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Biomedical subjects

N Symonds

Publications and source records attributed to N Symonds.

At least 19 recordsLinked to original sources
PubMed1990

Purification of overexpressed gam gene protein from bacteriophage Mu by denaturation-renaturation techniques and a study of its DNA-binding properties.

Recombinant Mu gam gene protein (Mu GAM) synthesized in Escherichia coli accumulates in the form of insoluble inclusion bodies which, after cell lysis and low-speed centrifugation, can be recovered in the pellet fraction. This property was utilized in a purification procedure for Mu GAM based on guanidine hydrochloride denaturation-renaturation followed by a single DEAE-cellulose chromatographic step. The purified Mu GAM was shown by nitrocellulose-filter-binding experiments to bind with high affinity to linear double-stranded DNA and more weakly to supercoiled and single-stranded forms. Mu GAM protects linear DNA from degradation by a variety of exonucleases, but only weakly inhibits endonuclease activity. These results are in accord with a model of Mu GAM conferring protection from exonuclease activity by binding to the ends of DNA.

Bacteriophage mu↗
PubMed1989

Identification of the bacteriophage Mu kil gene.

The kil gene encoded in bacteriophage Mu DNA was previously shown to reside between the end of the B gene at 4.3 kb and the EcoRI site at 5.1 kb from the left end. To precisely map the kil gene within this region, two series of BAL-31 deletion derivatives were created: one removed Mu DNA rightward from the Hpal site (4.2 kb) and the other removed Mu DNA leftward from the EcoRI site. The deleted Mu DNA was subcloned into the expression vector pUC19 under lac promoter control and tested for the expression of the killing function following IPTG induction. Using DNA sequencing analysis, the Mu DNA in Kil+ and Kil- clones was precisely determined, and the kil gene was mapped to the first open reading frame beyond the B gene. The expression of the kil gene was sufficient to induce dramatic morphological changes: cells became enlarged and predominantly spherical, reminiscent of the phenotype of certain cell mutants.

Bacteriophage mu↗
PubMed1986

Localization of the gam gene of bacteriophage mu and characterisation of the gene product.

Using cloning techniques in conjunction with an in vitro assay for activity of the gam-coded protein (pgam), the gam gene has been located on a 930-bp fragment immediately to the right of an AccI site situated 5.75 kb from the left-hand end of the phage Mu genome. An analysis of the properties of pgam obtained from an overproducing clone indicates that it is a non-specific DNA-binding protein which interacts with linear duplex plasmid DNA having a variety of different termini and confers protection against exonuclease action (Gam function). It also stimulates the frequency with which linear plasmid DNA transforms Escherichia coli to antibiotic resistance (Sot function). The preliminary results reported here suggest that pgam is potentially a useful 'tool' in molecular biology, although the molecular details of pgam activity require further clarification.

Bacterial Proteins↗
PubMed1982

Abnormal cointegrate structures mediated by gene B mutants of phage Mu: their implications with regard to gene function.

A system where the transposition of MupApl (a derivative of phage Mu carrying a determinant coding for ampicillin resistance) is followed from the small plasmid pML2 into the conjugative plasmid R388 has been used to investigate the influence on Mu transposition of B, an early Mu gene which is involved in normal phage DNA synthesis. In the absence of active B protein a low level (about 1% of normal) of transposition was detected. Roughly a third of these transpositional events was found to lead to the formation of cointegrate DNA structures which were shown to consist of R388, two complete copies of Mu and part only of pML2. The pML2 deletions vary in size but all those investigated appear to originate at an end of Mu. An explanation of these observations is proposed which envisages the B protein as part of the normal transposition complex.

Bacteriophage mu↗
PubMed1979

The isolation and characterisation of a plaque-forming derivative of bacteriophage Mu carrying a fragment of Tn3 conferring ampicillin resistance.

We have isolated a plaque-forming derivative of phage Mu which carries a determinant for ApR. The biological properties of this MuAp phage are similar to those of normal Mu. Its genome contains a 1.1 kb substitution where Mu DNA from the right end of the G region has been replaced by a similar length of DNA from the transposon Tn3. This fragment of Tn3 DNA carries the ApR gene, but is no longer capable of independent transposition.

Ampicillin↗