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At least 811 records · Page 45Linked to original sources
PubMed1991

Role of the Escherichia coli grpE heat shock protein in the initiation of bacteriophage lambda DNA replication.

Initiation of replication of lambda DNA requires assembly of the proper nucleoprotein complex consisting of the lambda origin of replication-lambda O-lambda P-dnaB proteins. The dnaJ, dnaK and grpE heat shock proteins destabilize the lambda P-dnaB interaction in this complex permitting dnaB helicase to unwind lambda DNA near ori lambda sequence. First step of this disassembling reaction is the binding of dnaK protein to lambda P protein. In this report we examined the influence of dnaJ and grpE proteins on stability of the lambda P-dnaK complex. Our results show that grpE alone dissociates this complex, but both grpE and dnaJ together do not. These results suggest that, in the presence of grpE protein, dnaK protein has a higher affinity for lambda P protein complexed with dnaJ protein than in the situation where grpE protein is not used.

Bacterial Proteins↗
PubMed2000

ATPase center of bacteriophage lambda terminase involved in post-cleavage stages of DNA packaging: identification of ATP-interactive amino acids.

Terminase is the enzyme that mediates lambda DNA packaging into the viral prohead. The large subunit of terminase, gpA (641 amino acid residues), has a high-affinity ATPase activity (K(m)=5 microM). To directly identify gpA's ATP-interacting amino acids, holoterminase bearing a His(6)-tag at the C terminus of gpA was UV-crosslinked with 8-N(3)-[alpha-(32)P]ATP. Tryptic peptides from the photolabeled terminase were purified by affinity chromatography and reverse-phase HPLC. Two labeled peptides of gpA were identified. Amino acid sequencing failed to show the tyrosine residue of the first peptide, E(43)SAY(46)QEGR(50), or the lysine of the second peptide, V(80)GYSK(84)MLL(87), indicating that Y(46) and K(84) were the 8-N(3)-ATP-modified amino acids. To investigate their roles in lambda DNA packaging, Y(46) was changed to E, A, and F, and K(84) was changed to E and A. Purified His(6)-tagged terminases with changes at residues 46 and 84 lacked the gpA high-affinity ATPase activity, though the cos cleavage and cohesive end separation activities were near to those of the wild-type enzyme. In virion assembly reactions using virion DNA as a packaging substrate, the mutant terminases showed severe defects. In summary, the results indicate that Y(46) and K(84) are part of the high-affinity ATPase center of gpA, and show that this ATPase activity is involved in the post-cos cleavage stages of lambda DNA packaging.

Adenosine Triphosphatases↗
PubMed1984

Fluorescence polarization study of tertiary structure of DNA within bacteriophage lambda.

The fluorescence polarization of acridine orange-stained, oriented lambda phages was measured. The parameters of DNA packing within the phage head cos2 theta and cos4 theta were calculated (theta, angle between the direction of a small segment of DNA and the phage axis). It is shown that simple models of lambda phage DNA tertiary structure are not consistent with calculated values. A new model is proposed.

Acridine Orange↗
PubMed1983

Retroregulation: control of integrase expression by the b2 region of bacteriophages lambda and 434.

Genetic fusions constructed by a combination of in vivo and in vitro techniques have been used to investigate the cis-regulatory role of a 250-base-pair segment of b2 DNA in the expression of int transcripts originating from three different promoters PI, PL, and PTRP. It has been established that (a) the PI and PTRP transcripts, which produce functional int protein, are efficiently terminated by the b2 segment, (b) in the same test system, the PL transcript, which does not result in functional integrase, is not terminated by the b2 segment, (c) this b2 (sib) effect does not depend on genes lying between int and N on the phage genome, (d) the sib effect is also observed with the b2 DNA of phage 434, which resembles that of lambda between -197 and att but diverges radically from it to the left of base -197.

Bacteriophage lambda↗
PubMed1982

The rex region of bacteriophage lambda: two genes under three-way control.

