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ecori cleavage of dna from bacillus subtilis phage spo1. 197896586
terminal redundancy of "high frequency of recombination" markers of bacillus subtilis phage spo1. 197896587
bacteriophage spo1 development: defects in a gene 31 mutant.spo1 temperature-sensitive mutant ts14-1, located in cistron 31, has a dd (dna synthesis-delayed) phenotype at 37 degrees c and produces progeny in a stretched program. at 44 degrees c it behaves as a do (dna synthesis-defective) mutant and shuts off the viral rna synthesis about 10 min after infection. the thermal sensitivity of this mutant is due to the inactivity of gp-31 (the product of gene 31) at 44 degrees c. however, gp-31 is synthesized at that temperature and partly recovers its activi ...1978100606
the nature of transcription selectivity of bacteriophage spo1-modified rna polymerase.escherichia coli and bacillus subtilis rna polymerase have almost identical transcription specificities on bacteriophage spo1 dna when assayed in a coupled transcription-translation cell free system. spo1-modified b. subtilis rna polymerase has altered transcription specificity. it is shown that rifampicin-inhibited e. coli rna polymerase can completely block transcription of spo1 dna by rifampicin resistant b. subtilis enzyme, whereas it has no effect on transcription by spo1-modified b. subtil ...1979113644
distinctive nucleotide sequences of promoters recognized by rna polymerase containing a phage-coded "sigma-like" protein.we report the nucleotide sequences of two promoters for bacteriophage sp01 "middle" genes. these promoters are recognized by a modified form of bacillus subtilis rna polymerase that contains a phage-coded "sigma-like" regulatory protein (gp28) in place of the bacterial sigma factor. both promoters shared the identical hexanucleotide 5'a-g-g-a-g-a at about 35 base pairs preceding the start point of transcription and the identical heptanucleotide 5'-t-t-t-a-t-t-t (t is the thymine analog 5-hydroxy ...1979118447
initiation and termination mutants of bacillus subtilis bacteriophage spo1.mutants affected in cistrons 21 and 32 of bacteriophage spo1 are defective specifically in the initiation of dna replication. mutations in cistron 32 also specifically affect the termination of replication.1977401896
selective screening procedure for the isolation of heat- and cold-sensitive, dna replication-deficient mutants of bacteriophage spo1 and preliminary characterization of the mutants isolated.a procedure is described for the selective isolation of temperature-sensitive replication-deficient mutants of bacillus subtilis phage spo1. a modification of the procedure permits the isolation of temperature-sensitive mutants in specific cistrons of interest. the applicability of these procedures to other viral systems is discussed. the mutations isolated were assigned to eight replication-deficient cistrons, with the cold-sensitive mutations showing a distribution strikingly different from th ...1977401897
specificity of the weak binding between the phage spo1 transcription-inhibitory protein, tf1, and spo1 dna.the interaction of the phage spo1 protein transcription factor 1 (tf1), with dna has been analyzed by membrane filter binding and by sedimentation methods. substantially specific binding of tf1 to helical spo1 dna can be demonstrated by nitrocellulose filter-binding assays at relatively low ionic strength (0.08). however, tf1-dna complexes dissociate and reequilibrate relatively rapidly and this makes filter-binding assays unsuitable for quantitative measurements of binding equilibra. accordingl ...1977402939
multiple origins of replication for bacillus subtilis phage spo1. 1977405794
restriction fragment analysis of the temporal program of bacteriophage spo1 transcription and its control by phage-modified rna polymerases. 1977412317
streptomycin-resistant, asporogenous mutant of bacillus subtilis.a streptomycin-resistant mutantant of bacillus subtilis that is also asporogenous, was isolated and partially characterized. this strain, srb15, sporulated at a frequency of about 1% compared to the wild type frequency of greater than 70%. the two phenotypes were inseparable by transformation, suggesting that this strain carries a single mutation that causes it to be both streptomycin-resistant and spore-minus. the mutation cotransduces with cysa, the closest auxotrophic marker to the "ribosomal ...1977414073
action of t4 endonuclease v on dna containing various photoproducts.the action of t4 endonuclease v on dna containing various photoproducts was investigated. (1) the enzyme introduced strand breaks in dna from ultraviolet-irradiated vegetative cells of bacillus subtilis but not in dna from irradiated spores of the same organism. dna irradiated with long wavelength (360 nm peak) ultraviolet light in the presence of 4,5',8-trimethylpsoralen was not attacked by the enzyme. these results indicate that 5-thyminyl 5,6-dihydrothymine (spore photoproduct) and psoralen m ...1977599143
bacteriophage spo1-induced macromolecular synthesis in minicells of bacillus subtilis.spo1 bacteriophage injects its dna into minicells produced by bacillus subtilis cu403 divivb1. the injected dna is partially degraded to small trichloracetic acid-precipitable material and trichloroacetic acid-soluble material. the injected dna is not replicated; however, it serves as a template for rna and protein synthesis. the rna produced specifically hybridizes to spo1 dna, and the amount of rna hybridized can be reduced by competition with rna isolated at all stages of the phage cycle from ...1975806703
bacteriophage spo1 dna- and rna-directed protein synthesis in vitro: the effect of tf1, a template-selective transcription inhibitor.transcription factor one (tf1), a protein synthesized after infection of b. subtilis by phage spo1, is a specific inhibitor of spo1 in vitro transcription. in this paper, we investigate the effect of tf1 on spo1-specific in vitro protein synthesis, using spo1 dna or messenger rna as templates. protein syhthesis is measured by incorporation of radioactive amino acids into acid insoluble form, synthesis of a phage-specific enzymatic activity, and analysis of radioactive polypeptides by acrylamide ...1975810655
