| phage phi 29 protein p6: a viral histone-like protein. | phage phi 29 protein p6 is one of the most abundant viral proteins in phi 29-infected b subtilis cells, constituting about 4% of the total cellular proteins (about 3 x 10(6) copies/cell) at late infection. electron microscopic studies showed that, in vitro, protein p6 forms heterogeneously-sized complexes all along phi 29 dna, suggesting that protein p6 may have a role in genome packaging and organization. the low stability of the protein p6-phi 29 dna complexes observed in vitro could reflect t ... | 1994 | 7748942 |
| a genetic approach to the identification of functional amino acids in protein p6 of bacillus subtilis phage phi 29. | protein p6 of the bacillus subtilis phage phi 29 is essential for in vivo viral dna replication. this protein activates the initiation of phi 29 dna replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. the n-terminal region of protein p6 is involved in dna binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. we report on the development of an in vivo functional assay for protein p6. this assay is based on the ... | 1994 | 7808404 |
| 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 ... | 1995 | 7831803 |
| primary structure and functional analysis of the lysis genes of lactobacillus gasseri bacteriophage phi adh. | the lysis genes of the lactobacillus gasseri bacteriophage phi adh were isolated by complementation of a lambda sam mutation in escherichia coli. nucleotide sequencing of a 1,735-bp dna fragment revealed two adjacent coding regions of 342 bp (hol) and 951 bp (lys) in the same reading frame which appear to belong to a common transcriptional unit. proteins corresponding to the predicted gene products, holin (12.9 kda) and lysin (34.7 kda), were identified by in vitro and in vivo expression of the ... | 1995 | 7836307 |
| transcription regulation in bacillus subtilis phage phi 29: expression of the viral promoters throughout the infection cycle. | transcription of the genome of bacillus subtilis phage phi 29 is tightly controlled, taking place in two stages, early and late. we have analyzed the abundance of the transcripts produced from each viral promoter throughout the infection cycle. we compare the relative strength of each promoter, as well as get a better understanding of the regulatory events, finding a new promoter regulated by the viral protein p4. the two strong early promoters, a2b and a2c, responsible for the expression of gen ... | 1995 | 7871731 |
| cloning, molecular characterization, and expression of the genes encoding the lytic functions of lactococcal bacteriophage phi lc3: a dual lysis system of modular design. | the genes encoding the lysis proteins of lactococcus lactis bacteriophage phi lc3 were cloned, sequenced, and expressed in escherichia coli. the phi lc3 lysis genes, lysa and lysb, encode a membrane-disrupting protein (lysa) of 88 amino acids, and a cell wall degrading protein (lysb) of 429 amino acids, which shares significant sequence similarity with lysins from the streptococcus pneumoniae phages cp-1, cp-7, and cp-9, and lactobacillus delbrueckii phage mv1. both lysa and lysb function in e. ... | 1994 | 7922887 |
| a new protein domain for binding to dna through the minor groove. | protein p6 of the bacillus subtilis phage phi 29 binds with low sequence specificity to dna through the minor groove, forming a multimeric nucleoprotein complex that activates the initiation of phi 29 dna replication. deletion analysis suggested that the n-terminal part of protein p6, predicted to form an amphipathic alpha-helix, is involved in dna binding. we have constructed site-directed mutants at the polar side of the putative alpha-helix. dna binding and activation of initiation of phi 29 ... | 1994 | 7925279 |
| in vivo functional relationships among terminal proteins of bacillus subtilis phi 29-related phages. | gene 3 of the bacillus subtilis phage phi 29 encodes the terminal protein (tp), which acts as a primer in the initiation of viral dna replication. we have developed an in vivo functional assay for the phi 29 tp based on the ability of tp-producing b. subtilis non-suppressor (su-) cells to support dna replication of a phi 29 sus3 mutant phage. this trans-complementation assay has been used to study in vivo functional relationships between the tp of phi 29 and related phages. our results demonstra ... | 1994 | 7926823 |
