Nuclear receptor and target gene mRNA abundance in duodenum and colon of dogs with chronic enteropathies.

Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR) and peroxisome proliferator-associated receptors alpha and gamma (PPARalpha, PPARgamma) are mediators of inflammation and may be involved in inflammatory bowel disease (IBD) and food responsive diarrhea (FRD) of dogs. The present study compared mRNA abundance of NR and NR target genes [multi drug-resistance gene-1 (MDR1), multiple drug-resistance-associated proteins (MRD2, MRD3), cytochrome P450 (CYP3A12), phenol-sulfating phenol sulfotransferase (SULT1A1) and glutathione-S-transferase (GST A3-3)] in biopsies obtained from duodenum and colon of dogs with IBD and FRD and healthy control dogs (CON; n=7 per group). Upon first presentation of dogs, mRNA levels of PPARalpha, PPARgamma, CAR, PXR and RXRalpha in duodenum as well as PPARgamma, CAR, PXR and RXRalpha in colon were not different among groups (P>0.10). Although mRNA abundance of PPARalpha in colon of dogs with FRD was similar in both IBD and CON (P>0.10), PPARalpha mRNA abundance was higher in IBD than CON (P<0.05). Levels of mRNA of MDR1 in duodenum were higher in FRD than IBD (P<0.05) or CON (P<0.001). Compared with CON, abundances of mRNA for MRP2, CYP3A12 and SULT1A1 were higher in both FRD and IBD than CON (P<0.05). Differences in mRNA levels of PPARalpha and MRP2 in colon and MDR1, MRP2, CYP3A12 and SULT1A1 in duodenum may be indicative for enteropathy in FRD and (or) IBD dogs relative to healthy dogs. More importantly, increased expression of MDR1 in FRD relative to IBD in duodenum may be a useful diagnostic marker to distinguish dogs with FRD from dogs with IBD.

Sequence, organization, transcription and evolution of RNA polymerase subunit genes from the archaebacterial extreme halophiles Halobacterium halobium and Halococcus morrhuae.

The genes for the four largest subunits, A, B', B" and C, of the DNA-dependent RNA polymerase were cloned from the extreme halophile Halobacterium halobium and sequenced and their transcription was analyzed. The downstream half of this gene cluster from another extreme halophile Halococcus morrhuae was also cloned, sequenced and its transcription products characterized. The H. halobium genes were transcribed into a common transcript from an upstream promoter in the order B", B', A and C. They are flanked by, and co-transcribed with, two smaller genes coding for 75 and 139 amino acid residues, respectively. Immediately downstream from these genes were two open reading frames that are homologous to ribosomal proteins S12 and S7 from Escherichia coli. In both extreme halophiles these genes were transcribed from their own promoter, but in Hc. morrhuae there was also considerable read-through from the RNA polymerase genes. Sequence alignment studies showed that the combined B" + B' subunits are equivalent to the B subunits of the eukaryotic polymerases I and II and to the eubacterial beta subunit, while the combined A + C subunits correspond to the A subunits of eukaryotic RNA polymerases I, II and III and to the eubacterial beta' subunit. The sequence similarity to the eukaryotic subunits was always much higher than to the eubacterial subunits. Conserved sequence regions within the individual subunits were located which are likely to constitute functionally important domains; they include sites associated with rifampicin and alpha-amanitin binding and two possible zinc binding fingers. Phylogenetic analyses based on sequence alignments confirmed that the extreme halophiles belong to the archaebacterial kingdom.

Heterologous in vitro transcription from two archaebacterial promoters.

A cell-free extract of Sulfolobus shibatae is able to specifically initiate transcription in vitro at the promoter of the plasmid-encoded gene for the major gas vesicle protein of Halobacterium halobium and at the promoter for the transcript T4 of the temperate H. halobium phage phi H. The corresponding promoter from the virulent phage mutant phi HL1 yields enhanced transcription in the heterologous system, in agreement with strongly increased in vivo expression.

Nuclear receptor and nuclear receptor target gene messenger ribonucleic acid levels at different sites of the gastrointestinal tract and in liver of healthy dogs.

