bluuebunny/crossref_metadata_2025 huggingface dataset:
Repeated Family of Genes Controlling Maltose Fermentation inSaccharomyces carlsbergensis
Authors: Needleman Richard B. , Michels Corinne
Published: September 2015

Maltose fermentation inSaccharomycesspp. requires the presence of any one of five unlinked genes:MAL1, MAL2, MAL3, MAL4, orMAL6.Although the genes are functionally equivalent, their natures and relationships to each other are not known. At least three proteins are necessary for maltose fermentation: maltase, maltose permease, and a regulatory protein. TheMALgenes may code for one or more of these proteins. Recently a DNA fragment containing a maltase structural gene has been cloned from aMAL6strain, CB11, to produce plasmid pMAL9-26. We have conducted genetic and physical analyses of strain CB11. The genetic analysis has demonstrated the presence of two crypticMALgenes in CB11,MAL1gandMAL3g(linked toMAL1and toMAL3, respectively), in addition to theMAL6locus. The physical analysis, which used a subclone of plasmid pMAL9-26 as a probe, detected threeHindIII genomic fragments with homology to the probe. Each fragment was shown to be linked to one of theMALloci genetically demonstrated to be present in CB11. Our results indicate that the cloned maltase structural gene in plasmid pMAL9-26 is linked toMAL6.Since theMAL6locus has previously been shown to contain a regulatory gene, theMAL6locus must be a complex locus containing at least two of the factors needed for maltose fermentation: the structural gene for maltase and the maltase regulatory protein. The absence of other fragments which hybridize to theMAL6-derived probe shows that eitherMAL2andMAL4are not related toMAL6, or the DNA corresponding to these genes is absent from theMAL6strain CB11.
Control of cellular gene expression during adenovirus infection: induction and shut-off of dihydrofolate reductase gene expression by adenovirus type 2.
Authors: Yoder S S , Robberson B L , Leys E J , Hook A G , Al-Ubaidi M , Yeung C Y , Kellems R E , Berget S M
Published: September 2015

Infection of human cells by adenovirus results in multiple alterations of host gene expression. To examine the effects of viral infection on the expression of a single gene, a line of human cells was developed which is resistant to growth in methotrexate and which contains amplified RNA and protein specific for dihydrofolate reductase (DHFR). Cytogenetic evidence indicated the presence of amplified DNA. Adenovirus infection of these cells caused an induction and subsequent decline in the synthesis of DHFR protein. The maximum DHFR induction occurred 16 to 19 h after infection and reached a level 2.5-fold greater than that observed in uninfected cells. Induction of DHFR protein synthesis was accompanied by concomitant increases in the level of steady-state DHFR-specific cytoplasmic RNA. The relative rate of DHFR mRNA production (i.e., the appearance of DHFR-specific mRNA sequences in the cytoplasm) also increased 2.5-fold during induction. Later in infection, the relative rate of DHFR protein synthesis declined, reaching a level below that observed in uninfected cells. This decline was accompanied by a similar decline in the steady-state levels of DHFR RNA and in the relative rate of synthesis of DHFR mRNA. These data suggest that adenovirus infection controls DHFR gene expression by increasing and subsequently decreasing the relative rate at which DHFR-specific mRNA sequences appear in the cytoplasm and enter the pool of mRNA available for translation.
Physiological control of repressible acid phosphatase gene transcripts in Saccharomyces cerevisiae.
Authors: Bostian K A , Lemire J M , Halvorson H O
Published: September 2015

We have examined the regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum], EC 3.1.3.2) in Saccharomyces cerevisiae at the physiological and molecular levels, through a series of repression and derepression experiments. We demonstrated that APase synthesis is tightly regulated throughout the growth phase and is influenced by exogenous and endogenous Pi pools. During growth in a nonlimiting Pi medium, APase is repressed. When external Pi becomes limiting, there is a biphasic appearance of APase mRNA and enzyme. Our data on APase mRNA half-lives and on the flux of intracellular Pi and polyphosphate during derepression are consistent with a mechanism of transcriptional autoregulation for the biphasic appearance of APase mRNA. Accordingly, preculture concentrations of Pi control the level of corepressor generated from intracellular polyphosphate degradation. When cells are fully derepressed, APase mRNA levels are constant, and the maximal linear accumulation rate of APase is observed. A scheme to integrate phosphorus metabolism and phosphatase regulation in S. cerevisiae is proposed.
Identification of two human beta-tubulin isotypes.
Authors: Hall J L , Dudley L , Dobner P R , Lewis S A , Cowan N J
Published: September 2015

The sequence of a human beta-tubulin cDNA clone (D beta-1) is described; our data revealed 95.6% homology compared with the sequence of a human beta-tubulin processed pseudogene derived by reverse transcription of a processed mRNA (Wilde et al., Nature [London] 297:83-84, 1982). However, the amino acid sequence encoded by this cDNA showed less homology with pig and chicken beta-tubulin sequences than the latter did to each other, with major divergence within the 15 carboxy-terminal amino acids. On the other hand, an independently isolated, functionally expressed genomic human beta-tubulin sequence (5 beta) possessed a very high degree of homology with chicken and pig beta-tubulins in this region. Thus, human cells appear to contain two distinct beta-tubulin isotypes. Both the intact beta-tubulin cDNA clone and a subclone containing only the 3' untranslated region detected two mRNA species in HeLa cells; these mRNAs were 1.8 and 2.6 kilobases long and were present in about equal amounts. Two independently subcloned probes constructed from the 3' untranslated region of the 5 beta genomic sequence also detected a 2.6-kilobase beta-tubulin mRNA. However, the 3'-untranslated-region probes from the cDNA clone and the genomic sequence did not cross-hybridize. Thus, at least two human beta-tubulin genes, each specifying a distinct isotype, are expressed in HeLa cells, and the 2.6-kilobase mRNA band is a composite of at least two comigrating beta-tubulin mRNAs.
Chitin synthesis and localization in cell division cycle mutants of Saccharomyces cerevisiae.
Authors: Roberts R L , Bowers B , Slater M L , Cabib E
Published: September 2015

