Showing papers on "Gene expression published in 1978"

Possible role of the 72,000 dalton DNA-binding protein in regulation of adenovirus type 5 early gene expression.

Abstract: Relative abundances of early virus RNA species in the cytoplasm of cells infected with wild-type adenovirus type 5 (WT Ad5) and a temperature-sensitive "early" mutant, H5ts125 (ts125), were compared by hybridization kinetics using separated strands of HindIII restriction endonuclease fragments of Ad5 DNA. 1-beta-D-Arabinofuranosylcytosine (ara-C) was used to limit transcription to early virus genes in cells infected by WT virus. At 40.5 degrees C, a restrictive temperature for ts125, three to seven times as much virus RNA from all four early regions of the genome accumulated in the cytoplasm of cells infected by the mutant as accumulated in cells infected by WT. At 32 degrees C, no such difference in the relative abundances of cytoplasmic virus RNA was observed. The capacity to synthesize a 72,000-dalton (72K) virus polypeptide, presumably the single-stranded DNA-binding protein that is defective in ts125 at restrictive temperatures, was compared in cells infected at 40.5 degrees C in the presence of ara-C with the mutant or WT Ad5. The rate of 72K polypeptide synthesis, measured by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis of [35S]methionine-labeled polypeptides and autoradiography, was greater at 15 h after infection in ts125-infected cells than in cells infected by WT. A time course experiment showed that the rate of synthesis of the 72K polypeptide increased continuously in ts125-infected cells during the first 15 h of infection, relative to the rate in WT-infected cells. These data are consistent with the hypothesis that Ad5 early gene expression is modulated by the product of an early gene, the 72K DNA-binding protein.

Ecdysone-inducible functions of larval fat bodies in Drosophila

Abstract: Late in the third instar larval stage of Drosophila melanogaster, the titer of the steroid hormone ecdysone increases sharply. This increase is blocked in the temperature-sensitive mutant ecd(1) after a temperature shift from 20 degrees C to 29 degrees C. The mutant was used to prepare three samples of late third instar larvae with different titers of ecdysone; the titer was low in one sample because of an earlier temperature shift, high in a second sample because the larvae were subsequently transferred to ecdysone-supplemented food, and also high in a third sample that was kept at 20 degrees C, providing a control for normal development. The effect of the high titer of ecdysone on proteins of the larval fat bodies was examined by comparing two-dimensional gel electrophoresis patterns of total proteins in stained gels. There were proteins at five positions in the gels for the high-ecdysone samples that were not detected at the corresponding positions in the gel for the low-ecdysone sample. The effect of ecdysone on these proteins was further studied by injecting [(35)S]methionine into the larvae at both early and late third instar stages, in order to label proteins synthesized before and after the increase in ecdysone titer. The results indicate that ecdysone induces two major responses in the fat bodies; certain proteins that were synthesized earlier in the fat bodies and secreted into the hemolymph are incorporated back into the fat bodies, and other proteins are newly synthesized. Attempts to induce prematurely the synthesis of the new proteins by exposing early third instar larvae to exogenous ecdysone were unsuccessful, suggesting that development must proceed further before the fat bodies can respond to ecdysone. By in vitro translation of RNA isolated from fat bodies of low-and high-ecdysone samples of larvae, it was shown that ecdysone greatly increases the amount of translatable messenger RNA for one of the newly synthesized proteins. A clone of DNA complementary to the induced messenger RNA has been isolated from a population of lambda bacteriophage carrying segments of the Drosophila genome. Using the cloned DNA to measure amounts of complementary poly(A)-RNA in the fat bodies by DNA.RNA hybridization, we detected about 50 times more complementary poly(A)-RNA in the high-ecdysone sample of larvae than in the low-ecdysone sample. This finding provides direct evidence that ecdysone induces an increase in the amount of the messenger RNA. The ecdysone-induced appearance of a major messenger RNA in late third instar larval fat bodies represents a developmental response to ecdysone that appears to be gene-specific, tissue-specific, and stage-specific, and it has exceptionally favorable features for further molecular studies of the control of gene expression by a steroid hormone.

