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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
unusual base sequence arrangement in phage phi 29 dna.susceptibility of bacillus subtilis phage phi 29 dna to 34 different restriction endoculceases was determined. three enzymes, bgli, xbai and bsteii, were found to cleave phi 29 dna only once at specific sites. the sites of these single cleavages have been mapped. thirteen enzymes did not cut phi 29 dna. phi 29 hindiii dna fragments inserted into pbr313 plasmid and propagated in escherichia coli, were resistant to these restriction endonucleases. this result suggests that the insusceptibility is ...1979107059
identification of the protein firmly bound to the ends of bacteriophage phi 29 dna. 1978203093
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
order of the two major head protein genes of bacteriophage phi 29 of bacillus subtilis.bacteriophage phi 29 mutation sus8(22) has been mapped by two-factor crosses between markers sus8(769) and ts8(93). whe sus8(22) infects bacillus subtilis su- proteins, hp1 (major head protein) and hp3 (fiber protein) are not synthesized; instead, a fragment with a molecular weight of 25,000 is produced. the tryptic peptides of the fragments overlap with corresponding peptides in protein hp1, but not with the peptides of protein hp3, showing that cistron 8 codes for the major head protein hp1.1977409855
comparison of the a-t rich regions and the bacillus subtilis rna polymerase binding sites in phage phi 29 dna.by using a modification of the bac spreading method for mounting the dna for electron microscopy, partial denaturation maps of protein-free phi 29 dna and of phi 29 dna containing protein p3 were obtained. in phi 29 p3-dna1 the protein does not seem to influence the melting of the ends of the molecules. the comparison of the partial denaturation map and the b. subtilis rna polymerase binding sites indicates that five of the seven early promoters (a1, a2, a3, b2 and c2) are located in a-t rich dn ...1979114982
isolation of a strong suppressor of nonsense mutations in bacillus subtilis.by 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
[infectivity of dna-protein complex: transfection of bacillus phage phi29 (author's transl)]. 1975806101
purification of bacillus subtilis rna polymerase with heparin-agarose. in vitro transcription of phi 29 dna.we have devised a new procedure for the purification of highly active preparations of bacillus subtilis rna polymerase holoenzyme. a column of heparin-agarose a-15m is used to rapidly and quantitatively adsorb rna polymerase from the initial crude extract fraction. this affinity procedure obviates the necessity of including nucleic acid precipitation or partitioning steps and allows for rapid separation of rna polymerase from proteolytic activity. the enzyme is further purified by preparative gl ...1979113409
transcription of the genome of bacteriophage phi 29: isolation and mapping of the major early mrna synthesized in vivo and in vitro.the phi29 early mrna's synthesized in infected bacillus subtilis were studied by using sedimentation velocity analysis, polyacrylamide gel electrophoresis, and hybridization of phi29 dna fragments generated by the restriction endonuclease eco ri. viral rnas synthesized in vivo in the resence of chloramphenicol were found to hybridize to eco ri-a, -c, and -d fragments, but not to eco ri-b and -e fragments, of the viral genome. major early mrna sedimenting as 16s material in neutral sucrose gradie ...1977408515
analysis of gene function of bacteriophage phi 29 of bacillus subtilis: identification of cistrons essential for viral assembly.restrictive infection of bacillus subtilis by suppressor-sensitive (sus) mutants of phi 29 has been used to search for cistrons that function in viral assembly. the products of cistrons 7, 9, 10, and 16 are necessary for head morphogenesis. the neck upper collar protein p10 and the tail protein p9 must be present for dna packaging to occur. the protein p7 must be present for phage-related particles to form. a prohead-like particle has been isolated during 16-restrictive infection. the particle i ...1976822175
initiation of phi 29 dna replication occurs at the second 3' nucleotide of the linear template: a sliding-back mechanism for protein-primed dna replication.bacteriophage phi 29 dna replication is initiated when a molecule of damp is covalently linked to a free molecule of the terminal protein, in a reaction catalyzed by the viral dna polymerase. we demonstrate that single-stranded dna molecules are active templates for the protein-primed initiation reaction and can be replicated by phi 29 dna polymerase. using synthetic oligonucleotides, we carried out a mutational analysis of the phi 29 dna right end to evaluate the effect of nucleotide changes at ...19921409668
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
dna conformational change induced by the bacteriophage phi 29 connector.translocation of viral dna inwards and outwards of the capsid of double-stranded dna bacteriophages occurs through the connector, a key viral structure that is known to interact with dna. it is shown here that phage phi 29 connector binds both linear and circular double-stranded dna. however, dna-mediated protection of phi 29 connectors against staphylococcus aureus endoprotease v8 digestion suggests that binding to linear dna is more stable than to circular dna. endoprotease v8-protection assay ...19921454519
genetic analysis of bacteriophage phi 29 of bacillus subtilis: integration and mapping of reference mutants of two collections.reference mutants of bacillus subtilis phage phi 29 of the madrid and minneapolis collections were employed to construct a genetic map. suppressor-sensitive and temperature-sensitive mutants were assigned to 17 cistrons by quantitative complementation. three-factor crosses were used to assign an unambiguous order for the 17 cistrons. recombination frequencies determined by two-factor crosses were used to construct a linear genetic map of 24.4 recombination units. the genes were numbered sequenti ...1976822174
morphogenesis of bacteriophage phi 29 of bacillus subtilis: preliminary isolation and characterization of intermediate particles of the assembly pathway.three classes of particles have been identified in restrictive phi 29 suppressor-sensitive (sus) mutant infections of bacillus subtilis, including dna-containing heads or phage, prohead, and empty heads. pulse-chase labeling experiments indicate that the prohead, the first particle assembled in 14-infected cells, is converted to dna-filled heads and phi 29. in addition to the proteins hd, p10, and f found in mature phi 29, the prohead contains a "core" protein p7 that exits as the prohead mature ...1976822176
