Biomedical subjects

P Boseley

Publications and source records attributed to P Boseley.

7 recordsLinked to original sources

PubMed2000

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA.

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.

Bacterial Proteins↗

PubMed1983

Expression of the human interferon-beta gene cloned in phage M13 mp7.

The single-stranded DNA phage, M13 mp7 was used in the construction of an expression vector containing the coding sequence for mature interferon-beta (IFN-beta). Two clones expressed a fused polypeptide showing the biological and physicochemical properties of IFN-beta, despite the fact that the N-terminal amino acid sequence had been changed; 10(6) I.U./l of culture were produced with a molecular weight of 20 000.

Amino Acid Sequence↗

PubMed1982

High-level expression of an interferon alpha 2 gene cloned in phage M13mp7 and subsequent purification with a monoclonal antibody.

A cloned interferon alpha 2 (IFN-alpha 2) gene was partially digestd with Pvu II to give a fragment that was inserted into the HincII site of the lacZ gene of bacteriophage M13mp7. Two recombinant phages containing the IFN-alpha 2 sequences in the correct orientation for expression from the lac promoter were characterized in detail. DNA sequence analysis showed that the inserted IFN-alpha 2 gene was in phase with the initiation codon of the lacZ gene. The polypeptide product has an additional 19 amino amino acids at the amino terminus of the mature IFN-alpha 2. The first 11 amino acids originate from the amino terminus of beta-galactosidase, and the remaining 8 amino acids are part of the signal sequence of pre-IFN-alpha 2. Infection of Escherichia coli with these phage followed by induction of the lac promoter with isopropyl thiogalactoside gives high yields (up to 10(9) units/liter with an average of 1.5 X 10(8) units/liter) of the modified IFN-alpha 2. This was purified to homogeneity in a single step by immunochromatography using the monoclonal antibody NK2. The nonreduced product had an apparent molecular weight of 20,500 and was shown by immunoradiometric assay to have the same specific activity as IFN made in Namalwa cells. It exhibited the characteristic cross-species antiviral activity of IFN-alpha 2.

Amino Acid Sequence↗

PubMed1982

The effect of hypertonic salt on interferon and interferon mRNA synthesis in human MG63 cells.

After infection with Sendai virus or Newcastle disease virus (NDV) strain F, human osteosarcoma MG63 cells produced large amounts of interferon-beta. Both interferon production and overall protein synthesis were strongly inhibited by hypertonic salt. Interferon mRNA synthesis, however, was little affected by hypertonic salt up to twice normal salt concentrations, although cellular RNA synthesis was inhibited under these conditions. The results are compared to those obtained with polyriboinosinic acid: polyribocytidylic acid copolymer [poly(rI) . poly(rC)] inductions of MG63 cells.

Cell Line↗

PubMed1979

Sequence organization of the spacer DNA in a ribosomal gene unit of Xenopus laevis.

A detailed restriction map was constructed for a cloned Xenopus laevis rDNA fragment containing the nontranscribed spacer (NTS) and external transcribed spacer (ETS) together with a portion of both the 18S and 28S rRNA genes. The NTS was found to contain at least three distinct repetitious areas. Region 1 has a repeating unit of approximately 100 bp. The primary structure of this unit has been determined by DNA sequencing. Region 2 is very similar in organization to region 3, and both have an alternating 81/60 bp arrangement as revealed by restriction with Alu I and DNA sequencing. It can be shown that the 81 and 60 bp canons are virtually identical to one another excepting a deletion/insertion of a 21 bp segment. Region 3 differs from region 2 in having sites for Sma I with its 81 bp units. Between these repeated DNA sequences there are two identical, nonrepetitive DNA sequences, each of which is centered around a Bam Hl site. Most of the ETS has been sequenced. It was found to be nonrepetitive and extremely rich in Cs. Close to the 5' end of the 18S coding sequence there is a DNA stretch very rich in purines. About 2.25 kb upstream from the Eco Rl restriction site bisecting the 18S structural gene there is a unique sequence which may be homologous to the 5' end of the 40S precursor RNA. Present evidence suggests that the boundaries between NTS and ETS occur farther downstream than was suggested by electron microscopic data. Sequencing has revealed that the spacer DNA of X. laevis contains different kinds of simple DNA sequences, but no evidence has been found that spacer DNA once arose by saltation of a 15 bp segment. The most surprising finding was that the spacer sequences around the Bam restriction sites (the Bam islands) show high homology with a sequence near the NTS/ETS interface. From the restriction and sequencing analyses it can be deduced that in recent evolutionary times the DNA sequences near the 5' end of the ribosomal transcription unit were reduplicated twice and displaced into spacer by saltation of an intervening short DNA sequence (the 60/81 bp canons). Possible implications of these evolutionary events for spacer functions are consisdered. The sequencing has also provided a molecular basis for a whole range of conclusions arrived at previously by indirect approaches, and these are discussed.

Animals↗

PubMed1977

Novel screening procedure for recombinant plasmids.

Lysed bacterial colonies containing potential recombinant plasmids were mixed with molten agar and sealed into slots of an agarose gel. After electrophoresis overnight, the gel was stained with ethidium bromide, which clearly reveals recombinant plasmids. Xenopus laevis ribosomal DNA and histone DNA of Psammechinus miliaris were ligated into pCRI plasmids and screened by this method.

Animals↗

PubMed1976

The arrangement of 18-S and 28-S ribosomal ribonucleic acids within the 40-S precursor molecule of Xenopus laevis.

The arrangement of 18-S rRNA and 28-S rRNA within their 40-S common precursor molecule (pre-rRNA) of Xenopus laevis was investigated by electron microscopic analysis of secondary structure of nascent pre-rRNA chains of oocytes, and by 5'-end analysis of 18-S rRNA and 28-S rRNA hybridized to the EcoRI fragment of rDNA cloned as plasmid pCD42. Secondary structure mapping of phenol-extracted RNA from nucleolar cores revealed complete pre-rRNA chains or molecules at various stages of processing and pre-rRNA molecules apparently lacking one end. In this latter group, which was regarded as representing nascent chains, more than 90% of the molecules had no 28-S rRNA REGION. This shows that the 28-S rRNA sequence is transcribed after the 18-S rRNA region and hence must be located nearer to the 3' end of the pre-rRNA molecule. For 5' end-group determination [3H]uridine-labelled 18-S rRNA and 28-S rRNA were hybridized, as fragments of about 200 nucleotides, to the plasmid pCD42 containing coding sequences for four-fifths of the 18-S rRNA sequence, the external transcribed spacer, the non-transcribed spacer and a tenth of the 28-S rRNA sequence. The RNA was recovered from the hybrids and analyzed for uridine 3',5'-bisphosphate (pUp) after alkaline hydrolysis. The pUp content of the hybridized 18-S rRNA fragments was 20-fold higher than in those of 28-S rRNA, THUS DEMONSTRATING THAT THE 5' END OF THE 18-S rRNA is located next to the external spacer region. From these results it is concluded that the 18-S rRNA is located close to the 5' end of the 40-S pre-rRNA molecule.

Animals↗