Human TAFII31 protein is a transcriptional coactivator of the p53 protein.

The p53 protein activates transcription of a target gene by binding to a specific DNA response element and interacting with the transcriptional apparatus of RNA polymerase II. The amino-terminal domain of p53 interacts with a component of the TFIID basal transcription complex. The human TATA-binding-protein-associated factor TAFII31, a component of TFIID, has been identified as a critical protein required for p53-mediated transcriptional activation. TAFII31 and p53 proteins bind to each other via amino acid residues in the amino-terminal domain of p53 that are essential for transcription. Antibodies directed against TAFII31 protein inhibit p53-activated but not basal transcription in vitro. These results demonstrate that TAFII31 is a coactivator for the p53 protein.

A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription.

Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II has been suggested to be critical for transcription initiation, activation, or elongation. A kinase activity specific for CTD is a component of the general transcription factor TFIIH. Recently, a cyclin-dependent kinase-activator kinase (MO15 and cyclin H) was found to be associated with TFIIH preparations and was suggested to be the CTD kinase. TFIIH preparations containing mutant, kinase-deficient MO15 lack CTD kinase activity, indicating that MO15 is critical for polymerase phosphorylation. Nonetheless, these mutant TFIIH preparations were fully functional (in vitro) in both basal and activated transcription. These results indicate that CTD phosphorylation is not required for transcription with a highly purified system.

Polyadenylylation destabilizes the rpsO mRNA of Escherichia coli.

The rpsO mRNA, encoding ribosomal protein S15, is only partly stabilized when the three ribonucleases implicated in its degradation--RNase E, polynucleotide phosphorylase, and RNase II--are inactivated. In the strain deficient for RNase E and 3'-to-5' exoribonucleases, degradation of this mRNA is correlated with the appearance of posttranscriptionally elongated molecules. We report that these elongated mRNAs harbor poly(A) tails, most of which are fused downstream of the 3'-terminal hairpin at the site where transcription terminates. Poly(A) tails are shorter in strains containing 3'-to-5' exoribonucleases. Inactivation of poly(A) polymerase I (pcnB) prevents polyadenylylation and stabilizes the rpsO mRNA if RNase E is inactive. In contrast polyadenylylation does not significantly modify the stability of rpsO mRNA undergoing RNase E-mediated degradation.

NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides.

A procedure for the enzymatic synthesis of uniformly 13C15N-labeled DNA oligonucleotides in milligram quantities for NMR studies is described. Deoxynucleotides obtained from microorganisms grown on 13C and 15N nutrient sources are enzymatically phosphorylated to dNTPs, and the dNTPs are incorporated into oligonucleotides using a 3'-5' exonuclease-deficient mutant of Klenow fragment of DNA polymerase I and an oligonucleotide template primer designed for efficient separation of labeled product DNA from unlabeled template. The labeling strategy has been used to uniformly label one or the other oligonucleotide strand in the DNA duplex dGGCAAAACGG.dCCGTTTTGCC in order to facilitate assignment and structure determination by NMR. Application of 15N and 13C heteronuclear NMR experiments to isotopically labeled DNA is presented.

Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription.

The human general transcription factor TFIIA is one of several factors involved in specific transcription by RNA polymerase II, possibly by regulating the activity of the TATA-binding subunit (TBP) of TFIID. TFIIA purified from HeLa extracts consists of 35-, 19-, and 12-kDa subunits. Here we describe the isolation of a cDNA clone (hTFIIA gamma) encoding the 12-kDa subunit. Using expression constructs derived from hTFIIA gamma and TFIIA alpha/beta (which encodes a 55-kDa precursor to the alpha and beta subunits of natural TFIIA), we have constructed a synthetic TFIIA with a polypeptide composition similar to that of natural TFIIA. The recombinant complex supports the formation of a DNA-TBP-TFIIA complex and mediates both basal and Gal4-VP16-activated transcription by RNA polymerase II in TFIIA-depleted nuclear extracts. In contrast, TFIIA has no effect on tRNA and 5S RNA transcription by RNA polymerase III in this system. We also present evidence that both the p55 and p12 recombinant subunits interact with TBP and that the basic region of TBP is critical for the TFIIA-dependent function of TBP in nuclear extracts.

DNA recombination is sufficient for retroviral transduction.

