Showing papers on "Transcription (biology) published in 1968"

Persistent synthesis of 5S RNA when production of 28S and 18S ribosomal RNA is inhibited by low doses of actinomycin D.

Abstract: To determine whether transcription of the genes coding for 5S ribosomal RNA depends upon the concurrent activity of the genes for 28S and 18S ribosomal RNA we studied the synthesis of 5S RNA in cultured L cells when the synthesis of the 45S precursor of 28 and 18S RNA was inhibited with low doses of actinomycin D. Synthesis of 5S RNA was measured by following the incorporation of 3H uridine into components which, upon acrylamide gel electrophoresis, co-migrated with markers of 5S RNA obtained from 32P labeled ribosomes. We observed that the synthesis of 5S RNA persists in the absence of detectable 28S and 18S RNA synthesis, continuing at the normal rate for several hours and at a reduced rate for at least a generation time. This strongly supports the concept that 45S RNA is not a precursor of 5S RNA, and indicates furthermore that neither the 45S component, nor any of the intermediates involved in its transition to 28S and 18S RNA, are involved in controlling the output of the 5S genes The 5S RNA which is made during actinomycin treatment is retained predominantly in the nucleoplasm and undergoes a turnover of about 3.5% per hour. It apparently cannot be utilized when ribosome synthesis resumes following removal of the actinomycin.

Molecular conformations and structure transitions of RNA complementary helices and their possible biological significance.

Abstract: RNA can exist in three distinct forms, all with a similar structure to that of the A form of DNA. The possible biological significance of these structures in protein synthesis and transcription mechanisms is discussed.

Messenger RNA in avian erythroblasts at the transcriptional and translational levels and the problem of regulation in animal cells.

Abstract: The RNA metabolism in immature duck erythrocytes has been investigated in order to determine the characteristics of messenger RNA (mRNA) in a highly differentiated animal cell. mRNA-like fractions were obtained from polysomes, on the one hand, and from pulse-labeled total cells or isolated nuclei, on the other, and were characterized by sedimentation, labeling kinetics, base composition, and hybridization to homologous DNA. At the translational level, the pulse-labeled RNA from polysomes consists of a predominant species sedimenting with about 9S and of a class of polydisperse material sedimenting between 6 and 28S. Very little ribosomal RNA (rRNA) is synthesized. The 9S RNA has been purified. Its base composition is relatively high in G + C (but different from rRNA or transfer RNA) – as determined, after alkaline hydrolysis, by 32P distribution or spectrophotometric analysis. The polydisperse RNA has a base composition characterized by relatively high proportions of U and A and is similar in this respect to nuclear RNA. Total polysomal RNA hybridizes to homologous DNA. The biological activity tested in a cell-free protein-synthesizing system of Escherichia coli is highest in the 16 to 18S zone of polysomal RNA. The rapidly labeled RNA synthesized at the transcriptional level in the nuclei sediments predominantly in the 30 to 80S zone. Base-composition analysis of this RNA reveals the presence of a predominant fraction of high-U-type RNA and of a small amount of 45 and 32S rRNA precursors. The former fraction — tentatively termed nascent, messenger-like RNA (nascent mlRNA), with respect to its base composition and capacity of selective hybridization — is metabolically more unstable than the precursor rRNA. Hybridization experiments demonstrate that up to 7% of the DNA is homologous to the nascent RNA fractions. Polysomal RNA hybridizes to a much smaller extent and competes only slightly with the heavy nuclear fractions. The significance of this heavy, nascent mlRNA and its eventual role in the regulation of protein synthesis in animal cells is discussed. We conclude that in a highly differentiated cell many more mRNA species are produced than would be expressed phenotypically through protein synthesis in the polysome. A surprisingly large part of the genome is activated, but an important fraction of the transcription products never reaches the sites of protein synthesis. Thus, the spectrum of functional RNA is not defined through synthesis only, but is restricted during metabolism. Under these conditions, control of differentiation is probably not limited exclusively to the transcription of the genome, but is subject to regulation mechanisms operating at the intermediate or translational level.

