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Biomedical subjects

R T Stone

Publications and source records attributed to R T Stone.

At least 55 records · Page 3Linked to original sources
PubMed1996

The linkage map of sheep Chromosome 6 compared with orthologous regions in other species.

The genetic linkage map of sheep Chromosome (Chr) 6 has been extended to include 35 loci with the addition of 11 RFLP and 12 microsatellite loci. The sex-averaged linkage map now spans 154 cM from phosphodiesterase cyclic GMP beta polypeptide (PDE6B) to OarCP125, an anonymous sheep microsatellite. The male and female map lengths, at 180 cM and 132 cM respectively, did not differ significantly. The physical assignment of PDE6B to Chr 6q33-qter orientates the linkage map on sheep Chr 6 with PDE6B near the telomere and OarCP125 towards the centromere. The order and genetic distances between loci are similar for the sheep Chr 6 and cattle Chr 6 maps, except for the position of the casein genes. The sheep Chr 6 linkage map is also comparable to portions of human Chr 4, mouse Chrs 5 and 3, and pig Chr 8. The synteny between sheep Chr 6 and human Chr 4 has been extended from PDE6B (4p16.3) to epidermal growth factor (EGF, 4q25-q27). However, a region from platelet-derived growth factor receptor alpha polypeptide (PDGFRA) to bone morphogenetic protein 3 (BMP3), which spans 19 cM on sheep Chr 6, appears to be inverted with respect to the human and mouse loci. Other differences in the gene order between sheep, pig, and mouse suggest more complex rearrangements.

Animals↗
PubMed1996

Cloning and characterization of the bovine Fas.

Fas (APO-1, CD95), a member of the tumor necrosis factor (TNF) receptor superfamily, is a cell membrane protein that mediates programmed cell death (apoptosis). In an effort to characterize the possible role of Fas-mediated apoptosis in some important physiological processes in livestock, bovine Fas (bFas) cDNA was isolated and its nucleotide sequence determined. The predicted amino acid sequence encodes a 323-amino-acid protein that contains a leader peptide, a transmembrane domain, and three domains of cysteine-rich motif within the extracellular region. At the amino acid level, bFas is 57% and 50% identical to human (hFas) and mouse (mFas), respectively. Its expression is abundant in peripheral blood lymphocytes, lung, spleen, thymus, and ovary, but minimal in heart, liver, and brain. A polyclonal anti-bFas serum raised against the carboxy-terminal half of cysteine-rich motif I recognized a single 46-kD protein in bovine MDBK cells by Western blot analysis. To investigate the apoptotic activity of bFas, MDBK and bFas-transfected L929 cells were exposed to a monoclonal anti-hFas IgM. Unlike other cell culture systems, the antibody failed to trigger cell death in MDBK and bFas transfected L929 cells.

Amino Acid Sequence↗
PubMed1996

Genomic organization and chromosomal mapping of the bovine Fas/APO-1 gene.

The cell-surface protein Fas (APO-1) is a member of the tumor necrosis factor receptor (TNFR) superfamily and transduces apoptosis following binding of Fas ligand or exposure to certain anti-Fas antibodies. We have isolated the bovine Fas (bFas) gene and determined its genomic organization and chromosomal location. Our data indicate that bFas is a single-copy gene that contains 9 exons and spans approximately 31.5 kb. The 5'-flanking region lacks conventional TATA and CCAAT elements, but contains several putative regulatory elements, including multiple copies of Sp1, AP-2, E-box, and N-box consensus sites. Linkage analysis of two (CA) dinucleotide repeat microsatellites within intron 1 and physical assignment by fluorescence in situ hybridization (FISH) placed the bFas gene on bovine chromosome 26. Collectively, these data provide a basis for understanding the regulatory mechanisms that control bFas gene expression.

Animals↗
PubMed1996

Cloning and chromosomal mapping of bovine interleukin-2 receptor gamma gene.