| an intrinsic-tryptophan-fluorescence study of phage phi 29 connector/nucleic acid interactions. | the protein p10 of bacteriophage phi 29 assembled into connectors exhibit an intrinsic fluorescence with an emission peak centered at 335 nm, which suggests a hydrophobic environment of the three tryptohan residues that the protein contains. upon incubation with linear dna (but not with circular dna), a decrease in the connector intrinsic fluorescence is measured which does not show any sequence specificity. the decrease in fluorescence is not observed when dna is incubated with proteolyzed conn ... | 1994 | 7957190 |
| terminal protein-primed dna amplification. | by using appropriate amounts of four bacteriophage phi 29 dna replication proteins--terminal protein, dna polymerase, protein p6 (double-stranded dna-binding protein), and protein p5 (single-stranded dna-binding protein)--it has been possible to amplify limited amounts of the 19,285-bp-long phi 29 dna molecule by three orders of magnitude after 1 hr of incubation at 30 degrees c. moreover, the quality of the amplified material was demonstrated by transfection experiments, in which infectivity of ... | 1994 | 7991606 |
| dna structure in the nucleoprotein complex that activates replication of phage phi 29. | initiation of phage phi 29 dna replication is activated by the viral protein p6 which forms a nucleoprotein complex at the replication origins, located at the linear genome ends. the complex consists of a dna right-handed superhelix wrapped around a multimeric protein core. we have determined the superhelical path of the dna in the complex, measuring the change in linking number induced by the protein, the surface-related helical repeat and the compaction of the dna. one superhelical turn has ap ... | 1994 | 8011933 |
| a highly sensitive system for the in vitro assembly of bacteriophage phi 29 of bacillus subtilis. | a sensitive system for the assay of bacteriophage phi 29 assembly in vitro was developed using 12 recombinant proteins and synthetic prna. this system detected in vitro assembled infectious phages up to 10(7) plaque forming units (pfu) per milliliter without any background. phi 29 dna-gp3 concentration dependence in phage assembly was found to be first order, while the dna-packaging protein gp16 dependence was higher order. the requirement for specific phi 29 prna for phi 29 dna packaging was co ... | 1994 | 8030206 |
| 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 ... | 1994 | 8034614 |
| the bacteriophage phi 29 head-tail connector shows 13-fold symmetry in both hexagonally packed arrays and as single particles. | the symmetry of the phi 29 head-tail connector is controversial: several studies of two-dimensional arrays of the connector have found a 12-fold symmetry, while a recent study of isolated particles has found a 13-fold symmetry. to investigate whether a polymorphism of the structure might explain these different results, electron microscopy and image analysis were used to study both isolated connectors and particles in hexagonally packed arrays. the hexagonally packed arrays have a p1 symmetry, a ... | 1994 | 8075347 |
| 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 ... | 1993 | 8096839 |
| characterization of the prohead-prna interaction of bacteriophage phi 29. | the small prohead rna (prna) of the bacillus subtilis bacteriophage phi 29 is essential for atp-dependent packaging of viral dna. the 174-, 124-, and 120-residue forms of prna produced in vitro using t7 rna polymerase were equivalent in prohead binding and dna packaging activity to prnas produced in phi 29-infected cells. prna binding to proheads, characterized by the use of northern hybridization and filter binding assays, was specific, rapid, and irreversible in the presence of 10 mm mg2+. pro ... | 1994 | 8106496 |
| requirement for an a-tract structure at the binding site of phage phi 29 transcriptional activator. | the bacillus subtilis phage phi 29 transcriptional activator, protein p4, binds to the 5'-aact-tttt-15 base-pair spacer-aaaatgtt-3' inverted repeat. in this communication, we study the influence in protein p4 binding of the dna helical structure within the protein p4 recognition sequences, 5'-aaaatag-3'. protein p4 could efficiently bind to a modified target in which the a-tracts had been changed into t-tracts (a different sequence with a similar structure). binding was lost when the structure o ... | 1994 | 8126731 |
