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Biomedical subjects

T M Wilson

Publications and source records attributed to T M Wilson.

At least 109 records · Page 6Linked to original sources
PubMed1987

A comparison of eukaryotic viral 5'-leader sequences as enhancers of mRNA expression in vivo.

The 5'-untranslated leader sequences of several plant RNA viruses, and a portion of the 5'-leader of an animal retrovirus, were tested for their ability to enhance expression of contiguous open reading frames for chloramphenicol acetyltransferase (CAT) or beta-glucuronidase (GUS) in tobacco mesophyll protoplasts, Escherichia coli and oocytes of Xenopus laevis. Translation of capped or uncapped transcripts was substantially enhanced in almost all systems by the leader sequence of either the U1 or SPS strain of TMV. All leader sequences, except that of TYMV, stimulated expression of 5'-capped GUS mRNA with the native prokaryotic initiation codon context, in electroporated protoplasts. Only the TMV leaders enhanced translation of uncapped GUS mRNAs in protoplasts and increased expression of uncapped CAT mRNA in microinjected X. laevis oocytes. In oocytes, the TYMV leader sequence was inhibitory. In transformed E. coli, the TMV-U1 leader enhanced expression of both the native and eukaryotic context forms of GUS mRNA about 7.5-fold, despite the absence of a Shine-Dalgarno region in any of the transcripts. The absolute levels of GUS activity were all about 6-fold higher with mRNAs containing the native initiation codon context. In E. coli, the leaders of AlMV RNA4 and TYMV were moderately stimulatory whereas those of BMV RNA3, RSV and the SPS strain of TMV enhanced GUS expression by only 2- to 3-fold.

Animals↗
PubMed1987

In vivo uncoating and efficient expression of foreign mRNAs packaged in TMV-like particles.

The ribonucleocapsids of many plant viruses are extremely stable. The protein coat protects the RNA genome against degradation during the accumulation and spread of progeny virions. Chimeric single-stranded RNA molecules were transcribed in vitro from recombinant plasmids and later encapsidated, in vitro, into ribonucleoprotein particles (pseudoviruses) 60 nanometers long that resembled tobacco mosaic virus. Transcripts encoding an assayable enzyme, chloramphenicol acetyltransferase (CAT), were packaged into pseudovirus particles to assess the utility of this single-stranded RNA delivery system in a wide range of cell types. In all cases, packaged CAT messenger RNA was uncoated and transiently expressed. Significantly higher levels of CAT activity were detected with packaged than with naked CAT messenger RNA after inoculation of plant protoplasts in the presence of polyethylene glycol or abrasive inoculation of intact leaf surfaces. Structural events that lead to the uncoating and expression of CAT messenger RNA showed no cell specificity. This observation may support the view that the comparatively restricted host range of a true plant virus results from events that occur later during the infection cycle.

Acetyltransferases↗
PubMed1987

The 5'-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo.

A 67-nucleotide portion of the non-coding, 5'-leader sequence of tobacco mosaic virus RNA [defined as omega' (Gr. omega prime)] has been shown to enhance the translation of contiguous foreign gene transcripts both in vitro and in vivo. Chemically-synthesized omega', containing convenient linker sequences, was inserted into derivatives of an in vitro transcription plasmid (pSP64) between the bacteriophage-SP6 promoter and sequences coding for either chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII). Run-off in vitro transcripts, with or without a 5'-cap structure (G(5')ppp(5')G) and/or the omega' sequence, were tested in mRNA-dependent cell-free translation systems derived from rabbit reticulocyte lysate, wheat germ extract or Escherichia coli (MRE 600). In all cases, the presence of omega' increased the translational expression of both reporter genes, typically between 2- to 10-fold. Electroporation of isolated mesophyll protoplasts from Nicotiana tabacum cv. Xanthi, or microinjection of oocytes from Xenopus laevis, with SP6-transcripts containing the CAT-coding region confirmed and extended the value of omega' as a potential translational enhancer of gene expression in vivo.

