Glycosylation of RNA polymerase II from wheat germ

The aim of this report was to study the cardiovascular autonomic tests in the evaluation of diabetic patients with gastroparesis. Forty diabetic subjects were divided into two groups: one group with gastroparesis (GP, n = 20) and another group paired by age and duration of diabetes without any complaint of autonomic neuropathy (DC, n = 20). They were evaluated clinically and submitted to a battery of five cardiovascular autonomic tests. The presence and severity of autonomic neuropathy were defined according to the number of normal cardiovascular tests. Each test had a score: zero (normal), one (borderline) and two (abnormal). The GP group showed a higher abnormal total score in the cardiovascular autonomic test than the group without any complaint (6.6 +/- 3.0 vs. 2.7 +/- 1.4, p < 0.01). These data suggest that diabetic with gastroparesis presents more abnormal cardiovascular autonomic tests than diabetic without autonomic neuropathy and these tests should be included in the evaluation of diabetic patients with gastroparesis.     

Rifampin resistance mutation of Bacillus subtilis altering the electrophoretic mobility of the beta subunit of ribonucleic acid polymerase

The rifampin-resistance mutation of LS3,an asporogenous mutant of Bacillus subtilis 3610, leads to altered mobility of the beta subunit of ribonucleic acid polymerase in sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. This finding argues that the rifampin-resistance mutation is located in the structural gene coding for the beta polypeptide.     

Sequence of the fifth largest subunit of RNA polymerase II from plants

An affinity-purified antibody raised against the fifth largest subunit of cauliflower (Brassica oleracea) RNA polymerase II was used to screen an expression library and isolate an Arabidopsis thaliana cDNA clone. This cDNA clone was used to isolate a soybean (Glycine max) cDNA clone, and both clones were sequenced. The open reading frames contain 176 amino acids and predict polypeptides of 19.5 and 19.6 kDa for Arabidopsis and soybean, respectively. The amino acid sequences of the Arabidopsis and soybean polypeptides are 91.5% identical. The fifth largest subunit in plant RNA polymerase II is present at unit stoichiometry in purified enzyme and does not dissociate from the holoenzyme during nondenaturing polyacrylamide gel electrophoresis. The gene encoding the 19.5-kDa subunit has been isolated and sequenced from Arabidopsis. The gene is single copy and contains five introns. The size of the mRNA encoding this RNA polymerase II subunit in Arabidopsis and soybean is approximately 1 kilobase. None of the published yeast or animal RNA polymerase subunit sequences show similarity to the fifth largest subunit in plants.     

Sequence analysis of the second largest subunit of tomato RNA polymerase II

We combined the retrograde transport of horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) technique and degenerating electron microscopic investigations to confirm the motor cortical area projection from the medial pallidal segment (GPm) via the thalamic nucleus ventralis lateralis pars oralis (VLo). We found first degenerated boutons arising from the GPm make synaptic contact with the somata and proximal dendrites of VLo neurons containing WGA-HRP reaction products transported retrogradely from motor area.     

Bacterial sporulation and regulation of dihydrodipicolinate synthase in ribonucleic acid polymerase mutants of Bacillus subtilis

The activity of dihydrodipicolinate synthase increased late in sporulation in Bacillus subtilis. Mutants blocked at several stages of sporulation due to having an altered ribonucleic acid polymerase failed to exhibit this increase.     

Autoimmunity to RNA polymerase II is focused at the carboxyl terminal domain of the large subunit

OBJECTIVE: Previous studies have demonstrated antibodies to the large (220 kd) polypeptide subunit of RNA polymerase II (Pol II) in sera from certain patients with scleroderma. In the present study, we sought to identify the autoantigenic region on this polypeptide. METHODS: A recombinant fusion protein, corresponding to the 52-heptapeptide repeat found in the carboxyl terminal domain (CTD) of the large Pol II subunit, was used to identify 15 patient sera that contained autoantibodies. Synthetic peptides CTD7 (representing a single heptapeptide) and CTD18 (representing 2 1/2 heptapeptide repeats) were used in a competitive inhibition assay to define the specificity of these sera and the importance of the CTD as an autoantigen. RESULTS: All 15 sera immunoprecipitated the Pol II subunit from radiolabeled cell extracts, and 11 of them bound the CTD fusion protein in immunoblots. Immunoprecipitation of Pol II was completely inhibited by CTD18 in 5 sera and partially inhibited in 4 additional sera. CONCLUSION: These results indicate that the CTD heptapeptide repeat is a focal point for autoimmune responses in scleroderma. It is likely that the repetitive sequence and high content of charged residues of this structure contribute to its role as an autoantigen.     

