Cloning of bacteriophage T5 DNA fragments in plasmid pBR322 and bacteriophage lambda gtWES

Bacteriophage T5 was digested with the restriction endonucleases HindIII and EcoRI and the resulting fragments were inserted into the plasmid pBR322 and the bacteriophage lambda gtWES as vectors. Approx. 15% of the phage genome was recovered in recombinant clones. The recombinants were characterized by restriction analysis, DNA/DNA hybridization employing Southern blots, and ability to complement or recombine with amber mutants of T5. The results obtained allow revisions of the physical map of the T5 genome and partial correlation of the physical map with the genetic map.     

In vitro construction of bacteriophage λ and plasmid DNA molecules containing DNA fragments from bacteriophage T4

Restriction endonucleases EcoRI and HindIII generated fragments of T4 cytosine-containing DNA were inserted into bacteriophage vector λgtSu_III and plasmid vectors pMB9 and pBR313. Resulting clones were screened for hybridization with ³²P labeled T4 tRNA. Recombinant bacteriophages and plasmids were isolated which contained a T4 fragment coding for T4 RNA species 1 and 2 and T4 tRNA^Arg. Selected λ-T4 hybrid bacteriophages were grown to high titer and their DNA analyzed by gel electrophoresis.     

Cloning of Herpes simplex virus 2 DNA fragments in a plasmid vector

DNA isolated from virions of Herpes simplex type 2 (HSV-2) strain 333 was digested with various restriction enzymes and joined to the EK2 plasmid vector pBR322. The viral DNA sequences present in the hybrids were analyzed by restriction enzyme mapping and hybridization to fragments of HSV-2 DNA. The collection of recombinant molecules represents approx. 75% of the HSV-2-genome. In most cases, the structure of the recombinants seemed identical to the organization of authentic fragments of HSV-2 DNA, however, a few hybrids contained rearrangements of viral and plasmid sequences.     

Molecular cloning of fragments of bacteriophage T4 DNA

Summary Non-glucosylated T4 DNA was digested with R. EcoRI and the resulting fragments covalently joined to vectors. The genetic content of each -T4 hybrid was determined by marker-rescue tests. The isolation of many recombinants containing partialdigestion products of T4 DNA provided the overlapping sequences necessary to order fragments within the T4 genome. The present analyses include parts of the early region between genes 42 and 46, and much of the late region between genes 50 and 29. T4 cytosine-DNA digested to completion by R.EcoRI was used to identify the fragments of DNA within the -T4 recombinants. The T4 cytosine-DNA was also sensitive to R.HindIII and R.Xho but not to R.BamH1.     

Cloning fragments of bacteriophage T5 DNA, determining expression of phage-dependent ligase

Cloning vèctor lambda gt-p MB9 has been used for cloning of DNA fragments of bacteriophage T5 produced by EcoR*I activity. One clone contains a DNA fragment of 2.2 Md which has been mapped at 67-71% on the physical map of the genome. Functional studies have shown that bacteriophage lambda gt-T5 can grow on E. coli lights 7. Infection of this E.coli strain with phage lambda gt-T5 induces DNA-ligase activity which has been previously observed in E. Coli infected with bacteriophage T5.     

Recombination between bacteriophage T4 and plasmid pBR322 molecules containing cloned T4 DNA

Reciprocal recombination between T4 DNA cloned in plasmid pBR322 and homologous sequences in bacteriophage T4 genomes leads to integration of complete plasmid molecules into phage genomes. Indirect evidence of this integration comes from two kinds of experiments. Packaging of pBR322 DNA into mature phage particles can be detected by a DNA--DNA hybridization assay only when a T4 restriction fragment is cloned in the plasmid. The density of the pBR322 DNA synthesized after phage infection is also consistent with integration of plasmid vector DNA into vegetative phage genomes. Direct evidence of plasmid integration into phage genomes in the region of DNA homology comes from genetic and biochemical analysis of cytosine-containing DNA isolated from mature phage particles. Agarose gel electrophoresis of restriction endonuclease-digested DNA, followed by Southern blot analysis with nick-translated probes, shows that entire plasmid molecules become integrated into phage genomes in the region of T4 DNA homology. In addition, this analysis shows that genomes containing multiple copies of complete plasmid molecules are also formed. Among phage particles containing at least one integrated copy, the average number of integrated plasmid molecules is almost ten. A cloning experiment done with restricted DNA confirms these conclusions and illustrates a method for walking along the T4 genome.     

The generation and analysis of clones containing bacteriophage T4 DNA fragments

Cytosine-containing DNA of bacteriophage T4 was digested with three restriction endonucleases: endo R · EcoRI, endo R · HindIII and endo R · PstI, and each digestion ligated with a cloning vector to generate three independent collections of T4 DNA-containing clones. The T4 clones were screened for their T4 genetic content by recombinational analysis using amber mutants of T4. Complementation of T4 amber mutant growth and labeling of proteins in vivo provided evidence of expression of specific (g30, g39, g44 and g46) cloned T4 genes.     

