Similarity between subunit 8 of yeast RNA polymerase II (RPB8) and the second-largest subunits of eukaryotic RNA polymerases.
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Amino Acid Sequence↗
Biomedical subjects
E K Bautz
Publications and source records attributed to E K Bautz.
Analysis of the promoter region of the housekeeping gene DmRP140 by sequence comparison of Drosophila melanogaster and Drosophila virilis.
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Animals↗
Identification of the genes coding for the second-largest subunits of RNA polymerases I and III of Drosophila melanogaster.
We have isolated cDNA and genomic clones of Drosophila melanogaster by cross-hybridization with a 658 bp fragment of the yeast gene coding for the second-largest subunit of RNA polymerase III (RET1). Determination of the sequence by comparison of genomic and cDNA regions reveals an ORF of 3405 nucleotides which is interrupted in the genomic sequence by an intron of 48 bp. The deduced polypeptide consists of 1135 amino acids with a calculated molecular weight of 128 kDa. The protein sequence shows the same conserved regions of homology as those observed for all the second-largest subunits of RNA polymerases cloned so far. The gene (DmRP128) obviously codes for a second-largest subunit of an RNA polymerase which is different from DmRP140 and DmRP135. We have purified three distinct RNA polymerase activities from D. melanogaster. By using specific RNA polymerase inhibitors in enzyme assays and by comparing their subunit composition we were able to distinguish between RNA polymerase I, II, and III. RNA polymerase preparations of D. melanogaster were blotted and the second-largest subunits were identified with antibodies raised against polypeptides expressed from DmRP128 and DmRP135. Anti-DmRP135 antibodies react strongly with the second-largest subunit of RNA polymerase I but do not react with the respective subunits of RNA polymerase II and III. The second-largest subunit of RNA polymerase III is only recognized by anti-DmRP128. Previously, we have claimed that DmRP135 codes for the second-largest subunit of RNA polymerase III.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Identification and characterization of a Hantavirus strain of unknown origin by nucleotide sequence analysis of the cDNA derived from the viral S RNA segment.
The genetic characterization of a serologically Hantaan-like virus but of unknown origin (termed DX) was carried out by molecular cloning and nucleotide sequencing of the corresponding cDNA of the viral S RNA segment. The S RNA was found to be 1765 nucleotides long with 3' and 5' termini being complementary for 24 bases. The virus messenger-sense RNA contains one major open reading frame (ORF) encoding 428 amino acids or a 50 kD polypeptide. A comparison of the DX S RNA segment to those of Sapporo rat, Hantaan, Puumala/Hällnäs B1, and Prospect Hill viruses reveals 95.4, 71.3, 55.3, and 60.9% homology at the nucleotide sequence level, and 94.7, 80.1, 58.4, and 59.8% at the deduced amino acid sequence level. Thus Hantavirus strain DX is very closely related to Sapporo rat virus. We also analyzed the S RNA segments of these Hantaviruses for the presence of a second ORF encoding a potential nonstructural NSs protein. All potential second ORFs detected in the different S RNA segments differ substantially in length and position among the viruses, despite the high conservation of the nucleotide sequences and the overall structure of the nucleocapsid proteins. This suggests that the nucleocapsid protein is the only polypeptide encoded by Hantavirus S RNA segments, setting them apart from the other members of the Bunyaviridae family.
Amino Acid Sequence↗
Primary structure of the large (L) RNA segment of nephropathia epidemica virus strain Hällnäs B1 coding for the viral RNA polymerase.
The L RNA segment of the nephropathia epidemica virus (NEV) strain Hällnäs B1 was characterized by molecular cloning of the corresponding cDNA and subsequent determination of the DNA nucleotide sequence. The L RNA segment is 6550 nucleotides long with complementarity of 20 bases at the 3' and 5' termini. The viral messenger sense RNA contains one major open reading frame (ORF) with a coding capacity of 2156 amino acid residues encoding a protein with a calculated molecular weight of 246 kDa and an IEP of pH 7.4. Comparison of the deduced amino acid sequences from NEV hantavirus and Bunyamwera virus (BWV) L segment messenger sense RNAs, revealed a high degree of diversity (overall amino acid identity, 17%). However, three clusters of 30-40% amino acid identity were detected. One of these domains, containing an Asp-Asp motif found in many RNA polymerases, also shares amino acid sequence homology with the PB1 polymerase component of influenza type A. These results indicate that the L RNA segment of the NEV codes for the viral RNA-dependent RNA polymerase. The data presented here complete our previous studies on the characterization of the NEV genome by cDNA sequencing of the viral M and S RNA segments.