phage phi 29 regulatory protein p4 stabilizes the binding of the rna polymerase to the late promoter in a process involving direct protein-protein contacts.transcription from the late promoter, pa3, of bacillus subtilis phage phi 29 is activated by the viral regulatory protein p4. a kinetic analysis of the activation process has revealed that the role of protein p4 is to stabilize the binding of rna polymerase to the promoter as a closed complex without significantly affecting further steps of the initiation process. electrophoretic band-shift assays performed with a dna fragment spanning only the protein p4 binding site showed that rna polymerase ...19921454827
role of the amino-terminal domain of bacteriophage phi 29 connector in dna binding and packaging.the connector of bacteriophage phi 29 is required for prohead assembly, binds dna, and drives dna packaging into viral proheads. limited proteolysis of the connector protein with endoproteinase glu-c from staphylococcus aureus v8 and chymotrypsin showed that a domain of the nh2-terminal region is involved in dna binding and in the subsequent packaging into preformed proheads, but not in prohead assembly. mutants in specific amino acids of the nh2-terminal domain, obtained by directed mutagenesis ...19921587868
a dna curvature can substitute phage phi 29 regulatory protein p4 when acting as a transcriptional repressor.binding of phage phi 29 regulatory protein p4 to its target sequences produces a strong bend in the dna that is important for activation of the late a3 promoter (pa3). protein p4 binding site in pa3 overlaps with the divergently transcribed main early promoter. pa2b, which suggested that p4 could also act as a repressor. we show that protein p4 both excludes bacillus subtilis sigma a-rna polymerase from pa2b and directs it to the divergently transcribed a3 promoter. although steric hindrance is ...19911655421
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
dna-independent deoxynucleotidylation of the phi 29 terminal protein by the phi 29 dna polymerase.in this paper, we show that the phi 29 dna polymerase, in the absence of dna, is able to catalyze the formation of a covalent complex between the phi 29 terminal protein (tp) and 5'-damp. like the reaction in the presence of phi 29 dna, tp.damp complex formation is strongly dependent on activating mn2+ ions and on the efficient formation of a tp/dna polymerase heterodimer. the nature of the tp-damp linkage was shown to be identical (a o-5'-deoxyadenylyl-l-serine bond) to that found covalently li ...19921730646
the bacteriophage phi 29 dna polymerase, a proofreading enzyme.the bacteriophage phi 29 dna polymerase, involved both in the protein-primed initiation and elongation steps of the viral dna replication, displays a very processive 3',5'-exonuclease activity acting preferentially on single-stranded dna. this exonucleolytic activity showed a marked preference for excision of a mismatched versus a correctly paired 3' terminus. these characteristics enable the phi 29 dna polymerase to act as a proofreading enzyme. a comparative analysis of the wild-type phi 29 dn ...19921733957
structural and functional studies on phi 29 dna polymerase.the bacillus subtilis phage phi 29 dna polymerase, involved in protein-primed viral dna replication, contains several amino acid consensus sequences common to other eukaryotic-type dna polymerases. using site-directed mutagenesis, we have studied the functional significance of a c-terminal conserved region, represented by the lys-x-tyr ("k-y") motif. single point mutants have been constructed and the corresponding proteins have been overproduced and characterized. measurements of the activity of ...19921291240
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
isolation of dna-dependent rna polymerase from streptomyces granaticolor and its binding to phage phi 29 dna.partially purified dna-dependent rna polymerase of streptomyces granaticolor was further separated on phosphocellulose in 50% glycerol and a single activity peak was obtained. the enzyme isolated in this way consisted of 4 main proteins with molar mass of 145, 132, 50 and 46 kg/mol. these four subunits represented 93% proteins of the active fraction. to test the ability of rna polymerase to recognize specific sites on dna, binding sites for rna polymerase on phage phi 29 dna were mapped by elect ...19911823645
mutant prohead rnas in the in vitro packaging of bacteriophage phi 29 dna-gp3.the 174-base prohead rna encoded by bacteriophage phi 29 of bacillus subtilis, essential for packaging of the dna-gp3 (dna-gene product 3) complex, was expressed efficiently from the cloned gene. computer programs for rna structure analysis were used to fold hypothetical rna mutants and thus to target mutagenesis of the rna for studies of structure and function. five mutants of the rna were then produced by oligonucleotide-directed mutagenesis that were altered in the primary sequence at selecte ...19921538407
characterization and mapping of the pyrophosphorolytic activity of the phage phi 29 dna polymerase. involvement of amino acid motifs highly conserved in alpha-like dna polymerases.the phi 29 dna polymerase, an alpha-like dna polymerase, shows an inorganic pyrophosphate-dependent degradative activity with similar requirements to the corresponding one of escherichia coli dna polymerase i: (a) it requires a high concentration of inorganic pyrophosphate and is reversed by polymerization; (b) like dna polymerization, it needs a duplex dna with protruding 5' single-strand; (c) it acts in the 3' to 5' direction releasing free dntps, thus, it can be considered as the reversal of ...19911850426
analysis of replicative intermediates produced during bacteriophage phi 29 dna replication in vitro.replication of bacteriophage phi 29 dna initiates at either end of its linear double-stranded dna molecule and proceeds by a strand-displacement mechanism. in the present paper we have used an in vitro phi 29 dna replication system to analyse by electron microscopy the replicative intermediates produced at different reaction times. two types of replicative intermediates were observed: type i (full-length double-stranded phi 29 dna molecules with one or more single-stranded dna branches) and type ...19911762160
