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Biomedical subjects

J Davison

Publications and source records attributed to J Davison.

At least 19 recordsLinked to original sources
PubMed1992

Cloning and sequencing of Pseudomonas genes determining sodium dodecyl sulfate biodegradation.

The nucleotide sequences of two genes involved in sodium dodecyl sulfate (SDS) degradation, by Pseudomonas, have been determined. One of these, sdsA, codes for an alkyl sulfatase (58,957 Da) and has similarity (31.8% identity over a 201-amino acid stretch) to the N terminus of a predicted protein of unknown function from Mycobacterium tuberculosis. The other gene, sdsB, codes for a positive activator protein (33,600 Da) that has extensive similarity with the lysR family of helix-turn-helix DNA-binding activator proteins.

Amino Acid Sequence↗
PubMed1991

Transposon vectors for stable chromosomal integration of cloned genes in rhizosphere bacteria.

A series of Tn5-based transposon-cloning vectors, in which many unique restriction sites lie within the transposon, have been constructed. These transposon vectors can be delivered, by conjugation, using a delivery vehicle containing a pBR322 replicon and the mobilization genes of plasmid RK2. In Pseudomonas sp., this delivery vehicle acts as a suicide plasmid, permitting transposition to the chromosome to be detected. To facilitate cloning into the transposon vector, the delivery vehicle has been simplified so that the useful cloning sites in the transposon are not duplicated. As a model system for the transposition of cloned genes, the xylE (coding for catechol-2,3-dioxygenase) has been transposed to a variety of Pseudomonads. The transposon vectors should be useful when a stable single copy of a cloned gene is desired. They should be particularly advantageous for the genetic engineering of soil bacteria for environmental studies (agriculture or pollution control) where the stability of the engineered strains, in the absence of continuous antibiotic selection, may be important.

Blotting, Southern↗
PubMed1991

Overexpression of the phage lambda lysozyme cloned in Escherichia coli: use of a degenerative mixture of synthetic ribosome binding sites and increase of the protein stability in vivo.

The R gene of the phage lambda coding for a lysozyme expressed at the end of an infection cycle in Escherichia coli has been cloned in a series of vector plasmids. Two methods for improving the efficiency of translation have been tested. First, the use of a bicistronic construction in which the ribosome binding site (RBS) of the first cistron is that of a highly expressed gene or the use of a degenerate mixture of synthetic oligonucleotides for the optimization of a RBS. The second strategy is more efficient: the analysis of a number of clones reveals that the LaL expression levels are increased by a factor between 3 and 6 times compared with the clone using the natural RBS. The expression levels are described by an approximately Gaussian histogram. The translation promoter that was found to afford the best expression (PL) is under the control of a thermolabile repressor. Under the expression conditions, the protein is partially proteolysed. The proteolysis is significantly decreased by adding salt to the growth medium. After optimization, an increase in expression by a factor of 40 is obtained compared with the initial conditions. An efficient purification protocol is described.

Base Sequence↗
PubMed1990

Malignant melanoma in England: risks associated with naevi, freckles, social class, hair colour, and sunburn.

In a case-control study in the Midlands of England, 195 subjects with superficial spreading or nodular melanoma were compared to age- and sex-matched controls chosen from all inpatients or outpatients in hospitals serving the defined source population. Significant increases in melanoma risk, after adjustment for other factors, were seen in association with total naevi on the arms (odds ratio (OR), for 15+ naevi compared to none = 3.8), intense freckling as an adult (OR = 6.2), and as a child (OR = 6.0), and higher social class (OR = 2.4). Positive single factor associations were also seen with light or red hair colour, tendency to sunburn easily, and a history of sunburn at ages 8-12, although these were not significant when adjusted for the other factors. No significant effect was seen with naevi greater than 6 mm, or with raised naevi, when adjusted for total number of naevi. Total arm naevi and density of freckling had independent effects, consistent with a multiplicative effect, the OR in those with 10+ naevi and heavy freckling being 20.8. The risk associated with red hair was independent of naevi, but associated with freckling. The strongest association with sunburn history was seen with a history of sunburn in childhood, with sunburn at later times, having smaller effects, or none. These results shows that freckling and social class as well as naevi are strong and independent risk indicators for melanoma. These associations were generally consistent by type of melanoma, sex, age, and extent of regular exposure of the body site affected, although the social class gradient was observed neither for nodular melanoma nor for melanomas occurring on the most exposed body sites.

Adult↗
PubMed1989

Bacteriophage T7 RNA polymerase-controlled specific gene expression in Pseudomonas.