Oncogenic retroviruses carry coding sequences that are transduced from cellular protooncogenes. Natural transduction involves two nonhomologous recombinations and is thus extremely rare. Since transduction has never been reproduced experimentally, its mechanism has been studied in terms of two hypotheses: (i) the DNA model, which postulates two DNA recombinations, and (ii) the RNA model, which postulates a 5' DNA recombination and a 3' RNA recombination occurring during reverse transcription of viral and protooncogene RNA. Here we use two viral DNA constructs to test the prediction of the DNA model that the 3' DNA recombination is achieved by conventional integration of a retroviral DNA 3' of the chromosomal protooncogene coding region. For the DNA model to be viable, such recombinant viruses must be infectious without the purportedly essential polypurine tract (ppt) that precedes the 3' long terminal repeat (LTR) of all retroviruses. Our constructs consist of a ras coding region from Harvey sarcoma virus which is naturally linked at the 5' end to a retroviral LTR and artificially linked at the 3' end either directly (construct NdN) or by a cellular sequence (construct SU) to the 5' LTR of a retrovirus. Both constructs lack the ppt, and the LTR of NdN even lacks 30 nucleotides at the 5' end. Both constructs proved to be infectious, producing viruses at titers of 10(5) focus-forming units per ml. Sequence analysis proved that both viruses were colinear with input DNAs and that NdN virus lacked a ppt and the 5' 30 nucleotides of the LTR. The results indicate that DNA recombination is sufficient for retroviral transduction and that neither the ppt nor the complete LTR is essential for retrovirus replication. DNA recombination explains the following observations by others that cannot be reconciled with the RNA model: (i) experimental transduction is independent of the packaging efficiency of viral RNA, and (ii) experimental transduction may invert sequences with respect to others, as expected for DNA recombination during transfection.

Modified PNA design and synthesis: a novel approach using Molecular Dynamics and Metadynamics

Gli acidi peptido nucleici sono potenti strumenti utilizzati in ambito biotecnologico per colpire DNA o RNA. PNA contenenti basi o backbone modificati sono attualmente studiati per migliorarne le proprietà in ambito biologico. Bersagliare i micro RNA (anti-miR) è particolarmente interessante nell’ottica di future applicazioni terapeutiche, ma strumenti computazionali che aiutino nel design di nuovi PNA anti-miR non sono stati ancora completamente sviluppati. Le proprietà conformazionali del singolo filamento di PNA (non modificato o recante modificazioni in γ) e dei duplex PNA:RNA e i processi di re-annealing e melting sono stati studiati tramite Dinamica Molecolare e Metadinamica. L’approccio computazionale consolidato, assieme a un programma modificato per la generazione delle strutture dei duplex contenenti PNA, è stato utilizzato per il virtual screening di PNA contenenti basi modificate. Sono state inoltre sintetizzate le unità per l’ottenimento del composto più promettente e una funzione idrolitica da legare al monomero finale.

Detecção e genotipagem de astrovírus em amostras fecais de aves de produção procedentes de diferentes criações comerciais brasileiras