Regulation of Transcription of the SV40 DNA in Productively Infected and in Transformed Cells

Abstract: The expression of genes of the oncogenic virus SV40 is regulated. In productive infection, some genes are expressed before viral DNA replication begins (“early” genes); others are expressed after it has begun (“late” genes).1 In transformed cells, which contain the complete viral genome,2 some genes are expressed; others are not. Whether either type of regulation is at the level of transcription of viral genes is the question raised in the work to be reported here. The basic technique was hybridization of RNA synthesized in infected or transformed cells to viral DNA, and hybridization competition experiments. Results partly similar to those reported here were recently published by Aloni et al.3

Mutant of E. coli containing an altered DNA-dependent RNA polymerase.

Abstract: IT is generally believed that genetic transcription is carried out by an enzyme, DNA-dependent RNA polymerase1, which is capable of copying DNA sequences into RNA sequences. The enzyme requires DNA and the four naturally occurring ribonucleotide triphosphates to produce RNA which is a faithful copy of the DNA template2 in terms of base composition, nearest neighbour analysis and DNA–RNA hybridization3.

Enzyme induction in embryonic retina: the role of transcription and translation

Abstract: The mechanisms of induction in embryonic cells and of the timing and stability of phenotypic changes are of major importance to the understanding of differentiation. Detailed studies of these problems have been hindered by the scarcity of suitable experimental systems. What is required is an embryonic tissue in which a chemically defined inducer promptly elicits a specific, measurable response characteristic of differentiation. The induction of glutamine synthetase in embryonic neural retina meets these requirements. The normal developmental pattern of this enzyme is representative of the progress of retinal differentiation and provides a quantitative indicator of this process; moreover, this pattern can be significantly modified by defined experimental conditions. Glutamine synthetase activity (GS) in the embryonic chick neural retina follows a characteristic developmental pattern that is typical for this tissue and is temporally and spatially correlated with other aspects of retinal development.1-4 During early embryonic development, GS activity in the retina increases at a slow rate, then rises very sharply after the sixteenth day during the period of rapid functional differentiation and maturation of the retina. Particularly important is the fact that the rapid rise of GS activity can be induced precociously, several days before the normal time, in cultures of embryonic retina with 1l1g-hydroxycorticosteroids (hydrocortisone, aldosterone, or corticosterone).5 6 A similar precocious induction of retinal GS can be elicited also in embryos by injecting one of these steroids. Induction of GS by steroids is specific for the embryonic neural retina; it does not involve cell proliferation and therefore is not due to differential growth. The induced rise in GS activity is accompanied or followed by additional developmental changes which normally occur later in development;2' 7, 8 thus, it is not an isolated response but one of a number of phenotypic changes accelerated by the steroid inducer. Since changes in retinal GS activity are an essential aspect of differentiation, the mode of regulation of GS activity is relevant to the mechanisms that control differentiation in this tissue. Previous work on control mechanisms in this system was limited to long-term cultures of the retina ;4, 9-12 however, the enzyme begins to rise very shortly after exposure of the tissue to the inducer.13 The present report is concerned with an analysis of the processes that control the early phases of the induction of retinal GS by steroid (hydrocortisone). Materials and Methods.-Organ cultures of retina: Standard procedures for the isolation and cultivation of embryonic chick neural retina in flask organ cultures were described before.3'7 In this study, retina tissue from 12-day chick embryos was used. Each culture contained one whole retina (approximately 101 cells; 2.5 mg protein) in 3 ml of culture medium in a 25-mi Erlenmeyer flask; the flasks were rotated (70 rpm) at 380C on a gyratory shaker. The cultures were maintained for various times, as described, up to 24 hr. The medium was 80% Eagle's medium (without glutamine) with 20% fetal bovine

Increased nucleic-acid synthesis in relation to the breaking of dormancy of hazel seed by gibberellic acid.

