Bacteriophage lambda-based expression vectors.
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Bacteriophage T7↗
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Distribution of sequences common to the 25--28S-ribonucleic acid genes of Xenopus laevis and Neurospora crassa.
The extent of homology between the nucleotide sequence of L-rRNA (the major RNA component of the larger ribosomal subparticle) of a lower eukaryote (Neurospora crassa) and an amphibian (Xenopus laevis) was investigated by utilizing rDNA (DNA coding for rRNA) of X. laevis cloned in plasmids pMB9 and pML2, and rDNA of N. crassa cloned in bacteriophage lambda. Hybridization studies revealed that sequences common to both N. crassa and X. laevis L-rRNA comprise a total of approx. 1050 /+- 200 nucleotides. The thermal stability of the X. laevis rDNA.N. crassa L-rRNA hybrid was 5 degrees C lower than that of the X. laevis rDNA.X. laevis L-rRNA duplex, indicating the presence of fewer than 10% mismatches in homologous sequences. X. laevis rDNA was analysed by means of restriction endonucleases and hybridization with 125I-labelled N. crassa L-rRNA. Most (at least 95%) of the conserved sequences were present in a 3000-base-pair fragment produced by restriction with endonucleases HindIII and BamHI. This fragment, which includes the 3'-OH terminus of the L-rRNA-coding region, was used as an adaptor in the construction of a bacteriophage-lambda recombinant. One section of the recombinant phage terminating in a HindIII-specific site was obtained from bacteriophage lambda plac5 (after restriction with endonuclease HindIII). A second section terminating in a BamHi-specific site was obtained from bacteriophage lambda 540 (after restriction with endonuclease BamHI). These two parts were joined by means of the X. laevis rDNA fragment. Further analysis of cloned rDNA by means of restriction endonucleases confirmed that conserved sequences were widely distributed throughout the 3000-base-pair fragment produced by HindIII and BamHi endonucleases. A 3400-base-pair fragment of N. crassa rDNA cloned in a bacteriophage lambda [Cox & Peden (1979) Mol. Gen. Genet. 174, 17--24] was restricted with endonucleases. The products were hybridized with 125I-labelled X. laevis L-rRNA. Conserved sequences were shown to be distributed over a range of approx. 1600--2700 base-pairs. Hence, in neither X. laevis nor N. crassa L-rRNA can be conserved sequences from a single block; instead regions of high and low (or no) homology must be intermingled. Both N. crassa rDNA and X. laevis rDNA were found to hybridize with Drosophila melanogaster L-rDNA sequences. Those rDNA fragments with sequences common to X. laevis and N. crassa L-rRNA also hybridized with D. melanogaster L-rRNA probe. Thus the same set of conserved sequences may be present in all three species.
Animals↗
Isolation and characterization of the complete chicken beta-globin gene region: frequent deletion of the adult beta-globin genes in lambda.
A library of bacteriophage lambda clones containing chicken chromosomal DNA was screened, using the adult beta-globin cDNA plasmid pHb 1001 as a probe. Sixteen overlapping clones were isolated containing 35 kilobase pairs (kbp) of chicken DNA. Characterization of these clones revealed four beta-like globin genes, some genomically repeated sequences, but no pseudo-genes. The four beta-like genes have an average intergenic distance of less than half of that found for the mammalian beta-like globin gene clusters so far characterized. The overall features of the map were confirmed by genomic Southern analysis. Frequent deletions were shown to occur between the various beta-like globin genes during phage propagation. The presumptive hatching gene in particular was always associated with abnormal lambda clones although we were able to find one such clone that did contain a normal copy of the hatching gene itself. Probably such deletions explain the failure to recover this gene in previous attempts.
Animals↗
Efficient isolation of cDNA clones encoding rheumatoid arthritis autoantigens by lambda phage surface display.
