae (bakers' yeast) have identified DNA sequences which permit extrachromosomal maintenance of recombinant DNA plasmids in transformed cells. It has been hypothesized that such sequences (called ARS for autonomously replicating sequence) serve as initiation sites for DNA replication in recombinant DNA plasmids and that they represent the normal sites for initiation of replication in yeast chromosomal DNA. We have constructed a novel plasmid called TRP1 R1 Circle which consists solely of 1,453 base pairs of yeast chromosomal DNA. TRP1 RI Circle contains both the TRP1 gene and a sequence called ARS1. This plasmid is found in 100 to 200 copies per cell and is relatively stable during both mitotic and meiotic cell cycles. Replication of TRP1 RI Circle requires the products of the same genes (CDC28, CDC4, CDC7, and CDC8) required for replication of chromosomaL DNA. Like chromosomal DNA, its replication does not occur in cells arrested in the B1 phase of the cell cycle by incubation with the yeast pheromone alpha-factor. In addition, TRP1 RI Circle DNA is organized into nucleosomes whose size and spacing are indistinguishable from that of bulk yeast chromatin. These results indicate that TRP1 RI Circle has the replicative and structural properties expected for an origin of replication from yeast chromosomal DNA. Thus, this plasmid is a suitable model for further studies of yeast DNA replication in both cells and cell-free extracts.
Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells.
Authors: Tischfield J A , Trill J J , Lee Y I , Coy K , Taylor M W
Published: September 2015

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.
Repression of the albumin gene in Novikoff hepatoma cells.
Authors: Capetanaki Y G , Flytzanis C N , Alonso A
Published: September 2015

Novikoff hepatoma cells have lost their capacity to synthesize albumin. As a first approach to study the mechanisms underlying this event, in vitro translation in a reticulocyte system was performed using total polyadenylated mRNA from rat liver and Novikoff hepatoma cells. Immunoprecipitation of the in vitro translation products with albumin-specific antibody revealed a total lack of albumin synthesis in Novikoff hepatoma, suggesting the absence of functional albumin mRNA in these cells. Titration experiments using as probe albumin cDNA cloned in pBR322 plasmid demonstrated the absence of albumin-specific sequences in both polysomal and nuclear polyadenylated and total RNA from Novikoff cells. This albumin recombinant plasmid was obtained by screening a rat liver cDNA library with albumin [32P]cDNA reverse transcribed from immuno-precipitated mRNA. The presence of an albumin-specific gene insert was documented with translation assays as well as by restriction mapping. Repression of the albumin gene at the transcriptional level was further demonstrated by RNA blotting experiments using the cloned albumin cDNA probe. Genomic DNA blots using the cloned albumin cDNA as probe did not reveal any large-scale deletions, insertions, or rearrangements in the albumin gene, suggesting that the processes involved in the suppression of albumin mRNA synthesis do not involve extensive genomic rearrangements.
Coordinate expression of parietal endodermal functions in hybrids of embryonal carcinoma and endodermal cells.
Authors: Howe W E , Oshima R G
Published: September 2015

A derivative, FOT5, of the F9 murine embryonal carcinoma cell line which is resistant to ouabain and thioguanine was fused with a near diploid parietal endodermal cell line, PFHR9, Hybrid clones (ENEC1 to ENEC5) were isolated in HAT Medium containing ouabain at a frequency of approximately 2 x 10(-4). The DNA contents and chromosome number of the ENEC hybrids were approximately the sum of those of the parents. Five hybrid cell lines examined in detail expressed the following parietal endodermal functions: plasminogen activator activity, basement membrane proteins, and endodermal cytoskeletal proteins. Embryonal carcinoma characteristic functions (tumorigenicity, a stage specific embryonic antigen, and high alkaline phosphatase activity) were extinguished in the hybrids. No hybrid clones with embryonal carcinoma morphology were observed among 1,358 hybrid clones examined. Hybrids, propagated for over 100 generations, continued to express endodermal functions and not embryonal carcinoma functions. The coordinate expression of endodermal functions and the extinction of embryonal carcinoma functions in the ENEC hybrids suggest that the parietal endodermal cells contain diffusible activities which extinguish embryonal carcinoma functions and possibly cause the embryonal carcinoma genome to express parietal endodermal characteristics.
Introduction and expression of a fetal human globin gene in mouse fibroblasts.
Authors: Hsiung N , Roginski R S , Henthorn P , Smithies O , Kucherlapati R , Skoultchi A I
Published: September 2015