characterization of a versatile in vitro dna-packaging system based on hybrid lambda/phi 29 proheads.we have studied the assembly of bacteriophage lambda head proteins on the phage phi 29 connector to produce in vitro chimeric proheads, whose ability to package different types of dna depends on the physical integrity of the phi 29 connector. terminal protein-free phi 29 as well as nonviral dnas have been shown to be efficiently packaged by this hybrid system. an rna, that can be provided by any of the extracts used in the complementation mixture, was required for dna packaging, both by the hybr ...19911827226
mechanism of stimulation of dna replication by bacteriophage phi 29 single-stranded dna-binding protein p5.protein p5 is a bacillus subtilis phage phi 29-encoded protein required for phi 29 dna replication in vivo. protein p5 has single-stranded dna binding (ssb) capacity and stimulates in vitro dna replication severalfold when phi 29 dna polymerase is used to replicate either the natural phi 29 dna template or primed m13 single-stranded dna (ssdna). furthermore, other ssb proteins, including escherichia coli ssb, t4 gp32, adenovirus dna-binding protein, and human replication factor a, can functional ...19911899235
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
regulation of the phage phi 29 prohead shape and size by the portal vertex.bacteriophage phi 29 of bacillus subtilis packages its double-stranded dna into a preformed prohead during morphogenesis. the prohead is composed of the scaffold protein gp7, the capsid protein pg8, the portal protein gp10, and the dispensable head fiber protein gp8.5. our objective was to elucidate the phi 29 prohead assembly pathway and to define the factors that determine prohead shape and size. the structural genes of the phi 29 prohead were cloned and expressed in escherichia coli individua ...19911905079
mutagenesis of conserved region i in the dna polymerase from human adenovirus serotype 2.the functional importance of the conserved region i (ygdtdslf) found in several prokaryotic, eukaryotic, and viral dna polymerases has been probed by site-directed mutagenesis of the adenovirus dna polymerase (ad pol). three different adenovirus-specific assays have been used to measure the in vitro activity of region i mutants of ad pol expressed in transiently transfected cmt-4 cells. in general, both conservative and nonconservative changes generally showed a greater than 5- to 10-fold reduct ...19911871969
deletions at the n terminus of bacteriophage phi 29 protein p6: dna binding and activity in phi 29 dna replication.deletions corresponding to the first 5 or 13 amino acids (aa), not counting the initial met, have been introduced into the n terminus of the phage phi 29 protein p6. the activity of such proteins in the in vitro phi 29 dna replication system, their capacity to interact with the phi 29 dna ends, and their interference with the wild type (wt) protein p6 activity have been studied. the initiation activity of protein p6 decreased considerably when 5 as were deleted and was undetectable when 13 aa we ...19901979302
bend induced by the phage phi 29 transcriptional activator in the viral late promoter is required for activation.transcription initiation from the bacillus subtilis phage phi 29 late a3 promoter requires the viral protein p4, a transcriptional activator. protein p4 binds to a region of the a3 promoter, located between nucleotides -50 and -100 relative to the transcription start site, that presents a sequence-directed curvature. this curvature is enhanced when protein p4 binds to the promoter. a number of deletion mutants at the carboxyl end of protein p4 have been constructed and their behavior as transcri ...19902107318
srna of phage phi 29 of bacillus subtilis mediates dna packaging of phi 29 proheads assembled in escherichia coli.the structural genes of the prohead of phage phi 29 of bacillus subtilis and a small phi 29 rna (srna) were cloned and expressed in escherichia coli individually or in combination to study the role of the srna in prohead assembly and the mechanism of prohead morphogenesis. the genes coding for the proteins of the scaffold (gp7), the capsid (gp8), the portal vertex (gp10), and the dispensable head fiber (gp8.5) were expressed in e. coli and the gene products were assembled, with and without the p ...19911926784
identification of the sequences recognized by phage phi 29 transcriptional activator: possible interaction between the activator and the rna polymerase.expression of bacillus subtilis phage phi 29 late genes requires the transcriptional activator protein p4. this activator binds to a region of the late a3 promoter spanning nucleotides -56 to -102 relative to the transcription start site, generating a strong bending tin the dna. in this work the target sequences recognized by protein p4 in the phage phi 29 late a3 promoter have been characterized. the binding of protein p4 to derivatives of the late a3 promoter harbouring deletions in the protei ...19911904153
structural and functional analysis of temperature-sensitive mutants of the phage phi 29 dna polymerase.the cloning and complete sequencing of gene 2 from four independently isolated temperature-sensitive mutants in the phage phi 29 dna polymerase (ts2 mutants) is reported. the results obtained indicate that, in vivo, the mutations only affect the initial steps of the replication process. interestingly, three of these mutations consist in the single amino acid change ala to val at position 492 of the protein. the ts2(24) and ts2(98) mutant phi 29 dna polymerases were expressed, purified and their ...19902118623
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
short n-terminal deletions in the phage phi 29 transcriptional activator protein impair its dna-binding ability.the expression of bacillus subtilis phage phi 29 late genes from the a3 promoter requires the viral protein p4. this protein is a transcriptional activator which binds to a region of the a3 promoter located between nucleotides -56 to -102, relative to the transcription start point. mutants at the n terminus of protein p4 have been constructed and their function investigated. the binding of these deletion mutants to the late a3 promoter has been analyzed by gel retardation and dnase i footprintin ...19902125015
