Showing papers on "Lambda phage published in 1985"
TL;DR: The results show that chemical induction of PL-containing plasmid expression vectors can serve as a convenient and useful alternative to the commonly used method of heat induction.
Abstract: We have constructed two plasmids in which transcription of the rho gene from Escherichia coli K-12 is under the control of the lambda phage PL promoter. In p31-356, the normal rho promoter is deleted, but the remainder of the rho leader region, including the ribosome binding site, is present. In p39-AS, the rho leader is completely absent, and the lambda cII ribosome binding site replaces that of rho. Under noninducing conditions, expression of rho protein from these plasmids is repressed by the lambda cI protein in hosts carrying lambda cryptic prophage. Induction using mitomycin C or nalidixic acid in a cryptic lysogen carrying the cI+ repressor resulted in the overproduction of rho protein to levels of 3%-5% of the total cellular protein with p31-356, and to levels of approximately equal to 40% with p39-AS. The overproduced protein is functionally indistinguishable from the rho protein isolated from the K-12 strain W3110, and it can be obtained from cells harboring p39-AS in yields of up to 25 mg of rho per g of cells. In contrast to chemical induction, heat induction in four cryptic lambda lysogens carrying the thermolabile cI857 repressor failed to yield the same high levels of rho protein (with either plasmid). Our results show that chemical induction of PL-containing plasmid expression vectors can serve as a convenient and useful alternative to the commonly used method of heat induction.
171 citations
TL;DR: It is shown that activated RecA protein plays a second role in SOS mutagenesis, as revealed by analyzing repair of UV-damaged phage lambda in host mutants with alterations in the SOS regulatory system.
Abstract: Induction of the Escherichia coli SOS system increases the ability of the cells to perform DNA repair and mutagenesis. Previous work has shown that this increased mutagenesis is the result of derepression of specific genes through a complex regulatory mechanism controlled by LexA and RecA proteins. One role of RecA protein in this process is to facilitate proteolytic cleavage of LexA protein (the repressor) in response to an inducing signal that reversibly activates RecA protein to perform this function. We show that activated RecA protein plays a second role in SOS mutagenesis, as revealed by analyzing repair of UV-damaged phage lambda in host mutants with alterations in the SOS regulatory system. First, phage mutagenesis was not expressed constitutively in a mutant that is derepressed through lack of functional LexA protein; activated RecA protein was still required. Second, phage mutagenesis was constitutively expressed in the presence of recA mutations that alter RecA protein so that it is activated in normally growing cells. There was also RecA-dependent constitutive expression of SOS mutagenesis in host mutants that lack functional LexA protein and carry plasmids. We discuss several possible biochemical mechanisms for this second role of activated RecA protein in SOS mutagenesis.
115 citations
TL;DR: It is concluded that the specialized nucleoprotein structure formed by phage lambda O protein and ori lambda provides for localized initiation of DNA replication by serving as the foundation for the assembly of the initial priming structure.
Abstract: The O protein of bacteriophage lambda is required for initiation of DNA replication at the lambda replicative origin designated ori lambda. The binding sites for O protein are four direct repeats, each of which is an inverted repeat. By means of electron microscopy, we have found that phage lambda O protein utilizes these multiple binding sites to form a specific nucleoprotein structure in which the origin DNA is inferred to be folded or wound. The phage lambda O and P proteins and host DnaB protein interact at ori lambda to generate a larger structure than that formed by O protein alone; P and DnaB proteins fail to form any observable complex when O protein is excluded from the reaction mixture. We conclude that the specialized nucleoprotein structure formed by phage lambda O protein and ori lambda provides for localized initiation of DNA replication by serving as the foundation for the assembly of the initial priming structure. Specialized nucleoprotein structures may be a general means to confer exceptional accuracy on DNA transactions requiring extraordinary precision.
112 citations
TL;DR: Heteroduplex analysis in the electron microscope of DNA from four phages revealed substantial secondary structure, including snap-back regions 200-500 base pairs in length, which were not found in phages from the same DNA library that grow in rec+ hosts.