An 8.5-kilobase segment of cloned human DNA including the complete G gamma-globin gene was introduced into LMTK- cells by the calcium phosphate precipitation method in the presence or absence of carrier DNA. Transfectants containing one or more copies of intact G gamma-globin genes were obtained either by ligation of the human DNA segment to a plasmid containing the herpes simplex virus thymidine kinase gene or by nonligated cotransfer. The integrity of the integrated gamma-globin gene was established by Southern blotting experiments. Expression of the herpes simplex virus thymidine kinase and human gamma-globin genes was evaluated by Northern blotting and solution hybridization. Of 23 transfectants analyzed, 21 produced a 9S gamma-globin RNA migrating like authentic gamma-globin mRNA on denaturing agarose gels. The gamma-globin RNA is polyadenylated and present in the cytoplasm of the transfected cells; it accumulates to a level 10 times that of thymidine kinase mRNA, or about 5 to 50 molecules per transfected cell. By using plasmids in which the gamma-gene is inserted in either transcriptional orientation with respect to the thymidine kinase gene, it was possible to show that transcription occurred from the gamma-gene promoter.
Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene.
Authors: Tabin C J , Hoffmann J W , Goff S P , Weinberg R A
Published: September 2015

We investigated the feasibility of using retroviruses as vectors for transferring DNA sequences into animal cells. The thymidine kinase (tk) gene of herpes simplex virus was chosen as a convenient model. The internal BamHI fragments of a DNA clone of Moloney leukemia virus (MLV) were replaced with a purified BamHI DNA segment containing the tk gene. Chimeric genomes were created carrying the tk insert in both orientations relative to the MLV sequence. Each was transfected into TK- cells along with MLV helper virus, and TK+ colonies were obtained by selection in the presence of hypoxanthine, aminopterin, and thymidine (HAT). Virus collected from TK+-transformed, MLV producer cells passed the TK+ phenotype to TK- cells. Nonproducer cells were isolated, and TK+ transducing virus was subsequently rescued from them. The chimeric virus showed single-hit kinetics in infections. Virion and cellular RNA and cellular DNA from infected cells were all shown to contain sequences which hybridized to both MLV- and tk-specific probes. The sizes of these sequences were consistent with those predicted for the chimeric virus. In all respects studied, the chimeric MLV-tk virus behaved like known replication-defective retroviruses. These experiments suggest great general applicability of retroviruses as eucaryotic vectors.
Oligoadenylate is present in the mitochondrial RNA of Saccharomyces cerevisiae.
Authors: Yuckenberg P D , Phillips S L
Published: September 2015

We examined Saccharomyces cerevisiae mitochondrial RNA for polyadenylate. Using hybridization to [3H]polyuridylate as the assay for adenylate sequences, we found adenylate-rich oligonucleotides approximately 8 residues long. Longer polyadenylate was not detected. Most of the adenylate-rich sequence is associated with the large mitochondrial rRNA. The remainder is associated with the 10-12S group of transcripts.
Isolation and characterization of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-resistant mutants of the Chinese hamster ovary cell line.
Authors: Funanage V L
Published: September 2015

