Search PubMed⌕ Search

Biomedical subjects

R A Collins

Publications and source records attributed to R A Collins.

At least 91 records · Page 5Linked to original sources
PubMed1990

A site-specific self-cleavage reaction performed by a novel RNA in Neurospora mitochondria.

We describe a novel DNA and RNA found in the mitochondria of the Varkud-1c natural isolate of Neurospora. The majority of the RNA, termed VSRNA, is an 881 nucleotide single-stranded circular molecule complementary to one strand of a low copy, double-stranded circular DNA, VSDNA. VSRNA combines some features of the human hepatitis delta virus, group I introns, retroelements, and plant viral satellite RNAs. VSRNA synthesized in vitro performs a self-cleavage reaction whose products terminate with a 5' hydroxyl and a 2',3' cyclic phosphate. This reaction may be involved in the natural processing pathway of multimeric VSRNA in vivo. VSRNA lacks a hammer-head structure or substantial sequence similarity to any other self-cleaving RNA, suggesting that the RNA structure involved in cleavage may be different from those in previously characterized catalytic RNAs.

Base Sequence↗
PubMed1990

Independent transfer of mitochondrial chromosomes and plasmids during unstable vegetative fusion in Neurospora.

The sporadic distribution of similar introns in organelle, nuclear ribosomal RNA and bacteriophage genes suggests that at least some of these introns are mobile genetic elements. Some plasmids in fungal mitochondria contain intron-like sequences and, like introns, they have scattered distributions within and among species. The occurrence and evolutionary importance of such horizontal transfer of DNA, not only between fungi, but among a wide range of organisms have been matters of much discussion. Here, we report experimental evidence for transfer of Neurospora mitochondrial plasmids from one mitochondrial genotype to another at high frequency during unstable vegetative cell fusion. Exchange of mitochondrial and nuclear genomes can also occur. These observations suggest that vegetative fusion may have a more important role in the mitochondrial genetic structure of natural populations than is generally thought, and may provide an explanation for the distribution of certain plasmids and possibly other mobile genetic elements.

DNA, Mitochondrial↗
PubMed1990

Behavior of the [mi-3] mutation and conversion of polymorphic mtDNA markers in heterokaryons of Neurospora crassa.

We have examined the behavior of the [mi-3] mitochondrial mutation and two physical mtDNA markers in heterokaryotic cultures of Neurospora crassa. Previous workers showed that a 1.2-kilobase insertion in the larger polymorphic form of EcoRI-5 restriction fragment is a site of high frequency and rapid unidirectional gene conversion. We have confirmed this observation and determined by DNA sequence analysis that the insertion in the EcoRI-5 fragment corresponds precisely to an optional intron that contains a long open reading frame in the ND1 gene. Thus, the conversion of the short, intron-lacking, form of EcoRI-5 to the longer, intron-containing, form may be analogous to the unidirectional gene conversion events catalyzed by intron-encoded proteins in other organisms. The resolution of two polymorphic forms of the mtDNA EcoRI-9 restriction fragment in our heterokaryons differs from that observed previously and suggests that this locus is not a site of gene conversion in our heterokaryon pair. The size polymorphism of the EcoRI-9 fragments is due to a tandemly reiterated 78-base-pair sequence which occurs two times in the short form and three times in the long form. One copy of the repeat unit and 66 base pairs following it have been duplicated from the ND2 gene which is located about 30 kilobases distant on the mtDNA. In contrast to the [poky] mitochondrial mutant, which was completely dominant over wild-type mitochondria in heterokaryons, the [mi-3] mutant was recovered in only seven of twenty heterokaryons after ten cycles of conidiation and subculturing. The resolution of the [mi-3] or wild-type phenotype in heterokaryons may depend solely on random factors such as allele input frequency, drift, and segregation rather than specific dominant or suppressive effects.

Base Sequence↗
PubMed1990

Production of bovine immunoregulatory molecules by xenogeneic hybrids.

