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PubMed1977

Base-unpaired regions in supercoiled replicative form DNA of coliphage M13.

Superhelical covalently closed circular replicative form DNA (RF I) of coliphage M13 appears as a relaxed molecule that has a base-unpaired region in the form of a bubble (100 to 200 base pairs long) seen in electron micrographs when spread in the presence of formaldehyde and formamide or after pretreatment with glyoxal. S1 endonuclease, specific for single-stranded DNA, converts superhelical M13 RF I DNA, but not nonsuperhelical M13 RF I to a significant extent, into unit-length linear molecules by sequential nicking of two strands. The locations of S1 nuclease-susceptible sites and glyoxal-fixed base-unpaired regions were both related to the five A-T-rich regions in M13 RF DNA. While S1 nuclease does not show preference for any of these sites, glyoxal-fixed bubbles occur predominantly at the major A-T-rich region in M13 RF DNA.

Coliphages↗
PubMed1978

The peculiar behaviour of coliphage P1vir mutants on restricting hosts.

The sensitivity of two vir coliphage P1 mutants, to the host-specificity systems A, K, RII, and B of Escherichia coli, and to the newly identified host-specificity systems Mir and 59 of Klebsiella pneumoniae has been studied. Both 1 vir phages were completely resistant to the E. coli K, but were restricted 10(-5) by the B, mir and 59 systems. The P1 parent, on the contrary, was restricted 10(-2) by all of the above mentioned hosts. The possible role of phage coded proteins in the altered sensitivity of the vir strains has been studied by analysing the sensitivity of both P1, and P1vir to the various systems in the presence of chloramphenicol. Protein synthesis inhibition by chloramphenicol did not significantly influence the sensitivity of P1 and P1vir to various restricting hosts. It is concluded that the altered sensitivity of vir mutants depends on the different affinities of the mutated phage DNA sequence for the enzymes of the various restriction and modification systems. The possibility that the phage repressor contributes to the regulation of the restriction and modification enzymes is also discussed.

Chloramphenicol↗
PubMed1983

Purification and characterization of coliphage N4 RNA polymerase II activity from infected cell extracts.

The soluble components of the RNA polymerase activity (N4 RNA polymerase II) required for coliphage N4 middle RNA synthesis have been purified to homogeneity using a complementation assay described elsewhere. These soluble components are found to exhibit the properties of a DNA-dependent RNA polymerase which is resistant to both rifampicin and streptolydigin and transcribes denatured N4 DNA with marked preference but with little selectivity for the middle region of the N4 genome. In its native form, the activity is composed of one Mr = 40,000 polypeptide (p4, the product of N4 cistron 4) and one Mr = 30,000 polypeptide (p7, the product of N4 cistron 3). Its physical properties and the mechanism of transcription selectivity are discussed.

Coliphages↗
PubMed1980

Evaluation of coliphages in sewage effluent of Faisalabad.

One hundred and ten samples of Sewage were collected from both underground sewage and open drain systems of Faisalabad for coliphage assay. It was observed that the samples from underground sewage system ranged from 8.43 X 10(3)--4.65 X 10(3) in mean plaque forming units (PFU) per ml, whereas the corresponding figures in open drain system varied from 8.66 X 10(3)--3.21 X 10(3) mean PFU per ml. In general, samples from congested areas of both the systems studied tended to be richest both in mean PFU per ml as well as plaque morphological variations. Overall 620 plaque morphological classes were isolated. It was also noted that the mean PFU per ml was higher in the summer than in the winter months and phage contents were increased after rain fall.

Coliphages↗
PubMed1994

A hammerhead ribozyme inhibits the proliferation of an RNA coliphage SP in Escherichia coli.

Ribozymes are potentially powerful tools for the suppression of intracellular gene expression. However, the few reports that exist of their activities in bacteria have described mixed success. Chuat and Galibert (Chuat, J.-C., and Galibert, F. (1989) Biochem. Biophys. Res. Commun. 162, 1025-1029) failed to detect any trans-activities of hammerhead ribozymes in Escherichia coli, while Sioud and Drlica (Sioud, M., and Drlica, K. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7303-7307) reported complete inhibition of expression of the gene for a nonbacterial protein, HIV-1 integrase, by trans-acting hammerhead ribozymes in E. coli. It is of interest to determine whether ribozymes can really be used in natural bacterial systems (Altman, S. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10898-10900). We now report that a ribozyme designed to cleave the A2 gene of RNA coliphage SP, when transcribed from a plasmid in E. coli caused failure of the proliferation of progeny phage. Inactive ribozymes with altered catalytic sequences did not affect phage growth. These results indicate that it is mainly the catalytic activity of the ribozyme and not its function as an antisense molecule that is responsible for suppressing the proliferation of the RNA phage. Moreover, an analysis based on numbers of plaque-forming units and the function of the A2 protein indicated that antisense RNA may successfully compete with ribosomes in targeting mRNA while ribozymes in this study may not compete with ribosomes in naturally occurring bacterial transcription/translation-coupled systems.