The nucleotide sequence of the phage lambda rex region consists of 1428 bp and codes for two genes, rexA and rexB. Hence the complete lambda immunity region codes for four genes and covers 2664 bp of sequence unique to lambda, as defined by the left and right boundaries of the imm434 region. Coordinate expression of both rexA and rexB, which are co-regulated with the cI repressor gene from promoters p rm and p re is responsible for the Rex phenotype, i.e. exclusion of a wide variety of superinfecting phage such as T4rII. The position of a third promoter, p lit, which overlaps the carboxy-terminal end of the rexA coding region, permits expression of rexB without rexA, from the resulting 470 nucleotide lit RNA. The lit transcript, therefore, must act as messenger for rexB in the noncoordinate expression of the rex genes that occurs late in lambda lytic infection. The coordinate and noncoordinate expression of rexB and rexA suggests a dual role for the very hydrophobic rexB protein. Studies of lambda early and late DNA replication implicate rexB as having auxiliary functions in both lysogenisation and lytic infection.

Bacteriophage lambda↗
PubMed1995

Optical mapping of lambda bacteriophage clones using restriction endonucleases.

Optical mapping is an emerging single molecule approach for the rapid generation of ordered restriction maps, using fluorescence microscopy. We have improved the size resolution of optical mapping by imaging individual DNA molecules elongated and fixed onto derivatized glass surfaces. Averaged fluorescence intensity and apparent length measurements accurately determined the mass of restriction fragments 800 basepairs long. We have used optical mapping to create ordered restriction maps for lambda clones derived from the mouse pygmy locus.

Animals↗
PubMed1980

Recombination between bacteriophage lambda and plasmid pBR322 in Escherichia coli.

Recombinant lambda phages were isolated that resulted from recombination between the lambda genome and plasmid pBR322 in Escherichia coli, even though these deoxyribonucleic acids (DNAs) did not share extensive regions of homology. The characterization of these recombinant DNAs by heteroduplex analysis and restriction endonucleases is described. All but one of the recombinants appeared to have resulted from reciprocal recombination between a site on lambda DNA and a site on the plasmid. In general, there were two classes of recombinants. One class appeared to have resulted from recombination at the phage attachment site that probably resulted from lambda integration into secondary attachment sites on the plasmid. Seven different secondary attachment sites on pBR322 were found. The other class resulted from plasmid integration at other sites that were widely scattered on the lambda genome. For this second class of recombinants, more than one site on the plasmid could recombine with lambda DNA. Thus, the recombination did not appear to be site specific with respect to lambda or the plasmid. Possible mechanisms for generating these recombinants are discussed.

Bacteriophage lambda↗
PubMed1999

Translational frameshift sites within bacteriophage lambda genes rexA and cI.

Phage lambda's cI-rexA-rexB operon displays an intriguing example of regulation by an unexplained mechanism of polarity. We have identified three potential -1 translational frameshift sites and present a model for translational frameshift suppression by lambda's CI repressor as a mechanism of regulating operon polarity, implying an additional role for CI self-regulation.

Bacteriophage lambda↗
PubMed1992

Inhibition of bacteriophage lambda development by the klaA gene of broad-host-range plasmid RK2.

The kil-kor regulon of broad-host-range plasmid RK2 is an unusual array of eight co-regulated operons that express at least 21 genes, including the plasmid replication initiator gene. Some of the operons were first identified as kil loci because uncontrolled expression in the absence of certain kor regulatory genes leads to death of the host cells. The functions of kilA, C and E are unknown, although co-regulation with the replication initiator gene suggests that they may have importance in the maintenance or host range of the plasmid. Here we report studies on the function of klaA, the first of three host-lethal genes in the kilA operon. We found that lambda pklaA-1, a lambda phage containing the klaA gene, is unable to form plaques unless the host expresses the KorA and KorB repressors needed to regulate transcription from the klaA promoter. The failure to form plaques depends on the klaA gene product and results from the inability of infected cells to produce viable phage particles. Transcription of early, delayed early and late genes or processing of lambda DNA are not affected by klaA overexpression, while cell lysis, lambda DNA replication and production of functional phage heads are reduced. However, the failure to produce viable phage is best explained by the inability to synthesize lambda tails. The finding that klaA strongly inhibits a specific morphogenetic step in the assembly of lambda phage particles has significance with respect to the function of klaA on plasmid RK2.