Bacteriophage lambda surface display was used to isolate cDNA clones encoding autoantigens recognized by synovial fluid (SF) or sera from patients with rheumatoid arthritis (RA). We constructed cDNA libraries from human synovial sarcoma cells and synovial tissue, using the surface display vector lambdafoo. The cDNA libraries were screened by affinity selection using 40 SF and 44 sera as probes separately immobilized in microtiter wells. Phage clones isolated encode 13 different autoantigens; an unknown protein, two proteins previously unanalyzed as autoimmune antigens, three proteins previously unknown to be recognized by RA sera, and seven known RA antigens. When analyzed their sensitivity and specificity for RA by phage enzyme-linked immunosorbent assay, frequencies of sera that recognize the newly-isolated autoantigens ranged from 20.5 to 6.8% of a panel of RA sera, and 13.6-0% of other autoimmune disease sera. These results indicate that the lambda phage surface display may be powerful for the isolation of cDNA clones encoding autoantigens recognized by SF or sera from patients with not only RA but also other autoimmune diseases.
Arthritis, Rheumatoid↗
Escherichia coli nusB mutations that suppress nusA1 exhibit lambda N specificity.
The bacteriophage lambda N protein regulates phage development by selectively suppressing transcription termination in its host, Escherichia coli. The E. coli nus mutants prevent N activity. To provide additional information on transcription termination, we have isolated pseudo-revertants of the nusA1 mutation that restore lambda N function. One series of pseudo-revertants lie in the E. coli nusB gene, whose product is normally required for lambda N activity. These mutations are N-specific: mutations that restore lambda N activity do not restore the activity of the analogous N protein of phage 21. Similarly, nusB mutations that restore phage 21 N function are deficient for lambda N function. Mapping of the two classes of mutation is consistent with their location in two distinct domains in the nusB protein. We discuss whether nusB is specific for N protein or for some other component of this regulation system, e.g. the phage site (nut) required for N action.
Bacteriophage lambda↗
Interactions between integrase and excisionase in the phage lambda excisive nucleoprotein complex.
Bacteriophage lambda site-specific recombination comprises two overall reactions, integration into and excision from the host chromosome. Lambda integrase (Int) carries out both reactions. During excision, excisionase (Xis) helps Int to bind DNA and introduces a bend in the DNA that facilitates formation of the proper excisive nucleoprotein complex. The carboxyl-terminal alpha-helix of Xis is thought to interact with Int through direct protein-protein interactions. In this study, we used gel mobility shift assays to show that the amino-terminal domain of Int maintained cooperative interactions with Xis. This finding indicates that the amino-terminal arm-type DNA binding domain of Int interacts with Xis.
Bacteriophage lambda↗
The beta protein of phage lambda promotes strand exchange.
Bacteriophage lambda encodes a 28 kDa protein called beta that binds to single-stranded DNA and promotes the renaturation of complementary single strands. beta Protein fails to bind directly to duplex DNA but remains bound to the DNA product of renaturation that beta itself catalyzes. These observations led to an examination of the ability of beta protein to promote strand exchange. beta Protein caused the replacement of a 43-mer oligonucleotide annealed to M13 circular single-stranded DNA by a homologous 63-mer whose 20 extra nucleotide residues were complementary to the adjacent 3' region of M13 DNA. The role of beta protein in this reaction was manifested in several ways: beta protein pushed the exchange through four to eight mismatches, which blocked exchange mediated by spontaneous renaturation and branch migration; beta imposed a polarity on the strand exchange that was lacking in the spontaneous reaction; and beta remained bound to the heteroduplex product of strand exchange. These observations reveal a mechanism by which a protein can drive strand exchange in one direction without using ATP or any other exogenous source of energy.
Bacteriophage M13↗
Characterization of the interaction between the lambda intasome and attB.
Bacteriophage lambda DNA integrates into the chromosome of Escherichia coli by first forming an intasome at the phage attachment site on the phage DNA with the integrase Int and integration host factor. This intasome searches the host chromosome for the bacterial attachment site (attB) and then orchestrates two sequential strand exchange reactions to achieve integration. This study characterizes the weak interaction of the intasome and attB. The hypothesis that all of the proteins necessary for integration are brought to the reaction site by the intasome is given additional support by showing that the concentration of phage attachment site and not attB determines the optimal concentration of proteins for integration. The value of the dissociation constant of the complex formed between the intasome and attB is determined in two different ways. First, the rate of the integration reaction is measured as a function of the attB DNA concentration. The saturation constant reflects the dissociation constant of the complex. Second, a recombination reaction is inhibited by the introduction of varying amounts of a second attB with a sequence change that blocks recombination with this site. The inhibition constant reflects the dissociation constant of the intasome and altered attB in this experiment. The two methods agree and give a dissociation constant of approximately 300 nM. attB contains two core binding sites for the intasome; it is shown that both are necessary for efficient capture. The value of the dissociation constants are considerably lower when a mutant integrase, IntE174K, is used. This increased affinity for core sites can explain how IntE174K can function in the absence of integration host factor. The inhibition constants also show dependence on the exact sequence of the inhibiting attB. Possible implications of this dependence are discussed.
