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PubMedDate not supplied

Host cell reactivation of CAT-expression vectors as a method to assay for cloned DNA-repair genes.

We demonstrate the feasibility of using passive host-cell reactivation of a shuttle-vector pRSVcat to detect cloned DNA-repair genes. As models, a transient expression vector, pRSVdenV, and a positive-selection vector, pRSVdenV/SVgpt, were constructed containing the T4 coliphage denV gene, coding for an ultraviolet-specific endonuclease, under promotion of the Rous sarcoma virus (RSV) long-terminal repeat. Cotransfection of one or three copies of pRSVdenV per UV-irradiated pRSVcat molecule into xeroderma pigmentosum (XP) cells (XP12Ro[M1]) resulted in a dramatic increase in transient expression of chloramphenicol acetyl transferase (CAT) activity. XP clones stable transformed by pRSVdenV/SVgpt but not the parent cell line rescued CAT activity from this UV-irradiated reporter gene. The ability to express CAT activity from a UV-irradiated pRSVcat correlated with the presence of the structural denV gene as detected by Southern blot analysis. Post-UV irradiation colony-forming ability and DNA nucleotide excision-repair synthesis were partially restored in XP clones which rescued CAT activity. These results demonstrate the feasibility of using the cloned denV gene with its well characterized pyrimidine cyclobutane dimer-specific endonuclease activity to reconstitute UV-induced DNA repair in human cells deficient in DNA repair. Measuring CAT expression from pRSVcat affords a rapid, sensitive procedure to screen for functional cloned DNA-repair genes and to test mutant cells for defects in DNA repair.

Amino Acid Sequence↗
PubMed1995

Simple concentration method for bacteriophages of Bacteroides fragilis in drinking water.

A membrane of inorganic material with a honeycomb pore structure was used to concentrate phages infecting Bacteroides fragilis from drinking water. Phages were removed from the membrane with 0.25 M glycine buffer pH 9.5. Phages were not inactivated by storage in this buffer neutralized to pH 7.0 for at least 9 days at 4 degrees C. The method allows recovery of around 50% in drinking water. When the turbidity of the water increased, the efficiency of the concentration decreased markedly. The efficiency of concentration was evaluated versus a presence/absence test in 317 water samples with turbidity level below the threshold of drinking water. Results obtained by concentration of 11 provided data which were significantly more informative than the presence/absence tests carried out on 100 ml. A number of additional tests carried out with both somatic and F-specific coliphages indicated that these conclusions can be extended to these groups of bacteriophages.

Bacteriophages↗
PubMed1986

Identification of sites of cysteine misincorporation during in vivo synthesis of bacteriophage T7 0.3 protein.

The 0.3 protein encoded by coliphage T7 does not normally contain cysteine residues. Incorporation of [35S]cysteine can therefore be used to assay mistranslation. We have purified 0.3 protein, synthesized in the presence of [35S]cysteine, from T7 infected cells of E. coli and determined the locations of misincorporated cysteine residues. Analysis of the molecular weights (Mr) of [35S]cysteine-labeled tryptic peptides of 0.3 protein demonstrated that cysteine (encoded by UGU or UGC) is not extensively misincorporated, as might be predicted by substitution for arginine residues (encoded by CGU or CGC). Edman degradation of the amino-terminal 50 residues of [35S]cysteine-labeled 0.3 protein determined that cysteine was most frequently misincorporated at position 15, which is correctly occupied by a tyrosine residue (encoded by UAC). There are four other tyrosine codons (1 UAU; 3 UAC) in the region of the 0.3 protein studied, but these were not mistranslated. The context in which a codon is located must therefore be more important in causing mistranslation than the sequence of the codon itself. Misincorporation of [35S]cysteine was also found at positions 9 (ACC, asparagine), 16 (GAA, glutamic acid), 41 (GCC, alanine) and 42 (GAU, aspartic acid). One mistranslation event appears to increase the likelihood that the following codon will also be mistranslated. This clustering of misincorporated [35S]cysteine residues was accentuated in 0.3 protein synthesized in the presence of streptomycin.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and expression of the xylanase gene from Chainia in Escherichia coli.

A complete genomic library of Chainia was constructed in coliphage lambda vector gt10 and was screened for the xylanase gene using an 18-mer mixed oligonucleotide probe corresponding to a six-amino acid sequence of low molecular mass Chainia xylanase. Inserts from 11 putative clones, showing hybridization with the oligonucleotide probe at medium stringency, were subcloned in pUC8 and screened for xylanase gene expression using anti-xylanase antibodies. The restriction map of the insert (1.4 kb) from one of the four immunopositive clones (PVX8) showing detectable xylanase activity was constructed. The xylanase activity of PVX8 was not induced by IPTG or xylan. Reorientation of the insert by directional cloning into pUC9 had no effect on the xylanase activity suggesting that an indigenous promoter from Chainia is responsible for the xylanase activity.