bacteriophage spo1 dna- and rna-directed protein synthesis in vitro: comparison with in vivo control.a cell free protein synthesizing system, derived from e. coli, is shown to be a quantitiative assay system for messenger rna extracted from b. subtilis infected with bacteriophage spo1. dna-directed protein synthesis in this system is shown to be limited mostly to those proteins whose messages are contained in early rna. a phage induced enzyme, dcmp deaminase, is shown to be dependent on appearance of a class mrna made in vivo in response to new initiations of transcription dependent on prior sy ...1975810656
synthesis of specific functional messenger rna in vitro by phage-sp01-modified rna polymerase of bacillus subtilis.rna polymerase (nucleosidetriphosphate: rna nucleotidyltransferase, ec 2.7.7.6) was purified from rifampicin-resistant bacillus subtilis, from both uninfected cells and cells infected with bacteriophage sp01. the enzyme from infected cells lacked all traces of the sigma subunit, contained several polypeptides absent from the enzyme made in uninfected cells, and had an altered template specificity in a transcription assay. a cell-free protein synthesizing system from escherichia coli, when poison ...1975813216
regulatory gene 28 of bacteriophage spo1 codes for a phage-induced subunit of rna polymerase. 1976815553
the virus-specified subunits of a modified b. subtilis rna polymerase are determinants of dna binding and rna chain initiation.the phage spo1-modified rna polymerase b-p can form rapidly initiating complexes with spo1 dna but not with heterologous phi1 dna. the b-p enzyme binds only weakly to heterologous phi29 dna: preincubation with phi29 dna does not substantially slow the formation of rapidly initiating complexes between polymerase b-p and subsequently added spo1 dna. in contrast, b. subtilis holoenzyme and core polymerase are substantially sequestered by preincubation with phi29 dna. the results show that at least ...1976821619
bacteriophage sp01 regulatory proteins directing late gene transcription in vitro. 1976822348
immunogenic properties of bacteriophage spo1 and t4 dna photooxidized in the presence of methylene blue, irradiated with ultra-violet light or containing 5-bromdesoxyuridine. 19761254318
the dna polymerase-encoding gene of bacillus subtilis bacteriophage spo1.the bacteriophage spo1 dna polymerase-encoding gene, which contains a self-splicing intron, has been sequenced and its amino acid (aa) sequence has been deduced. the aa sequence of spo1 dna polymerase shows a high degree of similarity with that of dna polymerase i from escherichia coli (po1i). alignment with the sequences of po1i, and the phi 29 and spo1 dna polymerases indicate that the aa residues that have been implicated in 3'----5' exonuclease activities are conserved.19921324872
sequence of the bacteriophage sp01 gene 30.the bacteriophage sp01 gene 30, whose function is essential for dna synthesis, has been analyzed for its primary structural features. conditionally lethal mutations in the gene 30 locus have been mapped and sequenced, and the wild-type amino acid (aa) sequence has been deduced along with that of a co-transcribed and possibly co-translated upstream unidentified reading frame (urf). the aa sequence deduced for gene 30 shares partial similarity with protein p of bacteriophage lambda, which particip ...19921587473
deoxyuridylate-hydroxymethylase of bacteriophage spo1.phage spo1 of bacillus subtilis carries hydroxymethyl-deoxyuridylate in place of thymidylate in its dna. the enzyme, responsible for the conversion of dump to hmdump, is a dump hydroxymethylase, encoded by the spo1 gene 29. here we describe the cloning and sequencing of the gene and the overexpression of the gene product. dna hybridization using the dna of bacteriophage t4 dcmp-hydroxymethylase gene as a probe, allowed us to identify and map g29 on a 3.9-kb restriction fragment, ecori*11. we det ...19921641983
dna-bending properties of tf1.transcription factor 1 (tf1) is the bacillus subtilis phage spo1-encoded member of the family of dna-binding proteins that includes escherichia coli hu and integration host factor, ihf. a gel electrophoretic retardation method has been used to show that a tf1 dimer binding to one of its preferred sites in (5-hydroxymethyl)uracil (hmura)-containing dna sharply bends the latter. in fact, the dna-bending properties of tf1 and e. coli ihf are indistinguishable. substitutions at amino acid 61 in the ...19911658334
bacteriophage spo1 middle transcripts.phage spo1 middle transcripts are known to fall into two classes, m and m1l. class m1l transcripts continue to be made late in the viral infection, while the synthesis of class m transcripts ceases soon after the onset of replication and late transcription. the experiments that are reported here deal with the regulatory nature of this diversity. the accumulation of transcripts associated with eight middle promoters was analyzed by s1 nuclease mapping. dna sequence surrounding these middle promot ...19911840706
bacteriophage spo1 middle transcripts. 19911909825
stoichiometry of dna binding by the bacteriophage sp01-encoded type ii dna-binding protein tf1.the stoichiometry of dna binding by the bacteriophage sp01-encoded type ii dna-binding protein tf1 has been determined. 3h-labeled tf1 was allowed to bind to a 32p-labeled dna fragment containing a tf1 binding site. multiple tf1-dna complexes were resolved from each other and from unbound dna by native gel electrophoresis. dna-protein complexes were cut from polyacrylamide gels, and the amounts of 3h and 32p contained in each slice were measured. a ratio of 1.12 +/- 0.06 tf1 dimer/dna molecule w ...19902113049
construction and properties of a temperature-sensitive mutation in the gene for the bacteriophage spo1 dna-binding protein tf1.the bacillus subtilis bacteriophage spo1 encodes the dna-binding protein tf1, a homolog of the ubiquitous type ii dna-binding proteins that are components of bacterial chromatin. the known three-dimensional structure of a related protein was used in devising a scheme of site-directed mutagenesis that led to the creation of a temperature-sensitive mutation in the tf1 gene. at the nonpermissive temperature, this mutation disrupted the temporal regulation of viral protein synthesis and processing, ...19902115873