Bacterial Proteins↗
DNA binding regions of Q proteins of phages lambda and phi80.
Bacteriophage lambda gene Q protein and the related proteins of other lambdoid phages are transcription antiterminators that interact both with DNA in the late gene promoter segment and with RNA polymerase subunits. Using hybrids between Q of lambda and the related Q of phage 80, we characterized elements of both Q and DNA that contribute to the DNA binding function. In particular, we found a C-terminal segment of the protein that is responsible for binding specificity and an approximately 15 residue segment on a predicted alpha helix within this segment at which alanine substitutions decrease DNA binding. We identified a six-nucleotide segment located between the -35 and -10 promoter elements that confers binding specificity and is the site of point mutants that impair binding, and we isolated suppressors in lambda Q that restore binding function by increasing the overall binding affinity. We also identified putative zinc finger structures in both proteins.
Amino Acid Sequence↗
Genetic and biochemical analysis of dimer and oligomer interactions of the lambda S holin.
Bacteriophage lambda uses a holin-endolysin system for host cell lysis. R, the endolysin, has muralytic activity. S, the holin, is a small membrane protein that permeabilizes the inner membrane at a precisely scheduled time after infection and allows the endolysin access to its substrate, resulting in host cell lysis. lambda S has a single cysteine at position 51 that can be replaced by a serine without loss of the holin function. A collection of 27 single-cysteine products of alleles created from lambda S(C51S) were tested for holin function. Most of the single-cysteine variants retained the ability to support lysis. Mutations with the most defective phenotype clustered in the first two hydrophobic transmembrane domains. Several lines of evidence indicate that S forms an oligomeric structure in the inner membrane. Here we show that oligomerization does not depend on disulfide bridge formation, since the cysteineless S(C51S) (i) is functional as a holin and (ii) shows the same oligomerization pattern as the parental S protein. In contrast, the lysis-defective S(A52V) mutant dimerizes but does not form cross-linkable oligomers. Again, dimerization does not depend on the natural cysteine, since the cysteineless lysis-defective S(A52V/C51S) is found in dimers after treatment of the membrane with a cross-linking agent. Furthermore, under oxidative conditions, dimerization via the natural cysteine is very efficient for S(A52V). Both S(A52V) (dominant negative) and S(A48V) (antidominant) interact with the parental S protein, as judged by oxidative disulfide bridge formation. Thus, productive and unproductive heterodimer formation between the parental protein and the mutants S(A52V) and S(A48V), respectively, may account for the dominant and antidominant lysis phenotypes. Examination of oxidative dimer formation between S variants with single cysteines in the hydrophobic core of the second membrane-spanning domain revealed that positions 48 and 51 are on a dimer interface. These results are discussed in terms of a three-step model leading to S-dependent hole formation in the inner membrane.
Alleles↗
Mutants of Escherichia coli that do not contain 1,4-diaminobutane (putrescine) or spermidine.
Strains of Escherichia coli K12 have been constructed which do not contain any of the polyamines normally present in a wild type strain, namely, 1,4-diaminobutane (putrescine) and spermidine. This phenotype arises as a consequence of the assembly into these strains of deletion mutations in speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), and speD (adenosylmethionine decarboxylase). The polyamine-deficient strains grow indefinitely in the absence of polyamines but with a growth rate one-third of that found in the presence of polyamines. These strains can act as hosts for bacteriophages T4, T7, and f2, although the latter phage is poorly adsorbed; they can also maintain F' factors, ColE1 and P1 plasmids, and lysogeny by bacteriophage lambda. In contrast, the production of bacteriophage lambda in the absence of polyamines is strikingly decreased (greater than 99%) either after infection of a nonlysogen or after induction of a lysogen. A polyamine-deficient Hfr strain can transfer its chromosome to a recipient at a normal rate, but the number of recombinants observed in a cross is decreased approximately 300-fold. No such effect is observed when only the F- recipient strain in a cross is polyamine deficient.
