PubMed Health⌕ Search

Biomedical subjects

R Evers

Publications and source records attributed to R Evers.

At least 19 recordsLinked to original sources
PubMed1995

Molecular coevolution of mammalian ribosomal gene terminator sequences and the transcription termination factor TTF-I.

Both the DNA elements and the nuclear factors that direct termination of ribosomal gene transcription exhibit species-specific differences. Even between mammals--e.g., human and mouse--the termination signals are not identical and the respective transcription termination factors (TTFs) which bind to the terminator sequence are not fully interchangeable. To elucidate the molecular basis for this species-specificity, we have cloned TTF-I from human and mouse cells and compared their structural and functional properties. Recombinant TTF-I exhibits species-specific DNA binding and terminates transcription both in cell-free transcription assays and in transfection experiments. Chimeric constructs of mouse TTF-I and human TTF-I reveal that the major determinant for species-specific DNA binding resides within the C terminus of TTF-I. Replacing 31 C-terminal amino acids of mouse TTF-I with the homologous human sequences relaxes the DNA-binding specificity and, as a consequence, allows the chimeric factor to bind the human terminator sequence and to specifically stop rDNA transcription.

Amino Acid Sequence↗
PubMed1995

Different domains of the murine RNA polymerase I-specific termination factor mTTF-I serve distinct functions in transcription termination.

Termination of mouse ribosomal gene transcription by RNA polymerase I (Pol I) requires the specific interaction of a DNA binding protein, mTTF-I, with an 18 bp sequence element located downstream of the rRNA coding region. Here we describe the molecular cloning and functional characterization of the cDNA encoding this transcription termination factor. Recombinant mTTF-I binds specifically to the murine terminator elements and terminates Pol I transcription in a reconstituted in vitro system. Deletion analysis has defined a modular structure of mTTF-I comprising a dispensable N-terminal half, a large C-terminal DNA binding region and an internal domain which is required for transcription termination. Significantly, the C-terminal region of mTTF-I reveals striking homology to the DNA binding domains of the proto-oncogene c-Myb and the yeast transcription factor Reb1p. Site-directed mutagenesis of one of the tryptophan residues that is conserved in the homology region of c-Myb, Reb1p and mTTF-I abolishes specific DNA binding, a finding which underscores the functional relevance of these residues in DNA-protein interactions.

3T3 Cells↗
PubMed1991

A transferrin-binding protein of Trypanosoma brucei is encoded by one of the genes in the variant surface glycoprotein gene expression site.

A transferrin-binding protein (TFBP) with an apparent molecular weight of 42 kd was purified from detergent-soluble membrane proteins of bloodstream forms of Trypanosoma brucei. The protein is not expressed in the insect-borne stage of the parasite's life-cycle. Purified TFBP can be converted from an amphiphilic to a hydrophilic form by cleavage with T.brucei glycosylphosphatidylinositol (GPI)-specific phospholipase C, demonstrating that the C-terminus is modified by a GPI-membrane anchor. The TFBP is encoded by an expression-site-associated gene [ESAG 6 in the nomenclature of Pays et al. (1989) Cell, 57, 835-845] which is under the control of the promoter transcribing the expressed variant surface glycoprotein gene. The possible function of TFBP as a receptor for the uptake of transferrin in bloodstream forms is discussed.

Amino Acid Sequence↗
PubMed1990

The Trypanosoma brucei protein phosphatase gene: polycistronic transcription with the RNA polymerase II largest subunit gene.

We have previously described the trypanosomal gene encoding the largest subunit of RNA polymerase II (RNAP II) and found that two almost identical genes are encoded within the Trypanosoma brucei genome. Here we show by Southern analyses that the 5' breakpoint between both loci is located approximately 7.5 kb upstream of the RNAP II genes. Northern analyses revealed that the 5' duplicated segment contains at least four other genes, which are transcribed in both bloodstream and procyclic trypanosomes. The gene located immediately upstream of the RNAP II gene in both loci was characterized by sequence analyses. The deduced amino acid sequences show a high degree of similarity to the catalytic subunit of protein phosphatase class 1 (PP1) genes. S1 mapping provided strong evidence in support of the fact that the PP1 and RNAP II genes belong to a single transcription unit.

Amino Acid Sequence↗
PubMed1990

Identification and sequence analysis of the ribosomal DNA promoter region of Crithidia fasciculata.

