Showing papers on "Lambda phage published in 1983"
TL;DR: A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes and was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel.
Abstract: A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal sequence of 20 amino acids. A mature polypeptide starting with either Met-Ala-Pro or Met-Pro was expressed in E. coli under control of the E. coli trp promoter or using a combination of the phage lambda PL promoter and a ribosome binding site derived from phage Mu. The bacterial IL2 polypeptide had a molecular weight of 15,000 daltons and accounted for more than 10% of the total E. coli proteins in fully induced cells; it was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel.
314 citations
TL;DR: A clone bearing the structural gene sacB, coding for the exoenzyme levansucrase, was isolated from a library of Bacillus subtilis DNA that was cloned in phage lambda charon 4A on the basis of the transforming activity of the chimeric DNA.
Abstract: A clone bearing the structural gene sacB, coding for the exoenzyme levansucrase, was isolated from a library of Bacillus subtilis DNA that was cloned in phage lambda charon 4A on the basis of the transforming activity of the chimeric DNA. This lambda clone also was found to contain the sacR and smo loci. Subcloning the sacB-sacR region in plasmid pBR325 resulted in a clone which directed levansucrase synthesis in Escherichia coli. The nucleotide sequence coding for the secreted protein was localized on the physical map of the cloned DNA.
263 citations
TL;DR: The dnaK protein possesses a weak DNA-independent ATPase activity, which results in the production of ADP and Pi, and participates in the initiation of DNA synthesis in an in vitro DNA replication system that is dependent on the O and P proteins.
Abstract: The Escherichia coli dnaK gene product, originally defined by mutations that blocked lambda phage DNA replication, is known to be necessary for E. coli viability. We have purified dnaK protein to homogeneity and have demonstrated that it possesses a weak DNA-independent ATPase activity, which results in the production of ADP and Pi. The proof that this ATPase activity is encoded by the dnaK+ gene relies primarily on the fact that the dnaK756 mutation results in the production of an ATPase activity with altered physical properties. The dnaK protein is phosphorylated in vitro and in vivo, probably as a result of an autophosphorylation reaction. The lambda O and P replication proteins were shown to interact in vitro with the dnaK protein. The ATPase activity of the dnaK protein was inhibited by purified lambda P protein and stimulated by purified lambda O protein. Moreover, the dnaK protein participates in the initiation of DNA synthesis in an in vitro DNA replication system that is dependent on the O and P proteins. Anti-dnaK protein immunoglobulin specifically inhibited DNA synthesis in this system.
237 citations
TL;DR: A stable lysogen is formed when an infecting viral particle succeeds both in repressing lytic functions and in inserting its DNA in a quiescent form of the viral chromosome called prophage.
Abstract: INTRODUCTION Cells lysogenic for λ carry a quiescent form of the viral chromosome called prophage The prophage differs from the DNA of viral particles in two important ways: (1) The ends of the prophage and of the viral particle DNA are at different points in the nucleotide sequence, and (2) the prophage ends are covalently joined to host DNA Campbell (1962) proposed that viral particle DNA is converted to prophage by the joining of the ends followed by insertion of the resulting circular molecule into the host DNA Insertion occurs by reciprocal recombination at specific sites (attachment or att sites) in each chromosome (Fig 1) This proposal has received extensive experimental support and, indeed, was generally accepted when the first edition of this book was written (see Gottesman and Weisberg 1971) A stable lysogen is formed when an infecting viral particle succeeds both in repressing lytic functions and in inserting its DNA The inserted prophage is then replicated as part of the bacterial chromosome In the rare event that repression is not followed by insertion (abortive lysogeny), the viral chromosome cannot replicate and so is lost by dilution as the host cell divides Excision of the prophage from the chromosome is rare during normal bacterial growth but occurs readily following repressor inactivation If the repressor is inactivated only briefly (abortive or transient derepression), prophage excision can occur without lytic growth The excised DNA is then frequently lost as the cell divides (prophage curing) Intricate controls ensure that insertion occurs only
193 citations
TL;DR: Seven mutants of Escherichia coli were isolated that are sensitive to methyl methane sulfonate but not to UV light and exhibited decreased host cell reactivation capacity for methyl methane sulphonate-treated phage lambda.
