Characterization of Three Species of Sordariomycetes Isolated from Freshwater and Soil Samples in Korea

During a survey of fungal diversity in the class Sordariomycetes, 3 fungal strains, CNUFC-KMHY6-1, CNUFC-MSW24-2-11, and CNUFC-GW2S-4 were isolated from soil and freshwater samples, respectively in Korea. The strains were analyzed both morphologically and phylogenetically on the basis of internal transcribed spacer and RNA polymerase II second largest subunit gene sequences. On the basis of their morphology and phylogeny, CNUFC-KMHY6-1, CNUFC-MSW24-2-11, and CNUFC-GW2S-4 isolates were identified as Arcopilus aureus, Memnoniella echinata, and Stachybotrys sansevieriae, respectively. To the best of our knowledge, Ar. aureus and M. echinata have not been previously recorded in Korea, and this is the first report of S. sansevieriae from freshwater niche.

Characterization of Paecilomyces variotii and Talaromyces amestolkiae in Korea Based on the Morphological Characteristics and Multigene Phylogenetic Analyses

During fungal diversity surveys of the order Eurotiales in Korea, two fungal strains, EML-DG33-1 and EML-NCP50, were isolated from samples of rat dung and fig tree leaf collected at a garden located in Gwangju in 2014. To complete the National Species List of Korea, it is a prerequisite to verify whether many questionable species, which were previously recorded but not confirmed, indeed present in Korea. Herein, the isolates were confirmed as undescribed species, Paecilomyces variotii and Talaromyces amestolkiae based on the combination of morphological and phylogenetic analyses of multigenes including the rDNA internal transcribed spacer, β-tubulin, and RNA polymerase II subunit 2.

New Records of Xylaria Species in Korea: X. ripicola sp. nov. and X. tentaculata

During a Korean mushroom diversity survey from 2011 to 2014, we found one new Xylaria species (X. ripicola sp. nov.) and one Xylaria species that had not been previously observed in Korea (X. tentaculata). To confirm the phylogenetic placement of the new species, we conducted a phylogenetic investigation based on internal transcribed spacer regions of ribosomal DNA sequences. Additionally, the new species, X. ripicola, was subsequently analyzed for RNA polymerase II subunit sequences. We also evaluated the macroscopic and microscopic features of this species. Herein, X. ripicola is described as a new species that was collected from a natural beach habitat and X. tentaculata is formally reported as newly found in Korea.

A phosphorylation pattern-recognizing antibody specifically reacts to RNA polymerase II bound to exons

The C-terminal domain of RNA polymerase II is an unusual series of repeated residues appended to the C-terminus of the largest subunit and serves as a flexible binding scaffold for numerous nuclear factors. The binding of these factors is determined by the phosphorylation patterns on the repeats in the domain. In this study, we generated a synthetic antibody library by replacing the third heavy chain complementarity-determining region of an anti-HER2 (human epidermal growth factor receptor 2) antibody (trastuzumab) with artificial sequences of 7–18 amino-acid residues. From this library, antibodies were selected that were specific to serine phosphopeptides that represent typical phosphorylation patterns on the functional unit (YSPTSPS)₂ of the RNA polymerase II C-terminal domain (CTD). Antibody clones pCTD-1stS2 and pCTD-2ndS2 showed specificity for peptides with phosphoserine at the second residues of the first or second heptamer repeat, respectively. Additional clones specifically reacted to peptides with phosphoserine at the fifth serine of the first repeat (pCTD-1stS5), the seventh residue of the first repeat and fifth residue of the second repeat (pCTD-S7S5) or the seventh residue of either the first or second repeat (pCTD-S7). All of these antibody clones successfully reacted to RNA polymerase II in immunoblot analysis. Interestingly, pCTD-2ndS2 precipitated predominately RNA polymerase II from the exonic regions of genes in genome-wide chromatin immunoprecipitation sequencing analysis, which suggests that the phosphoserine at the second residue of the second repeat of the functional unit (YSPTSPS)2 is a mediator of exon definition.