The rifampicin (Rif)-resistant RNA polymerase of phage T7 has proved invaluable for the exclusive over-expression, in Escherichia coli, of genes cloned downstream from the T7 phi 10 promoter [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 82 (1985) 1074-1078]. Here, we demonstrate that the system can be extended to Gram-negative bacteria other than E. coli, by the use of compatible wide host range plasmids. As an example, the Rif-resistant in vivo synthesis and specific radiolabelling of E. coli galactokinase in Pseudomonas ATCC19151, is demonstrated. The incidental observation that 30 min after treatment with Rif, two polypeptides continue to be synthesized in plasmid-free Pseudomonas ATCC19151, indicates that these proteins are produced by very stable mRNA species.

Cloning, Molecular↗
PubMed1989

Effects of a health education campaign for the earlier diagnosis of melanoma.

As part of a national campaign to combat the rising incidence of and mortality from cutaneous malignant melanoma, a programme of improved clinical services and professional and public education was set up in Nottingham in January to July 1987. The public education campaign in July led to an immediate increase in the weekly number of referrals to the pigmented lesion clinic from 10 to 54. The effect on general practitioner workload was less dramatic, the weekly number of consultations for discrete pigmented lesions rising from 0.5 to 3. In materials sent to GPs, we recommended that patients with three or more of seven specified signs should be referred for specialist opinion. Only 40% of the patients referred to the pigmented lesion clinic fulfilled this criterion, but 6% of these patients had a melanoma, compared to only 0.4% of those who did not meet the criterion. In the 6 months following the campaign, 64% of melanomas diagnosed in Nottingham residents had a Breslow thickness of less than 1.5 mm whereas only four (16%) were greater than 3.5 mm. However, this distribution was not significantly different from that seen in the three and a half years before the campaign. These results suggest that attempts to improve early diagnosis of the disease by health education are justified, but, in view of the service implications, full evaluation of such campaigns by large scale and long-term studies is essential. Future campaigns should give greater stress to referral criteria.

England↗
PubMed1988

Comparison of effects of a self management booklet and audiocassette for patients with asthma.

The effects on self management of asthma of a specially prepared book and audiocassette tape with similar contents were observed in a controlled study of 177 patients with asthma in general practice. After a run in period of six months patients were randomly given the book, the tape, both the book and tape, or neither. Patients' knowledge of the use of drugs, perceptions of their disability, skill in using an inhaler, consumption of drugs, consultations with their general practitioners, morbidity (from patients' entries on diary cards), and use of the educational material were measured. Knowledge about the use of drugs was significantly increased in the groups who received the material after three months and persisted after 12 months. Patients who had been given the tape or the book and tape increased their scores of knowledge of drugs more than patients given the book alone. Patients in all groups given the material considered that their disability was reduced. There were no other significant changes. Patients given both the book and the tape preferred the book. Patients with asthma can obtain useful information from such material. The paradoxical result whereby patients learnt more from the tape but preferred the book suggests that a distinction can be made between information that patients need, which may be acquired better from an audiocassette, and information that they want, which may be acquired better from a book.

Asthma↗
PubMed1988

Over-expression of natural and variant human H-chain ferritins in E. coli.

The natural human H-chain ferritin was expressed in E. coli using a multi-copy expression vector containing the lambda pL promoter. A variant H-ferritin, having an altered N-terminus, was also produced. These proteins are overproduced (greater than 30% of the soluble protein), correctly assembled into its 24-subunit shell, and able to bind iron. The identity of the products was confirmed using an antibody specific for H-ferritin.

Bacteriophage lambda↗
PubMed1988

Cloning and sequencing of Pseudomonas genes encoding vanillate demethylase.

A 2,598-base-pair (bp) SalI-HincII DNA fragment has been cloned which codes for vanillate demethylase, the enzyme responsible for the demethylation of vanillate (3-methoxy-4-hydroxybenzoate) to protocatechuate (3,4-dihydroxybenzoate). Complementation and insertional inactivation experiments have shown that this fragment carries two genes (vanA and vanB) which are predominantly cotranscribed from a promoter upstream of vanA. Nucleotide sequencing of the SalI-HincII fragment confirmed the genetic data: two open reading frames of 987 and 942 bp were present in the transcribed orientation. These had a very high G + C content in the third base of each codon, which is characteristic of Pseudomonas chromosomal genes. Expression of the genes in Escherichia coli with the T7 RNA polymerase-promoter system gave rise to two polypeptides of 36 and 33 kilodaltons which could be identified by deletion analysis as the products of vanA and vanB, respectively. A search of the protein sequence data bank indicated that the vanB gene product was related to the ferredoxin family.

