[Bacteriophage T5 Mutants Carrying Deletions in tRNA Gene Region].

A new series of heat-stable (st) mutants of bacteriophage T5, which contains deletions in the tRNA gene region, has been isolated. An accurate mapping of the deletion boundaries for more than 30 mutants of phage T5 has been carried out. As a result of the analysis of nucleotide sequences flanking the deleted regions in wild-type phage DNA, it has been shown that they all contain short, direct repeats of different lengths (2-35 nucleotide residues), and that only one repetition is retained in the mutant phage DNA. On the basis of the obtained results, it was suggested that deletion mutants of the phage T5 are formed as a result of illegal recombination occurring with the participation of short repeats in DNA (SHDIR). Based on the example of two mutants, it has been shown that the resistance to thermal inactivation depends on the size of the deleted region.

Transfer RNA(Phe) isoacceptors possess non-identical set of identity elements at high and low Mg2+ concentration.

Primary structures of phage T5- and Escherichia coli-encoded tRNA(Phe) are distinct at four out of 11 positions known as identity elements for E. coli phenylalanyl-tRNA synthetase (FRS). In order to reveal structural requirements for FRS recognition, aminoacylation of wild-type phage T5 tRNA(Phe) gene transcript and mutants containing substitutions of the identity elements at positions 20, 34, 35 and 36 was compared with E. coli tRNA(Phe) gene transcript. The wild-type phage T5 transcript can be aminoacylated with the same catalytic efficiency as the E. coli counterpart. However, the maximal aminoacylation rate for T5 and E. coli transcripts was reached at different Mg2+ concentrations: 4 and 15 mM, respectively. Aminoacylation assays with tRNA(Phe) mutants revealed that (i) phage transcripts with the substituted anticodon bases at positions 35 and 36 were efficient substrates for aminoacylation at 15 mM Mg2+ but not at optimal 4 mM Mg2+; (ii) any change of G34 in phage transcripts dramatically decreased the aminoacylation efficiency at both 4 and 15 mM Mg2+ whereas G34A mutation in the E. coli transcript exhibits virtually no influence on aminoacylation rate at 15 mM Mg2+; (iii) substitution of A20 with U in the phage transcript caused no significant change in the aminoacylation rate at both Mg2+ concentrations; (iv) phage transcripts with double substitutions A20U+A35C and A20U+A36C were very poor substrates for FRS. Collectively, the results indicate the non-identical mode of tRNA(Phe) recognition by E. coli FRS at low and high Mg2+ concentrations. Probably, along with identity elements, the local tRNA conformation is essential for recognition by FRS.

[The structure of 2 promoters and a transcription terminator from the region of early genes D10-D15 of bacteriophage T5]. / Struktura dvukh promotorov i terminatora transkriptsii iz oblasti rannikh genov D10-D15 bakteriofaga T5.

The primary structures of two promoters (T5 P31A and T5 P31B) and transcription terminator from the region of bacteriophage T5 early genes D10-D15 were determined. Both promoters (62% of the phage T5 DNA physical map) are located as a tandem at 70 bp distance from each other and are typical for the promoter-consensus structure of DNA of bacteriophage T5 early promoters. The transcription terminator is located in the region of the BamHI site (67% of the phage T5 DNA physical map) and is arranged in good coincidence with the consensus structure of the already known rho-independent terminators of various organisms. The promotor T5 P31A and the terminator were tested for the level of galactokinase synthesis using galK gene-carrying plasmids and D-14C-galactose as a substrate for galactokinase.

Cloning and DNA sequence of the 5'-exonuclease gene of bacteriophage T5.

The nucleotide sequence of the BalI-PstI fragment of T5 DNA, 1347 bp in length, coding for 5'-exonuclease (D15 gene), has been determined. A coding region of the gene contains 873 bp and is preceded by a typical Shine-Dalgarno sequence. The D15 gene belongs to a cluster, consisting of at least 3 genes, in which a termination codon of a preceding gene overlaps an initiation codon of the following one. The sequence contains an open reading frame for 291 amino acid residues. The molecular mass of the 5'-exonuclease calculated from the predicted amino acid sequence is 33 400 Da.

[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]. / Klonirovanie fragmentov DNK bakteriofaga T5 v plazmide pBR322. Analiz rekombinantnykh plazmid metodom sviazyvaniia s RNK-polimerazoi Escherichia coli na nitrotselliuloznykh fil'trakh.

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.

[Restriction mapping of T4 bacteriophage late gene region which contains the origins of DNA replication]. / Restriktsionnoe kartirovanie oblasti pozdnikh genov faga T4, soderzhashchei tochki initsiatsii replikatsii.

DNAs of lambda T4 recombinants 596-27 (genes 50-5), 596-30 (genes 50-8), 596-29 (genes 50-12), 591-16 (genes 6-8), 591-1 (genes 9-12), 596-13 (genes 13-16), 596-17 (genes 18-20) and 596-11 (genes 25-29) were mapped with the use of EcoRI, HindIII, SmaI, SalI and BamHI restriction enzymes. T4 dcDNA was digested with HindIII restriction endonuclease and resulting fragments were cloned into HindIII lambda vector 761. The recombinants 761-7, 761-17, 761-19, 761-24, 761-44, 761-50, 761-55 contained the region of genes 25-48 and 761-42, 761-26 and 761-16 contained a single HindIII-fragment with genes 6-12 in both orientations. Data obtained with the DNA of the latter recombinants allowed to show the correctness of the map established earlier which did not contain a full set of overlapping sequences. As a result of the experiments reported, the position of EcoRI and HindIII recognition sites in the region of genes 50-20 and 25-48 was determined and in the region of genes 25-48 BglII and XhoI restriction sites were mapped. The location of a single BamHI restriction site in the region of gene 8 was also established.

[Specific cleavage of glycosylyl denatured DNA of T2 and T4 phages adjacent to the oligothymidilic or oligoadenylic sequences]. / Fragmentatsiia gliukozilirovannykh odnotiazhevykh DNK fagov T2 i T4 vblizi oligotimidilovykh i oligoadenilovykh posledovatel'nostei.

A method of complementarily directed alkylation with following elimination of the alkylated bases and with DNA cleavage at apurinic sites was used for specific fragmentation of glucosylyl DNA of T2 and T4 phages. It was shown that denatured glucosylyl T2 and T4 DNA's are modified by alkylating derivatives of hexaadenylate and heptauridylilate. The extent of alkylation reached the maximum and then stopped. The extent of elimination and chain cleavage corresponded to that of alkylation. Treatment of DNA after alkylation with (Ap)5ARCl under condition of saturation at 20 degrees gives 572 +/- 28 fragments from T4 DNA with 200--25,000 nucleotides long and 578 +/- 33 fragments from T2 DNA. Alkylation under condition of DNA saturation with (Ap)5ARCl at 40 degrees leads to 138 +/- 15 fragments from T4 DNA and 170 +/- 16 fragments from T2 DNA. Characteristics of the fragments obtained are given.