a self-splicing group i intron in the dna polymerase gene of bacillus subtilis bacteriophage spo1.we report a self-splicing intron in bacteriophage spo1, whose host is the gram-positive bacillus subtilis. the intron contains all the conserved features of primary sequence and secondary structure previously described for the group ia introns of eukaryotic organelles and the gram-negative bacteriophage t4. the spo1 intron contains an open reading frame of 522 nucleotides. as in the t4 introns, this open reading frame begins in a region that is looped out of the secondary structure, but ends in ...19902119891
effects of mutations at amino acid 61 in the arm of tf1 on its dna-binding properties.transcription factor 1 (tf1) is the bacillus subtilis phage spo1-encoded member of the family of bacterial dna-binding proteins that includes escherichia coli hu and integration host factor (ihf). we have initiated a mutational analysis of the tf1 molecule to understand better its unique dna-binding properties and to investigate its physiological function. we report here the consequences of mutating the putative dna-binding "arms" of tf1. at position 61 in its primary sequence, tf1 contains a ph ...19902125081
tf1, a bacteriophage-specific dna-binding and dna-bending protein.we review and discuss the biological and dna-binding properties of the bacteriophage spo1 transcription factor 1 (tf1), a dna-binding protein belonging to the ubiquitous prokaryotic family of type ii dna-binding proteins. we review recent information on the effects of certain mutations in tf1 on dna-binding and on its ability to sharply bend dna. we also compare the dna-binding properties of the three best-studied type ii dna-binding proteins, escherichia coli hu, e. coli integration host factor ...19902128458
reduced dna flexibility in complexes with a type ii dna binding protein.we studied internal molecular motions in bacillus subtilis phage spo1 dna using the time-resolved fluorescence polarization anisotropy (fpa) of intercalated ethidium. the torsional flexibility of this (hydroxymethyl)uracil-containing dna is very similar to that of naturally occurring thymine-containing dnas, as judged from fits of the time-resolved fpa decay to an elastic dna model. binding of transcription factor 1 (tf1), a type ii procaryotic dna binding protein encoded by the phage spo1, enha ...19902340287
transcription of bacillis subtilis plasmid pbd64 and expression of bacteriophage spo1 genes cloned therein.plasmid pbd64, a vector which is useful for cloning in bacillis subtilis (t. j. gryczan, a. g. shivakumar, and d. dubnau (1980), j. bacteriol. 141, 246-253), has at least three substantial transcription units. two of these include the single ecori, xbai, and bamhi sites, while the other includes the single bglii site. each of these transcripts was synthesized in the counterclockwise direction, relative to the pbd64 restriction map. no transcripts were detected in the opposite direction. infectio ...19852414903
a protein structural motif that bends dna.the prokaryotic protein hu, integration host factor (ihf) from escherichia coli, and transcription factor 1 (tf1) from bacteriophage spo1 are closely related molecules. biochemical results suggest that the role of these proteins is to bind and bend dna. from the high-resolution structure of hu, we propose a model for this interaction with dna. crucial amino acid differences between the proteins can be rationalized in terms of their different specific functions.19892508086
a new isoschizomer, bnai, of the bamhi restriction endonuclease.by assaying the yield of phage spo1 we have identified a new restriction-modification activity in the bacillus natto b3364 strain. a class ii restriction endonuclease, bnai, isolated from the crude extract of b3364 cells was shown to be a true isoschizomer of the bamhi endonuclease. the mr, stability and optimal conditions required for dna digestion were determined for bnai. although both enzymes show the same specificity, bnai and bamhi differ from each other in all the properties specified abo ...19892583516
fluorescence studies of a single tyrosine in a type ii dna binding protein.we studied the fluorescence properties of a single tyrosine (tyr94) located in the c-terminal tail of transcription factor 1 (tf1), a type ii procaryotic dna binding protein encoded by the bacillus subtilis phage spo1. the time-resolved fluorescence intensity of tyr94 in free tf1 dimers decays as a single exponential, and this is consistent with a twofold symmetrical structure. the fluorescence is readily quenched by acrylamide, but it is less accessible to anionic quenchers (iodide and citrate) ...19892706265
distinct control sites located upstream from the levansucrase gene of bacillus subtilis.the sacr regulatory region, which modulates the expression of sacb, the structural gene for levansucrase, was separated into two parts: an upstream region which carries a constitutive promoter and a downstream region which carries a palindromic structure. three types of fusions were constructed in which the apha3 gene coding for kanamycin resistance of streptococcus faecalis was placed downstream from different deleted sacr regions. other fusions were constructed by inserting a promoter from pha ...19872835582
tf1, the bacteriophage spo1-encoded type ii dna-binding protein, is essential for viral multiplication.the lytic bacillus subtilis bacteriophage spo1 encodes an abundant, 99-amino-acid type ii dna-binding protein, transcription factor 1 (tf1). tf1 is special in this family of procaryotic chromatin-forming proteins in its preference for hydroxymethyluracil-containing dna, such as spo1 dna, and in binding with high affinity to specific sites in the spo1 chromosome. we constructed recessive null alleles of the tf1 gene and introduced them into spo1 chromosomes. segregation analysis with partially di ...19882841496