phylogenetic analysis and secondary structure of the bacillus subtilis bacteriophage rna required for dna packaging.an unusual rna molecule encoded by the bacillus subtilis bacteriophage phi 29 is a structural component of the viral prohead and is required for the atp-dependent packaging of dna. here we report a model of secondary structure for this prohead rna developed from a phylogenetic analysis of the primary sequences of prohead rnas of related phages. twenty-nine phages related to phi 29 were found to produce prohead rnas. these rnas were analyzed by their ability to replace phi 29 rna in in vitro phag ...19902125049
aphidicolin-resistant dna polymerase of bacteriophage phi 29 aphr71 mutant is hypersensitive to phosphonoacetic acid and butylphenyldeoxyguanosine 5'-triphosphate.bacteriophage phi 29 dna polymerase is sensitive to aphidicolin (aph). dna polymerase of the aph-resistant mutant, aphr71, was more sensitive to phosphonoacetic acid and butylphenyldeoxyguanosine 5'triphosphate than the wild type. nucleotide sequence analysis revealed a single transition of g at nucleotide 562 to a in the dna polymerase gene of aphr71, indicating that aphr71 dna polymerase (572 residues) had a single amino acid substitution from glycine at residue 188 to serine. the results sugg ...19902117830
site-directed mutagenesis of the ycdtds amino acid motif of the phi 29 dna polymerase.the bacillus subtilis phage phi 29 dna polymerase, involved in protein-primed viral dna replication, contains amino acid consensus sequences common to other alpha-like dna polymerases. using site-directed mutagenesis we have studied the functional significance of the most conserved c-terminal segment mainly represented by the ycdtds motif. a series of single point mutants has been constructed and the corresponding proteins have been overproduced and characterized. measurements, on crude fraction ...19902121621
the highly conserved amino acid sequence motif tyr-gly-asp-thr-asp-ser in alpha-like dna polymerases is required by phage phi 29 dna polymerase for protein-primed initiation and polymerization.the alpha-like dna polymerases from bacteriophage phi 29 and other viruses, prokaryotes and eukaryotes contain an amino acid consensus sequence that has been proposed to form part of the dntp binding site. we have used site-directed mutants to study five of the six highly conserved consecutive amino acids corresponding to the most conserved c-terminal segment (tyr-gly-asp-thr-asp-ser). our results indicate that in phi 29 dna polymerase this consensus sequence, although irrelevant for the 3'----5 ...19902191296
characterization of a dna binding protein of bacteriophage prd1 involved in dna replication.escherichia coli phage prd1 protein p12, involved in prd1 dna replication in vivo, has been highly purified from e. coli cells harbouring a gene xii-containing plasmid. protein p12 binds to single-stranded dna as shown by gel retardation assays and nuclease protection experiments. binding of protein p12 to single-stranded dna increases about 14% the contour length of the dna as revealed by electron microscopy. binding to single-stranded dna seems to be cooperative, and it is not sequence specifi ...19902251117
lysogenic phages of clostridium perfringens: mapping of the chromosomal attachment sites.the sites of insertion for two lysogenic bacteriophages have been mapped on the chromosome of clostridium perfringens strain cpn50 using two techniques based on pulsed field gel electrophoresis. phage phi 29 was mapped to the 1 mb region of the 3.6 mb genome, near nanh which encodes a potential virulence factor, while phi 59 was found to have inserted at 2.9 mb.19902323543
structural requirements in the herpes simplex virus type 1 transactivator vmw65 for interaction with the cellular octamer-binding protein and target taatgarat sequences.herpes simplex virus type 1 virion protein vmw65 forms a complex (trf.c) with taatgarat sequences and the cellular transcription factor oct-1, which has been implicated as an intermediate in the activation of gene expression by vmw65. to examine structural requirements within vmw65 for this interaction, we analyzed extracts of transfected cells that express mutant vmw65 proteins by gel retardation assay and identified two regions in the primary sequence of vmw65 which are necessary for in vitro ...19902335815
functional domain for priming activity in the phage phi 29 terminal protein.by site-directed mutagenesis we have changed into cys the ser232 of the phi 29 terminal protein (tp) involved in the covalent linkage to damp for the initiation of replication. the mutant tp, highly purified, had about 0.7% of the priming activity of the wild-type (wt) protein p3. the linkage between the mutant protein p3 and damp was more labile to piperidine treatment than the serine-damp linkage in the wt protein p3, suggesting the presence of a different kind of linkage, cys-damp. in the oth ...19902341040
conformational changes in bacteriophage phi 29 connector prevents dna-binding activity.in vitro dna packaging activity in a defined system derived from bacteriophage phi 29 depends upon the chemical integrity of the connector protein p10. proteolytic cleavage of p10 rendered the proheads inactive for dna packaging. a similar treatment on isolated connectors abolished the dna-binding activity of the native p10, but the general shape and size of the connector was not changed as revealed by electron microscopy. analytical ultracentrifugation showed that the proteolyzed connectors had ...19902342107
highly efficient dna synthesis by the phage phi 29 dna polymerase. symmetrical mode of dna replication.the results presented in this paper indicate that the phi 29 dna polymerase is the only enzyme required for efficient synthesis of full length phi 29 dna with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. analysis of phi 29 dna polymerase activity in various in vitro dna replication systems indicates that two main reasons are responsible for the efficiency of this minimal system: 1) the phi 29 dna polymerase is highly processive in the absence of ...19892498321
a novel nucleoprotein complex at a replication origin.the viral protein p6, required for the protein-primed initiation of replication of bacillus subtilis phage phi 29, forms a nucleoprotein complex at the viral replication origins that shows novel features. deoxyribonuclease i and hydroxyl radical footprinting data, as well as the induction of positive supercoiling, support a model in which a dna right-handed superhelix tightly wraps around a multimeric p6 core. the interaction occurs through the dna minor groove. the activity of p6 not only requi ...19902111580
