Mapping missense and nonsense mutations in genecI of bacteriophage lambda: Marker effects

Summary Amber and missense mutations in genecI of bacteriophage lambda were mapped by reciprocal four-factor crosses, selecting recombinants between the outside markers (N amber andO amber). Distances betweencI missense mutations were additive. SeveralcIamber mutants recombined with othercI mutations with a higher-frequency than expected from the map location. Multiple exchanges in theN-O region occurred at a frequency greater than expected by chance. This high negative interference was especially marked in crosses with thecIamber mutations that were strong recombiners.A newind mutation,ind2, was found neartsU51, to the left of the previously-knownindl mutation, which is located almost in the center of genecI. The mutationc50 maps to the right oftsU50 andc71. Mutationsc60, andts71, which differ in phenotype, are apparently at the same site.     

Purification and characterization of the N gene product of bacteriophage lambda

The N protein (pN) specified by bacteriophage λ is an antitermination factor and is required for phage development. pN can be assayed by making use of the observation that the in vitro synthesis of trp mRNA in a reaction programmed with DNA template from λtrp transducing phage bearing N$The N protein (pN) specified by bacteriophage λ is an antitermination factor and is required for phage development. pN can be assayed by making use of the observation that the in vitro synthesis of trp mRNA in a reaction programmed with DNA template from λtrp transducing phage bearing N and fed mutations is pN dependent (Ishii et al., 1980). The assay has been used to purify pN. We have observed that pN forms a complex with E. coll protein(s) and is dissociated in the presence of urea. The complex is not formed in host bacteria bearing thenusA_nusB_ mutations. pN is a basic protein and heat-stable. Using these characteristics, we have purified pN to virtual homogeneity as judged by polyacrylamide gel electrophoresis in the presence of SDS. pN is a monomeric protein and its mol. wt. is approx. 14 000. The antiterminating activity of pNappears to be enhanced by complex formation with host-encoded protein(s) depending on the nusA and/or nusB gene function.      7. The cis-specificity of the Q-gene product of bacteriophage lambda      D W Burt  W J Brammar 《Molecular & general genetics : MGG》1982,185(3):468-472 Summary A rtrp/lac W205 substitution, fused to the late ragion of bacteriophage lambda, provided a convenient assay for phage late gene expression in the presence or absence of lambda pQ. Comparison of lacZ expression from Q + and Q - phages showed that late gene expression was markedly Q-dependent (263-fold difference). A cis/trans comparison of lambda pQ action showed a 180-fold difference in lacZ expression. The results suggest that pQ is only significantly active when supplied in cis to its site of action.      8. Purification of the bacteriophage lambda xis gene product required for lambda excisive recombination   总被引：22，自引：0，他引：22   K Abremski  S Gottesman 《The Journal of biological chemistry》1982,257(16):9658-9662 Excision of the lambda prophage from the chromosome of its Escherichia coli host requires the products of the two viral genes int and xis. This paper reports a purification of the lambda xis gene product using a complementation assay in which functional Xis must be added to purified Int and an E. coli-derived host factor extract. Excisive recombination between a left (attL) and right (attR) prophage attachment site cloned on the same plasmid DNA substrate occurred efficiently under these conditions. Purified Int and Xis together could not carry out excision in vitro unless an extract derived from the E. coli host was added; purified integration host factor satisfied this requirement. Xis appears to have a molecular weight of 8800 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It possesses no detectable endonuclease or topoisomerase activities, does not appear to bind DNA to filters, and does not increase the ability of Int to bind DNA. The addition of Xis not only stimulated excisive recombination in vitro but also inhibited integrative recombination. Xis protected Int protein from heat inactivation, suggesting a possible interaction between the two proteins. In light of these observations, possible roles for Xis in recombination are discussed.      9. The Fi-gene product of bacteriophage lambda. Purification and properties      S Benchimol  A Becker 《European journal of biochemistry》1982,126(2):227-233 The FI gene product of bacteriophage lambda has been purified extensively using a biochemical assay that measures assembly of lambda phage particles in vitro. The molecular weight of the native protein was estimated to be 21700 with an S20, w of 2.1 S and a Stokes radius of 2.5 nm. The molecular weight in dodecylsulfate was estimated to be 19000. The protein is highly acidic with an isoelectric point less than 4.1.      10. Polarity suppression by the Q gene product of bacteriophage lambda   总被引：8，自引：0，他引：8   D Forbes  I Herskowitz 《Journal of molecular biology》1982,160(4):549-569       11. Control of bacteriophage lambda CII activity by bacteriophage and host functions   总被引：8，自引：4，他引：4       下载免费PDF全文 A Rattray  S Altuvia  G Mahajna  A B Oppenheim  M Gottesman 《Journal of bacteriology》1984,159(1):238-242 We have studied the regulation of the lambda cII gene in vivo using cloned lambda fragments. Lambda N protein stimulated cII expression. Surprisingly, although very high cII protein levels were detected by gel electrophoresis, little cII protein activity, measured as stimulation of the lambda pI and pE promoters, was observed. The half-life of cII protein depended critically on its initial level. At low concentrations its half-life was as short as 1.5 min, whereas at high cII protein levels, it could be as long as 22 min. The Escherichia coli mutant ER437 directs lambda towards lysogeny; cII protein was more stable in this strain than in the wild type. On the other hand, although cyclic AMP is required for efficient lysogeny, it did not appear to influence the synthesis, stability, or activity of cII protein.      