Growth of Saccharomyces cerevisiae cell cycle mutants cdc3, cdc4, cdc7, cdc24, and cdc28 at a nonpermissive temperature (37 degrees C) resulted in increased accumulation of chitin relative to other cell wall components, as compared with that observed at a permissive temperature (25 degrees C). Wild-type cells showed the same chitin/carbohydrate ratio at both temperatures, whereas mutants cdc13 and cdc21 yielded only a small increase in the ratio at 37 degrees C. These results confirm and extend those reported by B. F. Sloat and J. R. Pringle (Science 200:1171-1173, 1978) for mutant cdc24. The distribution of chitin in the cell wall was studied by electron microscopy, by specific staining with wheat germ agglutinin-colloidal gold complexes. At the permissive temperature, chitin was restricted to the septal region in all strains, whereas at 37 degrees C a generalized distribution of chitin in the cell wall was observed in all mutants. These results do not support a unique interdependence between the product of the cdc24 gene and localization of chitin deposition; they suggest that unbalanced conditions created in the cell by arresting the cycle at different stages result in generalized activation of the chitin synthetase zymogen. Thus, blockage of an event in the cell cycle may lead to consequences that are not functionally related to that event under normal conditions.
Myogenic differentiation of L6 rat myoblasts: evidence for pleiotropic effects on myogenesis by RNA polymerase II mutations to alpha-amanitin resistance.
Authors: Crerar M M , Leather R , David E , Pearson M L
Published: September 2015

To assess the functional role of RNA polymerase II in the regulation of transcription during muscle differentiation, we isolated and characterized a large number of independent alpha-amanitin-resistant (AmaR) mutants of L6 rat myoblasts that express both wild-type and altered RNA polymerase II activities. We also examined their myogenic (Myo) phenotype by determining their ability to develop into mature myotubes, to express elevated levels of muscle creatine kinase, and to synthesize muscle-characteristic proteins as detected by two-dimensional polyacrylamide gel electrophoresis. We found a two- to threefold increase in the frequency of clones with a myogenic-defective phenotype in the AmaR (RNA polymerase II) mutants as compared to control ethyl methane sulfonate-induced, 6-thioguanine-resistant (hypoxanthine, guanine phosphoribosyl transferase) mutants or to unselected survivors also exposed to ethyl methane sulfonate. Subsequent analysis showed that about half of these myogenic-defective AmaR mutants had a conditional Myo(ama) phenotype; when cultured in the presence of amanitin, they exhibited a Myo- phenotype; in its absence they exhibited a Myo+ phenotype. This conditional Myo(ama) phenotype is presumably caused by the inactivation by amanitin of the wild-type amanitin-sensitive RNA polymerase II activity and the subsequent rise in the level of mutant amanitin-resistant RNA polymerase II activity. In these Myo(ama) mutants, the wild-type RNA polymerase II is normally dominant with respect to the Myo+ phenotype, whereas the mutant RNA polymerase II is recessive and results in a Myo- phenotype only when the wild-type enzyme is inactivated. These findings suggest that certain mutations in the amaR structural gene for the amanitin-binding subunit of RNA polymerase II can selectively impair the transcription of genes specific for myogenic differentiation but not those specific for myoblast proliferation.
Saccharomyces cerevisiae cdc2 mutants fail to replicate approximately one-third of their nuclear genome.
Authors: Conrad M N , Newlon C S
Published: September 2015

Chromosomal DNA replication was examined in temperature-sensitive mutants of Saccharomyces cerevisiae defective in a gene required for the completion of S phase at the nonpermissive temperature, 37 degrees C. Based on incorporation of radioactive precursors and density transfer experiments, strains carrying three different alleles of cdc2 failed to replicate approximately one-third of their nuclear genome at 37 degrees C. Whole-cell autoradiography experiments demonstrated that 93 to 96% of the cells synthesized DNA at 37 degrees C. Therefore, all cells failed to replicate part of their genome. DNA isolated from terminally arrested cells was of normal size as measured on neutral and alkaline sucrose gradients, suggesting that partially replicated DNA molecules do not accumulate and that DNA strands are ligated properly in cdc2 mutants. In addition, electron microscopic examination of the equivalent of more than one genome's DNA from arrested cells failed to reveal any partially replicated molecules. The sequences which failed to replicate at 37 degrees C were not highly specific; eight different cloned sequences replicated to the same extent as total DNA. The 2-microns plasmid DNA and rDNA replicated significantly less well than total DNA, but approximately one-half of these sequences replicated at 37 degrees C. These observations suggest that cdc2 mutants are defective in an aspect of initiation of DNA replication common to all chromosomes such that a random fraction of the chromosomes fail to initiate replication at 37 degrees C, but that once initiated, replication proceeds normally.
Temperature-sensitive adult liver cell line dependent on glucocorticoid for differentiation.
Authors: Chou J Y
Published: September 2015

