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PubMed2006

The structural basis of cooperative regulation at an alternate genetic switch.

Bacteriophage lambda is a paradigm for understanding the role of cooperativity in gene regulation. Comparison of the regulatory regions of lambda and the unrelated temperate bacteriophage 186 provides insight into alternate ways to assemble functional genetic switches. The structure of the C-terminal domain of the 186 repressor, determined at 2.7 A resolution, reveals an unusual heptamer of dimers, consistent with presented genetic studies. In addition, the structure of a cooperativity mutant of the full-length 186 repressor, identified by genetic screens, was solved to 1.95 A resolution. These structures provide a molecular basis for understanding lysogenic regulation in 186. Whereas the overall fold of the 186 and lambda repressor monomers is remarkably similar, the way the two repressors cooperatively assemble is quite different and explains in part the differences in their regulatory activity.

Amino Acid Substitution↗

PubMed1987

The mapping of chromosomes in Saccharomyces cerevisiae. I. A cosmid vector designed to establish, by cloning into cdc-mutants, numerous start loci for chromosome walking in the yeast genome.

A series of vectors for cosmid cloning in yeast has been derived from cosmid pHC79. Vectors pMT4 through pMT6 contain two tandemly arranged cohesive end sites (cos) from the genome of bacteriophage lambda. Their design allows the rapid and simple preparation of cosmid arms by linearizing a vector at the unique PvuII-restriction site located between the two cos-sequences and then cutting the linearized molecule at one of its unique cloning sites for BamHI, ClaI, PvuI, SalI or ScaI. Cosmids generated with arms from the most advanced vector, pMT6, carry the origin of replication (ori) and the ApR gene from pBR322 and the TRP1/ARS1 and URA1 genes from Saccharomyces cerevisiae. A yeast genomic DNA library was established by packaging in vitro, into bacteriophage lambda preheads, of partially restricted yeast DNA fragments ligated to cosmid arms of vector pMT6. About 80% of the clones thus obtained comprise inserts of contiguous genomic DNA over 30 kb in length. Unique DNA probes for the yeast genes CDC10, CDC39, HIS4, LEU2, and PGK1 have successfully been applied when testing for completeness of this library by isolating a series of overlapping cosmid clones that carry the respective genes. The library will thus be useful for the selection of cosmid clones which carry CDC genes from yeast by complementing first, with the vectorial yeast gene URA1, the pyrimidine auxotrophy of most cdc-strains and then, with the respective CDC wild-type genes, of the temperature-sensitive mutant alleles. Most CDC clones thus obtained will provide unique DNA probes which serve as randomly distributed start sequences within the yeast genome for overlap hybridization screening in chromosome mapping studies.

Cell Cycle↗

PubMedDate not supplied

[Repair of the interstrand cross links in DNA: role of restriction system "K"].

The interstrand cross links formed in the DNA after psoralenplus light treatment of bacteria E. coli K12 and bacteriophage lambda are repaired by the restriction and modification system "K." Strain E. coli C, which does not have the restriction and modification system is 2 times more sensitive to psoralen plus light treatment, than the wild type E. coli K12. But these strains are equally sensitive to inactivation by UV-light (254 nm). By studing the sedimentation of bacterial DNA in alkaline sucrose it was found that cells of E. coli C are deficient in filling of gaps, appearing in parental DNA after enzymatic excision of the "arms" of cross links. In experiments with bacteriophage lambda (W-reactivation, prophage-reactivation), treated by psoralen plus light, it was shown that E. coli C and mutants of E. coli K12 rk-mk+/- are deficient in non-recombination lexA-dependent repair of cross links. The function of restriction and recognition of restriction and modification system are essential for repair of cross links. It is suggested that the restriction and modification system "K" acts either as a regulatory protein or as a protector of single-stranded regions of DNA against nuclease attack.

Coliphages↗

PubMed1990

Specificity determinants in the attachment sites of bacteriophages HK022 and lambda.

