A restriction endonuclease cleavage map of bacteriophage P2

Summary A restriction endonuclease cleavage map of phage P2 was constructed. The enzymes used and, within parenthesis, the number of their cleavage sites on the P2 lg cc DNA molecule were: AvaI(3), BalI(1), BamI(3), BglII(3), HaeIII (more than 40; only three were mapped), HindIII(0), HpaI(10), KpnI(3), PstI(3), SalI(2) and SmaI(1). The EcoRI cleavage sites (3), as determined earlier, were used as reference points for this study. The DNAs of a variety of P2 mutants carrying chromosomal aberrations (del1, del2, del3, del6, vir22, vir37(2), vir79 and vir94) were also similarly examined.     

Conditions for S. typhimurium transfection by isolated DNA from bacteriophage P22 H5]

Transfection of spontaneous S. typhimurium LT2 WTR mutant, sensitive to bacteriophages FO, Ffm, 6SR, and resistant to bacteriophages P22 H5, C-21 and P1 vir, by DNA of P22 H5 bacteriophage in the presence of Ca2+ ions was demonstrated. The following were conditions of infection providing maximal and reproducible results: concentration of the recipient cells of 6--7x 109 cells/ml, DNA concentration of 30 microng/ml, CaC12 concentration of 0,25 M. The efficacy of transfection was 5 x 10-8.     

Characteristics of inserted mutations obtained in the P-M hybrid dysgenesis system in the 9F12-10A7 region of the Drosophila melanogaster X-chromosome

19 new mutations in the 9F12-10A7 region of Drosophila melanogaster X chromosome was obtained in the system of P-M hybrid dysgenesis. They appeared to be lethals, as judged from viability of homo- or hemizygous females. In situ hybridization of P DNA with polytene chromosomes revealed P-element insertion in the 10A1-2 band in the majority of the mutants. As a result of complementation analysis, all these mutations were localized at previously known loci: l(1)BP1, l(1)BP5, l(1)BP8, l(1)BP7. No insertion mutations were found at the vermilion locus. This can imply for non-random distribution of insertion mutations in the region studied. Further comparison of these mutations with previously EMS-induced ones revealed that insertion mutations are predominantly hypomorph lethals which do not influence the viability, morphology and fertility of homozygous males and females, but drastically reduce viability of hemizygous females.     

Molecular cloning of bacterial DNA in vivo using a transposable R6K ori and a P1vir phage.

A new method of cloning in vivo using the P1vir phage and transposon Tn5-rpsL oriR6K was developed. The method relies upon recircularization of transducing DNA containing a transposon insertion in a recombination-deficient strain of Escherichia coli K-12 and subsequent stable replication of the recircularized DNA. Using this method, we were able to clone in vivo the chromosomal region located between approximately 7.1 and 9.2 min on the E. coli K-12 map in a 95-kb plasmid.     

Genome structure of Ri plasmid (3). Sequencing analysis of the vir region of pRi1724 in Japanese Agrobacterium rhizogenes.

The entire genome of the pRi1724 (217.6-kb) in the mikimopine type Agrobacterium rhizogenes strain MAFF03-01724 has been completely sequenced. The vir region covering 30.2-kb has found to be composed of 21 genes resembling virH1, virA, virB1-11, virG, virC1-2, and virD1-5. The structural organization of the pRi1724 vir operons in this study is exactly the same as that of the previously reported vir operons of other Ri or Ti plasmids, although the size of some ORFs showed little variations among the plasmids. We also found virE3 gene in the pRi1724 (1), but different from Ti plasmids, virE1 and virE2 that are also important for the virulence do not exist in the vir region of pRi1724.     