Abstract: The growth of clones of human genomic DNA fragments in a bacteriophage lambda vector has been examined in a number of different Escherichia coli hosts. A large proportion (8.9%) of the phages carrying different fragments of the human genome fail to grow on standard rec+ hosts but will grow on hosts carrying mutations in the recB, recC, and sbcB genes. Heteroduplex analysis in the electron microscope of DNA from four of these phages revealed substantial secondary structure, including snap-back regions 200-500 base pairs in length. Such structures were not found in phages from the same DNA library that grow in rec+ hosts. These results are interpreted in the light of prior observations [Leach, D.R.F. & Stahl, F. (1983) Nature (London) 305, 448-451] showing that inverted repetitions cloned in phage lambda can be propagated in recB recC sbcB hosts but not in rec+ hosts.
109 citations
TL;DR: High-resolution restriction maps derived from a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.
94 citations
TL;DR: A replication competent virus was derived that replicates to high titers in tissue culture cells and stably transduces the bacterial gene that was introduced into the long terminal repeat of the Moloney murine leukemia virus (Mo-MuLV) proviral genome to construct a retrovirus that allows easy cloning of the provirus with flanking host sequences.
Abstract: A bacterial suppressor tRNA gene was introduced into the long terminal repeat of the Moloney murine leukemia virus (Mo-MuLV) proviral genome to construct a retrovirus that allows easy cloning of the provirus with flanking host sequences. A replication competent virus, Mo-MuLV sup containing a tRNA amber suppressor gene, was derived that replicates to high titers in tissue culture cells and stably transduces the bacterial gene. The recombinant virus can efficiently replicate in vivo when microinjected into midgestation embryos or when injected into newborn mice and displays the same tissue tropism as wild-type Mo-MuLV. The suppressor gene in Mo-MuLV sup is functional in bacteria and allows efficient recovery of proviral genomes. This was shown by ligation of DNA from infected cells to phage lambda Charon 4A arms and selective growth of recombinant phages on su- host cells. All recovered phages contained Mo-MuLV proviral sequences and, because of the high cloning capacity of phage lambda, 1-11 kilobases of flanking host DNA. This virus should facilitate studying virus-host interactions in tissue culture cells and in animals.
93 citations
TL;DR: In this article, the phase variation control region is carried on the specialized transducing phage, which is not present on the phage but rather must be supplied by the recipient strain in the transduction.
Abstract: Expression of type 1 fimbriae in Escherichia coli exhibits phase variation, whereby individual cells can alternate between states of organelle expression (Fim+) and nonexpression (Fim-). Strains with a fimD-lac operon fusion, in which lac, rather than fimD, expression is under the control of the fimD promoter, undergo Lac+ in equilibrium Lac- phase variation, instead. After positioning a lambda prophage adjacent to the operon fusion, we were able to isolate specialized lambda phage carrying both the fimD-lac fusion and the phase variation control region. Introduction of such phage into an Fim+ strain resulted in construction of a strain with a double, independently switching phenotype (Fim+ in equilibrium Fim- and Lac+ in equilibrium Lac-), demonstrating that the region controlling phase variation is contiguous with the fimD-lac operon fusion and is cis acting. When the specialized lambda phage was propagated on a delta lac delta fim strain, phase variation occurred within the plaques, confirming that the phase variation control region is carried on the specialized transducing phage. All lysogens acquired the Lac+ in equilibrium Lac- phenotype, except for two nonswitching Lac+ recombinants, which acquired Lac+ in equilibrium Lac- phase variation only by trans complementation with fim. Phase variation of type 1 fimbriae, therefore, appears to involve both a cis-active element, which is cloned on a specialized lambda phage, and a trans-active permissive factor, which is not present on the phage, but rather must be supplied by the recipient strain in the transduction.
79 citations
TL;DR: The precise 3' junction between the bacterial and phage sequences in lambda gal8 is defined and it is demonstrated that this junction probably results from a homologous recombination event between identical 9 bp sequences common to the gal operon andphage lambda.
