Showing papers on "Lambda phage published in 1971"

Inactivation of Escherichia coli, F' episomes at transfer, and bacteriophage lambda by psoralen plus 360-nm light: significance of deoxyribonucleic acid cross-links.

Abstract: We have investigated some biological consequences of light-induced psoralen-deoxyribonucleic acid (DNA) adducts and find that for several Escherichia coli functions (killing of strain AB2480 recA13 uvrA6, inactivation of phage lambda plaque-forming ability in wild type and uvrA6 hosts, loss of ability to transmit intact Flac+ episomes), a light exposure sufficient for production of a single cross-link per DNA molecule correlates well with the biological consequence. Although one cross-link per genome is apparently lethal to recA13 uvr− strains, mutants carrying the recA13 or uvrA6 markers survive light exposures producing 6.7 and 16 cross-links per genome, respectively, and wild-type cells recover from 65 psoralen cross-links. Evidently, the excision and recombinational repair systems complement one another in reconstructing an intact genome from cellular DNA containing psoralen photoproducts. The above bacterial and phage strains, in which DNA repair processes are minimized, are also extremely sensitive to pyrimidine dimer-forming 254-nm UV light (without psoralen), and were expected to respond similarly to formation of psoralen-pyrimidine base monoadducts in their DNA. Since the biological inactivation by psoralen correlates well with cross-link formation, we suggest that the sensitizing action of this drug primarily derives from its ability to form DNA cross-links.

Replication Control in Phage Lambda

Abstract: Early in the productive growth cycle following infection, λ DNA can be found in doubly branched circular structures (Ogawa et al., 1968). The positions of branch points in these molecules suggest that each molecule carries two replication forks moving away from a locus in the region of genes O and P (Schnos and Inman, 1970). These suggestions have been substantiated and refined by measurements of replication in deletion prophages in the presence of helper (Stevens et al., this volume). The replication process can be divided into a step creating a replication fork (initiation) and the subsequent movement of the fork along the DNA template (progression or movement). Three λ genes ( N , O , and P ) are known to specify products that enter into the replication process. The host supplies the biosynthetic machinery for DNA precursors, and at least one gene product that is involved directly in replication of both the host chromosome and λ DNA beyond the precursor level (Hirota et al., 1968; Fangman and Feiss, 1969; Georgopoulos and Herskowitz, this volume). When λ DNA is repressed, it does not replicate autonomously (Wolf and Meselson, 1963), even if the products of genes N , O, and P are supplied by a heteroimmune hybrid of λ (Thomas and Bertani, 1964; Russo et al., 1970). Repressor not only prevents the synthesis of the products of genes N, O, and P ; it also blocks their action. The latter aspect of repression is to be called an epistatic block. A mutant, t 11, which is defective in...

Identification of Proteins Coded in Phage Lambda

Abstract: Phage λ has sufficient DNA to encode about 50 proteins of molecular weight 33,000, and about 35 genes are known. However, fewer than ten λ proteins have been studied in any detail, largely because of the lack of specific assays for the others, and because host protein synthesis continues after infection by λ . This paper describes the labeling, identification, and characterization of λ proteins by a technique devised by Ptashne which circumvents these problems. The proteins identified belong to both the early and late classes, and several are products of genes which have not been described previously. EXPERIMENTAL METHOD The basic method used here to characterize λ proteins is to label them with radioactive amino acids and to compare the electrophoretic patterns of radioactive proteins obtained from various mutants. These experiments depend on the fact, first reported by Ptashne, that infection by wild-type λ ( λ + ), of cells heavily irradiated with ultraviolet light (UV), stimulates the ability of the cells to incorporate radioactive leucine into TCA-insoluble material by 10–15-fold (Ptashne, 1967). This technique makes it possible to label phage-specific proteins without an intolerable level of labeling of Escherichia coli proteins. Synthesis of Early and Late λ Proteins in Irradiated Bacteria Figure 1 shows the dependence of total leucine incorporation on UV dose for uninfected cells and for cells infected with three different phages. The ratio of incorporation between λ + -infected and uninfected cells can be seen to increase with dose up to about 12,000 erg/mm 2 and then to remain approximately constant.