Regulation of the galactose pathway in Saccharomyces cerevisiae: Induction of uridyl transferase mRNA and dependency on GAL4 gene function

Abstract: In Saccharomyces cerevisiae, utilization of galactose requires four inducible enzyme activities. Three of these activities (galactose-1-phosphate uridyl transferase, EC 2.7.7.10; uridine diphosphogalactose 4-epimerase, EC 5.1.3.2; and galactokinase, EC 2.7.1.6) are specified by three tightly linked genes (GAL7, GAL10, and GAL1, respectively) on chromosome II, whereas the fourth, galactose transport, is specified by a gene (GAL2) located on chromosome XII. Although classic genetic analysis has revealed both positive and negative regulatory genes that coordinately affect the appearance of all four enzyme activities, neither the basic events leading to the appearance of enzyme activities nor the roles of the regulatory genes have yet been determined. Regulation of inducible enzyme activity could be mediated by events related to transcription, translation, or enzyme activation. For the purpose of studying galactose pathway induction and its regulation, we have developed an immunoprecipitation assay that enables us to detect the GAL7 specified uridyl transferase polypeptide in yeast extracts and among the polypeptides synthesized in an RNA-dependent in vitro translation system. Use of this immunoprecipitation assay in conjunction with in vivo labeling experiments demonstrates the presence of [(3)H]leucine-labeled transferase in extracts prepared from cells grown in galactose but not from cells grown in glucose. This galactose-specific induction of transferase polypeptide is mediated by the de novo appearance of a functional mRNA species whose synthetic capacity is detectable by the combination of in vitro translation and immunoprecipitation. The appearance of functional transferase mRNA depends on wild-type expression of the positive regulatory gene, GAL4. Cells carrying a nonsense (amber) mutation in the GAL4 gene fail to produce the transferase mRNA, whereas a nonsense suppressor of the GAL4 amber mutant regains the galactose-specific mRNA response. Our results establish that the induction of the GAL7 specified uridyl transferase activity is mediated by de novo appearance of a functional mRNA and that this galactose-specific response is dependent on a wild-type GAL4 gene product.

Molecular basis of reduced albumin synthesis in Morris hepatoma 7777.

Abstract: The level of albumin mRNA in the normal Buffalo rat liver and Morris hepatoma 7777 was compared by molecular hybridization with albumin complementary DNA (cDNA) and translational assays. Albumin mRNA was found to be 10% of the total rat liver poly(A)-containing RNA population but reduced approximately fourfold in the case of Morris hepatoma 7777. An equivalent decrease of albumin mRNA activity in the hepatoma was detected by translation in a mRNA-dependent cell-free protein-synthesizing system. A proportional increase in total hepatoma poly(A)-containing RNA was not observed, indicating that there was a true fourfold reduction of albumin synthesis in the hepatoma. DNA excess hybridization with albumin cDNA did not reveal any apparent change in albumin gene frequency in the hepatoma compared to normal liver. Complementary DNA copies of total liver and hepatoma poly(A)-containing RNA were synthesized and employed in homologous and heterologous hybridization reactions. Analyses of these reactions showed a high degree of homology between the poly(A)-containing RNA of the liver and hepatoma, with some difference in the relative sequence abundancy. However, qualitative differences were detected in hepatoma 7777 consistent with the concept of alterations in the control of gene expression upon neoplastic transformation.

Autoregulation of Adenovirus Type 5 Early Gene Expression II. Effect of Temperature-Sensitive Early Mutations on Virus RNA Accumulation

Abstract: The kinetics of accumulation of early virus RNA in the cytoplasm of KB cells infected at 40.5 degrees C by wild-type (WT) adenovirus type 5 and a temperature-sensitive "early" mutant, H5ts125 (ts125), were compared by hybridization of unlabeled RNA in solution to the (3)H-labeled l strand of Ad5 DNA HindIII restriction endonuclease fragment A. In the presence of 1-beta-d-arabinofuranosylcytosine, A(l) RNA accumulated in WT-infected cells for 9 h and then decreased in concentration to 6% of the 9-h concentration by 18 h. In ts125-infected cells, A(l) RNA accumulated for 12 h and then remained at the same concentration for at least 6 h thereafter. The concentrations of virus RNA from the four early transcription regions of the genome were measured at 15 h in cells infected at 40.5 degrees C in the presence of 1-beta-d-arabinofuranosylcytosine by: (i) ts125 and WT; (ii) two other ts early mutants, ts107 and ts149; and (iii) a revertant of ts125. The revertant and ts149, a mutant from a different complementation group than ts125, both accumulated all early virus cytoplasmic RNA species in amounts similar to, or less than, WT. However, both ts125 and ts107, independently isolated mutations in the 72,000-molecular-weight (72K) DNA-binding protein gene, accumulated cytoplasmic early RNA in excess of that found in WT infection. This pattern of RNA accumulation with the mutants and WT virus was the same in the nuclei as in the cytoplasm at 40.5 degrees C. At 32 degrees C, however, the abundance of nuclear virus RNA from all four early regions was the same in cells infected by either ts125 or WT. Differences in the relative abundance of nuclear RNA from the four early regions were observed in cells infected at 40.5 and 32 degrees C, but were not dependent upon the infecting virus genotype. These results are consistent with autoregulation of early gene expression by the 72K protein and support the hypothesis that the 72K protein either decreases the rate of early virus transcription or increases the rate of virus RNA degradation in the nucleus.