Abstract: Gibberellic acid breaks dormancy of hazel seeds. Markedly preceding growth of the embryonic axis is an increased RNA synthesis, as shown by total RNA determination and 32P incorporation into RNA. This increased RNA synthesis is accounted for by an increase in DNA template available for transcription and (subsequently) increased RNA polymerase activity. It is suggested that gibberellic acid breaks dormancy by a mechanism of gene derepression, as indicated by increased chromatin-directed RNA synthesis as measured in vitro.

Studies on the mechanism of action of progesterone in regulation of the synthesis of specific protein

Abstract: To study the process of hormone action, we have developed an in vitro system utilizing minced oviduct from estrogen-treated chicks incubated in tissue culture medium. Progesterone added to the medium induced synthesis of a specific protein, avidin, that continued for up to 96 hr. During this period there was no increase in total oviduct protein, ovalbumin, or lysozyme, which suggests the specificity of the progesterone effect. The induction process was dependent on new protein synthesis, since cycloheximide inhibited the induction completely. Actinomycin D in doses that prevented nuclear RNA synthesis, but not general protein synthesis, inhibited avidin production 70-90%. Avidin synthesis was not affected by 5-fluorouracil. The rate of DNA synthesis examined by thymidine-3H pulse labeling was not stimulated during avidin induction. Hydroxyurea (an inhibitor of DNA synthesis) and colchicine (a mitotic inhibitor) did not prevent induction. Studies utilizing uridine-3H pulses showed an effect on rapdly labeled nuclear RNA coincident with induction. Nuclear RNA polymerase activity increased before avidin induction. Since avidin was the only new protein synthesized in response to progesterone, the early stimulation of nuclear RNA synthesis and RNA polymerase activity would suggest a mechanism of action for this steroid at the transcription level of protein synthesis.

The transcription of the SV40 genome in productively infected and transformed cells.

Abstract: A particular feature of cells which have been transformed by DNA-containing tumor viruses is the restricted expression of the viral genome. These cells do not display any of the viral functions which occur late during a productive cycle of infection—such as extensive replication of the viral DNA, production of viral coat protein, and maturation of infectious viral progeny. They do contain, however, tumor antigen which is synthesized early after infection. Hence, it appears that in transformed cells only “early” but no “late” products are being synthesized.

Resolution of two factors required in the Q-beta-RNA polymerase reaction.

Abstract: THE Escherichia coli bacteriophage Qβ contains RNA as its genetic component. Qβ-RNA can be synthesized in vitro in the reaction catalysed by the Qβ-RNA polymerase isolated from Qβ infected E. coli1. The synthesis of infectious Qβ-RNA requires, in addition to the polymerase, the phage RNA to serve as template, ribonucleoside tri-phosphate substrates, Mg++, and a factor fraction obtained from both infected and uninfected E. coli2. Previous studies with the factor have suggested that it acts on Qβ-RNA at some early step in the reaction2. Although an association of the enzyme and Qβ-RNA occurs in the absence of this factor3, synthesis of the complementary minus strand is not detected. The requirement for this agent in the reaction seems to bear a quantitative relationship to Qβ-RNA but not to enzyme2. The factor fraction, furthermore, is not required for enzyme activity when synthetic polymers4, minus strands or other RNA molecules3 are used as template. These results suggest that the role of factor is to promote a step in the reaction occurring after association of the enzyme with Qβ-RNA, but before nucleotide polymerization.

Seapration of B. subtilis DNA into complementary strands. II. Template functions and composition as determined by transcription with RNA polymerase.