Amino Acids↗
Effects of condensing agent and nuclease on the extent of ejection from phage lambda.
We have recently demonstrated, that DNA ejection from bacteriophage lambda can be partially or completely suppressed in vitro by external osmotic pressure. This suggests that DNA ejection from phage is driven by an internal mechanical force consisting of DNA bending and DNA-DNA electrostatic repulsion energies. In the present work we investigate the extent to which DNA ejection is incomplete at zero osmotic external pressure when phage is opened with its receptor in vitro. The DNA fragment remaining in the capsid and the tail that is no longer bent or compressed -and hence for which there is no internal driving force for ejection- is shown not to be ejected. We also demonstrate that DNA can be "pulled" out from the capsid by DNase I acting as a DNA binding protein or spermine acting as a DNA condensing agent. In particular, cryo electron microscopy and gel electrophoresis experiments show the following: (i) DNA ejection from bacteriophage lambda incubated in vitro with its receptor is incomplete at zero external osmotic force, with several persistence lengths of DNA remaining inside the phage capsid, if no nuclease (DNase I) or DNA condensing agent (spermine) is present in the host solution; (ii) in the presence of both DNase I and spermine in the host solution, 60% (approximately 29 kbp) of wild-type lambda DNA (48.5 kbp) remains unejected inside the phage capsid, in the form of an unconstrained toroidal condensate; (iii) with DNase I added, but no spermine, the ejection is complete; (iv) with spermine, but without DNase I added, all the DNA is again ejected, and organized as a toroidal condensate outside.
Bacteriophage lambda↗
Isolation and characterization of recombinant lambda gt11 bacteriophages expressing four different Mycobacterium intracellulare antigens.
Four bacteriophages expressing different immunoreactive recombinant Mycobacterium intracellulare antigens were isolated from a lambda gt11 library with monoclonal antibodies to M. intracellulare. These four antibodies reacted with native M. intracellulare proteins of 54, 43, 40/38, and 22 kilodaltons. Southern blot hybridizations with DNA probes prepared from insert fragments of these bacteriophages confirmed the M. intracellulare derivation of the inserts. The physical maps of the immunoreactive phages were deduced by restriction enzyme digestions. The molecular weights of the expressed recombinant antigens were determined by Western (immuno-) blotting.
Antibodies, Bacterial↗
Structural studies of lambda transducing bacteriophage carrying bacterial deoxyribonucleic acid from the metBJLF region of the Escherichia coli chromosome.
The structures of several lambda dmet and related lambda darg transducing phage were studied by restriction fragment mapping and electron microscopic measurements of homoduplexes and heteroduplexes. A new transducing phage (lambda dmet141), in which metF is the only functional gene of the cluster, was isolated. In contrast, lambda dmet117, which expresses the entire metBJLF cluster, has only 3 kilobases more bacterial deoxyribonucleic acid (DNA) than lambda dmet141. An EcoRI restriction fragment of lambda dmet117, which carries the leftmost 6 kilobases of the bacterial DNA insert, was isolated and shown to contain a functional copy of metB. Small structural differences at the attachment sites of some of the phage were shown to result from different sites of lambda integration in the two parent insertion lysogens.
Attachment Sites, Microbiological↗
Engineering of a vaccinia virus bacterial artificial chromosome in Escherichia coli by bacteriophage lambda-based recombination.
The large capacity of vaccinia virus (VAC) for added DNA, cytoplasmic expression and broad host range make it a popular choice for gene delivery, despite the burdensome need for multiple plaque purifications to isolate recombinants. Here we describe how a bacterial artificial chromosome (BAC) containing the entire VAC genome can be engineered in Escherichia coli by homologous recombination using bacteriophage lambda-encoded enzymes. The engineered VAC genomes can then be used to produce clonally pure recombinant viruses in mammalian cells without the need for plaque purification.