role of rna in bacteriophage phi 29 dna packaging.a novel bacteriophage phi 29 rna of 174 nucleotides is essential for the in vitro packaging of the dna-terminal protein complex into proheads. the rna, bound to the prohead portal vertex (connector), participates in assembly and function of the dna translocating atpase and in recognition of the dna left-end during the course of the packaging reaction. the rna is present in related phages and varies widely in primary sequence, but its secondary structure, as deduced by phylogenetic analysis, is b ...19902150913
production of lambda-phi 29 phage chimeras.proheads of bacteriophage lambda which carry the connector of phage phi 29 instead of that of lambda have been produced in vitro. these hybrid proheads have a structure similar to that of normal lambda proheads. furthermore, the chimeric proheads can package both lambda and phi 29 dna. these data show that the connector domains involved in both head assembly and dna packaging are functionally similar. the dna-containing lambda-phi 29 proheads can be complemented in vitro with phi 29 tails to yie ...19902146805
characterization of the phage phi 29 protein p5 as a single-stranded dna binding protein. function in phi 29 dna-protein p3 replication.the phage phi 29 protein p5, required in vivo in the elongation step of phi 29 dna replication, was highly purified from escherichia coli cells harbouring a gene 5-containing plasmid and from phi 29-infected bacillus subtilis. the protein was characterized as the gene 5 product by amino acid analysis and nh2-terminal sequence determination. the purified protein p5 was shown to bind to single-stranded dna and to protect it against nuclease degradation. no effect of protein p5 was observed either ...19892499869
effects of internal deletions on the priming activity of the phage phi 29 terminal protein.a series of internal deletions of gene 3, coding for the phage phi 29 dna terminal protein, have been constructed and characterized. in addition, a substitution mutant in the sequence corresponding to amino acids (aa) 49-51 was obtained. the priming activity of the substitution mutant protein, in the formation of the protein p3-damp initiation complex, was drastically reduced suggesting that some of the aa present at position 49-51 are essential for p3 function. deletions of 8 to 33 aa, from aa ...19892511080
characterization, overproduction and purification of the product of gene 1 of bacillus subtilis phage phi 29.unit-length phi 29 dna was not synthesized after restrictive infection of bacillus subtilis with the phi 29 mutant sus1(629) indicating that the phage phi 29 protein p1 is needed for the viral dna replication. sequencing of the orf-6 of mutant sus1(629) showed that a c in the wild-type (wt) phage had been changed to a t at nt position 19 of the orf-6, giving rise to a taa ochre codon, indicating that this orf corresponds to gene 1. orf-6 was cloned in plasmid pplc28 under the control of the pl p ...19892526779
regions at the carboxyl end of bacteriophage phi 29 protein p6 required for dna binding and activity in phi 29 dna replication.series of deletions corresponding to the carboxyl end of the phage phi 29 protein p6 have been constructed and their activity in the initiation of phi 29 dna replication and their capacity to interact with the phi 29 dna ends have been studied. determination of the activity of the deletion mutants in phi 29 dna replication indicated the dispensability of the 14 carboxy-terminal amino acids of the protein. the activity of protein p6 decreased with deletions from 23 to 39 amino acids and was undet ...19892501757
prohead rna of bacteriophage phi 29: size, stoichiometry and biological activity.we previously demonstrated (guo et al., 1987. nucl. acids res. 15, 7081-7090) that purified proheads of bacteriophage phi 29 contain an rna of 120 bases which is essential for dna packaging. here we report that this rna exists primarily as a polymer of ca. 174 residues in phage-infected cells and that ca. 54 bases are cleaved from its 3'-terminus by adventitious nucleases during the purification of proheads. the long and short forms of the rna had similar activity in in vitro dna packaging and p ...19892498842
characterization of a new prokaryotic transcriptional activator and its dna recognition site.the expression of the bacillus subtilis phage phi 29 dna is controlled by the viral gene 4 product, which is required for the initiation of transcription at the unique late promoter a3. protein p4 binds specifically to a phi 29 dna fragment containing the a3 promoter. dnase i footprinting analysis has shown that the dna binding region for protein p4 is located between nucleotides -50 and -100 relative to the transcription start site. methylation interference assays suggest that two eight base-pa ...19892504924
in vitro packaging of bacteriophage phi 29 dna restriction fragments and the role of the terminal protein gp3.restriction fragments of bacteriophage phi 29 dna-gp3 (dna-gene product 3 complex) were packaged in a completely defined in vitro system that included purified proheads, the dna packaging protein gp16 and atp. both left and right end dna-gp3 fragments were packaged in this system, in contrast to the oriented and selective packaging of left end dna-gp3 fragments in extracts; left ends could be packaged quantitatively in the defined system, while the packaging efficiency of right ends was generall ...19892530357
a bacillus subtilis phage phi 29 transcription terminator is efficiently recognized in streptomyces lividans.a dna fragment from the bacillus subtilis phage phi 29, containing the bidirectional transcription terminator td1, where the right early transcription and late viral transcription terminate, has been inserted in one orientation between the aminoglycoside phosphotransferase (aph) gene (neo) and the phi 29 main early and late promoters present in derivative constructs of the streptomyces promoter-probe plasmid pij486. the td1 terminator is efficiently recognized in s. lividans and ends the transcr ...19872824291
cleaving the prohead rna of bacteriophage phi 29 alters the in vitro packaging of restriction fragments of dna-gp3.in vitro packaging of restriction fragments of the bacteriophage phi 29 dna-gp3 (dna-gene product 3 complex) in the defined system was dependent on prohead rna. truncated prohead rnas were obtained by in situ rnase a digestion, isolated and sequenced. proheads having the intact 174 base rna were compared to proheads having rnas of 120, 95, 71, 69 or 54 bases for the capacity to package the dna-gp3 left and right ends and internal (non-end) fragments generated by the restriction enzymes ecori, hp ...19892530354