Amino Acid Sequence↗
PubMed1988

A sports medicine clinic in the community.

We report the attempt to set up a mini sports medicine clinic in a health centre resourced by a Department of Community Medicine. The type of problems seen are similar to those reported by other clinics. On the results of this pilot project, we believe it would be possible to establish similar clinics in health centres elsewhere in the United Kingdom. The National Health Service resources required are minimal, but the benefits to local communities are considerable.

Adolescent↗
PubMed1987

The first-step transfer-DNA injection-stop signal of bacteriophage T5.

Bacteriophage T5 is different from most phages in that its DNA is injected in two steps during infection. The region containing the injection stop signal (iss) has been cloned and sequenced and found to contain numerous large repeats and inverted repeats which may be part of the iss. The most impressive of these are the 31-bp repeat units (rb) which are present three times in 99 bp. The rb repeats, themselves, contain inverted repeats so that mutually exclusive stem-and-loop structures may potentially form, not only within the repeats, but also between them. Another pair of repeats (21 bp each) contains two sequences resembling DnaA protein-binding sites. The region sequenced also contains one of the T5 site-specific strand interruptions and this was found to lie at the base of a perfect 9-bp palindrome.

Base Composition↗
PubMed1987

A 'phase-shift' fusion system for the regulation of foreign gene expression by lambda repressor in gram-negative bacteria.

A 'phase-shift' translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BclI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the lambda cro gene. The lambda cro gene is expressed from promoter pR and controlled by a thermosensitive (cI857) lambda repressor. The usefulness of the expression vector was demonstrated using a galK gene lacking the ATG start codon and fusing this to the pR promoter and ATG start codon of the lambda cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5'-GATC-3') at the N terminus (provided, for example, by a BamHI linker). The lambda cI857-pR-cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful lambda pR promoter and the efficient lambda repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.

Bacteriophage lambda↗
PubMed1987

Vectors with restriction site banks. V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites.

The construction of a new wide-host-range, restriction-site bank, cosmid-cloning vehicle (pJRD215) is described. The wide-host-range properties and the ability to be transferred by conjugation, extend genetic engineering to those Gram-negative species that cannot be transformed. The vector permits the cloning of genes from Gram-negative bacteria using a complementation screening procedure in a mutant host. This procedure is simplified by the possibility of construction of a cosmid gene bank so that only a few hundred clones need to be screened. Subsequent subcloning of the gene of interest is facilitated by the presence of at least 23 unique cloning sites.

Base Sequence↗
PubMed1987

Evaluation of enzyme inhibitors of pregnancy associated oxytocinase: application to the measurement of plasma immunoreactive oxytocin during human labour.

The presence of the human placental enzyme, oxytocinase, in blood samples taken during pregnancy causes major methological problems in the radioimmunoassay for plasma oxytocin. Inadequate inhibition of the enzyme activity may lead to spuriously high or low values of plasma oxytocin. This study systematically investigates a variety of enzyme inhibitors. The optimum inhibitory system was obtained by the addition of 10 microliters of cold 125 mmol/l 1.10 phenanthrolene and 1 mol/l EDTA per ml of whole blood into the syringe. Complete enzyme inhibition was maintained for up to 60 min, during which time the lithium heparinized plasma samples were extracted by the Florisil method. Following extraction there was no enzyme activity in the extract residue. Concentrations of phenanthrolene and EDTA necessary to eliminate enzyme activity were 50- and 10-fold greater, respectively, than in any previously reported method. Recovery of synthetic oxytocin added to pregnancy plasma with inhibitors was 80% or higher, over the concentration range 1-100 pmol/l. Extract residue could be stored at -20 degrees C for up to 7 weeks. Dilutions of pregnancy plasma extracts ran parallel to the oxytocin standard curve. Studies on plasma concentrations of oxytocin (OT) during the first stage of labour in 6 patients showed that 3 had pulsatile plasma OT, peak values ranging from 4-10 pmol/l in phase with uterine concentrations, but 2 who had regular uterine activity had no episodic changes in plasma OT. One patient with hypocontractile labour had low non-fluctuating plasma OT.

Aminopeptidases↗
PubMed1985

Structure of the galactokinase gene of Escherichia coli, the last (?) gene of the gal operon.

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.

Adenosine Triphosphate↗