A clonal rat adult hepatocyte cell line (RALA255-10G) was shown to be temperature sensitive (ts) for growth and differentiation. Glucocorticoid was necessary to maintain the maximal levels of differentiated functions in these cells. The RALA255-10G cell line was established by transforming primary adult hepatocytes with simian virus 40 tsA255 virus that is temperature sensitive for maintenance of transformation. At the permissive temperature (33 degrees C), RALA255-10G cells showed characteristics of malignant transformation, synthesized low levels of albumin and transferrin, and contained low levels of functional receptors for glucagon. At the nonpermissive temperature (40 degrees C), these cells regain the normal differentiated phenotype, and the levels of these three hepatic functions were increased. Induction of albumin and transferrin production by RALA255-10G cells at 40 degrees C was shown to be the result of the increase in the biosynthesis of these proteins. Furthermore, the albumin and transferrin produced by these cells were immunologically and electrophoretically indistinguishable from authentic rat albumin and transferrin. Glucocorticoid, which reduced the growth rate and saturation density of RALA255-10G cells at 33 degrees C, was absolutely required by these cells to synthesize albumin at both temperatures. This hormone also enhanced transferrin production and glucagon response. Our data indicate that glucocorticoid hormone is one of the factors that maintain adult hepatocytes in a differentiated state.
Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii.
Authors: Landfear S M , McMahon-Pratt D , Wirth D F
Published: September 2015

The arrangement of developmentally regulated alpha- and beta-tubulin genes has been studied in the parasitic protozoan Leishmania enriettii by using Southern blot hybridization analysis. The alpha-tubulin genes occur in a tandem repeat whose monomeric unit may be represented by a 2-kilobase PstI fragment. Similarly, the beta-tubulin genes probably occur in a separate tandem repeat consisting of approximately 4-kilobase units unlinked to the alpha-tubulin repeats.
Effect of the extracellular matrix on plasminogen activator isozyme activities of human mammary epithelial cells in culture.
Authors: Yang N S , Park C , Longley C , Furmanski P
Published: September 2015

Multiple molecular forms of plasminogen activator were detected in normal human mammary epithelial cells in culture. Cells derived from (normal) breast mammoplasty specimens and grown on the surface of collagen gels exhibited three major classes of plasminogen activator isozymes (Mr = 100,000 [100K], 75,000 [75K], and 55,000 [55K]). The activity of the 100K and 75K isozymes was greatly reduced when the cells were grown on conventional tissue-culture-grade plastic surfaces. MCF-7, a human mammary carcinoma cell line, exhibited predominantly or exclusively the 55K isozyme, irrespective of the cell growth substratum. The activity of the 55K isozyme was more than twofold higher for MCF-7 cells grown on collagen gels than for cells grown on plastic. Progesterone, diethylstilbestrol, and estrogen stimulated the activity of the 55K isozyme of MCF-7 cells, but only when the cells were grown on a plastic surface. The plasminogen activator activities of the normal human mammary epithelial cells were not stimulated by these hormones, irrespective of the growth substratum. These results show that the expression of plasminogen activator isozymes by human mammary epithelial cells is subject to modulation by the extracellular matrix. Normal and malignant cells may differ in their responsiveness to these effects.
Regulation of mRNA utilization in mouse erythroleukemia cells induced to differentiate by exposure to dimethyl sulfoxide.
Authors: Yenofsky R , Cereghini S , Krowczynska A , Brawerman G
Published: September 2015

Mouse erythroleukemia cells contain several abundant mRNA species that occur to a considerable extent as untranslated molecules. For two of these species, which code for polypeptides P40 and P21, the proportion of molecules engaged in translation decreases rapidly after exposure of the cells to dimethyl sulfoxide. The extent of utilization of a third species, the P36 mRNA, is not altered. The rate of production of the P40 mRNA does not appear to be affected in the dimethyl sulfoxide-treated cells. The P21 mRNA appears to be produced in increasing amounts, leading to a large accumulation of untranslated molecules in the cytoplasm. The mRNA for actin remains nearly fully utilized during this process, but its intracellular concentration decreases, thus resulting in a reduction in the amounts present in polysomes. The results indicate that some mRNA species in mouse tumor cells are subject to a translational repression process that can serve to regulate selectively the extent of expression of the corresponding genes.
Characterization of a phosphoprotein associated with the SS-B/La nuclear antigen in adenovirus-infected and uninfected KB cells.
Authors: Pizer L I , Deng J S , Stenberg R M , Tan E M
Published: September 2015

We have employed sera from patients with autoimmune disease to characterize the nuclear SS-B/La antigen in uninfected and adenovirus-infected KB cells. A 45,000-dalton phosphorylated polypeptide was specifically precipitated with anti-SS-B sera, and the level of phosphorylation was increased after infection. The increased phosphorylation appears to occur at the same amino acid residues phosphorylated in uninfected cells and results from increased phosphorylating activity rather than from altered enzyme specificity. A competition experiment between infected and uninfected cell extracts indicates that the antigen in the infected cell binds more strongly to SS-B/La antibodies. Fragments of adenovirus-induced virus-associated RNA as well as intact molecules complex with SS-B/La antigen and are immune precipitated with autoimmune sera.
Differential activation of the mouse beta-globin promoter by enhancers.
Authors: Berg P E , Yu J K , Popovic Z , Schumperli D , Johansen H , Rosenberg M , Anderson W F
Published: September 2015

