cDNA-derived amino acid sequence of the 86-kDa subunit of the Ku antigen

The Ku antigen is a DNA-associated nuclear protein recognized by sera from patients with autoimmune diseases. It consists of two polypeptides of 86 and 70 kDa. cDNA clones encoding the 86-kDa subunit of the Ku antigen were isolated by probing lambda gt11 recombinant cDNA expression libraries with a monoclonal antibody specific for this protein. The amino acid sequence deduced from the cDNA comprises 732 amino acids and corresponds to a protein with molecular weight of 81.914. Nineteen residues at the NH2 terminus determined by protein sequencing corresponded to the sequence deduced from the cDNA. The predicted amino acid sequence contains a region with repeating leucine residues similar to the leucine zipper structure observed in the c-myc, v-myc, and c-fos oncogene products. The largest cDNA hybridized to 2.7- and 3.4-kilobase poly(A)+ mRNAs from HeLa cells. The cDNA clones expressed fusion proteins immunoreactive with the monoclonal antibody and sera from patients with autoimmune diseases.     

An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains.

We have isolated the gene encoding the largest subunit of RNA polymerase II from Plasmodium falciparum. The RPII gene is expressed in the asexual erythrocytic stages of the parasite as a 9 kb mRNA, and is present as a single copy gene located on chromosome 3. The P. falciparum RPII subunit is the largest (2452 amino acids) eukaryotic RPII subunit, and it contains enlarged variable regions that clearly separate and define five conserved regions of the eukaryotic RPII largest subunits. A distinctive carboxyl-terminal domain contains a short highly conserved heptapeptide repeat domain which is bounded on its 5' side by a highly diverged heptapeptide repeat domain, and is bounded on its 3' side by a long carboxyl-terminal extension.     

The sequence of the largest subunit of RNA polymerase II is a useful marker for inferring seed plant phylogeny

We used RT-PCR to sequence approximately 3 kb of the gene coding for the largest subunit of RNA polymerase II (rpb1) from nine land plants. Our results show that plant rpb1 genes all have a similar GC-content and that their amino acid sequences evolve at a similar rate in most species we examined, except for the Arabidopsis thaliana and rice sequences which evolve faster. This gene also exists as a single copy in most species and contains enough phylogenetically informative sites to resolve the evolutionary relationships among seed plants. Protein maximum parsimony, as well as neighbor-joining and maximum likelihood analyses of DNA and protein sequences, all generated identical tree topologies with similar strong support values at each node. The angiosperms are a clade comprising Amborella as a sister group to all other angiosperms, followed by Nymphaea, Magnolia, Arabidopsis, and a monocot clade containing maize and rice. The gymnosperms also form a monophyletic clade with Welwitschia and pine grouped together and sister to a Cycas and Zamia clade. These findings concur with recent studies that refute the Anthophyte Hypothesis and place Amborella at the base of the angiosperm tree. These rpb1 sequences also give a more consistent picture of seed plant relationships than similar analyses performed on data sets made of 18S rDNA, atpB, and rbcL sequences from the same species. These sequences therefore show great promise to help further resolve the phylogenetic relationships of seed plants.     

A human RNA polymerase II subunit is encoded by a recently generated multigene family

Background  

Some origins in eukaryotic chromosomes fire more frequently than others. In the fission yeast, Schizosaccharomyces pombe, the relative firing frequencies of the three origins clustered 4-8 kbp upstream of the ura4 gene are controlled by a replication enhancer - an element that stimulates nearby origins in a relatively position-and orientation-independent fashion. The important sequence motifs within this enhancer were not previously localized.     

A human RNA polymerase II subunit is encoded by a recently generated multigene family

Background  

The sequences encoding the yeast RNA polymerase II (RPB) subunits are single copy genes.     

Vaults. II. Ribonucleoprotein structures are highly conserved among higher and lower eukaryotes.

Vaults are cytoplasmic ribonucleoprotein structures that display a complex morphology reminiscent of the multiple arches which form cathedral vaults, hence their name. Previous studies on rat liver vaults (Kedersha, N. L., and L. H. Rome. 1986. J. Cell Biol. 103:699-709) have established that their composition is unlike that of any known class of RNA-containing particles in that they contain multiple copies of a unique small RNA and more than 50 copies of a single polypeptide of 104,000 Mr. We now report on the isolation of vaults from numerous species and show that vaults appear to be ubiquitous among eukaryotes, including mammals, amphibians (Rana catesbeiana and Xenopus laevis), avians (Gallus Gallus), and the lower eukaryote Dictyostelium discoideum. Electron microscopy reveals that vaults purified from these diverse species are similar both in their dimensions and morphology. The vaults from these various species are also similar in their polypeptide composition; each being composed of a major polypeptide with an approximate mass of 100 kD and several minor polypeptides with molecular masses similar to those seen in the rat. Antibodies raised against rat vaults recognize the major vault protein of all species including Dictyostelium. Vaults therefore appear to be strongly conserved and broadly distributed, suggesting that their function is essential to eukaryotic cells.     