Evaluation of Cellular Proliferative Activity in Patients with Oral Lichen Planus and Hepatitis C through AgNOR Method

The objective of this study was to evaluate the cellular proliferative potential of oral lichen planus (OLP) lesions from patients without hepatitis C virus (HCV) by means of AgNOR method, as well as the cellular proliferative potential of the normal oral mucosa from patients with HCV, treated or untreated by interferon and ribavirin. A cross-sectional study was developed to investigate four groups: 10 HCV+ patients without clinical signs of OLP who had never been treated for HCV infection - Group 1; 10 HCV+ patients that were under interferon and ribavirin treatment - Group 2; 15 patients with reticular OLP lesions histopathologically confirmed, without HCV - Group 3; and 15 blood donors without HCV infection and no clinical signs of OLP GROUP 4 Control Group. The cytological material of all groups was collected by the liquid-based cytology technique. Then, the sedimented material from each patient was filled with the Nucleolar Organizer Regions impregnation by silver method (AgNOR). The count of NORs was performed on 100 epithelial cell nuclei per patient using the Image Tool(tm) software. The Tukey HSD test was used to compare the median value of NORs among the groups and showed that the oral mucosa of HCV+ patients previously treated with anti-HCV drugs (GROUP 2), presented a higher average number of NORs in relation to others (p<0.05). The anti-HCV treatment may be related to increased cell proliferation of oral mucosa, indicating a possible relationship between OLP and HCV+ patients treated with interferon and ribavirin.

O propósito deste estudo foi avaliar o potencial proliferativo celular das lesões de líquen plano bucal (LPB) de pacientes sem vírus da hepatite C (VHC) por meio do método AgNOR, comparando-o ao potencial proliferativo celular da mucosa bucal normal de portadores de VHC, tratados ou não com interferon e ribavirina. Um estudo transversal foi realizado para investigar 4 grupos: 10 pacientes VHC+ sem sinais clínicos de LPB que nunca haviam sido tratados para a infecção por VHC - Grupo 1; 10 pacientes VHC+ que estavam sob tratamento com interferon e ribavirina - Grupo 2; 15 pacientes com LPB reticular histopatologicamente confirmado, sem VHC - Grupo 3; e 15 doadores de sangue sem infecção por VHC e sem sinais clínicos de LPB (Grupo 4 - Grupo de Controle). O material celular de todos os grupos foi coletado pela técnica da citologia em base líquida. Então, o material sedimentado de cada paciente foi submetido ao método da impregnação das regiões organizadoras nucleolares pela prata (AgNOR). A contagem das NORs foi realizada em 100 núcleos celulares epiteliais por paciente por meio do programa Image Tool(r). O teste Tukey HSD foi utilizado para comparar o valor médio de NORs entre os grupos e mostrou que a mucosa bucal dos pacientes VHC+ previamente tratados com fármacos anti-VHC (Grupo 2) apresentou maior número médio de NORs por núcleo em relação aos outros (p<0,05). O tratamento anti-VHC pode estar relacionado ao aumento da atividade proliferativa celular da mucosa bucal, aventando uma possível relação entre LPB e pacientes VHC+ tratados com interferon e ribavirina.

Static body postural misalignment in individuals with temporomandibular disorders: a systematic review

BACKGROUND: The association between body postural changes and temporomandibular disorders (TMD) has been widely discussed in the literature, however, there is little evidence to support this association. OBJECTIVES: The aim of the present study was to conduct a systematic review to assess the evidence concerning the association between static body postural misalignment and TMD. METHOD: A search was conducted in the PubMed/Medline, Embase, Lilacs, Scielo, Cochrane, and Scopus databases including studies published in English between 1950 and March 2012. Cross-sectional, cohort, case control, and survey studies that assessed body posture in TMD patients were selected. Two reviewers performed each step independently. A methodological checklist was used to evaluate the quality of the selected articles. RESULTS: Twenty studies were analyzed for their methodological quality. Only one study was classified as a moderate quality study and two were classified as strong quality studies. Among all studies considered, only 12 included craniocervical postural assessment, 2 included assessment of craniocervical and shoulder postures,, and 6 included global assessment of body posture. CONCLUSION: There is strong evidence of craniocervical postural changes in myogenous TMD, moderate evidence of cervical postural misalignment in arthrogenous TMD, and no evidence of absence of craniocervical postural misalignment in mixed TMD patients or of global body postural misalignment in patients with TMD. It is important to note the poor methodological quality of the studies, particularly those regarding global body postural misalignment in TMD patients. .