Abstract: Seven mutants of Escherichia coli were isolated that are sensitive to methyl methane sulfonate but not to UV light. They exhibited decreased host cell reactivation capacity for methyl methane sulfonate-treated phage lambda. Five of the mutations were mapped in the same region as alkA (previously called alk) and may indeed be identical to known mutations. Another mutation was found near nalA, and the gene responsible was named alkB. Its phenotype was different from that of ada, since the alkB mutant exhibited a normal adaptive response to N-methyl-N'-nitro-N-nitrosoguanidine. A third type of mutation was mapped near polA, but this mutant contained an almost normal level of DNA polymerase I activity.
161 citations
TL;DR: The developed phage λ chromosome as a receptor for fragments of DNA was assured by the availability of well-characterized deletion mutants, by the means of selecting for the loss of restriction targets and by prior identification of the essential components of the phage transcriptional circuits and replication system.
Abstract: For more than 20 years, transducing derivatives of lambdoid phages have facilitated functional and structural analyses of bacterial genes. Restriction enzymes that make staggered cuts within specific DNA sequences (targets) produce discrete fragments with short cohesive ends (Hedgpeth et al. 1972; Mertz and Davis 1972; Bigger et al. 1973), and this discovery immediately indicated that fragments of DNA could be spliced into the severed arms of a λ vector molecule. The cohered fragments would then be joined covalently by DNA ligase (Gellert 1967), and the recombinant genomes, transducing phages, recovered by transfection of Escherichia coli (Mandel and Higa 1970). This goal was quickly realized (Murray and Murray 1974; Thomas et al. 1974), and detailed analyses of the type previously possible for only a few E. coli genes became generally applicable. The ability to insert DNA fragments from any source into plasmid and phage vectors, supplemented with the new techniques for rapid DNA sequence determination (Maxam and Gilbert 1977; Sanger et al. 1977), has already generated many exciting advances in diverse areas of molecular biology. The development of the phage λ chromosome as a receptor for fragments of DNA was assured by the availability of well-characterized deletion mutants, by the means of selecting for the loss of restriction targets and by prior identification of the essential components of the phage transcriptional circuits and replication system. The precise knowledge attained by 1972 showed the genetic engineer which regions of the λ genome could be sacrificed and, also, which special features could be...
159 citations
TL;DR: An Escherichia coli plasmid vector, pUN121, has been constructed which allows for positive selection of transformants harboring DNA inserts and has a functional ampicillin resistance gene and the new restriction sites, give rise to tetracycline resistant transformants.
Abstract: An Escherichia coli plasmid vector, pUN121, has been constructed which allows for positive selection of transformants harboring DNA inserts. The positive selection of transformants harboring DNA inserts. The vector is based on plasmid pTR262 (Roberts et al. Gene, 12, (1980), 123-127) in which the tetracycline resistance gene is under transcriptional control of the repressor protein coded by the phage lambda cI gene. This plasmid has been rearranged, using in vitro recombinant techniques including oligonucleotide mediated mutagenesis to yield a smaller plasmid (4.4 kb) with unique cloning sites for EcoRI, XmaI and SmaI in addition to the unique HindIII and BclI sites. The plasmid has a functional ampicillin resistance gene and the new restriction sites (EcoRI, XmaI and SmaI) when used for cloning, give rise to tetracycline resistant transformants.
159 citations
TL;DR: An Escherichia coli gene bank composed of large DNA fragments (about 40 kilobases) was constructed by using the small cosmid pHC79 and it was demonstrated that the structural gene, sodA, of manganese superoxide dismutase was cloned.
Abstract: An Escherichia coli gene bank composed of large DNA fragments (about 40 kilobases) was constructed by using the small cosmid pHC79. From it, a clone was isolated for its ability to overproduce superoxide dismutase. The enzyme overproduced was manganese superoxide dismutase, as determined by electrophoresis and antibody precipitation. Maxicell analysis and two-dimensional O'Farrell polyacrylamide gel electrophoresis demonstrated that the structural gene, sodA, of manganese superoxide dismutase was cloned. Subcloning fragments from the original cosmid located the sodA gene within a 4.8-kilobase EcoRI-BamHI fragment. This fragment was inserted into a lambda phage which was deleted for the att region and consequently could only lysogenize by recombination between the cloned bacterial DNA insertion and the bacterial chromosome. Genetic mapping of the prophage in such lysogens indicated that the chromosomal sodA locus lies near 87 min on the E. coli map.
