Mutational analysis of the bacteriophage φX174 replication origin

Abstract

Bacteriophage 4X174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis. A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained. The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type 4X174. Two +X174 ori mutants with a reduced growth ability spontaneously acquired additional mutations that enhanced the growth rate. The additional mutation was located at the same site as the original mutation or was located in the N-terminal part of the gene A protein. This latter secondary mutation is responsible for a better binding and/or recognition of the gene A protein to the mutated origin. In a Darwinian experiment wildtype 4X174 outgrows all 4X174 ori mutants, indicating the superiority of the wild-type ori sequence for the reproduction of bacteriophage 4X174.Insertions and deletions were constructed at different positions within the $X174 replication origin cloned in a plasmid. Small insertions and deletions in the A+T-rich spacer region do not inhibit #X174 gene A protein cleavage in vitro, but severely impair packaging of single-stranded plasmid DNA in viral coats.-

Lactobacillus hilgardii plasmid pLAB1000 consists of two functional cassettes commonly found in other gram-positive organisms

“…By analogy with pT181 and 4)X174, the nucleotides located downstream the nicking site are probably important for specificity of the Rep protein for the nicking site (3,8). Thus, our failure to demonstrate transcomplementation of pLAB1302 and pLAB1305 by pBD64 (K. Josson, unpublished data) may be due to a difference in this key binding sequence (Fig.…”

IS1294, a DNA Element That Transposes by RC Transposition

“…In its preference to act in cis, the IS1294 transposase resembles transposases encoded by a number of other elements, e.g., IS1, IS10, IS50, and IS903 (Mahillon and Chandler, 1998), a property shared with some replication proteins, notably the REP protein of bacteriophage X174 that initiates RC replication of the phage DNA (Heidekamp et al, 1981;Fluit et al, 1986;Baas, 1987;McKenna et al, 1994;Jones and Nakai, 1997) and which shows some identity to the IS1294 transposase. The transposase of IS1294 is shorter than either of its equivalents encoded by IS91 and IS801 (Fig.…”

Mutational analysis of the ?conserved region? of maize streak virus suggests its involvement in replication

“…It would be interesting to isolate second site mutations from the infectious MSV mutant. The location of such second site mutations would point to the interacting protein, as has been shown for ~X174 origin mutations and suppressing mutations located in the gene A protein [4].…”

“…The Below are the sequences of a stem-loop structure of the minus strand of CFDV [30] and part of the origin of replication of q~X174 [4], with the position of the phage-specific protein A-dependent single-stranded cut, marked with an arrow.…”