Valentine M. Kryukov

A phage T4 site‐specific endonuclease, SegE, is responsible for a non‐reciprocal genetic exchange between T‐even‐related phages

The bacteriophage T4 segE gene encoding site‐specific endonuclease lies between the hoc.1 and uvsW genes. The similar region of T‐even‐related phage RB30 lacks the segE gene. Here we demonstrate that the phage T4 segE gene is inherited preferably by progeny of mixed infection with RB30. The preferred inheritance of the segE gene depends on its own expression and is based on a non‐reciprocal homologous recombination event providing the transfer of the gene from the segE‐containing to the segE‐lacking allele. The SegE endonuclease cleaves DNA in a site located at the 5′ end of the uvsW gene in the RB30 genome. The T4 DNA is also cleaved by the enzyme, but less efficiently. The cleavage at the RB30 site appears to initiate the observed conversion, which is stimulated by DNA homology and accompanied by co‐conversion of flanking markers. Our findings provide a novel example of endonuclease‐dependent generation of genetic variation in prokaryotes.

Cloning and DNA sequence of the genes for two bacteriophage T5 tRNAsSer

Bacteriophage T5 BglII/HindIII DNA fragment (803 basepairs), containing the genes for 2 tRNAs and 2 RNAs with unknown functions, was cloned in the plasmid pBR322. The analysis of DNA sequence indicates that tRNA genes code isoacceptor tRNAsSer (tRNASer 1 and tRNASer 2) with anticodons UGA and GGA, respectively. The main unusual structural feature of these tRNAs is the presence of extra non‐basepaired nucleotides in the joinings of stem ‘b’ with stems ‘a’ and ‘c’.

The nucleotide sequence of the bacteriophage T5 ltf gene.

The nucleotide sequence of the bacteriophage T5 Bg/II‐BamHI fragment (4,835 bp in length) known to carry a gene encoding the LTF protein which forms the phage L‐shaped tail fibers was determined. It was shown to contain an open reading frame for 1,396 amino acid residues that corresponds to a protein of 147.8 kDa. The coding region of ltf gene is preceded by a typical Shine‐Dalgarno sequence. Downstream from the ltf gene there is a strong transcription terminator. Data bank analysis of the LTF protein sequence reveals 55.1% identity to the hypothetical protein ORF 401 of bacteriophage λ in a segment of 118 amino acids overlap.

Cloning of genes for bacteriophage T5 stable RNAs.

One EcoRI-generated fragment (440 basepairs) and two EcoRI/HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage lambda XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo 32P-labeled T5 4-5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI 440 fragment contains the gene for tRNA 10 (tRNAAsp), the EcoRI/HindIII 220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNAHis), and the EcoRI/HindIII 960 fragment contains only a part of the gene for tRNA 9 (tRNAGln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNAGln-tRNAHis-RNA III-RNA I-...-tRNAAsp.

Cloning of bacteriophage T5 promoters

SummaryBacteriophage T5 was subjected to combined hydrolysis with the restriction endonuclease PstI and HindIII and the resulting fragments were inserted into the plasmid pBR322. Selection of transformants for Aps-Tcr-phenotype made it possible to screen the hybrid plasmids that contained promoter sequences in the cloned fragments.Two PstI/HindIII fragment, 720 bp (51% of the T5 DNA length) and 1,200 bp (70%) were cloned in this study. Tcr levels for these plasmids were as high as 18 μg/ml and 75 μg/ml, respectively. The presence of Escherichia coli RNA polymerase binding sites on both fragments was shown using the nitrocellulose filter assay. These binding sites are situated between 35 bp and 95 bp from the HindIII cleavage site on the 1,200 bp fragment; and within 420 bp from the HindIII site on the 720 bp fragment.

The nucleotide sequence of bacteriophage T 5 leucine tRNA

This tRNA has anticodon sequence UAG, which can presumably recognize all the four leucine‐specific codons (CUN). The main feature of T5 tRNALeu is the absence of the A10‐C25 and C31‐Ψ39 pairing in the D and anticodon stems, respectively.