cloning and mapping of the spo1 genome.many of the xbai, ecori, kpni, and bglii fragments of bacteriophage spo1, accounting for about 65% of the genomic sequences, were cloned in bacillus subtilis. four of the ecori fragments were specifically refractory to cloning in both escherichia coli and b. subtilis, probably because of expression of deleterious genes carried on the spo1 fragments. to permit complete identification of the regions cloned, the spo1 restriction map has been extended to include the xbai fragments and the previously ...19852997983
activity of two strong promoters cloned into bacillus subtilis.two dna fragments, one encoding the escherichia coli trc promoter and the other encoding a sequence from the early region of bacillus subtilis phage spo1, were cloned into the b. subtilis promoter-probe vector ppl603. both fragments effected strong in vivo promoter activity in vegetative b. subtilis cells.19863086500
activity of a phage-modified rna polymerase at hybrid promoters. effects of substituting thymine for hydroxymethyluracil in a phage sp01 middle promoter.transcription of bacteriophage sp01 middle promoters is specifically initiated by a complex of the bacillus subtilis host's rna polymerase core (e) with the sp01 gene 28 transcription-regulating protein, gp28. normal sp01 dna contains hydroxymethyluracil (hmura) in place of thymine and e . gp28 preferentially transcribes hmura-containing dna. hybrid dna molecules containing an sp01 middle promoter, pm25 . 1, have been constructed in which one dna strand contains t and the other hmura. the major ...19863098985
[cloning in cells of bacillus subtilis and study of the characteristics of promoter regions of dna of bacillus mesentericus and phage spo2].11 fragments of bacillus mesentericus and phage spo1 dna containing promoters were cloned in bacillus subtilis. nucleotide sequences of these fragments were determined and s1 mapping of transcriptional start points was performed. it was found that some fragments contained tandem or overlapping promoters. nucleotide sequences of bacillus mesentericus promoters were compared with known promoters from bacilli. promoter activities were determined in different phases of cell growth. nucleotide sequen ...19883128731
the phage spo1-specific rna polymerase, e.gp28, recognizes its cognate promoters in thymine-containing dna.the bacteriophage spo1 gene 28-encoded protein, gp28, directs specific recognition of viral middle promoters in hydroxymethyluracil-containing dna by the bacillus subtilis host's rna polymerase core. using appropriately sensitive methods of detection, we have shown that discrimination against thymine-containing dna is not absolute and that the gp28-containing rna polymerase precisely initiates transcription at two thymine-containing spo1 middle promoters.19863739226
[bacillus subtilis mutant with altered ability for heterologous transformation].mutant strain of bacillus subtilis, which produced in certain conditions significantly reduced quantity of trnasformants during transformation by homologous dna, as compared with transformation by heterologous dna from bac. aterrimus, is isolated. the ability to transfection by phage spo1 dna and the efficiency of infection of the mutant by this phage are also decreased. the causes of such alterated properties are discussed.1978414961
dna binding by the bacteriophage spo1-encoded type ii dna-binding protein, transcription factor 1. formation of nested complexes at a selective binding site.the interactions of the phage spo1-encoded type ii dna-binding protein, transcription factor 1 (tf1), with one of its preferred binding sites in spo1 dna have been analyzed in detail. the results suggest that tf1 recognizes a high-affinity "core" binding site and that additional protein moieties can accrue to either side of the occupied core site to form higher order complexes. close contacts between tf1 and the core binding site as well as some of the steric requirements for recognition of the ...19863745213
dna binding by the bacteriophage spo1-encoded type ii dna-binding protein, transcription factor 1. site-specific binding requires 5-hydroxymethyluracil-containing dna.the bacteriophage spo1-encoded type ii dna-binding protein, transcription factor 1 (tf1), forms complexes with specific sites in spo1 dna. we have investigated the binding of tf1 to one of its preferred sites in which the normal 5-hydroxymethyluracil (hmura) of spo1 dna has been replaced by thymine and have also investigated the binding of a bacterial type ii dna-binding protein (from bacillus stearothermophilus) to the hmura- and thymine-containing forms of the same dna segment. our results sho ...19863745214
mapping the genes in the terminal redundancy of bacteriophage spo1 with restriction endonucleases.although most early transcription from spo1, a lytic dna bacteriophage of bacillus subtilis, is specified by the 12.6-kilobase region of the terminal redundancy, early genes from this region have not been identified by standard genetic means. we mapped genes to dna regions of the spo1 terminal redundancy by analyzing in vitro protein synthesis from isolated spo1 restriction fragments in an escherichia coli-coupled transcription-translation cell-free system. dna from the terminal redundancy direc ...19853928902
bacteriophage spo1 dna polymerase and the activity of viral gene 31.bacteriophage spo1 dna-negative (d0) mutants were tested for the induction of viral dna polymerase during bacillus subtilis infection. extracts from spo1-infected bacteria exhibited enzymatic activity when representative mutants of seven out of the nine known d0 genes were employed. this activity was undetectable in cells infected with mutants in genes 28 and 31. the product of gene 28 (gp28) is known to be responsible for turning on spo1 middle gene expression. results show that nonsense mutati ...19853933176
site-specific dna binding by the bacteriophage sp01-encoded type ii dna-binding protein.the bacteriophage sp01 genome encodes a virus-specific type ii dna-binding protein, tf1. the bacterial proteins of this ubiquitous and evolutionarily conserved class are thought to bind non-specifically to dna. in contrast, the experiments described here demonstrate that tf1 binds to specific sites in sp01 dna. several of these sites have been characterized by dnase i 'footprinting' and four of them have been shown to overlap strong phage promoters for bacillus subtilis rna polymerase holoenzyme ...19854040015