Amino Acid Sequence↗
Antigenicity of hantavirus nucleocapsid proteins expressed in E. coli.
DNA clones representing the small genomic segment of Nephropathia epidemica virus strain Hällnäs B1 (NEV) and Hantaan virus strain 76-118 (HTV) encoding their nucleocapsid proteins were inserted into the E. coli vector pIN-III-ompA for secretion of proteins into the periplasmic space. The complete HTV and NEV nucleocapsid proteins and two truncated versions of the NEV nucleocapsid proteins were expressed as fusion proteins. Unexpectedly, all products accumulated as insoluble aggregates. Most of the ompA signal peptide remained uncleaved. However, nucleocapsid fusion proteins could be purified from the insoluble fraction by extraction with 8 M urea followed by separation on SDS-PAGE and electroelution. Rabbits were immunized with the eluted proteins and the resulting antibodies reacted specifically with authentic viral nucleocapsid proteins of HTV and NEV. The recombinant nucleocapsid proteins were found to react specifically with various hantavirus-immune sera, but not with human control sera, indicating their suitability as potential diagnostic antigens. This is the first report on the expression of a protein of a NEV serotype strain of hantaviruses by use of recombinant DNA techniques.
Antigens, Viral↗
Analysis of the promoter region of the housekeeping gene DmRP140 by sequence comparison of Drosophila melanogaster and Drosophila virilis.
To analyze the transcriptional control regions of Drosophila melanogaster household genes, we have characterized the promoter of the gene coding for the second-largest subunit of RNA polymerase II (DmRP140). Analysis of cDNA revealed that the coding region of the protein extends beyond the originally assumed transcription start point (tsp) and deduced translation start codon [Falkenburg et al., J. Mol. Biol. 195 (1987) 929-937] and that the tsp determined previously corresponds to an intron/exon boundary of an additional intron. Upstream of the polII gene we found a transcription unit that is transcribed in the opposite direction. The initiating ATGs of the two genes are only 467 nucleotides (nt) apart. The untranslated region is extremely A + T-rich (88%) but none of the transcription units is preceded by a canonical TATA element. It does not feature any other known nt sequence motifs thought to be necessary for the basic transcriptional machinery; yet, this region functions as a bidirectional promoter: a central 309-bp fragment directs transcription of a reporter gene in transiently transfected Drosophila culture cells in both orientations. The gene coding for the second-largest subunit of RNA polymerase II of Drosophila virilis (DvRP140) was isolated and partially analyzed. The gene is located on the second chromosome at 22F/23A which corresponds to the position determined for D. melanogaster.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Molecular characterization of the RNA S segment of nephropathia epidemica virus strain Hällnäs B1.
The S segment RNA of nephropathia epidemica virus (NEV) strain Hällnäs B1 was isolated by molecular cloning of the corresponding cDNA. The RNA is 1785 nucleotides long with the 3' and 5' termini being complementary for 23 bases. The viral messenger-sense RNA contains one major open reading frame (ORF) with a coding capacity of 433 amino acids encoding a 49-kDa polypeptide. Compared to the Hantaan S segment cDNA sequence there is a nucleotide homology of 60 and 61% at the amino acid level. Many of the amino acid differences are conservative exchanges. The C-termini of the NEV and Hantaan nucleocapsid proteins are nearly identical and the hydrophilicity profiles are very similar. In contrast, the following differences are significant: The calculated isoelectric points of the NEV and Hantaan nucleocapsid proteins are 5.6 and 6.7, respectively. The most prominent antigenic determinants predicted by the hydrophilicity profiles are located close to the C-terminus of NEV and close to the N-terminus of Hantaan virus nucleocapsid polypeptides.
Amino Acid Sequence↗
Rapid detection of genomic variations in different strains of hantaviruses by polymerase chain reaction techniques and nucleotide sequence analysis.
The polymerase chain reaction (PCR) with subsequent nucleotide sequence analysis was employed to rapidly detect genomic variations among different Hantavirus strains. Using synthetic oligonucleotide primers derived from the M and S segment RNAs of nephropathia epidemica virus strain Hällnäs B1 (NEV) we succeeded in amplifying the corresponding sequences of Hantaan and Puumala viruses. The nucleotide sequences of the cDNAs derived from the Puumala M and S RNA segments were analyzed. It was found that the particular nucleotide sequences of Puumala M and S segments were 81% and 82% homologous to the corresponding genomic segments of NEV, respectively. The amino acid homology was 94% for both segments. In contrast, the degree of homology to the corresponding Hantaan M and S genomic RNA segments was 63% at the nucleotide level for both segments and 53 and 55% at the deduced amino acid level, respectively. This demonstrates that Puumala virus is very similar to NEV and significantly different from Hantaan virus at both the nucleotide and protein level.