Nuclear receptors (NR) are ligand-activated transcription factors that regulate different metabolic pathways by influencing the expression of target genes. The current study examined mRNA abundance of NR and NR target genes at different sites of the gastrointestinal tract (GIT) and the liver of healthy dogs (Beagles; n = 11). Samples of GIT and liver were collected postmortem and homogenized, total RNA was extracted and reverse transcribed, and gene expression was quantified by real-time reverse-transcription PCR relative to the mean of 3 housekeeping genes (beta-actin, glyceraldehyde-3-phosphate dehydrogenase, and ubi-quitin). Differences were observed (P < or = 0.05) in the mRNA abundance among stomach (St), duodenum (Du), jejunum (Je), ileum (Il), and colon (Col) for NR [pregnane X receptor (Du, Je > Il, Col > St), peroxisome proliferator-associated receptor gamma (St, Du, Col > Je, Il), constitutive androstane receptor (Je, Du > Il, Col), and retinoid x receptor alpha (Du > Il)] and NR target genes [glutathione-S-transferase A3-3 (Du > Je > St, Il; St > Col), phenol-sulfating phenol sulfotransferase 1A1 (Du, Je > Il, St; Col > St), cytochrome P450 3A12 (Du, Je > St, Il, Col), multiple drug resistance gene 1 (Du, Je, Il, Col > St), multiple drug resistance-associated protein 2 (Je, Du > Il > St, Col), multiple drug resistance-associated protein 3 (Col > St > Il; Du > Je, Il; St > Il), NR corepressor 2 (St > Il, Col), and cytochrome P450 reductase (St, Du, Je > Il, Col)], but not for peroxisome proliferator-associated receptor alpha. Differences (P > 0.05) in mRNA abundance in the liver relative to the GIT were also observed. In conclusion, the presence of numerous differences in expression of NR and NR target genes in different parts of the GIT and in liver of healthy dogs may be associated with location-specific functions and regulation of GIT regions.

Expression and regulation of Halobacterium halobium phage phi H genes.

In this paper we describe five distinct modes of phi H gene expression: (i) transcription of phage phi H during lytic growth on the sensitive host bacterium (Halobacterium halobium strain R1); (ii) transcription of the circularized prophage phi H1 in strain R(1)24; (iii) transcription of the L region of phi H present as 12-kilobase-plasmid in the immune strain R1L; (iv) transcription during the lytic growth of phage mutants containing an ISH23/50 in the immune strain R1L; (v) transcription during lytic growth of ISH23/50-insertion mutants in the sensitive host bacterium R1 showing enhancement of early transcripts. The sequential expression of the phage genome is described together with a detailed analysis of the transcription of early lytic, constitutive, and immune genes that map in the L region. The putative promoter sequences determined for several phage genes were compared with the upstream sequences of the H. halobium DNA-dependent RNA polymerase large subunit genes and with the gene for the ribosomal protein S12 homolog of H. halobium. The similarity of these putative promoter elements revealed conserved motifs that are discussed in relation to the TATA-box motif recognized by the eukaryotic DNA-dependent RNA polymerase II.

[The determination of von Willebrand factor activity in the plasma of dogs. 1. Experimental evaluation and methods]. / Die Bestimmung der Aktivität des von-Willebrand-Faktor im Plasma von Hunden. Teil 1: Versuchsansatz und Methodik.

Using a new aggregometer, the plasma activity of von Willebrand factor (vWF) in dogs was measured. The method is described. It has the advantage of being more precisely for the diagnosis of von Willebrand's disease than the determination of von Willebrand antigen (vWF: Ag) by using an ELISA. With the method described reproducible and reliable result can be obtained.

The bat gene of Halobacterium halobium encodes a trans-acting oxygen inducibility factor.