Tandem Genetic Duplications in a Derivative of Phage Lambda

Abstract: Gene duplication followed by independent evolution of the duplicated genes appears to be a basic feature of the genetics of higher organisms (see Bryson and Vogel, 1965). Such duplications are rare in Escherichia coli as judged by measurements of repeating DNA sequences (Britten and Kohne, 1968), although there are multiple copies of the ribosomal RNA genes in E. coli (Yankofsky and Spiegelman, 1962; Moore and McCarthy, 1967). There have been few studies of tandem duplications in phages. Duplications in the T4 r II region have been obtained by a strong selection technique (Weil et al., 1965; Parma and Ingraham, 1970). We have found that many of the spontaneous “revertants” of the deletion mutant t del 33 (Franklin, 1967) are duplication mutants, with changes at different locations on the phage chromosome. This phage has many advantages for studying the properties and mechanism of formation of duplication mutants. Some advantages which we have exploited in the present work are as follows. (1) Since the base sequence in 80- λ hy 1 DNA is nonpermuted and the phage chromosome can be extracted as a DNA molecule of definite length, a duplication mutant can be identified as such and the end points of the duplicated segment can be located by electron microscopy of DNA heteroduplexes. (2) The dependence of phage buoyant density on DNA length provides a nonspecific selection method for mutants which have longer DNA molecules. (Selection by plaque morphology is also possible in many cases.) (3) An independent method of identifying duplication mutants is based...

Mutations allowing growth on maltose of Escherichia coli K12 strains with a deleted malT gene

Abstract: Previous work suggests that the malT gene exerts a positive control on two operons. One operon (malB lamB) codes for maltose permease and a protein involved in the biosynthesis of cell wall receptors for phage lambda. The other operon (malP malQ) codes for two enzymes of maltose metabolism.

Purification and base composition analysis of phage lambda early promoters.

Abstract: RNA-polymerase of Escherichia coli was allowed to bind to DNA of phage lambda in the absence of precursors. The resulting complex was excised by nuclease digestion and the protected DNA was recovered by phenol-extraction and ethanol precipitation. Acrylamide gel electrophoresis of protected DNA fragments reveals the existence of two distinct oligonucleotide peaks corresponding, respectively, to 45-52 and 7-10 nucleotide residues along with species of intermediate sizes. Peak I molecules have two properties: (a) their existence is dependent on the presence of sigma factor during the initial binding step, and (b) they are considerably enriched in A-T (up to 67%). On the contrary, peak II molecules have the same base composition as DNA of phage lambda, whether obtained in the presence or absence of sigma factor. Peak I molecules are thus believed to contain DNA sequences involved in promoter recognition, whether they are the promoters themselves, adjacent, or related sequences.

The Morphogenesis of the Phage Lambda Head: the Step Controlled by Gene F

Abstract: AN OUTLINE OF LAMBDA MORPHOGENESIS Eighteen genes, all located in the left arm of the chromosome, control the morphogenesis of phage λ (Campbell, 1961; Parkinson, 1968; Mount et al., 1968). These can be divided into two distinct gene clusters by electron microscopic investigations (Mount et al., 1968; Kemp et al., 1968) and by utilization of the observation by Weigle (1966) that λ heads and tails can join in vitro to form infectious phage (Weigle, 1966; Parkinson, 1968). One cluster containing seven genes ( A , W , B , C , D , E , and F ), controls the formation of phage heads, and the other, containing eleven genes ( Z , U , V , G , T , H , M , L , K , I , J ), controls the synthesis of phage tails. Thus, the maximum number of phage-specified proteins present in the mature phage particle could be 18; however physicochemical studies of phage particles (Villarejo et al., 1967; Casjens et al., 1970; Buchwald et al., 1970; Murialdo and Siminovitch, this volume) have revealed only 3 major proteins and up to 8 minor protein species which can be reproducibly identified by sodium dodecylsulfate (SDS) gel electrophoresis (Table 1). A more detailed description of the process of morphogenesis requires knowledge of the function performed by the products of each of these genes. Some progress toward this aim has been made by the identification of the products of several of these genes as components of the phage particle. Gene E appears to code for the synthesis of the major head protein (h2). Mutants in gene E ...