A series of plasmids was constructed to study the effect of two enhancers, the simian virus 40 72-base-pair repeat and the Harvey sarcoma virus 73-base-pair repeat, on the mouse beta maj-globin promoter. These plasmids contain the mouse beta maj-globin promoter linked to the Escherichia coli galK gene, thus allowing galactokinase enzyme activity to be used as a measure of promoter function. In CV-1 (primate) cells, it was found that an enhancer is required for optimal promoter activity and that the simian virus 40 (primate) enhancer increases galactokinase fourfold more than the Harvey sarcoma virus (mouse) enhancer. In L (mouse) cells, however, the Harvey sarcoma virus enhancer is 1.3-fold stronger than the simian virus 40 enhancer. These data support the hypothesis that enhancer activity can be species specific. Furthermore, when both enhancers are present on the same plasmid, their effect is additive on the beta-globin promoter whether the plasmid is in CV-1 cells or L cells.
Amplification of a cloned Chinese hamster dihydrofolate reductase gene after transfer into a dihydrofolate reductase-deficient cell line.
Authors: Milbrandt J D , Azizkhan J C , Hamlin J L
Published: September 2015

We have transformed a dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary cell line to the DHFR+ phenotype with a recombinant cosmid (cH1) containing a functional Chinese hamster DHFR gene (J.D. Milbrandt et al., Mol. Cell. Biol. 3:1266-1273, 1983). After exposure of cells to successive increases in methotrexate, we have isolated a resistant cell line (JSH-1) that grows in 1 microM methotrexate. We show here that JSH-1 contains 300 to 500 copies of the integrated cosmid and that these copies are located predominantly at one position on a chromosome identified as Z5a. Hybridization analysis of restriction digests of genomic DNA indicates that the cosmid has been integrated intact into the genome and that upon amplification, the original cosmid/genomic junction fragments are also amplified in JSH-1. Furthermore, the pattern of amplified bands observed in ethidium bromide-stained gels indicates that the unit amplified sequence (amplicon) may be as large as 120 to 135 kilobases and therefore includes considerable amounts of flanking DNA in addition to the 45 kilobases of integrated cosmid. We also show that the protein overproduced by the amplified cosmid in JSH-1 comigrates with the 21,000-dalton polypeptide characteristic of the methotrexate-resistant cell line (CHOC 400) from which cH1 was cloned. However, the DHFR mRNA species overproduced in JSH-1 appear to be larger than those detected in CHOC 400, indicating that not all of the normal transcription and processing signals are preserved in the integrated recombinant cosmid.
Transient expression of a mouse alpha-fetoprotein minigene: deletion analyses of promoter function.
Authors: Scott R W , Tilghman S M
Published: September 2015

The constitutive transcription of a mouse alpha-fetoprotein (AFP) minigene was examined during the transient expression of AFP-simian virus 40-pBR322 recombinant DNAs introduced into HeLa cells by Ca3(PO4)2 precipitation. We tested three constructs, each of which contains the AFP minigene and pBR322 DNAs inserted in the late region of simian virus 40 and found that the relative efficiency of AFP gene expression was dependent on the arrangement of the three DNA elements in the vector. The transcripts begin at the authentic AFP cap site and are properly spliced and polyadenylated. To define a sequence domain in the 5' flanking region of the AFP gene required for constitutive expression, sequential 5' deletion mutants of the AFP minigene were constructed and introduced into HeLa cells. All AFP deletion mutants which retained at least the TATA motif located 30 base pairs upstream from the cap site were capable of directing accurate and efficient AFP transcription. However, when the TATA sequence was deleted, no accurately initiated AFP transcripts were detected. These results are identical to those obtained from in vitro transcription of truncated AFP 5' deletion mutant templates assayed in HeLa cell extracts. The rate of AFP transcription in vivo was unaffected by deletion of DNA upstream of the AFP TATA box but was greatly affected by the distance between the simian virus 40 control region and the 5' end of the gene. The absence of any promoter activity upstream of the TATA box in this assay system is in contrast to what has been reported for several other eucaryotic structural genes in a variety of in vivo systems. A sequence comparison between the 5' flanking region of the AFP gene and these genes suggested that the AFP gene lacks those structural elements found to be important for constitutive transcription in vivo. Either the AFP gene lacks upstream promoter function in the 5' flanking DNA contained within the minigene, or the use of a viral vector in a heterologous system precludes its identification.
Immunological selection of variant mouse lymphoid cells with altered glucocorticoid responsiveness.
Authors: Danielsen M , Peterson D O , Stallcup M R
Published: September 2015

We have devised an immunological procedure to separate cells on the basis of expression of mouse mammary tumor virus (MMTV) gene products. Plastic petri dishes coated with specific antibodies against MMTV proteins bind cells with an efficiency that correlates with the level of MMTV gene expression. Glucocorticoid-sensitive mouse thymoma cell line W7 was infected with MMTV. Clones from the infected population retain the relatively slow cytolytic glucocorticoid response and, in addition, exhibit a rapid induction of MMTV-specific RNA and proteins. By combining our immunological selection with the selection for resistance to hormone-mediated cytolysis, we have isolated variant cells which are resistant to the cytotoxic effect of glucocorticoids but which retain the induction of viral gene products and must therefore have a functional glucocorticoid receptor protein.
Industrial yeasts display tandem gene iteration at the CUP1 region.
Authors: Welch J W , Fogel S , Cathala G , Karin M
Published: September 2015

