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At least 595 records · Page 33Linked to original sources
PubMed2000

Antitermination in bacteriophage lambda. The structure of the N36 peptide-boxB RNA complex.

The solution structure of a 15-mer nutRboxB RNA hairpin complexed with the 36-mer N-terminal peptide of the N protein (N36) from bacteriophage lambda was determined by 2D and 3D homonuclear and heteronuclear magnetic resonance spectroscopy. These 36 amino acids include the arginine-rich motif of the N protein involved in transcriptional antitermination of phage lambda. Upon complex formation with boxB RNA, the synthetic N36 peptide binds tightly to the major groove of the boxB hairpin through hydrophobic and electrostatic interactions forming a bent alpha helix. Four nucleotides of the GAAAA pentaloop of the boxB RNA adopt a GNRA-like tetraloop fold in the complex. The formation of a GAAA tetraloop involves a loop-closing sheared base pair (G6-A10), base stacking of three adenines (A7, A8, and A10), and extrusion of one nucleotide (A9) from the loop, as observed previously for the complex of N(1-22) peptide and the nutLboxB RNA [Legault, P., Li, J., Mogridge, J., Kay, L.E. & Greenblatt, J. (1998) Cell 93, 289-299]. Stacking of the bases is extended by the indole-ring of Trp18 which also forms hydrophobic contacts to the side-chains of Leu24, Leu25, and Val26. Based on the structure of the complex, three mutant peptides were synthesized and investigated by CD and NMR spectroscopy in order to determine the role of particular residues for complex formation. These studies revealed very distinct amino-acid requirements at positions 3, 4, and 8, while replacement of Trp18 with tyrosine did not result in any gross structural changes.

Amino Acid Sequence↗
PubMed1975

Role of the receptor for bacteriophage lambda in the functioning of the maltose chemoreceptor of Escherichia coli.

Chemotaxis towards maltose is specifically defective in many strains of Escherichia coli carrying mutations affecting lamB, the gene coding for the outer membrane receptor for bacteriophage lambda. However, with one exception, the most extreme effect of lamB mutants on the maltose response as determined in the capillary assay is a shift to higher sugar concentrations and a reduction in the number of bacteria accumulated to about 25% of the wild-type level. The severity of the taxis defect is strongly correlated with reduced ability of the cells to take up the maltose present at 1 and 10 muM. Evidence presented here and in the accompanying paper indicates that the lambda receptor is involved in the transport of maltose at these concentrations. The effects of lamB mutations on maltose taxis can be explained by postulating that the high-affinity maltose transport system in which the lambda receptor participates transfers maltose from the surrounding medium across the outer membrane and into the periplasmic space. If the maltose chemoreceptor detects sugar present in the periplasmic space, and not molecules external to the outer membrane, then defective transport of low concentrations of maltose into the periplasm would result in the observed apparent reduction in the sensitivity of the maltose receptor. Thus, the lambda receptor protein would participate in maltose chemorecepton only indirectly through its role in maltose transport.

Binding Sites↗
PubMed1978

Identification of the Escherichia coli cell division gene sep and organization of the cell division-cell envelope genes in the sep-mur-ftsA-envA cluster as determined with specialized transducing lambda bacteriophages.

From a lysogen with lambda integrated in the leu operon, specialized transducing phages that carry the cell division, murein biosynthesis, and envelope permeability genes located about 0.5 min to the right of leu were isolated. These phages were used to identify the previously undiscovered cell division gene sep. A genetic map proves that sep is located in the sequence leuA sep murE murF murC ddl ftsA envA. A physical map of this region was prepared by heteroduplex analysis of the phage DNAs. Overlapping segments of host DNA extended rightward for as much as 26.4 kilobase pairs from the prophage insertion point (thought to be in leuA) to include all the genes through envA.

Cell Division↗
PubMed1991

A Salmonella typhimurium virulence protein is similar to a Yersinia enterocolitica invasion protein and a bacteriophage lambda outer membrane protein.