113 citations
TL;DR: It appears that high level expression of genes containing a relatively large proportion of minor codons can be obtained under the appropriate conditions, and this technology can be used to obtain other viral proteins in pure form for structural, functional, and immunological studies.
Abstract: RNA segment 8 of the influenza A virus genome codes for two nonstructural proteins, NS1 and NS2, for which the functions are unknown. Cloned cDNA copies of this gene from three different influenza A virus strains were inserted into an Escherichia coli plasmid expression vector, pAS1, carrying the strong regulatable lambda phage promoter, PL. After induction, the NS1 proteins were overproduced to levels of 20-25% of total cellular protein. This was surprising in that the codon composition for these eukaryotic genes is similar to that for weakly expressed proteins in E. coli. Thus, under the appropriate conditions, it appears that high level expression of genes containing a relatively large proportion of minor codons can be obtained. The NS1 protein produced in bacteria from a cloned cDNA copy of the A/PR/8/34 virus NS gene was purified to apparent homogeneity and used to generate a high-titer monospecific rabbit antiserum. Immunoprecipitation studies showed this antibody to be crossreactive against the NS1 proteins produced by several different influenza A virus strains. Immunofluorescence experiments in Madin-Darby canine kidney cells showed the NS1 proteins to be located in the nucleoplasm early in infection for all strains examined. With some of the strains, NS1-specific immunofluorescence was observed predominantly in the nucleoli later in infection. This technology can be used to obtain other viral proteins in pure form for structural, functional, and immunological studies.
102 citations
TL;DR: High level synthesis in Escherichia coli of mature human fibroblast interferon is obtained using a plasmid vector designed to allow easy coupling of a DNA coding region to the initiator AUG of the replicase gene of the RNA phage MS2 cloned downstream of phage lambda's leftward promoter.
Abstract: We have obtained high level synthesis in Escherichia coli of mature human fibroblast interferon using a plasmid vector that was designed to allow easy coupling of a DNA coding region to the initiator AUG of the replicase gene of the RNA phage MS2 cloned downstream of phage lambda's leftward promoter. The activity of the promoter can be regulated by temperature. Induced cells accumulated the interferon up to 4% of the total cellular protein. The biological activity of the product amounted to 4 X 10(9) international units per litre of culture. The synthesis of human fibroblast interferon was shown to drastically inhibit the growth rate of the bacterial host.
100 citations
TL;DR: A novel dye-staining method has been developed to detect β-glucanase activity in colonies on agar plates and directs the synthesis in E. coli of a β- GLUCanase which specifically degrades barley glucan and lichenan.
TL;DR: It is concluded that DNA helicase II is the uvrD gene product, which has single stranded DNA-dependent ATPase activity and ATP-dependent DNA halicase activity, and both activities are specifically inactivated by antibodies raised against DNA helicases II.
Abstract: We have shown that the uvrD gene product, previously identified in maxicell extracts as a 73 kilodalton protein, copurifies with single stranded DNA-dependent ATPase and ATP-dependent DNA helicase activities. This protein is specifically precipitated from maxicell extracts by antibodies raised against DNA helicase II. In order to facilitate purification of the UvrD protein we have sub-cloned the uvrD gene into a plasmid vector in which its transcription is under the control of the phage lambda leftward promoter. Using cells harbouring this recombinant plasmid as a source of elevated levels of the UvrD protein we have purified this protein to homogeneity by a simple, rapid procedure. The purified protein has single stranded DNA-dependent ATPase activity and ATP-dependent DNA halicase activity, and both activities are specifically inactivated by antibodies raised against DNA helicase II. We conclude that DNA helicase II is the uvrD gene product.
TL;DR: A plasmid, pJL6, was constructed that contains a unique ClaI site twelve codons beyond the bacteriophage lambda cII gene initiation codon that allowed us to fuse the carboxy-terminal sequences of the avian myelocytomatosis virus (MC29) v-myc gene to the amino- terminal portion of the c II gene.
