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PubMed1976

Restriction assay for integrative recombination of bacteriophage lambda DNA in vitro: requirement for closed circular DNA substrate.

A novel assay has been developed for in vitro genetic recombination of DNA. Substrate and product DNAs are cleaved with a restriction endonuclease and the resulting fragments are separated by electrophoresis in agarose gels. The substrate DNA has been chosen so that the recombination to be studied deletes a segment of DNA. The remaining DNA gives rise to a unique restriciton fragment, as does the DNA segment that has been removed. The method provides a convenient and physical, rather than genetic, assessment of the conversion of parental to recombinant DNA. This method has been applied to an in vitro system that carries out integrative recombination of bacteriophage lambda. We find that, different molecular forms of DNA tested, closed circular DNA is the only efficient substrate. Linear DNA and three kinds of circular DNA containing interruptions are at best very poor substrates. The implications of this surprising result are discussed. In addition, we show that the in vitro recombination system completes the breaking and rejoining steps of recombination. No stable DNA intermediates involving chiasmata or broken end structures are found.

Chromosome Mapping↗
PubMed2006

Display of aggregation-prone ligand binding domain of human PPAR gamma on surface of bacteriophage lambda.

AIM: To display the aggregation-prone ligand binding domain (LBD) of the human peroxisome proliferator-activated receptor gamma (PPARgamma) on the surface of bacteriophages to establish an easy screening assay for the identification of PPARgamma ligands. METHODS: Plasmids were constructed for the expression of the PPARgamma LBD as a fusion to the N-terminus of the g3p protein of filamentous phage or the C-terminus of the capsid protein D (pD) of phage lambda. The fusion proteins were expressed in E coli and solubility characteristics were compared. Polyclonal antibodies against the LBD as well as the pD protein were prepared for Western blot analysis and phage capture assay. RESULTS: The pD-LBD fusion protein was partially soluble, whereas the LBD-g3p fusion protein was detected only in the insoluble fraction. The pD-LBD fusion protein was efficiently incorporated in phage particles. Furthermore, the LBD was shown to be displayed on the surface of bacteriophage lambda. On average, the pD-LBD fusion protein accounted for 28% of the total pD protein in the lambda head capsid. CONCLUSION: The hydrophobic PPARgamma LBD was expressed as a soluble form of fusion protein in E coli and displayed on the surface of bacteriophage lambda when it was fused to the lambda pD protein. The lambda pD fusion system could be used for improving the solubility of proteins that tend to form inclusion bodies when expressed in E coli. The lambda phage particles displaying the LBD of PPARgamma may be of great value for the identification of novel PPARgamma ligands.

Bacteriophage lambda↗
PubMed1980

Extraordinary stability of the receptor of bacteriophage lambda.

The phage lambda receptor, previously shown to be a major outer membrane protein of Mr = 47 000 in maltose-grown Escherichia coli K12 (Braun, V. and Krieger-Brauer, H.J. (1977) Biochim. Biophys. Acta 469, 89--98), was found to be unusually resistant against the denaturants sodium dodecyl sulfate, urea and guanidine hydrochloride. A new isolation procedure for active phage lambda receptor, based on its resistance against pronase and hot sodium dodecyl sulfate, is described. The electrophoretic mobility of phage lambda receptor in sodium dodecyl sulfate polyacrylamide gels is abnormal even after heating in 2% sodium dodecyl sulfate, as is demonstrated by a Ferguson analysis. Phage lambda receptor in its active form sediments with a Svedberg constant of 8.4 +/- 0.4, which indicates an oligomeric structure of the phage lambda receptor.

Bacteriophage lambda↗
PubMed1994

Bipartite DNA recognition by the human Oct-2 POU domain: POUs-specific phosphate contacts are analogous to those of bacteriophage lambda repressor.

The POU motif defines a family of eukaryotic transcription factors broadly involved in tissue-specific gene expression and developmental regulation. The motif contains two DNA-binding domains: an N-terminal POU-specific domain (POUs) and C-terminal homeodomain (POUHD). Surprisingly, POUs has recently been found to be similar in structure to helix-turn-helix (HTH) domains of phage repressor and Cro proteins [Assa-Munt, N., Mortishire-Smith, R., Aurora, R., Herr, W., & Wright, P.E. (1993) Cell 73, 193-205; Dekker, N., Cox, M., Boelens, R., Verrijzer, C.P., van der Vliet, P.C., & Kaptein, R. (1993) Nature 362, 852-855]. Because POUHD and POUs are expected to bind DNA differently, we have used "methylphosphonate interference" to investigate the alignment of their HTH elements in a specific DNA complex. This neutral phosphate analogue, originally developed for applications in antisense drug design [Miller, P. S., & Ts'o, P. O. P. (1987) Anti-Cancer Drug Des. 2, 117-128], is shown to provide a sensitive probe for sites of backbone-specific protein-DNA interaction. Inferred POUs-phosphate contacts are in striking accord with cocrystal structures of bacteriophage repressor and Cro proteins. Alignment of POUHD and POUs in successive major grooves in each case predicts unique HTH-adenine contacts. This prediction is verified using DNA base analogues to effect interchange of AT functional groups by the method of 2'-deoxyinosine/5-methyl-2'-deoxycytosine substitution [McLaughlin, L. W., Benseler, F., Graeser, E., Piel, N., & Scholtissek, S. (1987) Biochemistry 26, 7238-7245]. Our results strongly support the hypothesis that the DNA-binding properties of POUs are analogous to those of bacteriophage lambda repressor.

5-Methylcytosine↗
PubMed2006

[Bacteriophage lambda DNA replication--new discoveries made using an old experimental model].

Bacteriophage lamda is a model in molecular biology studies since over fifty years. Nevertheless, studies of recent years (similarly to previous time periods) resulted in many new experimental results which not only expanded our knowledge on molecular mechanisms of functions of this virus, but also shed new light on general rules of the transduction and transfer of genetic information. In this review, we present recent achievements of studies on mechanisms of regulation of bacteriophage lamda DNA replication. Between others, these studies led to determination of the composition of lamda inherited replication complex, indication of the biological role of rapid degradation of free lamda omicron protein, description of the proposal of regulation of the switch from early (theta) to late (sigma) lamda DNA replication mode, elucidation of the mechanism of transcription regulation by a replication protein DnaA and demonstration of the activity of transcription stimulator by another replication regulator--the SeqA protein. These results may be important to better understand regulation of DNA replication of not only bacteriophage lamda but also other organisms.

Bacterial Proteins↗
PubMed1987

An open reading frame in the Escherichia coli bacteriophage lambda genome encodes a protein that functions in assembly of the long tail fibers of bacteriophage T4.

Assembly of the long tail fibers of the Escherichia coli bacteriophage T4 requires the catalytic action of two auxiliary proteins. It was found that a gene of the entirely unrelated phage lambda codes for a protein which can substitute for one of these T4 polypeptides, protein 38. The lambda gene was designated tfa (tail fiber assembly). Protein 38 consists of 183 residues, and the Tfa protein consists of 194 residues; the two polypeptides are about 40% homologous. Although the tfa gene is dispensable for the growth of phage lambda, these results indicate that it may have a function in lambda morphogenesis.

Amino Acid Sequence↗