Base Sequence↗
PubMed1977

Sequence of the A-protein of coliphage MS2. I. Isolation of A-protein, determination of the NH2- and COOH-terminal sequences, isolation and amino acid sequence of the tryptic peptides.

The A-protein of coliphage MS2 was purified to a state of sufficient homogeneity to study its primary structure. The NH2-terminal sequence was determined for the first 8 residues. Comparison with the reported sequence of R17 protein (Weiner, A. M., Platt, T., and Weber, K. (1972) J. Biol. Chem. 247, 3242-3251) shows a difference at position 6 where alanine in R17 is replaced by threonine in MS2. The COOH-terminal sequence was shown to be -Arg-Leu-Ser-Arg, confirming the existence of UAG as the termination codon of the maturation protein (Comtreras, R., Ysebaert, M., Min Jou, W., and Fiers, W. (19731 Nature New Biol. 241, 99-101; Vandekerckhove, J., Nolf, F., and Van Montagu, M. C. (1973) Nature New Biol. 241, 102; Remaut E., and Fiers, W. (1972) J. Mol. Biol. 71, 243-261). Peptides obtained by enzymatic hydrolysis with trypsin were fractionated by a combination of gel filtration and paper electrophoresis and chromatography. Thirty-eight peptides were analyzed for amino acid composition and sequence. They provide information for 312 of the 393 residues of the A-protein polypeptide chain.

Amino Acid Sequence↗
PubMed1998

[COS-region of temperate coliphage N15].

The cohesive termini including the cos region (altogether 414 bp) of the DNA of the temperate coliphage N15 are sequenced. The termini are complementary 12-nucleotide single-stranded 5'-extended DNAs. The sequence of the left terminus is 5'-GGGCGGCGTCCG-3', that of the right 5'CGGACGCCGCCC-3'. Ten nucleotides of the N15 termini are identical to those of phage lambda. The N15 and lambda sequences are notably homologous only within the 50 bp region from the left and right ends. Phage N15 has a region with the nucleotide sequence identical to the R4 site of phage lambda, presumably reacting with terminase. This region is situated in the same site with regard to the cohesive sequence as in phage lambda. The cos region of N15 has no sequences similar to R1, R2, and R3 of lambda. N15 has a sequence similar to IHF of phage lambda, but in N15 this sequence is located near the right but not left (as in phage lambda) terminus. Computer analysis revealed palindromes and repeats within 450 bp of N15, including the cohesive termini.

Bacteriophage lambda↗
PubMed1988

PL of coliphage lambda: an alternative solution for an efficient promoter.

Promoter PL of coliphage lambda is highly active in vivo although it is recognized 15-30 times less efficiently by RNA polymerase when compared with promoters of similar strength. Moreover, it differs significantly from the consensus sequence for Escherichia coli promoters. Sequence variants of PL which are more homologous to consensus promoters bind RNA polymerase with increased efficiency. They are nevertheless significantly reduced in their in vivo strength. High activity can be restored by a downstream sequence of a typical consensus-like promoter. Evidently, such elements are required for the efficient release of a stably bound RNA polymerase into a transcriptional elongation complex. We propose that the functional programme encoded in a promoter sequence can be optimized in alternative ways.

Bacteriophage lambda↗
PubMed1994

Alterations of receptor specificities of coliphages of the T2 family.

The T-even type coliphage M1 uses the outer membrane protein OmpA as a receptor. Host range mutants were isolated in two sequential steps, the first resulting in strains able to use the porin OmpC, the second in mutants using the outer membrane protease OmpT as receptor. The mutational alterations in the receptor-recognizing protein of the phage (a component of the long tail fibers) have been determined. Their character and that of such previously described alterations suggest an antibody-type of ligand binding.

Amino Acid Sequence↗
PubMed1997

Translational activation in coliphage Qbeta: on a polycistronic messenger RNA, repression of one gene can activate translation of another.