Mutants resistant to the RNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofurano-sylbenzimidazole (DRB) have been isolated in the Chinese hamster ovary cell line CHO-K1. Three independently isolated mutants, DRB6 DRB10, and DRB13, were 3-, 5-, and 3.5-fold, respectively, more resistant to DRB than the parental cell line WTCHO. The DRB-resistant mutations were expressed codominantly in somatic cell hybrids of DRB-resistant and DRB-sensitive cell lines. In vivo treatment of CHO-K1 cells with DRB resulted in specific inhibition of endogenous RNA polymerase II activity in cell lysates. Whereas DRB inhibited RNA polymerase II activity in WTCHO cells by a maximum of 60% at concentrations as low as 60 microM, 300 microM DRB was required to inhibit 60% of the RNA polymerase II activity in DRB10 cells. However, the inhibition of the DRB-sensitive RNA polymerase II activity in DRB10 was biphasic. About half (53 to 56%) of this activity was inhibited by 90 microM DRB and thus showed a DRB sensitivity similar to the wild-type RNA polymerase II activity; the remaining DRB-sensitive RNA polymerase II activity was maximally inhibited by 300 microM DRB. These results indicated that there were two copies of the drbR locus (drb+ and drbR-10) in DRB10 and confirmed that the drbR-10 mutation was expressed codominantly. Somatic cell hybrids of DRB-resistant and alpha-amanitin-resistant cell lines grew in medium containing both DRB and alpha-amanitin, demonstrating that the drbR and amaR mutations were not in the same gene. Thus, the drbR mutations may define an additional component of the RNA polymerase II transcriptional complex in mammalian cells.
Massive heat-shock polypeptide synthesis in late chicken embryos: convenient system for study of protein synthesis in highly differentiated organisms.
Authors: Voellmy R , Bromley P A
Published: September 2015

In cultured eucaryotic cells, heat treatments specifically induced the rapid synthesis of the so-called heat-shock polypeptides. To ascertain the physiological importance of this phenomenon for highly differentiated organisms, we attempted to determine whether the heat-shock response occurs in a living endothermic organism at extreme temperatures, and if so, whether the response is organ specific. We developed a procedure to label proteins efficiently in 5- to 18-day-old chicken embryos. Heat-shock polypeptides of identical sizes of 85,000, 70,000, and 25,000 daltons were synthesized predominantly in chicken embryo fibroblasts and in many different organs of 18-day-old embryos at 42.5 to 44 degrees C.
Phosphorylation of the major heat shock protein of Dictyostelium discoideum.
Authors: Loomis W F , Wheeler S , Schmidt J A
Published: September 2015

The major heat shock protein, hsp 70, of Dictyostelium discoideum was found to be rapidly phosphorylated. Analysis of [35S]methionine- and 32Pi-labeled hsp 70 revealed that two similar but distinct proteins of about 70,000 daltons each are synthesized at a high rate after a heat shock, and that each has a phosphorylated member. The phosphorylation chiefly modifies threonine residues. Rapid turnover of the phosphate group occurs, resulting in a steady-state condition in which only about half of the hsp 70 is phosphorylated at a given time.
Dependence of nucleus-directed rRNA synthesis upon mitochondrial protein synthesis in Tetrahymena.
Authors: Ruben L , Hooper A B
Published: September 2015

The antibiotic chloramphenicol selectively inhibited mitochondrial protein synthesis in the ciliate protozoan Tetrahymena pyriformis GL. Secondary to the inhibition of mitochondrial protein synthesis was an inhibition of nuclear RNA synthesis at a time before inhibition of cellular protein and DNA synthesis. Of the stable non-polyadenylated RNA species in Tetrahymena, the addition of chloramphenicol resulted specifically in the inhibition of synthesis of 28S + 17S and 5S rRNA transcripts. By contrast, syntheses of 4S tRNA and 21S mitochondrial rRNA were not as extensively inhibited. The addition of 60 microM hemin before the addition of chloramphenicol partially protected against the inhibition of RNA synthesis. These data indicate that continued synthesis of nucleus-directed rRNA is linked to the synthesis of mitochondrial proteins in Tetrahymena.
Chinese hamster ovary cell mutants defective in the internalization of ricin.
Authors: Ray B , Wu H C
Published: September 2015

Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.
Human beta interferon gene localization and expression in somatic cell hybrids.
Authors: Pitha P M , Slate D L , Raj N B , Ruddle F H
Published: September 2015