Abstract: We present the nucleotide sequence of the galactokinase gene (galK) of Escherichia coli including its 5' and 3' flanking regions. This DNA sequence derives from the lambda gal8 transducing phage and is identical to the sequence present in the galK gene fusion vectors, pKO and pKG, commonly used to study transcriptional regulatory elements. We define the precise 3' junction between the bacterial and phage sequences in lambda gal8 and demonstrate that this junction probably results from a homologous recombination event between identical 9 bp sequences common to the gal operon and phage lambda. Moreover, we examine the 300 bp region located immediately beyond galK for transcription termination function and find no gal operon terminator. Lastly, we compare the galK genes of E. coli and the yeast S. cerevisiae and find several regions of strong homology among which is a potential ATP-binding site homology shared by a variety of ATP-binding proteins including protein kinases encoded by mammalian oncogenes.
72 citations
TL;DR: Cylindrical DNA projections were used to model O protein binding to the lambda origin and compare it with the lambda repressor‐operator interaction, and a few non‐symmetrical contacts are found, indicating that O protein may distort the helix.
Abstract: We have characterized the binding of lambda phage replication initiation protein O to the phage origin of replication. The minimal DNA segment required for O binding is the single iteron, a 19-bp sequence of hyphenated dyad symmetry that is repeated with variations four times in the origin. The isolated amino terminus of O protein is also sufficient to bind DNA. Electrophoretic studies show that the amino terminus of O protein induces bending of a single iteron. The DNA-protein interaction was characterized by ethylation interference, dimethyl sulfate protection and neocarzinostatin footprinting. Points of DNA-protein contact are largely concentrated in two areas symmetrically disposed with respect to the dyad symmetry of the iteron. This suggests the protein interacts as a dimer with half sites in the DNA. However, a few non-symmetrical contacts are found, indicating that O protein may distort the helix. This may correlate with the bending effects demonstrated electrophoretically. Cylindrical DNA projections were used to model O protein binding to the lambda origin and compare it with the lambda repressor-operator interaction. Whereas bound repressor nearly encircles the DNA in the major groove, O protein leaves the major groove on the opposite side exposed.
72 citations
TL;DR: It is shown that the E. coli p40mos binds ATP analog p-fluorosulfonylbenzoyladenosine and exhibits ATPase activity.
Abstract: The nucleotide sequence of the Moloney murine sarcoma virus strain HT-1 (HT1MSV) mos gene differs from that of the cellular mos gene in three positions, but these are silent changes, and the amino acid sequence of the v-mos and c-mos open reading frames are identical. We have overproduced the mos HT1MSV (equivalent to c-mos) in Escherichia coli under the control of phage lambda promoter (pL). The E. coli p40mos protein thus obtained was partially purified and examined for several biochemical activities. We show that the p40mos binds ATP analog p-fluorosulfonylbenzoyladenosine and exhibits ATPase activity.
49 citations
TL;DR: A general method for transferring cloned DNA segments onto bacteriophage lambda was developed and the method involves the use of in vivo recombination with a selection and was used to construct two derivatives of lambda SE4.
Abstract: A bacteriophage lambda cloning vector was designed to facilitate the isolation of genes from procaryotic organisms by complementation of Escherichia coli mutants. This vector, lambda SE4, was constructed by attaching a very-low-copy-number replication system (from the plasmid NR1) and a spectinomycin resistance gene to the left arm of lambda 1059 (Karn et al., Proc. Natl. Acad. Sci. U.S.A. 77:5172-5176, 1980). This phasmid cloning vector is capable of growing lytically as a phage in a nonimmune host or lysogenically as a phasmid in an immune host. This phasmid utilizes the Spi- selection for insertions of DNA into the vector and has the ability to accept 2- to 19-kilobase Sau3A1, BamHI, BglII, BclI, or XhoII fragments; recombinants lysogenize immune hosts as single-copy-number selectable plasmids at 100% frequency. An E. coli library was constructed by using the initial vector lambda SE4, and clones of a number of representative genes were identified. A typical clone, lambda ant+, was shown to be readily mutagenized by a mini-Tn10 transposon. A general method for transferring cloned DNA segments onto bacteriophage lambda was developed. The method involves the use of in vivo recombination with a selection and was used to construct two derivatives of lambda SE4. Possible uses of these vectors and of the method for transferring cloned DNA onto phage lambda are discussed.
