order of assembly of the lower collar and the tail proteins of bacillus subtilis bacteriophage phi 29.extracts obtained after restrictive infection of bacillus subtilis with mutants in cistron 11 of bacteriophage phi 29 are complemented in vitro by extract donors of the lower collar protein (p11). purified 11- heads, containing the major capsid protein (p8), the fiber protein (p8.5), the upper collar protein (p10), and the virus dna, can be also complemented in vitro to produce infective virus. this result suggests that 11- heads are intermediates in phage phi 29 morphogenesis. the order of asse ...1979107325
morphogenesis of bacteriophage phi 29 of bacillus subtilis: mapping and functional analysis of the head fiber gene.a set of mutants of bacillus subtilis bacteriophage phi29 unable to synthesize the head fiber protein has been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. infectious phage are produced during restrictive infection. we have focused on mutant sus 8.5(900) because the mutation is suppressible by both the su(+3) and su(+44) hosts, and it can be mapped by three- and four-factor crosses. after restrictive infection with mutant sus 8.5(900), a fragment a ...1977409854
[infectivity of dna-protein complex: transfection of bacillus phage phi29 (author's transl)]. 1975806101
isolation of a strong suppressor of nonsense mutations in bacillus treatment of bacillus subtilis mo-101-p spoa- met thr- su- with ethyl methanesulfonate, a strong suppressor strain of nonsense mutations, b. subtilis mo-101-p spoa- [met-]+thr- su+44, was isolated. this strain does not suppress phage phi 29 mutant susb47, selected on a b. subtilis strain containing the su+3 suppressor isolated by georgopoulos. a revertant from this mutant, susb610, was isolated, being suppressed by both the su+3 and su+44 suppressor strains. the efficiency of suppression by s ...1976819269
supercoiled dna wraps around the bacteriophage phi 29 head-tail connector.supercoiled pbr322 dna wraps around the outside of the isolated bacillus subtilis bacteriophage phi 29 head-tail connector, the crux of the dna packaging machine of the viral precursor capsid or prohead. the contour length of the supercoiled dna, determined by em, decreased by approximately 180 base pairs for each connector bound. mass and radial density determinations by scanning transmission em showed that the increased mass of the connector-dna complex relative to the connector alone was equi ...19921438237
solid-phase synthesis of the nucleopeptide fragment h-asp-ser[paaagtaagcc]-glu-oh from the nucleoprotein of bacillus subtilis phage phi 29.the naturally occurring dna-nucleopeptide h-asp-ser[5'-paaagtaagcc-3']-glu-oh was prepared via a solid-phase phosphite triester approach using n-2-(tert-butyldiphenylsilyloxymethyl)benzoyl protected nucleosides. the oligonucleotide was linked via the extremely base-labile oxalyl ester anchor to the solid support.19921508685
rna dependence of the bacteriophage phi 29 dna packaging atpase.the activity of the dna packaging adenosine triphosphatase (atpase) of the bacillus subtilis bacteriophage phi 29 is dependent upon prohead rna. the 174 nucleotide viral-encoded rna is positioned on the head-tail connector at the portal vertex of the phi 29 precursor shell (prohead). here, the rna interacts with the atp-binding gene 16 product (gp16) to constitute the dna-packaging atpase and initiate dna packaging in vitro. both the prohead connector (gene 10 product, gp10) and gp16 may utilize ...19901700132
transcription regulation in bacillus subtilis phage phi 29. 19911784815
analysis of an mrna exhibiting anomalous translational specificity.gene 6 mrna of bacillus subtilis phage phi 29 is inefficiently translated under standard in vitro conditions by escherichia coli, while it is efficiently translated by the in vitro system derived from b. subtilis. this is a rare example of the inability of e. coli to translate mrna translated by b. subtilis. the ionic condition in the translation systems was the key component in the differential recognition of the gene 6 message by e. coli and b. subtilis ribosomes. its translation by e. coli ri ...19911898927
transcription activation at a distance by phage phi 29 protein p4. effect of bent and non-bent intervening dna sequences.protein p4 of the bacillus subtilis phage phi 29 switches on the transcription of the viral late genes by binding to the viral late promoter at a region close to the rna polymerase binding site. gel retardation and dnase i footprinting assays show that the presence of protein p4 is required for rna polymerase recognition of the late promoter. the protein p4 and rna polymerase dna binding sites have been separated by the insertion of bent and non-bent dna sequences of different lengths. these mut ...19911904941