Actinomycetales↗
PubMed1980

Site-specific mutagenesis using oligodeoxyribonucleotides: isolation of a phenotypically silent phi X174 mutant, with a specific nucleotide deletion, at very high efficiency.

A decadeoxyribonucleotide, pAAATCCCTCA, was synthesized to complement nucleotides 2920--2930 of coliphage phi X174 viral DNA except that the nucleotide corresponding to position 2925 is deleted. The phage DNA sequence in this region codes for the ribosome-binding site of gene H. The oligodeoxyribonucleotide was integrated into double-stranded phi X174 DNA by using it as a primer for DNA polymerase with wild-type DNA template followed by ligation. The resultant heteroduplex DNA was used to transfect Escherichia coli spheroplasts and progeny bacteriophage were isolated. The synthetic decadeoxyribonucleotide was then used to enrich mutant viral DNA from which nucleotide 2925 had been deleted. After three cycles of enrichment and spheroplast transfection only mutant DNA was detectable. The deletion of nucleotide 2925 from the phi X174 genome seriously disrupts a sequence complementary to the 3'-terminus of the 16S rRNA of E. coli and also eliminates a translation termination codon from that sequence. However, the mutant grows normally with no readily perceptible phenotype. Thus, a short synthetic oligodeoxyribonucleotide has been used to construct, and to isolate with 100% efficiency, a mutant for which there is no biological selection procedure.

Bacteriophage phi X 174↗
PubMed1983

Use of lambda phasmids for deletion mapping of non-selectable markers cloned in plasmids.

A nonselectable gene carried on a poorly selectable recombinant plasmid has been physically mapped by deletion analysis. Our method involved cloning the plasmid into a coliphage lambda vector and treating the recombinant phage with a chelator. Virtually all particles surviving this treatment carried large deletions within the plasmid insert. Further deletion analysis was done by inserting a selectable lambda sequence into one such deletion derivative and repeating the chelator selection. Chelator selection was also used to isolate deletions constructed in vitro. The deleted phage are readily characterized by restriction mapping, and the gene in question scored after infection of a mutant host strain. These techniques have enabled us to physically assign the cyclopropane fatty acid synthase gene of Escherichia coli to 0.8 kb of a 16-kb segment after characterizing only a small number of isolates. This approach should be generally useful in the mapping of plasmids for which no convenient method exists for selecting or scoring the gene in question.

Bacteriophage lambda↗
PubMed1984

Sequence analysis of a region from the early right operon in phage P22 including the replication genes 18 and 12.

A comparison of a 3000-bp sequence of Salmonella phage P22, coding for gene c1 and the replication genes 18 and 12, with the analogous cII-O-P region of coliphage lambda permits the localization of transcriptional signals, an oop RNA species, and the origin of replication (ori) within gene 18. Gene c1 and the amino terminal region of gene 18 show homology to the respective lambda genes. In the ori domain of the replicator proteins the homology to phi 82 is most pronounced. Of two lambda:repP22 hybrids (lambda with replication genes of P22) analysed, one codes for a hybrid O/18 protein with 30 N-terminal amino acids coded for by lambda. Gene 12 is nonhomologous to its lambda counterpart (gene P), but closely related to dnaB of Escherichia coli. A ren gene is missing, whereas two open reading frames (ORFs) distal to gene 12 are almost identical to those in the lambda nin region. We try to account for the occurrence and location of highly conserved sequences among the lambdoid phages by assigning them a role in recombinational reassortment of functional units during the evolution of this phage family.

Bacteriophage lambda↗
PubMed1984

Thermo-inducible gene expression in Bacillus subtilis using transcriptional regulatory elements from temperate phage phi 105.

A Bacillus subtilis/Escherichia coli shuttle plasmid vector containing a transcriptionally silent chloramphenicol acetyl transferase gene (cat-86) was constructed by ligation of pPL603 (Williams et al., 1981a) and pUC8 (Vieira and Messing, 1982) at their unique EcoRI sites. Using this "promoter probe" vector we have obtained, by direct Cm resistance selection, a collection of cloned Sau3A fragments from the temperate phage phi 105 genome exhibiting promoter activity in B. subtilis. 18 promoter plasmids were subsequently transferred to an acceptor cell containing a functional repressor gene of phage phi 105 inserted into the temperature-sensitive replicon pE194. A repressor-controlled promoter was identified on the basis of its ability to confer thermo-inducible Cm resistance. The promoter is located on a 650-bp Sau3A fragment, mapping within the 3.2-kb EcoRI-F fragment, which also contains the phi 105 repressor gene. By assaying cloned subfragments of EcoRI-F for expression of immunity against phi 105 infection, the repressor gene could be assigned to a 1.1-kb EcoRI-HindIII fragment, which partially overlaps the promoter fragment. Taken together, these results suggest that, like the cI-coded repressor in coliphage lambda, the phi 105 repressor interacts with an operator sequence mapping very close to its own gene.

