Two large subunits of the fission yeast RNA polymerase II provide platforms for the assembly of small subunits

BACKGROUND: We analyse whether the tuberculin skin test is a good survival marker in a cohort of pulmonary tuberculosis patients with HIV infection (PTB/HIV). In all, 494 PTB/HIV patients were enrolled in Barcelona (Spain) between January 1992 and December 1994 in the Tuberculosis Program of Barcelona. The main data problem was the large proportion of missing values in the covariates percentage of T CD4+ lymphocytes and the tuberculin test results: only 157 patients (31.8%) had both covariates recorded. METHODS: Patients were dichotomized into two groups according to their level of immunosuppression (< or = 14 and >14% T CD4+ cells). First, we carried out the semiparametric and parametric complete case analysis. After this, we analysed the data assuming a missing at random non-response pattern. We developed a bootstrap approach where missing data in the markers are imputed via a two-way linear model. Using Weibull regression estimation, we used a multiple imputation scheme to estimate the parameters of interest. RESULTS: We found significative differences for the most immunosuppressed group when comparing positive tuberculin patients with those who were tuberculin negative. From a complete case approach and through a multivariate Cox analysis, we obtained a significant relative hazard of 0.3657 (95% CI: 0.13-1.02; P = 0.054). When a Weibull model was fitted, we estimated a constant relative percentile value of pR = 4.1329 (95% CI: 0.97-17.59). From a missing data approach, we obtain a higher constant relative percentile 5.48 (P = 0.079). CONCLUSIONS: The imputation method allows us to assess the protective character of positivity for the tuberculin test for the lowest CD4+ level. These findings strongly suggest the value of the tuberculin skin test as a qualitative measure of the immunological response and its interest for developing countries where specific laboratory tests are not affordable.     

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.     

Transcriptional sequencing: A method for DNA sequencing using RNA polymerase

We have developed a sequencing method based on the RNA polymerase chain termination reaction with rhodamine dye attached to 3'-deoxynucleoside triphosphate (3'-dNTP). This method enables us to conduct a rapid isothermal sequencing reaction in <30 min, to reduce the amount of template required, and to do PCR direct sequencing without the elimination of primers and 2'-dNTP, which disturbs the Sanger sequencing reaction. An accurate and longer read length was made possible by newly designed four-color dye-3'-dNTPs and mutated RNA polymerase with an improved incorporation rate of 3'-dNTP. This method should be useful for large-scale sequencing in genome projects and clinical diagnosis.     

An RNA binding protein negatively controlling differentiation in fission yeast

The fission yeast Schizosaccharomyces pombe starts sexual development when starved for nutrients and simultaneously activated by mating pheromones. We have identified a new gene regulating the onset of this process. This gene, called nrd1(+), encodes a typical RNA binding protein that preferentially binds poly(U). Deletion of nrd1(+) causes cells to initiate sexual development without nutrient starvation. We have found that the biological role of nrd1(+) is to block the onset of sexual development by repressing the Ste11-regulated genes essential for conjugation and meiosis until cells reach a critical level of starvation.     

Rpb3, stoichiometry and sequence determinants of the assembly into yeast RNA polymerase II in vivo

Stoichiometry of the third largest subunit (Rpb3) of the yeast RNA polymerase II is a subject of continuing controversy. In this work we utilized immunoaffinity and nickel-chelate chromatographic techniques to isolate the RNA polymerase II species assembled in vivo in the presence of the His6-tagged and untagged Rpb3. The distribution pattern of tagged and untagged subunits among the RNA polymerase II molecules is consistent with a stoichiometry of 1 Rpb3 polypeptide per molecule of RNA polymerase. Deletion of either alpha-homology region (amino acids 29-55 or 226-267) from the Rpb3 sequence abolished its ability to assemble into RNA polymerase II in vivo.