Showing papers on "Transcription (biology) published in 1973"

Organization, transcription, and regulation in the animal genome.

Abstract: This review concern recent experimental information areas of animal cell molecular biology which are relevant to the mechanism of gene regulation. New data regarding interspersion and clustering of repetitive sequence elements in DNA are considered Molecular characteristics of animal structural genes and mRNAs are discussed, with particular reference the frequency of structural gene sequences, mRNA turn over and the interpretation of dipteran complementation groups. The molecular characteristics of nuclear RNAs, the primary transcription products, are reviewed. Evidence for transcription level regulation is summarized and the relation of nuclear and mRNA examined. The protein activator branch of the Britten-Davidson model for gene regulation is further developed and considered in light of current knowledge.

Synthesis of globin ribonucleic acid from duck-reticulocyte chromatin in vitro.

Abstract: The proteins of chromatin serve to restrict the transcription of DNA. The relevance of these findings to the control of gene expression is contingent upon the demonstration that this restriction is specific and mirrors the patterns of RNA synthesis observed in vivo. In this study we demonstrate by RNA-DNA hybridization that the vast majority of the chromatin-directed RNA is synthesized from the unique regions of the reticulocyte genome. Furthermore, by use of the DNA complement of globin mRNA as a probe in annealing reactions, de novo synthesis of globin RNA was detected in RNA transcripts from duck reticulocyte chromatin. No globin sequences were detected in similar preparations of RNA in vitro either from liver chromatin or from DNA freed of protein. These results show that the proteins of chromatin serve to restrict transcription in a very specific manner and provide convincing evidence for the existence of transcriptional control factors in eukaryotes.

The Nucleotide Sequence of the Lactose Messenger Ribonucleic Acid Transcribed from the UV5 Promoter Mutant of Escherichia coli

Abstract: I have sequenced the first 63 bases of mRNA transcribed in vitro from the UV5 promoter mutant of the E. coli lactose operon. Sonic fragments of DNA, 1000 base pairs long and purified to contain only the lac operator-promoter region, were used as template. The UV5 promoter mutation allows transcription of the lac operon in the absence of catabolite activator protein and cAMP; lac repressor controls the synthesis of this RNA. I find that during synthesis, RNA polymerase pauses at particular sites along the DNA, naturally generating several discrete sizes of RNA that provide overlaps useful for sequencing. The UV5 lac mRNA initiates within the lac operator and copies the operator sequence. The AUG initiator codon for β-galactosidase occurs at position 39 of the message. The sequence is: pppA-A-U-U-G-U-G-A-G-C-G-G-A-U-A-A-C-A-A-U-U-U- C-A-C-A-C-A-G-G-A-A-A-C-A-G-C-U-A-U-G-A-C-C-A-U- G-A-U-U-A-C-G-G-A-U-U-C-A-C-U-G-G.

T7 Early RNAs are Generated by Site-Specific Cleavages

Abstract: Transcription of T7 DNA by purified Escherichia coli RNA polymerase without added factors produces long RNA molecules that begin near the left end of T7 DNA and terminate at the end of the early region. An endonuclease has been isolated from uninfected E. coli that cleaves these long RNAs at five specific sites to generate RNA molecules essentially the same as the early T7 RNAs observed in vivo. This sizing factor, which may be RNase III, can act during or after RNA synthesis. Synthesis of early RNA chains has been shown to start at three strong initiators, spaced about 150-200 base-pairs apart near the left end of T7 DNA. Thus, the five cleavages by sizing factor generate the five early messenger RNAs of T7 plus three overlapping RNAs from the promoter region. RNA chains that are started at two of the strong initiators begin with A; those started at the third begin with G. A few minor initiators have also been observed, from which only short chains seem to be synthesized. Their locations in T7 DNA have not been mapped. Rho factor does not appear to be needed to generate any of these early T7 RNAs.

Tissue-Specific Transcription of the Globin Gene in Isolated Chromatin

Abstract: RNA was transcribed from chrmation from mouse fetal liver or brain, by use of DNA-dependent RNA polymerase of Escherichia coli. Globin messenger RNA sequences in the transcript were measured with complementary DNA copied from globin messenger RNA with RNA-dependent DNA polymerase. Globin messenger RNA sequences were found in RNA newly transcribed from chromatin from erythropoietic tissue but not in that transcribed from brain chromatin.