We present evidence for translational activation of the Qbeta coliphage maturation cistron, mediated by the presence of Qbeta replicase. This activation does not require RNA replication, translation of a second gene, or any direct protein-RNA binding at the maturation gene initiation site. Our data support a model in which the Qbeta maturation gene remains translationally "off" by two means: (1) the thermodynamic stability of an RNA structure that greatly discourages, but does not eliminate, ribosome access at the maturation start site; and (2) the presence of the stronger, proximal coat gene ribosome binding site. Moreover, maturation gene expression is switched "on" when ribosome entry at the coat initiation site, present on the same polycistronic RNA molecule, is repressed by Qbeta replicase, thereby allowing ribosomes to compete for the weaker, upstream maturation start site.

Allolevivirus↗
PubMed1998

Structural plasticity in RNA and its role in the regulation of protein translation in coliphage Q beta.

We have analyzed both conformational and functional changes caused by two large cis-acting deletions (delta 159 and delta 549) located within the read-through domain, a 850 nucleotide hairpin, in coliphage Q beta genomic RNA. Studies in vivo show that co-translational regulation of the viral coat and replicase genes has been uncoupled in viral genomes carrying deletion delta 159. Translational regulation is restored in deletion delta 549, a naturally evolved pseudorevertant. Structural analysis by computer modeling shows that structural features within the read-through domain of delta 159 RNA are less well determined than they are in the read-through domain of wild-type RNA, whereas predicted structure in the read-through domain of evolved pseudorevertant delta 549 is unusually well determined. Structural analysis by electron microscopy of the genomic RNAs shows that several long range helices at the base of the read-through domain, that suppress translational initiation of the viral replicase gene in the wild-type genome, have been destabilized in delta 159 RNA. In addition, the structure of local hairpins within the read-through region is more variable in delta 159 RNA than in wild-type RNA. Stable RNA secondary structure is restored in the read-through domain of delta 549 RNA. Our analyses suggest that structure throughout the read-through domain affects the regulation of viral replicase expression by altering the likelihood that long-range interactions at the base of the domain will form. We discuss possible kinetic and equilibrium models that can explain this effect, and argue that observed changes in structural plasticity within the read-through domain of the mutant genomes are key in understanding the process. During the course of these studies, we became aware of the importance of the information contained in the energy dot plot produced by the RNA secondary structure prediction program mfold. As a result, we have improved the graphical representation of this information through the use of color annotation in the predicted optimal folding. The method is presented here for the first time.

Allolevivirus↗
PubMed1995

Protection of coliphage lambda O initiator protein from proteolysis in the assembly of the replication complex in vivo.

We have shown previously that, in contrast to the free coliphage lambda O initiator protein rapidly degraded by ClpP/ClpX protease, the lambda present in the replication complex (RC) is protected from proteolysis. Now we asked at which step of the pathway of RC assembly in vivo does the stabilization of lambda O occur. In accordance with the in vitro established order we found that lambda P and DnaB helicase functions are, but those of DnaJ and GrpE chaperones are not, required for the protection of lambda O from proteolysis. Therefore, our results suggest that the first lambda O protecting structure of the pathway of RC assembly is the lambda O-lambda P-DnaB preprimosome. The next step of the pathway, the chaperone-mediated rearrangement of the preprimosome, is not essential for lambda O stabilization. However, in contrast to other chaperones, the DnaK function was required for the protection of lambda O from proteolysis, suggesting an earlier access of DnaK to the pathway of RC assembly in vivo, in accordance with current models by which molecular chaperones facilitate protein assembly.

Bacterial Proteins↗
PubMed1998

A vibriophage, KVP40, with major capsid protein homologous to gp23* of coliphage T4.

The mcp gene encoding the major capsid protein (Mcp) of vibriophage KVP40, a large-tailed DNA phage, was cloned and sequenced. The nucleotide sequence of the mcp gene was 64.4% similar to that of gene 23 of coliphage T4. Analysis of the N-terminal amino acid sequence of purified native Mcp revealed that the mcp gene actually coded for a precursor, pro-Mcp, whose 62 N-terminal amino acids must be removed upon maturation to Mcp. Thus, mature Mcp would consist of 452 amino acid residues and have a calculated molecular mass of 47,561 Da. Comparison of amino acid sequences of Mcp and gp23*, the major capsid protein of T4, demonstrated 61.8% identity and 89.7% similarity between them. In addition, a sequence, TATAAATA, identical to a typical T4 late promoter sequence was seen in the region upstream of the mcp gene. These findings, together with their morphological similarity, suggest that KVP40 and T4 are phylogenetically related.

