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Lambda cin-1, a new mutation which enhances lysogenization by bacteriophage lambda, and the genetic structure of the lambda cy region.

Seven lambda cy mutants have been mapped within a small region located approximately halfway between the rightward boundary of the imm434 region and the lambda cII gene. The seven mutants lie at four sites separated by a total distance of about 12 nucleotide pairs, as estimated from recombination frequencies. Six of the seven mutants lie on the right side of the cy fine structure map, spanning a total distance of about 3-5 nucleotide pairs. Lying approximately 11-21 nucleotide pairs to the left of the leftmost cy mutant is a newly described mutation called cin-1, for c independent. The cin-1 mutation allows some lysogenization when coupled with any cy, cII or cIII mutant, but not when coupled with a defective cI gene. The cin-1 mutation, like cy mutants, has a cis-dominant action upon the cI gene in mixed infections. The observation that gammaimm434 cin-1-cy2001 lysogenizes efficiently, but not gammaimm434 cin-1 cy2001 cII68 nor any other gammaimm434 cin-1 cy derivative, is interpreted to mean that all of the cy mutants on the right side of the cy fine structure map inactivate a binding site for cII/cIII function, but that cy2001, the single mutant on the left side of the cy fine structure map, does not inactivate that binding site.

Chromosome Mapping↗

Structure and function of the repressor of bacteriophage lambda. III. Molecular cloning of the high-affinity mutant cI gene of lambda and studies of the properties of the clones.

The high-affinity mutant cI gene of lambda cIha (Nag et al. 1984) was cloned in the multicopy plasmid pBR322. In the resulting plasmid, pMD 102, a lacUV5 promoter was inserted giving the lacUV5-cIha fusion plasmid pMD 205. Bacteria carrying pMD 102 and pMD 205 contain 2.5 and 15 times, respectively, the level of repressor in a monolysogen of lambda cIha. Results of the study of certain properties of the bacteria carrying these plasmids suggest that the ha repressor also has a higher affinity for the virulent mutant operators as well as the prm promoter of lambda.

Bacteriophage lambda↗

Tandem transcription-termination sites in the late rightward operon of bacteriophage lambda.

A transcription termination site (designated as t'R2) is located between the rightward late t'R1 terminator and the S gene of phage lambda. This t'R2 terminator is rightward and absolutely dependent on the rho factor, being about 45% effective in rho+ E. coli and only 6% in rho- cells at 30 degrees C. This 7.5-fold rho dependence of t'R2 is in contrast to that of tR1 (4.5 fold, from about 81% to 18%) and the partial rho dependence of t'R1 (1.4 fold, from 96% to 67%). At the elevated temperature of 42 degrees C, t'R2 becomes 1.5 times more leaky (with about 2.5-fold reduction in termination efficiency) than tR1 or t'R1 (with only 1.1-fold reduction) in rho+ hosts. The calculated joint efficiencies of t'R1 and t'R2 are 98% in rho+ cells at 30 degrees C. t'R2 is also active in vitro, but only in the presence of rho factor, whereas t'R1 is active both in the presence and absence of rho. However, the in vitro termination at t'R1 is enhanced about 1.7-fold by the rho factor. The properly oriented lambda nutR site together with the N gene function bring about almost complete antitermination at t'R2 (96% effective), but incomplete at t'R1 (72%). The termination points at t'R2 are located around 532-534 bp to the right of the s'R startpoint of the p'R-initiated RNA on lambda DNA (or 338-340 bp downstream of t'R1) and 66-68 bp to the left of the S gene, as determined by S1 mapping. The t'R2 termination points are located within a dyad symmetry region which, in the transcript, is able to form a hairpin structure consisting of 16 bp in the stem and 6 bases in the loop. It is proposed that t'R2 acts as a second terminator to block any readthrough transcription initiated at the late promoter p'R into the late genes of phage lambda.

Bacteriophage lambda↗

Location of ribosome-binding sites in the nin5 region of bacteriophage lambda.

Seven ribosome-binding sites on DNA have been located within the region defined by the nin5 deletion as well as several ribosome-binding sites on each side of the nin5 region. These were mapped by electron microscopy relative to the end points of the nin5 deletion and two Tn903 transposons, one inserted into gene Rz and another inserted near gene Q. These ribosomes binding sites within the nin5 region may correspond to polypeptide initiation sites for up to seven new dispensible lambda genes.

Bacteriophage lambda↗

Sepcific fragmentation of DNA heteroduplex molecules of two bacteriophage lambda mutants with endonuclease Si from Aspergillus oryzae.

Heteroduplex DNA molecules of two bacteriophage mutants (lambda b2 and lambda i434ct68) were obtained by the method of molecular hybridization. These heteroduplexes possessed two types of loops formed as a result of: a) deletion in one of the DNA strands; and b) substitution of a DNA fragment for nonhomological one. The digestion of heteroduplexes with single-stranded specific nuclease SI from Aspergillus oryzae produced two fragments at 37 degrees C and three ones at 55 degrees C. The separation of fragments and determination of their molecular weight were carried out by means of electrophoresis in agarose. The molecular weights both measured and preliminarily calculated proved to be close. One of the fragments was identificated by its biological activity in CaCl2-dependent infectious system with helperphage.

Aspergillus oryzae↗

Physical and kinetic characterization of the DNA packaging enzyme from bacteriophage lambda.

Terminases are enzymes common to complex double-stranded DNA viruses and are required for packaging of the viral genome into a preformed capsid. The overexpression of bacteriophage lambda-terminase in Escherichia coli has been previously reported (Chow, S., Daub, E., and Murialdo, H. (1987) Gene (Amst.) 60, 277-289), and we present here a purification scheme for the isolation of milligram quantities of protein which is homogenous ( > 97%) as determined by SDS-polyacrylamide gel electrophoresis. lambda-Terminase is composed of the gene products of Nu1 and A. Using N-terminal amino acid sequence analysis of the purified protein, we have determined a subunit stoichiometry of 2 gpNu1 polypeptides/gpA molecule in terminase holoenzyme. The circular dichroism spectrum for the purified holoenzyme has been obtained and is consistent with a protein complex composed primarily of alpha-helical structure. The endonucleolytic activity of the enzyme (the TER reaction) has been optimized with respect to pH, salt, and polyamine concentrations. Divalent metal ion is strictly required for the reaction and may be satisfied by either magnesium or manganese, but not by any of the other metals examined. E. coli integration host factor in amounts stoichiometric with the DNA substrate stimulates the TER reaction, but only when the enzyme is present in limiting amounts. Increasing the enzyme/DNA ratio attenuates the observed stimulation by integration host factor. A kinetic analysis of the TER reaction suggests that the assembly of multiple terminase promoters is required for efficient cleavage of viral DNA and that this reaction appears to be stoichiometric, rather than catalytic under the reaction conditions utilized. The implications of these results with respect to the packaging of viral DNA by terminase enzymes are discussed.

Bacterial Proteins↗