Interleukin-2 receptor (IL-2R) gamma chain, a member of the cytokine receptor superfamily, forms a high-affinity receptor with IL-2R alpha and beta chains that plays an important role in interleukin-2 (IL-2) signal transduction. We have cloned and characterized the bovine IL-2Rgamma gene and corresponding cDNA. Bovine IL-2Rgamma is a single-copy gene that contains 8 exons and spans approximately 3.8 kb. The promoter region lacks conventional TATA and CCAAT consensus sites, but contains several regulatory elements that are recognition sites for the GATA binding proteins, AP-1 and AP-2. Physical assignment by fluorescence in situ hybridization (FISH) placed the bovine IL-2Rgamma gene on chromosome Xq23.

Amino Acid Sequence↗
PubMed1996

Report of the first workshop on the genetic map of bovine chromosome 23.

A report of the first workshop on the genetic map of bovine chromosome 23 (BTA23) is given. Five laboratories contributed data from 29 loci, including a total 11586 informative genotypes. The combined pedigrees represented 1930 potentially informative meioses. Eighteen of the 29 loci were common to two or more data sets and were used to construct a framework linkage map of BTA23. Twelve of the 18 could be ordered on the linkage map with a likelihood ratio of greater than 1000:1. Thus, a low resolution consensus map was constructed with a high level of support for order. The sex-averaged, female and male maps span 54.5, 52.7 and 55.8 cM, respectively. Sex-specific differences in recombination frequency were identified for eight pairs of framework loci. Average genetic distance between framework loci on the sex-averaged map is 5.0 cM.

Animals↗
PubMed1996

Characterization of a single-locus minisatellite DNA in Xenopus laevis.

We have cloned a minisatellite tandem array (XTA) from Xenopus laevis that contains approximately 200 copies of the 20-bp repeat 5'-CCAACAGCCTGCCCATCCAT-3'. The XTA sequence is present only once per haploid genome and is polymorphic with respect to repeat number and location of flanking restriction endonuclease sites. Although the 20-bp repeat has not previously been described, flanking sequences suggest that it lies proximal to coding regions in the Xenopus genome.

Animals↗
PubMed1996

Evaluation of a fully automated end-tidal carbon monoxide instrument for breath analysis.

We evaluated a novel, portable breath sampler/CO-quantifying instrument [Baby's Breath Carbon Monoxide Analyzer (BB); Natus Medical], developed for use at the bedside or with gas samples collected into bags. Bench tests demonstrated that the CO measurements were linear, accurate, and precise when compared with gas chromatography (GC) results. In vivo tests (n = 30) performed with adults showed excellent correlation between end-tidal breath CO measurements (ETCO) corrected for inhaled CO (ETCOc) as determined by BB and GC. Breath sampling efficiency was 96%. ETCOc measurements and blood carboxyhemoglobin fractions (% of total hemoglobin) corrected for inhaled CO (COHbc) correlated strongly: COHbc = 0.25 ETCOc--0.01 microL/L CO (r2 = 0.97, Sy/x = 0.47, SE slope = 0.01, n = 30). The imprecision, assessed by the mean of the population's CV for triplicate determinations, was 11%. Measurements with healthy and hemolytic term newborns showed that ETCOc values of > 3 microL/L correlated with known hemolytic conditions. We conclude that this instrument is clinically reliable and can be used to noninvasively measure ETCO in neonates and adults.

Adult↗
PubMed1995

A small-insert bovine genomic library highly enriched for microsatellite repeat sequences.

A bovine genomic phagemid library was constructed with randomly sheared DNA. Enrichment of this single-stranded DNA library with CA or GT primers resulted in 45% positive clones. The 14% of positive clones with (CA.GT) > 12, and not containing flanking repetitive elements, were sequenced, and the efficiency of marker production was compared with random M13 bacteriophage libraries. Primer sequences and genotyping information are presented for 390 informative bovine microsatellite markers. The genomic frequency for 11 tri- and tetranucleotide repeats was estimated by hybridization to a lambda genomic library. Only GCT, GGT, and GGAT were estimated to have a frequency of > 100 per genome. Enrichment of the phagemid library for these repeats failed to provide a viable source of microsatellite markers in the bovine. Comparison of map interval lengths between 100 markers from the enriched library prepared from randomly sheared DNA and M13 bacteriophage libraries prepared from Mbo1 restriction digests suggested no bias in skeletal genomic coverage based on source of small insert DNA. In conclusion, enrichment of the bovine phagemid library provides a sufficient source of microsatellites so that small repeat lengths and flanking repetitive sequences common in the bovine can be eliminated, resulting in a high percentage of informative markers.