Niemann-Pick type C disease: mutations of NPC1 gene and evidence of abnormal expression of some mutant alleles in fibroblasts

We analyzed Niemann-Pick type C disease 1 (NPC1) gene in 12 patients with Niemann-Pick type C disease by sequencing both cDNA obtained from fibroblasts and genomic DNA. All the patients were compound heterozygotes. We found 15 mutations, eight of which previously unreported. The comparison of cDNA and genomic DNA revealed discrepancies in some subjects. In two unrelated patients carrying the same mutations (P474L and nt 2972del2) only one mutant allele (P474L), was expressed in fibroblasts. The mRNA corresponding to the other allele was not detected even in cells incubated with cycloheximide. The promoter variants (-1026T/G and -1186T/C or -238 C/G), found to be in linkage with 2972del2 allele do not explain the lack of expression of this allele, as they were also found in control subjects. In another patient, (N1156S/Q922X) the N1156S allele was expressed in fibroblasts while the expression of the other allele was hardly detectable. In a fourth patient cDNA analysis revealed a point mutation in exon 20 (P1007A) and a 56 nt deletion in exon 22 leading to a frameshift and a premature stop codon. The first mutation was confirmed in genomic DNA; the second turned out to be a T-->G transversion in exon 22, predicted to cause a missense mutation (V1141G). In fact, this transversion generates a donor splice site in exon 22, which causes an abnormal pre-mRNA splicing leading to a partial deletion of this exon. In some NPC patients, therefore, the comparison between cDNA and genomic DNA may reveal an unexpected expression of some mutant alleles of NPC1 gene.     

Restrictase BamHI, HindII, EcoRI and SmaI mapping of hybrid lambda-phi 80 phages with recombination in the structural gene region

Hybrids lambda H lambda T80 with recombination in the region of structural genes have lambda head and phi 80 tail genes. In this paper the molecular structure of 5 independently isolated hybrids was established using restriction endonucleases. It has been shown that all of them have a recombinant head or tail. A deletion of 4,8% lambda was demonstrated in the immunity region of phi 80vir phage. Co-ordinates of restriction sites for BamHI, HindIII, EcoRI and SmaI restriction endonucleases on phi 80 DNA were calculated.     

Homologous DNA sequences on the virulence plasmids of pathogenic Yersinia and Salmonella dublin Lane

Yersinia and Salmonella harbour plasmids that encode traits important for virulence, enabling both pathogenic genera to survive and grow in cells of the reticulo-endothelial organs during systemic infections. We have detected DNA homology between the Salmonella dublin virulence plasmid pSDL2 and the plasmids of the pathogenic Yersinia species pestis, pseudotuberculosis, and enterocolitica. Three regions of pSDL2 were found to share homology with the virulence plasmid pIB1 of Yersinia pseudotuberculosis. Two separate hybridizing segments mapped within the previously characterized 6.4 kb vir region of pSDL2 in the SalI B fragment. The third homologous region involved the replicon of pIB1, which hybridized to the SalI C2 fragment of pSDL2. The virulence plasmid pCD1 from Y. pestis showed similar homology with the three regions of pSDL2. Homologies to the vir and SalI C2 regions of pSDL2 were also found on plasmids from Yersinia enterocolitica serotypes 0:9, 0:3 and 0:5, 27. The discovery of separate homologous regions on the virulence plasmids of Salmonella and Yersinia suggests a distant evolutionary relationship.     

Physical mapping of the immunity region of phage phi 80

Mapping of the sites of cleavage of five restriction endonucleases (BglI, BglII, EcoRV, KpnI and PvuII) in the immunity region of bacteriophage phi 80 DNA is described. The order of the 27 restriction sites was established. This helped to localize the phi 80 cI gene within the 640 bp fragment of the immunity region. Recombinant plasmids carrying phi 80 "kil" function were constructed. The function is suppressed by the cI-coded repressor. The deletion AB43 which is present in the phi 80 vir mutant is located within the phi 80 DNA fragment carrying the cI gene.     

Further physical characterization of deletion and substitution mutants affecting the control of lysogeny in bacteriophage P2

Summary A deletion of phage P2, del6 (L.E. Bertani, 1980), thought to remove the structural gene int, and a deletion/substitution, vir94, thought to remove genes int, C and cox, were mapped by electron microscopy, using the heteroduplex technique.Four independent deletion/substitution mutations, all affecting the regulatory region of P2, were compared in all possible combinations with the same technique: two showed sequence homology in their substitution DNA. The results confirm the model proposed for the origin of these mutants, analogous to that for the origin of transducing variants in phage , but suggest in first approximation that the exchange between the P2 DNA and the chromosome of the host bacterium may occur at several different bacterial sites.A map of the regulatory region of P2, based on all data available from the study of deletions and insertions, is presented.     

