Biomedical subjects

D R Sargan

Publications and source records attributed to D R Sargan.

50 records · Page 3Linked to original sources

PubMed1992

Expression of maedi-visna virus major core protein, p25: development of a sensitive p25 antigen detection assay.

The gene for the major core protein, p25, of maedi-visna virus (MVV) was cloned using a PCR (polymerase chain reaction) strategy employing primers designed for the insertion of the gene directly into yeast Ty-VLP expression vectors. In this system p25 is expressed as a fusion protein which self-assembles into virus-like particles (VLPs) due to interaction of the Ty A fusion partner. High levels (50-60 mg/l) of p25 fusion protein were produced, and p25 was recovered in soluble and highly pure form following cleavage from the Ty particle by Factor Xa protease digestion. The p25 protein produced in yeast is antigenically authentic, as defined by its reactivity with p25-specific antisera and its ability to elicit antibodies reactive with native viral p25 protein; although the cleaved, soluble form of p25 was found to be considerably more antigenic than the hybrid Ty-p25 VLP. Using this reagent anti-p25 monoclonal and polyclonal antibodies were generated. These sera and the p25 protein have been used to develop a sensitive MVV p25 detection assay. These reagents and assays will facilitate further studies of viral replication and immune response to the virus.

Animals↗

PubMed1992

Characterisation of visna virus reverse transcriptase: a micro scale reverse transcriptase assay adapted for use with an automated cell harvester.

The reverse transcriptase of the sheep lentivirus visna/maedi virus has been characterised. Optima for magnesium ion concentration (5-10 mM), potassium ion concentration (150 mM) and pH (8.25) for this enzyme are very similar to those previously described for the human immunodeficiency viruses. The assay used for this work makes use of a cell harvester to speed up the processing of multiple samples. It is small scale, requiring 15 microliters of sample, is rapid, and is able to detect virus at titres below 10(3)/ml. Harvesting the assay onto either DEAE paper or using TCA and glass fibre mats make it suitable for use with either tissue culture media or infected cell lysates, but not with body fluids. It has been used to detect cell-associated reverse transcriptase in choroid plexus cells within 36 h of visna infection.

Animals↗

PubMed1992

A simplified method for the detection of maedi-visna virus RNA by in situ hybridization.

A simplified in situ hybridization method for the detection of maedi-visna virus (MVV) RNA in cultured cells using 35S-labelled DNA probes is described. The protocol currently used in this laboratory for the in situ detection of MVV RNA involves paraformaldehyde fixation followed by extensive cellular pretreatment prior to hybridization. It was found that substitution of paraformaldehyde fixation with brief acetone treatment and the removal of subsequent pretreatment steps gave a similar level of hybridization signal to that of our standard protocol. Acetone fixed, non-pretreated samples were used to develop a double labelling procedure in which immunocytochemistry and in situ hybridization were combined to allow the simultaneous detection of visna virus antigens and RNA within the same cell.

Acetone↗

PubMed1992

Cloning, expression and characterization of ovine interleukins 1 alpha and beta.

Ovine interleukin 1 alpha (IL-1 alpha) c-DNA, obtained by polymerase chain reaction, has been cloned into pTZ18R and pTZ19R. The resulting DNA sequence shows close homology with the bovine sequence. The derived amino-acid sequence shows conserved motifs similar to those observed in all species studied so far. No signal peptide is seen. Northern blots of RNA from lipopolysaccharide (LPS)-stimulated ovine alveolar macrophages show IL-1 beta m-RNA to be produced earlier than and to be more transient than IL-1 alpha m-RNA. c-DNAs coding for the IL-1 alpha proprotein and IL-1 alpha and IL-1 beta mature proteins have been cloned and expressed in the yeast Ty-VLP system as fusion proteins. The resultant IL-1 protein preparations, cleaved from their fusion partners by the action of activated coagulation Factor Xa, are 80-95% pure and show biological activity in standard thymocyte co-mitogen and cartilage degradation assays for IL-1. Some species specificity is observed in that sheep thymocytes are more responsive to ovine rIL-1 than are mouse thymocytes. The presence of a Factor Xa cleavage site in the IL-1 alpha proprotein suggests that Factor Xa may be involved in the processing of ovine IL-1 alpha to its mature form.