RNA transcription and chromatin structure during meiotic and postmeiotic stages of spermatogenesis.

Abstract: Autoradiographic procedures for the study of RNA and protein synthesis during spermatogenesis have been complemented with electron microscope techniques for visualization of gene activity. These procedures have enabled us to determine that RNA transcription is highly selective with respect to RNA species, timing of synthesis, types of chromosomes (autosomes and sex chromosomes), segments of chromosomes (i.e., the lampbrush segment), and chromatin structure. In mouse and human spermatocytes, a peak production of ribosomal RNA (rRNA) occurs during leptotene-zygotene, preceding nonnucleolar RNA synthesis, which is at a peak in middle pachytene. Transcription in late spermatids decreases in coincidence with changes in chromatin structure and high incorporation rates of [3H]arginine. In these cells, a particulate repeating pattern of chromatin is replaced by chromatin fibers of uniform diameter as highly arginine-rich proteins replace somatic histones. In spermatogonia, spermatocytes and Sertoli cells, the products of transcription are mainly heterogeneous nuclear RNA (hnRNA) and rRNA, whereas spermatids transcribe predominantly hnRNA during early spermiogenesis. Persistent long-lived [3H]-uridine-labeled RNA species in pachytene spermatocyte nuclei contrast with a fast turnover of [3H]uridine-labeled RNA in Sertoli cells as detected at the same pulse labeling time (8--12 days). From these results one can postulate a still undefined control mechanism of gene expression during spermatogenesis for modulating a cascade of events required for male gamete formation.

Biochemical genetics of the alpha-keto acid dehydrogenase complexes of Escherichia coli K12: genetic characterization and regulatory properties of deletion mutants.

Abstract: Twenty-eight spontaneous auxotrophic aroP mutants with deletions in the azi-nadC-aroP-aceE-aceF-lpd region of the Escherichia coli K 12 chromosome were characterized genetically with respect to various azi, nadC, ace and lpd markers by P1-mediated transduction. One mutant (K δ18; aroP-lpdδ) had a deletion which extended through the aceE and aceF genes to end within the lpd gene. The polarity of the ace operon (aceE to aceF) was confirmed. It was concluded that 10 out of 15 deletions generating a strict requirement for acetate terminated in the aceE gene. Of the ten, three mutants (K δ22, cδ41 and cδ42) synthesized detectable dihydrolipoamide acetyltransferase (the aceF gene product) and seven were assumed to possess deletions generating polar effects on aceF gene expression. Five deletions appeared to extend into the aceF gene. A further five deletions, which limited the expression of the ace operon without generating an Ace- phenotype or a complete Ace- phenotype, ended closest to the aroP-proximal aceE markers. The opposite ends of all these deletions appeared to terminate before (10), within (2) or extend beyond (9) the nadC gene. There was no obvious correlation between the deletion end-points and the corresponding lipoamide dehydrogenase activities, which ranged from 30 to 95% of parental levels in different deletion strains. The remaining seven deletions simply extended between the aroP and nadC genes (nad-aroPδ) without affecting expression of the ace operon. Regulation of the synthesis of the pyruvate and α-ketoglutarate dehydrogenase complexes was investigated in some of the parental and deletion strains under different physiological conditions including thiamin-deprivation. The results indicate that the syntheses of the two dehydrogenase complexes are independently regulated. Expression of the lpd gene appears to be coupled to complex synthesis but can be dissociated under some conditions. Mechanisms for regulating lpd gene expression are discussed and an autogenous mechanism involving uncomplexed lipoamide dehydrogenase functioning as a negatively acting repressor at the operator site of an independent lpd gene is proposed as the simplest mechanism which is consistent with all available information.

Identification of the Mtv-2 gene responsible for the early appearance of mammary tumors in the GR mouse by nucleic acid hybridization.