The Int proteins of bacteriophages HK022 and lambda promote recombination between phage and bacterial attachment sites. Although the proteins and attachment sites of the two phages are similar, neither protein promotes efficient recombination between the pair of attachment sites used by the other phage. To analyze this difference in specificity, we constructed and characterized chimeric attachment sites in which segments of one site were replaced with corresponding segments of the other. Most such chimeras recombined with appropriate partner sites in vivo and in vitro, and their differential responses to the Int proteins of the two phages allowed us to locate determinants of the specificity difference in the bacterial attachment sites and a central segment of the phage attachment sites. The location of these determinants encompasses three of the four core-type binding sites for lambda Int: C, B, and most importantly, B'. The regions corresponding to the C' core binding site and the arm-type binding sites of lambda Int play no role in the specificity difference and, indeed, are well conserved in the two phages. We found, unexpectedly, that the effect of replacement of an Int-binding region on the recombinational potency of one chimeric site was reversed by a change of partner. This novel context effect suggests that postsynaptic interactions affect the specificity of recognition of attachment sites by Int.

Bacteriophage lambda↗

PubMed1985

Recognition sequences of restriction endonucleases and methylases--a review.

The properties and sources of all known endonucleases and methylases acting site-specifically on DNA are listed. The enzymes are crossindexed (Table I), classified according to homologies within their recognition sequences (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the DNA of the bacteriophages lambda, phi X174 and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328 and the microorganisms from which they originate. Other tabulated properties of the restriction endonucleases include relaxed specificities (Table III), the structure of the restriction fragment ends (Table IV), and the sensitivity to different kinds of DNA methylation (Table V). Table VI classifies the methylases according to the nature of the methylated base(s) within their recognition sequences. This table also comprises those restriction endonucleases, which are known to be inhibited by the modified nucleotides. Furthermore, this review includes a restriction map of bacteriophage lambda DNA based on sequence data. Table VII lists the exact nucleotide positions of the cleavage sites, the length of the generated fragments ordered according to size, and the effects of the Escherichia coli dam- and dcmI-coded methylases M X Eco dam and M X Eco dcmI on the particular recognition sites.

Base Sequence↗

PubMed2003

Immunity profiles of wild-type and recombinant shiga-like toxin-encoding bacteriophages and characterization of novel double lysogens.

The pathogenicity of Shiga-like toxin (stx)-producing Escherichia coli (STEC), notably serotype O157, the causative agent of hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura, is based partly on the presence of genes (stx(1) and/or stx(2)) that are known to be carried on temperate lambdoid bacteriophages. Stx phages were isolated from different STEC strains and found to have genome sizes in the range of 48 to 62 kb and to carry either stx(1) or stx(2) genes. Restriction fragment length polymorphism patterns and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles were relatively uninformative, but the phages could be differentiated according to their immunity profiles. Furthermore, these were sufficiently sensitive to enable the identification and differentiation of two different phages, both carrying the genes for Stx2 and originating from the same STEC host strain. The immunity profiles of the different Stx phages did not conform to the model established for bacteriophage lambda, in that the pattern of individual Stx phage infection of various lysogens was neither expected nor predicted. Unexpected differences were also observed among Stx phages in their relative lytic productivity within a single host. Two antibiotic resistance markers were used to tag a recombinant phage in which the stx genes were inactivated, enabling the first reported observation of the simultaneous infection of a single host with two genetically identical Stx phages. The data demonstrate that, although Stx phages are members of the lambdoid family, their replication and infection control strategies are not necessarily identical to the archetypical bacteriophage lambda, and this could be responsible for the widespread occurrence of stx genes across a diverse range of E. coli serotypes.

Coliphages↗

PubMed1978

Functional organization of the kdp genes of Escherichia coli K-12.

The kdp genes code for a high-affinity and repressible K+ transport system. The regulation and organization of the kdp genes were analyzed by studies of constitutive mutants and of strains in which bacteriophage lambda is integrated into the kdp genes. The polar effects of lambda integration demonstrate that three of the kdp genes form an operon, kdpABC, read from A to C. The kdpD gene is a separate transcription unit and is the site of mutations making expression of the kdp genes partially constitutive. The constitutive mutants are dominant to kdpD+ in diploids. These findings, the fact that kdpD mutations identified previously are Kdp-, and the existence of intracistronic complementation between some kdpD mutations indicate that the kdpD gene product is an oligomeric positive regulator of the kdp genes. Deletions extending clockwise from kdp as far as the gltA locus were isolated from strains with bacteriophage lambda integrated into kdpD. Plaque-forming transducing lambda phages carrying the kdpABC operon were isolated.

Biological Transport, Active↗

PubMed1982

Expression of the replication region of phage lambda DNA cloned into pBR322 in E. coli minicells.

