PubMed Health⌕ Search

Biomedical subjects

W Widner

Publications and source records attributed to W Widner.

5 recordsLinked to original sources
PubMed1991

Interference with phage lambda development by the small subunit of the phage 21 terminase, gp1.

Bacteriophage lambda development is blocked in cells carrying a plasmid that expresses the terminase genes of phage 21. The interference is caused by the small subunit of phage 21 terminase, gp1. Mutants of lambda able to form plaques in the presence of gp1 include sti mutants. One such mutation, sti30, is an A. T-to-G.C transition mutation at base pair 184 on the lambda chromosome. The sti30 mutation extends the length of the ribosome-binding sequence of the Nul gene that is complementary to the 3' end of the 16S rRNA from GGA to GGAG. The sti30 mutation causes a approximately 50-fold increase in the level of expression of a Nul-lacZ reporter gene, indicating that the sti30 mutation overcomes the gp1 inhibition by increasing the level of expression of gpNul. Although the Nul and A genes of lambda overlap, the sti30 mutation has little effect on the level of gpA expression, indicating that translational coupling does not occur.

Bacteriophage lambda↗
PubMed1983

Structure of the bacteriophage lambda cohesive end site: location of the sites of terminase binding (cosB) and nicking (cosN).

The extents of the sites for nicking (cosN) and binding (cosB) of bacteriophage lambda DNA by terminase have been determined by studying cos cleavage and terminase binding in vitro. The cosN site is located in the segment from -22 to +24 bp (numbered from the center of the cohesive end sequence in the circular lambda genome). The cosB site is located in the segment from +51 to +120 (the +120 boundary determined by Miwa and Matsubara, 1983). Additional sequences are necessary for packaging into infectious phage particles, including regions to the left (Rz gene side) of cosN, and between cosN and cosB. Small deletions (7 and 11 bp) between cosN and cosB abolish packaging in vivo without affecting cos binding and cleavage in vitro, whereas a large deletion (26 bp) abolishes packaging in vivo and cleavage in vitro.

Bacteriophage lambda↗
PubMed1983

Separate sites for binding and nicking of bacteriophage lambda DNA by terminase.

The cohesive end site (cos) is the site of action of bacteriophage lambda terminase, the enzyme that introduces staggered nicks to generate the 12-base cohesive ends of mature lambda DNA. Deletion mutations that remove the lambda cohesive end sequence have been isolated after in vitro mutagenesis. The deletions were obtained by digesting the DNA of a cos duplication phage with S1 nuclease to remove the cohesive ends and adjacent base pairs, followed by blunt end ligation and DNA packaging into phage particles. cos2 is the result of a 22-base-pair deletion that exactly removes the segment of rotational symmetry that includes the cohesive end sequence. The cos2 mutation abolishes nicking by terminase but does not affect terminase binding. We conclude that cos contains two sites that interact with terminase: cosN, the nicking site; and cosB, a binding site for terminase.

Bacteriophage lambda↗
PubMed1982

Bacteriophage lambda DNA packaging: scanning for the terminal cohesive end site during packaging.

Bacteriophage lambda packages the DNA of the related phage 21 poorly [Hohn, B. (1975) J. Mol. Biol. 98, 93--106]. To understand the nature of the packaging defect, the interaction of the cohesive end site (cos) specific for phage 21 (cos phi 21) with phage lambda terminase has been investigated. The ability of lambda terminase to cleave cos phi 21 was studied in vitro; lambda terminase cleaved cos phi 21 only 1% as well as it cleaved the phage lambda cohesive end site (cos lambda). In vitro packaging experiments showed that the lambda and 21 packaging specificities observed in vivo are also found in vitro. The cos cleavage reaction was modified so that competition experiments could be performed; these experiments showed that cos phi 21 was unable to bind lambda terminase, thus identifying the nature of the defect. Previous work [Feiss, M., Fisher, R. A., Siegele, D. A., Nichols, B. P. & Donelson, J. E. (1979) Virology 92, 56--67] has shown that the base pairs giving lambda or 21 packaging specificity are at the left end of the chromosome, outside the 22-base-pair symmetry region that includes the annealed cohesive ends. Therefore, terminase binding to cos requires interactions with base pairs to the Nu1 side of the cohesive end symmetry segment. The evidence supports the proposition that cos consists of adjacent sites for binding of terminase and for nicking by terminase. Because cos phi 21 can be cut by lambda terminase to terminate DNA packaging, it is proposed that the terminase that binds and nicks at the initial cos site is brought into contact with the terminal cos site by the packaging process. Terminase recognizes and nicks the cohesive end sequence of the terminal cos without requiring the binding site.

Bacteriophage lambda↗