in vitro transcription of bacteriophage phi 29 dna: inhibition of early promoters by the viral replication protein p6.the effect of the phi 29 protein p6 on the in vitro initiation of transcription from the main phi 29 promoters was studied. protein p6 interfered with the transcription from the early promoters c1 and c2, located at the right end of the phi 29 genome. transcription initiated at the early promoters a1, a2b, and a2c and at the late promoter a3, located at the left region of the viral genome, was not affected by protein p6. these results suggest that protein p6 might play a dual role, acting as a n ...19892908927
functional domains in the bacteriophage phi 29 terminal protein for interaction with the phi 29 dna polymerase and with dna.deletion mutants at the amino- and carboxyl-ends of the phi 29 terminal protein, as well as internal deletion and substitution mutants, whose ability to prime the initiation of phi 29 dna replication was affected to different extent, have been assayed for their capacity to interact with dna or with the phi 29 dna polymerase. one dna binding domain at the amino end of the terminal protein has been mapped. two regions involved in the binding to the dna polymerase, an internal region near the amino ...19892602154
characterization of a 3'----5' exonuclease activity in the phage phi 29-encoded dna polymerase.purified protein p2 of phage phi 29, characterized as a specific dna polymerase involved in the initiation and elongation of phi 29 dna replication, contains a 3'----5' exonuclease active on single-stranded dna, but not on double-stranded dna. no 5'----3' exonuclease activity was found. the 3'----5' exonuclease activity was shown to be associated with the dna polymerase since 1) the two activities were heat-inactivated with identical kinetics and 2) both activities, present in purified protein p ...19852987819
signals at the bacteriophage phi 29 dna replication origins required for protein p6 binding and activity.protein p6 of bacillus subtilis phage phi 29 binds specifically to the ends of the viral dna that contain the replication origins, giving rise to a nucleoprotein structure. dna regions recognized by protein p6 have been mapped by deletion analysis and dnase i footprinting. main protein p6-recognition signals have been located between nucleotides 62 and 125 at the right phi 29 dna end and between nucleotides 46 and 68 at the left end. in addition, recognition signals are also present at other sit ...19892767056
the complete sequence of the bacillus phage phi 29 right early region.we have sequenced the rightmost 2216 bp of the bacillus phage phi 29 genome. this region encompasses the right early region and completes the sequence of the phi 29 early functions. the sequence of gene 17, an early gene implicated in the replication process, is presented. from these results we predict that gene 17 encodes a 19.1-kdal protein. further analysis of the sequence revealed five previously undetected potential genes, encoding 12.6-, 12.4-, 15.2-, 6.2- and 4.6-kdal proteins. the biolog ...19853007295
nucleotide sequence of gene f of bacillus phage nf.the nucleotide sequence of bacillus phage nf gene f has been determined. the deduced amino acid sequence of gpf is very similar to that of gp4, the transcriptional activator of phage phi 29. both proteins contain the consensus structure that is conserved for the dna-protein interacting domain of dna-binding proteins such as the repressor, cro and cii proteins of phage lambda.19863015737
cloning and template activity of the origins of replication of phage phi 29 dna.a 73-bp fragment from the left end of phi 29 dna and a 269-bp fragment from the right end have been cloned in plasmids pplc28 and pkk223-3, respectively, after removal of the terminal protein p3 by treatment with piperidine. in addition, the 73- and 269-bp fragments were cloned together in plasmid pkk223-3 in such a way that the two termini of phi 29 dna were joined. treatment of the latter recombinant plasmid with ahaiii releases several fragments, two of which contain the phi 29 dna terminal s ...19863019829
initiation of phage phi 29 dna replication by mutants with deletions at the carboxyl end of the terminal protein.series of deletions at the c end of p3, the phage phi 29 dna terminal protein (tp), have been constructed and characterized. measurements of the activity of those deletion mutants in the formation of the p3-damp initiation complex in vitro indicate the need of an intact c-end for the normal tp primer function in dna replication. it appears that the region at the c-end between aa 240 and 262 of p3, or part of it, might be essential for the normal tp function.19863019830
in vivo transcription of bacteriophage phi 29 dna: transcription initiation sites.the initiation sites of the rna transcripts synthesized in vivo in bacillus subtilis infected with bacteriophage phi 29 have been mapped by s1 protection experiments. nine transcription initiation sites were localized along the entire phi 29 genome, close to previously reported b. subtilis and escherichia coli rna polymerase-binding sites. eight of these sites corresponded to early transcription and only one corresponded to late transcription. by using 5'-end-labeled rna, four of the early sites ...19863023677
nucleotide sequence of bacillus phage phi 29 genes 14 and 15: homology of gene 15 with other phage lysozymes.the nucleotide sequence of bacillus phage phi 29 genes 14 (g14) and 15 (g15) have been determined and shown to encode proteins with molecular weights of 15,014 and 28,022, respectively. the g14 open reading frame (orf) was confirmed by sequencing a sus14(1241) mutant. gene product 15 (gp15) has considerable homology with salmonella phage p22 lysozyme and lesser homology with escherichia coli phage t4 lysozyme. putative translation signals are identified. in addition, the role of a previously des ...19863027653
in vivo transcription of bacteriophage phi 29 dna: transcription termination.the main early and late transcription termination sites in vivo in bacteriophage phi 29 dna were determined by nuclease s1 mapping. transcription of the phi 29 early genes located at the left end of the viral genome terminated at the very end of the dna molecule and within the hindiii g fragment of the viral dna. transcription termination of the early genes located at the right end of the genome and that of the late viral genes overlapped in a specific region of the phi 29 dna within the ecori d ...19873033305