Replication region of bacteriophage lambda DNA was cloned into pBR322 plasmid by the use of two restriction enzymes--PstI and HindIII. The restriction analysis of four obtained plasmids revealed that lambda DNA was cloned in both orientations. Recombinant plasmids were transferred to the minicell-producing strain of E. coli and synthesis of the plasmid-mediated proteins was analysed by polyacrylamide-gel electrophoresis. All four recombinant plasmids produced lambda DNA replication proteins pO and pP as well as some proteins specific for pBR322. The orientation of cloned fragment did not affect the synthesis of lambda DNA replication proteins.

Bacterial Proteins↗

PubMed2002

Catalytic efficiency and phenotype of HIV-1 proteases encoding single critical resistance substitutions.

We have shown that a bacteriophage lambda genetic screen system may be useful in predicting the activity and phenotype of HIV-1 protease in the course of viral infection and antiretroviral therapy. This simple and rapid genetic screening system has been used here to characterize HIV-1 proteases encoding single primary resistance substitutions. Except for proteases with amino acid changes at positions 46 and 84, proteases containing single-resistance substitutions displayed a lower catalytic efficiency than the WT enzyme. Single mutants could be identified by their efficiency, demonstrating that modest differences in protease activity can be monitored with this simple assay. Overall, drug susceptibility could be reduced by introduction of single mutations. However, high-level protease inhibitor (PI) resistance was only achieved by multiple mutated proteases. The small but reproducible increase in resistance displayed by single mutants also demonstrated the ability of this genetic screen system for detecting minor reductions in drug susceptibility. These results show that the bacteriophage lambda genetic screen system used here is a useful tool in the analysis of specific contribution of mutations in the HIV protease-coding region or in specific cleavage sites that affect the process of PI resistance.

Amino Acid Substitution↗

PubMed1982

A rho-independent termination caused by the cloned inverted nut L site of phage lambda.

For studying the termination activity of inverted nutL site of bacteriophage lambda, we have constructed a plasmid carrying the nutL fragment oriented reversely with respect to cloned lambda promoter p'R-directed transcription. The results of in vitro transcription on this plasmid template and S1 mapping assay reveal that the termination of p'R-promoted transcription at inverted nutL site is a rho-independent event. This nutL terminator shares several features with the other known sites of transcription termination, including (i) a uridine-rich 3' terminal RNA sequences,--UUAAUUUUU-OH, (ii) a GC-rich region in the DNA immediately preceding the site of termination, (iii) a region of dyad symmetry in the DNA which, in transcript, is capable of forming a stable hairpin containing four GC base pairs and one AU base pair in its stem.

Bacteriophage lambda↗

PubMed1993

Identification and characterization of the Escherichia coli RecT protein, a protein encoded by the recE region that promotes renaturation of homologous single-stranded DNA.

Recombination of plasmid DNAs and recombination of bacteriophage lambda red mutants in recB recC sbcA Escherichia coli mutants, in which the recE region is expressed, do not require recA. The recE gene is known to encode exonuclease VIII (exoVIII), which is an ATP-independent exonuclease involved in the RecE pathway of recombination. A 33,000-molecular-weight (MW) protein was observed to be coexpressed with both exoVIII and a truncated version of exoVIII, pRac3 exo, when they were overproduced under the control of strong promoters. We have purified this 33,000-MW protein (p33) and demonstrated by protein sequence analysis that it is encoded by the same coding sequence that encodes the C-terminal 33,000-MW portion of exoVIII. p33 is expressed independently of exoVIII but is probably translated from the same mRNA. p33 was found to bind to single-stranded DNA and also to promote the renaturation of complementary single-stranded DNA. It appears that p33 is functionally analogous to the bacteriophage lambda beta protein, which may explain why RecE pathway recombination does not require recA.

Amino Acid Sequence↗

PubMed2005

Genotoxicity of the Yamuna River water at Okhla (Delhi), India.