The phoP-phoQ-regulated pagC locus is essential for full virulence and survival within macrophages of Salmonella typhimurium. The protein product, DNA sequence, and transcript of pagC were determined. The pagC locus encodes a single 188-amino-acid membrane protein that is similar to the ail-encoded eucaryotic cell invasion protein of Yersinia enterocolitica and the lom-encoded protein of bacteriophage lambda. The similarity of PagC and Ail to Lom leads us to hypothesize that Lom is a virulence protein and that bacteriophage gene transfer and lysogeny could have led to the development of proteins essential to survival within macrophages and eucaryotic cell invasion.

Adhesins, Bacterial↗
PubMed1978

Mechanism of action of the cro protein of bacteriophage lambda.

The mechanism of action of cro protein was probed by measuring its ability to protect DNA against methylation by dimethyl sulfate and its effect on transcription in vitro. The cro protein binds to the same three sites in the right operator (OR) of bacteriophage lambda DNA as does the lambda repressor. Dimethyl sulfate protection experiments reveal major groove contacts for both proteins, and cro protein protects from methylation a subset of those purines protected by lambda repressor. These experiments also show that the relative affinity of these two proteins for the three operator sites is different: whereas lambda repressor binds with an affinity OR1 greater than OR2 greater than OR3, the order for cro protein is OR3 greater than (OR1, OR2). As predicted by these results, cro protein, like the lambda repressor, blocks in vitro transcription of cI and cro from the two divergent promoters that overlap OR. Also as predicted, transcription of cI is turned off at lower cro protein concentrations than is transcription of cro, whereas the opposite order of repression is obtained with lambda repressor. These results describe the molecular mechanism of cro protein action and show that two regulatory proteins can bind to the same three adjacent sites in DNA with markedly different consequences.

Base Sequence↗
PubMed1977

Cloning specific segments of the mammalian genome: bacteriophage lambda containing mouse globin and surrounding gene sequences.

We have developed a general approach to the cloning of specific segments of the mammalian genome that involves a two-step purification of EcoRI fragments of mammalian DNA and their in vitro insertion into a suitably constructed EK2 derivative of bacteriophage lambda. The combination of fragment purification, exclusion of parental-type recombinants, and simple phage screening techniques permits the isolation of virtually any gene segment for which there is an identifying hybridization probe. We illustrate the approach by describing the cloning of an approximately 7000-base-long segment of mouse DNA containing globin and surrounding gene sequences.

Animals↗
PubMed1973

Nucleotide sequence analysis with polynucleotide kinase and nucleotide "mapping" methods. 5'-Terminal sequences of deoxyribonucleic acid from bacteriophages lambda and 424.

The polynucleotide kinase reaction was used in analyses of complex mixtures of oligodeoxynucleotides which were fractionated by various two-dimensional nucleotide ;mapping' procedures. Parallel ionophoretic analyses on DEAE-cellulose paper, pH2, and AE-cellulose paper, pH3.5, of venom phosphodiesterase partial digests of 5'-terminally labelled oligonucleotides enabled the sequence of the nucleotides to be deduced uniquely. A ;diagonal ionophoresis' method has been used with mixtures of nucleotides. Application of these methods to 5'-terminally labelled DNA from bacteriophage lambda gave the terminal sequences pA-G-G-T-C-G and pG-G-G-C-G. Identical 5'-terminal sequences were found with DNA from bacteriophage 424.

Adenine Nucleotides↗
PubMed1976

Second-site mutations in capR (lon) strains of Escherichia coli K-12 that prevent radiation sensitivity and allow bacteriophage lambda to lysogenize.

capR (lon) mutants of Escherichia coli K-12 are mucoid and sensitive to ultraviolet (UV) and X-ray radiation as well as to nitrofurantoin. The mutants form filaments after exposure to these agents. capR mutants are also conditionally lethal since they die when plated on complex medium even without UV treatment; this phenomenon is designated "complex medium-induced killing". Furthermore, capR mutants are poorly lysogenized by bacteriophage lambda. Second-site revertants were isolated by plating on media containing nitrofurantoin. All 17 of the independent revertants studied were still mucoid but resistant to UV radiation. Sixteen of the 17 revertants contained a mutation, sulA, that cotransduced with pyrD (21 min). A second locus, sulB, was also found that cotransduced with leu (2 min). Studies with partial diploids (F'pyrD+ sulA+/pyrD36 sulA17 capR9 (lon) demonstrated that sulA+ is dominant to sulA; thus the indicated partial diploid is UV sensitive, whereas the haploid parent is UV resistant. Furthermore, two other phenotypic traits of capR (lon) mutants were reversed by the sul mutation:complex medium-induced killing and the inability of lambda phage to efficiently lysogenize capR strains. On the basis of these and other results, the following model is suggested to explain capR (lon) and sul gene interactions. capR (lon) is a regulator gene for the structural genes sulA+ and sulB+. Depression of both sul operons results in UV sensitivity and decreased ability of lambda to lysogenize, whereas inactivation of either sul+ protein by mutation to sul prevents these phenomena.