Abstract: In a very receint commulicationl' it was showin that denatured DN'A of B. subtilis can be separated oni a methylated albumini-kieselguhr (MAK) column into two fractionis. These fractions, designated L (light) and H (heavy), were considered to represent the two complementary strands of the native duplex; an attribution based principally on biological properties (bacterial transformation) as well as on a few physicochemical criteria. Additional evidence in support of this view will be presented in this study, which shows that the polyribonlucleotides synthesized enizymically with the separated fractions as the templates exhibit a mutually complementary base composition. The discussion will also offer the opportunity of pointing out an uinexpected compositionial regularity evideniced by the single strands. Previous work in m'any laboratories2" as well as in this laboratory7-9 has indicated that the polyribonucleotide product of the action of RNA polymerase is a more or less faithful complementary copy of the DNA serving as the template. Since it was knowni that denatured templates could be copied without the alteration of the expected base ratios of the product,7' 9 it was of interest to deduce the base compositiont of the separated DNA strands from the anialysis of the polyribonucleotides to which they gave rise. Because of the great sensitivity of the method, only about 30 Ag of DNA are required for a complete analysis in triplicate.

Bacteriophage Coat Protein as Repressor

Abstract: It seems that viral coat protein acts as a repressor of protein synthesis at the level of transcription rather than translation.

RNA synthesis in synchronously growing populations of HeLa S3 cells. II. Rate of synthesis of individual RNA fractions.

Abstract: The rates of synthesis of various species of RNA were examined in synchronously growing HeLa cells as a function of stage in the cell generation cycle. The synthesis of each RNA species examined occurs throughout interphase, but undergoes a two-fold increase in rate during early S which is dependent on the duplication of DNA; blocking the initiation of DNA synthesis also blocks the acceleration of RNA synthesis. To explain the data, a model is discussed in which the acceleration of RNA synthesis during early S is regulated by the number of active cistrons present; thus, as the genome is duplicated, more template is available for transcription, and the rate of RNA synthesis increases. Some implications of the model, and experimental evidence bearing on them, are also discussed.

Inhibition of host protein synthesis during infection of Escherichia coli by bacteriophage T4. I. Continued synthesis of host ribonucleic acid.

Abstract: The ribonucleic acid (RNA) synthesized at specified intervals during infection of Escherichia coli K-12 by bacteriophage T4 was hybridized to denatured E. coli or T4 deoxyribonucleic acids (DNA). The reactions were performed under conditions that maximized the yield and at RNA/DNA inputs such that excess DNA sites were available for all RNA species. Most of the RNA synthesized at any time during the first 3 min of infection was host-specific. The fraction declined rapidly as infection progressed; host RNA represented about half that made between 3 and 4 min. It is unlikely that this represented RNA synthesized by bacteria that had escaped infection, as judged by the kinetics of adsorption and killing as well as by the rapid inhibition of beta-galactosidase induction after infection. The nature of the host RNA was also examined. Part of the RNA synthesized during infection of cells rendered sensitive to actinomycin was stable in the presence of this inhibitor. This RNA was essentially all host-specific and it sedimented as ribosomal and transfer RNA; most of the ribosomal RNA was incorporated into 30S and 50S ribosomes. Hybridization analyses suggested that unstable E. coli messenger RNA was also synthesized for several minutes after infection; the proportion of unstable to stable host RNA synthesized appeared to be similar in infected and uninfected cells. Thus, it is concluded that significant amounts of E. coli RNA are synthesized during the first minutes of T4 infection. Host messenger RNA initiated after infection may not be translated into enzymes; alternatively, it is conceivable that continued bacterial messenger RNA synthesis only reflects the completion of transcription of operons whose reading was initiated prior to infection.

Isolation of Chromatin-free RNA Polymerase from Mammalian Cell Nuclei

Abstract: MAMMALIAN RNA polymerase1 has been obtained in a soluble form from tumours2,3, chick embryo4, rat testis5 and rat liver nuclei6,7. The extraction procedures used involved the disruption of cell nuclei by sonication or exposure either to a hypotonic solution or alkaline pH. As a consequence, extracts were grossly contaminated with nuclear DNA and other cellular components and the yields of the enzyme were usually very low. We describe here a procedure for the rapid extraction from liver nuclei of fairly large yields of soluble RNA polymerase with fairly high specific activity.