Control of short leftward transcripts from the immunity and ori regions in induced coliphage lambda.

Abstract: The patterns of (1) leftward transcription from the repressed lambda prophage and (2) post-induction changes in the initiation of RNA synthesis within the immunity-ori region (which contains several regulatory elements including the λ repressor gene) were studied in detail. In the noninduced prophage about 80% of the leftward transcription originates from within the immunity region (cI-rex mRNA), 2% is from the ori segment (oop RNA) and the remainder is evenly distributed between the int and b2 regions (Fig.1). The sc startpoint for the 1880 nucleotide-long cI-rex transcript, which codes for the λ repressor and the rex product, in 325 nucleotides from the right imm434 endpoint (Figs. 2 and 3). Upon induction of Tof+ λ lysogens, the cI-rex transcription is rapidly turned off. After a brief lag, a 600 nucleotide-long transcript, denoted lit, appears in the left part of gene rex. The lit and cI-rex transcripts both terminate at the same ti site. No RNA synthesis is detected in the 400-nucleotide segment between the left imm434 endpoint and the ti terminator. This DNA segment contains the pL-oL promoter-operator region for the major leftward transcription. The increase in lit transcription parallels the increase in synthesis of oop RNA, as if both transcripts originated from a common promoter or were positively regulated by a common factor at their promoters. The oop startpoint so is located at least 2000 nucleotides upstream from the lit startpoint si. The synthesis of the oop and lit RNAs is coordinately stimulated up to 100-fold by host and phage DNA replication factors. The short 4S oop RNA is thought to prime leftward λ DNA replication initiated at the ori site.

Effect of Rho on transcription of bacterial operons.

Abstract: In vitro experiments suggest that termination of transcription by the E. coli rho factor probably accounts for the natural polarity observed in certain bacterial operons, arrest of transcription at the end of operons and the severe polar properties of some insertion mutations.

Stabilization of Interferon Messenger RNA Activity by Treatment of Cells with Metabolic Inhibitors and Lowering of the Incubation Temperature

Abstract: Interferon production was induced in a strain of human diploid foreskin cells with poly(I)·poly(C). Cycloheximide was included in the culture medium at the time of addition of the inducer. Actinomycin D was added to the cultures 4 or 5 hr later, before the inhibition of protein synthesis was reversed at 6 hr. Thus, subsequent interferon synthesis had to be directed by messenger RNA synthesized before addition of actinomycin D. The amount of interferon produced after such treatment was about 50-fold greater than in cells induced with poly(I)·poly(C) but not treated with inhibitors. Experiments using cordycepin suggested that in spite of the continued presence of the inducer the synthesis of the bulk of interferon messenger RNA was completed within the first 2 hr of exposure of cells to poly(I)·poly(C) and cycloheximide. Transcription of interferon messenger RNA was apparently not affected when interferon synthesis was suppressed to various degrees by different inhibitors of protein synthesis, indicating the independence of transcription and translation. The high rate of interferon synthesis after the reversal of cycloheximide action was more sustained at 32° than at 37°. The rate of decrease of overall protein synthesis in cells treated with actinomycin D and then incubated either at 32° or 37° showed a similar dependence on incubation temperature, suggesting that the stability of messenger RNA (or of another actinomycin D-sensitive component required for protein synthesis) was greater at the lower temperature.

Effect of Circularity and Superhelicity on Transcription from Bacteriophage λ DNA

Abstract: We studied RNA synthesis in vitro from closed-circular λ DNA molecules with varying degrees of superhelicity. The four circular templates examined had 0, -50, -110, and -160 superhelical turns under the conditions of the transcription assay. We found that the total amount of RNA synthesis increases as the template acquires more negative superhelical turns. This increased transcription results from more frequent initiation of RNA chains. Transcription of circular DNA with no superhelical turns appears to mimic RNA synthesis in vivo more closely than transcription from either highly superhelical or linear DNA with regard to two criteria: preferential transcription of the region corresponding to early genes and sensitivity to repression by λ cI protein. We suggest that the physical basis for the increased initiation of RNA chains from superhelical DNA is the fact that unwinding events are energetically favored on a DNA molecule with negative superhelical turns. Possible general mechanisms are: (a) RNA polymerase must unwind the DNA duplex as a prelude to initiation; (b) the DNA itself must assume a new conformation at the promoter site which requires an unwinding of the DNA duplex.

