Showing papers on "Gene expression published in 1975"

of Mouse Mammary Tumor Virus RNA: a System for Studying Glucocorticoid Action"

Abstract: Gordon M. Ringold, Keith R. Yamamoto, Gordon M. Tomkins, J. Michael Bishop, and Harold E. Varmus Departments of Microbiology and Biochemistry and Biophysics University of California San Francisco, California 94143 Summary We have investigated the mechanisms by which dexamethasone (a synthetic glucocorticoid) stimu- lates the production of mouse mammary tumor virus (MMTV) by cell cultures derived from mam- mary carcinomas of GR mice. Treatment of these cells with dexamethasone stimulates a rapid accu- mulation of intracellular virus-specific RNA which is dependent upon RNA synthesis but not upon DNA or protein synthesis. The effect of dexametha- sone is probably mediated by a specific and satura- ble glucocorticoid receptor. We conclude that the accumulation of MMTV RNA is a primary response to dexamethasone and that the rate of synthesis of MMTV RNA is probably accelerated by treatment with dexamethasone. Introduction Steroid hormones have an important role in regulat- ing specific gene expression in metazoan orga- nisms. It is commonly believed that the various steroids function via a unified mechanism (for re- view, see King and Mainwaring, 1974) in which the hormone diffuses into the target cell and becomes tightly associated with a soluble, steroid-specific re- ceptor protein; the complex then binds to DNA- containing nuclear sites (Harris, 1971; Yamamoto and Alberts, 1972) to initiate the biological response. Thus steroids may act as allosteric effec- tors, altering receptor properties to increase the af- finity of the steroid-receptor complex for the nu- clear binding sites (Jensen et al., 1969; Samuels and Tomkins, 1970). Although evidence consistent with this model has been reported, the initial molecular events subse- quent to interaction of the steroid-receptor complex with the nuclear sites remain obscure. In no case has it been unambiguously shown that the first event detected biochemically actually represents the primary biological effect of the hormone. In fact, in several systems, there is evidence that the pri- mary event is not being directly monitored. For example, the induction of specific proteins by glu-

Use of a specific probe for ovalbumin messenger RNA to quantitate estrogen-induced gene transcripts.

Abstract: DNA complementary to purified ovalbumin messenger RNA (cDNA ov) was synthesized in vitro using RNA-directed DNA olymerase from avian myeloblastosis virus. This cDNAov was then employed in hybridization assays to determine the effect of estrogen on the number of ovalbumin mRNA (MRNAov) molecules per tubular gland cell of the chick oviduct. The changes in mRNAov were measured in immature chicks during primary stimulation, after hormone withdrawal and again following secondary stimulation of the chick oviduct with estrogen. The number of mRNAov per tubular gland cell was also determined for egg-laving hen. Daily estrogen administration to the immature chick resulted in growth of the oviduct, differentiation of epithelial cells to tubular glands, and a corresponding increase in the concentration of mRNAov in the tubular gland cell from essentially zero before estrogen administration to 48,000 molecules per cell after 18 days of estrogen treatment. Upon withdrawal of estrogen from the chick, the mRNAov concentration decreased to a level of 0-10 molecules/tubular gland cell after 12 days. Readministration of a single dose of estrogen to these chicks resulted in a dramatic and rapid increase in the concentration of mRNAov. Within 30 min, the mRNAov concentration approximately doubled and by 29 hr the tubular gland cell concentration had reached 17,000 molecules. The initial transcription rate for the ovalbumin gene was 12 mRNAov molecules/min. With these data, we have calculated that the half-life of the ovalbumin messinger RNA should be on the order of 40-60 hr and that the steady-state concentration of mRNAov per tubular gland cell was 50,000 molecules. Similarly, each messenger RNA molecule was translated approximately 50,000 times during its lifetime in order to effect the necessary quantity of ovalbumin required for egg production. These data substantiate the hypothisis that estrogen exerts its primary action at the level of transcription to effect the synthesis of nascene mRNA molecules which in turn code for synthesis of hormone-induced proteins.