The study of bovine cytokines and lymphocyte surface markers has been restricted by the lack of stable, long-lived, easily maintained cell lines. To address this problem, bovine mononuclear leucocytes were fused with the mouse thymoma cell line BW5147 to produce interspecies hybrid cell lines and clones. Hybridomas were produced which contained more than 40 chromosomes and expressed one or more bovine leucocyte surface markers, they grew well and were cloned without the use of exogenous factors. Fusion rates were improved when exogenous factors, including recombinant human interleukin-2 (rhIL-2), were added. Some hybrids secreted immunoregulatory factors able to maintain the growth of either an IL-2-dependent bovine T-cell line (C625) and/or preactivated normal bovine B cells. Anti-human IL-2 partially blocked the T-cell growth factor (TCGF) activity produced by these hybrids. The release of growth factors by the hybrid clones 5/AA6/16 and 5/AA6/19 was augmented by concanavalin A stimulation.

Animals↗
PubMed1989

A group II intron in the Neurospora mitochondrial coI gene: nucleotide sequence and implications for splicing and molecular evolution.

The temperature-sensitive Neurospora nuclear mutant cyt18-1 is deficient in splicing many Group I mitochondrial introns when grown at its non-permissive temperature; however, splicing of intron 1 in the coI gene of the Adiopodoume (formerly called North Africa) strain is unaffected (R.A. Collins and A.M. Lambowitz, J. Mol. Biol. 184: 413-428, 1985). Here we show that coI intron 1 is a typical Group II intron, the only one identified to date in Neurospora. The differential effect of the cyt18-1 mutation suggests that splicing of certain introns could be regulated independently of others by nuclear-encoded proteins. The intron contains a long open reading frame (ORF) resembling that of the Neurospora Mauriceville mitochondrial plasmid. The intron and plasmid ORFs share unusual features of codon usage that suggest both evolved outside of the Neurospora mitochondrial genetic system.

Amino Acid Sequence↗
PubMed1989

Mutations in nuclear gene cyt-4 of Neurospora crassa result in pleiotropic defects in processing and splicing of mitochondrial RNAs.

The nuclear cyt-4 mutants of Neurospora crassa have been shown previously to be defective in splicing the group I intron in the mitochondrial large rRNA gene and in 3' end synthesis of the mitochondrial large rRNA. Here, Northern hybridization experiments show that the cyt-4-1 mutant has alterations in a number of mitochondrial RNA processing pathways, including those for cob, coI, coII and ATPase 6 mRNAs, as well as mitochondrial tRNAs. Defects in these pathways include inhibition of 5' and 3' end processing, accumulation of aberrant RNA species, and inhibition of splicing of both group I introns in the cob gene. The various defects in mitochondrial RNA synthesis in the cyt-4-1 mutant cannot be accounted for by deficiency of mitochondrial protein synthesis or energy metabolism, and they suggest that the cyt-4-1 mutant is defective in a component or components required for processing and/or turnover of a number of different mitochondrial RNAs. Defective splicing of the mitochondrial large rRNA intron in the cyt-4-1 mutant may be a secondary effect of failure to synthesize pre-rRNAs having the correct 3' end. However, a similar explanation cannot be invoked to account for defective splicing of the cob pre-mRNA introns, and the cyt-4-1 mutation may directly affect splicing of these introns.

Blotting, Northern↗
PubMed1989

Pathological changes following implantation of intramammary devices (IMD) and immunological mediator release by cells on recovered IMDs.

Implantation of abraded polyethylene intramammary devices (IMD) for six months in the mammary glands of cows resulted in macroscopic and microscopic pathological changes in udder tissue. These changes were characterised by hyperplasia and metaplasia of the epithelial cells and hypertrophy of the subepithelial connective tissue examined by light and electron microscopy (EM). Quarters containing IMDs also had increased numbers of neutrophils and macrophages in the subepithelial stroma compared with control quarters. IMDs recovered six months after implantation were shown by transmission and scanning EM to be covered with plaque and cells. These cells were mainly macrophages, although other leucocytes were also present. In vitro culture of recovered IMDs in the presence of lipopolysaccharide resulted in the release of neutrophil chemotactic factor, or factors, and interleukin-1. Some quarters with IMDs also had concurrent infections at the time of slaughter. In these cases both the pathological changes seen in the tissues and the release of soluble mediators following in vitro culture of the IMDs were significantly increased compared with sterile quarters containing IMD.

Animals↗
PubMed1988

Independent evolution of structural and coding regions in a Neurospora mitochondrial intron.

