Expression of Bacteriophage M13 DNA in vivo. Isolation, Identification and Characterization of Phage-Specific mRNA Species (original) (raw)
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Initiation and termination signals for transcription in bacteriophage M13
Nucleic Acids Research, 1984
Transcription of the infrequently expressed phage M13 genome domain, comprising genes III, VI, I and IV, has been studied in detail by hybridization and SI-nuclease mapping studies. The contiguous genes III and VI are transcribed via an 1800 nucleotide-long RNA molecule that is initiated at a promoter which overlaps with the Rho-independent termination signal between genes III and VIII. Its synthesis is terminated at a Rho-dependent terminator in the proximal part of gene I. Transcription of gene I is not mediated by an independent promoter but most probably by read-through of RNA-polymerase through this terminator. Transcription of gene IV is accomplished by synthesis of four distinct RNAs of about 1500 to 1680 nucleotides long which are initiated at a promoter located immediately in front of gene IV. Termination of these transcripts is generated at at least four different sites located in tandem within the intergenic region between genes IV and II. phage morphogenesis and the replication origins for (+) and (-) strands are located .
Expression of bacteriophage M13 DNA in vivo
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1978
It is demonstrated that after infection of the appropriate minicell-producing strain of Escherichia coli with the filamentous bacteriophage M13, its replicative form DNA is segregated into miniceUs. Consequently these minicells have acquired the capability to direct the synthesis of phage-specific RNA and protein. Comparison of the electrophoretic mobilities of phage-specific RNA species made in vitro with those made in M13 replicative form DNA harbouring minicells, have indicated that almost all in vitro synthesized G-start RNAs have an equivalent among the in vivo synthesized RNA products. Furthermore it could be demonstrated that in M13 replicative form DNA harbouring minicells the phage-specific proteins encoded by genes III, IV, V and VIII are made. In addition the synthesis of a phage-specific polypeptide (molecular weight approx. 3000) co-migrating with the recently discovered capsid protein (designated C-protein) could be demonstrated. The meaning of these results for the resolution of the regulatory mechanisms operative during the life cycle of this phage will be discussed.
Mapping of Promoter Sites on the Genome of Bacteriophage M13
European Journal of Biochemistry, 1976
With the aid of transcription studies on restriction fragments of bacteriophage M13 DNA it has been demonstrated that at least eight promoter sites are located on the M13 genome. Five of these promoters initiate the synthesis of RNA chains which contain at their 5'-terminal end pppG (G promoters), while the other three promoters initiate RNA chains which start exclusively with pppA (A promoters). The positions of these promoter sites on the physical map are : 0.82 (G0.82), 0.88 0.94 (G0.94), 0.01 (Go.oI), 0.08 (G 0. 0 8) , 0.36 (A0.36), 0.51 (A0.51) and 0.56 (A0.56). The G promoters were found to be clustered within a distance of one-third of the genome length from the central termination site for transcription (map position 0.77). The A promoters, however, were found at greater distances from this termination signal. Based upon the incorporation of [y-32P]ATP or [ Y-~~P I G T P , the capacity of these promoters to initiate the synthesis of RNA chains varies. The strongest G promoters are G0.82, G0.94 and G0.08 and the strongest A promoter is A0.36. As judged from their position on the genetic map, it is postulated that two promoters, namely G0.94 and Go.01, are located within gene 11. The other promoters are most probably located immediately in front of the gene VIII/VII boundary (Go.xz), and immediately in front of gene V gene I1 (G0.08), gene IV (A0.36), gene I (A0.51) and gene VI (A0.56). No evidence has been obtained so far for the existence of a promoter immediately in front of gene 111.