Bacteriophage lambda↗
Analysis of the different molecular forms of penicillin-binding protein 1B in Escherichia coli ponB mutants lysogenized with specialized transducing lambda (ponB+) bacteriophages.
Penicillin-binding protein (pbp) 1b, the main DD-transpeptidase/transglycosylase of Escherichia coli, is normally present in the cell in three molecular forms alpha, beta and gamma, differentiated by their mobility in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The three molecular forms are enzymatically active in vitro and their relative amounts are kept fairly constant in most labelling experiments with radioactive beta-lactam antibiotics. In this paper, we have analyzed the expression of ponB (mrcB), the structural gene for pbp 1b, and the relation among the three forms of pbp 1b in ponB strains lysogenyzed by lambda 540 (ponB+) recombinant bacteriophages. Our data indicate that ponB is transcribed anti-clockwise on the E. coli chromosome and suggest that pbp 1b alpha is the first membrane-bound form of pbp 1b able to bind labelled beta-lactams, and is the precursor of pbp 1b beta which is, in turn, the precursor of pbp 1 beta gamma.
Acyltransferases↗
Initiation of lambda DNA replication with purified host- and bacteriophage-encoded proteins: the role of the dnaK, dnaJ and grpE heat shock proteins.
Based on previous in vivo genetic analysis of bacteriophage lambda growth, we have developed two in vitro lambda DNA replication systems composed entirely of purified proteins. One is termed 'grpE-independent' and consists of supercoiled lambda dv plasmid DNA, the lambda O and lambda P proteins, as well as the Escherichia coli dnaK, dnaJ, dnaB, dnaG, ssb, DNA gyrase and DNA polymerase III holoenzyme proteins. The second system includes the E.coli grpE protein and is termed 'grpE-dependent'. Both systems are specific for plasmid molecules carrying the ori lambda DNA initiation site. The major difference in the two systems is that the 'grpE-independent' system requires at least a 10-fold higher level of dnaK protein compared with the grpE-dependent one. The lambda DNA replication process may be divided into several discernible steps, some of which are defined by the isolation of stable intermediates. The first is the formation of a stable ori lambda-lambda O structure. The second is the assembly of a stable ori lambda-lambda O-lambda P-dnaB complex. The addition of dnaJ to this complex also results in an isolatable intermediate. The dnaK, dnaJ and grpE proteins destabilize the lambda P-dnaB interaction, thus liberating dnaB's helicase activity, resulting in unwinding of the DNA template. At this stage, a stable DNA replication intermediate can be isolated, provided that the grpE protein has acted and/or is present. Following this, the dnaG primase enzyme recognizes the single-stranded DNA-dnaB complex and synthesizes RNA primers. Subsequently, the RNA primers are extended into DNA by DNA polymerase III holoenzyme. The proposed model of the molecular series of events taking place at ori lambda is substantiated by the many demonstrable protein-protein interactions among the various participants.
Bacterial Proteins↗
Effects of recB21, recF143, and uvrD152 on recombination in lambda bacteriophage-prophage and Hfr by F- crosses.
The effects of the mutation pairs recB21 recF143 and recB21 uvrD152 on the frequency of genetic recombination were investigated in lambda phage-prophage crosses under homoimmune conditions. To prevent recombinants from being formed by the phage red system, these experiments were performed with phages and prophages carrying red and gam mutations. Both spontaneous and damage-induced recombination was measured, the phages being either undamaged or treated with trimethylpsoralen and 360-nm light to cross-link the phage DNA. Control and damaged phages were allowed to infect lysogenic host cells under conditions in which phage gene expression was repressed and phage DNA replication was blocked by lambda immunity. Although the double mutations recB21 recF143 and recB21 uvrD152 reduced recombination in Hfr by F- crosses to 0.3 to 0.02% of the wild-type controls, the presence of these pairs of mutations in the host lysogens had relatively little effect on the results of the phage-prophage crosses. In the latter system, recB21 recF143 reduced spontaneous and damaged-induced recombination by less than threefold whereas recB21 uvrD152 increased it to three times the wild-type level, the increase being attributable to the uvrD mutation. Evidently, the gene products of recB,C uvrD, and recF wee not needed for lambda phage-prophage recombination under repressed conditions.
Bacteriophage lambda↗