Animals↗
PubMed1987

Anderson Fabry disease, a close linkage with highly polymorphic DNA markers DXS17, DXS87 and DXS88.

Anderson Fabry disease is an X-linked lysosomal storage disorder caused by alpha-galactosidase A deficiency. Hemizygous males and some heterozygous females develop renal failure and cardiovascular complications in early adult life. We have investigated six large UK families to assess the possible linkage of five polymorphic DNA probes to the Anderson Fabry locus, previously localised to Xq21-24. No recombination was found between Anderson Fabry disease and DXS87, DXS88 and DXS17, which gave lodmax = 6.4, 6.4 and 5.8 respectively at theta = 0.10, (upper confidence limit 0.10). DXS3 gave lodmax 2.9 at theta = 0.10 (upper confidence limit 0.25). DXYS1 was excluded from linkage. The best fit map (DXYS1/DXS3) theta = 0.192 (DXS17/DXS87/DXS88/Anderson Fabry locus) provided no information about the order of loci in parentheses due to the absence of recombinants. The close linkage of DXS17, DXS87 and DXS88, together with alpha-galactosidase A estimation, can be used for antenatal diagnosis and carrier detection until the application of a gene specific probe has been evaluated.

DNA↗
PubMed1987

Anderson-Fabry disease--family linkage studies using two polymorphic X-linked DNA probes.

Anderson-Fabry disease is an X-linked lysosomal storage disorder due to alpha-galactosidase A deficiency. In affected males there is a high mortality in early adult life due to renal failure and cardiovascular complications. We describe our preliminary results from genetic linkage studies in five families using two polymorphic DNA probes, DXS17 and DXYS1, mapping to an area on the long arm of the X chromosome between Xq13-22. DXS17 identified a Taql polymorphism closely linked to the disease locus in three families (lodmax Z = 4.23. at a recombination fraction decreases theta = 0.0). Restriction fragment length polymorphisms detected by DXYS1 were not linked.

Blotting, Southern↗
PubMed1987

Immunogold localization of the intracellular sites of structural and nonstructural tobacco mosaic virus proteins.

Antibodies raised against the 126K nonstructural protein (replicase) encoded by tobacco mosaic virus (TMV) RNA or the viral coat protein have been used to localize these proteins within virus-infected tobacco leaf cells by an immunogold labeling technique. A protocol is given for low-temperature fixation to facilitate immunogold labeling. In cells of TMV-infected leaf tissue, the 126K protein immunogold label was found almost exclusively in "viroplasms" in the cytoplasm and in pockets of virus particles at the viroplasmic periphery. When utilizing the coat protein antiserum, very little labeling was seen within the viroplasms, although virus particles throughout the cytoplasm were heavily labeled. Viroplasms contained electron-dense rope-like structures embedded in a ribosome-rich matrix. In their "mature" form, viroplasms are the well-known "X body" inclusions. The rope-like structures were up to 1.2 micron long and appear twisted, undergoing several revolutions throughout their length, but were not of a constant pitch. In transverse section, they appeared to be composed of several hollow, radially segmented cylinders 21 nm in diameter, with a 9-nm hole. Antibody labeling showed them to be composed, at least in part, of the 126K protein. Clusters of virus particles at the edge of or within the viroplasms were also labeled with the 126K antiserum, in contrast to virus particles in other areas of the cell, which were not. TMV-infected tobacco mesophyll protoplasts cultured for up to 27 hr did not contain the rope-like ribbons. Instead, isolated protoplasts contained amorphous cytoplasmic areas which were labeled with 126K antibody. Since the 126K protein is most probably a constituent of the TMV RNA-replicating enzyme (replicase), its intracellular location is considered to be indicative of the site of replication of TMV RNA. Therefore these results suggest that replication occurs at the edges of the viroplasms.

Antibody Specificity↗
PubMed1987

The effect of multiple dispersed copies of the origin-of-assembly sequence from TMV RNA on the morphology of pseudovirus particles assembled in vitro.