Amino Acid Sequence↗

PubMed1991

Sequence of the cDNA encoding ovine tumor necrosis factor-alpha: problems with cloning by inverse PCR.

We have cloned and sequenced the ovine tumor necrosis factor-alpha (TNF-alpha)-encoding cDNA, using gene amplification by polymerase chain reaction (PCR) technology, to aid studies of assorted diseases in this species. We used primers selected from published TnfA sequences of other species on a cDNA template prepared from lipopolysaccharide-stimulated ovine alveolar macrophages, to generate a product representing the central region of the molecule. We then used a novel method based on 'inverse PCR' to generate a product containing the 5' and 3' ends of the molecule. Here, we present the complete sequence of the ovine TNF-alpha cDNA and compare it with other published TNF sequences. The cloned cDNA has a leader sequence of 156 bp followed by a protein-coding sequence of 702 bp and a 3'-untranslated region of 800 bp. The protein product of the gene is a protein of Mr = 25,586, 79% homologous to human TNF-alpha. An mRNA produced by alveolar macrophages, which hybridises to the cloned gene, is induced greatly, with a peak induction time of approx. 135 min, in response to stimulation by lipopolysaccharide and to plating on plastic. We also discuss the resolution of some artefacts of the inverse PCR technique.

Amino Acid Sequence↗

PubMed1991

Nucleotide sequence of EV1, a British isolate of maedi-visna virus.

We have isolated a maedi-visna-like virus from the peripheral blood mononuclear cells of a British sheep displaying symptoms of arthritis and pneumonia. After brief passage in fibroblasts this virus (designated EV1) was used to infect choroid plexus cells. cDNA clones of the virus were prepared from these cells and sequenced. Gaps between non-overlapping clones were filled using gene amplification by the polymerase chain reaction. The genome structure is similar to that described for visna virus strain 1514, and differs from that described for visna virus strain SA-OMVV in not having a W reading frame. Overall the genome differs by about 20% between each of these strains, but there is fivefold variation in the amount of divergence of derived amino acid sequences of different open reading frames. Two sequenced EV1 clones each contain only one copy of the 43 bp repeat, with paired AP-1 sites, which is a feature of other ruminant lentiviral long terminal repeats (LTRs). However, analysis of viral DNA in infected cells by gene amplification shows that LTRs with two repeats do occur, albeit at a relatively low frequency.

Amino Acid Sequence↗

PubMed1989

A transcriptional map of visna virus: definition of the second intron structure suggests a rev-like gene product.

Visna virus is the prototype lentivirus, with a genome structure similar to that of the human immunodeficiency viruses HIV-1 and HIV-2. We have analysed in vitro the transcription pattern of this virus in lytic infections of choroid plexus cells. Northern blot analysis shows the presence of spliced subgenomic mRNA species of 4.9, 4.3, 4.0, 1.7 and 1.4 kb. Use of appropriate subgenomic probes shows that the first three of these species encode envelope protein (but also potentially the small open reading frames Q and S). The 1.7 kb RNA could contain S. In order to elucidate the translational coding potential of the smallest RNA, and to characterize further all the transcripts, S1 mapping was performed across those parts of the genome which were close to exon/intron boundaries. This allowed the definition of acceptor splice sites following both introns 1 and 2 as well as donor sites preceding intron 2. The data suggest that the 1.4 kb RNA encodes a protein derived from the F reading frame that may form part of a precursor protein and also contains sequences with some degree of homology to the rev (trs/art) protein of HIV, as well as a typical protease cleavage site between these sequences and the F protein.