Water samples from the Yamuna River at Okhla (Delhi), India, were concentrated using XAD resins (XAD-4 and XAD-8) and liquid-liquid extraction procedures. Gas chromatographic analysis of liquid-liquid extracted water samples revealed the presence of the pesticides DDT, BHC, dieldrin, endosulfan, aldrin, 2,4-D, dimethoate, methyl parathion, and malathion at concentrations of 14, 25, 2.1, 114, 0.9, 0.6, 0.9, 1.7, and 1.9 ng/L, respectively. The genotoxicity of the extracted water samples was evaluated with the Ames Salmonella/mammalian microsome test, DNA repair-defective mutants, and bacteriophage lambda systems. The results of the Salmonella test demonstrated that the XAD-concentrated water samples had maximum mutagenicity with the TA98 strain both with and without metabolic activation. However, the liquid-liquid-extracted water samples were also found to be mutagenic with one or more of the Ames tester strains, but to a lesser extent as compared with XAD extracts. The damage brought about in the DNA repair-defective mutants in the presence of XAD-concentrated water samples was found to be markedly high as compared with that liquid-liquid-extracted water samples at the dose level of 20 microl/mL culture. All mutants invariably exhibited significant declines in their colony-forming units as compared with their isogenic wild-type counterparts. Survival decreased by 86.3 and 75.5% in the polA- strain after 6 h of treatment with XAD-concentrated and liquid-liquid-extracted water samples, respectively. A significant decrease was also observed in the survival of bacteriophage lambda when treated with the test samples. Mutagenic responses of the liquid-liquid-extracted water samples may not necessarily reflect the mutagenicity of existing pesticides in the test water, because some other organic pollutants might accompany the pesticides in the extract.

Animals↗

PubMed2003

Genetics of cosQ, the DNA-packaging termination site of phage lambda: local suppressors and methylation effects.

The cos site of the bacteriophage lambda chromosome contains the sites required for DNA processing and packaging during virion assembly. cos is composed of three subsites, cosQ, cosN, and cosB. cosQ is required for the termination of chromosome packaging. Previous studies have shown cosQ mutations to be suppressed in three ways: by a local suppressor within cosQ; by an increase in the length of the lambda chromosome; and by missense mutations affecting the prohead's portal protein, gpB. In the first study reported here, revertants of a set of cosQ mutants were screened for suppressors, and cis-acting suppressors of cosQ mutations were studied; these included second-site cosQ point mutations, base-pair insertions within cosQ, and an additional genome-lengthening suppressor. The 7-bp-long cosQ, with the sequence 5'-GGGTCCT-3', coincides exactly with the recognition site for the EcoO109I restriction/methylation system, which has the consensus sequence 5'-PuGGNCCPy-3'. In a second study, EcoO109I methylation was found to strongly interfere with the residual cosQ function of leaky cosQ mutants. cis-acting suppressors that overcome methylation-associated defects, including a methylation-dependent suppressor, were also isolated. Models of cosQ suppression are presented.

Bacteriophage lambda↗

PubMed1987

Aberrant regulation of synthesis and degradation of viral proteins in coliphage lambda-infected UV-irradiated cells and in minicells.

The patterns of bacteriophage lambda proteins synthesized in UV-irradiated Escherichia coli cells and in anucleate minicells are significantly different; both systems exhibit aberrations of regulation in lambda gene expression. In unirradiated cells or cells irradiated with low UV doses (less than 600 J/m2), regulation of lambda protein synthesis is controlled by the regulatory proteins CI, N, CII, CIII, Cro, and Q. As the UV dose increases, activation of transcription of the cI, rexA, and int genes by CII and CIII proteins fails to occur and early protein synthesis, normally inhibited by the action of Cro, continues. After high UV doses (greater than 2,000 J/m2), late lambda protein synthesis does not occur. Progression through the sequence of regulatory steps in lambda gene expression is slower in infected minicells. In minicells, there is no detectable cII- and cIII-dependent synthesis of CI, RexA, or Int proteins and inhibition of early protein synthesis by Cro activity is always incomplete. The synthesis of early b region proteins is not subject to control by CI, N, or Cro proteins, and evidence is presented suggesting that, in minicells, transcription of the early b region is initiated at a promoter(s) within the b region. Proteolytic cleavage of the regulatory proteins O and N and of the capsid proteins C, B, and Nu3 is much reduced in infected minicells. Exposure of minicells to very high UV doses before infection does not completely inhibit late lambda protein synthesis.

Bacteriophage lambda↗

PubMed1982

Transcription terminates at lambda tR1 in three clusters.