TL;DR: Immune electron microscopy, performed with antiserum against the hybrid MS2-mycoplasma protein produced in E. coli and corresponding to P90, showed that its antigenic determinant is associated with the myCoplasma surface.
Abstract: A genomic library of Mycoplasma hyopneumoniae was constructed by cloning random DNA fragments approximately 300 base pairs long in a fusion expression plasmid, pEx29, containing the N terminus of the phage MS2 polymerase under the control of the PL promoter of phage lambda. Clones that produced fusion proteins carrying surface-specific antigenic determinants were identified by using antiserum raised in a pig by intranasal inoculation of viable mycoplasmas. Rabbit antisera produced against gel-purified fusion proteins synthesized in Escherichia coli were analyzed by Western blotting to identify antigenically related mycoplasma components. Distinct mycoplasma proteins termed P90, P68, P50, P30, and P26 were identified. Evidence for the surface location of P90, P68, and P50 was provided by their sensitivity to trypsin and their comigration with lactoperoxidase-catalyzed iodinated proteins of intact mycoplasmas. Immune electron microscopy, performed with antiserum against the hybrid MS2-mycoplasma protein produced in E. coli and corresponding to P90, also showed that its antigenic determinant is associated with the mycoplasma surface.
TL;DR: The sizes of the OmpC protein fragments and the results obtained with the hybrid proteins very strongly suggest that the protein is cleaved from outside the cell at a region involving amino acid residues 150-178 of the 346-residue protein, which shows homology to two regions of theOmpA protein which are involved in its phage receptor site.
Abstract: The Escherichia coli K12 outer-membrane proteins OmpA, OmpC, OmpF, PhoE, and LamB (all of transmembrane nature) can serve as phage receptors. We have shown previously that one OmpA-specific phage, Ox2, can give rise to the host range mutants Ox2h10 and Ox2h12, with the latter being derived from the former [Morona, R. & Henning, U. (1984) J. Bacteriol. 159, 579-582]. Unlike Ox2, both host range phages can use the OmpA and OmpC proteins as receptors and Ox2h12 is better adapted to the OmpC protein than Ox2h10. In a search for the site(s) of OmpC protein involved in phage recognition, it was found that proteinase K is able to cleave all of the proteins mentioned above. OmpC protein (Mr = 38306) could be cleaved from outside the cell by proteinase K resulting in two fragments of Mr approximately equal to 21000 and Mr approximately equal to 17500. The use of OmpC-PhoE hybrid proteins allowed us to assign the approximately equal to 21000-Mr fragment to the CO2H-terminal moiety of the protein. Proteinase K treatment of intact cells abolished their activity to neutralize the OmpC-specific phage Tulb and reduced this ability towards phage Ox2h12. The OmpA, OmpF, PhoE and LamB proteins were cleaved by the protease not in intact cells but only when acting on cell envelopes. The sizes of the OmpC protein fragments and the results obtained with the hybrid proteins very strongly suggest that the protein is cleaved from outside the cell at a region involving amino acid residues 150-178 of the 346-residue protein, which shows homology to two regions of the OmpA protein which are involved in its phage receptor site (loc. cit.). These areas also exhibit some homology to a region of the LamB protein which is thought to be part of this protein's receptor site [Charbit et al. (1984) J. Mol. Biol. 175, 395-401]. This suggests that there is a common denominator for proteinaceous phage receptor site because the LamB-specific phage lambda and phage Tulb are of completely different nature. We conclude that the region of the OmpC protein in question is cell-surface-exposed and acts as a phage receptor site.
TL;DR: It is concluded that PaQ functions in concert with PRE and PI to regulate the lysogenic growth response of phage lambda, and presume that the PaQ directed anti-sense Q RNA transcript functions to down-regulate the expression of the Q gene, which is needed for theexpression of all phage late genes during lytic growth.
TL;DR: The presence of repC protein was absolutely essential for the initiation of replication of pT181 and its derivatives in vitro, which is consistent with the size predicted from the DNA sequence data.
TL;DR: The extent of DNA sequence required to form a bacterial attachment site (attB) that functions in bacteriophage lambda integration is investigated and it is found that the common core sequence plus the adjoining 4-bp sequences of both the B and B' arms are required for full activity.