TL;DR: A 1.6-kilobase-pair DNA fragment derived from the Escherichia coli chromosome was analyzed by Tn3 transposon insertion and deletion mapping to locate a mutator gene, dnaQ, and the rnh gene that codes for RNase H, suggesting that the two genes are transcribed in opposite directions and that their promoters are located in a narrow region between the genes.
Abstract: A 1.6-kilobase-pair DNA fragment derived from the Escherichia coli chromosome was analyzed by Tn3 transposon insertion and deletion mapping to locate a mutator gene, dnaQ (mutD), and the rnh gene that codes for RNase H. When a strong promoter, PL of lambda phage, was placed at the right- and left-side of the cloned DNA fragment, the dnaQ protein and RNase H, respectively were overproduced. These results suggested that the two genes are transcribed in opposite directions and that their promoters are located in a narrow region between the genes. Nucleotide sequence analysis confirmed this and further revealed that transcriptional and translational initiation signals for the two genes overlap. From the sequence data it was deduced that the dnaQ protein and RNase H consist of 243 and 155 triplets and have molecular weights of 27,500 and 17,500, respectively. dnaQ81 amber mutant showed two codon alterations, CAG(glutamine-195) leads to TAG(amber) and ACA(threonine-193) leads to ATA(isoleucine). The dnaQ-lacZ and the rnh-lacZ fused genes were constructed and hybrid proteins with beta-galactosidase activity were produced. From beta-galactosidase levels it was estimated that the promoter for dnaQ is 5 times more active than that for rnh.
TL;DR: Phage λ’s accessory genes are reviewed and their role with respect to both λ and Escherichia coli is discussed.
Abstract: INTRODUCTION Phage λ contains some 50 kbp of DNA, and approximately 50 genes have been revealed by analysis of mutants and phage proteins. Most of these genes are essential for perpetuation of the virus in either the lytic or the lysogenic mode of growth. For example, 27 genes are required for the phage to produce mature progeny and form plaques. Five other genes are required for repression, integration, and excision. Several other genes, which form the subject of this paper, are not absolutely required for either lysis or lysogeny. These genes are distributed in several places along the λ genome, and the regions carrying them can be deleted with minimal effects on phage development. Because they are not essential for “normal” phage development in standard hosts and under standard laboratory conditions these genes have been collectively designated as nonessential or dispensable. However, it is becoming clear that these genes are not functionless but, rather, they often have interesting and even essential roles in certain circumstances. Consequently, we prefer to call them accessory genes. In this paper, we review λ ’s accessory genes and discuss their role with respect to both λ and Escherichia coli . Since these genes are dispensable for “normal” λ development, they have not been studied extensively, and our understanding of their expression and function is limited. Nevertheless, we can make a few general statements about their roles. Some appear to be nonessential for phage growth because an analogous or compensating function exists in the host. Others are essential...
TL;DR: The techniques and genetic constructions described support the feasibility of employing similar amplification methods to prepare highly purified thymidylate synthetases from other sources.
TL;DR: MutD5 plasmid or mutD5 transducing lambda phage can be used to convert a wild-type strain to a highly mutable strain and suggested that the dnaQ49 and mutD 5 mutator are mutations that have arisen in a single gene, though they differ in many respects.
TL;DR: Results suggest that P22 repressor, like the related cI repressor of phage lambda, contains two structural domains which mediate different functions of the intact molecule.
TL;DR: This work has identified a minimal region of bacteriophage λ DNA that is necessary for packaging into phage particles and a second region, which is called the enhancing region for packaging, stimulates cutting at cos λ as as packaging.
TL;DR: The cloned bacteriophage T4 pin gene functions to stabilize several different kinds of proteins in Escherichia coli bacteria.
Abstract: The cloned bacteriophage T4 pin gene functions to stabilize several different kinds of proteins in Escherichia coli bacteria. Incomplete proteins such as puromycyl polypeptides, abnormal but complete proteins such as the lambda phage tsO protein, and labile eukaryotic proteins encoded by genes cloned in E. coli such as mature human fibroblast interferon are stabilized in cells in which the T4 pin gene is expressed. The cloned T4 pin gene does not seem to affect the turnover of normal E. coli proteins.