immunogenic specificity of bacillus subtilis phage spo1-dna. 19714107659
deoxyribonucleic acid synthesis in bacteriophage sp01-infected bacillus subtilis. ii. purification and catalytic properties of a deoxyribonucleic acid polymerase induced after infection. 19734200923
transcription specificity of an rna polymerase fraction from bacteriophage sp01-infected b. subtilis. 19734201173
minicells of bacillus subtilis: a new bacteriophage-blocking agent.bacteriophage sp01, sp17, and phi29 rapidly absorb irreversibly to minicells of bacillus subtilis but do not produce a lytic cycle in minicells.19734204693
bacillus subtilis bacteriophage sp01, sp82, and phi e require host lysyl- and tryptophanyl-trna synthetases for phage development.infection of bacillus subtilis mutants having temperature-sensitive lysyl- or tryptophanyl-trna synthetases with bacteriophage sp01, sp82, or phie indicates that both host enzymes are essential for phage development. no apparent modification of the temperature-sensitive phenotype of the mutant host enzymes occurs during phage infection.19744211168
new phage-spo1-induced polypeptides associated with bacillus subtilis rna polymerase.rna polymerase was precipitated from extracts of bacillus subtilis infected with phage sp01 by antiserum prepared against core rna polymerase. as shown by sodium dodecyl sulfate gel electrophoresis, the precipitates contained at least five new polypeptides not present in uninfected bacteria, in addition to the known subunits of rna polymerase. the molecular weights of these polypeptides are (1) 85,000; (ii) 40,000; (iii) 28,000; (iv) 25,000; and (v) 23,000. four of the polypeptides (i, iii, iv, ...19744212197
purification of tf1--a template-specific dna-binding protein and transcription inhibitor from bacteriophage spo1-infected bacillus subtilis. 19744212291
deoxyribonucleic acid synthesis in bacteriophage spo1-infected bacillus subtilis. i. bacteriophage deoxyribonucleic acid synthesis and fate of host deoxyribonucleic acid in normal and polymerase-deficient strains.the effect of bacteriophage spo1 infection of bacillus subtilis and a deoxyribonucleic acid (dna) polymerase-deficient (pol(-)) mutant of this microorganism on the synthesis of dna has been examined. soon after infection, the incorporation of deoxyribonucleoside triphosphates into acid-insoluble material by cell lysates was greatly reduced. this inhibition of host dna synthesis was not a result of host chromosome degradation nor did it appear to be due to the induction of thymidine triphosphate ...19724622590
specific inhibition of bacteriophage spo1 dna-directed protein synthesis by the spo1 transcription factor, tf 1. 19724622613
purification of the bacteriophage sp01 transcription factor 1. 19724624121
the genetics of bacteriophage spo1. 19724628367
effect of nalidixic acid on deoxyribonucleic acid synthesis in bacteriophage spo1-infected bacillus subtilis.the effect of nalidixic acid on deoxyribonucleic acid (dna) synthesis in bacillus subtilis cells infected with bacteriophage spo1 was studied. nalidixic acid had little inhibitory effect on spo1 dna synthesis at concentrations that drastically inhibited b. subtilis dna synthesis. inhibition of dna synthesis, appropriate to the concentration used, was imposed within 1 min after addition of nalidixic acid, suggesting that it acts directly on dna synthesis in both infected and uninfected cells. the ...19694976473
a template-selective inhibitor of in vitro transcription.a macromolecular factor, tf1, has been isolated from bacteriophage sp01-infected b. subtilis. tf1 selectively inhibits in vitro transcription of sp01 and related viral dna. evidence is presented regarding these properties of the repressor-like tf1: (1) template and conformational specificity; (2) interaction with dna rather than rna polymerase; (3) trypsin sensitivity; (4) reversibility of action; and (5) ability to block initiation, but not propagation, of rna synthesis.19694978743
rna synthesis during bacteriophage spo1 development: six classes of spo1 rna. 19714996233
transcription during bacteriophage spo1 development: mutations affecting the program of viral transcription. 19714996235
effects of the positively regulating product of gene 28 of the b. subtilis phage spo1 on in vitro transcription.some of the properties of the rna polymerase purified from spo1-infected bacillus subtilis have been compared with the properties of rna polymerase from uninfected cells (core + sigma). the two enzymes synthetize rna from nonoverlapping regions on spo1 dna, and they lead to the retention of different restriction fragments of spo1 dna on cellulose-nitrate filters. the action of the positively regulating product of gene 28 of spo1 (gp 28) has been analyzed. the isolated gp 28 has been shown to be ...19816174844
a transcriptional map of the bacteriophage spo1 genome. iii. a region of early and middle promoters (the gene 28 region). 19816266141
dna gyrase inhibitors block development of bacillus subtilis bacteriophage sp01.sp01 development was inhibited by nalidixic acid and novobiocin in the sensitive host bacillus subtilis 168m. inhibition by novobiocin was prevented by a novr mutation in the cellular dna gyrase gene. nalidixic acid inhibition persisted in hosts carrying a nalr gyrase, but could be overcome by phage mutation. we conclude that sp01 requires for its development subunit b of the host dna gyrase, but replaces or modifies subunit a.19816270354
interaction of bacillus subtilis rna polymerase core with two specificity-determining subunits. competition between sigma and the spo1 gene 28 protein.gene activity in the development of phage spo1 is transcriptionally regulated. early viral genes are transcribed by the major vegetative cell rna polymerase (e. sigma). middle viral genes are transcribed by rna polymerase core (e) bearing the spo1 gene 28 protein (gp28) instead of sigma. this paper deals with the competitive interactions of sigma and gp28 with e which must, at least in part, be involved in the ability of viral middle gene expression to succeed early gene expression. an in vitro ...19826281274