Supercoiled plasmid DNA was used as a template to transcribe long concatameric RNA molecules containing several dispersed copies of the origin-of-assembly sequence (OAS) from tobacco mosaic virus (TMV) RNA. When incubated with TMV coat protein "disks" in vitro, these RNAs spontaneously assembled into TMV-like pseudovirus particles. However, as each OAS initiated assembly more or less simultaneously, the concatameric RNA species generated complex nucleoprotein structures of predictable morphology. Similar structures were proposed some time ago (M. E. Taliansky, I. B. Kaplan, L. V. Yarvekulg, T. I. Atabekova, A. A. Agranovsky, and J. G. Atabekov, 1982, Virology 118, 309-316) to account for the RNase-sensitive phenotype of a ts mutant of TMV, Ni2519. These results extend the utility of our RNA-packaging vector system and confirm many of the predictions based on the current model for the self-assembly of TMV.

Base Sequence↗
PubMed1987

Characterisation of the 5'-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro.

Uncapped messenger RNAs (mRNAs) encoding calf preprochymosin, chicken prelysozyme, or Escherichia coli beta-glucuronidase (GUS) were synthesized in vitro, with or without a 5'-terminal 67-nucleotide sequence (omega') derived from the untranslated 5'-leader (omega) of tobacco mosaic virus (TMV) RNA. Messenger RNAs were translated in vitro, in messenger-dependent systems derived from rabbit reticulocytes (MDL), wheat-germ (WG) or E. coli (EC). The omega' sequence enhanced expression of each mRNA in almost every translation system. While MDL was the least responsive to omega', this sequence proved particularly efficient in permitting translation of the eukaryotic mRNAs in EC, despite the absence of a consensus Shine-Dalgarno sequence in either the mRNAs or omega'. The local context of the initiation codon (AUG) in two GUS mRNA constructs did not influence the relative enhancement caused by the omega' sequence. These findings extend the utility of omega' as a general enhancer of translation for both prokaryotic and eukaryotic mRNAs in either 80S- or 70S-ribosome-based systems.

Acetyltransferases↗
PubMed1987

Ultrasonic backscatter from bovine tissues: variation with pathology.

This is the second in a series of two papers reporting results collected on ultrasonic propagation properties, namely, velocity, attenuation, and backscatter of various mammalian tissues. In the first paper [J. Acoust. Soc. Am 78, 871 (1985)], the experimental results of five different types of normal bovine tissues were given. In this paper, results obtained for the most common bovine disease in the liver, liver abscess, and in the heart, lymphosarcoma, will be presented. These data show that the ultrasonic properties of tissues with these two types of pathologies are significantly deviated from those of normal tissues, thus providing further evidence that ultrasonic properties may be used as quantitative indicators of a certain disease for diagnostic purposes.

Animals↗
PubMed1986

Porcine proliferative enteritis: serological, microbiological and pathological studies from three field epizootics.

Three outbreaks of porcine proliferative enteritis were evaluated clinically, pathologically, microbiologically and serologically. The disease was characterized by a chronic intermittent diarrhea. Pathological lesions included a thickened, turbid ileum with the microscopic appearance of proliferating ileal crypt epithelial cells. Comma shaped intracytoplasmic organisms were observed in the apical portions of the proliferating crypt epithelial cells with a Warthin-Starry silver stain. Microbiologically, both Campylobacter sputorum subspecies mucosalis and Campylobacter hyointestinalis, were cultured from ileal specimens of seven pigs with lesions of porcine proliferative enteritis. Microagglutination antibody titers were determined on sera from 12 of 14 pigs with porcine proliferative enteritis and on sera from 91 clinically normal swine. Pigs with porcine proliferative enteritis had a low antibody titer to subspecies mucosalis that ranged from 1-3 with a mean of 2.17. A varied C. hyointestinalis titer from 3-7 with mean of 4.83 was determined. Titers to either subspecies mucosalis and C. hyointestinalis were higher in non-porcine proliferative enteritis pigs. The results indicate that the presence of a positive titer to either C. hyointestinalis or subspecies mucosalis in swine is not indicative of clinical disease. The isolation of C. hyointestinalis from diseased ileal specimens (porcine proliferative enteritis) confirms previous reports implicating this agent in the disease.