Abstract: We have investigated the extent of DNA sequence required to form a bacterial attachment site (attB) that functions in bacteriophage lambda integration. A DNA fragment carrying attB of Escherichia coli was trimmed, recloned and tested for recombination proficiency. We found that the common core sequence plus the adjoining 4-bp sequences of both the B and B' arms are required for full activity, while plasmids with an even shorter attB sequence retain some capacity to function as attB in vivo. We also found that the nonspecific DNA that is joined to the required attachment site sequence does not significantly influence the rate of the recombination reaction.
TL;DR: A 713-base-pair Hae III fragment from bacteriophage T4 encompassing the denV gene with its preceding promoter has been cloned in a pBR322-derived positive-selection vector and introduced into a variety of DNA repair-deficient uvr and rec and uvr,rec Escherichia coli strains.
Abstract: A 713-base-pair Hae III fragment from bacteriophage T4 encompassing the denV gene with its preceding promoter has been cloned in a pBR322-derived positive-selection vector and introduced into a variety of DNA repair-deficient uvr and rec and uvr,rec Escherichia coli strains. The denV gene was found to be expressed, probably from its own promoter, causing pyrimidine dimer incision-deficient uvrA, uvrB, uvrC strains to be rescued by the denV gene. A uvrD (DNA helicase II) strain was also complemented, but to a lesser extent. A wild-type strain did not seem to be affected at the UV doses tested. Surprisingly, all recA, recB, and recC strains tested also showed an increased UV resistance, perhaps by reinforcement of the intact uvr system in these strains. Complementation of denV- T4 strains and host-cell reactivation of lambda phage was also observed in denV+ E. coli strains. Equilibrium sedimentation showed that DNA repair synthesis occurred in a UV-irradiated uvrA E. coli strain carrying the cloned denV gene. Southern blotting confirmed our earlier results [Valerie, K., Henderson, E. E. & de Riel, J. K. (1984) Nucleic Acids Res. 12, 8085-8096] that the denV gene is located at 64 kilobases on the T4 map. Phage T2 (denV-) did not hybridize to a denV-specific probe.
TL;DR: Results show that in this system, the lesion inducing transitions (the major type of u.v.-induced mutation) is not the cyclobutyl pyrimidine dimer; a strong candidate for a mutagenic lesion is the Pyr(6-4)Pyo photoproduct.
TL;DR: It is found that there is no significant homology to Nu1 protein, which appears to have an analogous or similar function in the DNA maturation of Escherichia coli phage lambda.
Abstract: DNA packaging of Salmonella phage P22 starts at a defined site on a concatemer of P22 genomes. The molecular ends formed at the packaging initiation site (pac) map within a region of ca. 120 base pairs and may contain any of the four nucleotides at their 5' end. The determination of the positions of the cuts within the sequence demonstrates a characteristic distribution of cut sites which apparently cannot be attributed to the sequence organization of the involved regions. Symmetric elements of the sequence might serve as signals for a recognition event(s) at pac in a separate process preceding the cutting reaction. The region of packaging initiation is located within the sequence coding for gene 3. The 3 protein is responsible for the site specificity of this process. We find no significant homology to Nu1 protein, which appears to have an analogous or similar function in the DNA maturation of Escherichia coli phage lambda.
Patent•
10 Oct 1985TL;DR: In this paper, a vector derived from lambda phage is used in combination with a high frequency lysogenic host to clone genes using antibodies as probes and isolating unknown proteins encoded by cloned DNA.
Abstract: Systems are provided for the dual purpose of cloning genes using antibodies as probes and isolating unknown proteins encoded by cloned DNA. The method employs a vector derived from phage which is used in combination with a high frequency lysogenic host. The vector is further characterized by having an inducible promoter regulating expression of a gene into which the foreign DNA may be introduced to produce a fused protein, and controlled induction of the prophage with rapid increase in copy number and high level transcription of foreign DNA. The technique is exemplified with a specific lambda phage construct in conjunction with the β-galactosidase structural gene lacZ.