The gene copy number at the CUP1 locus and the resistance level to external copper was directly correlated in five wild-type commercial Saccharomyces strains. An increased copy number of the CUP1 gene leads to increased accumulation of chelatin mRNA, which codes for a low-molecular-weight, copper-binding protein. The enhanced production of this rapidly inducible protein mediates resistance of the cell to copper. Industrial yeasts exhibit homologies to the amplified copper resistance repeat unit found in laboratory strains. However, the extent of tandem iteration is strain dependent, and the repetitious unit is either 1.7 or 1.5 kilobases in length compared with the 2.0-kilobase unit in laboratory strains. Strain 522 (Montrachet) contains two chromosome VIII segments distinguishable by their numbers of repeat units (2 and 11) and the size of the units (1.5 and 1.7 kilobases). Distillers yeast 513 carries a 1.5-kilobase repeat unit on each homologous chromosome, although they contain nine and five iterations, respectively.
Secretion ofSaccharomyces cerevisiaeKiller Toxin: Processing of the Glycosylated Precursor
Authors: Bussey H. , Saville D. , Greene D. , Tipper D. J. , Bostian K. A.
Published: September 2015

Killer toxin secretion was blocked at the restrictive temperature inSaccharomyces cerevisiae secmutants with conditional defects in theS. cerevisiaesecretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (sec1). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in allsecmutants at the restrictive temperature. Insec18the protoxin was stable after a chase; but insec7andsec1the protoxin was unstable, and insec111K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-l-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and insec7andsec1cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-sec18and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined twokexmutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable inkex1but unstable inkex2. Protoxin stability inkex1 kex2double mutants indicated the orderkex1→kex2in the protoxin processing pathway. TPCK did not block protoxin instability inkex2mutants. This suggested that theKEX1- andKEX2-dependent steps preceded thesec7Golgi block. We attempted to localize the protoxin inS. cerevisiaecells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.
A splice junction deletion deficient in the transport of RNA does not polyadenylate nuclear RNA.
Authors: Villarreal L P , White R T
Published: September 2015

A late region deletion mutant of simian virus 40 (dl5) was previously shown to be deficient in the transport of nuclear RNA. This is a splice junction deletion that has lost the 3' end of an RNA leader, an intervening sequence, and the 5' end of the splice acceptor site on the body of the mRNA. In this report, we analyzed the steady-state structure of the untransported nuclear RNA. The 5' ends of this RNA are heterogeneous but contain a prominent 5' end at the normal position (nucleotide 325) in addition to several other prominent 5' ends not seen in wild-type RNA. The 3' end of this RNA does not occur at the usual position (nucleotide 2674) of polyadenylation; instead, this RNA is non-polyadenylated, with the 3' end occurring either downstream or upstream of the normal position.
A cytomegalovirus DNA sequence containing tracts of tandemly repeated CA dinucleotides hybridizes to highly repetitive dispersed elements in mammalian cell genomes.
Authors: Jeang K T , Hayward G S
Published: September 2015

A single 880-base-pair region within the genome of simian cytomegalovirus strain Colburn contains sequences that hybridize intensely with both human and mouse total genome DNA probes. This sequence was also found in a second simian cytomegalovirus isolate and was retained in both plaque-purified virus subclones and in plasmid DNA clones containing the SalI P fragment. Cleaved genomic DNAs from several mammalian species all exhibited strong dispersed hybridization with the SalI-P probes, and over 70% of the lambda clones in a mouse genomic library plus several selected clones containing globin, 45S rDNA, or 5S rDNA genes all formed hybrids with SalI-P. The appropriate region of cytomegalovirus SalI-P contains relatively A + T-rich unique sequences interrupted by three stretches of the simple alternating dinucleotides, (CA)15, (CA)22, and (CA)21, which we show to be responsible for most of the cell-virus homology. We conclude that discrete, tandemly repeated (CA) dinucleotide tracts capable of forming left-handed Z-DNA helices punctuate mammalian genomes at greater than 10(5) copies per cell and that three adjacent copies of what appear to be a family of interspersed repetitive elements containing these (CA)n stretches are carried in the genomes of simian cytomegaloviruses.
Location of the initial cleavage sites in mouse pre-rRNA.
Authors: Bowman L H , Goldman W E , Goldberg G I , Hebert M B , Schlessinger D
Published: September 2015