| identification of bacteriophage phi 29 prohead rna domains necessary for in vitro dna-gp3 packaging. | functional domains of the bacteriophage phi 29 prohead rna (prna) that are essential for in vitro packaging of dna-gp3 into the prohead were mapped using prna mutants. oligonucleotide-directed mutant prnas were produced that contained deletions and sequence alterations but were predicted to retain the overall secondary structure of wild-type prna. mutant prnas were compared to wild-type prna for prohead binding in a competition assay and for dna packaging in the defined in vitro system. the proh ... | 1994 | 8132646 |
| the proximate 5' and 3' ends of the 120-base viral rna (prna) are crucial for the packaging of bacteriophage phi 29 dna. | in vitro mutagenesis was performed to identify the dna packaging domain of the 120-base prna essential and specific for dna encapsidation by bacteriophage phi 29 of bacillus subtilis. all deletions and mutations targeted the 5' and 3' ends of the prna. dna templates of a control or mutant prnas used for in vitro transcription with t7 rna polymerase were generated by pcr. fourteen mutant prna molecules were synthesized from dna templates either directly after pcr or after cloning the pcr fragment ... | 1994 | 8178491 |
| complex formation between phage phi 29 single-stranded dna binding protein and dna. | bacteriophage phi 29 gene 5 encodes a single-stranded dna (ssdna) binding protein (ssb) which stimulates viral dna replication. in the present study, a structural characterization of the complex between ssdna and the phi 29 ssb was carried out using electron microscopy, band-shift assays and nuclease digestion as well as by monitoring changes in the intrinsic fluorescence of phi 29 ssb upon binding. phage phi 29 ssb behaves as a monomer in solution and forms complexes with ssdna which have a hom ... | 1994 | 8196055 |
| rna-mediated specificity of dna packaging into hybrid lambda/phi 29 proheads. | a small rna (prna, 174 nt) is known to be essential for dna packaging in bacteriophage phi 29. however, in an in vitro dna packaging system based on hybrid lambda/phi 29 proheads (made up of head proteins from phage lambda and connectors from phage phi 29), the specificity of dna packaging is lost, and different rna molecules fulfil the requirements for dna packaging, albeit with less efficiency than phi 29 prna. competition assays with rnas from different sources have shown that phi 29 connecto ... | 1993 | 8223455 |
| complementation assay of primer protein: gene expression systems of plasmid vectors support the infection of suppressor sensitive mutant phages m2 and phi 29. | two different expression systems of genes of primer proteins (pe for phage m2, and p3 for phi 29) were constructed to study the protein primed dna replication of bacillus phages m2 and phi 29. in one system, expression of the genes was under the control of the inducible spac promoter, whereas in the other system, the expression was under the control of the constitutive promoter in plasmid pub110. complementation tests in vivo were performed between the primer proteins expressed by these systems ... | 1993 | 8292388 |
| assembly of phage phi 29 genome with viral protein p6 into a compact complex. | the formation of a multimeric nucleoprotein complex by the phage phi 29 dsdna binding protein p6 at the phi 29 dna replication origins, leads to activation of viral dna replication. in the present study, we have analysed protein p6-dna complexes formed in vitro along the 19.3 kb phi 29 genome by electron microscopy and micrococcal nuclease digestion, and estimated binding parameters. under conditions that greatly favour protein-dna interaction, the saturated phi 29 dna-protein p6 complex appears ... | 1994 | 8306969 |
| bacteriophage nf dna region controlling late transcription: structural and functional homology with bacteriophage phi 29. | the putative region for the control of late transcription of the bacillus subtilis phage nf has been identified by dna sequence homology with the equivalent region of the evolutionary related phage phi 29. a similar arrangement of early and late promoters has been detected in the two phages, suggesting that viral transcription could be regulated in a similar way at late times of the infection. transcription of late genes requires the presence of a viral early protein, gpf in phage nf and p4 in p ... | 1993 | 8332494 |