Amino Acid Sequence↗
Molecular organization of RNP complexes containing P11 antigen in heat-shocked and non-heat-shocked Drosophila cells.
Immunofluorescence analysis of polytene chromosomes of Drosophila melanogaster using the monoclonal antibody P11 has shown that after heat-shock the 38-kDa P11 antigen almost exclusively localizes at heat-shock puff 93D where it is part of giant puff-specific RNP granules. The biochemical experiments reported here show that, independent of growth temperature, the P11 antigen is a component of nuclear 10S RNP particles. The P11-containing 10S snRNPs can be stabilized in CsCl with 20 mM Mg2+ and possess a buoyant density of rho = 1.4 g/cm3. Sucrose gradient analysis of nuclear RNP extracts of heat-shocked Schneider's S-3 tissue culture cells shows that, after a 37 degree C heat-shock, the 10S RNPs associate with large RNP complexes sedimenting at 170-220S. The change in distribution is a temperature-dependent process with intermediate forms at 29 degrees C and 33 degrees C. In thermotolerant cells this observed change in distribution is strongly reduced. DEAE-Sephacel column chromatography and sucrose gradient analysis of nuclear RNP, followed by Northern blot analysis using 93D-specific probes of the TaqI repeat and immunoblotting experiments, show that the P11-containing 10S snRNPs are distinct from the RNP complexes formed by the 93D transcripts, suggesting an indirect association after heat-shock. Our experiments demonstrate that, despite the fact that a 37 degrees C heat-shock does not affect the overall integrity of nuclear RNP, it imposes changes on the general organization and interaction of the nuclear RNP population, resulting in the formation of large nuclear RNP aggregates and complexes. Such changes may be important for the survival strategy of the cell and for hnRNA processing and storage events which are effected by heat-shock.
Animals↗
Immunoblot analysis of the serological response in Hantavirus infections.
Sera from patients with nephropathia epidemica (NE) or Korean hemorrhagic fever (KHF) were tested for specific antibody response to antigens of Hällnäs virus and Hantaan virus strain 76-118. A Vero E6 derived cell line persistently infected with Hällnäs virus strain B1, and Vero E6 cells freshly infected with Hantaan virus type strain 76-118 were used as antigens in the immunofluorescence assay (IFA) and the immunoblot. Blots were prepared from whole cell lysates. The convalescent-phase sera of NE patients tested in this study regularly revealed a marked reaction with a 52 kilodalton (Kd) protein of Hällnäs virus and a 50 Kd protein of Hantaan virus. A convalescent serum from a patient with Korean hemorrhagic fever and a rat antiserum against Hantaan virus could recognize the 50 Kd band of Hantaan virus but showed no apparent reactivity with the 52 Kd component of Hällnäs virus in the standard dilutions. Some sera could additionally identify minor bands in the 55 Kd and/or 67 Kd region of the blots. A one-way cross reactivity between Hantaan and Hällnäs viruses was also evident from the results of the immunofluorescence assays in that NE convalescent sera reacted with both viruses, whereas KHF convalescent or anti-Hantaan sera gave strongly positive results with Hantaan virus but only faint reaction with Hällnäs virus.
Antibodies, Viral↗
Primary structure and functional aspects of the gene coding for the second-largest subunit of RNA polymerase III of Drosophila.
We have cloned and sequenced the gene coding for the second-largest subunit of RNA polymerase III of Drosophila melanogaster (DmRP135). The gene, interrupted by two introns of 62 and 59 bp, respectively, codes for an mRNA of 3.6 kb. As for other housekeeping genes transcription initiates at several sites (between positions -98 and -76) none of which is preceded by a clear TATA sequence. The deduced polypeptide consists of 1129 amino acids with an aggregate molecular weight of 128 kDa. The protein sequence features the same regions of similarity as observed for the corresponding subunits of RNA polymerase II of Drosophila and yeast and the Escherichia coli beta subunit. As in the second-largest subunit of RNA polymerase II there is a zinc-binding motif which is absent in the beta subunit of E. coli. Antibodies directed against a fusion protein expressing 164 amino acids of the DmRP135 polypeptide cross-react with the second-largest subunit of RNA polymerase III of yeast and generate a distinct banding pattern on Drosophila polytene chromosomes distinguishable from that obtained with anti-RNA polymerase II antibodies.
Amino Acid Sequence↗
Telomeric sequences derived from laser-microdissected polytene chromosomes.