Temperature Sensitivity of Maltose Utilization and Lambda Resistance in Escherichia coli B

Abstract: Escherichia coli B strains that have acquired the malB region from E. coli K-12 are able to utilize maltose and to adsorb phage lambda when grown at 30 C, but when grown at 40 C they do not absorb phage lambda and are devoid of amylomaltase activity. These Mal(ts) Lam(ts) cells can be mutated or transduced to become able to grow on maltose at 40 C, but they still have no detectable amylomaltase activity nor functional lambda receptors at that temperature. This Mal(40) phenotype is governed by a gene located near or at malA. It is suggested that the temperature sensitivity of both characters results from a defect in malT. However, transduction of malA from E. coli B to E. coli K-12 results in a wild-type phenotype, whereas E. coli B cells that have acquired malA from E. coli K-12 donors are still temperature sensitive for both amylomaltase and lambda-receptor production.

Chapter 1 Introduction to Lambda

Abstract: The bacteriophages form a diverse collection of viruses that multiply in bacterial cells. This book describes the variety called λ and some of its relatives, which multiply in Escherichia coli. Chapter 1 presents general characteristics of the λ group, and explains how λ came to be the subject of intensive study. The classical phages destroy their host cells by lysis. Lambda causes lysis too, but can also propagate in a form that permits joint multiplication of phage and host. Phages possessing this added potentiality are called temperate, in distinction from the broad class of intemperate or virulent species, which cannot reproduce without destroying their host cells. Lambda is also one of a few well known genetic elements, called episomes, that are able to multiply in the cell either autonomously or as part of the bacterial chromosome. In fact λ and the fertility agent F of E. coli are historical prototypes of the class. Campbell (1969) presents the comparative biology of episomes. For newcomers to phage research, Stent’s Molecular Biology of Bacterial Viruses (1963) can be recommended as a source book. THE LIFE CYCLE Lambda phage particles are about half protein and half DNA. Each contains one double-stranded DNA molecule encapsulated in an icosahedral head, 0.05 μ in diameter, from which projects a tubular tail, 0.15 μ long (Chapter 14). The DNA molecule is made from the usual four bases and weighs about 31 million daltons. Phage particles can be prepared in milligram quantities for analysis of various sorts. Lambda is...

Formation of concatemers of lambda phage DNA in a recombination-deficient system.

Abstract: Concatenand molecules of lambda DNA were formed even in a recombination dificient system (Int-Red-Rec-) in the late stage of phage growth. No significant difference was observed in the formation of concatemers between recombination deficient and proficient systems. These results suggest that concatemer formation is an intrinsic nature of replication in the late stage. The possibility of concatemer formation by molecular exchanges which do not contribute genetic recombination is not excluded.

Protection of irradiated lambda phage by methylated proflavine.

Abstract: The lethal action of U.V. on λ bacteriophage is diminished by pretreatment with methylated proflavine.

Control of leftward transcription in the immunity region of Escherichia coli phage lambda.

Abstract: It was shown by various RNA-DNA hybridization techniques that the l-strand transcription of the λ immunity region, including genes c I and rex (see i 434, Fig 1), of Escherichia coli phage λ rapidly decreases after induction of lysogenic cells This decrease appears to depend on protein synthesis, since the c I transcription was not reduced after heat induction of a λc I,857 lysogen in the presence of chloramphenicol Upon infection of a sensitive E coli host with λbio256, an immediate, but very low and transient synthesis of l-strand specific mRNA from the immunity region was observed