12. Role of S gene product of bacteriophage lambda in host cell lysis      S. Barik  N. C. Mandal 《Journal of biosciences》1982,4(4):527       13. Role ofS gene product of bacteriophage lambda in host cell lysis      S. Barik  N. C. Mandal 《Journal of biosciences》1982,4(3):361-368 Studies with the induced lysogens of λS +R+, λS-R+, λS+R- and λS-R- phages have shown that while theS gene product is essential for the action of intracellularR gene product to release the periplasmic alkaline phosphatase in the presence of EDTA, the latter gene product can bring about this effect while acting onEscherichia coli cells from outside, in the absence of functionalS gene product; chloroform, could help the intracellularR gene product in effecting bacterial lysis in the absence ofS gene product. These result support the premise that theS gene product facilitates theR gene product in crossing the cytoplasmic membrane into the periplasmic space such that the latter can act on the peptidoglycan layer of the host cell thus causing both the release of alkaline phosphatase and cell lysis. An erratum to this article is available at .      14. Orientation-dependent recombination hotspot activity in bacteriophage lambda.   总被引：14，自引：0，他引：14   D Faulds  N Dower  M M Stahl  F W Stahl 《Journal of molecular biology》1979,131(4):681-695 Promoters of genetic exchange by the Escherichia coli Rec system, Chi elements, have been analyzed in λ phages carrying bacterial EcoRI restriction fragments. Some fragments confer Chi+ phenotype in one orientation and Chi? in the opposite orientation. The inactivity of Chi in one orientation explains why all active Chi elements in λ manifest a certain recombinational bias of the same sense.When these studies were undertaken, we rather expected to find two classes of Chi, one class which stimulated recombinant formation stronger to its left and one class stimulating recombinant formation more strongly to its right. The failure to find the second class is now understandable by supposing that the orientation of Chi which would have permitted it to act rightward is the orientation in which Chi has no activity at all. Several models are proposed for the orientation dependence of Chi activity.      15. The R gene product of bacteriophage lambda is the murein transglycosylase   总被引：14，自引：0，他引：14   K Bienkowska-Szewczyk  B Lipinska  A Taylor 《Molecular & general genetics : MGG》1981,184(1):111-114 Summary The radioactively labeled proteins synthesised in Escherichia coli minicells infected by bacteriophage R and R + were compared by polyacrylamide gel electrophoresis. R mutants, which have lost the ability to lyse host cells, lack a polypeptide of molecular weight 17.5 kD corresponding to the molecular weight of murein transglycosylase — a bacteriolytic enzyme from lysates which we have described previously. It has been shown by direct comparison using radio-labeled enzyme that transglycosylase comigrates with the R gene product. The enzyme was endetectable in induced cultures of E. coli W3350 su o (cI857 Ram5) and C600 (cI857 acR301), while it was present in a R – + mutant lysate. We conclude that the transglycosylase is the R gene product.Abbreviations Muropeptide CA GlcNac-1-4-1,6-anhydro-MurNac-L-Ala-D-Glu-msA2pm-D-Ala - muropeptide CB GlcNac-MurNac-GlcNac-1,6-anhydro-MurNac in which the carboxyl groups of MurNac and 1,6-anhydro-MurNac are substituted by the tetrapeptide L-Ala-D-Glu-msA2pm-D-Ala - muropeptide C3 dimer of the two units GlcNac-MurNac-L-Ala-D-Glu-msA2pm-D-Ala which are connected by D-D peptide bond between D-Ala and msA2pm - GlcNac N-acetyl-D-glucosamine - MurNac N-acetylmuramic acid - msA2pm meso-diaminopimelic acid - rivanol 6,9-diamino-2-ethoxyacridine lactate - SDS sodium dodecyl sulfate      16. Cohesion and the biological activity of bacteriophage lambda DNA   总被引：16，自引：0，他引：16   A D Kaiser  R B Inman 《Journal of molecular biology》1965,13(1):78-91       17. Biological activity of different forms of bacteriophage lambda DNA]      I Fodor  N I Matvienko  L P Tikhomirova  V I Taniashin  A A Baev 《Molekuliarnaia biologiia》1975,9(2):179-189 Hershey circles and linear tandem aggregated forms of DNA have been obtained in vitro and treated with polynucleotide ligase to form phosphodiester bond. Using zone centrifugation in glycerol gradient covalently closed circles and linear dimers have been purified and their biological activity investigated. It was found that closed circular molecules lost most, if not all, of their activity in CaCl2-dependent system. In order to investigate the biological activity of tandem dimer molecules, hybrid dimers consisting of DNA's from lambda C1857 and lambda 1434 have been obtained. In plaque assay with the appropriate non-permissive strains of E. coli the efficiency of infectivity of hybrid dimers was measured. Biological activity of dimer molecules sealed with ligase was about 5% of the activity of linear monomers. Ig has been suggested that tandem dimers of lambda DNA joined by phosphodiester bond are able to penetrate into the CaCl2-treated host cells and both components of dimers are active during subsequent multiplication.      18. The role of cIII product and anti-immunity in zygotic induction of bacteriophage lambda      Vladimír Špelina  Zdeněk Neubauer  Vojtěch Závada 《Molecular genetics and genomics : MGG》1973,123(4):355-361       19. Crypticogenicity of bacteriophage lambda   总被引：3，自引：0，他引：3   S Adhya  A Campbell 《Journal of molecular biology》1970,50(2):481-490       20. RecA protein-dependent proteolysis of bacteriophage lambda repressor Characterization of the reaction and stimulation by DNA-binding proteins   总被引：11，自引：0，他引：11   G M Weinstock  K McEntee 《The Journal of biological chemistry》1981,256(21):10883-10888       $