The human fibroblast interferon gene beta 1 was mapped to human chromosome 9. Sequence homology with a beta 1 cDNA clone was detected in both genomic DNA and induced mRNA of human/mouse or human/hamster somatic cell hybrids containing human chromosome 9, but not in lines lacking this chromosome or those retaining a complex translocation involving chromosomes 9 and 11. Interferon mRNA that did not share sequence homology with the beta 1 cDNA clone was detected in lines containing human chromosomes 2 and 5 but lacking chromosome 9, suggesting the presence of other unlinked interferon sequences in the human genome.
General method for cloning amplified DNA by differential screening with genomic probes.
Authors: Brison O , Ardeshir F , Stark G R
Published: September 2015

Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite.This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or more can be cloned, even if no function for the region is known. The method for removing highly repetitive sequences from genomic DNA probes should also be of general use.
Saccharomyces cerevisiae spheroplasts are sensitive to the action of diphtheria toxin.
Authors: Murakami S , Bodley J W , Livingston D M
Published: September 2015

Diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae but not the intact yeast cells. After 2 h of exposure to ca. 10(-7) M toxin, less than 1% of spheroplasts were able to regenerate into intact cells. The same high levels of toxin inhibited the rate of protein synthesis by more than 90% within 1 h, whereas RNA and DNA synthesis were not inhibited until 4 h or exposure. Both killing and protein synthesis inhibition were dependent on toxin concentration. The nature of the toxin-cell interaction was also studied by using fragments of intact toxin and mutant toxin proteins. Neither toxin fragment A nor CRM45 nor CRM197 affected spheroplasts, but CRM197 and ATP prevented the inhibitory action of intact toxin. These results suggest that toxin acts on S. cerevisiae spheroplasts in much the same manner as it acts on sensitive mammalian cells.
Heterogeneity in the Rate of Benzo[a]pyrene Metabolism in Single Cells: Quantitation Using Flow Cytometry
Authors: Miller Arthur G. , Whitlock James P.
Published: September 2015

We describe a method for quantitating heterogeneity in the rate of benzo[a]pyrene metabolism in single cells by using flow cytometry. We have used the technique to study the response of Hepa-1c1c7 mouse hepatoma cells to the microsomal enzyme inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cells responded in a relatively homogeneous fashion at different times of induction with a maximally inducing concentration of the inducer. However, the induction response could be heterogeneous at a submaximal inducer concentration. We found even higher heterogeneity of enzyme activity among low-activity variants derived from the Hepa-1c1c7 cell line. When cells of either high or low activity were isolated from such a clonal population, propagated, and reanalyzed, they displayed average enzyme activity and heterogeneity identical to the parental cells; therefore, the heterogeneity represents transient, nonheritable differences between cells within the population.
Role for Endocytosis in Conjugation inTetrahymena
Authors: Rotheim Minna B. , Love Bruce
Published: September 2015

We examined the effect of inhibitors of receptor-mediated endocytosis on cell pair formation during conjugation inTetrahymena thermophila.Dansylcadaverine (20 μM), methylamine (20 mM), and bacitracin (2 mg/ml) prevented cell pair formation even when added poststarvation, after mixing of cells of opposite mating types (during the prepairing interaction). Chloroquine (10 and 25 μM) did not inhibit cell pair formation, leading to the conclusion that inhibition by dansylcadaverine, methylamine, and bacitracin is not due to an alkalinization of the lysosome. These results did not allow us to define the time in the prepairing interaction at which inhibition occurs, nor to identify the cellular components involved, but they did support the hypothesis that an endocytotic event(s) plays a role in the cell contact-mediated recognition which occurs during the prepairing interaction.
Quantitation of a 55K cellular protein: similar amount and instability in normal and malignant mouse cells.
Authors: Mora P T , Chandrasekaran K , Hoffman J C , McFarland V W
Published: September 2015