Os astrovírus são agentes virais associados com enteropatias e infecções extra-intestinais em aves, ocasionando prejuízos no desenvolvimento produtivo e saúde animal. Porém, são escassos os estudos no Brasil que visem a detecção e caracterização deste vírus com maior amplitude. Nesse contexto, detectaram-se e caracterizaram-se cepas de astrovírus a partir de 60 amostras fecais de aves procedentes de diferentes criações comerciais brasileiras (postura, corte e matrizes), mediante o emprego de uma reação PanAstrovírus como triagem inicial, que consistiu numa reação de RT-hemi-nested-PCR, visando a amplificação e posterior sequenciamento de um segmento parcial do gene RdRp (RNA polimerase dependente de RNA) do gene ORF1b, uma região gênica conservada entre todos os astrovírus. Subsequentemente, nas amostras positivas na triagem, realizou-se a amplificação, clonagem e sequenciamento do gene ORF2 codificante do capsídeo viral, analisado mediante análise filogenética. Os dados obtidos demonstraram uma frequência de infecção por astrovírus em 48,3% (29/60) das amostras analisadas. As espécies virais detectadas foram as seguintes: Avastrovírus 2 (Avian Nephritis Virus, ANV) em 72,4% (21/29), Chicken Astrovírus (CAstV) em 6,8% (2/29) e Avastrovírus 1 (Turkey Astrovírus tipo 1, TAstV-1) em 20,8% (6/29) das amostras positivas. O sequenciamento total do gene ORF2 foi possível em oito amostras (8/21) positivas a ANV, em uma amostra (1/6) positiva a TAstV-1 e uma amostra (1/2) positiva para CAstV. A análise deste gene revelou, nas sequências ANV, a presença de três genótipos bem diferenciados, segundo critérios do Astroviridae Study Group, sendo que um deles, agrupou-se dentro do genótipo 5. Além disso, quando analisado com sequências procedentes de outras regiões, as sequências deste estudo formaram seis clusters, dois deles, relacionados com sequências procedentes de Austrália e Reino Unido. O análise do ORF2 em CAstV, revelou também um agrupamento com cepas procedentes do Reino Unido. Já o análise da ORF2 de TAstV-1, revelou divergência genética com cepas isoladas em Estados Unidos, que foram as únicas sequências disponíveis no GenBank. O presente estudo traz a luz vários dados epidemiológicos concernentes aos astrovírus aviário de origem brasileira o que permitirá a realização de outros estudos relacionados a epidemiologia deste vírus, seus possíveis riscos potenciais em saúde pública e a adoção de medidas de controle direcionadas a este agente

Insuficiência hepática aguda provocada por Amanita phalloides

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Microcin J25 has a threaded sidechain-to-backbone ring structure and not a head-to-tail cyclized backbone

Microcin J25 is a 21 amino acid bacterial peptide that has potent antibacterial activity against Gram-negative bacteria, resulting from its interaction with RNA polymerase. The peptide was previously proposed to have a head-to-tail cyclized peptide backbone and a tight globular structure (Blond, A., Peduzzi, J., Goulard, C., Chiuchiolo, M. J., Barthelemy, M., Prigent, Y., Salomon, R. A., Farias, R. N., Moreno, F. & Rebuffat, S. Eur. J. Biochem. 1999, 259, 747-755). It exhibits remarkable thermal stability for a peptide of its size lacking disulfide bonds and in part this was previously proposed to derive from its macrocyclic structure. We show here that in fact the peptide does not have a head-to-tail cyclic structure but rather a side chain to backbone cyclization between Glu8 and the N-terminus. This creates an embedded ring that is threaded by the C-terminal tail of the molecule, forming a noose-like feature. The three-dimensional structure deduced from NMR data suggests that slippage of the noose is prevented by two aromatic residues flanking the embedded ring. Unthreading does not occur even when the molecule is enzymatically digested with thermolysin. The new structural interpretation fully accounts for previously reported NMR and biophysical data and is consistent with the remarkable stability of this potent antimicrobial peptide.

Menin associates with a trithorax family histone methyltransferase complex and with the Hoxc8 locus

The cellular function of the menin tumor suppressor protein, product of the MEN1 gene mutated in familial multiple endocrine neoplasia type 1, has not been defined. We now show that menin is associated with a histone methyltransferase complex containing two trithorax family proteins, MLL2 and Ash2L, and other homologs of the yeast Set1 assembly. This menin-associated complex methylates histone H3 on lysine 4. A subset of tumor-derived menin mutants lacks the associated histone methyltransferase activity. In addition, menin is associated with RNA polymerase II whose large subunit carboxyl-terminal domain is phosphorylated on Ser5. Men1 knockout embryos and cells show decreased expression of the homeobox genes Hoxc6 and Hoxc8. Chromatin immunoprecipitation experiments reveal that menin is bound to the Hoxc8 locus. These results suggest that menin activates the transcription of differentiation-regulating genes by covalent histone modification, and that this activity is related to tumor suppression by MEN1.

Guideline to reference gene selection for quantitative real-time PCR

Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and P-actin have been used for that purpose. However, it has been reported that these genes as well as alternatives, like rRNA genes, are unsuitable references, because their transcription is significantly regulated in various experimental settings and variable in different tissues. Therefore, quantitative real-time PCR was used to determine the mRNA transcription profiles of 13 putative reference genes, comparing their transcription in 16 different tissues and in CCRF-HSB-2 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and ionomycin. Our results show that Classical reference genes are indeed unsuitable, whereas the RNA polymerase II gene was the gene with the most constant expression in different tissues and following stimulation in CCRF-HSB-2 cells. (C) 2003 Elsevier Inc. All rights reserved.

