A simplified reconstitution of mRNA-directed peptide synthesis: activity of the epsilon enhancer and an unnatural amino acid.

The study of the early events in translation would be greatly facilitated by reconstitution with easily purified components. Here, Escherichia coli oligopeptide synthesis has been reconstituted using five purified recombinant His-tagged E. coli initiation and elongation factors. Highly purified ribosomes are required to yield products with strong dependencies on the translation factors. Based on HPLC separation of radiolabeled translation products from an mRNA encoding a tetrapeptide, approximately 80% of peptide products are full length, and the remaining 20% are the dipeptide and tripeptide products resulting from pausing or premature termination. Oligopeptide synthesis is enhanced when a commonly used epsilon (enhancer of protein synthesis initiation) sequence is included in the mRNA. The system incorporates a selectable, large, unnatural amino acid and may ultimately form the basis of a pure translation display technology for the directed evolution of peptidomimetic ligands and drug candidates. The recombinant clones can be exploited to prepare initiation factors and initiation complexes for structural studies, to study initiation and elongation in ribosomal peptide synthesis, and to screen for eubacterial-specific drugs.

Transformation and trends in preventive and social medicine education at the undergraduate level in a Brazilian medical school.

In the present study we discuss some transformations in undergraduate training in Preventive and Social Medicine in the Department of Social Medicine of the Faculty of Medicine of Ribeiro Preto, University of So Paulo, from 1993 to 1999. Aspects of the relationship between medical training and the reorganization of local services of the Brazilian national health system, and between graduate teaching in Preventive and Social Medicine and medical education as a whole are discussed. The crisis in Preventive and Social Medicine and its influence of medical training are evaluated. Trends for the application of a body of knowledge of the specialty and for the relationship between the department and the medical school are discussed.

Assistência e avaliaçäo: um exercício na implantaçäo de um ambulatorio primario de ginecologia - Centro de Saude escola de Ribeiräo Preto, 1985 / Medical care and evaluation: an exercise in the implementation of a gynaecology primary health care - Ribeiräo Preto Medical School Health Center , 1985

A tarefa de programar em saude tambem implica em estar atento ao processo de execuçäo e avaliaçäo das açöes de saude. Nessa orientaçäo foi proposto um serviço de atençäo primaria em ginecologia na Unidade de Saude de Vila Tiberio do Centro de Saude Escola de Ribeiräo Preto, no ano de 1985. Este estudo revelou que 36,5% do total de 148 mulheres atendidas possuiam entre 15 e 25 anos de idade; as cervicopatias e doenças sexualmente transmitidas foram os diagnosticos mais frequentes (16,4%). Encontraram-se taxas de alta e abandono de 48,6% e 33,8%, respectivamente, enquanto 17,6% das pacientes continuaram em seguimento. Receberam encaminhamento a outro nível de assistencia 28,4% do total delas, ressaltando-se que em 40,5% deles o motivo foi a falta de eletrocauterio nessa Unidade de Saude

External guide sequences for an RNA enzyme.

Ribonuclease P (RNase P) from Escherichia coli or its catalytic RNA subunit can efficiently cleave small RNA substrates that lack the conserved features of natural substrates of RNase P if an additional small RNA is also present. This additional RNA must contain a sequence complementary to the substrate [external guide sequence (EGS)] and a 3'-proximal CCA sequence to ensure cleavage. The aminoacyl acceptor stem and some additional 5'- and 3'-terminal sequences of a precursor transfer RNA are sufficient to allow efficient cleavage by RNAase P, and the 2'-hydroxyl group at the cleavage site is not absolutely necessary for cleavage. In principle, any RNA could be targeted by a custom-designed EGS RNA for specific cleavage by RNase P in vitro or in vivo.

Self-cleaving viroid and newt RNAs may only be active as dimers.

Avocado sunblotch viroid (ASBV) is a 247-nucleotide, single-stranded, circular RNA. It is considered to replicate via a rolling-circle mechanism in which circular, monomeric plus and minus RNAs act as templates for the synthesis of longer-than-unit-length precursor RNAs. Processing of these RNAs in vivo may occur by a self-cleavage reaction, as indicated by ability of dimeric, linear plus and minus ASBV RNAs to specifically self-cleave in vitro with the excision of a monomeric RNA with 5'-hydroxyl and 2',3'-cyclic phosphodiester termini. A similar self-cleavage reaction has also been reported to occur in an RNA transcript containing a dimeric copy of a tandemly repeated, 330-base-pair sequence of the newt genome. Based on comparisons with self-cleaving plant viral satellite RNAs, hammerhead-shaped active structures, each containing one self-cleavage site, were proposed for the plus and minus ASBV RNAs and the newt RNA, but the stability of these hammerheads has been questioned. Here, more stable active structures that contain two self-cleavage sites are proposed and data supporting these models are presented.