Telomeric fragments from salivary gland squashes of Drosophila melanogaster Oregon R. were produced by a new microdissection technique, UV laser microbeam dissection. Microdissection, an essential step in microcloning procedures, is usually performed using micromanipulators and microneedles. Recently it has been shown that microdissection can be improved to very high precision if a laser coupled into a microscope is used. A laser microbeam, generated by an excimer pumped dye laser, allows chromosomes to be cut into slices of less than 0.5 micron. Here it is shown, that single copy DNA probes prepared from Drosophila chromosomes by laser microdissection and microcloning relocalize to the chromosomal regions from which they are derived. The combination of laser technique and microcloning provides an advantageous approach for rapid genetic analysis with potential for the study of genetic diseases and genome mapping.
Animals↗
Determination of the coding capacity of the M genome segment of nephropathia epidemica virus strain Hällnäs B1 by molecular cloning and nucleotide sequence analysis.
The M genome RNA segment of nephropathia epidemica virus (NEV) strain Hällnäs B1 was characterized by molecular cloning and DNA nucleotide sequencing of the corresponding cDNA clones. The size of the M RNA segment is 3682 nucleotides. The 3' and 5' terminal sequences are complementary for 21 bases and their predicted secondary structure is very stable. The viral complementary messenger RNA possesses a single long open reading frame with a coding capacity of 1148 amino acids (polypeptide of 126 kDa). A comparison of the NEV M segment to that of Hantaan virus strain 76-118 reveals 61% sequence homology at the nucleotide level and 53% at the deduced amino acid level. Four out of five potential asparagine-linked glycosylation sites of the encoded glycoproteins have been conserved between NEV and Hantaan M. The isoelectric points (IEP) are nearly identical. Furthermore it was found that 90% of all cysteine residues have been conserved. Putative NEV G1 and G2 are preceded by a short hydrophobic sequence as shown for G1 and G2 of Hantaan virus. Hydrophilicity profiles of the two segments are of striking similarity. These data indicate that NEV- and Hantaan virus M-encoded polypeptides seem to be very similar in structure and function despite the relatively low amino acid sequence homology.
Amino Acid Sequence↗
RNA-binding properties of hnRNP proteins.
The RNA-binding properties of the hnRNP monoparticle proteins were examined using a renaturing blotting procedure. All 'core' proteins are able to bind single-stranded nucleic acids, probably not sequence-specific. The core proteins C1 and, in one case A2 and B2, are able to bind nucleic acids which are double-stranded or which show a high degree of base-paired regions, among them U1 snRNA, whereas A1, B1 and C2 are unable to bind base-paired nucleic acids. The characteristics of C1 in binding base-paired nucleic acids are especially interesting, since the involvement of C1 in the splicing process has been described.
Base Composition↗
Developmental regulation of a constitutively expressed mouse mRNA encoding a 72-kDa heat shock-like protein.
Multiple heat shock cognate (hsc70) cDNA clones were isolated from the mouse embryonal carcinoma cell line F9. They all encode a single 72-kDa protein, which is constitutively expressed in all mouse cell lines and tissues tested, and which is only slightly induced by hyperthermia. hsc70 RNA is very abundant in F9 stem cells and brain, but very little is found in 14-day-old embryos. Upon differentiation of F9 stem cells induced by retinoic acid and cyclic AMP, expression of the hsc70 gene decreases only slightly, suggesting that hsc70 is highly expressed in early mouse development and is then down-regulated towards the end of embryogenesis. In adult tissues only the brain retains the high level of hsc70 gene expression found in F9 stem cells. We also show that expression of hsc70 protein and clathrin is uncoupled in F9 cells, indicating that the uncoating activity of coated vesicles may not be the only function of hsc70 protein.
Amino Acid Sequence↗
Nucleotide sequence of a processed human hsc70 pseudogene.
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Base Sequence↗
RNA polymerase II of Drosophila. Relation of its 140,000 Mr subunit to the beta subunit of Escherichia coli RNA polymerase.
We have determined the nucleotide sequence of the gene coding for the 140,000 Mr subunit of the DNA-dependent RNA polymerase II from Drosophila melanogaster. This analysis revealed features that are typical for a household-function gene. The codon usage is relaxed and there is no apparent TATA box upstream from the transcription start site. A comparison of the deduced amino acid sequence with that of the Escherichia coli RNA polymerase beta subunit shows a total of nine regions of homology. These regions are also conserved in chloroplast DNA of tobacco. This supports the notion that the two large subunits of the eukaryotic RNA polymerases are the structural and functional equivalents of E. coli beta' and beta subunits.
Amino Acid Sequence↗