TL;DR: The DNA sequence alterations of 17 lamB point mutations which result in resistance to phage lambda h+.
Abstract: Gene lamB encodes the outer membrane receptor for phage lambda in Escherichia coli K12. We have determined the DNA sequence alterations of 17 lamB point mutations which result in resistance to phage lambda h+. The mutations correspond to four phenotypic classes according to the pattern of growth of three phages which use the lambda receptor: lambda h (a one-step host-range derivative of lambda h+), lambda hh* (a two-step host-range derivative of lambda h+) and K10 (another lambdoid phage). Fourteen mutations are of the missense type and correspond to Gly to Asp changes distributed as follows. One class I mutation is at position 382 of the mature lambda receptor. Seven class I* mutations, four of which at least are independent, are at position 401. Six independent class II mutations are at position 151. The three other (class III) mutations are of the nonsense type. They change codons TGG (Trp) into TAG (amber) at positions 120 (two mutations) and 351 (one mutation). Implications of these results for the topological organization of the lambda receptor as well as possible reasons for the limited number of altered sites detected are discussed.
TL;DR: This chapter describes the purification of Int and discusses the properties of the purified protein, including how a crossover can be carried out by a topoisomerase.
Abstract: Publisher Summary In living systems, recombinant DNA is often formed by a crossover between specific sequences on DNA. The integration of bacteriophage lambda DNA into the chromosome of Escherichia coli is a particularly well-studied example of such site-specific recombination. Cell-free extracts carry out integrative recombination between the specialized recombining site of the phage, attP, and that of the bacterium, attB. In vitro recombination provides a functional assay for the components involved in the integration of phage lambda DNA. This approach has led to the discovery of two proteins that are encoded by the E. coli host: DNA gyrase and integration host factor (IHF). The role of DNA gyrase in integrative recombination is simply to provide a suitably supercoiled substrate DNA. Recombination of supercoiled DNA requires only IHF and a viral protein, Int, the product of the phage int gene. Recent studies indicate that Int carries the active site responsible for the breakage and reunion of DNA at the crossover; IHF plays an important but secondary role. This chapter describes the purification of Int and discusses the properties of the purified protein. A model is also prepared that suggests how a crossover can be carried out by a topoisomerase.
TL;DR: The ability of Gin to catalyze deletions was investigated by inverting in vitro one of the two recombination sites using restriction enzymes and genetically marking the DNA between those sites, although at a lower frequency than inversions.
TL;DR: These two experiments show that, if the DNA of each phage is packaged identically, then the curved-spool model is ruled out and the straight spools model is unlikely, and the manner of packaging the DNA may vary from one phage to the next.
TL;DR: The 7.8-kilobase HindIII insert in phage lambda NM589thyA was confirmed as originating from Escherichia coli by hybridization analysis and was shown to encode the thymidylate synthetase (5,-10-methylenetetrahydrofolate:dUMP C-methyltransferase EC 2.1.45) of E. coli K-12 by using biochemical, structural, and immunologic criteria.
Abstract: The 7.8-kilobase HindIII insert in phage lambda NM589thyA [Borck, K., Beggs, J.D., Brammar, W.J., Hopkins, A.S. & Murray, N. (1976) Mol. Gen. Genet. 146, 199] was confirmed as originating from Escherichia coli by hybridization analysis and was shown to encode the thymidylate synthetase (5,-10-methylenetetrahydrofolate:dUMP C-methyltransferase EC 2.1.1.45) of E. coli K-12 by using biochemical, structural, and immunologic criteria. The 7.8-kilobase insert was reduced in size to a quasi-random population of DNA subfragments by partial digestion with the 4-base-pair recognition enzymes Alu I and Hae III. A clone containing a 1.1- to 1.2-kilobase fragment that encompassed the gene was obtained from this mixture by selecting for Thy+ recombinants. Fusion of this DNA fragment to the phage lambda rho L promoter in plasmid pKC30 revealed the direction of transcription of the thyA gene, and, in a phage lambda lysogen containing a thermolabile repressor, intracellular synthetase levels were increased about 700-fold. The enzyme was purified to homogeneity from this source by affinity chromatography, and some of its properties are described.
