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 genetic methods. Promoter candidates in the right early region of PZA were found and compared to phi 29 promoters. The sequenced region together with previously determined sequences [Paces et al., Gene 38 (1985) 45-56 and 44 (1986) 107-114] completes the entire 19,366-bp sequence of phage PZA genome.     

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 described promoter, B2, is discussed.     

X-ray scattering of the non-isometric Bacillus subtilis phage phi29.

The non-isometric virus φ29 and its empty capsid have enough elements of symmetry so that their size and approximate shape can be determined by low-angle X-ray scattering. The scattering curve of the virus can be simulated by a cylinder of 390 Å diameter and 460 Å height. The protein coat has a thickness of about 30 Å. The DNA is packed tightly and regularly in the phage head.     

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 due to the absence of the nucleotide sequences on phi 29 recognized by the enzymes, and not to the presence of modified nucleotides.     

Nucleotide sequence of the right early region of Bacillus phage phi 15 and comparison with related phages: reorganization of gene 17 during evolution

The rightmost 2016 bp of the Bacillus subtilis phage phi 15 genome were sequenced. The nucleotide sequence was compared with the homologous regions of the related phages PZA and phi 29. There are six open reading frames (ORFs) in this region of the phi 15 genome; all of them are present in the PZA and phi 29 genomes. One of the ORFs was assigned to gene 17, which is involved in the replication of the phage DNA. Gene 17 has undergone reorganization during the evolution of this phage family. Comparison of the nucleotide sequence of its mRNA-like strand in phi 15, PZA and phi 29 showed that deletions in its central and 3'-end-proximal parts are tolerated and do not interfere with the gene 17 product function. It seems that the only portion of gene 17 that has to be conserved to encode the functional product is its 5'-end-proximal part.     

A novel DNA polymerase induced by Bacillus subtilis phage phi 29.

A novel DNA polymerase induced by Bacillus subtilis bacteriophage phi 29 has been identified. This polymerase can be separated from host DNA polymerase, by fractionation of extracts prepared from phage infected cells, using phosphocellulose chromatography. The isolated polymerase prefers poly(dA)oligo(dT) as template. The DNA polymerase isolated from the cells infected with a gene 2 temperature sensitive mutant (ts2) showed greater heat-lability than that induced by wild type phi 29. The ts2 DNA polymerase was also thermolabile for its activity in the formation of a covalent complex between phi 29 terminal protein and dAMP, the initiation step of phi 29 DNA replication. These findings indicate that gene 2 is the structural gene for a phi 29 DNA polymerase required for the complex formation step of DNA initiation.     

Nucleotide sequence of Bacillus phage Nf terminal protein gene.

The nucleotide sequence of Bacillus phage Nf gene E has been determined. Gene E codes for phage terminal protein which is the primer necessary for the initiation of DNA replication. The deduced amino acid sequence of Nf terminal protein is approximately 66% homologous with the terminal proteins of Bacillus phages PZA and luminal diameter 29, and shows similar hydropathy and secondary structure predictions. A serine which has been identified as the residue which covalently links the protein to the 5' end of the genome in luminal diameter 29, is conserved in all three phages. The hydropathic and secondary structural environment of this serine is similar in these phage terminal proteins and also similar to the linking serine of adenovirus terminal protein.     

Overproduction and purification of the connector protein of Bacillus subtilis phage phi 29.

A phi 29 DNA fragment containing genes 10 and 11, coding for the connector protein and the lower collar protein, respectively, has been cloned in the pBR322 derivative plasmid pKC30 under the control of the PL promoter of phage lambda. Two polypeptides with the electrophoretic mobility of proteins p10 and p11 were labelled with 35S-methionine after heat induction. The proteins were characterized as p10 and p11 by radioimmunoassay and they represented about 10% and 7%, respectively, of the total E. coli protein after 4 hours of induction. These proteins represent less than 1% of the B. subtilis protein in phi 29-infected cells. Protein p10 has been highly purified from the E. coli cells carrying the recombinant plasmid. Antibodies raised against the purified protein p10 reacted with the connector protein produced in phi 29-infected B. subtilis.     

Nucleotide sequence of the thymidylate synthetase gene (thyP3) from the Bacillus subtilis phage phi 3T

The thyP3 gene, encoding thymidylate synthetase, from the Bacillus subtilis phage phi 3T has been cloned and the nucleotide sequence determined. The derived amino acid sequence indicates a subunit Mr of 32 748. The primary amino acid sequence is compared with the sequences of the analogous proteins specified by Escherichia coli (thyA), Lactobacillus casei, (thyA) and phage T4 (td). Extensive conservation exists in all four sequences implying a shared tertiary structure.     

Nucleotide sequence analysis of two simian virus 40 mutants with deletions in the late region of the genome.

Two mutants of simian virus 40, dl-1261 and dl-1262, have deletions that map between coordinated 0.90 and 0.95 (Cole et al., J. Virol 24:277--294, 1977). Both affect the structure of the two minor proteins VP2 and VP3. The precise location and size of the deletions have now been determined by nucleotide sequence analysis. Mutant dl-1261 is deleted of 54 base pairs, is temperature sensitive for the protein defined by the D complementation group, and promotes the synthesis of shorter VP2 and VP3 polypeptides. Mutant dl-1262 is viable irrespective of temperature and has a deletion of 36 base pairs, 23 of which overlap the deletion in dl-1261. Since these mutants produce normal VP1, the deleted regions probably have no function in the splicing of precursor RNA to the VP1 mRNA.     

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 as a rigid, rod-like, homogeneous structure. Complex formation was analysed also by a psoralen crosslinking procedure that did not disrupt complexes. The whole phi 29 genome appears, under saturating conditions, as an irregularly spaced array of complexes approximately 200-300 bp long; however, the size of these complexes varies from approximately 2 kb to 130 bp. The minimal size of the complexes, confirmed by micrococcal nuclease digestion, probably reflects a structural requirement for stability. The values obtained for the affinity constant (K(eff) approximately 10(5) M-1) and the cooperativity parameter (omega approximately 100) indicate that the complex is highly dynamic. These results, together with the high abundance of protein p6 in infected cells, lead us to propose that protein p6-DNA complexes could have, at least at some stages, during infection, a structural role in the organization of the phi 29 genome into a nucleoid-type, compact nucleoprotein complex.     

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 DNA regions whereas the other two early promoters (B1 and C1) are located in less A-T rich sites.