Identification of the vaccinia virus gene encoding nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase.

Vaccinia virus encapsidates a DNA-dependent ATPase known as nucleoside triphosphate phosphohydrolase I (NPH I). A bacteriophage lambda gt11 expression library of poxvirus DNA was screened with antibodies specific for NPH I. Positive clones were used to probe restriction fragments of vaccinia virus genomic DNA to locate the NPH I gene. The identity of the open reading frame (ORF) was confirmed by placing it downstream of a bacteriophage T7 promoter, transcribing the ORF in vitro, and translating the RNA in a reticulocyte lysate. A polypeptide of the correct molecular weight, which was recognized by anti-NPH I antibody, was synthesized. Inspection of the deduced amino acid sequence of the NPH I ORF revealed consensus ATP-binding sites.     

Promoters and processing sites within the transforming region of bovine papillomavirus type 1.

The mRNAs present in bovine papillomavirus type 1 (BPV-1)-transformed C127 cells were studied by primer extension. The results show that two internal promoters are present in the E region of BPV-1 in addition to the previously identified promoter at coordinate 1 (H. Ahola, A. Stenlund, J. Moreno-López, and U. Pettersson, Nucleic Acids Res. 11:2639-2650, 1983). One, located at coordinate 31, generated a set of mRNAs with heterogeneous 5' ends, which may encode the major transforming protein of BPV-1, the E5 protein. The second promoter, which is located at coordinate 39, generates colinear mRNAs which encode either the E4 protein or a truncated form of the E2 protein. Unlike the cottontail rabbit papillomavirus (O. Danos, E. Georges, G. Orth, and M. Yaniv, J. Virol. 53:735-741, 1985), BPV-1 appears to lack a separate promoter for expression of the E7 protein. The major splice sites in the transforming region (E region) of the BPV-1 genome were also identified by nucleotide sequence analysis.     

Intermolecular recE4-independent recombination in Bacillus subtilis: formation of plasmid pKBT1

Summary The plasmid pKBT1 was derived by in vivo recE4-independent recombinational event(s) yielding a structure containing regions of plasmid and chromosomal origin. BamHI digests of plasmid pUB110 (Kan^r/Neo^r) and Bg/II digests of pTL12 (Tmp^r, leuA) were mixed, ligated and used to transform competent cells of a recE4 strain of Bacillus subtilis. Kanamycin-resistant transformants were electrophoretically screened for hybrid plasmids. Plasmid pKBT1 (8.0 kb) was smaller than pTL12 (10.4 kb) but larger than monomeric pUB110 (4.5 kb). Plasmid PKBT1 was stably maintained in recE4 strains of B. subtilis and conferred kanamycin resistance but did not specify trimethoprim resistance or leucine prototrophy. At least 86% of the pUB110 monomer length was present in pKBT1 and was completely contained within a single 5.58 kb HindIII fragment. The other segment of pKBT1 was of chromosomal origin as evidenced by lack of homology to pTL12 and strong hybridization to B. subtilis chromosomal DNA. At least one of the in vivo recE4-independent event(s) which produced pKBT1 must have involved intermolecular recombination between transforming and chromosomal DNA. This finding differs from previous reports in which recE4-independent recombination involving pUB110 sequences was a strictly intramolecular event.     

Chromosomal initiation in Bacillus subtilis may involve two closely linked origins

Summary When the dnaB37 initiation mutant of Bacillus subtilis is returned to a permissive temperature following a period at 45° C, a synchronous round of DNA replication immediately ensues. Using this system we have been able to analyse the first fragments to be replicated while avoiding the use of thymine starvation or inhibitors of DNA replication. Such treatments are necessary to achieve even modest synchrony in germinating spores. Our results showed that the first fragment to be replicated was a 4kb BamHI-SalI restriction fragment, BS6. In contrast, when the analysis was performed out in the presence of novobiocin, an inhibitor of DNA gyrase, replication from BS6 was inhibited and the first fragment to be replicated was BS5, a 5.6 kb fragment located 1.7 kb to the right of BS 6. Replication from both putative origins was suppressed by rifamycin and was dependent upon dnaB. The results are discussed in relation to previous attempts to identify the first replicating fragment in germinating spores. We also discuss the possibility that B. subtilis contains two origins and suggest that either can act as the primary origin under certain conditions, or alternatively that both origins may act in concert in normal bidirectional replication, each site being required for the leading strand in each direction.     

Cloning of the cysB gene of Erwinia carotovora subsp. carotovora, and the identification of its product

Summary The suicide vector pJB4JI was used to generate a range of Tn5-induced mutants of Erwinia carotovora subsp. carotovora (Ecc). One mutant, HC500, was a cysteine auxotroph which had a non-pectolytic, non-cellulolytic, non-proteolytic phenotype when grown under sulphate-limitation. The cysteine lesion of HC500 was shown to be analogous to the cysB mutation of Escherichia coli. The Ecc-cysB ⁺ gene product was identified as a protein of M_r 36000.     

Expression of heterologous genes for wall teichoic acid in Bacillus subtilis 168

Summary A localized region of low DNA sequence homology was revealed in two strains of Bacillus subtilis by a specific 100-fold reduction in transformation by W23 DNA of the tag1 locus, a teichoic acid marker of strain 168. Fifty nine rare recombinants, hybrid at this locus, had all acquired donor-specific phage resistance characters, while losing those specific to the 168 recipient. Chemical analysis of isolated cell walls showed that these modifications are associated with major changes in the wall teichoic acids. Genetic analysis demonstrated that determinants for the ribitol phosphate polymer of strain W23 had been transferred to 168, replacing those for the glycerol phosphate polymer in the recipient. All W23 genes coding for poly(ribitol phosphate) in the hybrids and those specifying anionic wall polymers in strain 168 are clustered near hisA. In addition to tag1, the region exchanged extends just beyond gtaA in some hybrids, whereas in others it may include the more distant gtaB marker, encompassing a region sufficient to contain at least 20 average-sized genes. Surface growth, flagellation, transformability and sporulation all appeared normal in hybrids examined. Recombinants without a major wall teichoic acid from either strain were not found, suggesting that an integral transfer of genes for poly(ribitol phosphate) from W23 had occurred in all hybrids isolated. We interpret these results as indicating an essential role for anionic wall polymers in the growth of B. subtillis.