Transcription of the rightward operon of bacteriophage lambda terminates in the presence of rho factor at a region known as tR1, located downstream of the structural gene for the lytic repressor cro. We demonstrate by nuclease Sl mapping that transcription termination at lambda tR1 occurs in vitro over a stretch of 60 nucleotides. End points of transcripts are clustered in three distinct regions, which we refer to as termination sites I, II, and III. Termination at site I is inhibited by L factor, whereas termination at sites II and III is not affected by L factor. The sensitivities of these sites to rho factor are in the order III greater than II greater than I. The cin-lcnc-l mutations abolish termination at site II but not at sites I and III; this result may explain the failure of these mutations to alleviate the phage requirement for N function for growth. Although possible stem-and-loop structures in the RNA transcript immediately upstream of each of the three termination sites can be found, no consistent correlation exists between the strengths of these stem-and-loop structures and the termination efficiencies of their respective sites.

Bacteriophage lambda↗

PubMed1987

The control of lambda DNA terminase synthesis.

Nu1 and A, the genes coding for bacteriophage lambda DNA terminase, rank among the most poorly translated genes expressed in E. coli. To understand the reason for this low level of translation the genes were cloned into plasmids and their expression measured. In addition, the wild type DNA sequences immediately preceding the genes were reduced and modified. It was found that the elements that control translation are contained in the 100 base pairs upstream from the initiation codon. Interchanging these upstream sequences with those of an efficiently translated gene dramatically increased the translation of terminase subunits. It seems unlikely that the rare codons present in the genes, and any feature of their mRNA secondary structure play a role in the control of their translation. The elimination of cos from plasmids containing Nu1 and A also resulted in an increase in terminase production. This result suggests a role for cos in the control of late gene expression. The terminase subunit overproducer strains are potentially very useful for the design of improved DNA packaging and cosmid mapping techniques.

Bacteriophage lambda↗

PubMed1990

Protein-DNA conformational changes in the crystal structure of a lambda Cro-operator complex.

The structure of a complex of bacteriophage lambda Cro protein with a 17-base-pair operator has been determined at 3.9-A resolution. Isomorphous derivatives obtained by the synthesis of site-specific iodinated DNA oligomers were of critical importance in solving the structure. The crystal structure contains three independent Cro-operator complexes that have very similar, although not necessarily identical, conformations. In the complex, the protein dimer undergoes a large conformational change relative to the crystal structure of the free protein. One monomer rotates by about 40 degrees relative to the other, this being accomplished primarily by a twisting of the two beta-sheet strands that connect one monomer with the other. In the complex, the DNA is bent by about 40 degrees into the shape of a boomerang but maintains essentially Watson-Crick B-form. In contrast to other known protein-DNA complexes, the DNA is not stacked end-to-end. The structure confirms the general features of the model previously proposed for the interaction of Cro with DNA.

Amino Acid Sequence↗

PubMed1990

RNase III-dependent hydrolysis of lambda cII-O gene mRNA mediated by lambda OOP antisense RNA.

The 77-nucleotide OOP antisense RNA of bacteriophage lambda complements lambda cII-O mRNA in a region that includes 55 nucleotides at the 3' end of the cII gene and 22 nucleotides in the intercistronic region between the cII and O genes. OOP RNA, produced from multicopy plasmids, inhibits lambda cII gene expression by approximately 100-fold through an RNase III-dependent mechanism. Using primer extension analysis of cellular RNA isolated from an induced lambda lysogen that contains an OOP DNA plasmid, we have identified a cleavage site in cII-O mRNA within the region of complementarity with OOP RNA, at 13 nucleotides from the 3' end of that region. Ribonuclease protection experiments demonstrate that almost all cII-O mRNA in this overlap region is cleaved when OOP RNA is overproduced in RNase III+ cells but not in RNase III- cells. RNA fragments are detected that extend into the O gene from the cleavage sites, while the sister fragments that extend into the cII gene cannot be detected and must be eliminated by additional hydrolytic events. Differences in levels of uncleaved mRNA between RNase III+ and RNase III- cells are much less at several hundred nucleotides to either side of the target region. An alternate OOP RNA-dependent hydrolytic process occurs in RNase III- cells that results in cleavages in one of two regions, one close to the cleavage site observed in RNase III+ cells, and the second several nucleotides beyond the end of the complementary region between OOP RNA and cII-O mRNA. In this latter case, the fragments that extend into the cII gene are stable, while the sister O gene fragments are destroyed, in direct contrast to the RNase III-dependent process.

Bacteriophage lambda↗