Abstract: In the mouse strain GR, the Mtv-2 gene controls the expression of large amounts of mammary tumor virus (MTV) antigens in the milk at first lactation. It also controls the early appearance of mammary tumors. We have investigated the number of MTV proviral sequences associated with this Mtv-2 gene by nucleic acid hybridization between MTV [3H]cDNA and DNA from GR, B10, and GR-Mtv-2- mice. B10 and GR-Mtv-2- mice lack Mtv-2 gene expression. The molecular hybridizations revealed that the DNA of GR mice contains 12 copies of MTV proviral sequences, whereas only 4 copies are present in the DNA of B10 and GR-Mtv-2- mice. We therefore conclude that the Mtv-2 gene in the GR mouse strain is associated with eight additional MTV proviral sequences. The four Mtv proviral sequences in the GR-Mtv-2- DNA might represent another Mtv gene in the GR mouse. Different amounts of MTV RNA are detected in mammary glands at first lactation of B10 and GR-Mtv-2- mice, even though both contain four copies of MTV proviral sequences. This indicates a difference between these two mouse strains either in the regulation of expression of these MTV proviral sequences or in the location of these sequences in the murine genome.

Inhibition of host protein synthesis in vaccinia virus-infected cells in the presence of cordycepin (3'-deoxyadenosine).

Abstract: Cordycepin inhibited efficiently viral mRNA and polyadenylic acid syntheses in vaccinia virus-infected cells, but allowed the shutoff of host protein synthesis to occur. Therefore, cordycepin was used to study this shutoff in the absence of gene expression. Ribosome transit time was increased in infected cells, revealing an inhibition at the level of elongation and/or release of polypeptide chains. However, the disappearance of heavy polysomes in vaccinia virus-infected cells showed that the inhibition of host protein synthesis resulted predominantly from a block at the stage of initiation. This conclusion was confirmed by the recovery of heavy polyribosomes when low levels of cycloheximide were added to slow down ribosome release from the mRNA. Similar amounts of cellular mRNA (present in the polyribosomes) were found in vaccinia virus-infected cells and in mock-infected cels (exposed to cordycepin), showing that the cellular mRNA was not inactivated in these conditions. It was concluded that a component of the vaccinia virion inhibits, in the absence of viral RNA and polyadenylic acid syntheses, host protein synthesis at the level of initiation and, to a lesser extent, at the level of elongation (and/or release) of polypeptide chains.

Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B

Abstract: The synthesis of ovalbumin mRNA sequences was studied in isolated nuclei from hen oviduct. Two different methods of analysis were used to distinguish in vitro synthesized from preexisting mRNA sequences: (i) Mercurated ribonucleotides were used for in vitro RNA synthesis, and the newly synthesized RNA was purified by chromatography on sulfhydryl-agarose and hybridized to radioactive ovalbumin cDNA. (ii) [3H]UTP was used to label the in vitro synthesized RNA. Hybridization to unlabeled mercurated cDNA, RNase A digestion, and subsequent purification of the hybrids on SH-agarose allowed the quantitation of newly synthesized ovalbumin mRNA sequences. Approximately 0.1% of the newly synthesized RNA was identified as ovalbumin RNA by both methods. The synthesis of ovalbumin RNA progressed during the incubation of nuclei and was sensitive to actinomycin D and low concentrations of alpha-amanitin. The preferential in vitro transcription of the ovalbumin gene (1000-fold over random transcription of the chicken genome) by RNA polymerase B (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) suggests that the specificity of in vitro RNA synthesis is retained in isolated nuclei.

Regulation of murine cytomegalovirus gene expression. I. Transcription during productive infection.

Abstract: Murine cytomegalovirus RNA synthesis in productively infected mouse embryo cultures was measured by reassociation kinetics with iodinated viral DNA. The data were analyzed by a computer program and indicated the following: before DNA replication approximately 25% of the genome was transcribed into asymmetric transcripts, of which slightly fewer than half of the sequences were recovered from the cytoplasm. After viral DNA replication, approximately 38% of the genome was transcribed (5% as symmetric transcripts), and again less than half of the sequences appeared in the cytoplasm. Both early and late RNA comprised two abundance classes differing about 8- to 10-fold in concentration. Early RNA was a subset of late RNA. The RNE sequences synthesized in late-infected cells in the presence of cytosine arabinoside or cycloheximide were similar to early RNA. Thus, murine cytomegalovirus displays temporal, quantitative, and post-transcriptional controls over gene expression, but the pattern differs considerably from herpes simplex virus.

Inactivation of interferon mRNA in the shutoff of human interferon synthesis.

Abstract: INDUCTION of interferon synthesis in cultured cells by rIn.rCn has been intensely studied because of its attractiveness as a model for gene expression in mammalian cells and its possible significance to medicine. Exposure of human fibroblast FS-4 cells to rlu.rCn results in interferon (IF) synthesis which persists for 6 h and then abruptly shuts off1,2. The rapidity of this shutoff indicates that the cessation of IF mRNA transcription is unlikely to be the primary control. Moreover, studies with metabolic inhibitors3–7 have suggested that a post-transcriptional event is involved. To evaluate the role of post-transcriptional control in the shut-off of IF production, we measured directly the level of IF mRNA activity in a strain of human fibroblast HF 926 cells during the IF induction process. This was achieved by the development of a highly efficient and reproducible heterologous whole cell transnational system. We report here that, using this system, we have found a significant decrease in the IF mRNA activity during the shutoff of IF protein synthesis.