Amino Acid Sequence↗

PubMed1987

Steroid hormone regulation of the gene encoding the chicken heat shock protein hsp 108.

We have previously isolated a partial cDNA clone encoding a heat shock protein which has been termed hsp 108 (Zarucki-Schulz, T., Kulomaa, M. S., Headon, D. R., Weigel, N. L., Baez, M., Edwards, D. O., McGuire, W. L., Schrader, W. T., and O'Malley, B. W. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6358-6362; Sargan, D. R., Tsai, M.-J., and O'Malley, B. W. (1985) Biochemistry 25, 6252-6258). Here we examine the expression of the hsp 108 gene in steroid-stimulated chick oviducts. After 16 h of secondary stimulation with estrogen or progesterone, a 20-50-fold increase in hsp 108 mRNA is detected above unstimulated levels. RNA quantitation by Rot analysis shows that in these oviducts there are 75 molecules of hsp 108 mRNA/oviduct cell. Nuclear "run-off" assays indicate only a 2-4-fold increase in the rate of transcription of the gene in response to either sex steroid, suggesting that the gene is regulated both at the transcriptional level and by mRNA stabilization. On hormone withdrawal, the concentration of hsp 108 mRNA in the oviduct falls to unstimulated control levels within 4 days. Chronic stimulation of the chicks with estrogen (or high acute doses of estrogen) attenuates specifically the inductive response of the hsp 108 gene, but not of ovalbumin, under these conditions. This is not due to a significant reduction of the transcription rate of the gene. We have previously shown that hsp 108 is expressed constitutively in many tissues of the chick (Sargan, D. R., Tsai, M.-J., and O'Malley, B. W. (1985) Biochemistry 25, 6252-6258). In tissues which are not responsive to hormones, no short-term effects of hormone administration on the gene were observed. In the spleen there is a reproducible slow activation of the gene, but the kinetics of this response suggest that it is not a primary response to the hormone. Thus, this hsp 108 gene codes for an interesting new eucaryotic heat shock protein which is regulated also by steroid hormones in a tissue-specific manner.

Animals↗

PubMed1986

hsp108,,,,,, a novel heat shock inducible protein of chicken.

cDNA clones encoding a protein that copurifies with the progesterone receptor B subunit but does not bind progesterone have been described [Kulomaa, M. S., Weigel, N. L., Kleinsek, D. A., Beattie, W. G., Conneely, O. M., March, C., Zarucki-Schulz, T., Schrader, W. T., & O'Malley, B. W. (1986) Biochemistry (preceding paper in this issue)]. A full-length sequence for these clones was derived and was found to encode a protein that is structurally unrelated to the progesterone receptor but that contains significant homologies to the previously described heat shock proteins hsp90 of yeast and hsp83a of Drosophila melanogaster. In this paper it is shown that this protein is indeed a heat shock protein. Though the apparent molecular weight of the protein is 108,000 on sodium dodecyl sulfate-polyacrylamide gels, the molecular weight of the polypeptide backbone is 92,000. The steady-state level of gene transcripts as well as the level of protein is inducible by heat shock, but the gene is constitutively expressed in a number of tissues. A previously undescribed heat shock protein of molecular weight 78,000 in these preparations is also reported.

Animals↗

PubMed1985

Eukaryotic ternary transcription complexes: transcription complexes of RNA polymerase II are associated with histone-containing, nucleosome-like particles in vivo.

Using a psoralen crosslinking, radioactive labelling technique, we have previously been able to study ternary transcription complexes containing DNA-dependent RNA polymerases I and II which are released from rat liver nuclei by endogenous nuclease digestion [Sargan and Butterworth, refs 1 and 2]. Although the DNA component of these complexes was found to have a 'nucleosome-like' size profile and although the experimental conditions for autodigestion were designed to minimise histone rearrangement, it is necessary to provide further evidence that the periodicity of nuclease cutting around these transcription complexes is conferred by histones. Studies using secondary nuclease digestion of the released transcription complexes now show a digestion barrier characteristic of that conferred by nucleosomal histones which is lost if histones are removed from the complexes. Furthermore, antibodies raised against histones are effective in precipitating transcription complexes of RNA polymerase II and, to a lesser extent, of RNA polymerase I. The data suggest that, in rat hepatic tissue, transcription complexes are in very close proximity (within a few hundred base pairs) of histone-containing, nucleosome-like particles in vivo.