| residues of the bacillus subtilis phage phi 29 transcriptional activator required both to interact with rna polymerase and to activate transcription. | regulatory protein p4 from bacillus subtilis phage phi 29 activates transcription from the viral late promoter, pa3, by stabilizing the binding of rna polymerase to the dna as a closed complex. protein p4-induced dna bending and direct contacts between p4 and rna polymerase have been proposed to play a role in p(a3) activation. by site-directed mutagenesis at the carboxyl end of protein p4 we have identified residues that are critical both to interact with rna polymerase and to activate transcri ... | 1993 | 8411175 |
| the missing link in phage lysis of gram-positive bacteria: gene 14 of bacillus subtilis phage phi 29 encodes the functional homolog of lambda s protein. | in most bacteriophages of gram-negative bacteria, the phage endolysin is released to its murein substrate through a lesion in the inner membrane. the lesion is brought about by a second phage-encoded lysis function. for the first time, we present evidence that the same strategy is elaborated by a phage of a gram-positive bacterium. thus, there appears to be an evolutionarily conserved lysis pathway for most phages whether their host bacterium is gram negative or gram positive. phage phi 29 gene ... | 1993 | 8432697 |
| complete inhibition of virion assembly in vivo with mutant procapsid rna essential for phage phi 29 dna packaging. | a highly efficient method for the inhibition of bacteriophage phi 29 assembly was developed with the use of mutant forms of the viral procapsid (or packaging) rna (prna) indispensable for phi 29 dna packaging. phage phi 29 assembly was severely reduced in vitro in the presence of mutant prna and completely blocked in vivo when the host cell expressed mutant prna. addition of 45% mutant prna resulted in a reduction of infectious virion production by 4 orders of magnitude, indicating that factors ... | 1996 | 8523569 |
| reversed metal replicas of freeze-dried proteins to be visualized with the scanning tunneling microscope. | scanning tunneling microscopy of metal-coated specimens has become a reliable technique that permits direct three-dimensional visualization of structural details at a level at which individual subunits in protein complexes or even single domains of proteins can be resolved. we describe in this paper a variation of the freeze-drying metal coating procedure that allows us to image with the stm the inner side of the metal replica, previously in contact with the protein molecules. we have tested thi ... | 1995 | 8533174 |
| electron microscopy of bacillus subtilis groesl chaperonin and interaction with the bacteriophage phi 29 head-tail connector. | the bacillus subtilis groesl chaperonin was isolated by sucrose density gradient centrifugation and the constituent groes and groel moieties were purified by electrophoresis in agarose. electron microscopic images of negatively stained groel and groes oligomers and groesl complexes were averaged using a reference-free alignment method. the groel and groes particles had the sevenfold symmetry characteristic of their escherichia coli counterparts. groesl complexes, reconstituted efficiently in vit ... | 1995 | 8573469 |
| bacteriophage phi 29 early protein p17. self-association and hetero-association with the viral histone-like protein p6. | gene 17 of the bacillus subtilis phage phi29 is expressed early after infection, and it has been shown to be required at the very beginning of phage replication under conditions of low but not high multiplicity of infection. it has been proposed that, at the beginning of the infection, protein p17 could be recruiting limiting amounts of initiation factors at the viral origins. once the infection process is established and the replication proteins reach optimal concentration, protein p17 becomes ... | 2003 | 12480935 |
| mutational analysis of bacteriophage phi 29 dna polymerase. | | 1995 | 8594354 |
| purification of bacteriophage phi 29 dna polymerase. | | 1995 | 8594366 |
| confirmation of the helical structure of the 5'/3' termini of the essential dna packaging prna of phage phi 29. | bacteriophage phi 29 is typical of double-stranded dna viruses in that its genome is packaged into a preformed procapsid during viral assembly. an intriguing feature of phi 29 is the presence of a 120-base virus-encoded rna (prna) that is indispensable for dna packaging. phylogenetic comparison of similar rnas in numerous phages has revealed that the secondary structure of the prna is well conserved. computer analysis predicts the presence of an extensive segment of helix with three single-base ... | 1995 | 8595559 |