Dynamic structure of Agrobacterium tumefaciens Ti plasmids.

Agrobacterium tumefaciens C58F is a variant of strain C58 which generates a high proportion of avirulent mutants in the presence of the virulence (vir) gene inducer acetosyringone. These mutants are altered in the Ti plasmid and do not respond to the acetosyringone signal (C. Fortin, E. W. Nester, and P. Dion, J. Bacteriol. 174:5676-5685, 1992). The physical organization of the Ti plasmid was compared in strain C58 and its variant. One feature distinguishing pTiC58F from its parent plasmid was the presence of the insertion element IS426. Three copies of this element were detected in the strain C58 chromosome, whereas two additional copies were found in strain C58F, including one copy in the Ti plasmid. This particular copy of IS426 was associated with the region of arginine and nopaline catabolism of pTiC58F. Most of the avirulent mutants recovered following growth of strain C58F in the presence of acetosyringone were complemented by clones carrying either virA or virG. Element IS426 was no longer found in the arginine and nopaline catabolism region of the Ti plasmids from the virA and virG mutants, but it resided in the particular KpnI fragment containing the modified vir locus. Behavior of a strain C58F derivative, which was inactivated in a chromosomal component required for the response to acetosyringone, was consistent with the possibility that vir gene induction is essential to the massive production of avirulent mutants.     

Cloning and expression of the Erwinia carotovora subsp. carotovora gene encoding the low-molecular-weight bacteriocin carocin S1

The purpose of this study was to clone the carocin S1 gene and express it in a non-carocin-producing strain of Erwinia carotovora. A mutant, TH22-10, which produced a high-molecular-weight bacteriocin but not a low-molecular-weight bacteriocin, was obtained by Tn5 insertional mutagenesis using H-rif-8-2 (a spontaneous rifampin-resistant mutant of Erwinia carotovora subsp. carotovora 89-H-4). Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of the contiguous 2,280-bp region were determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequence fragment. ORF2 and ORF3 were identified with the carocin S1 genes, caroS1K (ORF2) and caroS1I (ORF3), which, respectively, encode a killing protein (CaroS1K) and an immunity protein (CaroS1I). These genes were homologous to the pyocin S3 gene and the pyocin AP41 gene. Carocin S1 was expressed in E. carotovora subsp. carotovora Ea1068 and replicated in TH22-10 but could not be expressed in Escherichia coli (JM101) because a consensus sequence resembling an SOS box was absent. A putative sequence similar to the consensus sequence for the E. coli cyclic AMP receptor protein binding site (-312 bp) was found upstream of the start codon. Production of this bacteriocin was also induced by glucose and lactose. The homology search results indicated that the carocin S1 gene (between bp 1078 and bp 1704) was homologous to the pyocin S3 and pyocin AP41 genes in Pseudomonas aeruginosa. These genes encode proteins with nuclease activity (domain 4). This study found that carocin S1 also has nuclease activity.     

Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir.

The vir locus of Bordetella pertussis apparently encodes a trans-acting positive regulator that is required for the coordinate expression of genes associated with virulence: pertussis toxin, filamentous hemagglutinin (FHA), hemolysin, and adenylate cyclase toxin. DNA clones of vir and of genes required for the synthesis of some of the factors under vir control were obtained with DNA probes from the chromosomal DNA surrounding sites of Tn5 insertion mutations that inactivated those genes. Two vir clones were found which also contained genes required for the proper expression of FHA in B. pertussis. The plasmids which contained both the fha and vir genes expressed immunologically reactive FHA in Escherichia coli, as detected by colony blots, whereas plasmids which contained only fha or vir were negative in this assay. The regulation of FHA production in E. coli, as in B. pertussis, was temperature dependent and inhibited by high concentrations of either magnesium ions or nicotinic acid, indicating that the sequences cloned in E. coli contained the information required to preserve the physiological responses seen in B. pertussis. Further characterization of the vir-fha clones by Tn5 mutagenesis in E. coli and by the return of cloned sequences to B. pertussis in trans and to the B. pertussis chromosome led to the localization of the vir locus, the structural gene for FHA, and genes that are possibly required for the synthesis and export of FHA.