aphidicolin-resistant mutants of bacteriophage phi 29: genetic evidence for altered dna polymerase.aphidicolin-resistant mutants (aphr) of bacillus subtilis bacteriophage phi 29 were isolated after mutagenesis with hydroxylamine. efficiency of plating (e.o.p.) of the resistant mutants was not reduced at 500 microm aphidicolin, although e.o.p. of wild type phi 29 was less than 10(-5) at the same concentration of aphidicolin. by recombination and complementation analyses, both sites of the mutations, aph-71 and aph-101, of aphr71 and aphr101, respectively, were mapped in gene 2 which encodes ph ...19863087058
in vitro expression of a tn9-derived chloramphenicol acetyltransferase gene fusion by using a bacillus subtilis system.a coupled in vitro protein-synthesizing system has been developed with components derived totally from bacillus subtilis. the system synthesized specific gene products from various exogenous dna templates, including b. subtilis phage phi 29, plasmid pub110, and a heterologous b. subtilis-escherichia coli gene fusion containing the transposon tn9-derived chloramphenicol acetyltransferase (cat) gene. the gene fusion product was able to show cat activity, bind specifically to a sephacryl-chloramphe ...19873102458
initiation events in in-vitro packaging of bacteriophage phi 29 dna-gp3.initiation events in the packaging of bacteriophage phi 29 dna-gp3 (dna-gene product 3 complex) were studied in a completely defined in-vitro system that included purified proheads, dna-gp3 and the dna packaging protein gp16. in the sequential interactions, gp16 first bound to, and was modified by, the prohead. the prohead-gp16 complex then bound to dna-gp3, resulting in a second modification of gp16 that permitted binding of atp. dna-gp3 aggregates were produced, and the hydrolysis of atp accom ...19873119862
initiation of phage phi 29 dna replication by mutants with deletions at the amino end of the terminal protein.series of deletions at the amino end of protein p3, the phage phi 29 dna terminal protein (tp), have been constructed and characterized. measurements of the activity of the deletion mutants in the formation of the protein p3-damp initiation complex in vitro indicate the dispensability of the first 13 amino acids (aa) of the protein. the activity of protein p3 decreased considerably when 17 or more aa were deleted. the results on the in vitro phi 29 dna replication primed by the p3 deletion mutan ...19883133284
proteins induced in bacillus subtilis infected with bacteriophage phi 29. 19734200881
antigenic properties of bacteriophage phi 29 structural proteins.serological methods and electron microscopy were used to study the structural proteins of the small bacillus subtilis bacteriophage phi29. this virus has a large number of fibers attached at both ends of its prolate head. a complex neck assembly is comprised of 12 symmetrically arranged appendages as the outer component. head fibers, neck appendages, and the head surface bind anti-phi29 antibodies. immune sera absorbed with defective lysates of suppressor-sensitive (sus) mutants have been used t ...19734202619
viral protein synthesis in bacteriophage phi 29-infected bacillus subtilis.twenty-three (14)c-labeled phage phi29-specific proteins in lysates of uv-irradiated bacillus subtilis have been resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified by autoradiography. included in this group of proteins are the six major structural proteins of the virion. analysis of the temporal sequence of viral protein synthesis indicates that three groups of proteins can be identified by time of appearance, beginning at 2 to 4, 4 to 6, or 8 to 10 min after in ...19734203085
cloning, nucleotide sequence and high level expression of the gene coding for the connector protein of bacillus subtilis phage phi 29.the phi 29 dna restriction fragment hindiii-d, shown to contain gene 10 coding for the connector protein, has been cloned in plasmid pplc28 under the control of the pl promoter of phage lambda. after heat induction to inactivate the lambda repressor, a protein with the electrophoretic mobility of the connector protein p10 was synthesized, accounting for about 30% of the total escherichia coli protein after 3 h of induction. the 2205 nucleotide-long sequence of the cloned hindiii-d fragment has b ...19846096227
