ABSTRACT
The cDNA cloning of the human polII 14-kDa subunit, hRPB14, and the comparison of its aa sequence with those of other pol subunits are described. The aa sequence of hRPB14 has homology to yeast poIII subunit RPB11 (44%), to a common subunit of yeast polI and polIII AC19 (24%) and to a Caenorhabditis elegans sequence (33%). hRPB14 contains a 19-aa motif, located in its N terminus, which was also found in human polII 33-kDa subunit hRPB33, yeast pol subunits (AC40, AC19, RPB3 and RPB11), and in the bacterial pol alpha subunit, which was involved in subunit assembly. This motif was also conserved in the conjugation-specific gene products of Tetrahymena (CnjC), Merchantia polymorpha chloroplast DNA (RNLVA) and C. elegans DNA (CEF58A4; deduced from the nucleotide sequence and of unknown function). The evolutionary emergence of a probable eukaryotic heterodimer, hRPB14/hRPB33, from a prokaryotic homodimer, alpha 2, is hypothesized.
Subject(s)
DNA-Directed RNA Polymerases/classification , RNA Polymerase II/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA-Directed RNA Polymerases/genetics , Humans , Molecular Sequence Data , Protein Conformation , RNA Polymerase II/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
Two composite constructs, pNHA and pCHA, are described. These plasmids are designed for the expression of cDNA in mammalian cells to produce proteins which are tagged with the hemagglutinin epitope sequence, YPYDVPDYA (HA1). The insertion of cDNA into the multiple cloning sites of these vectors has the advantage of 'tagging' the produced protein at either the N terminus or C terminus. To demonstrate the utility of these vectors, pNHA, containing a full-length cDNA (pNHRP33), was expressed in HeLa cells to produce a HA1-tagged peptide. The resulting peptide was purified from the whole-cell extracts by immunoprecipitation with an antibody to the tag (mAb12CA5). The mRNA was transcribed from a T7 promoter of the pNHRP33 construct, translated in a rabbit reticulocyte assay, and the protein product was purified using mAb12CA5 for the HA1 epitope. Among other possibilities, these vectors can be used to: (1) study protein-protein interactions in a mammalian transcription unit, (2) co-purify associated transcription factors, and (3) purify produced proteins when antibodies are not available.
Subject(s)
DNA , Epitopes/genetics , Genetic Vectors , Plasmids , Recombinant Proteins/genetics , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Base Sequence , Cytomegalovirus/genetics , DNA, Recombinant , Epitopes/immunology , HeLa Cells , Hemagglutinin Glycoproteins, Influenza Virus , Hemagglutinins, Viral/genetics , Hemagglutinins, Viral/immunology , Humans , Mammals , Molecular Sequence Data , Polymerase Chain Reaction , Precipitin Tests , Protein Biosynthesis , RNA Polymerase II/genetics , RNA Polymerase II/isolation & purification , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Transcription, Genetic , TransfectionSubject(s)
Gene Expression , Receptors, Steroid , Transcription Factors , Animals , Congresses as Topic , HumansABSTRACT
We have cloned and sequenced a cDNA of 1766 base pairs in length encoding the 275 amino acids of hRPB 33, the third largest subunit of human RNA polymerase II. The DNA was isolated by screening of a human lambda gt11 cDNA library with oligonucleotides designed on the basis of the amino acid residue analysis of the bovine material. The hRPB 33 amino acid sequence is highly conserved between Saccharomyces cerevisiae and human. Overall, 45% of the amino acid residues are identical with the yeast homologue RPB 3, and 65% of the amino acids are identical in the two major conserved regions at residues 0-103 and 151-197. hRPB 33 is also homologous to yeast RPC 5. The amino acid sequence of hRPB 33 showed no obvious homology with bacterial RNA polymerase or with any of its sigma factors.
Subject(s)
RNA Polymerase II/genetics , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , DNA/genetics , Gene Library , Humans , Macromolecular Substances , Molecular Sequence Data , Oligonucleotide Probes , Restriction Mapping , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Sequence Homology, Nucleic AcidABSTRACT
In a continuing effort to synthesize cardiac glucoside analogs with modified 17 beta-functional groups for pharmacologic testing, we used 3 beta-benzyloxy-5 beta-androst-15-en-17-one as an efficient intermediate. This report describes a preparation of 17 beta-(3'-thiophenyl)-5 beta-androstane-3 beta,14 beta-diol 3-D-glucopyranoside.
Subject(s)
Androstanols , Cardiac Glycosides/chemical synthesis , Chemical Phenomena , Chemistry , Magnetic Resonance Spectroscopy , Myocardial Contraction/drug effects , Stimulation, ChemicalABSTRACT
We have shown that antibodies against native calf thymus RNA polymerase II and antibodies against its 23-kDa subunit cross-reacted with the 23-kDa subunit of human RNA polymerase II. Immunoglobin G (IgG) against the 23-kDa subunit of calf thymus RNA polymerase II inhibited transcription in vitro from the adenovirus major late promoter. By immunoscreening of a human placenta lambda gt11 cDNA library with IgG against native CT RNA polymerase II and with IgG against its 23-kDa subunit, we isolated and characterized a full length 1.2-kilobase cDNA. We also generated oligonucleotide probes from a sequence of amino acid residues obtained by a modified peptide microsequencing procedure. The cDNAs isolated both from oligoscreening and immunoscreening were identical. The amino acid sequence deduced from the nucleotide sequence analysis indicates a polypeptide of 197 amino acid (23 kDa). The in vitro translation product of human cDNA HP-23 was precipitated by IgG against the 23-kDa subunit of CT RNA polymerase II. The amino acid sequence deduced from HP-23 showed no obvious homology with Escherichia coli RNA polymerase subunits or with any of its sigma factors.