comparison of bacillus subtilis phages pza, phi 29 and phi 15.morphologically identical phages pza, pze , phi 29, and phi 15 can be distinguished by the neutralization test with rabbit antisera and by host range specificity. each member of this phage group contains 18 kb double-stranded linear dna carrying proteins covalently attached to its 5' ends. physical maps of their dna constructed with the use of restriction endonucleases ecori, hpai, hindiii, bspri, and xbai and dna-dna hybridization experiments show a closer relationship between phi 29 and pze th ...19846327410
overproduction and purification of a bacteriophage spo1-encoded rna polymerase sigma factor.gene 28 of bacteriophage spo1 encodes an rna polymerase sigma factor sigma gp28, which replaces the host bacillus subtilis sigma subunit sigma 55, to alter the promoter recognition specificity of rna polymerase. a fragment of spo1 dna containing gene 28 was placed under the control of the pl promoter of bacteriophage lambda in an escherichia coli expression vector. when transcription of gene 28 was induced by derepression of pl, the sigma gp28 synthesized constituted several per cent of total ce ...19846327692
ribonucleoside triphosphate concentration-dependent termination of bacteriophage sp01 transcription in vitro by bacillus subtilis rna polymerase.several sites specifying transcription termination in the bacteriophage sp01 terminal repeat have recently been located and characterized. some of these were identified as partial terminators. further characterization of three of the partial terminators leads to the conclusion that they are not sites of inefficient transcriptional termination by bacillus subtilis rna polymerase. rather, these are sites where termination is either completely efficient or fails to occur at all, depending upon the ...19846330985
bacteriophage spo1 structure and morphogenesis. i. tail structure and length regulation.bacteriophage spo1, a structually complex phage with hydroxymethyl uracil replacing thymine, has been studied by structural and chemical methods with the aim of defining the virion organization. the contractile tail of spo1 consists of a complex baseplate, a tail tube, and a 140-nm-long sheath composed of stacked disks (4.1 nm repeat), each containing six subunits of molecular weight 60,300. the subunits are arranged in six parallel helices, each with a helical screw angle (omega 0) of 22.5 degr ...19836402605
bacteriophage spo1 structure and morphogenesis. ii. head structure and dna size.the capsid of bacteriophage spo1 is icosahedral, and the subunit arrangement on the 87-nm-diameter head suggests the triangulation number t = 16. the major capsid protein (45,700 daltons) is cleaved from a 47,700-dalton precursor. tubular heads (polyheads) are produced by mutations in genes 5 and 8 and contain cores as well as capped ends. the lattice constant of these structures is 13.4 nm; diameter is 109.5 nm. the size of the double-stranded spo1 dna (containing 5' hydroxymethyl uracil in pla ...19836402606
structure of a bacillus subtilis bacteriophage spo1 gene encoding rna polymerase sigma factor.gene 28 of bacillus subtilis bacteriophage spo1 codes for a regulatory protein, a sigma factor known as sigma gp28, that binds to the bacterial core rna polymerase to direct the recognition of phage middle gene promoters. middle promoters exhibit distinctive and conserved nucleotide sequences in two regions centered about 10 and 35 base pairs upstream from the start point of mrna synthesis. here we report the cloning of gene 28 and its complete nucleotide sequence. we infer that sigma gp28 is a ...19836402778
regions specifying transcriptional termination and pausing in the bacteriophage sp01 terminal repeat.we have determined the nucleotide sequences of four termination sites recognized by bacillus subtilis rna polymerase. these sites are located in the terminally repeated segment of the bacteriophage sp01 genome, where most early phage transcription occurs. the sp01 terminators have structures that are similar to those recognized by escherichia coli rna polymerase, containing a region of dyad symmetry followed by a stretch of hmu residues in the noncoding dna strand (hmu is substituted for t in sp ...19836408611
bacteriophage spo1 gene 27: location and nucleotide sequence.bacteriophage spo1 gene 27, whose product is required for late gene transcription and dna replication, has been cloned in escherichia coli, and its complete nucleotide sequence has been determined. we infer that the product of gene 27 is a highly basic 17,518-dalton protein of 155 amino acids. the gene for this regulatory protein is transcribed from two promoters: an early promoter situated before the adjacent upstream gene 28 and a middle promoter located between genes 28 and 27.19836413701
sequence of the bacteriophage sp01 gene coding for transcription factor 1, a viral homologue of the bacterial type ii dna-binding proteins.the bacillus subtilis phage sp01, whose dna contains 5-hydroxymethyluracil (hmura) in place of thymine, codes for an abundant, small, basic protein called tf1. tf1 binds preferentially to hydroxymethyluracil-containing dna and thereby selectively inhibits transcription of such dna in vitro. the gene for tf1 has been sequenced. we find that this viral protein is a homologue of the ubiquitous bacterial type ii dna-binding proteins. the three-dimensional structure of one of these bacterial proteins ...19846438630
bacteriophage spo1 genes 33 and 34. location and primary structure of genes encoding regulatory subunits of bacillus subtilis rna polymerase.bacteriophage spo1 gene 33 and 34 products are required for spo1 late gene transcription. both proteins bind to the core rna polymerase of the bacillus subtilis host to direct the recognition of spo1 late gene promoters, whose sequences differ from those of spo1 early and middle gene promoters. we have located and cloned the genes for these two regulatory proteins, and have engineered their expression in escherichia coli by placing them under the control of the bacteriophage lambda pl promoter. ...19846441846