Chromosome Mapping↗
PubMed1976

Suppression of polarity of insertion mutations in the gal operon and N mutations in bacteriophage lambda.

Bacterial mutations (psuA and psu) known for their ability to suppress the polarity on nonsense mutations are shown to suppress the polarity of certain insertion mutations in the gal operon. The short insertion, IS1 (800 nucleotide pairs), is about 15 to 50% suppressed, whereas longer insertions, IS2 (1,400 nucleotide pairs), and IS3 (1,200 nucleotide pairs), are not. Some of the polarity suppressor mutations (psu-1, psu-2, and psu-3) are at least partially permissive for N-gene mutations (N7 and N53) of bacteriophage lambda, suggesting a relationship between natural and mutational polar signals. That this relationship may be complex is indicated by the fact that other suppressor mutations, effective in suppressing nonsense or insertion polarity, fail entirely to permit the growth of lambda N mutants.

Cell-Free System↗
PubMed1983

p lambda CM system: observations on the roles of transposable elements in formation and breakdown of plasmids derived from bacteriophage lambda replicons.

Transduction with phage derived from a 2-year-old lysate of lambda cam105 (lambda::Tn9) gave rise to chloramphenicol-resistant (Cm(r)) transductants harboring a plasmid (plambdaCM1) formed from lambda cam105 by a Tn9-mediated adjacent deletion to position 36.07 kilobases in the N cistron of lambda. The plambdaCM element can replicate as a plasmid, insert into the bacterial genome, or reproduce lytically as a phage on cells that provide N function. The feasibility of obtaining high titers in encapsidated form and the ease of synchronous introduction into and recovery from bacterial populations make plambdaCM very suitable for quantitative studies of recombination involving transposable elements. Replicon fusions between plambdaCM1 and RSF1596 (pMB8::Tn3Delta596) occur by duplication of either IS1 (at low rate in the absence of TnpA activity) or Tn3Delta596 (in the presence of TnpA activity). At 24 or 32 degrees C, the rate of increase of TnpA-mediated fusions per plambdaCM is about 2% per cell doubling. RSF103 contains the deleted Tn1DeltaAp (which lacks intact beta-lactamase and TnpR resolvase coding sequences) adjacent to a streptomycin resistance (Sm(r)) determinant. We observed that Tn1DeltaAp mediates insertions of external RSF103 sequences into the R388 plasmid. R388::Tn1DeltaAp plasmids show transposition immunity in cells lacking TnpR activity. Using the plambdaCM system, we isolated adjacent transpositions of the RSF103 Sm(r) determinant. The resulting plambdaCM-Sm cosmids contain Sm(r) genetic material flanked by direct repeats of Tn1DeltaAp, and all are deleted for some RSF103 or plambdaCM sequences. The plambdaCM-Sm constructs will fuse into R388 by duplication of a single Tn1DeltaAp element. In the presence of tnpR(+) (but not tnpR) Tn1 or Tn3 elements, all Tn1DeltaAp-mediated complex replicons break down completely and rapidly to simple Tn1DeltaAp inserts. The equilibrium for resolution is at least 10(5):1, and resolution is more than 90% complete after 40 min of exposure to a tnpR(+) cytoplasm. In the absence of TnpR, Rec, and Red activities, Tn1DeltaAp-mediated complex replicons yield simple Tn1DeltaAp inserts at a lower rate. The presence of intact RSF103 replication determinants between direct Tn1DeltaAp repeats appears to accelerate this precise TnpR- and Rec-independent breakdown.

Bacteriophage lambda↗