The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase

The E3 ubiquitin-protein ligases play an important role in controlling substrate specificity of the ubiquitin proteolysis system. A biochemical approach was taken to identify substrates of Rsp5, an essential hect (homologous to E6-AP carboxyl terminus) E3 of Saccharomyces cerevisiae. We show here that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II (Rpb1) in vitro. Stable complex formation between Rsp5 and Rpb1 was also detected in yeast cell extracts, and repression of RSP5 expression in vivo led to an elevated steady-state level of Rpb1. The amino-terminal domain of Rsp5 mediates binding to Rpb1, while the carboxyl-terminal domain of Rpb1, containing the heptapeptide repeats characteristic of polymerase II, is necessary and sufficient for binding to Rsp5. Fusion of the Rpb1 carboxyl-terminal domain to another protein also causes that protein to be ubiquitinated by Rsp5. These findings indicate that Rsp5 targets at least a subset of cellular Rpb1 molecules for ubiquitin-dependent degradation and may therefore play a role in regulating polymerase II activities. In addition, the results support a model for hect E3 function in which the amino-terminal domain mediates substrate binding, while the carboxyl-terminal hect domain catalyzes ubiquitination of bound substrates.     

Growth hormone and drug metabolism. Acute effects on nuclear ribonucleic acid polymerase activity and chromatin

Adult male rats, subjected either to sham operation or to hypophysectomy and adrenalectomy were maintained for 10 days before treatment with growth hormone. Results of the acute effects of growth hormone on the rat liver nuclear RNA polymerase I (nucleolar) and II (nucleoplasmic) activities as well as the chromatin template capacity were then studied and compared with the growth-hormone effects on the drug metabolism described in the preceding paper (Wilson & Spelsberg, 1976). 2. Conditions for isolation and storage of nuclei for maintenance of optimal polymerase activities are described. It is verified that the assays for polymerase activities require a DNA template, all four nucleoside triphosphates, and a bivalent cation, and that the acid-insoluble radioactive product represents RNA. Proof is presented that under high-salt conditions DNA-like RNA (polymerase II) is synthesized, and that under low-salt conditions in the presence of alpha-amanitin, rRNA (polymerase I) is synthesized. 3. In the livers of hypophysectomized/adrenalectomized rats, growth hormone increases the activity of both RNA polymerase enzymes and the chromatin template capacity within 1h after treatment. The effects last for 12h in the case of polymerase II but for only 6h in the case of polymerase I. Sham-operated rats respond to growth hormone in a manner somewhat similar to that shown by hypophysectomized/adrenalectomized rats. These results, which demonstrate an enhancement of RNA polymerase I activity in response to growth hormone, support those from other laboratories. 4. Growth-hormone enhancement of the chromatin template capacity in the liver of hypophysectomized/adrenalectomized rats contrasts with previous reports. The growth-hormone-induced de-repression of the chromatin DNA could represent the basis of the growth-hormone-induced enhancement of RNA polymerase II activity in the hypophysectomized/adrenalectomized rats, although some effect of growth-hormone on the polymerase enzymes is still suggested.     

Microsporidia are related to Fungi: evidence from the largest subunit of RNA polymerase II and other proteins

We have determined complete gene sequences encoding the largest subunit of the RNA polymerase II (RBP1) from two Microsporidia, Vairimorpha necatrix and Nosema locustae. Phylogenetic analyses of these and other RPB1 sequences strongly support the notion that Microsporidia are not early-diverging eukaryotes but instead are specifically related to Fungi. Our reexamination of elongation factors EF-1alpha and EF-2 sequence data that had previously been taken as support for an early (Archezoan) divergence of these amitochondriate protists show such support to be weak and likely caused by artifacts in phylogenetic analyses. These EF data sets are, in fact, not inconsistent with a Microsporidia + Fungi relationship. In addition, we show that none of these proteins strongly support a deep divergence of Parabasalia and Metamonada, the other amitochondriate protist groups currently thought to compose early branches. Thus, the phylogenetic placement among eukaryotes for these protist taxa is in need of further critical examination.     

DNA-dependent DNA polymerase species in male germ cells of the mouse

Quasi-homogeneous fractions of male mouse germ cells at definite stages of meiosis and spermiogenesis were obtained by using a separation method based on sedimentation velocity in an albumin gradient. In the various cell types, the total DNA-dependent DNA polymerase activity was determined, and the major enzymatic forms were characterized. The DNA polymerase species present in premeiotic, meiotic and post-meiotic cells were analyzed by glycerol gradient sedimentation. Two types of DNA polymerase were identified in fractions enriched in spermatogonia and preleptotene spermatocytes. One showed a sedimentation coefficient of about 7.5 S and was sensitive to N-ethylmaleimide (NEM); the other exhibited a sedimentation coefficient between 3 and 4 S and was resistant to NEM. On the basis of their sedimentation coefficients, their sensitivity to NEM and their template specificities, these 2 enzymes were identified respectively as alpha and beta DNA polymerases as reported in mammals. The gradient analysis performed on fractions enriched in meiotic and post-meiotic cells revealed the presence of DNA polymerase beta only. A quantitative analysis showed that the activity of the DNA polymerase beta reaches a maximum at middle-late pachytene stage and then drops gradually during spermiogenesis. Although any conclusion as to the biological role of this high level of DNA polymerase activity in pachytene spermatocytes is premature, it is tempting to suggest that this enzyme is involved in meiotic recombination.     