Acetyltransferases↗
PubMed1991

Promoter sequence analysis in Bacillus and Escherichia: construction of strong promoters in E. coli.

Many derivatives of the nprM promoter of Bacillus stearothermophilus and the strong early promoter, A3, of coliphage T3 were designed and chemically synthesized. These promoters consisted of some or all of the AT box, consensus sequence, tac promoter sequence, spacer, and lac operator. The promoter activities were assessed by their ability to express the cat gene in Escherichia coli. One of the derivatives of the A3 promoter, which contained the lac operator, was much stronger (about 3.5 times) than the tac promoter. The promoter activities in E. coli were considerably modified by the substitutions in the -43 (AT box) and -35 regions.

Base Sequence↗
PubMed1991

Bacillus subtilis DNA polymerase III: complete sequence, overexpression, and characterization of the polC gene.

Genomic DNA encompassing polC, the structural gene specifying Bacillus subtilis DNA polymerase III (PolIII), was sequenced and found to contain a 4311-bp open reading frame (ORF) encoding a 162.4-kDa polypeptide of 1437 amino acids (aa). The ORF was engineered into an Escherichia coli expression plasmid under the control of the coliphage lambda repressor. Derepression of E. coli transformants carrying the recombinant vector resulted in the high-level synthesis of a recombinant DNA polymerase indistinguishable from native PolIII. N-terminal aa sequence analysis of the recombinant polymerase unequivocally identified the 4311-bp ORF as that of polC. Comparative aa sequence analysis indicated significant homology of the B. subtilis enzyme with the catalytic alpha subunit of the E. coli PolIII and, with the exception of an exonuclease domain, little homology with other DNA polymerases. The respective sequences of the mutant polC alleles, dnaF and ts-6, were identified, and the expression of specifically truncated forms of polC was exploited to assess the dependence of polymerase activity on the structure of the enzyme's C terminus.

Alleles↗
PubMed1991

A surface expression vector for antibody screening.

To select specific antibodies (Ab) from large recombinant libraries using small amounts of antigen, we have constructed a phagemid that expresses a single-chain Ab fused to pIII, a coliphage protein product of gene III that initiates infection by binding to F pili. Surprisingly, the production of the fusion protein (Ab::pIII) was induced by wild-type (wt) phage fd in the absence of IPTG. Ab::pIII was identified by a monoclonal Ab to an epitope in the linker sequence between the heavy and light chains, and by antisera to their N-terminal sequences. It is able to bind antigen and be assembled into infectious phagemid particles that can be enriched on columns of immobilised antigen. The phagemid DNA is even smaller than that of wt fd phages and can easily be propagated in plasmid form. Most importantly, its Ab::pIII-encoding gene can be tightly repressed so that Ab libraries can be amplified without risk of being dominated by deletion mutants. After induction, however, large quantities of the fusion protein can be produced, thus greatly facilitating its analysis.

Amino Acid Sequence↗
PubMed1992

The Escherichia coli Mu/D108 phage ner homologue gene (nlp) is transcribed and evolutionarily conserved among the Enterobacteriaceae.

The Escherichia coli nlp gene is highly homologous to the regulatory ner genes of transposable coliphages, Mu and D108. It was discovered, via its action when overexpressed, as a positive activator of mal gene expression in a cya- crp*1 strain. Chromosomal disruption of the nlp gene by insertion of a promoterless luxAB reporter gene revealed that nlp is nonessential for E. coli viability. Light measurements from the resulting nlp::luxAB transcriptional fusion, plus RNA dot blot analysis, suggest that nlp is transcribed. Southern-blot analyses of DNAs from several bacterial species were performed and indicate that nlp is evolutionarily conserved, but only among closely related Enterobacteriaceae.

Bacteriophage mu↗
PubMed1993

A revised strategy for cloning antibody gene fragments in bacteria.

The ability to clone and overexpress genes encoding mouse Fab (antigen-binding fragment) proteins in bacteria led to the development of a methodology which has the potential to replace traditional hybridoma technology [Huse et al., Science 246 (1989) 1275-1281]; however, several observations have suggested that clones with desirable chemical properties may be missed in immunoscreens of large combinatorial libraries due to low levels of functional Ab protein. To increase the efficiency of cloning and characterization of Ab gene fragments, we have reconsidered several features of the original cloning vehicles. These studies show that at the present time a unique expression system cannot adequately accommodate the requirements of plaque-lift immunoassays for clonal selection and biochemical assays for further characterization in vitro. A monocistronic arrangement of heavy- and light-chain-encoding genes using two lacP promoters produces sufficient amounts of functional Ab protein for clonal selection from phage lambda libraries and minimizes interference with the lytic cycle of recombinant vectors. In liquid culture, a strong coliphage promoter and a relatively abundant RNA polymerase can be used to produce quantities of Ab protein sufficient for further characterization in vitro. A rapid purification protocol obviates the need for fusing heavy-chain protein to a decapeptide sequence, an affinity-tail sequence which slows the folding and assembly of the Ig heterodimer. These results have been used to formulate a new strategy for cloning and characterization of Ab gene fragments in bacteria.