Characterization and Pathogenicity of Alternaria vanuatuensis, a New Record from Allium Plants in Korea and China

Alternaria from different Allium plants was characterized by multilocus sequence analysis. Based on sequences of the beta-tubulin (BT2b), the Alternaria allergen a1 (Alt a1), and the RNA polymerase II second largest subunit (RPB2) genes and phylogenetic data analysis, isolates were divided into two groups. The two groups were identical to representative isolates of A. porri (EGS48-147) and A. vanuatuensis (EGS45-018). The conidial characteristics and pathogenicity of A. vanuatuensis also well supported the molecular characteristics. This is the first record of A. vanuatuensis E. G. Simmons & C. F. Hill from Korea and China.

Delimitation of Russula Subgenus Amoenula in Korea Using Three Molecular Markers

Distinguishing individual Russula species has been difficult due to extensive phenotypic plasticity and obscure morphological and anatomical discontinuities. Due to highly similar macroscopic features, such as the presence of a red-cap, species identification within the Russula subgenus Amoenula is particularly difficult. Three species of the subgenus Amoneula have been reported in Korea. We used a combination of morphology and three molecular markers, the internal transcribed spacer (ITS), 28S nuclear ribosomal large subunit (LSU), and RNA polymerase II gene (RPB2), for identification and study of the genetic diversity of Russula subgenus Amoenula in Korea. We identified only two species in Korea (R. mariae and R. violeipes); these two species were indistinguishable according to morphology and LSU, but were found to be reciprocally monophyletic species using ITS and RPB2. The markers, ITS, LSU, and RPB2, have been tested in the past for use as DNA barcoding markers, and findings of our study suggest that ITS and RPB2 had the best performance for the Russula subgenus Amoneula.

The RNA Pol II sub-complex hsRpb4/7 is required for viability of multiple human cell lines

The evolutionarily conserved RNA Polymerase II Rpb4/7 sub-complex has been thoroughly studied in yeast and impacts gene expression at multiple levels including transcription, mRNA processing and decay. In addition Rpb4/7 exerts differential effects on gene expression in yeast and Rpb4 is not obligatory for yeast (S. cerevisiae) survival. Specialised roles for human (hs) Rpb4/7 have not been extensively described and we have probed this question by depleting hsRpb4/7 in established human cell lines using RNA interference. We find that Rpb4/7 protein levels are inter-dependent and accordingly, the functional effects of depleting either protein are co-incident. hsRpb4/7 exhibits gene-specific effects and cells initially remain viable upon hsRpb4/7 depletion. However prolonged hsRpb4/7 depletion is cytotoxic in the range of cell lines tested. Protracted cell death occurs by an unknown mechanism and in some cases is accompanied by a pronounced elongated cell morphology. In conclusion we provide evidence for a gene-specific role of hsRpb4/7 in human cell viability.

Biogênese, estabilidade e localização sub-celular de RNAs não-codificadores longos expressos em regiões intrônicas do genoma humano / Biogenesis, stability and sub-cellular localization of long non-coding RNAs expressed in intronic regions of the human genome

