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.     

Sequence and cloning of bacteriophage T4 gene 63 encoding RNA ligase and tail fibre attachment activities

The sequence of gene 63 of bacteriophage T4 was determined by a shotgun approach. Small DNA fragments, derived by sonication of a restriction fragment that encompasses the region of gene 63, were cloned in M13 vectors and sequenced by the 'dideoxy' method. The position of the gene was established by comparison with the sequence of a gene 63 amber mutant. Knowledge of the DNA sequence of gene 63 and surrounding regions has allowed the construction of a clone of gene 63 in which RNA ligase production is under the control of the lac promoter of bacteriophage M13mp8. Infected E. coli cells can be induced to produce a protein indistinguishable from commercially available RNA ligase.     

Cloning of the Dam methyltransferase gene from Haemophilus influenzae bacteriophage HP1

The putative product of orf13 from the genome of Haemophilus influenzae HP1 bacteriophage shows homology only to bacteriophage T1 Dam methyltransferase, and a weak similarity to the conserved amino acids sequence motifs characteristic of m6A-methyltransferases. Especially interesting is lack of characteristic motif I responsible for binding of S-adenosylmethionine. Despite this fact, a DNA sequence of HP1 bacteriophage of Haemophilus influenzae encoding methyltransferase activity was cloned and expressed in Escherichia coli using pMPMT4 omega expression vector. The cloned methyltransferase recognizes the sequence 5'-GATC-3' and methylates an adenine residue. The enzyme methylates both double- and single-stranded DNA substrates.     

Chemical synthesis and biochemical reactivity of bacteriophage lambda PR promoter

By a combination of chemical and enzymatic methods, a 75 base pair DNA duplex containing the sequence of the lambda PR promoter including the OR1 and OR2 cI repressor binding sites was synthesized. The solid support phosphite triester procedure (Caruthers, M. H. et al., Cold Spring Harbor Symposia on Quantitative Biology XLVII, in press) was used for the synthesis of oligonucleotides comprising the sequence. We report here an adaptation of the method of DNA synthesis in test tubes. Assembly of the oligonucleotides involved the use of T4 polynucleotide kinase and T4 DNA ligase. We show that the synthetic DNA is recognized by RNA polymerase and cI repressor in a manner identical to the same control region contained on a restriction fragment isolated from bacteriophage lambda DNA. Our synthetic approach using chemically synthesized promoter variants is thus suitable for studies probing the function of promoters.     

Cloning, sequencing and overexpression of the mannitol-specific enzyme-III-encoding gene of Staphylococcus carnosus

The gene which encodes the mannitol-specific enzyme III (EIIImtl) of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus, has been cloned. Genomic libraries of S. carnosus DNA were constructed using the expression vector pUC19 and EIIImtl-producing clones were identified using rabbit polyclonal antiserum. A 700-bp Dde I fragment, containing the complete gene encoding EIIImtl, was sequenced by the dideoxy chain-termination technique. Upstream from the ORF for EIIImtl one can find a sequence analogous to that of the Escherichia coli promoter. This region acts as a strong promoter when subcloned into the promoter test vector M13HDL17. EIIImtl was overproduced using the inducible T7 polymerase system and purified to homogeneity. Amino acid sequence comparison confirmed a 38% similarity to the hydrophilic enzyme-III-like portion of enzyme IImtl of E. coli. There is also a 36% similarity to the N terminus of the fructose-specific phospho-carrier protein from E. coli.