Animals↗
PubMed1986

Sperm counts and reproductive tract lesions in male Syrian hamsters exposed in utero to diethylstilbestrol.

Pregnant hamsters were administered diethylstilbestrol (DES) orally on day 14 of gestation. Sperm counts from F1 adult males were lower than controls. Significant and consistent lesions were confined to the epididymides of DES exposed males, in which epididymal granulomas were the most common lesion. Epididymal granulomas of the tail have been associated with a bacterial etiology while granulomas of the head may be related to blind ductules or a lack of ductule connection.

Animals↗
PubMed1985

Hepatic neoplastic nodules, adenofibrosis, and cholangiocarcinomas in male Fisher 344 rats fed corn naturally contaminated with Fusarium moniliforme.

Twelve male Fisher 344 rats were fed corn naturally contaminated with Fusarium moniliforme. This corn sample was obtained from feed being fed horses during an outbreak of leukoencephalomalacia. All rats necropsied from 123 to 176 days post feeding had multiple hepatic nodules and pale depressed hepatic areas. Histological examination revealed multiple hepatic neoplastic nodules and large areas of adenofibrosis and cholangiocarcinomas. The corn only diet was deficient in many nutrients including choline and methionine. Aflatoxins at a level of greater than 0.9 p.p.b. were not detected. A discussion is presented regarding the significance of these lesions and possible relationship to the contaminating fungus and the choline, methionine deficient diet.

Adenoma, Bile Duct↗
PubMed1985

The screening of patients with suspected thyrotoxicosis using a sensitive TSH radioimmunoassay.

A radioimmunoassay was optimized to measure serum TSH with maximum sensitivity (sTSH). With this assay sTSH was less than 0.5 mIU/l in 100% of 64 patients with proven thyrotoxicosis and greater than 0.5 mIU/l in 92% of 76 normal subjects. sTSH correctly predicted the TSH response to TRH (delta TSH) in 93% of 125 patients with suspected thyrotoxicosis. In this group thyrotoxicosis was excluded by an sTSH greater than 0.5 mIU/l in 52% of patients thus avoiding the need for further testing.

Humans↗
PubMed1984

First-trimester diagnosis of Lesch-Nyhan syndrome.

Chorionic villi were sampled at 8-9 weeks' gestation in four women whose fetuses were at risk for Lesch-Nyhan syndrome. Radiochemical assay of hypoxanthine phosphoribosyl transferase activity and fetal sexing (in two fetuses by means of a Y chromosome specific cDNA probe) showed that three fetuses were affected. The biochemical findings were confirmed after therapeutic abortion in two cases and spontaneous abortion in the third. Chorion biopsy and ultramicroscale enzymology may be a suitable alternative to recombinant-DNA-based methods for first-trimester diagnosis in selected diseases where the abnormal gene product is an enzyme expressed in the chorion by the 8th week of pregnancy.

Chorionic Villi↗
PubMed1984

An encapsidated, subgenomic messenger RNA encodes the coat protein of carnation mottle virus.

The translation strategy of carnation mottle virus (CarMV) in vitro has been generally assumed to involve internal initiation events on full-length, genomic RNA (4.3 kb). We suggest that this is, at least in part, incorrect. Encapsidated RNA, fractionated on denaturing sucrose gradients, or total RNA from CarMV-infected leaves, fractionated under non-denaturing conditions, was translated in an mRNA-dependent rabbit reticulocyte cell-free system. Evidence for subgenomic RNAs which encode a polypeptide of Mr 38 000 was found. This product was shown to be related to authentic CarMV coat protein by partial proteolysis with alpha-chymotrypsin and SDS/polyacrylamide-gel electrophoresis.

Capsid↗