Trabalhos recentes indicam que a maior parte do transcriptoma de células de mamíferos é composto por RNAs não-codificadores de proteínas (ncRNAs). Nosso grupo tem identificado e caracterizado ncRNAs longos (>200 nt), sem splicing, expressos em regiões intrônicas de genes codificadores de proteína. Contudo, a biogênese, processamento e localização sub-celular desta classe de RNAs permanecem desconhecidos. Este trabalho teve como objetivos i) investigar a contribuição da RNA Polimerase II (RNAP II) na transcrição de ncRNAs intrônicos, ii) avaliar a meia-vida destes ncRNAs em relação a mRNAs, e iii) verificar a distribuição sub-celular de ncRNAs intrônicos. Os resultados obtidos indicaram que ncRNAs intrônicos são predominantemente transcritos pela RNAP II a partir de regiões promotoras funcionalmente semelhantes as que controlam a transcrição de mRNAs. Ensaios de estabilidade revelaram que, em média, ncRNAs intrônicos possuem meia-vida igual ou maior (3,4h a 4,2h) do que mRNAs (3,1h). A maior parte dos ncRNAs intrônicos possui estrutura cap 5', sugerindo que sejam estabilizados para desempenhar papéis na biologia da célula que não dependam de um rápido turnover. A maior parte dos ncRNAs intrônicos é exportada para o citoplasma, indicando que devam exercer alguma função biológica neste compartimento. Em conjunto, este trabalho fornece informações novas a respeito da biogênese, estabilidade e localização sub-celular ncRNAs intrônicos expressos em células humanas, contribuindo para avançar o conhecimento sobre esta classe de transcritos celulares.

Recent studies have shown that most of the mammalian transcriptome is comprised of non-coding RNAs (lncRNAs). Our group has identified and characterized long (>200 nt), unspliced lncRNAs expressed in intronic regions of protein coding genes. However, the biogenesis, processing, stability and subcellular localization of members from this RNA class remain unknown. The aims of this work were i) to investigate the contribution of RNA Polymerase II (RNAP II) to the transcription of intronic, ii) to evaluate the half-life of these ncRNAs relative to mRNAs, and iii) determine their subcellular distribution. Our results indicate that intronic ncRNAs are predominantly transcribed by RNAP II from promoter regions functionally similar to those that control the transcription of mRNAs. Stability assays revealed that intronic ncRNAs have an average half-life equal or greater (3.4h to 4.2h) than mRNAs (3.1h). The majority of intronic ncRNAs have 5' cap modification suggesting that these transcripts are stabilized, possibly to exert roles in the biology of the cell that does not depend on a rapid turnover. Although intronic ncRNAs do not encode proteins, most of these transcripts are transported to the cytoplasm which indicates that they may perform some biological function in this compartment. Altogether, this study reveals with novel information regarding the biogenesis, stability and subcellular localization of intronic ncRNAs expressed in human cells, thus contributing to advance the knowledge on this class of cellular transcripts.

Immunohistochemical localization of galectin-3 in the granulomatous lesions of paratuberculosis-infected bovine intestine

The presence of galectin-3 was immunohistochemically quantified in bovine intestines infected with paratuberculosis (Johne's disease) to determine whether galectin-3 was involved in the formation of granulation tissue associated with the disease. Mycobacterium avium subsp. paratuberculosis infection was histochemically confirmed using Ziehl-Neelsen staining and molecularly diagnosed through rpoB DNA sequencing. Galectin-3 was detected in the majority of inflammatory cells, possibly macrophages, in the granulomatous lesions within affected tissues, including the ileum. These findings suggest that galectin-3 is associated with the formation of chronic granulation tissues in bovine paratuberculosis, probably through cell adhesion and anti-apoptosis mechanisms.

Active transcription and ultrastructural changes during Trypanosoma cruzi metacyclogenesis