Specific binding of CAP factor to lac promoter DNA.

Abstract: CATABOLITE repression in Escherichia coli is mediated by the cyclic AMP dependent catabolite activator protein (CAP). Growth conditions where the induced catabolism of certain sugars is energetically favourable, primarily in the absence of glucose, promote the intracellular induction of a high cyclic AMP concentration; CAP binds cyclic AMP, is activated, and stimulates mRNA synthesis from promoters of catabolite repressible operons1–4. The mechanism by which CAP influences the interaction between RNA polymerase and promoter DNA in unknown. Purified CAP preparations exhibit a cyclic AMP-dependent DNA binding activity, but efforts to detect specific binding to promoters of catabolite repressible genes have been unsuccessful; all DNAs tested have shown similar binding affinities5,6. The only evidence for a specific interaction between CAP and promoter DNA is a fluorescent probe study which showed that a lac transducing phage DNA molecule induces a conformational change in CAP that is not seen with the wild-type phage molecule7; but no effort was made to correlate this difference with in vivo CAP behaviour. Here I report experiments which demonstrate that the CAP factor binds specifically to a site within the lac promoter. This binding is sensitive to mutations which affect the ability of CAP to stimulate in vivo and in vitro lac gene expression.

Effects of estrogen on gene expression in chick oviduct: nuclear receptor levels and initiation of transcription.

Abstract: Estrogen (diethylstilbesterol) was administered in vivo to chicks for various time periods. Chromatin was then prepared from oviduct nuclei and assayed for its capacity to support initiation of RNA chain synthesis in vitro in the presence of saturating levels of Escherichia coli RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6). These same nuclei were also assayed by a [3H]estradiol exchange assay for their endogenous receptor content. The number of available initiation sites for RNA synthesis on chromatin was shown to correlate with the endogenous levels of nuclear estrogen receptor. A decrease in the nuclear concentration of estrogen receptor molecules and the concentration of initiation sites for RNA synthesis occurred during withdrawal of estrogen from previously stimulated chicks. Both parameters declined with a similar half-life. When estrogen was readministered to withdrawn chicks, the number of initiation sites increased 2-fold as early as 30 min and approached a maximal level (3-fold) by 1 hr. During the same period of restimulation with estrogen, the number of estrogen receptor molecules bound to nuclei increased to a maximum at 20 min and then declined at 1 hr to a steady-state level 2-fold higher than the withdrawn chicks. Simultaneous measurements of RNA chain length and RNA chain propagation rate demonstrated that parameters remained relatively constant throughout estrogen withdrawal as well as secondary stimulation. The temporal correlation between changes in the levels of nuclear-bound estrogen receptor and the number of RNA chain initiation sites on chromatin prepared from these same nuclei strongly suggested that the hormone receptor complexes act on chromatin to mediate these changes in genetic transcriptional activity.

Transcription of simian virus 40. V. Regulattion of simian virus 40 gene expression.

Abstract: RNA "exhaustion type" hybridization was used to measure the complementarity of nuclear and cytoplasmic viral RNA to the early (E) and late (L) simian virus 40 (SV40) DNA strands. This type of hybridization measures the amount of labeled RNA complementary to each of the two DNA strands, rather than the fraction of each SV40 DNA strand that is homologous to SV40 RNA. At 48 h after infection, about 5% of the nuclear newly synthesized viral RNA was complementary to the E-strand (- strand) and 95% was complementary to the L-strand (+ strand). This proportion was independent of the labeling time, indicating similar accumulation of the E- and L-RNA transcripts in the nucleus. The nuclear E- and L-viral RNA transcripts sedimented in a similar manner on sucrose gradients. Of the cytoplasmic viral RNA only about 1% was complementary to the E-strand, these molecules sedimenting at 19S, whereas 99% were complementary to the L-strand and sedimented at 19S and 16S. The abundance of E-RNA transcripts in nuclei of cells infected with serially passaged virus was about four times higher than that in nuclei of cells infected with plaque-purified virus; however, the size and proportion of the corresponding cytoplasmic E- and L-RNA transcripts was independent of the type of virus used to infect the cells. According to these results at least two control mechanisms regulate viral gene expression in productively infected cells, one operates at the trnascriptional level and the second at the post-transcriptional level.