Bacterial Proteins↗
PubMed1998

Embryonic stem cell gene targeting using bacteriophage lambda vectors generated by phage-plasmid recombination.

Targeted mutagenesis is an extremely useful experimental approach in molecular medicine, allowing the generation of specialized animals that are mutant for any gene of interest. Currently the rate determining step in any gene targeting experiment is construction of the targeting vector (TV). In order to streamline gene targeting methods and avoid problems encountered with plasmid TVs, we describe the direct application of lambda phage in targeted mutagenesis. The recombination-proficient phage vector lambda2TK permits generation of TVs by conventional restriction-ligation or recombination-mediated methods. The resulting lambdaTV DNA can then be cleaved with restriction endonucleases to release the bacteriophage arms and can subsequently be electroporated directly into ES cells to yield gene targets. We demonstrate that in vivo phage-plasmid recombination can be used to introduce neo and lacZ - neo mutations into precise positions within a lambda2TK subclone via double crossover recombination. We describe two methods for eliminating single crossover recombinants, spi selection and size restriction, both of which result in phage TVs bearing double crossover insertions. Thus TVs can be easily and quickly generated in bacteriophage without plasmid subcloning and with little genomic sequence or restriction site information.

Animals↗
PubMed1982

Molecular cloning of the spo0B sporulation locus in bacteriophage lambda.

The stage 0 sporulation locus spo0B has been mapped by transformation between the pheA and spoIVF loci. Analysis of the behavior of alleles of the spo0B locus in trpE26 merodiploid strains indicates that all of the known alleles of this locus comprise a single complementation group. The spoIVF88 mutation was found to reside in a separate complementation group. The chromosomal region surrounding and including the spo0B locus was cloned in the lambda vector Charon 4A. Extensive restriction endonuclease analyses of the inserts in these phage revealed that an EcoRI fragment of DNA of 2.3 kilobases had transforming activity for spo0B mutations. Examination of the physical and genetic maps of the locus suggested that the entire spo0B locus is contained within this fragment. Subcloning of restriction endonuclease fragments of the lambda inserts and transformation analyses allowed assignment of surrounding genetic loci to specific DNA fragments.

Alleles↗
PubMed1984

High-level expression of human interferon gamma in Escherichia coli under control of the pL promoter of bacteriophage lambda.

Several recombinant plasmids have been constructed which direct high-level synthesis of mature human interferon gamma (IFN-gamma) in Escherichia coli using the inducible leftward promoter pL of phage lambda followed by a translational initiator region derived either from the phage MS2 replicase gene or the E. coli tryptophan attenuator region. Under these conditions, IFN levels of up to 25% of the total cellular protein can be achieved. The highest levels were obtained when a terminator of transcription was cloned downstream from the IFN-gamma sequence. IFN-gamma was almost entirely found in the initial pellet fraction and not in soluble extracts. Co-induction of the lysis genes derived from phage MS2 or from phage lambda, inserted downstream from the IFN-gamma sequence, did not enhance the biological activity present in the supernatant fraction.

Bacteriophage lambda↗
PubMed1980

On inactivation of bacteriophage lambda by hydroxylamine.

Hydroxylamine is a mutagen which is much more active on single-stranded DNA than on double-stranded DNA. It is shown here that the cohesive ends of lambda DNA, with 10 cytidine residues, constitute a hydroxylamine target roughly equal in magnitude to the entire duplex part of the molecule, which contains ca. 25 000 cytidine residues.

Bacteriophage lambda↗