The locations of three cleavages that can occur in mouse 45S pre-rRNA were determined by Northern blot hybridization and S1 nuclease mapping techniques. These experiments indicate that an initial cleavage of 45S pre-rRNA can directly generate the mature 5' terminus of 18S rRNA. Initial cleavage of 45S pre-rRNA can also generate the mature 5' terminus of 5.8S rRNA, but in this case cleavage can occur at two different locations, one at the known 5' terminus of 5.8S rRNA and another 6 or 7 nucleotides upstream. This pattern of cleavage results in the formation of cytoplasmic 5.8S rRNA with heterogeneous 5' termini. Further, our results indicate that one pathway for the formation of the mature 5' terminus of 28S rRNA involves initial cleavages within spacer sequences followed by cleavages which generate the mature 5' terminus of 28S rRNA. Comparison of these different patterns of cleavage for mouse pre-rRNA with that for Escherichia coli pre-rRNA implies that there are fundamental differences in the two processing mechanisms. Further, several possible cleavage signals have been identified by comparing the cleavage sites with the primary and secondary structure of mouse rRNA (see W. E. Goldman, G. Goldberg, L. H. Bowman, D. Steinmetz, and D. Schlessinger, Mol. Cell. Biol. 3:1488-1500, 1983).
Isolation of the catalase T structural gene of Saccharomyces cerevisiae by functional complementation.
Authors: Spevak W , Fessl F , Rytka J , Traczyk A , Skoneczny M , Ruis H
Published: September 2015

The catalase T structural gene of Saccharomyces cerevisiae was cloned by functional complementation of a mutation causing specific lack of the enzyme (cttl). Catalase T-deficient mutants were obtained by UV mutagenesis of an S. cerevisiae strain bearing the cas1 mutation, which causes insensitivity of catalase T to glucose repression. Since the second catalase protein of S. cerevisiae, catalase A, is completely repressed on 10% glucose, catalase T-deficient mutant colonies could be detected under such conditions. A cttl mutant was transformed with an S. cerevisiae gene library in plasmid YEp13. Among the catalase T-positive clones, four contained overlapping DNA fragments according to restriction analysis. Hybridization selection of yeast mRNA binding specifically to one of the cloned DNAs, translation of this mRNA in cell-free protein synthesis systems, and demonstration of catalase T protein formation by specific immunoadsorption showed that the catalase T structural gene had been cloned. By subcloning, the gene was located within a 3.5-kilobase S. cerevisiae DNA fragment. As in wild-type cells, catalase T synthesis in cttl mutant cells transformed with plasmids containing this fragment is sensitive to glucose repression. By DNA-RNA hybridization, catalase T transcripts were shown to be present in oxygen-adapting cells but absent from heme-deficient cells.
Proteins tightly bound to HeLa cell DNA at nuclear matrix attachment sites.
Authors: Bodnar J W , Jones C J , Coombs D H , Pearson G D , Ward D C
Published: September 2015

DNA-protein complexes have been isolated from HeLa cell nuclei and nuclear matrix preparations. Two proteins, 55 and 66 kilodaltons in size, remain bound to HeLa DNA after treatment at 80 degrees C in 2% sodium dodecyl sulfate and purification by exclusion chromatography on Sepharose 2B-CL in the presence of 0.3% sodium dodecyl sulfate. These proteins appear to be tightly bound but not covalently linked to the DNA, and they are distributed over the DNA with an average spacing of 40 kilobase pairs. This spacing distribution remains essentially constant throughout the cell cycle. The proteins are bound to the residual 2% of HeLa cell DNA which remains attached to the nuclear matrix after extensive nuclease digestion, a condition which reduces the average size of the DNA to approximately 150 base pairs. Our results suggest that these tightly bound proteins are involved in anchoring cellular DNA to the nuclear matrix. These tightly bound proteins are identical by partial peptide mapping to proteins found tightly bound to the DNA of mammalian, plant, and bacterial cells (D. Werner and C. Petzelt, J. Mol. Biol. 150:297-302, 1981), implying that these proteins are involved in the organization of chromosomal domains and are highly conserved in both procaryotic and eucaryotic cells.
Growth-dependent expression of dihydrofolate reductase mRNA from modular cDNA genes.
Authors: Kaufman R J , Sharp P A
Published: September 2015

Dihydrofolate reductase (DHFR) synthesis is regulated in a growth-dependent fashion. Dividing cells synthesize DHFR at a 10-fold-higher rate than do stationary cells. To study this growth-dependent synthesis. DHFR genes have been constructed from a DHFR cDNA segment, the adenovirus major late promoter, and fragments of simian virus 40 (SV40) which provide signals for polyadenylation. These genes have been introduced into Chinese hamster ovary cells. The DHFR mRNAs produced in different transformants are identical at their 5' ends, but differ in sequences in their 3' ends as different sites are utilized for polyadenylation. Three transformants that utilize either DHFR polyadenylation signals or the SV40 late polyadenylation signal exhibit growth-dependent DHFR synthesis. The level of DHFR mRNA in growing cells is approximately 10 times that in stationary cells for these transformants. This growth-dependent DHFR mRNA production probably results from posttranscriptional events. In contrast, three transformants that utilize the SV40 early polyadenylation signal and another transformant that utilizes a cellular polyadenylation signal do not exhibit growth-dependent DHFR synthesis. In these three cell lines, the fraction of mRNAs polyadenylated at different sites in a tandem array shifts between growing and stationary cells. These results suggest that the metabolic state of the cell is important in determining either the efficiency of polyadenylation at various sites or the stability of mRNA polyadenylated at various sites.
Isolation of the ARO1 cluster gene of Saccharomyces cerevisiae.
Authors: Larimer F W , Morse C C , Beck A K , Cole K W , Gaertner F H
Published: September 2015