Comparisons of physical separation methods of Kunjin virus-induced membranes

The two sets of connected membranes induced in Kunjin virus-infected cells are characterized by the presence of NS3 helicase/protease in both, and by RNA-dependent RNA polymerase (RdRp) activity plus the associated double-stranded RNA (dsRNA) template in vesicle packets (VP), or by the absence of both the VP-specific markers in the convoluted membranes/paracrystalline arrays (CM/PC). Attempts were made to separate flavivirus-induced membranes by sedimentation or flotation analyses in density gradients of sucrose or iodixanol, respectively, after treatment of cell lysates by sonication, osmotic shock, or tryptic digestion. Only osmotic shock treatment provided suggestive evidence of separation. This was explored by flow cytometry analysis (FCA) of RdRp active membrane fractions from a sucrose gradient, using dual fluorescent labelling via antibodies to NS3 and dsRNA. FCA revealed the presence of a dual labelled membrane population indicative of VP, and in a faster sedimenting fraction a membrane population able to be labelled only in NS3, representative of CM/PC and associated (R)ER. It was postulated that osmotic shock ruptured the bounding membrane of the VP, releasing the enclosed small vesicles associated with the Kunjin virus replication complex characterized previously. Notably, the presence of the full spectrum of nonstructural proteins in some membrane fractions was not a reliable marker for RdRp activity. These experiments may provide the opportunity for isolation of relatively pure flavivirus replication complexes in their native membrane-associated state by fluorescence-activated cell sorting. (C) 2004 Elsevier B.V. All rights reserved.

Structure of thermolysin cleaved microcin J25: Extreme stability of a two-chain antimicrobial peptide devoid of covalent links

sThe structure of a two-chain peptide formed by the treatment of the potent antimicrobial peptide microcin J25 (MccJ25) with thermolysin has been characterized by NMR spectroscopy and mass spectrometry. The native peptide is 21 amino acids in size and has the remarkable structural feature of a ring formed by linkage of the side chain of Glu8 to the N-terminus that is threaded by the C-terminal tail of the peptide. Thermolysin cleaves the peptide at the Phe10-Val11 amide bond, but the threading of the C-terminus through the N-terminal ring is so tight that the resultant two chains remain associated both in the solution and in the gas phases. The three-dimensional structure of the thermolysin-cleaved peptide derived using NMR spectroscopy and simulated annealing calculations has a well-defined core that comprises the N-terminal ring and the threading C-terminal tail. In contrast to the well-defined core, the newly formed termini at residues Phe10 and Val11 are disordered in solution. The C-terminal tail is associated to the ring both by hydrogen bonds stabilizing a short beta-sheet and by hydrophobic interactions. Moreover, unthreading of the tail through the ring is prevented by the bulky side chains of Phe19 and Tyr20, which flank the octapeptide ring. This noncovalent two-peptide complex that has a remarkable stability in solution and in highly denaturing conditions and that survives in the gas phase is the first example of such a two-chain peptide lacking disulfide or interchain covalent bonds.

Cyclization of pyrrhocoricin retains structural elements crucial for the antimircobial activity of the native peptide

Pyrrhacoricin is a naturally occurring antimicrobial peptide from the European fire bug Pyrrhocoris apterus. It has submicromolar activity against a range of Gram-negative bacterial strains and has created recent interest as a lead for the development of novel antibiotic compounds. In this study, we have used NMR spectroscopy to determine the solution structures of pyrrhocoricin and a synthetic macrocyclic derivative that has improved in vivo pharmaceutical properties. Native pyrrhocoricin is largely disordered in solution, but there is evidence of a subpopulation with ordered turn regions over residues 2-5, 4-7, and 16-19. The macrocyclic derivative incorporates a nine amino acid linker joining the N- and C-termini, which does not adversely affect the antimicrobial potency but leads to a broader spectrum of activity. The NMR data suggest that the turn conformations in the cyclic derivative are similar to those in the native form, thus implicating them in the biological function. (C) 2004 Wiley Periodicals, Inc.