Oxygen and light affect the expression of the bacterioopsin gene (bop), which encodes a light-driven proton pump in the purple membrane of Halobacterium halobium. This response is thought to be mediated by a set of genes located adjacent to the bop gene. DNA fragments containing either the bop gene or the entire bop gene cluster reversed the phenotype of purple membrane-deficient strains with mutations in the bop gene. Purple membrane synthesis was constitutive in one of these strains transformed with the bop gene alone. The same strain transformed with the bop gene cluster was inducible by low oxygen tension. Moreover, another strain that constitutively expresses purple membrane remained constitutive when transformed with the bop gene alone but the phenotype of the strain changed to inducible when transformed with the bop gene cluster. Additional experiments have confirmed that one of the genes of the bop gene cluster, the bat gene, encodes a trans-acting factor that is necessary and sufficient to confer inducibility of purple membrane synthesis by low oxygen tension.

Association of the halobacterial 7S RNA to the polysome correlates with expression of the membrane protein bacterioopsin.

The sedimentation behavior of the halobacterial 7S RNA and bacterioopsin mRNA was assessed after application of total cell lysates to sucrose gradients. These two RNAs cosedimented predominantly with membrane-bound polysomes, and the quantity of 7S RNA bound to the ribosomes was directly correlated with the expression of bacterioopsin. Puromycin treatment released the 7S RNA from the polysomes, indicating that it is transiently associated with protein translation. We suggest that halobacteria contain a signal-recognition-like particle involved in translation of membrane-associated proteins.

Effects of upstream deletions on light- and oxygen-regulated bacterio-opsin gene expression in Halobacterium halobium.

The bacterio-opsin gene (bop) of Halobacterium halobium is located within a cluster with three other genes. Growth conditions of high light intensity and low oxygen tension induce bop gene cluster expression. To identify putative regulatory factor binding sites upstream of the bop gene, we have compared sequences upstream of the bop gene with the corresponding sequences from two other genes in the bop gene cluster. Conserved sequence motifs were observed which may mediate the effect of high light intensity and/or low oxygen tension on bop gene expression. Based on these motifs, a set of mutants was constructed which contained deletions upstream of the bop gene. These constructs were tested in a host strain where bop gene expression is independent of oxygen regulation and in another strain where it is regulated by oxygen and light. The minimal upstream sequence required for both light- and oxygen-regulated bop gene expression was determined to be 54 bp.

Comparative evaluation of gene expression in archaebacteria.

Gene organization, gene structure, especially regarding transcription and translation signals, and the structure of essential components of the gene expression machinery of archaebacteria are compared with those of eubacteria and eukaryotes. Many features of the genetic machinery of archaebacteria are shared either with eubacteria or with eukaryotes. For example, the translation signals including ribosome-binding sites are the same as in eubacteria, but the consensus sequence of archaebacterial promoters closely resembles that of the eukaryotic polymerase II promoters. Archaebacterial genes can be organized in transcription units resembling those of eubacteria. But the sequences of several protein components of the genetic machinery have strikingly more homology with those of their eukaryotic than with those of their eubacterial correspondents. The sequences of the large components of DNA-dependent RNA polymerases of archaebacteria closely resemble those of the eukaryotic RNA polymerases II and, somewhat less, III. In a dendrogram calculated from percentage homology data, the eukaryotic RNA polymerase I component A shares a branching point with the eubacterial component. The implications of these findings for the origin and the evolution of the eukaryotic ancestry are discussed.

The immunity-conferring plasmid p phi HL from the Halobacterium salinarium phage phi H: nucleotide sequence and transcription.

The complete nucleotide sequence of the plasmid p phi HL, composing the central 12,041-bp L-region from the temperate phage phi H of Halobacterium salinarium is presented. Transcripts mapped to the p phi HL and the L-region produced under immune conditions, under lytic growth or constitutively, are described. The sequences upstream of the transcription start points show homology to the consensus sequence for archaeal (formerly archaebacterial) promoters. Lytic transcription is shown to be strictly time-dependent, with an early gene product required for the expression of late genes.

Plasmid-related anaerobic autotrophy of the novel archaebacterium Sulfolobus ambivalens.