Quantitative expression of a specific 55,000 (55K)-molecular-weight cellular protein was studied in two groups of mouse embryo fibroblast (clonal) cells originating from two parent clones, one of which possessed high tumorigenicity and the other of which possessed very low tumorigenicity. From the clone with low tumorigenicity, tumor lines and clones were obtained by selecting rare spontaneously transformed highly tumorigenic (mutant) cells. Cells were labeled during exponential growth for 3 h at 37 degrees C, with [35S]methionine, and the cellular 55K protein was immunoprecipitated with a monoclonal antibody and quantitated. There were low and approximately equal amounts of 55K protein in cells (clones) with both low and high tumorigenicity from both groups of cells, and there was no correlation at all between quantitative expression of 55K protein and of cellular tumorigenicity. There was approximately 10- to 20-fold more 55K protein in all simian virus 40-transformed T antigen-positive derivative clones, as shown previously. The T antigen-negative revertant tumor lines and clones obtained by an immunological in vivo selection method had low amounts of 55K protein, similar to the parent cell before simian virus 40 transformation. In all of the T antigen-negative cells, including the highly tumorigenic cells, degradation (turnover?) of the 55K protein was rapid, and a half-life of 15 to 60 min was estimated from pulse-chase experiments. In all of the T antigen-positive cells the 55K protein was stable (half-life greater than 10 h). In primary cells established from the tumors induced by highly tumorigenic cells there was a very low or no detectable amount of the 55K protein. This is in contrast to the primary cells obtained from early murine embryos in which we have reported high amounts of (stable) 55K proteins.
Genetics of the mammalian oxidative phosphorylation system: characterization of a new oligomycin-resistant Chinese hamster ovary cell line.
Authors: Breen G A
Published: September 2015

The properties of a new type of oligomycin-resistant Chinese hamster ovary (CHO) cell line (Olir 2.2) are described in this paper. Olir 2.2 cells were approximately 50,000-fold more resistant to oligomycin than were wild-type CHO cells when tested in glucose-containing medium, but only 10- to 100-fold more resistant when tested in galactose-containing medium. Olir 2.2 cells grew with a doubling time similar to that of wild-type cells both in the presence or absence of oligomycin. Oligomycin resistance in Olir 2.2 cells was stable in the absence of drug. In vitro assays indicated that there was approximately a 25-fold increase in the resistance of the mitochondrial ATPase to inhibition by oligomycin in Olir 2.2 cells, with little change in the total ATPase activity. The electron transport chain was shown to be functional in Olir 2.2 cells. Olir 2.2 cells were cross-resistant to other inhibitors of the mitochondrial ATPase (such as rutamycin, ossamycin, peliomycin, venturicidin, leucinostatin, and efrapeptin) and to other inhibitors of mitochondrial functions (such as chloramphenicol, rotenone, and antimycin). Oligomycin resistance was expressed codominantly in hybrids between Olir 2.2 cells and wild-type cells. Cross-resistance to ossamycin, peliomycin, chloramphenicol, antimycin, venturicidin, leucinostatin, and efrapeptin was also expressed codominantly in hybrids. Fusions of enucleated Olir 2.2 cells with wild-type cells and characterization of the resulting cybrid clones indicated that resistance to oligomycin and ossamycin results from a mutation in both a nuclear gene and a cytoplasmic gene. Cross-resistance to efrapeptin, leucinostatin, venturicidin, and antimycin results from a mutation in only a nuclear gene.
Deletion mutants which affect the nuclease-sensitive site in simian virus 40 chromatin.
Authors: Gerard R D , Woodworth-Gutai M , Scott W A
Published: September 2015

A short segment of simian virus 40 (SV40) chromatin on the late side of the origin of replication is hypersensitive to nuclease cleavage. The role of DNA sequence information in this nuclease-sensitive feature was examined by constructing deletion mutations in this region. Deletions were introduced into the inserted segment of in(Or)-1411 (a viable, partially duplicated variant of SV40), and nuclease sensitivity of the inserted segment was compared with that of the unaltered sequences in their normal location in the viral genome. Extended deletions (118 to 161 base pairs) essentially abolished nuclease sensitivity within the inserted segment. Shorter deletions (21 to 52 base pairs) at separate locations retained the nuclease-sensitive feature. In some short-deletion mutants nuclease susceptibility was substantially reduced. We conclude that more than one genetic element in this region contributes to the organization of the nuclease-sensitive feature and that the SV40 72-base repeat is not, in itself, sufficient signal for this feature.
Selective inhibition of adenovirus type 2 early region II and III transcription by an anisomycin block of protein synthesis.
Authors: Shaw A R , Ziff E B
Published: September 2015