TL;DR: The bacteriophage Mu transposase (the Mu A gene product), which is absolutely required for both integration of Mu and replicative transposition during the lytic cycle, has been overproduced by cloning the gene on a plasmid under the control of the phage lambda PL promoter.
TL;DR: It is shown that a phage lambda cI+ carrying operator mutations v2 and v3 displays an Inds phenotype, as doeslambda cI inds-1, which confirms that RecA+ protein must be activated to promote prophage induction.
Abstract: Mutants of the cI gene of prophage lambda have been defined phenotypically in a recA+ host as noninducible (Ind-), inducible (Ind+), or induction sensitive (Inds). We showed that a phage lambda cI+ carrying operator mutations v2 and v3 displays an Inds phenotype, as does lambda cI inds-1. We characterized a fourth induction phenotype called induction resistant (Indr). Using these four prophage types, we tested the influence of bacterial recA mutations on prophage induction. Indr prophages were fully induced in recA441 bacteria whose RecA441 protein is activated constitutively. Indr prophages were not induced in a mutant overproducing RecA+ protein, confirming that RecA+ protein must be activated to promote prophage induction. Inds prophages were induced in recA142 and recA453-441 lysogens, previously described as deficient in prophage induction.
TL;DR: It is concluded that the structural gene for the NmpC porin protein is located within the defective qsr' prophage at 12.5 min on the E. coli K-12 map and that this gene can be activated by loss of an insertion element.
Abstract: Recombinants between phage lambda and the defective qsr' prophage of Escherichia coli K-12 were made in an nmpC (p+) mutant strain and in the nmpC+ parent. The outer membrane of strains lysogenic for recombinant qsr' phage derived from the nmpC (p+) strain contained a new protein identical in electrophoretic mobility to the NmpC porin and to the Lc porin encoded by phage PA-2. Lysogens of qsr' recombinants from the nmpC+ strain and lysogens of lambda p4, which carries the qsr' region, did not produce this protein. When observed by electron microscopy, the DNA acquired from the qsr' prophage showed homology with the region of the DNA molecule of phage PA-2 which contains the lc gene. Relative to that of the recombinant from the nmpC (p+) mutant, the DNA molecule of the recombinant from the nmpC+ parent contained an insertion near the lc gene. These results were supported by blot hybridization analysis of the E. coli chromosome with probes derived from the lc gene of phage PA-2. A sequence homologous to the lc gene was found at the nmpC locus, and the parental strains contained an insertion, tentatively identified as IS5B, located near the 3' end of the porin coding sequence. We conclude that the structural gene for the NmpC porin protein is located within the defective qsr' prophage at 12.5 min on the E. coli K-12 map and that this gene can be activated by loss of an insertion element.
TL;DR: The PtsM/Pel protein does not seem to play a role through its transport function, although it is confirmed that it must be present for a successful lambda DNA injection, suggesting that DNA is entering the cell cytoplasm by diffusion.
TL;DR: When a recA-lacZ protein fusion was cloned into phage lambda, the resulting transducing phage grew normally on wild-type Escherichia coli, but its growth was severely inhibited in lexA(Def) mutant strains that express recA constitutively at high levels.
Abstract: When a recA-lacZ protein fusion was cloned into phage lambda, the resulting transducing phage grew normally on wild-type Escherichia coli, but its growth was severely inhibited in lexA(Def) mutant strains that express recA constitutively at high levels. Mutants of the transducing phage that grew on the lexA(Def) strains were isolated and were found to affect production of the RecA-beta-galactosidase hybrid protein. Most mutants, including a number of nonsense mutants, were phenotypically LacZ-. LacZ+ mutants were also isolated; most of these expressed lower basal and induced levels of beta-galactosidase activity. DNA sequence analysis revealed that some of the LacZ+ mutations were in the recA promoter. One of these was found to prevent induction. Unexpectedly, three of the mutations that reduced expression were located in the recA structural gene, at codons 10, 11, and 12. Further analysis of the codon 10 mutant showed that it most likely affected translation since it had little effect on transcription as measured by beta-galactosidase synthesis from a recA-lacZ operon fusion. This expression defect was not limited to the protein fusion, since the codon 10 mutation also reduced synthesis of RecA protein when present in a complete recA gene. Analysis of the recA DNA sequence in the fusion revealed that each of the mutations at codons 10, 11, and 12 increases the homology between this region of the mRNA and a sequence found at codons 1 to 4. Thus, the secondary structure of the mutant recA mRNAs may be affecting translation.