The differentiation of proliferating epimastigote forms of Trypanosoma cruzi , the protozoan parasite that causes Chagas’ disease, into the infective and non-proliferating metacyclic forms can be reproduced in the laboratory by incubating the cells in a chemically-defined medium that mimics the urine of the insect vector. Epimastigotes have a spherical nucleus, a flagellum protruding from the middle of the protozoan cell, and a disk-shaped kinetoplast - an organelle that corresponds to the mitochondrial DNA. Metacyclic trypomastigotes have an elongated shape with the flagellum protruding from the posterior portion of the cell and associated with a spherical kinetoplast. Here we describe the morphological events of this transformation and characterize a novel intermediate stage by three-dimensional reconstruction of electron microscope serial sections. This new intermediate stage is characterized by a kinetoplast compressing an already elongated nucleus, indicating that metacyclogenesis involves active movements of the flagellar structure relative to the cell body. As transcription occurs more intensely in proliferating epimastigotes than in metacyclics, we also examined the presence of RNA polymerase II and measured transcriptional activity during the differentiation process. Both the presence of the enzyme and transcriptional activity remain unchanged during all steps of metacyclogenesis. RNA polymerase II levels and transcriptional activity only decrease after metacyclics are formed. We suggest that transcription is required during the epimastigote-to-metacyclic trypomastigote differentiation process, until the kinetoplast and flagellum reach the posterior position of the parasites in the infective form.

A diferenciação de formas epimastigotas (proliferativas) do Trypanosoma cruzi, parasita protozoário causador da doença de Chagas, em formas metacíclicas tripomastigotas (infectivas e não proliferativas), pode ser reproduzida em laboratório incubando-se as células em um meio quimicamente definido que imita a urina do inseto vetor deste parasita. Os epimastigotas têm um núcleo esférico, o flagelo se projeta da metade do corpo do protozoário e o cinetoplasto (organela que possui o DNA mitocondrial) possui formato de disco. Os tripomastigotas metacíclicos têm um núcleo alongado com o flagelo emergindo da extremidade posterior da célula associado ao cinetoplasto esférico. Neste trabalho descrevemos as mudanças morfológicas que ocorrem durante essa transformação e caracterizamos uma nova forma intermediária do parasita usando reconstrução tridimensional de cortes seriados, visualizados por microscopia eletrônica de transmissão. Essa nova forma intermediária é caracterizada pela compressão do cinetoplasto contra o núcleo alongado, indicando que a metaciclogênese envolve movimentos ativos do cinetoplasto associado à estrutura flagelar em relação ao corpo celular. Como tripomastigotas metacíclicos transcrevem menos que as formas epimastigotas proliferativas, verificamos a presença da RNA polimerase II e medimos a atividade transcricional durante o processo de diferenciação. A presença da enzima e a atividade transcricional permanecem inalteradas durante todas as etapas da metaciclogênese, desaparecendo apenas quando as formas metacíclicas são formadas. Sugerimos que a diferenciação requer uma atividade transcricional, necessária para uma intensa remodelação da célula, que acontece até o cinetoplasto e o flagelo atingirem uma posição posterior do corpo do tripomastigota metacíclico.

The rescue of H1N1 subtype swine influenza virus / 生物工程学报

The swine influenza virus (SIV) strain A/Swine/TianJin/01/2004(H1N1) (A/S/TJ/04) was rescued successfully by an eight-plasmid rescue system. The cDNAs of SIV 8 gene segments were synthesized by RT-PCR and cloned into the RNA polymerase I/II bidirection expression vector PHW2000 independently, resulting in 8 recombinant plasmids. The 8 recombinant plasmids were cotransfected into COS-1 cell, 30 h later TPCK-trypsin was added to 0.5 microg/mL. The COS-1 cell and supernatant were harvested 48 h after cotransfection and were inoculated into the allantoic cavity of 9-day-old specific-pathogen free (SPF) chicken eggs. The allantoic fluid of dead eggs was harvested and passaged 3 generations in SPF chicken eggs to get infective virus. The successful rescue of A/S/TJ/04 SIV was identified by hemagglutination assay, hemagglutination inhibition assay, sequence analysis and electron microscope observation. The successful rescue of SIV built a platform for the research of the relationship between genome structure and function of SIV, the mechanisms of trans-species transmission of influenza virus and for the generation of new SIV as vaccine.