Snythesis and differentiation of plasma proteins in cultured embryonic chicken liver cells: a system for study of regulation of protein synthesis.

Abstract: A new system is described for studying the control of protein synthesis. In a monolayer culture of chick embryo liver cells, plasma proteins are synthesized for three days at in vivo rates. The plasma proteins are secreted into the culture medium and without concentration are detected there simply and sensitively by a modified Laurell electronimmunoassay. Secretion of the newly synthesized plasma proteins occurs within 30 min of their synthesis. Thus, rates of synthesis of the plasma proteins can be followed readily from rates of their accumulation in the culture medium. This system has the following advantages for the study of protein synthesis: cells do not have to be disrupted for the assay; the cell population can be followed over several days; it is not necessary to label the proteins radioactively; and turnover of plasma proteins is negligible and need not be taken into account. The usefulness of the system is illustrated by a number of findings. The spectrum of plasma proteins synthesized in culture changed qualitatively and quantitatively. Albumin synthesis steadily decreased with culture time and stopped at the third day, whereas the synthesis of some new plasma proteins ("adult") was induced. These qualitative changes suggest differential gene expression in culture and a special control of albumin synthesis in vivo, different from the synthesis of the other plasma proteins. Quantitative changes in the rates of synthesis of specific plasma proteins suggest a competition among their messenger RNAs for components of the translational machinery. Insulin has a differential effect on the synthesis of specific plasma proteins at concentrations within the physiological range of the hormone.

Functional inactivation of bacteriophage lambda morphogenetic gene in RNA.

Abstract: THE synthesis of the morphogenetic proteins of bacteriophage λ seems to be regulated at the post-transcriptional level. This conclusion is based on the observation that the ratio of protein to DNA along the left arm of the λ genome varies from gene to gene by as much as 870-fold (Fig. 1), while the ratio of mRNA to DNA in this region varies less than twofold1, reflecting its transcription from a single promoter as a polycistronic mRNA2–5. These large variations in the molar ratios of the morphogenetic proteins could be explained by three different control mechanisms. (1) The initiation of protein synthesis could be controlled at the level of ribosome binding either by initiation factor complexes or by mRNA secondary structure6,7. (2) Some morphogenetic proteins could act to regulate their own translation or that of neighbouring genes (N. Sternberg, personal communication). (3) The differential translation of the late gene transcripts could be achieved by selectively inactivating some transcripts but not others. Our previous experiments using RNA:DNA hybridisation ruled out differential chemical decay of the mRNA derived from the late region of the genome, but did not exclude the possibility that differential functional inactivation of morphogenetic gene transcripts could account for the late protein to DNA variation1. Recent experiments indicate that functional and chemical decay are different processes which can vary widely in rate8 and temperature dependence9. Functional decay in a polycistronic message generally seems to involve an endonucleolytic attack near the 5′-end of each gene transcript8,10 at a specific target either in or near the ribosome-binding site11. Thus mRNA inactivation is not primarily a function of the length of a transcript and can vary from transcript to transcript within the same cell11. Because of this variation, differential functional decay has been invoked as a possibly significant mechanism of post-transcriptional control11,12.

Early ribonucleic acid synthesis during the germination of rye (Secale cereale) embryos and the relationship to early protein synthesis.

Abstract: Incorporation studies with radioactive precursors showed that synthesis of protein and RNA is initiated in germinating embryos of rye within the first hour of imbibition of water. By polyacrylamide-gel fractionations of radioactive nucleic acid components, the appearance of products of transcription of the genome was shown to follow the sequence: heterogeneous (ribonuclease-sensitive) RNA, 4S and 5S RNA by 20min, 31S and 25S rRNA by 40min, and 18S RNA by 60min. "Fingerprint' analysis of T1-ribonuclease digests show that all the large oligonucleotides present in 25S and 18S RNA are present in the 31S species, indicating that 31S RNA is the precursor rRNA molecule to both 25S and 18S RNA. The importance of these early RNA syntheses and in particular the possible template function of the heterogeneous RNA is discussed in relation to the concept of long-lived mRNA and the coding for protein synthesis in the first hours of germination.

