Interactions between the human RNA polymerase II subunits

As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione S-transferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.     

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.     

Growth-related changes in phosphorylation of yeast RNA polymerase II

The largest subunit of RNA polymerase II contains a unique C-terminal domain (CTD) consisting of tandem repeats of the consensus heptapeptide sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Two forms of the largest subunit can be separated by SDS-polyacrylamide gel electrophoresis. The faster migrating form termed IIA contains little or no phosphate on the CTD, whereas the slower migrating II0 form is multiply phosphorylated. CTD kinases with different phosphoryl acceptor specificities are able to convert IIA to II0 in vitro, and different phosphoisomers have been identified in vivo. In this paper we report the binding specificities of a set of monoclonal antibodies that recognize different phosphoepitopes on the CTD. Monoclonal antibodies like H5 recognize phosphoserine in position 2, whereas monoclonal antibodies like H14 recognize phosphoserine in position 5. The relative abundance of these phosphoepitopes changes when growing yeast enter stationary phase or are heat-shocked. These results indicate that phosphorylation of different CTD phosphoacceptor sites are independently regulated in response to environmental signals.     

RNA polymerase II transcription complex assembly in nuclear extracts

Variation in superovulatory responses in cattle may be related to the stage of follicular growth at the time of gonadotropin treatment. Waves of follicle growth are regulated by both follicle-stimulating hormone (FSH) and oestradiol. The objective of experiment 1 was to determine the dynamics of follicle wave emergence and the relationship with FSH and oestradiol concentrations, after treatment of heifers with oestradiol benzoate (ODB) in the presence of an intravaginal progesterone-releasing device (CIDR-B). Experiment 2 examined the superovulatory response, embryo yield and quality following treatment with porcine follicle-stimulating hormone (pFSH) at different times relative to ODB injection. In experiment 1, 28 beef heifers were treated with a CIDR for 9 days and allocated at random to one of four groups to receive either: (I) CIDR only, or 5 mg ODB given as a single intramuscular injection at (II) day 0 (d0); (III) day 1.5 (d1.5); or (IV) day 3 (d3) post CIDR insertion. Ovaries were examined using daily ultrasound and blood samples were collected twice daily for 11 days. In experiment 2, 96 heifers were treated with a CIDR and 5 mg ODB as in experiment 1, and were allocated using a 4 x 3 factorial design plan to a superovulation programme using three doses (400 IU; 600 IU; 800 IU) of pFSH. FSH was given for 4 days at 12-h intervals beginning 6.5 days after CIDR insertion. Heifers received prostaglandin analogue 12 h before CIDR removal and were inseminated (AI) at 48 and 60 h post CIDR withdrawal and embryos were recovered 7 days after AI. In experiment 1, the interval from CIDR insertion to follicle wave emergence (FWE) was longer (P < 0.05) in heifers treated with ODB at d1.5 (5.4 +/- 0.4 days) and d3 (5.1 +/- 0.6 days) compared to heifers treated with CIDR only (2.4 +/- 0.4 days). On the basis of time to proposed injection of pFSH heifers would have had follicle emergence 4.4, 2.3, 1.5 and 1.4 days prior to pFSH for groups I, II, III and IV, respectively. In experiment 2, heifers treated with ODB at d1.5 had a higher (P < 0.05) superovulatory response (18.2 +/- 1.7) than heifers treated at d3 (12.8 +/- 1.7), but superovulatory response in both groups did not differ (P > 0.05) from heifers treated at d0 (14.4 +/- 2.0) or with CIDR only (15.0 +/- 1.8). There were fewer (P < 0.05) freezable-grade embryos recovered from heifers treated with ODB at d0 (1.5 +/- 0.7) and d3 (2.1 +/- 0.5) compared to heifers treated at d1.5 (3.0 +/- 0.6) or in heifers treated with CIDR only (3.4 +/- 0.7). Increasing the dose of pFSH caused a linear increase in the superovulatory response (11.7 +/- 1.0, 15.8 +/- 1.4 and 18.0 +/- 1.9) and in the number of embryos recovered (5.8 +/- 0.9, 7.0 +/- 0.8 and 9.1 +/- 1.0) for 400 IU, 600 IU and 800 IU, respectively. In conclusion, heifers treated with ODB had wide variation in time to follicle wave emergence and there was not a consistent beneficial effect of pretreatment with ODB on embryo yield and quality following superovulation.     

RNA polymerase II transcription initiation: a structural view

The emergence of new strains of Vibrio Cholerae has added a new dimension to the variability in pathogenicity and potential virulence of the organisms precipitating diarrhoeal diseases. Considering the shifting patterns of V. cholerae 01 there is a continuous need to monitor the strain characteristics. In this study total 541 stool specimens of acute secretory diarrhoea were investigated between May 1992 and November 1994 for strains of Vibrio Cholerae and anti-microbial susceptibility testing of all the confirmed V. Cholerae strains. In 1992, 50 of the 125 strains (40%) were positive for V. cholerae 01 predominantly biotype El Tor serotype ogawa, and 10 (80%) of non 01 type, with most strains susceptible to tetracycline (100%), chloramphenicol (98%) and Cotrimoxazole (98%), but all resistant to polymyxin B and furazolidine. In 1993, 44 (43.6%) of the 010 strains were positive for V. cholerae 0139 and the rest V. cholerae 01. In 1994, another sero group of V. cholerae 010 emerged, with 42 (13.3%) being positive. Isolates did not agglutinate with any of these antisera and have been labelled as 'other than non-01 vibrio cholerae'.     

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.     

An alpha-amanitin-resistant DNA-dependent RNA polymerase II from the fungus Aspergillus nidulans

An alpha-amanitin-resistant DNA-dependent RNA polymerase II has been purified from the lower eukaryote Aspergillus nidulans to apparent homogeneity by extraction of the enzyme at low salt concentration, polymin P (polyethylene imine) fractionation, binding to ion-exchangers and density gradient centrifugation. By this procedure 0.4 mg of RNA polymerase II can be purified over 6,000-fold from 500 g (wet weight) of starting material with a yield of 25% and a specific activity of 550 units/mg. The subunit composition has been resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate and by two-dimensional gel electrophoresis using a non-denaturing gel in the first dimension and a dodecylsulphate slab gel in the second dimension. The putative subunits have molecular weights of 170,000, 150,000, 33,000, 27,000, 24,000, 19,000, 18,000 and 16,000. Only one form of RNA polymerase II could be resolved by electrophoresis. The chromatographic and catalytic properties and the subunit composition of the purified RNA polymerase II are clearly different from RNA polymerase I from A. nidulans but throughout comparable with other class II enzymes. It differs from all other class II enzymes by its insensitivity towards the toxin alpha-amanitin, even at concentrations up to 400 micrograms/ml, and appears to be unable to bind O-[14C]methyl-gamma-amanitin at a concentration of 10 micrograms/ml of the toxin. We conclude that the purified RNA polymerase from A. nidulans is a real, but exceptional, type of the class II RNA polymerases.     

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.