related functional domains in virus dna polymerases.analysis of the lesions in several drug-resistant dna polymerase mutants of herpes simplex virus along with comparative analysis of the published polymerase sequences of other human herpesviruses has shown that most lesions (five out of six) are substitutions at amino acid residues conserved in all four polymerases. furthermore, the majority of lesions are in regions of the polypeptide where there are marked clusterings of conserved residues. on the basis of these data we have identified several ...19873034575
characterization and cloning of gene 5 of bacillus subtilis phage phi 29.sequencing of the phi 29 dna region [open reading frames (orfs) 12, 11 and 10] between genes 6 and 4 of the mutant ts5(219) showed that a g in the wild-type phage had been changed to an a in the mutant at position 218 of orf 10 indicating that this orf corresponds to gene 5. orf 10 was cloned in plasmid pplc28 under the control of the pl promoter of phage lambda and, after heat induction of the escherichia coli cells carrying the recombinant plasmid pgm26, a 12-kda protein was overproduced, acco ...19882971594
primary structural relationships may reflect similar dna replication strategies.the primary structures of several proteins of bacterial and viral origin involved in the initiation of dna synthesis and its subsequent elongation were compared. it was found that the known sequences of dna polymerases and the single-stranded dna binding proteins from phage t7 and escherichia coli aligned well. furthermore, segmental homologies were found in the phage phi 29 and adenovirus polymerases as well as in their dna binding proteins. these results suggest similar mechanisms of dna synth ...19863004026
prohead and dna-gp3-dependent atpase activity of the dna packaging protein gp16 of bacteriophage phi 29.the atpase activity of the dna packaging protein gp16 (gene product 16) of bacteriophage phi 29 was studied in the completely defined in-vitro assembly system. atp was hydrolyzed to adp and pi in the packaging reaction that included purified proheads, dna-gp3 and gp16. approximately one molecule of atp was used in the packaging of 2 base-pairs of phi 29 dna, or 9 x 10(3) atp molecules per virion. the hydrolysis of atp by gp16 was both prohead and dna-gp3 dependent. gp16 contained both the "a-typ ...19872960820
effect of aphidicolin and nucleotide analogs on the phage phi 29 dna polymerase.the drugs aphidicolin and the nucleotide analogs butylanilino datp, butylphenyl dgtp, and butylphenyl rgtp inhibited the protein-primed replication of phi 29 dna-protein p3 in the presence of purified terminal protein p3 and phi 29 dna polymerase p2. the effect of aphidicolin was mainly on the polymerization reaction by decreasing the rate of elongation. the nucleotide analogs inhibited both the formation of the p3-damp initiation complex and its further elongation, the latter being also due to ...19863090778
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 transcription of bacteriophage phi 29 dna. correlation between in vitro and in vivo promoters.the phi 29 dna in vitro transcription initiation sites have been accurately mapped by s1 protection experiments. the results obtained indicated that the b. subtilis rna polymerase containing the sigma 43 subunit basically recognized the same set of phi 29 promoters in vitro as those used in vivo. in addition, the sequence of the phi 29 early a2a promoter used both in vitro and in vivo has been determined as well as the precise nucleotide where initiation of transcription from the c2 promoter occ ...19863088543
replication of phage phi 29 dna in vitro: role of the viral protein p6 in initiation and elongation.the phi 29 protein p6 stimulates the formation of the protein p3-damp initiation complex when added to a minimal system containing the terminal protein p3, the phi 29 dna polymerase p2 and phi 29 dna-protein p3 complex, by decreasing about 5 fold the km value for datp. in addition, protein p6 stimulates elongation of the p3-damp initiation complex. whereas the effect of protein p6 on initiation is similar with protein p3-containing fragments from the right or left phi 29 dna ends, the stimulatio ...19863088545
nucleotide sequence of the right early region of bacillus subtilis phage pza completes the 19366-bp sequence of pza genome. comparison with the homologous sequence of phage phi 29.we have sequenced the rightmost 2079 bp of the bacillus subtilis phage pza genome. this region encompasses the right early region. we compared it with the homologous region of phage phi 29. six open reading frames (orfs) were found in this region of pza and one of them was assigned to gene 17. analysis of putative ribosome-binding sites and comparison with phi 29 orfs indicate that at least some of the remaining orfs could encode proteins. corresponding genes were not identified so far by geneti ...19863095189
in vivo transcription of bacteriophage phi 29 dna early and late promoter sequences.the in vivo transcription initiation sites of eight putative phi 29 promoters have been accurately determined: seven of them correspond to early promoters, including the four main ones, and the other corresponds to the only late promoter found in vivo. comparison of the phi 29 promoter sequences with the consensus sequence for the bacillus subtilis sigma 43 rna polymerase suggests that the sigma 43 enzyme is involved in the recognition of the viral early promoters, whereas the late promoter sequ ...19863100809
modulation of in vivo and in vitro transcription of bacteriophage phi 29 early genes.the majority of early transcripts of the phi 29 bacteriophage are produced throughout the lytic cycle but the levels of a class of transcripts from the right end of the phi 29 genome are significantly reduced late in the infection. we have isolated a phage early protein which selectively interferes with the initiation in vitro of transcription from promoters at the right end of the phi 29 genome. the amino acid sequence of the purified inhibitory protein correlates to the sequence predicted from ...19863097957
signals in the phi 29 dna-terminal protein template for the initiation of phage phi 29 dna replication.the protein-free terminal fragments hindiii b and l, from the left and right ends of phi 29 dna, respectively, but not internal fragments of similar size, were active as templates in the formation of the p3-damp initiation complex in an in vitro system containing purified phi 29 terminal protein p3 and dna polymerase p2, although the activity was lower than that obtained with the phi 29 dna-p3 complex. these results indicate the existence of specific sequences at the ends of phi 29 dna that allo ...19863097958