The intervening sequence of the ribosomal RNA gene is highly conserved between two Tetrahymena species.

The entire intervening sequence of Tetrahymena thermophila ribosomal DNA has been determined. It is 413 nucleotides long and has the same splice junctions as those in T. pigmentosa. There is 93% homology between the intervening sequences in the two species, and 100% homology between their adjacent 26S RNA coding regions.     

Functional substitution of an essential yeast RNA polymerase subunit by a highly conserved mammalian counterpart.

We isolated the cDNA encoding the homolog of the Saccharomyces cerevisiae nuclear RNA polymerase common subunit RPB6 from hamster CHO cells. Alignment of yeast RPB6 with its mammalian counterpart revealed that the subunits have nearly identical carboxy-terminal halves and a short acidic region at the amino terminus. Remarkably, the length and amino acid sequence of the hamster RPB6 are identical to those of the human RPB6 subunit. The conservation in sequence from lower to higher eukaryotes also reflects conservation of function in vivo, since hamster RPB6 supports normal wild-type yeast cell growth in the absence of the essential gene encoding RPB6.     

Conditional mutations occur predominantly in highly conserved residues of RNA polymerase II subunits.

Conditional mutations in the Saccharomyces cerevisiae RNA polymerase II large subunit, RPB1, were obtained by introducing a mutagenized RPB1 plasmid into yeast cells, selecting for loss of the wild-type RPB1 gene, and screening the cells for heat or cold sensitivity. Sequence analysis of 10 conditional RPB1 mutations and 10 conditional RPB2 mutations revealed that the amino acid residues altered by these distinct mutations are nearly always invariant among eucaryotic RPB1 and RPB2 homologs. These results suggest that RNA polymerase mutants might be obtained in other eucaryotic organisms by alteration of these invariant residues.     

Gene co-regulation is highly conserved in the evolution of eukaryotes and prokaryotes

Differences between species have been suggested to largely reside in the network of connections among the genes. Nevertheless, the rate at which these connections evolve has not been properly quantified. Here, we measure the extent to which co-regulation between pairs of genes is conserved over large phylogenetic distances; between two eukaryotes Caenorhabditis elegans and Saccharomyces cerevisiae, and between two prokaryotes Escherichia coli and Bacillus subtilis. We first construct a reliable set of co-regulated genes by combining various functional genomics data from yeast, and subsequently determine conservation of co-regulation in worm from the distribution of co-expression values. For B.subtilis and E.coli, we use known operons and regulons. We find that between 76 and 80% of the co-regulatory connections are conserved between orthologous pairs of genes, which is very high compared with previous estimates and expectations regarding network evolution. We show that in the case of gene duplication after speciation, one of the two inparalogous genes tends to retain its original co-regulatory relationship, while the other loses this link and is presumably free for differentiation or sub-functionalization. The high level of co-regulation conservation implies that reliably predicted functional relationships from functional genomics data in one species can be transferred with high accuracy to another species when that species also harbours the associated genes.     

Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif.

RNA-dependent RNA polymerases contain a highly conserved region of amino acids with a core segment composed of the amino acids YGDD which have been hypothesized to be at or near the catalytic active site of the molecule. Six mutations in this conserved YGDD region of the poliovirus RNA-dependent RNA polymerase were made by using oligonucleotide site-directed DNA mutagenesis of the poliovirus cDNA to substitute A, C, M, P, S, or V for the amino acid G. The mutant polymerase genes were expressed in Escherichia coli, and the purified RNA polymerases were tested for in vitro enzyme activity. Two of the mutant RNA polymerases (those in which the glycine residue was replaced with alanine or serine) exhibited in vitro enzymatic activity ranging from 5 to 20% of wild-type activity, while the remaining mutant RNA polymerases were inactive. Alterations in the in vitro reaction conditions by modification of temperature, metal ion concentration, or pH resulted in no significant differences in the activities of the mutant RNA polymerases relative to that of the wild-type enzyme. An antipeptide antibody directed against the wild-type core amino acid segment containing the YGDD region of the poliovirus polymerase reacted with the wild-type recombinant RNA polymerase and to a limited extent with the two enzymatically active mutant polymerases; the antipeptide antibody did not react with the mutant RNA polymerases which did not have in vitro enzyme activity. These results are discussed in the context of secondary-structure predictions for the core segment containing the conserved YGDD amino acids in the poliovirus RNA polymerase.     