The AROl cluster gene was isolated by complementation in Saccharomyces cerevisiae after transformation with a comprehensive yeast DNA library of BamHI restriction fragments inserted into the shuttle vector YEp13. Most of the transformants exhibited the expected episomal inheritance of the ARO+ phenotype; however, one stable transformant has been shown to be an integration of the AROl fragment and the vector YEp13 at the arol locus. The insert containing AROl is a 17.2-kilobase pair (kbp) BamHI fragment which complements both nonsense and missense alleles of arol. Subcloning by Sau3AI partial digestion further locates the AROl segment to a 6.2-kbp region. An autonomously replicating sequence (ars) was found on the 17.2-kbp fragment. Yeast arol mutants transformed with the AROl episome express 5 to 12 times the normal level of the five AROl enzyme activities and possess elevated amounts of the AROl protein. The yeast AROl fragment also complemented aroA, aroB, aroD, and aroE mutants of Escherichia coli. The expression of AROl in both S. cerevisiae and E. coli was independent of the orientation of the fragment with respect to the vector.
Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves.
Authors: Nover L , Scharf K D , Neumann D
Published: September 2015

Biochemical and electron microscopic analyses of heat-shocked suspension cultures of Peruvian tomato (Lycopersicon peruvianum) revealed that a considerable part of the dominant small heat shock proteins (hsps) with an Mr of approximately 17,000 are structural proteins of newly forming granular aggregates in the cytoplasm (heat shock granules), whose formation strictly depends on heat shock conditions (37 to 40 degrees C) and the presence or simultaneous synthesis of hsps. However, under certain conditions, e.g., in preinduced cultures maintained at 25 degrees C, hsps also accumulate as soluble proteins without concomitant assembly of heat shock granules. Similar heat shock-induced cytoplasmic aggregates were also observed in other cell cultures and heat-shocked tomato leaves and corn coleoptiles.
Functional role of laminin carbohydrate.
Authors: Howe C C
Published: September 2015

Previous work showed that tunicamycin suppresses glycosylation of laminin. In the present work, the role of glycosylation in the secretion of laminin and in the disulfide bonding of laminin subunits was studied, using tunicamycin to inhibit glycosylation. Tunicamycin inhibited extensively the secretion of laminin into culture medium and extracellular matrix even though the treated cells contained higher concentrations of laminin than the control cells. The laminin subunits synthesized in the presence of tunicamycin were disulfide bonded. Thus, suppression of glycosylation did not adversely affect disulfide bonding of the subunits, but did decrease the secretion of laminin. Glycosidases were also used to remove the carbohydrate of laminin to study the role of carbohydrate in the stability of laminin and in its interaction with another extracellular matrix component, heparin. The glycosidases removed about 73% of [3H]glucosamine. Both glycosidase-treated and untreated laminin were stable when incubated with cell lysate or culture medium. The glycosidase-treated laminin bound as efficiently as the untreated laminin to heparin. These results suggest that the presence of a carbohydrate moiety, at least at the level found in untreated laminin, is not essential in binding to heparin or in protecting laminin from proteolytic degradation in the cell or culture medium.
Inducible expression of amplified human beta interferon genes in CHO cells.
Authors: McCormick F , Trahey M , Innis M , Dieckmann B , Ringold G
Published: September 2015

Plasmid DNA containing the human beta-interferon (IFN-beta) gene and mouse dihydrofolate reductase cDNA was transfected into dihydrofolate reductase-negative Chinese hamster ovary cells. Dihydrofolate reductase-positive transformants were obtained, and cells containing amplified copies of mouse dihydrofolate reductase were selected by exposure to increasing methotrexate concentrations. These cells were found to express high levels of human IFN-beta after polyriboinosinic acid-polyribocytidylic acid superinduction or NDV infection; this was a result of coamplification of the IFN-beta gene. Levels of expression of 1 U/cell per day were achieved on superinduction, giving corresponding titers of up to 10(10) U/liter medium in culture supernatants. Constitutive production of IFN-beta rates of about 0.5% of superinduced rates was observed; cells producing these levels of IFN-beta had acquired resistance to cytotoxic antiviral effects of IFN-beta. Two forms of human IFN-beta were produced; a major glycosylated 23,000-dalton form and an unglycosylated 18,500-dalton form. The latter had greatly reduced antiviral activity. IFN-beta production was very sensitive to cellular growth rate; the highest levels were produced by density-arrested cultures. Regulation of IFN-beta production by polyriboinosinic acid-polyribocytidylic acid or by cell density effects required the presence of DNA sequences 5' to the IFN-beta-coding sequences; replacement of these sequences with the simian virus 40 early promoter resulted in uninducible, density-independent production of IFN-beta.
Diverse mitogenic agents induce the phosphorylation of two related 42,000-dalton proteins on tyrosine in quiescent chick cells.
Authors: Cooper J A , Sefton B M , Hunter T
Published: September 2015

Incubation of quiescent chicken embryo cells with platelet-derived growth factor, epidermal growth factor, or serum was found to stimulate phosphorylation of two proteins of ca. 42,000 daltons on tyrosine. These proteins are structurally related to each other and to two proteins phosphorylated on tyrosine under similar conditions in mitogen-treated mouse fibroblasts. Three other very different mitogenic agents, the protease trypsin and the chemically unrelated tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate and teleocidin, stimulated phosphorylation of the same proteins. In all cases, phosphotyrosine was detected in these phosphoproteins. Although additional changes in protein phosphorylation were evident, no other proteins were observed by two-dimensional gel electrophoresis which contained increased amounts of phosphotyrosine in mitogen-treated chicken embryo cells. One of these 42,000-dalton proteins was shown previously to be phosphorylated on tyrosine in chicken embryo cells transformed with various retroviruses whose transforming proteins possess tyrosine protein kinase activity. Phosphorylation of the 42,000-dalton proteins could be important in the regulation of cell division.
Structure and expression of the SNF1 gene of Saccharomyces cerevisiae.
Authors: Celenza J L , Carlson M
Published: September 2015