Animals↗
PubMed1995

Porcine SINE-associated microsatellite markers: evidence for new artiodactyl SINEs.

Approximately 24% (170/710) of porcine (dG-dT)n.(dC-dA)n microsatellites isolated in our laboratory are associated with a previously described porcine Short Interdispersed Element (SINE) termed PRE-1 SINE. Another 5.6% (40/710) of the microsatellites were adjacent to two previously unidentified SINE sequences, which we have designated ARE-1P (Artiodactyl Repetitive Element-1 Porcine) and ARE-2P. The ARE repeats were also found in bovine microsatellite and genomic sequences in the GenBank database. Genotypic information was obtained from 68.9% of primers where at least one primer sequence was obtained from the PRE-1 SINE and 66.6% of primer pairs designed from the ARE SINEs. The use of primers derived from SINEs significantly increases the number of primer pairs available for genetic linkage studies in swine.

Animals↗
PubMed1995

Bovine fast-twitch myosin light chain 1: cloning and mRNA amount in muscle of cattle treated with clenbuterol.

The cDNA clone encoding the fast-twitch isoform of myosin light chain 1 (MLC-1f) was isolated from bovine longissimus dorsi muscle and sequenced in M13 and pUC8. An 0.8-kb subclone, produced by digestion of the cDNA with EcoRI, contained the portion of the molecule common to MLC-1f and MLC-3f. The cDNA in pUC8 contained an additional 81 bp upstream of the EcoR I digestion site, which was unique to MLC-1f. The cDNA clone was used to measure MLC-1f mRNA in longissimus dorsi muscle of cattle chronically administered the beta-adrenergic agonist clenbuterol. Treatment with clenbuterol for 50 days increased succinic dehydrogenase negative (type IIB) and positive (types I and IIA) myofiber cross-sectional areas by 25%. After the 50-day treatment period, the amount of MLC-1f mRNA was 90% greater in longissimus dorsi muscle of treated animals than in the initial group. This effect was lost when clenbuterol treatment was withdrawn for a 78-day period, during which time muscle growth in the treated animals stopped completely. We conclude that we have cloned the bovine cDNA for MLC-1f, which has provided additional evidence that beta-adrenergic agonists increase myofibrillar gene expression.

Amino Acid Sequence↗
PubMed1994

The Booroola fecundity (FecB) gene maps to sheep chromosome 6.

The Booroola (FecB) mutation in sheep is linked to markers from a region of syntenic homology to human chromosome HSA4q, but the chromosomal location in sheep has not been determined. Analysis of linkage in Booroola half-sib pedigrees and 17 full-sib families identified genetic linkage between platelet-derived growth factor receptor-alpha (PDGFRA) and alpha s1-casein (CSN1S1) at 12 cM (Zmax = 9.14) and between PDGFRA and the microsatellite markers BM143 and OarHH55 (Zmax = 6.28 and 3.83, respectively). The microsatellite markers OarAE101 and BM143 and genes from the linkage group (PDGFRA, SPP1, and EGF) were mapped in a partial sheep x hamster somatic cell hybrid panel. All markers identified bands specific to somatic cell hybrids containing the sheep chromosome t1 (rob6;24) or t1q (chromosome 6). In sheep the casein genes alpha s1 (CSN1S1), alpha s2 (CSN1S2), beta (CSN2), and kappa (CSN3) are tightly linked, and CSN2 has been mapped to sheep chromosome 6q23-q31. We conclude that the Booroola mutation is located within a conserved syntenic group that maps to sheep chromosome 6.

Animals↗
PubMed1994

Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells.

The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and beta-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.

Animals↗