Photobiotin-labeled DNA and RNA hybridization probes.

Nucleic acid hybridization, the formation of a duplex between two complementary nucleotide sequences, is being increasingly utilized in the research laboratory and for the routine diagnosis of disease. Biotin-labeled nucleic acid hybridization probes have advantages over radioactively labeled probes in terms of stability, safety, and time of detection. The hybridization of a biotin-labeled probe to a target nucleic acid is carried out on a nitrocellulose or nylon filter; the bound probe is usually detected by the binding of an avidin-enzyme conjugate or a streptavidin-enzyme complex (both avidin and streptavidin have a high affinity for biotin), followed by a colorimetric reaction in which the bound enzyme converts a colorless substrate into a colored product.

Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active site.

Virusoids are circular single-stranded RNAs dependent on plant viruses for replication and encapsidation. Recently, we showed that an in vitro-synthesized RNA containing 273 nucleotides of the 324-nucleotide virusoid of lucerne transient streak virus self-cleaves at a unique site. The reaction requires heating and snap cooling of the RNA and the subsequent addition of magnesium ions. Here, we test the 55-nucleotide, hammerhead-shaped, structural model proposed for the active site by preparing RNAs with both 5' and 3' terminal deletions. Results indicate that the hammerhead structure is sufficient and necessary for self-cleavage, that certain sequences prevent the formation of the hammerhead structure in the native virusoid RNA, and that an RNA molecule containing only 52 nucleotides is capable of an RNA-mediated reaction.

Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites.

Virusoids are circular single-stranded RNAs dependent on plant viruses for replication and encapsidation. Virusoid replication appears to involve longer-than-unit-length plus and minus RNAs, indicating that unit-length plus RNA is generated by specific cleavage reactions. Here, we synthesize plus and minus partial-length RNAs of the 324-nucleotide virusoid from lucerne transient streak virus in vitro. Both RNAs self-cleave at a unique site in the presence of magnesium ions to give 5' hydroxyl and 2',3' cyclic phosphodiester termini. Conformations other than the native structures are necessary for cleavage. Similar secondary structures with considerable sequence homology are proposed for the active sites of these and other plant pathogenic RNAs. Our results are consistent with certain rolling-circle replication models.

Self-cleavage of RNA in the replication of viroids and virusoids.

Viroids are infectious, circular RNA molecules of 246 to 375 nucleotides found in plants. Virusoids are of similar size and structure but they are dependent on, and encapsidated in, a helper virus. A rolling circle mechanism of replication is considered to account for the presence of greater-than-unit-length plus and minus RNAs of both viroids and virusoids found in infected plants. An essential feature of this mechanism is the specific processing or cleavage of high molecular weight intermediates to produce linear monomers which are then ligated to circular monomers. We have investigated the putative processing cleavage reactions using in vitro-synthesized RNA transcripts of dimeric cDNA clones of the 247-nucleotide avocado sunblotch viroid (ASBV) and of partial cDNA clones of the 324-nucleotide virusoid of lucerne transient streak virus (vLTSV). In both cases, there is a specific, non-enzymic, self-cleavage of plus as well as minus transcripts. The plus and minus sites of cleavage are in neighbouring parts of ASBV and of vLTSV and highly conserved two-dimensional structures can be drawn around the cleavage sites as well as around the putative or demonstrated cleavage sites of precursors of the virusoids of three other viruses and of the linear satellite RNA of tobacco ringspot virus. The results also indicate that the sole function of about one-third of the ASBV and vLTSV molecules is provision of sequences that allow the formation of the self-cleavage structures of both 'plus' and 'minus' RNA precursors during the replication cycle. Similar self-cleavage of 'plus' RNA transcripts of a dimeric cDNA clone of citrus exocortis virus (CEV) was not observed. However, the putative processing site for CEV precursors was located within three nucleotides by site-directed mutagenesis. No two-dimensional structures similar to those found for ASBV and vLTSV were found around the processing site. It is possible that a different type of self-cleavage or enzymic processing event occurs during the replication cycle of CEV and related viroids.

Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid.

Self-cleavage of both plus and minus RNA transcripts of the 247-residue avocado sunblotch viroid (ASBV), prepared from tandem dimeric cDNA clones, occurs specifically at two sites in each transcript to give monomeric plus and minus species. The cleavage reaction occurs both during transcription and on incubation of purified transcripts at pH 8 and 37 degrees C in the presence of magnesium ions to give a 3'-terminal 2',3'-cyclic phosphate and a 5'-terminal hydroxyl group. Although the self-cleavage occurs at different sites in the ASBV molecule for the plus and minus species, very similar secondary structures with high sequence homology can be drawn at each site. The results are considered to provide further evidence that ASBV is replicated in vivo by a rolling circle mechanism involving non-enzymic cleavage of high molecular weight RNA precursors of ASBV.

Infectivity and in vitro mutagenesis of monomeric cDNA clones of citrus exocortis viroid indicates the site of processing of viroid precursors.

Monomeric cDNA clones of citrus exocortis viroid (CEV) were constructed in the plasmid vector pSP6-4 and the infectivity of the clones plus in vitro-synthesized RNA transcripts determined by inoculation onto tomato seedlings. Infectivity was dependent on the site of the viroid molecule used for cloning and the orientation of the cDNA insert. Only the plus BamHI cDNA clone was infectious and produced progeny viroid with wild-type sequence at the region corresponding to the BamHI cloning site. Infectivity correlated with the terminal repetition of 11 nucleotides of viroid sequence, 5'GGATCCCCGGG 3', in the vector adjacent to the insert. The 11-nucleotide sequence lies within the highly conserved central region of viroids. Site-directed mutagenesis of a single nucleotide in the repeat at the 5'-end of the CEV insert to 5' GGATCCCC(T,A)GG 3' gave two point mutants. The two mutant CEV inserts, when excised from the vector, were not infectious. However, plasmid DNA and RNA transcripts from non-excised mutant CEV inserts were infectious. The progeny of one of these clones was examined and contained wild-type sequence. It was concluded that in vivo processing of longer-than-unit-length CEV occurs at one of three adjacent sites in the 11 nucleotide sequence and that the G nucleotide at position 97 is important for viroid replication.

Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin.

A photo-activatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3- propanediamine (photobiotin), has been synthesized and used for the rapid and reliable preparation of large amounts of stable, non-radioactive, biotin-labelled DNA and RNA hybridization probes. Upon brief irradiation with visible light, photobiotin formed stable linkages with single- and double-stranded nucleic acids yielding probes which were purified from excess reagent by 2-butanol extraction and ethanol precipitation. Using single-stranded phage M13 DNA probes chemically labelled with one biotin per 100-400 residues and dot-blot hybridization reactions on nitrocellulose, as little as 0.5 pg (6 X 10(-18) mol) of target DNA was detected colorimetrically by avidin or streptavidin complexes with acid or alkaline phosphatase from three commercial sources. The sensitivity of detection of target RNA in dot-blots and Northern blots was equivalent to that obtained with 32p-labelled DNA probes. Photobiotin was also used for the labelling of proteins with biotin.

Avaliacao da utilizacao de exames laboratoriais multiplos num hospital universitario. / Evaluation of the multiple laboratory tests utilization in an university hospital

Os autores estudam a existencia de exames sorologicos multiplos, e seus resultados, em uma amostra casual simples de prontuarios medicos de pacientes de 3 grandes clinicas de um hospital universitario.Mais da metade dos prontuarios amostrados tinha ao menos um exame de perfil 1 e 2 realizado. O numero de resultados normais foi grande. Mais ou menos 30% dos exames tinham apenas 1 resultado alterado e 23% tinham 2 ou 3 exames anormais. Os resultados sugerem que as baterias de exames realizados nao tinham por objetivo elucidar hipoteses diagnosticas dos pacientes; por outro lado, se a utilizacao dirigia-se ao controle de saude e diagnostico precoce de doencas em populacao usuaria de servico terciario, a proporcao dos achados foi muito baixa. As queixas dos pacientes, referidas no prontuario, nao justificam, na maioria dos casos, os exames realizados. E preciso desenvolver o pensamento critico a respeito da utilizacao de recursos tecnologicos a fim de propiciar seu uso mais criterioso