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At least 505 records · Page 28Linked to original sources
PubMed1975

Isolation of coliphage lambda ghosts able to adsorb onto bacterial cells.

We have examined three methods of lambda ghost production, starting with the [3H]leucine-labelled phage, purified by CsCl density gradient sedimentation. Ghosts obtained by the osmotic shock or by incubation in 5 M LiCl do not adsorb on bacteria. Ghosts obtained by the treatment with the chelating agent EDTA and purified by CsCl density gradient sedimentation possess well preserved adsorption properties and are virtually free of DNA and infectious phage particles.

Adsorption↗
PubMed1977

Purification and cloning of a mouse ribosomal gene fragment in coliphage lambda.

We have found and characterized a recombinant between the EK2 vector lambdagtWES.lambdaC and a portion of the mouse ribosomal genes. A 6.6 kb endoR.Eco RI fragment was purified from total mouse DNA using RPC-5 ion exchange chromatography and then cloned and detected twice among 183 hybrid phage screened. In situ hybridization of restriction fragments of the hybrid phage DNA revealed that the inserted fragment contained both 18S and 25S RNA sequences. Electron microscopic analysis further suggested that most, if not all, of the 28S RNA sequence was present in the insert. The orientation of the 28S sequences in the hybrid phage was such that the "sense" of the inserted fragment should be under the control of the leftward promoter of lambda.

Animals↗
PubMed1978

Current status of coliphage lambda EK2 vectors.

This article summarizes the rationale behind the design of standardized laboratory tests for certification of bacteriophage lambda EK-2 vector systems. A discussion and description of the six vector systems which have been certified by the U.S. National Institutes of Health are also included. An appendix describes the officially approved laboratory tests in detail.

Coliphages↗
PubMed1978

Isolation and characterization of transducing coliphage fd carrying a kanamycin resistance gene.

The DNA segment (Tn903) with a size of 3100 nucleotide pairs which carries a gene specifying kanamycin resistance derived from a chimeric plasmid pML21 (Hershfield et al., 1976) was transposed to various sites on the filamentous phage fd DNA. Wild type fd can be restored by excision of Tn903 from the resulting hybrid DNA molecule. The fd DNA carrying Tn903 when converted to the mature phage particle, was capable of transducing the kanamycin marker, and its replicative form DNA could be maintained in a bacterial cell like a plasmid.

Coliphages↗
PubMed1978

Plasmids useable as gene-cloning vectors in an in vitro packaging by coliphage lambda: "cosmids".

A plasmid which contains a cos site of lambda and can be packaged into lambda bacteriophage particles is termed a "cosmid". Such plasmids can be used as gene cloning vectors in conjunction with an in vitro packaging system. The properties of a new series of cosmids based on the ColE1 replicon are described, including small temperature-sensitive plasmids which have lost mobilisation functions and carry no IS sequences. Amongst these plasmids are vectors for XmaI, BglII, BamHI, HindIII, PstI, KpnI, SalI and EcoRI. It is demonstrated that by using cosmids in particular size ranges these plasmids provide a high efficiency cloning system which yields essentially only hybrid clones without resort to a second selection or screening step, and without prior modification (e.g. phosphatase) treatment of the DNA. Attempts were made to optimise the cloning properties of the cosmid system. An Escherichia coli "gene bank" was obtained with an efficiency of 5 . 10(5) clones per microgram of E. coli DNA, and in which any particular unselected marker may be found in about one out of every 400 clones. It was demonstrated that deletion of mobilisation functions leads to loss of ability to form relaxation-complex without affecting copy number or segregation properties of the temperature-sensitive derivatives. The vectors are amplifiable in chloramphenicol to make up about 50% of the total cellular DNA.

Coliphages↗
PubMed1979

A coliphage lambda vector with enhanced biological containment: lambda gtALO.lambda B.

The biological containment of the lambda gt family of cloning vectors has been enhanced by conditionally blocking DNA replication as well as head and tail morphogenesis. The vector, lambda gtALO.lambda B, was constructed by crossing the Oam29, Aama1 and Lam439 mutations into lambda gt.lambda B. The mutation blocking phage DNA replication, Oam29, is suppressed by suII+ or suIII+. The head gene mutation, Aama1, is suppressed by suIII+ but not by suII+ and the tail gene mutation, Lam439, is suppressed by suII+ but not by suIII+. This allows the option of increasing the biological containment by producing heads when a large amount of cloned DNA is being prepared from an individual isolate. A model recombinant, lambda gt Aama1 Lam439 Oam29.KmR' (lambda gtALO.KmR') was constructed and the containment of the vector was evaluated by the series of standardized experiments required for EK2 certification.

Coliphages↗
PubMed1980

Construction and characterization of new coliphage M13 cloning vectors.

New single-stranded DNA cloning vectors have been constructed by the insertion of additional DNA fragments into a HaeII restriction site in the bacteriophage M13 duplex replicative form (RF). These inserts into the M13 genome bring a single restriction sites useful for cloning, including PstI, XorII, EcoRI, SstI, XhoI, KpnI, and PvuII. Drug-resistance genes cloned into M13 include the beta-lactamase (bla) gene and the chloramphenicol acetyl transferase (cat) gene. These vectors provide a convenient means of easily obtaining the separated strands of a cloned duplex DNA fragment by cloning the fragment in each of the two possible orientations. Standard cloning techniques commonly applied to double-stranded DNAs can be utilized to insert foreign DNAs into the duplex RF DNAs of these vectors. Cells transformed by chimeric DNAs extrude filamentous phage particles carrying a circular single-stranded copy of the chimeric viral strand. Because M13-infected cells continue to grow and divide, cells can be transformed to yield either plaques or drug-resistant colonies. Specific inserts are readily detected by plaque hybridization techniques using an appropriate probe. Chimeric viral single strands from virus particles in the supernatant of small volumes of infected cultures can be rapidly and sensitively analyzed by agarose gel electrophoresis to determine the size of an insert.