| analysis of the complete nucleotide sequence and functional organization of the genome of streptococcus pneumoniae bacteriophage cp-1. | cp-1, a bacteriophage infecting streptococcus pneumoniae, has a linear double-stranded dna genome, with a terminal protein covalently linked to its 5' ends, that replicates by the protein-priming mechanism. we describe here the complete dna sequence and transcriptional map of the cp-1 genome. these analyses have led to the firm assignment of 10 genes and the localization of 19 additional open reading frames in the 19,345-bp cp-1 dna. striking similarities and differences between some of these pr ... | 1996 | 8648702 |
| transcriptional activator of phage phi 29 late promoter: mapping of residues involved in interaction with rna polymerase and in dna bending. | phage phi 29 regulatory protein p4 activates transcription from the late a3 promoter by stabilizing sigma a-rna polymerase at the promoter as a closed complex. activation requires interaction between both proteins. protein p4 bends the dna upon binding. we have performed a detailed mutagenesis study of the carboxyl end of the protein, which is involved in both transcription activation and dna bending. the results indicate that arg-120 is the most critical residue for activation, probably mediati ... | 1996 | 8733227 |
| protein p4 represses phage phi 29 a2c promoter by interacting with the alpha subunit of bacillus subtilis rna polymerase. | regulatory protein p4 from bacillus subtilis phage phi 29 represses the strong viral a2c promoter (pa2c) by preventing promoter clearance; it allows rna polymerase to bind to the promoter and form an initiated complex, but the elongation step is not reached. protein p4 binds at pa2c immediately upstream from rna polymerase; repression involves a contact between both proteins that holds the rna polymerase at the promoter. this contact is held mainly through p4 residue arg120, which is also requir ... | 1996 | 8799127 |
| the interaction of dna with bacteriophage phi 29 connector: a study by afm and tem. | the connector of bacteriophage phi 29 is involved in dna packaging during viral morphogenesis and we have studied its in vitro binding to dna using either linear or circular dna. the protein-dna complexes have been analyzed by transmission electron microscopy (tem) and by atomic force microscopy (afm) of samples directly deposited on mica. tem showed the presence of a specific binding due to the interaction of the protein with the free ends of the dna. the study of these samples by afm showed tw ... | 1996 | 8812997 |
| analysis of structural variability within two-dimensional biological crystals by a combination of patch averaging techniques and self organizing maps. | we study in this work the use of self organizing maps to analyze the structural variability that can be found along two-dimensional crystals of biological macromolecules. small areas of the crystals, termed "patches" by previous researchers, are used to obtain local average images that are then used as the input of a self organizing map. this procedure allows for a fast and accurate image classification. multivariate statistical analysis is then used on the resulting code vectors producing a ver ... | 1996 | 8961548 |
| substitution of the c-terminal domain of the escherichia coli rna polymerase alpha subunit by that from bacillus subtilis makes the enzyme responsive to a bacillus subtilis transcriptional activator. | regulatory protein p4 of bacillus subtilis phage phi 29 activates transcription from the viral late a3 promoter by interacting with the c-terminal domain (ctd) of the b. subtilis rna polymerase alpha subunit, thereby stabilizing the holoenzyme at the promoter. protein p4 does not interact with the escherichia coli rna polymerase and cannot activate transcription with this enzyme. we have constructed a chimerical alpha subunit containing the n-terminal domain of the e. coli alpha subunit and the ... | 1998 | 9466901 |
| gene 1, one of the dna genes of bacteriophage phi 29, represses other dna genes through binding to mrna. | the dna genes, essential for protein priming dna replication of bacteriophage phi 29, are transcribed as a long polycistronic mrna. in the previous study, gene 1 product (gp1) was shown to repress the expression of the upstream dna genes for dna polymerase and primer protein. to investigate the details of the repression by gp1, we have examined the amount and integrity of polycistronic mrna encoding dna polymerase and primer protein by agarose gel electrophoresis and nuclease s1 protection assay ... | 1998 | 9480849 |