The cis-specificity of the Q-gene product of bacteriophage lambda

Summary A rtrp/lac W205 substitution, fused to the late ragion of bacteriophage lambda, provided a convenient assay for phage late gene expression in the presence or absence of lambda pQ. Comparison of lacZ expression from Q ⁺ and Q ^- phages showed that late gene expression was markedly Q-dependent (263-fold difference). A cis/trans comparison of lambda pQ action showed a 180-fold difference in lacZ expression. The results suggest that pQ is only significantly active when supplied in cis to its site of action.     

Purification of the bacteriophage lambda xis gene product required for lambda excisive recombination

Excision of the lambda prophage from the chromosome of its Escherichia coli host requires the products of the two viral genes int and xis. This paper reports a purification of the lambda xis gene product using a complementation assay in which functional Xis must be added to purified Int and an E. coli-derived host factor extract. Excisive recombination between a left (attL) and right (attR) prophage attachment site cloned on the same plasmid DNA substrate occurred efficiently under these conditions. Purified Int and Xis together could not carry out excision in vitro unless an extract derived from the E. coli host was added; purified integration host factor satisfied this requirement. Xis appears to have a molecular weight of 8800 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It possesses no detectable endonuclease or topoisomerase activities, does not appear to bind DNA to filters, and does not increase the ability of Int to bind DNA. The addition of Xis not only stimulated excisive recombination in vitro but also inhibited integrative recombination. Xis protected Int protein from heat inactivation, suggesting a possible interaction between the two proteins. In light of these observations, possible roles for Xis in recombination are discussed.     

The Fi-gene product of bacteriophage lambda. Purification and properties

The FI gene product of bacteriophage lambda has been purified extensively using a biochemical assay that measures assembly of lambda phage particles in vitro. The molecular weight of the native protein was estimated to be 21700 with an S20, w of 2.1 S and a Stokes radius of 2.5 nm. The molecular weight in dodecylsulfate was estimated to be 19000. The protein is highly acidic with an isoelectric point less than 4.1.     

Control of bacteriophage lambda CII activity by bacteriophage and host functions

We have studied the regulation of the lambda cII gene in vivo using cloned lambda fragments. Lambda N protein stimulated cII expression. Surprisingly, although very high cII protein levels were detected by gel electrophoresis, little cII protein activity, measured as stimulation of the lambda pI and pE promoters, was observed. The half-life of cII protein depended critically on its initial level. At low concentrations its half-life was as short as 1.5 min, whereas at high cII protein levels, it could be as long as 22 min. The Escherichia coli mutant ER437 directs lambda towards lysogeny; cII protein was more stable in this strain than in the wild type. On the other hand, although cyclic AMP is required for efficient lysogeny, it did not appear to influence the synthesis, stability, or activity of cII protein.     