TL;DR: The gene for the Harvey murine sarcoma virus (Ha-MuSV) p21ras protein was fused to the amino-terminal portion of the bacteriophage lambda cII gene on the expression vector pJL6, such that transcription was controlled by the well-regulated phage lambda pL promoter and translation continued in frame into the ras gene sequences that code for p21.
Abstract: The gene for the Harvey murine sarcoma virus (Ha-MuSV) p21ras protein was fused to the amino-terminal portion of the bacteriophage lambda cII gene on the expression vector pJL6. The fusion was such that transcription was controlled by the well-regulated phage lambda pL promoter, and translation initiated in the cII gene continued in frame into the ras gene sequences that code for p21. When the pL promoter was derepressed, the Escherichia coli cells harboring the fusion plasmid synthesized 23,000-dalton protein, which represented more than 10 percent of the total cellular protein. This protein was chimeric and contained 14 residues, which were specified by the vector; these residues were followed by all of the amino acids that make up Ha-MuSV p21ras except for four residues at the amino-terminal end. The protein appears similar to Ha-MuSV p21ras in that it undergoes immunoprecipitation by monoclonal antibodies directed toward that protein, binds guanosine diphosphate, and is capable of autophosphorylation.
TL;DR: Complementation analysis with plasmids carrying various DNA fragments derived from pLC3-13 showed that the lSpA locus is between the rpsT and ileS loci, and the wildtype allele was dominant over the lspA allele.
Abstract: A mutation (lspA, prolipoprotein signal peptidase) rendering the prolipoprotein signal peptidase temperature-sensitive in Escherichia coli has been analyzed. The mutation was mapped in the dnaJ-rpsT-ileS-dapB region by interrupted mating with various Hfr strains and P1 phage transduction. lambda transducing phage lambda ddapB2 that carries the rpsT-ileS-dapB region was shown to complement the lspA mutation. Plasmid pLC3-13 which had been isolated from Clarke and Carbon's collection as a plasmid carrying the lspA locus was shown to carry the dnaJ and rpsT loci. Complementation analysis with plasmids carrying various DNA fragments derived from pLC3-13 showed that the lspA locus is between the rpsT and ileS loci. The wildtype allele was dominant over the lspA allele.
TL;DR: This book describes the variety called λ and some of its relatives, which multiply in Escherichia coli, and explains how λ came to be the subject of intensive study.
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...
Patent•
12 Jan 1983
TL;DR: A plasmid cloning vector containing both transcriptional and translational regulatory sequences derived from the bacteriophage lambda genome was constructed to achieve high level expression of prokaryotic and eukaryotic genes.
Abstract: A plasmid cloning vector containing both transcriptional and translational regulatory sequences derived from the bacteriophage lambda genome was constructed to achieve high level expression of prokaryotic and eukaryotic genes. The system utilizes a plasmid vehicle carrying the strong, regulatable lambda promoter, P L , and host lysogens into which this vector can be stabily transformed. The lysogen synthesizes sufficient repressor (cI) to control P L expression and thereby stabilize plasmids which carry such a highly efficient promoter. Use of a temperature sensitive repressor permits simple, rapid induction of P L transcripts at any given time. Efficient transcription of essentially any coding sequence is assured by providing the phage lambda antitermination factor, N, and a site on the transcription unit for its utilization (Nut site). This pAS1 plasmid closely resembles the earlier constructed pKC30cII, also a regulatory protein which activates promoters for lysogenic development.
TL;DR: Investigation of the effect of umuC mutations on error prone repair of singleand double-stranded DNA phages suggests that induced error prone replication of phage DNA can occur viaUmuC dependent (transdimer synthesis) and umUC independent mechanisms.
Abstract: The umuC product of Escherichia coli has been suggested to have a central role in SOS induced error prone replication of DNA (Kato and Shinoura 1977) To investigate this possibility, we examined the effect of umuC mutations on error prone repair of single and double-stranded DNA phages No Weigle reactivation of M13 phage was detected in a umuC mutant Reactivation of lambda phage was reduced but still evident However mutagenesis occurred in both cases These results suggest that induced error prone replication of phage DNA can occur via umuC dependent (transdimer synthesis) and umuC independent mechanisms