Amino Acid Sequence↗
PubMed1993

Circularly permuted DNA, RNA and proteins--a review.

Circular permutation represents a form of macromolecular isomerization when the normal termini are covalently linked and new termini introduced by breaking the backbone elsewhere. Here, we describe implications of circular permutation on the folding and function of biologically relevant macromolecules. A method permitting the analysis of the folding of all circularly permuted isomers of RNA is presented that has been successfully applied for a tRNA and the binding site of the coliphage R17 coat protein.

Animals↗
PubMed1995

Identification of a gene encoding a bacteriophage-related integrase in a vap region of the Dichelobacter nodosus genome.

Dichelobacter nodosus is the principal causative agent of ovine footrot. Nucleotide (nt) sequences from the D. nodosus genome have been isolated and a series of overlapping lambda clones defining vap (virulence-associated protein) regions 1, 2 and 3 have been reported [Katz et al., J. Bacteriol. 176 (1994) 2663-2669]. In the present study, the limits of the virulence-associated (va) DNA around vap regions 1 and 3 were determined by dot-blot hybridization experiments using plasmid subclones to probe genomic DNA from the D. nodosus virulent strain A198 and the benign strain C305. This va region was found to be approx. 11.9 kb in length, and to be interrupted by a short DNA segment which is also found in the benign D. nodosus strain. Sequence analysis of the entire region revealed an ORF, intA, which is very similar to the integrases of bacteriophages phi R73, P4 and Sf6. Bacteriophages phi R73 and P4 integrate into the 3' ends of tRNA genes, with the integrase genes adjacent to the tRNA genes. A similar arrangement was found in the D. nodosus va region. A 19-bp nt sequence was found to be repeated at the ends of the va region, and may represent the bacteriphage attachment site. These findings suggest that D. nodosus may have acquired these DNA sequences by the integration of a bacteriophage, or an integrative plasmid that contains a bacteriophage-related integrase gene. The high similarity of the D. nodosus integrase to integrases from coliphages suggests that these va sequences may be transferred between distantly related bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

Genetic and sequence analysis of the cos region of the temperate Pseudomonas aeruginosa bacteriophage, D3.

The location and structure of the cos ends of bacteriophage D3, which infects Pseudomonas aeruginosa strain PAO, has been determined using a combination of deletion analysis, transposon mutagenesis, and sequencing directly off the phage DNA. Phage D3 was found to have 9-bp 3' cos ends, making it the first phage of a Gram-negative organism known to have 3' cos ends. A 700-bp region flanking the cos site was necessary for efficient transduction of D3 cosmid derivatives. This region was found to contain incomplete inverted repeat sequences flanking the cos site, along with adenine-rich repeats homologous to coliphage gama Ter binding sites. Possible IHF binding sites were also present.

Base Sequence↗
PubMed1996

Genetic analysis of the replication region of the Lactobacillus plasmid vector pPSC22.

The sequence and genetic organization of the 1,600-bp replication region of the Lactobacillus vector pPSC22, a plasmid derived from a 7-kb cryptic plasmid of L. plantarum used for the cloning of heterologous genes in several lactobacilli, were determined. Sequence analysis revealed the presence of a plus origin of replication containing the two functional elements nic and bind, required for initiation of the leading strands typical of the rolling circle (RC)-replicating plasmids belonging to the pLS1 family. Two open reading frames (copA and repA) were located within the Lactobacillus portion of pPSC22. The repA gene product, a 234-amino acid protein, showed homologies with the Rep protein of the streptococcal plasmid pLS1 and contained the three conserved domains detected in most Rep proteins of RC-replicating plasmids and ss-coliphages. The genetic organization of the replication region of pPSC22 shared relevant homologies with the lactococcal plasmids pWVO1 and pFX2.

Base Sequence↗
PubMed1992

Peptide screening.

Since late 1990, there have been several advances in preparing and screening large numbers of various peptides. Developments have continued in methods of peptide screening based on peptides exposition on coat proteins, produced via fusion coliphage constructs. Further developments have been made in increasing the multitude of peptides produced by the chemical synthetic strategy, including light-directed, spatially addressable chemical synthesis, single-bead, single-peptide synthesis, as well as iterative peptide selection and synthesis.

Amino Acid Sequence↗