Maharani Chakravorty | PubMed Health

PubMed2005

Cloning and characterization of a gene encoding 22 kDa functional protein of bacteriophage MB78.

Functional protein of MB78 bacteriophage having apparent molecular weight of 22 kDa is expressed from 1.7 kb HindIII G fragment. The nucleotide sequence of this fragment showed two open reading frames of 222 and 196 codons in tail-to-tail orientation separated by a 62-nucleotide intercistronic region. The ORF of 22 kDa protein is present in opposite orientation, i.e. in the complementary strand, preceded by a strong ribosomal binding site and a promoter sequence. Another ORF started from the beginning of the fragment whose promoter region and translational start site lies in the 0.45kb HincII U fragment which is located next to the HindIII G fragment, that has the sequence for DNA bending. 3'end of the fragment has high sequence homology to the EaA and EaI proteins of bacteriophage P22, a close relative of MB78 phage.

Amino Acid Sequence↗

PubMed2003

Restriction-modification system in bacteriophage MB78.

Restriction-modification system is present in bacteria to protect the cells against phage infection. Interestingly, the bacteriophage MB78, a virulent phage of Salmonella typhimurium possesses restriction-modification system. Permissive host transformed with plasmid having the genomic fragment of MB78 carrying the putative restriction-modification genes severely restrict the growth of the phage 9NA. Growth of phage MB78 is also restricted to some extent. However, the temperate phage P22 is not restricted at all. Cloning of the the putative restriction-modification genes has been done in both orientations in different vectors. The clones carrying the genes in the same orientation as that of the lacZ in pUC19 are mostly unstable. However, those are stable when cloned in opposite orientation. Viability of the transformants is strain-, orientation-, and medium-dependent. The two genes have also been cloned individually/separately. Hosts carrying only the modification gene do not restrict growth of phages while the hosts carrying only the restriction gene do. The former produces stable transformants while the latter produces very unstable transformants which were viable only upto 36 h or so. The colonies carrying modification gene were normal looking while those carrying the restriction gene were tiny, flat, and looked distressed resembling very much the clones carrying bacterial restriction-modification system. Amplification of the genes and subsequent cloning in expression vector will be carried out for characterization of the enzymes.

Base Sequence↗