The discovery of intervening sequences (introns) in eukaryotic genes has raised questions about the origin and evolution of these sequences. Hypotheses concerning these topics usually consider the intron as a unit that could be lost or gained over time, or as a region within which recombination can occur to facilitate the production of new proteins by exon shuffling. Additional complexities are observed in introns of mitochondrial and chloroplast genes which contain secondary structures required for messenger RNA splicing and open-reading frames encoding proteins. Here we describe differences in the organization of protein-coding sequences in the intron of the mitochondrial ND1 gene in two closely related species of Neurospora. These differences show that intron sequences involved in secondary structure formation and in protein coding can evolve as physically distinct elements. Indeed, the secondary structure elements of the ND1 intron can contain two different coding sequences located at two different positions within the intron.

Amino Acid Sequence↗
PubMed1988

Evidence of natural selection to maintain a functional domain outside of the 'core' in a large subclass of group I introns.

Comparison of three closely-related, homologous Group I introns reveals conservation of RNA secondary structure and some primary sequence outside of the characteristic Group I core structure. Further examination of forty Group I introns showed that all can be placed into one of two categories based on the length of the "loop L5" region (subtended by the base-paired sequences P and Q): short (21 to 38 bases) or long (59 to 295 bases). Despite the large variation in size and sequence, all nineteen of the long L5 introns share a common structure whose features include an adenine-rich bulge at a fixed distance from the P-Q pairing. This bulge is flanked by base-paired regions of greater than or equal to 6 base pairs on the core-proximal side and greater than or equal to 3 base pairs on the distal side. In the core-proximal helix there are a large number and high proportion of deviations from the consensus sequence that maintain base-pairing. These naturally-occurring compensatory base substitutions provide compelling phylogenetic support for the existence of this pairing and indicate that the conserved structure has a function in vivo.

Aspergillus↗
PubMed1988

The self-splicing intron in the Neurospora apocytochrome b gene contains a long reading frame in frame with the upstream exon.

We have determined the DNA sequence of intron 1 and flanking exons in the mitochondrial apocytochrome b gene of the Neurospora laboratory strain 74A and the natural isolate North Africa. In contrast to a previous report, we find that this intron contains an open reading frame (ORF) of 951 bases in frame with the upstream exon. The putative intron-encoded protein resembles those of other intron ORFs with respect to length, calculated isoelectric point, and proportion of basic, acidic, polar, and non-polar amino acids; however, no amino acid sequences resembling the "decapeptides" characteristic of maturase-like ORFs were found. Coupled with the previous finding that this intron is capable of self-splicing in vitro in the absence of proteins, the observations discussed here raise the possibility that other introns with long, in-frame ORFs may also be capable of RNA-catalyzed splicing in vitro.

Amino Acid Sequence↗
PubMed1988

Histochemical localization and possible antibacterial role of xanthine oxidase in the bovine mammary gland.

Xanthine oxidase (XO) was demonstrated to be present in the teat canal and secretory tissue of the bovine mammary gland by histochemical techniques. Homogenates of these tissues were able to replace XO in an antibacterial assay with Streptococcus uberis. The action of XO on its substrate hypoxanthine was shown to provide an essential component for anti-streptococcal activity mediated by lactoperoxidase. A mechanism is proposed whereby the interaction of XO, lactoperoxidase and thiocyanate may provide antibacterial activity in the teat canal.

Animals↗
PubMed1986

Proliferative responses and IL-2 production by mononuclear cells from bovine mammary secretions, and the effect of mammary secretions on peripheral blood lymphocytes.

Mammary gland mononuclear cells (MGM) were compared with peripheral blood mononuclear cells (PBM) for their ability to proliferate in response to phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM), and to produce interleukin-2 (IL-2) when cultured with Con A. MGM proliferative responses to all three mitogens were at a level only just above background. Following removal of adherent cells by passage of the MGM population over Sephadex G10, their ability to proliferate increased but these responses were still significantly lower (P less than 0.001) than those of PBM. Mammary secretions from non-lactating cows and colostrum, but not milk, were found to suppress the proliferative responses to PHA and Con A of PBM following a 24-hr preincubation period. There was no difference in IL-2 production in response to Con A by Sephadex G10-treated MGM and PBM, and dry-cow secretions had no effect on IL-2 production.

Animals↗
PubMed1986

Antibody-containing cells and specialised epithelial cells in the bovine teat.