Amino Acid Sequence↗
PubMed1985

Transcription termination regions of coliphage T7 DNA: the effects of nusA1.

We report the effects in vivo of four segments of coliphage T7 DNA upon expression, from an upstream promoter, of galK in plasmids of the pKO family. Three of the segments carry the known major or putative distal terminators of host-dependent T7 early transcription. The fourth carries a novel terminator and maps in the late region of T7. We report the efficiencies of termination in these regions: evidence, based on studies with the E. coli nusA1 mutation, for an involvement of the transcription factor NusA in events at the major early and novel terminators: and the nature of the latter transcription signal.

Base Sequence↗
PubMed2005

Site-specific recombination in Arabidopsis plants promoted by the Integrase protein of coliphage HK022.

The gene encoding the wild type Integrase protein of coliphage HK022 was integrated chromosomally and expressed in Arabidopsis thaliana plants. Double-transgenic plants cloned with the int gene as well as with a T-DNA fragment carrying the proper att sites in a tandem orientation showed that Int catalyzed a site-specific integration reaction (attP x attB) as well as a site-specific excision reaction (attL x attR). The reactions took place without the need to provide any of the accessory proteins that are required by Int in the bacterial host. When expressed in tobacco plants a GFP-Int fusion exhibits a predominant nuclear localization.

Arabidopsis↗
PubMed1992

Stability of coliphage lambda DNA replication initiator, the lambda O protein.

The initiator of coliphage lambda DNA replication, lambda O protein, may be detected among other 35S-labeled phage and bacterial proteins by a method based on immunoprecipitation. This method makes it possible to study lambda O proteolytic degradation in lambda plasmid-harboring or lambda phage-infected cells; it avoids ultraviolet (u.v.)-irradiation of bacteria, used for depression of host protein synthesis, prior to lambda phage infection. We confirm the rapid decay of lambda O protein (half-time of 80 s), but we demonstrate the existence of a stable lambda O fraction. In the standard five minute pulse-chase experiments, 20% of synthesized lambda O is stable. The extension of the [35S]methionine pulse, possible in lambda plasmid-harboring cells, leads to a linear increase of this fraction, as if a part of the synthesized lambda O was constantly made resistant to proteolysis. Less than 5% of lambda O protein synthesized during one minute is transformed into a stable form. We presume that the stable lambda O is identical with lambda O present in the normal replication complex and thus protected from proteases. We cannot find any stable lambda O in Escherichia coli recA+ cells that were irradiated with u.v. light prior to lambda phage infection, but their recA- counterparts behave normally, suggesting that recA function interferes in the assembly of a normal replication complex in u.v.-irradiated bacteria. The stable lambda O found in lambda plasmid-harboring, amino acid-starved relA cells is responsible for the lambda O-dependent lambda plasmid replication that occurs in this system in the absence of lambda O synthesis. The existence of stable lambda O raises doubt concerning its role as the limiting initiator protein in the control of replication. Another significance of lambda O rapid degradation is proposed.

Bacteriophage lambda↗
PubMed1987

The attachment sites of T5-host range temperate coliphages.

The attachment sites of 13 temperate coliphages were determined. Specialized transduction of proAB mutants was shown by eight isolates and of a his mutant by another two. Two isolates were concluded to integrate at atthtt and the integration site of one isolate remained undetermined.

Base Sequence↗
PubMed1989

Repression of transcription from the b2-att region of coliphage lambda by integration host factor.

The central b2-att region of coliphage lambda is known to be transcriptionally active in vitro, but silent in vivo in lambda lysogens. To explain such in vivo repression of transcription originating in the b2-att region, we explored the effect of the Escherichia coli integration host factor (IHF), the product of E. coli genes himA and himD, especially since the att region contains several IHF-binding sites. Using various lambda DNA templates, we mapped the transcripts which are initiated in vitro in the attP region by the RNA polymerase and found that there are three rightward (RI, RII, and RIII) and one leftward (LI) transcripts. All four of them are repressed by a factor of about 10 by 10 micrograms IHF/ml. Moreover, in in vivo experiments we found that plasmids carrying the attP fragment cannot be established and maintained in IHF-hosts. These results indicate that IHF may play a significant auxiliary role in repressing transcription in the prophage state.

Attachment Sites, Microbiological↗