We have identified the promoter region of the large ribosomal DNA repeat unit of Crithidia fasciculata by northern blotting and nuclear run-on analyses. These data show that transcription starts approximately 1 kb upstream of the 18S rRNA gene. S1 protection experiments and sequence analysis of this area resulted in a precise localization of the start site. We have been unable to identify conserved sequence element(s) by a direct comparison of the crithidial RNA polymerase I promoter region and similar promoter regions of other eukaryotes; not even to the promoter region of the more closely related kinetoplastid species, Trypanosoma brucei. The absence of homology within the primary sequence of the promoter region, which is also found in other eukaryotes, might explain the observed species specificity of in vivo and in vitro rDNA transcription, since this resides in the interaction of initiation factor(s) and the core promoter domain.

Animals↗
PubMed1990

Phylogenetic analysis of the RNA polymerases of Trypanosoma brucei, with special reference to class-specific transcription.

We have sequenced the genes encoding to largest subunits of the three classes of DNA-dependent RNA polymerases of Trypanosoma brucei. The nucleotide and deduced amino acid sequences were compared and aligned with the corresponding sequences of other eukaryotes. Phylogenetic relationships were subsequently calculated with a distant matrix, a bootstrapped parsimony and a maximum-likelihood method. These independent calculations resulted in trees with very similar topologies. The analyses show that all the largest subunits of T. brucei are evolutionarily distant members within each of the three RNA polymerase classes. An early separation of the trypanosomal subunits from the eukaryotic lineage might form the fundamental basis for the unusual transcription process of this species. Finally, all dendrograms show a separate ramification for the largest subunit of RNA polymerase I, II and III. RNA polymerase II and/or III form a bifurcation with the archaebacterial lineage, RNA polymerase I, however, arises separately from the eubacterial beta' lineage. This suggests that the three eukaryotic RNA polymerase classes are not simply derived by two gene duplications of an ancestral gene with subsequent differentiation.

Amino Acid Sequence↗
PubMed1989

Unusual C-terminal domain of the largest subunit of RNA polymerase II of Crithidia fasciculata.

The C-terminal domain of the largest subunit of RNA polymerase II in higher eukaryotes is present in the protozoan parasite Trypanosoma brucei in a strongly modified form. To determine whether this is a general feature of the Kinetoplastida and to determine the role of this domain in RNA polymerase II transcription, we have analysed the C-terminal domain of the distantly related species Crithidia fasciculata. No positional identity of amino acid residues between the C-termini of C. fasciculata and T. brucei can be found. Moreover, both domains lack the heptapeptide repeat structure present in higher eukaryotes. The two domains are, however, very similar in amino acid composition, being rich in acidic residues as well as serine and tryosine. The latter observation is compatible with the concept that in vivo phosphorylation of the C-terminus activates RNA polymerase II.

Amino Acid Sequence↗
PubMed1989

Trypanosoma brucei contains two RNA polymerase II largest subunit genes with an altered C-terminal domain.

We have identified and cloned four trypanosomal RNA polymerase largest subunit genes. Here, we present the molecular analysis of two genes, Trp4.8 and Trp5.9. The sequence of these genes shows that they are almost identical to each other and indicates that they encode the largest subunit of RNA polymerase II. Both genes contain a C-terminal extension that is clearly distinct from that of other eukaryotic RNA polymerase II genes, because it lacks the common tandemly repeated heptapeptide sequence and is rich in acidic amino acids. It shares many potential phosphorylation sites, however, with the C-terminal extension of other eukaryotic RNA polymerase II large subunits. The presence of two RNA polymerase II loci suggests that a fourth RNA polymerase could be formed. Interestingly, the fourth gene is only found in species exhibiting antigenic variation.

Amanitins↗
PubMed1989

Characterization of the RNA polymerases of Trypanosoma brucei: trypanosomal mRNAs are composed of transcripts derived from both RNA polymerase II and III.

To analyze transcription in Typanosoma brucei, we have characterized the trypanosomal RNA polymerases. Here we present our results, which allow a discrimination between the different classes of RNA polymerases in nuclear run-on experiments by polymerase inhibitors and Mn2+ dependence. We also describe the separation of trypanosomal RNA polymerases by chromatography, demonstrating that T. brucei contains RNA polymerases I-III. The outcome of our experiments suggests that the VSG genes of T. brucei are not transcribed by RNA polymerase I, as previously reported, but by RNA polymerase II. We propose that an additional factor modifies RNA polymerase II, resulting in the alpha-amanitin-resistant transcription of VSG genes. Our data also suggest that the mini-exon genes, which encode the 5' end of each trypanosomal mRNA, are probably transcribed by RNA polymerase III.