The transcription of adenovirus type 2 genes proceeds through a broad three-phase program. From 1 to 4 h postinfection six early transcription units (EIa, EIb, EII, EIII, EIV, and the promoter-proximal segment of the late transcription unit) are activated. From 4 to 6 h postinfection transcription of the early genes is depressed. After the onset of viral DNA replication at approximately 6 h postinfection, the transcript from the late promoter is antiterminated, and this transcript dominates viral RNA synthesis. The early activation period also proceeds through a series of stages; early regions EIa and EIV are activated first, followed by early region EII. We show that in the presence of anisomycin, a stringent inhibitor of protein synthesis, nuclear RNA and cytoplasmic polyadenylated RNA from regions EIa and EIV accumulate at normal rates, whereas RNAs from regions EII and EIII do not accumulate. We also show that failure to accumulate RNAs from regions EII and EIII is due to reduction of the rate of transcription by greater than 90%. We conclude that the regulation of the promoters for early regions EII and EIII is distinct from the mechanism that operates on the other early transcription units. The promoters for early regions EII and EIII diverge and lie approximately 500 nucleotides apart. We examined the structure of viral chromatin in this region early during infection by mild DNase I digestion in isolated nuclei and indirect end labeling with a DNA probe near these promoters. In control, drug-free cells where EII and EIII are transcribed and in anisomycin-treated cells where EII and EIII are not transcribed we detect the same regular DNase I pattern. We suggest that regulation of EII and EIII is not mediated through a change in gross chromatin structure, but rather by a viral effector, possibly a product of region EIa.
Post-translational processing of urea amidolyase in Saccharomyces cerevisiae.
Authors: Sumrada R A , Chisholm G , Cooper T G
Published: September 2015

Urea amidolyase catalyzes the two reactions (urea carboxylase and a allophanate hydrolase) associated with urea degradation in Saccharomyces cerevisiae. Past work has shown that both reactions are catalyzed by a 204-kilodalton, multifunctional protein. In view of these observations, it was surprising to find that on induction at 22 degrees C, approximately 2 to 6 min elapsed between the appearance of allophanate hydrolase and urea carboxylase activities. In search of an explanation for this apparent paradox, we determined whether or not a detectable period of time elapsed between the appearance of allophanate hydrolase activity and activation of the urea carboxylase domain by the addition of biotin. We found that a significant portion of the protein produced immediately after the onset of induction lacked the prosthetic group. A steady-state level of biotin-free enzyme was reached 16 min after induction and persisted indefinitely thereafter. These data are consistent with the suggestion that sequential induction of allophanate hydrolase and urea carboxylase activities results from the time required to covalently bind biotin to the latter domain of the protein.
DNA content, kinetic complexity, and the ploidy question in Candida albicans.
Authors: Riggsby W S , Torres-Bauza L J , Wills J W , Townes T M
Published: September 2015

Candida albicans is a dimorphic fungus that is pathogenic for humans. No sexual cycle has been reported for this fungus, and earlier reports have differed on whether typical strains of C. albicans are haploid or diploid. Previous estimates of the DNA content of C. albicans varied by one order of magnitude. We used three independent methods to measure the kinetic complexity of the single-copy DNA from a typical strain of C. albicans (strain H317) to determine the DNA content per haploid genote; we obtained values of 15 and 20 fg per cell by using S1 nuclease and hydroxyapatite assays, respectively. Optical assays for DNA reassociation kinetics, although not definitive in themselves, yielded values in this range. Chemical measurements of the DNA content of several typical strains, including strain H317, yielded values clustered about a mean of 37 fg per cell. We concluded that these strains are diploid.