| transcription activation and repression by interaction of a regulator with the alpha subunit of rna polymerase: the model of phage phi 29 protein p4. | regulatory protein p4, encoded by bacillus subtilis phage phi 29, has proved to be a very useful model to analyze the molecular mechanisms of transcription regulation. protein p4 modulates the transcription of phage phi 29 genome by activating the late a3 promoter (pa3) and simultaneously repressing the two main early promoters, a2b and a2c (or pa2b and pa2c). this review describes in detail the regulatory mechanism leading to activation or repression, and discusses them in the context of the re ... | 1998 | 9594570 |
| function of hexameric rna in packaging of bacteriophage phi 29 dna in vitro. | a cyclic hexamer of the 120-base prohead rna (prna) is needed for efficient in vitro packaging of the b. subtilis bacteriophage phi 29 genome. this capacity of prna to form higher multimers by intermolecular base pairing of identical subunits represents a new rna structural motif. dimers of prna are likely intermediates in formation of the cyclic hexamer. a three-dimensional model of the prna hexamer is presented. | 1998 | 9702201 |
| polymerization of bacteriophage phi 29 replication protein p1 into protofilament sheets. | protein p1 (85 amino acids) of the bacillus subtilis phage phi29 is a membrane-associated protein required for in vivo viral dna replication. in the present study, we have constructed two fusion proteins, maltose-binding protein (male)-p1 and male-p1deltan33. by using both sedimentation assays and negative-stain electron microscopy analysis, we demonstrated that male-p1 molecules self-associated into long filamentous structures, which did not assemble further into larger arrays. these structures ... | 1998 | 9774353 |
| sequence-specific 1h, 13c and 15n assignment of the single-stranded dna binding protein of the bacteriophage phi 29. | | 1999 | 10212988 |
| dynamic relocalization of phage phi 29 dna during replication and the role of the viral protein p16.7. | we have examined the localization of dna replication of the bacillus subtilis phage phi 29 by immunofluorescence. to determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage dna. on initiation of replication, the phage dna localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. at later stages of the infection cyc ... | 2000 | 10921898 |
| superhelical path of the dna in the nucleoprotein complex that activates the initiation of phage phi 29 dna replication. | initiation of bacteriophage phi 29 dna replication is activated by protein p6, a viral double-stranded dna-binding protein that forms a nucleoprotein complex at the viral replication origins. this complex consists of a dna right-handed superhelix wrapped around a multimeric protein p6 core with protein p6 dimers regularly bound every 24 base-pairs (bp). in this paper, we have constructed a concatemer formed by direct repeats of a 24 bp sequence previously proposed to act as a signal for protein ... | 1993 | 8450539 |
| 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 ... | 1993 | 8451193 |
| construction and characterization of a bacteriophage t4 dna polymerase deficient in 3'-->5' exonuclease activity. | bacteriophage t4 dna polymerase has a proofreading 3'-->5' exonuclease that plays an important role in maintaining the accuracy of dna replication. we have constructed a t4 dna polymerase deficient in this exonuclease by converting asp-219 to ala. the exonuclease activity of the mutant t4 dna polymerase has been reduced by a factor of at least 10(7), but it retains a polymerase activity whose kinetic parameters, kcat, kd dna, and kd datp, are very close to those of the wild-type enzyme. bacterio ... | 1993 | 8464864 |
| the bacillus subtilis phage phi 29 protein p16.7, involved in phi 29 dna replication, is a membrane-localized single-stranded dna-binding protein. | the functional role of the phi 29-encoded integral membrane protein p16.7 in phage dna replication was studied using a soluble variant, p16.7a, lacking the n-terminal membrane-spanning domain. because of the protein-primed mechanism of dna replication, the bacteriophage phi 29 replication intermediates contain long stretches of single-stranded dna (ssdna). protein p16.7a was found to be an ssdna-binding protein. in addition, by direct and functional analysis we show that protein p16.7a binds to ... | 2002 | 11741949 |
| bacteriophage phi 29 dna packaging. | | 2002 | 12205781 |