Cloning, Molecular↗
PubMed1981

DNA sequence of the att region of coliphage 434.

Phages lambda and 434 are related phages that insert at the same site on the Escherichia coli chromosome. A 5.9-kb SalI-BamHI fragment derived from phage 434 was shown to hybridize to a 0.5-kb probe carrying attP-lambda. A 0.8-kb Bam HI-TaqI fragment subcloned into pBR327 was used for sequencing. The sequence of the 500 bp around the insertion site is given here, Comparison of the lambda and 434 sequence shows that the following regions are conserved: the coding sequence for the integrase protein (only 162 bp have been sequenced corresponding to the carboxy terminus), the 15-bp common core at the insertion site, and the three integrase-binding sites flanking the insertion site. The lambda and 434 sequences diverge radically to the left of base-197, suggesting that DNA to the left of that point plays no specific role in insertion or its regulation.

Attachment Sites, Microbiological↗
PubMed1984

CII-dependent activation of the pRE promoter of coliphage lambda fused to the Escherichia coli galK gene.

Using a cloning vector designed for the study of prokaryotic promoters by fusion to the Escherichia coli galactokinase gene (galK), we have constructed a plasmid in which the lambda pRE promoter controls galactokinase expression. A galK- host containing this plasmid has a Gal- phenotype since transcription from pRE requires activation by the lambda CII protein. When CII protein is provided by a prophage, galactokinase is synthesized at a rate dependent on the concentration of CII protein. A second plasmid was constructed in which the pRE promoter from phage 21 controls galactokinase expression. Transcription of the galK gene in this plasmid requires the phage 21 CII protein. Using this system, we demonstrate that the lambda and 21 pRE promoters are highly selective for their corresponding CII proteins. However, a cross-reaction between 21 pRE and the lambda CII protein was observed. In addition, we transferred the pRE-galK fusion unit from the plasmid to a phage, and then to the host chromosome in single copy. Galactokinase expression in this single copy pRE-galK system is also dependent on CII protein, which may be provided from a multicopy plasmid. The high concentration of CII protein provided by the plasmid results in maximal expression of the pRE-galK transcription unit. In this second system low levels of CII activity from CII- mutants are amplified and can be readily detected.

Bacterial Proteins↗
PubMed1984

The construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites of the cI gene of coliphage 434.

A new plasmid vector, pNS1, is described that allows positive selection for bacterial transformants carrying recombinant plasmids. It is a derivative of pBR327, and it includes a regulatory region from the lambdoid phage 434. The expression of the TcR gene of pNS1 is under the control of the ORpR operator-promoter of phage 434, which is regulated by the repressor gene cI. The cloning sites of pNS1 (StuI, NdeI, HpaI, HindIII, AsuII and EcoRI) are situated within cI; hence insertion of foreign DNA into these sites causes depressed expression of the TcR gene from pR thus conferring the TcR phenotype on the harboring Escherichia coli strain. The use of pNS1 is facilitated by the presence of another selectable marker, ApR, its small size, and its known nucleotide sequence; no special host strain is required.

Base Sequence↗
PubMed2006

Intra-laboratory validation of a concentration method adapted for the enumeration of infectious F-specific RNA coliphage, enterovirus, and hepatitis A virus from inoculated leaves of salad vegetables.

Salad vegetables exposed to fecal contamination may cause outbreaks of hepatitis or gastro-enteritis if they are eaten raw. A procedure, based on elution with phosphate-buffered saline and concentration by filtration through membrane filters, was developed for the recovery of enteric viruses from salad leaves. The method was evaluated using lettuce leaves inoculated with hepatitis A virus (HAV), poliovirus, and MS2 bacteriophage. In addition, this method was validated by an intra-laboratory study using leaves of various salad vegetables inoculated with MS2 phage. The French standard NF V 03-110 was used to establish the general principle and the technical protocol of the validation procedure. Linear regression models describing the quantitative reactions were good fits to data in the whole range of viral concentrations tested, which was from about 1 to 4 log plaque-forming units (PFU) per 25 g of lettuce. The fractions of inoculated viruses recovered were estimated to be about 64% for HAV, 18% for poliovirus, and 29% for MS2. No significant effect of the food matrix was found using various types of salad vegetable (butter lettuce, iceberg lettuce, romaine lettuce, witloof chicory, curly endive, corn salad, rocket and watercress). Moreover, the variance of the results was constant for all levels of virus contamination within the experimental range. Intermediate reproducibility experiments were also performed to allow calculation of the uncertainty factor, which was found to be 0.58 log PFU/25 g. When used in association with phage enumeration, this validated procedure is rapid enough to be used for screening salad vegetables for evaluation of the efficacy of processes for control of pathogenic microorganisms on such foods.

Clinical Laboratory Techniques↗