The amino acid sequence of the beta subunit of allophycocyanin

The complete amino acid sequence of the beta subunit of Anabaena variabilis allophycocyanin is: H2N-Ala-Gln-Asp-Ala-Ile-Thr-Ala-Val-Ile-Asn-Ser-Ala-Asp-Val-Gln-Gly-Lys-Tyr-Leu-Asp-Thr-Ala-Ala-Leu-Glu-Lys-Leu-Lys-Ala-Tyr-Phe-Ser-Thr-Gly-Glu-Leu-Arg-Val-Arg-Ala-Ala-Thr-Thr-Ile-Ser-Ala-Asn-Ala-Ala-Ala-Ile-Val-Lys-Glu-Ala-Val-Ala-Lys-Ser-Leu-Leu-Tyr-Ser-Asp-Ile-Thr-Arg-Pro-Gly-Gly-Asn-Met-Tyr-Thr-Thr-Arg-Arg-Tyr-Ala-Ala-Cys-Ile-Arg-Asp-Leu-Asp-Tyr-Tyr-Leu-Arg-Tyr-Ala-Thr-Tyr-Ala-Met-Leu-Ala-Gly-Asp-Pro-Ser-Ile-Leu-Asp-Glu-Arg-Val-Leu-Asn-Gly-Leu-Lys-Glu-Thr-Tyr-Asn-Ser-Leu-Gly-Val-Pro-Val-Gly-Ala-Thr-Val-Gln-Ala-Ile-Gln-Ala-Ile-Lys-Glu-Val-Thr-Ala-Ser-Leu-Val-Gly-Ala-Asp-Ala-Gly-Lys-Glu-Met-Gly-Ile-Tyr-Leu-Asp-Tyr-Ile-Ser-Ser-Gly-Leu-Ser-COOH Phycocyanobilin is attached though a thioether linkage to cysteinyl residue 81, indicated by an asterisk. Comparison of this sequence with those of C-phycocyanins shows that there are 60 identities between corresponding subunits of these two biliproteins. Of the region between residues 79 and 120, 29 residues are identical in the beta subunits of allophycocyanin and phycocyanin. The character of all 10 charged residues in this region of the beta subunit sequences is completely conserved.     

A novel subunit of axonemal dynein conserved among lower and higher eukaryotes

To elucidate the subunit composition of axonemal inner-arm dynein, we examined a 38 kDa protein (p38) co-purified with a Chlamydomonas inner arm subspecies, dynein d. We found it is a novel protein conserved among a variety of organisms with motile cilia and flagella. Immunoprecipitation using specific antibody verified its association with a heavy chain, actin and a previously identified light chain (p28). Unexpectedly, mutant axonemes lacking dynein d and other dyneins retained reduced amounts of p38. This finding suggests that p38 is involved in the docking of dynein d to specific loci.     

The amino acid sequence of the rabbit lutropin beta subunit

The amino acid sequence of the beta subunit of rabbit lutropin (lLH) has been determined. The amino terminus of about 97% of the beta subunit has a two amino acid extension (pyro-Glu-Pro) compared to other lutropin beta sequences. Overlapping peptides from trypsin and chymotrypsin digestions of the performic acid-oxidized beta subunit and trypsin digestion of the S-aminoethylated cysteine beta subunit were isolated by chromatography on TSK Fractogel 40F and high-pressure liquid chromatography (HPLC). Sequencing was by a combination of the dansyl-Edman method and the direct Edman method. Amide placements were established by HPLC analysis of the PTH amino acid derivatives. The proposed sequence of lLH subunit is: This sequence is highly homologous to the other known lutropin beta subunits, especially rat and pig lutropin beta (91%). Partial cleavage of the peptide bond between Asp-79 and Pro-80 was observed during cyanogen bromide treatment. Rabbit thyrotropin and thyrotropin beta subunit copurified with lLH and lLH except at a final chromatography on Sephadex G-100.     

The 24,000 Da subunit is not required for the RNA synthesis activity of chicken leukemia RNA polymerase II

The partially purified RNA polymerase II from chicken leukemia cells (Chuang R. Y., Chuang L. F. & Israel M. (1986) Biochem. Pharmacol. 35, 1293-1297) contained multiple subunits with molecular masses (in Da) ranging from 220,000 to 24,000, as shown by SDS-polyacrylamide gel electrophoresis. The enzyme was further purified through phosphocellulose column and fractions containing the enzyme activity were collected and concentrated 400-fold through a microconcentrator. The microconcentrator contained a membrane with a molecular weight cutoff around 30,000 and, hence, removed the 24,000 Da polypeptide from the enzyme. It was found that the resulting enzyme retained all the catalytic activity as compared to the enzyme preparation before the concentration step, suggesting that the stoichiometric amount of the 24,000 Da polypeptide is not required for RNA synthesis activity with a denatured DNA template.