| three bacillus anthracis bacteriophages from topsoil. | three bacillus anthracis bacteriophages from iowa topsoil are characterized as to latent period, morphology, structural proteins, dna size, and restriction endonuclease digestion. electron micrographs indicate that the three isolates include two members of the myoviridae and one smaller phage belonging to the podoviridae. phages nk and db resemble myoviridae phage sp50 in morphology, but host range studies, protein, and dna analysis indicate that both differ from sp50. phage mh is very similar t ... | 2003 | 12783194 |
| in vivo assembly of phage phi 29 replication protein p1 into membrane-associated multimeric structures. | the mechanisms underlying compartmentalization of prokaryotic dna replication are largely unknown. in the case of the bacillus subtilis phage 29, the viral protein p1 enhances the rate of in vivo viral dna replication. previous work showed that p1 generates highly ordered structures in vitro. we now show that protein p1, like integral membrane proteins, has an amphiphilic nature. furthermore, immunoelectron microscopy studies reveal that p1 has a peripheral subcellular location. by combining in ... | 2003 | 12904294 |
| genotyping: a new application for the spent dialysate in peritoneal dialysis. | the dialysate of patients on peritoneal dialysis (pd) is used to determine the concentration of growth factors and cytokines, and as a source of resident peritoneal cells for subsequent culture experiments. we hypothesized that the cells contained in spent dialysate samples obtained at the time of the peritoneal equilibration test (pet) and subsequently stored may represent a source of dna from a given pd patient. | 2004 | 14993500 |
| domain structures and roles in bacteriophage hk97 capsid assembly and maturation. | head assembly in the double-stranded dna coliphage hk97 involves initially the formation of the precursor shell prohead i from approximately 420 copies of a 384-residue subunit. this is followed by proteolytic removal of residues 2-103 to create prohead ii, and then reorganization and expansion of the shell lattice and covalent cross-linking of subunits make head ii. here, we report and structurally interpret solution raman spectra of prohead i, prohead ii, and head ii particles. the raman signa ... | 2004 | 15122908 |
| bacteriophage capsids: tough nanoshells with complex elastic properties. | the shell of bacteriophages protects the viral dna during host-to-host transfer and serves as a high-pressure container storing energy for dna injection into a host bacterium. here, we probe the mechanical properties of nanometer-sized bacteriophage phi 29 shells by applying point forces. we show that empty shells withstand nanonewton forces while being indented up to 30% of their height. the elastic response varies across the surface, reflecting the arrangement of shell proteins. the measured y ... | 2004 | 15133147 |
| mechanism of force generation of a viral dna packaging motor. | a large family of multimeric atpases are involved in such diverse tasks as cell division, chromosome segregation, dna recombination, strand separation, conjugation, and viral genome packaging. one such system is the bacillus subtilis phage phi 29 dna packaging motor, which generates large forces to compact its genome into a small protein capsid. here we use optical tweezers to study, at the single-molecule level, the mechanism of force generation in this motor. we determine the kinetic parameter ... | 2005 | 16143101 |
| complementary strands of bacteriophage phi29 deoxyribonucleic acid: preparative separation and transcription studies. | bacillus subtilis phage phi29 has a nonpermuted, duplex deoxyribonucleic acid (dna) with cohesive ends and a molecular weight of 11 x 10(6). denaturation of this dna yielded two intact polynucleotide chains. preferential binding of the polyribonucleotide polyuridylic-guanylic acid (poly ug) to the complementary strands of denatured phi29 dna permitted separation of the strands in neutral cscl gradients. in analytical cscl density gradient centrifugation, the separated strands with poly ug appear ... | 1970 | 16789128 |
| ionic effects on viral dna packaging and portal motor function in bacteriophage phi 29. | in many viruses, dna is confined at such high density that its bending rigidity and electrostatic self-repulsion present a strong energy barrier in viral assembly. therefore, a powerful molecular motor is needed to package the dna into the viral capsid. here, we investigate the role of electrostatic repulsion on single dna packaging dynamics in bacteriophage phi 29 via optical tweezers measurements. we show that ionic screening strongly affects the packing forces, confirming the importance of el ... | 2007 | 17556543 |