Three different species of the genus Sulfolobus, S. acidocaldarius, S. solfataricus (= Caldariella) and S. brierleyi, have been distinguished by the conditions required for optimal growth, by the component patterns of their DNA-dependent RNA polymerases and by DNA sequence data. Many isolates of these species are able to grow chemolithoautotrophically using CO2 as the sole carbon source and the oxidation of S(0) with O2 yielding sulphuric acid, as the energy source, though a few others grow only heterotrophically. We show here that a strain of a novel Sulfolobus species, S. ambivalens, is alternatively able to live by an anaerobic mode of chemolithoautotrophy, also using CO2 as the sole carbon source, but using reduction of S(0) with H2, yielding H2S as the energy source. This mode of growth is correlated with the amplification of a plasmid, pSL10.

Presence of poly (ADP-ribose) polymerase and poly (ADP-ribose) glycohydrolase in the dinoflagellate Crypthecodinium cohnii.

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase have been detected in chromatin extracts from the dinoflagellate Crypthecodinium cohnii. Poly(ADP-ribose) glycohydrolase was detected by the liberation of ADP-ribose from poly(ADP-ribose). Poly(ADP-ribose) polymerase was proved by (a) demonstration of phosphoribosyl-AMP in the phosphodiesterase digest of the reaction product, (b) demonstration of ADP-ribose oligomers by fractionation of the reaction product on DEAE-Sephadex. The (ADP-ribose)-protein transfer is dependent on DNA; it is inhibited by nicotinamide, thymidine, theophylline and benzamide. The protein-(ADP-ribose bond is susceptible to 0.1 M NaOH (70%) and 0.4 M NH2OH (33%). Dinoflagellates, nucleated protists, are unique in that their chromatin lacks histones and shows a conformation like bacterial chromatin [Loeblich, A. R., III (1976) J. Protozool. 23, 13--28]; poly(ADP-ribose) polymerase, however, has been found only in eucaryotes. Thus our results suggest that histones were not relevant to the establishment of poly(ADP-ribose) during evolution.

Archaebacterial DNA-dependent RNA polymerases testify to the evolution of the eukaryotic nuclear genome.

Genes for DNA-dependent RNA polymerase components B, A, and C from the archaebacterium Sulfolobus acidocaldarius and for components B", B', A, and C from the archaebacterium Halobacterium halobium were cloned and sequenced. They are organized in gene clusters in the order above, which corresponds to the order of the homologous rpoB and rpoC genes in the corresponding operon of the Escherichia coli genome. Derived amino acid sequences of archaebacterial components A and C were aligned with each other and with the sequences of corresponding (largest) subunits from the archaebacterium Methanobacterium thermoautotrophicum, with sequences of various eukaryotic nuclear RNA polymerases I, II, and III, and with the sequence of the beta' component from E. coli polymerase. The archaebacterial genes for component A are homologous to about the first two-thirds of genes for the eukaryotic component A and the eubacterial component beta', and the archaebacterial genes for component C are homologous to the last third of the genes for the eukaryotic component A and the eubacterial component beta'. Unrooted phylogenetic dendrograms derived from both distance matrix and parsimony analyses show the archaebacteria are a coherent group closely related to the eukaryotic nuclear RNA polymerase II and/or III lineages. The eukaryotic polymerase I lineage appears to arise separately from a bifurcation with the eubacterial beta' component lineage.

The phylogenetic relations of DNA-dependent RNA polymerases of archaebacteria, eukaryotes, and eubacteria.

Unrooted phylogenetic dendrograms were calculated by two independent methods, parsimony and distance matrix analysis, from an alignment of the derived amino acid sequences of the A and C subunits of the DNA-dependent RNA polymerases of the archaebacteria Sulfolobus acidocaldarius and Halobacterium halobium with 12 corresponding sequences including a further set of archaebacterial A+C subunits, eukaryotic nuclear RNA polymerases, pol I, pol II, and pol III, eubacterial beta' and chloroplast beta' and beta" subunits. They show the archaebacteria as a coherent group in close neighborhood of and sharing a bifurcation with eukaryotic pol II and (or) pol IIIA components. The most probable trees show pol IA branching off from the tree separately at a bifurcation with the eubacterial beta' lineage. The implications of these results, especially for understanding the possibly chimeric origin of the eukaryotic nuclear genome, are discussed.