TL;DR: The transformation efficiency of human fibroblast cells by pSV2-gpt DNA trapped in erythrocyte membrane vesicles was less than that of L cells by HSV-TK DNA, but almost the same as that of rat cells byHSV- TK DNA.
TL;DR: The sequence of the mutated pyrI gene is reported and it is shown that, during the generation of this pyrBI-bearing phage, six codons from lambda DNA have been substituted for the eight terminal codons of the wild-type gene.
TL;DR: The results demonstrate that the structural gene coding for type 5 streptococcal M protein which was inserted into lambda DNA expresses immunoprotective epitopes, some of which are shared with human heart tissue.
Abstract: The immunochemical properties of type 5 M protein antigens that were expressed in Escherichia coli K-12 by recombinant lambda bacteriophages isolated from a gene bank of serotype 5 Streptococcus pyogenes have been analyzed in detail. M proteins from partially purified bacteriophage lysates displayed precipitin lines of identity with a purified peptic extract of type 5 M protein (pep M5) in immunodiffusion assays. Immunoblot analyses of the M protein-positive lysates demonstrated that the cloned M protein component resided in five polypeptides with relative molecular weights of 57,900 (57.9K), 55.4K, 52.9K, 40.0K, and 32.6K. The hybrid lambda phage (lambda M5)-produced M protein contained immunoprotective epitopes; lambda M5 protein inhibited opsonization of type 5 streptococci by pep M5 antibodies, and antiserum raised against lambda M5 lysates opsonized type 5 streptococci. Each of the five antigenic polypeptides of the recombinant phage M protein also shared epitopes with human heart tissue, as demonstrated by the reactivity of immunoblots of lambda M5 antigens separated on sodium dodecyl sulfate gels with anti-pep M5 antibodies absorbed to and eluted from human heart sarcolemmal membranes. Moreover, antiserum raised against the lambda M5 lysates reacted with sarcolemmal membrane proteins with relative molecular weights of 200K, 59K, 55K, 53K, and 27K as determined by immunoblot analyses. These results demonstrate that the structural gene coding for type 5 streptococcal M protein which was inserted into lambda DNA expresses immunoprotective epitopes, some of which are shared with human heart tissue.
TL;DR: Observations support the hypothesis, previously based on nut site DNA sequence homology, that the phage lambda and rRNA antitermination systems are related.
Abstract: Transcription of the bacteriophage lambda nutL region from the PL promoter on a multicopy plasmid in Escherichia coli causes a reduction in growth rate and in transcription of rRNA relative both to total transcription and to transcription of tRNAs that are not encoded in rRNA operons. These observations support the hypothesis, previously based on nut site DNA sequence homology, that the phage lambda and rRNA antitermination systems are related.
TL;DR: A DNA fragment carrying 85% of the coding sequence of regA has been cloned into the phage lambda leftward promoter PL expression vector pAS1, and a high level of truncated regA protein was produced by nalidixic acid induction.
Abstract: The bacteriophage T4 regA gene codes for a regulatory protein that controls the expression of a number of T4 early genes, apparently at the level of translation. Restriction fragments containing the regA structural gene have been cloned into phage M13, and the nucleotide sequence has been determined. Translation of the DNA sequence predicted that regA protein contains 122 amino acids, with a Mr of 14,620. A DNA fragment carrying 85% of the coding sequence of regA has been cloned into the phage lambda leftward promoter PL expression vector pAS1, and a high level of truncated regA protein was produced by nalidixic acid induction. Protein chemical studies of the truncated regA protein gave results consistent with the nucleotide sequence of the regA gene. Subsequently, an intact regA gene was cloned into plasmid pAS1 and overexpressed. The regA protein produced in this way regulates the level of T4 45 and 44 proteins when their corresponding genes are carried on the same plasmid as the regA gene.