in vitro initiation of bacteriophage phi 29 and m2 dna replication: genes required for formation of a complex between the terminal protein and 5'damp.cell-free extracts prepared from phi 29 and m2-infected bacillus subtilis cells catalyse the formation of complexes between terminal protein and [alpha-32p]-damp in the presence of [alpha-32p]-datp, mgcl2, atp, and phage dna with terminal protein covalently linked at both the 5'ends. the complex formation does not take place when proteinase k-treated dna is added or when uninfected extract is used. the phi 29 complex thus formed is smaller than the m2 complex, primarily due to the different mole ...19836310350
structural localization of the proteins of the head to tail connecting region of bacteriophage phi 29.the head to tail connector of bacteriophage phi 29 has been studied to locate its two structural proteins (p10 and p11). treatment with trypsin led to proteolysis of p10 while p11 remained intact. computer filtration of electron micrographs of crystals of trypsinized necks showed a change in the external 12-fold area of the neck when compared with control necks. proteolized necks completely released p10 after treatment with low concentrations of an ionic detergent. the resulting structures, cont ...19836401885
factors involved in the initiation of phage phi 29 dna replication in vitro: requirement of the gene 2 product for the formation of the protein p3-damp study the requirements for the in vitro formation of the protein p3-damp complex, the first step in phi29 dna replication, extracts from b. subtilis infected with phi29 mutants in genes 2, 3, 5, 6 and 17, involved in dna synthesis, have been used. the formation of the initiation complex is completely dependent on the presence of a functional gene 2 product, in addition to protein p3 and phi29 dna-protein p3 as template. atp is also required, although it can be replaced by other nucleotides. t ...19836402761
protein-primed initiation of phage phi 29 dna replication.we recently reported the development of an in vitro replication system for bacteriophage phi 29 dna. we have used this system for the isolation of replication activity associated with gene 3 protein (terminal protein) from phi 29-infected bacillus subtilis cells. we utilized two assay systems: (i) dna replication dependent on phi 29 dna with the 5' end covalently linked to terminal protein (dna-protein) and (ii) the formation of complex between the terminal protein and damp. the dna-replication ...19836410387
characterization and purification of a phage phi 29-encoded dna polymerase required for the initiation of replication.the phage phi 29 protein p2, required for the formation of the protein p3-damp initiation complex, has been purified from escherichia coli cells harboring a gene 2-containing recombinant plasmid. the purified protein p2, of molecular weight 68,000, had a specific dna polymerase activity that elongated the p3-damp initiation complex when phi 29 dna-protein p3 was used as template. in addition, the purified protein p2 was active in catalyzing the initiation reaction when complemented with phi 29 m ...19846433348
the protein covalently linked to the 5' termini of the dna of bacillus subtilis phage phi 29 is involved in the initiation of dna replication. 19806771916
initiation of phage phi 29 dna replication in vitro: formation of a covalent complex between the terminal protein, p3, and 5'-damp.incubation of extracts of phi 29-infected bacillus subtilis with [alpha-32p]datp produced a labeled protein having the electrophoretic mobility of p3, the 5'-terminal protein of phi 29 dna. the reaction product was resistant to treatment with micrococcal nuclease, phosphatase, and rnases a and t1 and sensitive to proteinase k. incubation of the 32p-labeled protein with piperidine under conditions in which the phi 29 dna-protein p3 linkage is hydrolyzed released 5'-damp. the reaction with [alpha- ...19826813861
nucleotide sequence and transcription of the left early region of streptococcus pneumoniae bacteriophage cp-1 coding for the terminal protein and the dna polymerase.cp-1 is a small virulent bacteriophage infecting streptococcus pneumoniae. it has a linear, double-stranded genome of about 19 kb that replicates by a protein-priming mechanism. we have determined the nucleotide sequence of the leftmost 4780 bp of the dna of this bacteriophage; computer analysis revealed that this fragment contains seven open reading frames (orfs) which could encode polypeptides containing more than 50 amino acids. the orfs are clustered in two groups separated by noncoding inte ...19957645213