Nuclear receptors for thyroid hormone: evidence for nonrandom distribution within chromatin.

Abstract: Chromatin receptor proteins appear to mediate some actions of thyroid hormone. In this study, sheared mammalian chromatin containing [125I]triiodothyronine (T3) bound by these receptors was separated using sucrose gradient velocity sedimentation. T3-receptor complexes were distributed throughout the chromatin fractions, but were enriched in the slowly sedimenting fractions. The latter contain most of the template capacity for RNA synthesis and most of the endogenous RNA polymerase activity but a minor portion of the total DNA. Formaldehyde treatment of chromatin containing receptor-bound [125 I ]T3 resulted in fixation of radioactivity, as evidenced by its migration with chromatin after equilibrium density gradient sedimentation in both cesium chloride and Conray. This fixation implies that the T3 receptor protein is closely associated with chromatin. These results suggest that proteins involved in the regulation of gene function may be nonrandomly distributed within chromatin subfractions, and are consistent with a direct role for thyroid hormone in regulating genetic expression.

Progressive decrease in protein synthesis accuracy induced by streptomycin in Escherichia coli

Abstract: THE passage of genetic information from gene to polypeptide does not proceed with perfect fidelity1–3, but it is not known what short and long-term cellular responses may be induced by decreases in the accuracy of protein synthesis. Errors in gene expression may tend to increase their own rate of production4, and thus could constitute a limiting instability of cellular life. The synthesis of inaccurate RNA, RNA polymerases, tRNAs, tRNA modifying enzymes, tRNA charging enzymes and ribosomal proteins are potential pathways for the perpetuation or amplification of errors. Such errors may be inducible in vivo by external chemical agents and may constitute an indirect pathway for mutagenesis5–8. An enhanced rate of such errors may be the mechanism by which certain deleterious mutations exert their effects. Here we report the results of experiments with Escherichia coli designed to detect evidence of self amplification of errors in protein synthesis. If such error feedback is quantitatively significant, a slight increase in the pre-existing error rate should be followed by a progressive decrease in the accuracy of protein synthesis. While this process could lead to a new stable level and thus need not precipitate a catastrophic breakdown, some late effects should be observable if the mechanisms are at all as hypothesised.

Gene transfer to human cells: transducing phage lambda plac gene expression in GMI-gangliosidosis fibroblasts.

Abstract: Genetic information from the bacterium Escherichia coli was transferred to human cells by means of the specialized transducing phage lambda plac carrying the bacterial z gene for the enzyme beta-galactosidase (geta-D-galactoside galactohydrolase, EC 3.2.1.23). As recipient cells, cultured skin fibroblasts from a patient with generalized gangliosidosis (GMI-gangliosidosis Type I) characterized by a severe deficiency of beta-galactosidase activity were used. The deficient human cells were incubated with the bacteriophage lambda plac or lambda plac DNA and beta-galactosidase activity was measured in order to detect gene transfer and acceptance of the prokaryotic information in the mammalian system for transcription and translation. The expression of the phage genome in the deficient fibroblasts could be demonstrated by detection of higher beta-galactosidase activity after incubation with phage lambda plac in three out of 19 experiments and in four out of 16 experiments after treatment with lambda plac DNA. Lambda plac DNA induced much higher enzyme activities than infective phage particles. Immunochemical and physicochemical assays could not distinguish the induced beta-galactosidase activity from that of the z-gene product of E. coli.

Strand-selective transcription of globin genes in rabbit erythroid cells and chromatin.