Tissue from the ends of teats of dry, periparturient and lactating cows were studied using light and electron microscopy. Accumulations of infiltrating leucocytes mainly in the folds of the distal rosette of the teat cistern (Furstenberg's rosette) were detected; plasma cells predominated. The latter were classified by the type of immunoglobulin (Ig) which they synthesised. Plasma cells synthesising IgG1 were found to be the major antibody producing cell type of the teat. Neither the number of stromal plasma cells present nor the class of Ig which they synthesised was significantly altered by changes in mammary gland secretory activity. Scanning electron microscopy revealed areas of epithelium of Furstenberg's rosette that contained cells differing in surface characteristics from epithelial cells of adjacent areas of the teat cistern.

Animals↗
PubMed1985

Influence of liver fat on experimental Escherichia coli mastitis in periparturient cows.

Eleven cows with a wide range of liver fat (5.7 to 51.4 per cent) at seven days post partum were experimentally infected in a single quarter with a capsular Escherichia coli at 10 days post partum. The results suggested that a fatty liver in itself does not influence the severity of mastitis. All animals had clinical mastitis 10 hours after infection but no animals became severely ill and no treatment was given. Four out of five animals in the group with less than 20.2 per cent liver fat had bacteria in their milk at 10 hours after infection but these bacteria were eliminated by 12 hours. The six animals in the group with more than 28.3 per cent fat in their liver retained viable bacteria in the udder for much longer; with two animals bacteria were shed and abnormal milk was secreted for up to four months despite antibiotic therapy.

Animals↗
PubMed1985

RNA splicing in Neurospora mitochondria. Defective splicing of mitochondrial mRNA precursors in the nuclear mutant cyt18-1.

cyt18-1 (299-9) is a nuclear mutant of Neurospora crassa that has been shown to have a temperature-sensitive defect in splicing the mitochondrial large rRNA intron. In the present work, we investigate the effect of the cyt18-1 mutation on splicing of mitochondrial mRNA introns. Two genes were studied in detail; the cytochrome b (cob) gene, which contains two introns, and a "long form" of the cytochrome oxidase subunit I (coI) gene, which contains four introns. We found that splicing of both cob introns and splicing of at least two of the coI introns are strongly inhibited in the mutant, whereas splicing of coI intron 1, which is excised as a 2.6 X 10(3) base circle, is relatively unaffected. The rRNA intron and both cob introns are group I introns, whereas the circular coI intron may belong to another structural class. Control experiments showed that the degree of inhibition of splicing is greater in the mutant than can be accounted for by severe inhibition of mitochondrial protein synthesis. Finally, experiments in which mutant cells were shifted from 25 degrees C to 37 degrees C showed that splicing of the large rRNA precursor and splicing of the coI mRNA precursor are inhibited with similar kinetics. Considered together, our results suggest that the cyt18 gene encodes a trans-acting component that is required for the splicing of group I mitochondrial DNA introns or some subclass thereof. Since Neurospora cob intron 1 has been shown to be self-splicing in vitro, defective splicing of this intron in cyt18-1 indicates that an essentially RNA-catalyzed splicing reaction must be facilitated by a trans-acting factor, presumably a protein, in vivo.

Electrophoresis, Agar Gel↗
PubMed1985

Histological measurement of fat content of liver of dairy cows.

A total of 275 liver biopsies were performed on dairy cows in 6 Friesian and one Guernsey herd during the first 2 weeks after calving. Liver samples were processed by 2 independent histological procedures: (a) formalin-fixed frozen sections were stained with oil-red O (ORO), or (b) samples fixed in glutaraldehyde-osmium tetroxide were embedded in plastic and sections stained with toluidine blue (TOLB). The sections were then subjected to stereological point-counting procedures to assess the quantities of stainable fat present within the liver cells. Estimates of liver cell fat by the 2 techniques were highly correlated, although those obtained by the TOLB method were consistently lower than those obtained by the ORO method. The analytical sampling error was slightly lower in the TOLB method. The simpler ORO method should prove an acceptable alternative in the routine histology laboratory to the tedious and technically demanding TOLB method.

Adipose Tissue↗
PubMed1984

Urokinase perfusion for axillary-subclavian vein thrombosis.

We report a single case of urokinase perfusion of axillary-subclavian vein thrombosis with the restoration of normal venous outflow of the arm. It is hoped that this will halt the usual sequelae causing chronic venous insufficiency.

Angioplasty, Balloon↗