a small viral rna is required for in vitro packaging of bacteriophage phi 29 dna.a small rna of bacillus subtilis bacteriophage phi 29 is shown to have a novel and essential role in viral dna packaging in vitro. this requirement for rna in the encapsidation of viral dna provides a new dimension of complexity to the attendant protein-dna interactions. the rna is a constituent of the viral precursor shell of the dna-packaging machine but is not a component of the mature virion. studies of the sequential interactions involving this rna molecule are likely to provide new insight ...19873107124
characterization of the small rna of the bacteriophage phi 29 dna packaging machine.the prohead connector of the bacteriophage luminal diameter 29 dna packaging machine was reconstructed with the small rna that regulates dna packaging in vitro. the complete sequence of the 120 nucleotide rna proved its origination from the promoter pe1(a1) of the left early region of phi 29 dna, the end packaged first during assembly. the prohead rna was clearly distinct from eubacterial 5s rrna in sequence and composition.19873116499
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
interaction of the bacteriophage phi 29 connector protein with the viral dna.the protein that forms the connector of phage phi 29, p10, binds to dna. apparently, p10 binding is not sequence specific. nevertheless, the presence of the terminal protein (p3) covalently attached to the ends of phi 29 dna produces a significant increase of p10 molecules bound to the dna ends, thus suggesting a terminal protein-mediated recognition of dna ends by the phage connector. as the p3-dna complex is the substrate for phage phi 29 dna packaging, these results may reflect a direct impli ...19863095983
effect of the delta subunit of bacillus subtilis rna polymerase on initiation of rna synthesis at two bacteriophage phi 29 promoters.initiation of rna synthesis by bacillus subtilis rna polymerase (sigma-43) has been examined at two early promoters of phage phi 29: the a2 promoter, which is a weak promoter, and the g2 promoter, which is a strong promoter. the delta subunit of the polymerase inhibits the rate of initiation at a2, but not g2. in addition, formation of stable complexes by the polymerase at a2, but not at g2, requires the presence of the first two nucleotides of the a2 transcript.19873126800
initiation of dna replication by primer proteins: bacteriophage phi 29 and its relatives. 19883131070
bacillus subtilis phage phi 29 main promoters are efficiently recognized in vivo by the streptomyces lividans rna polymerase.a dna fragment from the bacillus subtilis phage phi 29, containing the main early and late viral promoters, has been inserted upstream of the aminoglycoside phosphotransferase gene (neo) derived from the transposon tn5 and present in a streptomyces lividans promoter-probe plasmid. the phi 29 promoters are specifically recognized by the s. lividans rna polymerase which initiates transcription in vivo at the same sites utilized in b. subtilis. moreover, the viral promoters efficiently direct the s ...19863106159
site-directed mutagenesis in the dna linking site of bacteriophage phi 29 terminal protein: isolation and characterization of a ser232----thr mutant.by site-directed mutagenesis we have changed the serine residue 232 of the phi 29 terminal protein, involved in the covalent linkage to damp for the initiation of replication, into a threonine residue. the mutant terminal protein has been purified to homogeneity and shown to be inactive in the formation of the initiation complex; nevertheless, the mutant protein retains its ability to interact with the phi 29 dna polymerase and with the dna. the results obtained indicate a high specificity in th ...19883135531
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
overproduction and purification of protein p6 of bacillus subtilis phage phi 29: role in the initiation of dna replication.a phi 29 dna fragment containing gene 6, required for dna replication, has been cloned in plasmid pplc28 under the control of the pl promoter of phage lambda. a polypeptide with an electrophoretic mobility close to that of p6 was labelled with 35s-methionine after heat induction. this protein, representing about 4% of the total e. coli protein after 1 h of induction, was obtained in a highly purified form. the protein was characterized as p6 by amino acid analysis and nh2-and cooh-terminal seque ...19853158884
symmetrical transcription in bacteriophage phi 29 dna.transcription of some early genes occurring during phi 29 infection in the absence but not in the presence of chloramphenicol has been shown to depend upon the synthesis of the viral protein p4, the positive regulator of late transcription. in addition, the early promoter b1, responsible for early transcription on the late region of the phi 29 genome, has been accurately mapped by nuclease s1 protection experiments. the deduced promoter sequence shares homology with that of the other early phi 2 ...19883139079
interaction of the bacteriophage phi 29 protein p6 with double-stranded dna.the bacillus subtilis bacteriophage phi 29 protein p6 binds to double-stranded dna, but not to single-stranded dna, as determined by a gel retardation assay. the nature of the interaction was further studied by dnase i "footprinting" experiments. protein p6 binds to fragments containing the right or left terminal sequences of phi 29 dna, producing a characteristic pattern of hypersensitive bands spaced about 24 nucleotides apart along most of the fragment, flanking protected regions. binding of ...19883124105
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
bacteriophage t3 connector: three-dimensional structure and comparison with other viral head-tail connecting regions.the bacteriophage t3 connector, which consists of 12 copies of protein gp8, has been studied by image processing of electron micrographs from negatively stained ordered aggregates. a three-dimensional reconstruction of t3 connectors was obtained by collection of tilted views and using the direct fourier method, up to 2.3 nm resolution. the reconstructed unit cell contains two connectors whose main structural features are essentially identical, but facing in opposite directions. the t3 connector ...19883262165
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