Amanitins↗
PubMed1989

Production and characterization of monoclonal antibodies against specific serotypes of Mycobacterium avium and the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex.

Serotype-specific and Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex (MAIS complex)-specific monoclonal antibodies (MAbs) were prepared. A series of MAbs were obtained, five specific for serotype 2, three specific for serotype 4, eight against a strain of serotype 19, two specific for the MAIS complex, and two against a common glycolipid shared by all the mycobacteria tested so far. The serotype-specific and the MAIS complex-specific MAbs reacted in immunofluorescence with intact mycobacteria and in enzyme-linked immunosorbent assay and immuno-thin-layer chromatography with lipid extracts of mycobacteria. The two MAbs against a common mycobacterial glycolipid reacted only in lipid enzyme-linked immunosorbent assay and immuno-thin-layer chromatography. All MAbs were directed against glycopeptidolipids (GPLs), except for four MAbs against proteins of serotype 19. The serotype- and MAIS complex-specific epitopes on GPLs are exposed on the mycobacterial cell wall, in contrast with the common mycobacterial glycolipid, which is probably located inside the cell wall. The serotype-specific MAbs reacted with native as well as deacetylated GPLs, in contrast with the MAIS complex-specific MAbs, which reacted only with native GPLs. The MAbs will be useful for the identification of MAIS complex and M. avium serotypes 2 and 4 and a strain of serotype 19, GPL analyses with immuno-thin-layer chromatography, and the localization of GPL epitopes in mycobacteria.

Animals↗
PubMed1988

Structure and sequence of the gene for the largest subunit of trypanosomal RNA polymerase III.

As the first step in the analysis of the transcription process in the African trypanosome, Trypanosoma brucei, we have started to characterise the trypanosomal RNA polymerases. We have previously described the gene encoding the largest subunit of RNA polymerase II and found that two almost identical RNA polymerase II genes are encoded within the genome of T. brucei. Here we present the identification, cloning and sequence analysis of the gene encoding the largest subunit of RNA polymerase III. This gene contains a single open reading frame encoding a polypeptide with a Mr of 170 kD. In total, eight encoding a polypeptide with a Mr of 170 kD. In total, eight highly conserved regions with significant homology to those previously reported in other eukaryotic RNA polymerase largest subunits were identified. Some of these domains contain functional sites, which are conserved among all eukaryotic largest subunit genes analysed thus far. Since these domains make up a large part of each polypeptide, independent of the RNA polymerase class, these data strongly support the hypothesis that these domains provide a major part of the transcription machinery of the RNA polymerase complex. The additional domains which are uniquely present in the largest subunit of RNA polymerase I and II, respectively, two large hydrophylic insertions and a C-terminal extension, might be a determining factor in specific transcription of the gene classes.

Amino Acid Sequence↗
PubMed1988

The topogenic signal of the glycosomal (microbody) phosphoglycerate kinase of Crithidia fasciculata resides in a carboxy-terminal extension.

To determine how microbody proteins enter microbodies, we have previously compared the genes for the cytosolic and glycosomal (microbody) phosphoglycerate kinases (PGKs) of Trypanosoma brucei and found the microbody enzyme to differ from other PGKs and the cytosolic form in two respects: a high net positive charge and a C-terminal extension of 20 amino acids (Osinga et al., 1985). Here we present the comparison of the genes for the cytosolic and glycosomal PGKs of Crithidia fasciculata, another kinetoplastid organism. The amino acid sequences of the two Crithidia isoenzymes are virtually identical, except for a C-terminal extension of 38 amino acids. We conclude that this extension must direct the glycosomal PGK to the glycosome. The extensions of the Crithidia and Trypanosoma enzymes are both rich in small hydrophobic and hydroxyl amino acids.

Amino Acid Sequence↗
PubMed1977

Bronchodilator effect of hexoprenaline aerosol in bronchial asthma and chronic bronchitis.

The bronchodilator effects of aerosols of hexoprenaline (200 microgram and 400 microgram), and salbutamol (200 microgram) were compared in 15 patients with asthma, and nine patients with chronic bronchitis. In both groups of patients, hexoprenaline and salbutamol produced a similar increase in the forced expiratory volume in one second (FEV1), and mean percentage increase in FEV1. Neither drug caused tachycardia or arrhythmia. It was concluded that hexoprenaline aerosol was a safe and effective bronchodilator.

Adult↗