The SNF1 gene of Saccharomyces cerevisiae is essential for normal regulation of gene expression by glucose repression. A functional SNF1 gene product is required to derepress many glucose-repressible genes in response to conditions of low external glucose. In the case of the SUC2 structural gene for invertase, SNF1 acts at the RNA level. We have reported the isolation of a cloned gene that complements the snf1 defect in S. cerevisiae and that is homologous to DNA at the SNF1 locus (J. L. Celenza and M. Carlson, Mol. Cell. Biol. 4:49-53, 1984). In this work we identified a 2.4-kilobase polyadenylate-containing RNA encoded by the SNF1 gene and showed that its level is neither regulated by glucose repression nor dependent on a functional SNF1 product. The position of the SNF1 RNA relative to the cloned DNA was mapped, and the direction of transcription was determined. The cloned DNA was used to disrupt the SNF1 gene at its chromosomal locus. Gene disruption resulted in A Snf1- phenotype, thereby proving that the cloned gene is the SNF1 gene and showing that the phenotype of a true null mutation is indistinguishable from that of previously isolated snf1 mutations.
Carbon source dependence of transposable element-associated gene activation in Saccharomyces cerevisiae.
Authors: Taguchi A K , Ciriacy M , Young E T
Published: September 2015

Seven cis-dominant mutations leading to the overproduction of the glucose-repressible alcohol dehydrogenase isozyme ADHII (structural gene, ADH2) in Saccharomyces cerevisiae have previously been shown to be due to insertion of a transposable element, Ty, in the 5' regulatory region of the ADH2 gene. We showed that although mating-competent cells (a, alpha, a/a, or alpha/alpha cells) overproduced both ADHII enzyme and ADH2 mRNA, mating-incompetent cells (a/alpha or ste-cells) produced much less ADHII enzyme and ADH2 mRNA. This mating type effect on ADH2 expression was greatest in the presence of a normally derepressing carbon source, glycerol, and much less apparent in the presence of a repressing carbon source, glucose. In addition, Ty insertion led to an aberrant carbon source response in mating-incompetent cells--the normally glucose-repressible ADHII becomes glycerol repressible. The mating type effect and aberrant carbon source response in mating-incompetent cells was specific for Ty-associated mutations in the 5' flanking region of the ADH2 gene in that a non-Ty mutation in the same region did not show these effects. Finally, Ty1 RNA levels also showed a/alpha, suppression, which was apparent only during growth on a nonfermentable carbon source such as glycerol. This suggests that Ty-mediated gene expression is subject to regulation by both mating competence and carbon catabolites.
Moderate-level gene amplification in methotrexate-resistant Chinese hamster ovary cells is accompanied by chromosomal translocations at or near the site of the amplified DHFR gene.
Authors: Flintoff W F , Livingston E , Duff C , Worton R G
Published: September 2015

In previous studies, we have described several classes of methotrexate-resistant Chinese hamster ovary cell lines. Although the RI class is resistant because of an altered target enzyme, dihydrofolate reductase, the RIII class derived from RI cells is somewhat more resistant because of a moderate amplification of the altered dhfr structural gene (Flintoff et al., Mol. Cell. Biol. 2:275-285, 1982). In one RIII line, a translocation between the short arm (p) of chromosome 2 and the long arm (q) of chromosome 5 was observed, and the amplified RIII gene complex was mapped to the p arm of the 2p-marker chromosome derived from the translocation (Worton et al., Mol. Cell. Biol. 1:330-335, 1981). We tested the hypothesis that chromosomal translocation is a general feature of RIII cells and that such translocation involves a site at or near the dhfr structural gene. Thus, we examined four independently derived RIII-type mutants and found that each had a moderate amplification of the dhfr gene sequences, and karyotype analysis revealed that each carried a translocation involving the 2p arm at or near band 2p25. That this chromosomal rearrangement involves a site near the dhfr locus was demonstrated by mapping the altered but unamplified structural gene coding for the RI phenotype to the short arm of an unaltered chromosome 2. This suggests that a highly specific rearrangement involving an exchange at or near the site of the unamplified gene is a necessary prerequisite for the amplification process. A model for gene amplification involving chromosomal rearrangements and sister chromatid exchange is described.
Isolation and characterization of the centromere from chromosome V (CEN5) of Saccharomyces cerevisiae.
Authors: Maine G T , Surosky R T , Tye B K
Published: September 2015

We have cloned a functional centromeric DNA sequence from Saccharomyces cerevisiae. Using the 2 mu chromosome-loss mapping technique and meiotic tetrad analysis, we have identified this DNA sequence as the centromere of chromosome V (CEN5). The CEN5 sequence has been localized on an 1,100-base-pair BamHI-BglII restriction fragment. Plasmids containing CEN5 and an autonomously replicating sequence are mitotically stable in S. cerevisiae and segregate in a Mendelian fashion during meiosis.