Abstract: In order to investigate the symmetry of globin gene transcription, complementary RNA (cRNA) was synthesized using rabbit globin complementary DNA (cDNA) as a template for Escherichia coli DNA-dependent RNA polymerase (RNA nucleotidyltransferase). The cRNA hybridized specifically to its own cDNA template but not to sheep cDNA, rabbit globin mRNA, or poly(dT). Hybridization studies with cRNA demonstrated that RNA sequences transcribed from the DNA strand complementary to the globin gene region (anti-strand) were not present in cellular, total nuclear, or fractionated nuclear RNA from rabbit marrow. Such sequences were detected in RNA transcribed from rabbit marrow chromatin by E. coli or sheep liver RNA polymerases, but amounted to less than 50% of the globin mRNA sequences present in the same transcript. The evidence indicates that globin mRNA transcription is predominantly DNA strand specific.

Effect of estrogen on gene expression: purification of vitellogenin messenger RNA.

Abstract: We report initial studies on estrogen-mediated regulation of egg yolk protein synthesis in the rooster. Egg yolk proteins are normally synthesized as a large precursor, vitellogenin, in the liver of the laying hen; roosters synthesize vitellogenin only when treated with estrogen. Polysomal RNA from the liver of estrogen-treated roosters was translated in a reticulocyte cell-free system, and the newly synthesized proteins were identified by a highly specific and sensitive indirect immunoprecipitation reaction. The messenger RNA that specifies vitellogenin has been purified more than 800-fold from rooster liver polysomal RNA by a combination of methods, including immunoprecipitation of polysomes and chromatography of RNA on poly(U)-Sepharose.

Parameters of gene expression in the bipolar argECBH operon of E. coli K12. The question of translational control.

Abstract: The pattern of divergent transcription of the argECBH cluster of genes previously demonstrated by the hybridization of RNA to the separated strand of a ϕ80 drag transducing phage, is confirmed with the DNA of a set of different λdarg phages. The accurate determination of argE and argCBH m-RNA levels in different steady states of expression of the arg regulon supports the following conclusions:

Preliminary evidence for the effects of environmental complexity on hybridization of rat brain RNA to rat unique DNA.

Abstract: Hybridization to unique DNA by RNA from brain or liver of rats given varying degrees of experience was investigated. RNA from brain of environmentally enriched rats hybridized to more unique DNA than did brain RNA from nonenriched controls. No significant differences were observed with liver RNA. This provides preliminary evidence for an increased transcription of the unique sequences of DNA in the enriched animals. The technique used has great promise for the investigation of experientially-induced alterations in gene expression.

Inactive complex formation between E. coli RNA polymerase and inhibitor protein purified from T7 phage infected cells

Abstract: THE “host shut-off” function of T7 phage has been thought to result from an inhibition of the host Escherichia coli RNA polymerase, which transcribes host RNA and T7 early mRNA, by an early T7 protein presumably the product of gene 0.7, or the “host shut-off” gene1–4. When T7 infection proceeds, early mRNA synthesis is shut off and the T7-specific RNA polymerase5, the product of an early gene of T7 (gene 1), transcribes late mRNA. Thus, “host shut-off” is another control factor involved in the early–late switch of T7 gene expression in addition to the switch of two RNA polymerases from the host to the phage-coded enzyme transcribing early mRNA and late mRNA, respectively, at different times of infection3.

THE INJECTION OF RNA INTO LIVING CELLS: THE USE OF FROG OOCYTES FOR THE ASSAY OF mRNA AND THE STUDY OF THE CONTROL OF GENE EXPRESSION

Abstract: Publisher Summary This chapter discusses the use of frog oocytes for the assay of mRNA and the study of the control of gene expression To identify mRNA, it should be demonstrated that it codes for a characteristic protein This means showing that synthesis of the particular protein is caused by adding the putative messenger to a system derived from cells that neither express nor even contain the genetic information for the protein in question In practical terms, this is most simply achieved by adding mRNA from one species to a translational system derived from another species The disadvantage of this approach is that translational systems from one species may be restricted in their ability to handle foreign messengers Restrictions are less likely to exist in a translational system derived from embryonic cells, for these, unlike differentiated cells, give rise to an enormous variety of cell types The sensitivity of such an assay for a particular messenger depends upon the ease of detecting one particular translation product among a wide variety of others and against a background of protein synthesis by the oocyte or egg