Identificação e análise de expressão de RNAs intrônicos não codificadores humanos / Identification and expression analysis of human intronic noncoding RNAs

Nesse trabalho, nós mostramos estudos em larga-escala de RNAs não codificadores antisenso que são transcritos em regiões intrônicas de genes humanos. Alguns destes transcritos intrônicos possuem níveis de expressão correlacionados ao grau de diferenciação tumoral de câncer de próstata, apontando para uma relevância biológica destas mensagens em doenças complexas como o câncer. Nós também avaliamos a existência de um mecanismo comum de regulação de transcrição, compartilhado por mRNAs codificadores de proteína e RNAs intrônicos, através de análises de perfís de expressão de uma linhagem tumoral de próstata estimulada por andrógeno. A análise de ESTs e mRNAs depositados em bancos públicos de seqüência revelou mais de 55 mil RNAs Totalmente Intrônicos Não-codificadores (TIN), transcritos dos íntrons de 74% de todos os genes RefSeq únicos. Guiados por esta informação, nós desenhamos uma plataforma de oligonucleotídeos contendo sondas senso e antisenso para cada um de 7.520 transcritos TIN selecionados aleatoriamente, além de sondas para os genes codificadores de proteína correspondentes. Nos identificamos assinaturas intrônicas e exônicas de expressão tecido-específicas em fígado, próstata e rim.Os RNAs TIN antisenso mais altamente expressos eram transcritos de íntrons de genes codificadores de proteína enriquecidos na categoria “Regulação da transcrição...

RNA polymerase II carboxy-terminal domain with multiple connections

The largest subunit of eukaryotic RNA polymerase II contains a unique domain at its carboxy-terminus, which is referred to as the carboxy-terminal domain (CTD). The CTD is made up of an evolutionarily conserved heptapeptide repeat (YSPTSPS). Over the past decade, there has been increasing attention on the role of the CTD in transcription regulation in the view of mRNA processing and chromatin remodeling. This paper provides a brief overview of the recent progress in the dynamic changes in CTD phosphorylation and its role in integrating multiple nuclear events.

Molecular characterization of mutation associated with rifampicin and isoniazid resistance in Mycobacterium tuberculosis isolates.

Nucleotide changes in catalase peroxidase (Kat G) gene and gene encoding the beta subunit of RNA polymerase (rpo B), responsible for isoniazid and rifampicin drug resistance were determined in the clinical isolates of Mycobacterium tuberculosis by PCR-RFLP, Line probe assay and DNA sequencing. PCR-RFLP test was performed by HapII cleavage of an amplified fragment of Kat G gene to detect the transversion 315AGC-->ACC(Ser-->Thr) which is associated with INH drug resistance. The Line probe assay kit was evaluated to detect the mutation in 81bp RMP resistance determining region of rpo B gene associated with RMP drug resistance. These results were validated by DNA sequencing and drug susceptibility test. Kat G S 315 T mutation was found in 74.19% strains of M. tuberculosis from Delhi. This mutation was not found in any of the susceptible strains tested. The line probe assay kit and DNA sequencing identified 18 isolates as RMP resistant with specific mutation, while one of the RMP resistant strain was identified as RMP susceptible, with a concordance of 94.73% with the phenotypic drug susceptibility result. Majority (8 of 19, 42.1%) of resistant isolates involved base changes at codon 531 of rpo B gene. Both PCR-RFLP and Line probe assay test can be used in many of the clinical microbiology laboratories for early detection of isoniazid and rifampicin drug resistance in clinical isolates of M. tuberculosis.

Paracoccidioides brasiliensis RNA biogenesis apparatus revealed by functional genome analysis