| 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 ... | 2008 | 18394643 |
| kinc/d-mediated heterogeneous expression of spo0a during logarithmical growth in bacillus subtilis is responsible for partial suppression of phi 29 development. | the host of the lytic bacteriophage phi 29 is the spore-forming bacterium bacillus subtilis. when infection occurs during early stages of sporulation, however, phi 29 development is suppressed and the infecting phage genome becomes trapped into the developing spore. recently, we have shown that spo0a, the key transcriptional regulator for entry into sporulation, is directly responsible for suppression of the lytic phi 29 cycle in cells having initiated sporulation. surprisingly, we found that ph ... | 2008 | 18410285 |
| structural organization of bacillus subtilis phage phi29. a model. | phage phi29 is a nonisometric virus producing several types of morphological variants in normal infections. the study of these variants by electron microscopy, and their comparison with those from t-even phages, suggest that the capsid of phage phi29 is a prolate icosahedron. phage phi29 capsid consists of a major protein, p8, and an additional protein, p8.5, making up the fibers. we have determined the number of subunits of each structural protein per viral particle taking into account the phag ... | 1981 | 18635054 |
| molecular characterization of a distinct begomovirus species from vernonia cinerea and its associated dna-beta using the bacteriophage phi 29 dna polymerase. | vernonia cinerea plants with yellow vein symptoms were collected around crop fields in madurai. a portion (550 bp) of the av1 gene amplified using degenerate primers from the total dna purified from diseased leaf sample was cloned and sequenced. specific primers derived from the above sequence were used to amplify 2,745 nucleotides with the typical genome organization of begomoviral dna a (embl accession no. am182232). sequence comparison with other begomoviruses revealed the greatest identity ( ... | 2010 | 20401528 |
| molecular characterization of podoviral bacteriophages virulent for clostridium perfringens and their comparison with members of the picovirinae. | clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages th ... | 2012 | 22666499 |
| 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 ... | 1998 | 10943379 |
| phage phi 29 dna polymerase residues involved in the proper stabilisation of the primer-terminus at the 3'-5' exonuclease active site. | three highly conserved amino acid residues have been characterised here as ssdna ligands at the 3'-5' exonuclease active site of o29 dna polymerase. the functional role of tyr59, his61 and phe69 residues of o29 dna polymerase (belonging to exo ii motif, previously described as containing an invariant catalytic aspartate residue and two highly conserved ssdna ligands) was assayed by biochemical analysis of six site-directed mutants at those residues. these studies revealed that the mutations intr ... | 2000 | 11071805 |
| a mutation in the c-terminal domain of the rna polymerase alpha subunit that destabilizes the open complexes formed at the phage phi 29 late a3 promoter. | regulatory protein p4 from bacillus subtilis phage phi29 activates the viral late a3 promoter mainly by stabilizing the binding of rna polymerase (rnap) to it as a closed complex. this requires an interaction between protein p4 residue arg120 and the c-terminal domain (ctd) of the rnap alpha subunit. several acidic residues of the alpha-ctd, considered as plausible targets for p4 residue arg120, were individually changed into alanine. in addition, a truncated alpha subunit lacking the last four ... | 2001 | 11254377 |
| repression of bacteriophage phi 29 early promoter c2 by viral protein p6 is due to impairment of closed complex. | bacillus subtilis phage phi 29 encodes a very abundant protein, p6, which is a non sequence-specific dna-binding protein. protein p6 has the potential to bind cooperatively to the phage genome, forming a nucleoprotein complex in which the dna adopts a right-handed toroidal conformation winding around a protein core. the formation of this complex at the right end of the phage genome where the early promoter c2 is located affects local topology, which may contribute to the promoter repression, alt ... | 2001 | 11384991 |