hydroxymethyluracil dna glycosylase in mammalian cells.an activity has been purified 350-fold from extracts of mouse plasmacytoma cells that forms 5-hydroxymethyluracil (alpha-hydroxythymine) and apyrimidinic sites with phage spo1 dna, which contains this base in place of thymine. this dna glycosylase presumably functions to eliminate hydroxymethyluracil, a major thymine-derived dna lesion produced by ionizing radiation and oxidative damage. the enzyme has no cofactor requirement and is active in edta. neither intermediate formation nor hydrolysis o ...19846588376
dissection of ha20, a double mutant of bacteriophage spo1.ha20, a mutant of bacillus subtilis phage spo1, is deficient in both dna replication and late transcription. ha20 contains mutations in two different genes, which suggested that the two effects might be caused independently. however, single-mutation derivatives, affected only in gene 27, were deficient for both activities. thus, a single mutation apparently affects both dna replication and late transcription.19846690720
purification and dna-binding properties of rna polymerase from bacillus subtilis.four rna-polymerizing activities having different subunit composition can be purified from uninfected and from spo1-infected bacillus subtilis. lysozyme and sodium deoxycholate are used for lysing the cells. polymin p is used for precipitating nucleic acids and deae-cellulose chromatography allows separation of enzymatic activity from the residual polymin p. after these common steps, one can purify core + sigma + delta by chromatography on single-stranded dna-agarose followed by gel filtration w ...19806772439
inhibitory action of erythromycin on bacteriophage spo1 multiplication in sporulating cells of bacillus subtilis 168.erythromycin (2--4 microgram/ml) was found to inhibit specifically multiplication of spo1 in sporulating cells of an erythromycin-resistant, conditional asporogenous mutant of bacillus subtilis 168 thy- trp-, ery1040. in contrast, streptomycin (150--200 microgram/ml) which inhibits protein synthesis to a similar extent as erythromycin did not inhibit spo1 multiplication severely, suggesting that the inhibition of spo1 multiplication by erythromycin is not caused by an overall inhibition of prote ...19806777627
predominance of bacteriophage sp82 over bacteriophage sp01 in mixed infections of bacillus subtilis.in mixed infections with bacillus subtilis phages sp82 and sp01, the sp82 genotype is predominant among the progeny. this predominance is determined by a specific region of the genome, the pos region, which apparently is located near genes 29 to 32 (by the sp01 numbering system). recombination between sp82 and sp01 yields phage which have both the sp82 pos region and an sp01 mutation. this mutation then behaves in mixed infection as if it were part of an sp82 genome.19816787215
changes in the association between bacillus subtilis rna polymerase core and two specificity-determining subunits during transcription.the bacillus subtilis rna polymerase sigma subunit and the phage spo1-coded gene 28 protein are responsible for selective binding of rna polymerase to early and middle spo1 promoters, respectively. the association of the rna polymerase core with each of these subunits weakens during the elongation of rna chains. similar changes are known to be an essential part of the escherichia coli rna polymerase sigma cycle.19816796116
transcriptional regulation of bacteriophage spo1 protein synthesis in vivo and in vitro.there are six classes of spo1 transcripts which are, at least partially, regulated independently of each other. analysis of proteins made in infections by phage mutants defective in dna synthesis, or in genes which positively control transcription, permitted each protein to be assigned to one transcription class. most of the late proteins belong to transcription class m2l. there seem to be few, if any, phage proteins in the l class whose mrna synthesis depends absolutely on phage dna synthesis, ...19826808157
recombination and expression of a cloned fragment of the dna of bacillus subtilis bacteriophage sp01. 19826808761
the localization of spo1 phage resistance in the genome of bacillus subtilis as revealed by fusion of protoplasts.we localized the gene for resistance to phage spo1 relatively to the markers pur b 34 and ura by means of the polyethylene-glycol induced fusion of bacterial protoplasts of three-fold auxotrophic bacillus subtilis strains s3 and s13. by this same method, the site of some auxotrophic markers was tentatively determined. the application of the protoplast fusion technique to exact genetic analysis will not be possible until the exo- and endogenous factors influencing cell wall regeneration are stand ...19826812297
bacteriophage spo1 structure and morphogenesis. iii. spo1 proteins and synthesis.the virion proteins of spo1 have been determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods on purified phage components and on phage lysates. the phage head contains 16 proteins, and the connector or neck structure has an additional 3 proteins not found in the head. the proximal part of the tail, composed of sheath, tube and connecting components, contains six proteins. the distal baseplate is the most complex structure, with 28 proteins identifiable on sodium dodecyl ...19836827651
genes that protect against the host-killing activity of the e3 protein of bacillus subtilis bacteriophage spo1.a cloned rpob gene, specifying an apparently mutant rna polymerase beta subunit, protected escherichia coli against the cytocidal effects of the e3 protein of bacteriophage spo1, suggesting that rna polymerase is the primary cellular target of the e3 protein. two segments of the wild-type e. coli genome, one of which specifies a suppressor of dnak mutations, and thus, possibly, a molecular chaperone, also provided protection when overexpressed, but wild-type rpob did not.19957751311
restriction, methylation and ligation of 5-hydroxymethyluracil-containing dna.oxidation of dna and its components can cause genetic mutations and chromosomal instability. these changes have generally been implicated in aging. oxidation of the methyl group of thymidine residues in dna is known to result in the formation 5-hydroxymethyl-2'-deoxyuridine (5hmdurd). we have utilized bacillus subtilis phage spo1 dna as a model of oxidatively damaged dna. in this phage, all thymine (thy) residues are replaced by 5-hydroxymethyluracil (5hmura), but the species is naturally devoid ...19957862175