The RNA biogenesis machinery of Paracoccidioides brasiliensis was assessed by comparative analyses of PbAESTs (P. brasiliensis assembled expressed sequence tags (ESTs)) with sequences from Saccharomyces cerevisiae MIPS database. PbAESTs related to almost all categories of S. cerevisiae RNA biogenesis were found. Two of the 12 S. cerevisiae RNA Pol II core subunits, Rpb3 and Rpb7, were found, probably reflecting the growth phase from which the cDNA libraries used in ESTs generation were constructed, as well as the low abundance of some of these transcripts. We have also found orthologs to TATA-box-binding protein (TBP), and at least one subunit of each TBP-associated factors (TFII) in P. brasiliensis transcriptome, except TFIIB. Genes associated to the chromatin remodeling complex, as well as transcription factors probably involved in the control of genes associated to a sexual cycle and virulence, were also identified. With respect to the pre-mRNA processing, 65 PbAEST orthologs to S. cerevisiae basal splicing machinery and 21 orthologs of 5'- and 3'-end formation processes were found. Components involved in RNA interference were detected, suggesting that this gene expression regulation mechanism is probably used by P. brasiliensis. Twelve PbAESTs related to Pol I and Pol III machineries were assigned as S. cerevisiae orthologs. Finally, 25 and 10 PbAESTs associated to rRNA and tRNA processing, respectively, were detected. Taken together, our results enable us to depict, for the first time, a global view of transcription and RNA processing in P. brasiliensis.

Organização nuclear da RNA polimerase II e da transcrição em Trypanosoma cruzi / RNA polymerase II nuclear organization and transcription in Trypanosoma cruzi

A geração de um mRNA maduro nas células de eucariotos é um processo altamente regulado que envolve etapas como iniciação, elongação, capping, splicing, poliadenilação e transporte da mensagem para o citoplasma. A organização espacial do núcleo necessária para acomodar estes processos já há algum tempo tem sido alvo de vários estudos. Os processos de iniciação de transcrição e capping, elongação, terminação, e processamento dos mRNAs estão acoplados e são dependentes do domínio carboxi- terminal (CI'D) da subunidade maior da RNA polimerase 11 (RNA Pol 11), que tem como papel recrutar de maneira regulada as diversas maquinarias necessárias para estes processos. Citologicamente, este acoplamento é percebido, pois os componentes destas maquinarias se localizam nos mesmos sítios no nucleoplasma. Nos protozoários tripanossomatídeos, organismos causadores de diversas doenças parasitárias, a transcrição gera mensagens policistrônicas e a maturação destes transcritos se dá pela adição em 5' de uma seqüência de 30-40 nucleotídeos chamada de spliced leader (SL), evento que leva o nome de trans-splicing, e por poliadenilação na porção 3' de cada gene, gerando os mRNAs maduros. A regulação dos níveis dos mRNAs se dá principalmente pelo controle da estabilidade e tradução das mensagens, não sendo conhecidas seqüências promotoras de genes transcritos pela RNA pol II, exceto aquela dos genes SL. Assim, existem também evidências de que os proCessos de transcrição e processamento dos RNAs não sejam acoplados, como é observado na maioria dos eucariotos já estudados. Devido a estes peculiares mecanismos, nesta tese estudamos a organização nuclear da RNA Pol II e da transcrição no T. cruzi. Observamos que a distribuição da RNA Pol II no núcleo de formas epimastigotas de T. cruzi não é homogênea, acumulando-se numa região central do núcleo próxima do nucléolo. Outras regiões do núcleo também contêm RNA Pol II distribuída em micro-regiões. A concentração da RNA Pol II é dependente do...

The DPE, a core promoter element for transcription by RNA polymerase II

The core promoter is an important yet often overlooked component in the regulation of transcription by RNA polymerase II. In fact, the core promoter is the ultimate target of action of all of the factors and coregulators that control the transcriptional activity of every gene. In this review, I describe our current knowledge of a downstream core promoter element termed the DPE, which is a TFIID recognition site that is conserved from Drosophila to humans. The DPE is located from +28 to +32 relative to the +1 transcription start site, and is mainly present in core promoters that lack a TATA box motif. Moreover, in Drosophila, the DPE appears to be about as common as the TATA box. There are distinct mechanisms of basal transcription from DPE- versus TATA-dependent core promoters. For instance, NC2/Dr1-Drap1 is a repressor of TATA-dependent transcription and an activator of DPE-dependent transcription. In addition, DPE-specific and TATA-specific transcriptional enhancers have been identified. These findings further indicate that the core promoter is an active participant in the regulation of eukaryotic gene expression.