Studies on the control region of the bipolar argECBH operon of Escherichia coli. I. Effect of regulatory mutations and IS2 insertions.

Abstract: Several mutations affecting the control or the potential of gene expression in the argECBH bipolar operon have been characterized by enzyme assays, genetic mapping, dominance tests and pulse labelled RNA determinations. None of the mutations involves DNA rearrangements detectable by heteroduplex analysis (Charlier et al., 1978). Partially constitutive transcription of both argE and argCBH has been observed in mutant L10 while constitutive argE transcription and normal argCBH control characterize mutants L9, LL13 and LL2. The control region thus appears to contain two overlapping operators, as suggested previously (Elseviers et al., 1972). Two mutants (L2, LL1) and strain 6-8 from Bretscher and Baumberg (1976) display an increase in acetylornithinase specific activity (argE product) without concommittant increased argE transcription. In addition, they exhibit a decreased argCBH transcription. It is suggested that in these organisms, argE translation and argCBH transcription may be affected by the same genetic event; this explanation is compatible with present working hypothesis for the structure of the control region. An interpretation in terms of messenger attenuation also appears possible. From the properties of two strains harbouring an IS2 insertion in the control region (Charlier et al., 1978) the following conclusion may be drawn: 1. When inserted in orientation I close to the proximal end of a silent gene IS2 appears to promote a low but detectable transcription readthrough into that gene. 2. Insertion of an IS2 element in orientation II close to a neighbouring gene is not a sufficient condition to express that gene at a high rate. The properties of the two insertions appear compatible with the structure proposed for the control region.

Purification of virus-specific RNA from chicken cells infected with avian sarcoma virus: identification of genome-length and subgenome-leghth viral RNAs.

Abstract: Avian sarcoma virus (ASV)-specific RNA was purified from ASV-infected cells by using hybridization techniques which employ polydeoxycytidylic acid-elongated DNA complementary to ASV RNA as well as chromatography on polyinosinic acid-Sephadex columns. The purity and nucleotide sequence composition of purified, virus-specific RNA were established by rehybridization experiments and analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. Polyadenylic acid-containing RNA purified from ASV-infected cells contained approximately 1 to 4% virus-specific RNA, compared with 0.06 to 0.15% observed in uninfected cells. Sucrose gradient analysis of virus-specific RNA isolated from ASV-infected cells revealed two major classes of polyadenylated viral RNA with sedimentation values of 36S and 26-28S. Cells infected with transformation-defective ASV (virus containing a deletion of the sarcoma gene) contained 34S and 20-22S viral RNA species. Double-label experiments employing infected cells labeled initially for 48 h with [3H]uridine and then for either 30, 60, or 240 min with [32P]phosphate showed that the intracellular accumulation of genome-length RNA (36S) was significantly faster than that of the 26-28S viral RNA species.

Early T7 gene expression: rates of RNA synthesis and degradation, protein kinase dependent termination of transcription, and efficiency of translation.

Abstract: The mode of transcription of early T7 genes starting from one promotor region and generating a unique polycistronic RNA species suggests the appearance of equimolar amounts of the monocistronic species after RNA processing. Rate measurements revealed, however, a disproportion in the generation of the individual early RNA species. The rate of appearance of the promotor-proximal M gene message (nomenclature see in Table 1) is 4–5x the rate of appearance of all other species. This rate pattern is caused by termination behind the M gene because i) the rate of RNA degradation is fairly similar for most RNA species and ii) termination behind the M gene is released in a T7 mutant lacking protein kinase or in wild type infections in the absence of protein synthesis. Then, the RNA species are produced in equimolar amounts. The rate of degradation is similar for all early RNA species except for the protein kinase message. As measured by two independent methods, the physical halflives of M, POL, 1.1, and LIG (nomenclature see Table 1) message were 7–8 min (30°), while KIN RNA was degraded with a halflife of 4 min. The functional halflives were around 50% of the physical halflives. There is apparently no relationship between size of RNA and halflife, and the data suggest specific signals on each RNA which determine the rate of degradation. The monocistronic RNA species are utilized with different rates in translation. The M gene is not only transcribed more often, it is also translated with highest efficiency. The in vivo translation of the POL gene message occurred with the lowest rate.