Role ofS gene product of bacteriophage lambda in host cell lysis

Studies with the induced lysogens of λS ⁺R⁺, λS^-R⁺, λS⁺R^- and λS-R^- phages have shown that while theS gene product is essential for the action of intracellularR gene product to release the periplasmic alkaline phosphatase in the presence of EDTA, the latter gene product can bring about this effect while acting onEscherichia coli cells from outside, in the absence of functionalS gene product; chloroform, could help the intracellularR gene product in effecting bacterial lysis in the absence ofS gene product. These result support the premise that theS gene product facilitates theR gene product in crossing the cytoplasmic membrane into the periplasmic space such that the latter can act on the peptidoglycan layer of the host cell thus causing both the release of alkaline phosphatase and cell lysis. An erratum to this article is available at .     

Orientation-dependent recombination hotspot activity in bacteriophage lambda.

Promoters of genetic exchange by the Escherichia coli Rec system, Chi elements, have been analyzed in λ phages carrying bacterial EcoRI restriction fragments. Some fragments confer Chi⁺ phenotype in one orientation and Chi^? in the opposite orientation. The inactivity of Chi in one orientation explains why all active Chi elements in λ manifest a certain recombinational bias of the same sense.When these studies were undertaken, we rather expected to find two classes of Chi, one class which stimulated recombinant formation stronger to its left and one class stimulating recombinant formation more strongly to its right. The failure to find the second class is now understandable by supposing that the orientation of Chi which would have permitted it to act rightward is the orientation in which Chi has no activity at all. Several models are proposed for the orientation dependence of Chi activity.     

The R gene product of bacteriophage lambda is the murein transglycosylase

Summary The radioactively labeled proteins synthesised in Escherichia coli minicells infected by bacteriophage R and R ⁺ were compared by polyacrylamide gel electrophoresis. R mutants, which have lost the ability to lyse host cells, lack a polypeptide of molecular weight 17.5 kD corresponding to the molecular weight of murein transglycosylase — a bacteriolytic enzyme from lysates which we have described previously. It has been shown by direct comparison using radio-labeled enzyme that transglycosylase comigrates with the R gene product. The enzyme was endetectable in induced cultures of E. coli W3350 su ^o (cI857 Ram5) and C600 (cI857 acR301), while it was present in a R _– ⁺ mutant lysate. We conclude that the transglycosylase is the R gene product.Abbreviations Muropeptide CA GlcNac-1-4-1,6-anhydro-MurNac-L-Ala-D-Glu-msA₂pm-D-Ala - muropeptide CB GlcNac-MurNac-GlcNac-1,6-anhydro-MurNac in which the carboxyl groups of MurNac and 1,6-anhydro-MurNac are substituted by the tetrapeptide L-Ala-D-Glu-msA₂pm-D-Ala - muropeptide C3 dimer of the two units GlcNac-MurNac-L-Ala-D-Glu-msA₂pm-D-Ala which are connected by D-D peptide bond between D-Ala and msA₂pm - GlcNac N-acetyl-D-glucosamine - MurNac N-acetylmuramic acid - msA₂pm meso-diaminopimelic acid - rivanol 6,9-diamino-2-ethoxyacridine lactate - SDS sodium dodecyl sulfate     

Biological activity of different forms of bacteriophage lambda DNA]

Hershey circles and linear tandem aggregated forms of DNA have been obtained in vitro and treated with polynucleotide ligase to form phosphodiester bond. Using zone centrifugation in glycerol gradient covalently closed circles and linear dimers have been purified and their biological activity investigated. It was found that closed circular molecules lost most, if not all, of their activity in CaCl2-dependent system. In order to investigate the biological activity of tandem dimer molecules, hybrid dimers consisting of DNA's from lambda C1857 and lambda 1434 have been obtained. In plaque assay with the appropriate non-permissive strains of E. coli the efficiency of infectivity of hybrid dimers was measured. Biological activity of dimer molecules sealed with ligase was about 5% of the activity of linear monomers. Ig has been suggested that tandem dimers of lambda DNA joined by phosphodiester bond are able to penetrate into the CaCl2-treated host cells and both components of dimers are active during subsequent multiplication.