Animals↗

PubMed1982

A possible novel interaction between the 3'-end of 18 S ribosomal RNA and the 5'-leader sequence of many eukaryotic messenger RNAs.

A novel interaction between the 5'-untranslated region of eukaryotic messenger RNAs and non-contiguous sequences in the 18 S ribosomal RNA is proposed. The small ribosomal RNA contains, at its 3'-terminus, a heavily conserved hairpin structure. It is suggested that mRNA 5'-leader sequence stabilises this structure by interacting with other conserved nucleotides which flank it. Sequences closely related to the required sequence (A-U-C-C-A-C-C) occur quite commonly in eukaryotic mRNAs and are often found immediately upstream from the AUG-codon. This interaction may have a role in the events which lead up to the initiation of protein synthesis.

Animals↗

PubMed1982

Eukaryotic ternary transcription complexes. I. The release of ternary transcription complexes of RNA polymerases I and II by the endogenous nucleases of rat liver nuclei.

Autodigestion of rat liver nuclei in magnesium-containing buffers leads to the release of about 80% of DNA-dependent RNA polymerases I and II, together with 4 to 8% of the DNA. The RNA polymerases are at least partially DNA bound as judged by the effect on in vitro transcription of (1) Actinomycin D, (2) preirradiation of the enzymes in the presence of 8-methoxypsoralen and (3) heparin. The released DNA migrates as ladders of nicked nucleosome-size fragments on electrophoresis. On sucrose gradients, most RNA polymerase activity sediments as two peaks: one slightly smaller than the 11S mononucleosome and the other with the dinucleosome. The released material can act as a source of ternary transcription complexes for further structural studies.

Animals↗

PubMed1982

Eukaryotic ternary transcription complexes. II. An approach to the determination of chromatin conformation at the site of transcription.

Digestion of rat liver nuclei by endogenous nucleases or micrococcal nuclease releases a chromatin fraction containing RNA polymerases I and II bound to DNA fragments in ternary transcription complexes. To label the DNA in these transcription complexes, the polymerases were allowed to add radioactively labelled ribonucleotides in vitro to in vivo-initiated RNA chains. During this transcription step, nucleic acids were photochemically cross-linked using 8-methoxypsoralen. Nucleic acids in transcription complexes were then sized by gel electrophoresis. Under conditions where RNA polymerases I and II were active in vitro, most of the labelled DNA was found in a series of fragments of sizes which were multiples of approximately 200 base-pairs. When polymerase I alone was active, the smallest member of this series carried the bulk of the label; when polymerase II also was active, a significant proportion of the label was carried on the dimer and higher oligomers. Proteins other than polymerase alone are shown to be responsible for the pattern of DNA fragments protected from nucleases. Therefore active RNA polymerases I and II in vivo are in close proximity to structures protecting DNA fragments, the sizes of which are similar to those found in nucleosomes. We have yet to establish that these structures are composed of histones.

Animals↗

PubMed1981

A transcriptionally enriched chromatin fraction released by a Mg++ -dependent endogenous nuclease from rat liver.

When rat liver nuclei are subjected to endogenous nuclease digestion in the presence of 5 mM Mg++, they release a chromatin fraction containing DNA of well defined fragment lengths and DNA-dependent RNA polymerases. The DNA is enriched 4.4-fold for sequences complementary to cDNA prepared to cytoplasmic poly-A-containing RNA when compared with whole cell DNA. This method of obtaining a transcriptionally enriched chromatin fraction is compared with other reported nuclease methods.

Animals↗