expression of the flagellin gene in borrelia is controlled by an alternative sigma factor.the flagellin genes from six borrelia species were cloned, sequenced and characterized at the molecular level. the flagellin genes of two relapsing fever borrelia species, b. hermsii and b. crocidurae, three lyme disease genomic species, b. burgdorferi, b. afzelii and b. garinii, and the avian borreliosis agent, b. anserina, were compared and showed an 85-93% sequence identity to each other. comparison of the fla genes from the different lyme borreliosis spirochaetes revealed that they were 94-9 ...19957894724
the dna polymerase genes of several hmu-bacteriophages have similar group i introns with highly divergent open reading frames.a previous report described the discovery of a group i, self-splicing intron in the dna polymerase gene of the bacillus subtilis bacteriophage spo1 (1). in this study, the dna polymerase genes of three close relatives of spo1: sp82, 2c and phi e, were also found to be interrupted by an intron. all of these introns have group i secondary structures that are extremely similar to one another in primary sequence. each is interrupted by an open reading frame (orf) that, unlike the intron core or exon ...19947937082
interrelations of secondary structure stability and dna-binding affinity in the bacteriophage spo1-encoded type ii dna-binding protein tf1.tf1, a homodimeric dna-binding and -bending protein with a preference for hydroxymethyluracil-containing dna is the bacillus subtilis-encoded homolog of the bacterial hu proteins and of the e. coli integration host factor. a temperature-sensitive mutation at amino acid 25 of tf1 (l25-->a) and two intragenic second site revertants at amino acids 15 (e15-->g) and 32 (l32-->i) were previously identified and their effects on virus development were examined. the dna-binding properties of these protei ...19948107099
determinants of affinity and mode of dna binding at the carboxy terminus of the bacteriophage spo1-encoded type ii dna-binding protein, tf1.the role of the carboxy-terminal amino acids of the bacteriophage spo1-encoded type ii dna-binding protein, tf1, in dna binding was analyzed. chain-terminating mutations truncating the normally 99-amino-acid tf1 at amino acids 96, 97, and 98 were constructed, as were missense mutations substituting cysteine, arginine, and serine for phenylalanine at amino acid 97 and tryptophan for lysine at amino acid 99. the binding of the resulting proteins to a synthetic 44-bp binding site in 5-(hydroxymethy ...19948113176
some properties of hu are modified after the infection of escherichia coli by bacteriophage t4.escherichia coli hu, an abundant, nucleoid-associated, dna-binding protein, plays a role in several biological processes including dna replication. many other bacteria have well-conserved hu homologs, and there are several more-distantly related members of the family, including tf1, encoded by bacillus subtilis phage spo1. we have asked whether coliphage t4, like spo1, encodes an hu homolog or whether it alters the properties of host hu. we have been unable to detect a t4-specified hu homolog, b ...19948132451
a cytotoxic early gene of bacillus subtilis bacteriophage spo1.some of the early genes of bacillus subtilis bacteriophage spo1 were hypothesized to function in the shutoff of host biosyntheses. two of these genes, e3 and e22, were cloned and sequenced. e22 showed no similarity to any known protein, while e3, a highly acidic protein, showed significant similarity only to other similarly acidic proteins. each gene was immediately downstream of a very active early promoter. each was expressed actively during the first few minutes of infection and was then rapi ...19938253678
an african swine fever virus gene with similarity to bacterial dna binding proteins, bacterial integration host factors, and the bacillus phage spo1 transcription factor, tf1. 19938464748
a 1h-nmr study of the transcription factor 1 from bacillus subtilis phage spo1 by selective 2h-labeling. complete assignment and structural analysis of the aromatic resonances for a 22-kda homodimer.1h-nmr experiments have been performed on transcription factor 1 (tf1) encoded by bacillus subtilis phage spo1. to study this 22-kda homodimeric dna-binding protein, a selective 2h-labeling strategy has been employed. complete sequence-specific assignments of all the resonances from the five aromatic residues were determined by a modified standard sequential-assignment procedure. the reduced contribution of spin diffusion upon the long-mixing-time nuclear-overhauser-enhancement spectroscopy for ...19938477755
cloning, expression, purification, and characterization of 2'-deoxyuridylate hydroxymethylase from phage spo1.2'-deoxyuridylate hydroxymethylase (dump-hmase) from phage spo1 has been cloned and expressed in escherichia coli. in crude extracts, the enzyme represents about 25% of the soluble protein and has a higher specific activity than the most purified preparation yet reported. the enzyme was purified to homogeneity by ion-exchange and hydrophobic chromatography. the subunits of dump-hmase are 45 kda by sds-page and form dimers with a molecular mass of 89.2 kda by analytical centrifugation. in additio ...19958527927
a structural dna binding protein of african swine fever virus with similarity to bacterial histone-like proteins.here we describe an african swine fever virus (asfv) protein encoded by the open reading frame 5-ar that shares structural and functional similarities with the family of bacterial histone-like proteins which include histone-like dna binding proteins, integration host factor, and bacillus phage spo1 transcription factor, tf1. the asfv 5-ar gene was cloned by pcr and expressed in e. coli. monospecific antiserum prepared to the 5-ar bacterial expression product specifically immunoprecipitated a pro ...19968634022
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