RNA Synthesis of Vesicular Stomatitis Virus V. INTERACTIONS BETWEEN TRANSCRIPTION AND REPLICATION

Abstract: The temperature-sensitive mutant ts114 of vesicular stomatitis virus is temperature-sensitive in both primary and secondary transcription, but not in the replication of 40S RNA. The synthesis of 40S RNA is specifically inhibited when protein synthesis is shut off. The addition of cycloheximide to cells infected by ts114, rapidly inhibits RNA replication at the permissive and nonpermissive temperatures. However, the addition of cycloheximide at the nonpermissive temperature also results in almost complete recovery of transcription to the level found at the permissive temperature. Other inhibitors of protein synthesis applied at the nonpermissive temperature result in the same recoverability of the apparent temperature-sensitive lesion. Ribonucleic acid products synthesized by ts114 at the nonpermissive temperature in the presence of cycloheximide are identical to virus-specific messenger RNA by the criteria of size and complementarity to virion RNA. When mutant-infected cells are shifted to the nonpermissive temperature, incubated for a period of time, and then treated with cycloheximide, the ratio of transcriptive to replicative activity varies depending on the time at which radioactive precursor is added to cells. This suggests an interdependence between replication and transcription during virus-specific RNA synthesis.

Relationship of replication and transcription of Simian Virus 40 DNA.

Abstract: RNA produced by the Simian Virus 40 (SV40) mutant tsA30 during lytic infection of kidney cells of African green monkeys was examined by RNA-DNA competition-hybridization. This mutant is temperature-sensitive in a function (gene A) that regulates synthesis of viral DNA. No detectable difference between mutant RNA synthesized at the permissive temperature (33°) and wild-type viral RNA was found. During continuous infection with the mutant at the restrictive temperature (41°) only early viral RNA was produced. When mutant DNA and late RNA synthesis were initiated at the permissive temperature, a shift to the restrictive temperature rapidly terminated synthesis of viral DNA but not that of late viral RNA. The data indicate that the function of gene A is required before synthesis of late viral RNA and that after initiation, the production of late RNA continues without further expression of gene A or concomittant viral DNA synthesis.

Transcription of high-molecular-weight RNA from hen-oviduct chromatin by bacterial and endogenous form-B RNA polymerases.

Abstract: 1 With the aid of the ribonuclease inhibitor, heparin, techniques were developed to isolate high-molecular-weight RNA products synthesised by Micrococcus and homologous form B RNA polymerases using hen oviduct chromatin as template. These methods are suitable for analysis of RNA transcripts synthesised in vitro by a variety of enzymes and templates. 2 A highly sensitive assay for ribonuclease, based on the ability of the latter to degrade ovalbumin mRNA, confirmed that chromatin and enzyme samples contained significant amounts of ribonuclease activity. 3 Heparin completely inhibits initiation of both bacterial and homologous enzymes, but has no effect on elongation. The average rates of elongation of RNA polymerases on chromatin DNA could therefore be determined. These values were 3–6 nucleotides per second for bacterial enzyme and 1–2 nucleotides per second for homologous enzyme. 4 By allowing initiation of bacterial enzyme prior to the addition of heparin, RNA chains approaching 2000 nucleotides in length (molecular weight 0.7 X 106) were synthesised after 1 min; at later times, these chains exceeded 5000 nucleotides in length. 5 Hen oviduct chromatin contains endogenous RNA polymerase activity which represents predominantly the elongation of form B enzymes. RNA chains ranging from 100 to 2000 nucleotides long are still attached to these enzymes in isolated chromatin. By allowing elongation of these chains in vitro, products ranging from 2000 to 10000 nucleotides in length (molecular weight 0.7 to 3.0 X 106) were synthesised. 6 Estimates of the number of endogenous RNA polymerases bound to chromatin DNA suggest that approximately 10% of the total form B RNA polymerase molecules per cell are retained in the isolated chromatin fraction.