Functional map of the alpha subunit of Escherichia coli RNA polymerase: amino acid substitution within the amino-terminal assembly domain

The alpha subunit of Escherichia coli RNA polymerase plays a key role in assembly of the core enzyme. In previous studies the amino-terminal domain consisting of 215 amino acid residues between positions 21 and 235 was identified to be involved in this assembly, and the sites for beta and beta' association were suggested to be located within or near the two conserved regions in this amino-terminal assembly domain of alpha. For detailed functional mapping, Ala was substituted for 26 highly conserved amino acids around residues 40, 80 and 170 to 210. The alpha-point mutants were analyzed in vitro for their abilities to form dimers and to assemble beta beta' subunits. New types of assembly-deficient mutants were identified: alpha-R45A (having substituted Ala for Arg at residue 45) dimerized but did not assemble beta (and beta') subunits; and alpha-L48A showed a decreased level of alpha 2 beta subassembly formation, indicating that this region (residues 45 to 48) is responsible for beta-binding. Isolation of two mutants, alpha-K86A and alpha-V173A, both forming alpha 2 beta but not alpha 2 beta beta' complex, confirmed our previous conclusion that two separated regions participate in beta'-binding.     

Rpb4, a subunit of RNA polymerase II, enables the enzyme to transcribe at temperature extremes in vitro

Rpb4 is a subunit of Saccharomyces cerevisiae RNA polymerase II (Pol II). It associates with the polymerase preferentially in stationary phase and is essential for some stress responses. Using the promoter-independent initiation and chain elongation assay, we monitored Pol II enzymatic activity in cell extracts. We show here that Rpb4 is required for the polymerase activity at temperature extremes (10 and 35 degreesC). In contrast, at moderate temperature (23 degreesC) Pol II activity is independent of Rpb4. These results are consistent with the role previously attributed to Rpb4 as a subunit whose association with Pol II helps Pol II to transcribe during extreme temperatures. The enzymatic inactivation of Pol II lacking Rpb4 at the nonoptimal temperature was prevented by the addition of recombinant Rpb4 produced in Escherichia coli prior to the in vitro reaction assay. This finding suggests that modification of Rpb4 is not required for its functional association with the other Pol II subunits. Sucrose gradient and immunoprecipitation experiments demonstrated that Rpb4 is present in the cell in excess over the Pol II complex during all growth phases. Nevertheless, the rescue of Pol II activity at the nonoptimal temperature by Rpb4 is possible only when cell extracts are obtained from postlogarithmic cells, not from logarithmically growing cells. This result suggests that Pol II molecules should be modified in order to recruit Rpb4; the portion of the modified Pol II molecules is small during logarithmic phase and becomes predominant in stationary phase.     

The subunit structure of thyroglobulin

Human and rat thyroglobulin were reduced and alkylated in aqueous alkaline conditions in the absence of denaturants; the product of reduction in both cases has been found to have mol.wt. about 165000, or one-quarter that of the native molecule.     

Studies towards the synthesis of the hypermodified nucleoside of rat liver phenylalanine transfer ribonucleic acid: improved synthesis of the base beta-hydroxywybutine

An improved synthesis of the key intermediates (3 and 8) for the synthesis of beta-hydroxywybutines [[R-(R*,S*)]- and [S-(R*,R*)]-4], the most probable structures for the minor base from rat liver tRNA(Phe), has been achieved by the Wittig reaction between 1-benzyl-7-formylwye (1) and the phosphorane derived from (R)-2-[(methoxycarbonyl)amino]-3-(triphenylphosphonio)propanoate (10), followed by methylation, OsO4 oxidation, and cyclocondensation with COCl2 in the presence of pyridine. The racemic forms of beta-hydroxywybutines [(R*,S*)- and (R*,R*)-4], which were required for the determination of the optical purity of [R-(R*,S*)]- and [S-(R*,R*)]-4 by means of chiral HPLC, were conveniently prepared through pyrolysis of the cyclic carbonate 3 followed by NaBH4 reduction and catalytic hydrogenolysis. The samples of [R-(R*,S*)]- and [S-(R*,R*)]-4 were thus shown to be optically pure.