characterization of an rna-binding domain in the bacteriophage phi 29 connector.the connector of bacteriophage phi 29 is known to promote the viral prohead assembly, to bind dna, and to drive dna packaging into preformed viral shells in an rna-dependent process. in this report, the phi 29 connector protein, p10, is shown to bind rna in a sequence-independent fashion, and to possess an rna recognition motif comprised approximately the region between residues 21 and 94 of the p10 sequence. substitution mutants in specific amino acids of the rna-binding domain obtained by site ...19937690751
dual translational start motif evolutionarily conserved in the holin gene of bacillus subtilis phage phi 29.holins represent phage encoded lysis functions required for transit of the phage murein hydrolases to the periplasm. the lambda s, phage 21 s, and p22 13 holin genes contain a dual translational start motif, beginning with met1-lys2-x-met3. in all cases both start codons at the 5' end of the respective holin gene are utilized. the resulting polypeptides have opposing functions, with the longer product acting as an inhibitor of the shorter one. the 131-codon gene 14 of bacillus subtilis phage phi ...19957831803
probing the structure of bacteriophage phi 29 prohead rna with specific mutations.bacteriophage phi 29 of bacillus subtilis packages its double-stranded dna genome into a preformed prohead in an atp-dependent reaction. a 174-residue phi 29-encoded rna molecule (prna) is a structural component of the prohead and is essential for dna packaging. the secondary and tertiary structures of the prohead binding site on prna have been probed using a series of specific mutant prnas and by measuring binding to rna-free proheads and in vitro packaging of the dna-gene product 3 (dna.gp3) c ...19948034614
bacillus subtilis mutants defective in bacteriophage phi 29 head assembly.virus assembly mutants of asporogenous bacillus subtilis defective in bacteriophage phi 29 head assembly were detected by the use of antibodies that reacted strongly with the free dodecameric phi 29 portal vertex composed of gene product 10 (gp10) but weakly with the portal vertex assembled into proheads or phage. phage adsorption and the synthesis of phage proteins, dna-gene product 3, and prohead rna were normal in these mutants, but prohead and phage production was greatly reduced. the assemb ...19938096839
the main early and late promoters of bacillus subtilis phage phi 29 form unstable open complexes with sigma a-rna polymerase that are stabilized by dna supercoiling.most escherichia coli promoters studied so far form stable open complexes with sigma 70-rna polymerase which have relatively long half-lives and, therefore, are resistant to a competitor challenge. a few exceptions are nevertheless known. the analysis of a number of promoters in bacillus subtilis has suggested that the instability of open complexes formed by the vegetative sigma a-rna polymerase may be a more general phenomenon than in escherichia coli. we show that the main early and late promo ...19938451193
control mechanisms of bacteriophage phi 29 dna expression.the phage phi 29 regulatory protein p4 activates the late promoter a3 by stabilizing the binding of bacillus subtilis rna polymerase (rnap) as a closed complex. interaction between the two proteins occurs through amino acid arg120 in protein p4 and the c-terminal domain of the rnap alpha subunit (alpha-ctd). in addition to its role as activator of the late transcription, protein p4 represses early transcription from the a2b and a2c promoters, that are divergently transcribed. binding of p4 to it ...199810943379
multifunctional roles of a bacteriophage phi 29 morphogenetic factor in assembly and infection.low copy number proteins within macromolecular complexes, such as viruses, can be critical to biological function while comprising a minimal mass fraction of the complex. the bacillus subtilis double-stranded dna bacteriophage phi 29 gene 13 product (gp13), previously undetected in the virion, was identified and localized to the distal tip of the tail knob. western blots and immuno-electron microscopy detected a few copies of gp13 in phi 29, dna-free particles, purified tails, and defective part ...200818394643
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