Physical mapping of the restriction fragments obtained from bacteriophage T4 dC-DNA with the restriction endonucleases SmaI, KpnI and BglII.

Summary The cytosine-containing DNA of a mutant of bacteriophage T4 was digested with restriction endonucleases SmaI, KpnI and BglII producing 5, 7 and 13 fragments respectively. Complete physical maps of the T4 genome were constructed with the enzymes SmaI and KpnI and an almost complete map with the enzyme BglII.     

Nucleotide sequence of gene 31 of the T4 bacteriophage

We have determined the nucleotide sequence of a region of 656 nucleotides comprising the 31 gene of bacteriophage T4. The coding region consisted of 333 nucleotides directing the synthesis of a polypeptide of 111 amino acids, with a calculated molecular weight of 12,060. The upstream sequence contains the consensus sequences for T4 early and two middle promoters. The downstream sequence contains the consensus sequence for T4 late promoter and the inverted repeats. In addition, there are two incomplete open reading frames in the sequenced region.     

Cloning of restriction fragments of DNA from staphylococcal bacteriophage phi 11.

EcoRI fragments of Staphylococcus aureus bacteriophage phi 11 DNA were cloned in vector plasmid pSA2100 in S. aureus. The clones were analyzed in marker rescue experiments with suppressor- and temperature-sensitive mutants of phi 11 to correlate the genetic and physical map. Several mutants could be identified on the physical map, and a clone containing fragment EcoRI-B of phi 11 DNA expressed immunity to phage infection. In addition, it was found that recombinant plasmids containing phi 11 DNA sequences can be transferred by high-frequency transduction after phage phi 11 infection of host cells.     

Cloning high molecular weight DNA fragments by the bacteriophage P1 system.

The cloning of high molecular weight genomic DNA promises to provide the means of mapping chromosomes, isolating genes, and understanding long-range effects on gene expression. This review describes the background and some recent advances in cloning of high molecular weight DNA using the bacteriophage P1 system.     

Molecular arrangement of DNA in bacteriophage T4

The degree of preferred orientation and the coiling of the deoxyribonucleic acid within phage T₄ was studied by two independent techniques, namely, polarization of fluorescence and uv linear dichroism. A correlation between the two kinds of data was obtained, which indicated that a significant proportion (about 30%) of total phage DNA is aligned preferentially along the long axis of phage heads. Analyses of the data suggest that all of the phage DNA cannot be in a highly supercoiled helical configuration. A few models of the DNA arrangement in T₄ have been discussed in which linear sidewise packings of DNA would be predominant and may explain the observed longitudinal orientation of intraphage DNA.     

Characterization of proteolytic fragments of bacteriophage T7 DNA ligase.

Treatment of T7 DNA ligase with a range of proteases generates two major fragments which are resistant to further digestion. These fragments, of molecular weight 16 and 26 kDa, are derived from the N- and C-termini of the protein, respectively. The presence of ATP or a non-hydrolysable analogue, ADPNP, during limited proteolysis greatly reduces the level of digestion. The N-terminal 16 kDa region of the intact T7 ligase is labelled selectively in the presence of [alpha-32P]ATP, confirming that it contains the active site lysine residue. In common with the intact enzyme, the C-terminal portion of the protein retains the ability to band shift DNA fragments of various lengths, implicating it in DNA binding. It can also inhibit ligation by the intact protein, apparently by competing for target sites on DNA. We conclude that the N-terminal region, which contains the putative active site lysine, plays a role in the transfer of AMP from the enzyme-adenylate complex to the 5'phosphate at the nick site, while the C-terminal 26 kDa fragment appears to position the enzyme at the target site on DNA.     

Cloning of bacteriophage T5 DNA fragments in the plasmid pBR322. Analysis of recombinant plasmids by the method of bonding with RNA-polymerase from Escherichia coli on nitrocellulose filters

DNA of bacteriophage T5 was hydrolyzed with restriction endonucleases HindIII and BamHI, and subjected to the combined hydrolysis with BamHI+EcoRI and BamHI+ +HindIII. Fragments obtained were cloned in the plasmid pBR322. About 17% of T5 genome were recovered in recombinant plasmids. Cloned fragments were localized on the physical map of the phage by restriction analysis and Southern hybridization. With the aim of direct cloning of T5 promoters, PstI/HindIII fragments were inserted into pBR322 followed by selection of recombinants on ApsTCr phenotype. Binding of BsuRI and AluI fragments of hybrid plasmids with E. coli RNA polymerase was studied by nitrocellulose filter assay. The fragments, which were capable to form heparin resistant complexes were identified.