Biochemical and Biophysical Research Communications, 1989
The transcription of phage Mu DRA during the lysogenic state has been quantitatively analysed. For this purpose pulse-labelled RNA from two lysogens and from their nonlysogenic parental strains were hybridized to non-overlapping Mu DRA restriction fragments covering the whole phage genome. The data revealed that all regions of the prophage are transcribed at low rates and that phage promoters are involved in this transcription. For this study an improved assay for quantitative filter hybridization was employed. The high sensitivity and reproducibility that can be obtained with the assay make it suitable for the quantitative analysis of minute amounts of mRRA. 0 1989 Academic FTe**, Inc. development by hybridization of pulse-labelled REA to plasmids that define Wu DBA segments covering the whole phage genome. In those experiments an assay for quantivative filter hybridization, that we derived from classical
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1987
The initial stages of transcription have been characterized using a template containing the gene I1 promoter region of M13 phage. Initiation of transcription in the presence of all four nucleotides gives rise to the 140-residue runoff transcript, with a minor pause at the RNA hexamer stage. Cycling, leading to the accumulation of significant amounts of short oligonucleotides [1], was not observed. An RNA hexamer GUUUUU was the sole product when GpU and UTP were used and the ternary complex with the hexamer was stable and resistant to high salt (0.4 M) and S1 nuclease attack. After direct ultraviolet photocrosslinking of the RNA hexamer to RNA polymerase in the ternary complex, the radioactive label incorporation into various subunits was determined by autoradiography after sodium tetradecyl sulfate gel eleetrophoresis to be as follows: o, 86%; t, 14%; fl' and a, negligible. Both electrophoresis and sucrose gradient centrifugation experiments indicate that the o subunit is not released from the ternary complex when either the RNA hexamer or the 140-residue RNA is synthesized on this template, even though the complexes are stable.
On the control of transcription of bacteriophage Mu
MGG Molecular & General Genetics, 1974
The transcription pattern of bacteriophage Mu has been studied with the use of Mu-lcts62, a thermo-inducible derivative of wild-type Mu. The rate of transcription at various times after induction was measured by pulse-labeling the RNA during synthesis and determining the fraction of Mu-specific RNA by hybridization with the separated strands of Mu-DNA. Transcription was found to take place predominantly from the heavy strand of Mu-DNA, as was found previously by Bade (1972). A study of the kinetics of this process revealed four phases. Initially after the induction the rate of transcription increases and reaches a maximum after four minutes. In the second phase during five minutes the rate falls down. During the third phase, up to 25 minutes after induction, the rate of transcription rises slowly, followed by a very rapid increase in the final phase, at the end of the lytic cycle. Phage Mu can be integrated in the host chromosome in two opposite orientations. The strand specificity, rate and time-course of transcription appeared not to be influenced by the orientation. The presence of chloramphenicol during the induction of the phage does not have an effect on the initial phase of transcription, but it prevents the decrease in the second phase. This suggests that in the early phase a Mu-speeific protein is synthesized which acts as a negative regulator of trancription. In non-permissive strains, lysogenic for a phage with an amber mutation in gene A or B, the transcription during the first and the second phase is the same as with wild-type phage; in the third phase, however, there is much less transcription than with wild type phage, whereas in the final phase the increase of the transcription rate is completely absent. Control experiments showed that DNA synthesis does not take place when a non-permissive strain is infected with a phage with an amber mutation in gene A or B. Therefore we conclude that the products of the genes A and B are required, directly or indirectly, for the autonomous replication of phage DNA. Since these amber mutants arc also impaired in the integration process, we conclude that the genes A and B code for regulator proteins with a crucial role in the development of bacteriophage Mu.
MGG Molecular & General Genetics, 1983
Fractionation of pulse-labeled RNA extracted from E. coli cells infected with phage fl and hybridization of this RNA to fl DNA reveals that very large species are synthesized on the phage genome. Hybridization of the RNA to specific fragments of fl DNA shows that, in the infected cell, at least one mRNA is present into which the sequences of genes III, VI, and I are all transcribed together. This result fully explains the polar effect shown by gene III mutants on the expression of genes VI and I .