Benchmarking pathway interaction network for colorectal cancer to identify dysregulated pathways

Different pathways act synergistically to participate in many biological processes. Thus, the purpose of our study was to extract dysregulated pathways to investigate the pathogenesis of colorectal cancer (CRC) based on the functional dependency among pathways. Protein-protein interaction (PPI) information and pathway data were retrieved from STRING and Reactome databases, respectively. After genes were aligned to the pathways, each pathway activity was calculated using the principal component analysis (PCA) method, and the seed pathway was discovered. Subsequently, we constructed the pathway interaction network (PIN), where each node represented a biological pathway based on gene expression profile, PPI data, as well as pathways. Dysregulated pathways were then selected from the PIN according to classification performance and seed pathway. A PIN including 11,960 interactions was constructed to identify dysregulated pathways. Interestingly, the interaction of mRNA splicing and mRNA splicing-major pathway had the highest score of 719.8167. Maximum change of the activity score between CRC and normal samples appeared in the pathway of DNA replication, which was selected as the seed pathway. Starting with this seed pathway, a pathway set containing 30 dysregulated pathways was obtained with an area under the curve score of 0.8598. The pathway of mRNA splicing, mRNA splicing-major pathway, and RNA polymerase I had the maximum genes of 107. Moreover, we found that these 30 pathways had crosstalks with each other. The results suggest that these dysregulated pathways might be used as biomarkers to diagnose CRC.

Splicing factor SF3B1 mutations and ring sideroblasts in myelodysplastic syndromes: a Brazilian cohort screening study

ABSTRACT Background: Myelodysplastic syndromes (MDS) comprise a group of malignant clonal hematologic disorders characterized by ineffective hematopoiesis and propensity for progression to acute myeloid leukemia. Acquired mutations in the gene encoding RNA splicing factor 3B subunit 1 (SF3B1) are highly associated with the MDS subtypes presenting ring sideroblasts, and represent a specific nosological entity. The effects of these mutations on clinical outcomes are diverse and contrasting. Methods: A cohort of 91 Brazilian MDS patients, including patients with ring sideroblasts in the bone marrow, were screened for mutations in the SF3B1 hotspots (exons 12-15) by direct Sanger sequencing. Results: SF3B1 heterozygous mutations were identified in six patients (7%), all of them with ring sideroblasts, thus confirming the association between SF3B1 mutations and myelodysplastic syndrome subtypes bearing this morphologic feature (frequency of 6/13, p-value < 0.0001). Conclusion: This is the first screening of SF3B1 mutations in a cohort of Brazilian myelodysplastic syndrome patients. Our findings confirm that mutations in this splicing gene correlate with bone marrow ringed sideroblasts.

Inhibición parcial de dos genes que codifican para proteínas del empalmosoma en Giardia intestinalis / Partial inhibition of two genes that encode spliceosomal proteins in Giardia intestinalis

Introducción. Giardia intestinalis es un organismo tempranamente divergente en el que recientemente se demostró la presencia de intrones. La maquinaria responsable de la remoción de intrones en organismos eucariotas superiores es el empalmosoma, el cual está conformado por cinco ribonucleoproteínas, cada una de las cuales tiene un ARN pequeño nuclear, un set de siete proteínas Sm (B, D1, D2, D3, E, F y G) y varias proteínas específicas. En G. intestinalis se han identificado los genes de algunas proteínas del empalmosoma por bioinformática. Aunque se asume que este es el responsable del empalme en el parásito, su caracterización bioquímica no se ha hecho. Objetivo. Inhibir dos genes que codifican para proteínas del empalmosoma de G. intestinalis con el fin de determinar si esta inhibición afecta el crecimiento o el enquistamiento del parásito. Materiales y métodos. En un vector específico para G. intestinalis se clonaron secuencias antisentido de los genes que codifican para las proteínas SmB y SmD3 del empalmosoma del parásito. Posteriormente, se transfectó G. intestinalis con los vectores recombinantes y se seleccionaron aquellos parásitos que lo incorporaron. Se confirmó la disminución del mensajero mediante reacción en cadena de la polimerasa (PCR) en tiempo real, y se evaluaron el crecimiento y el enquistamiento en parásitos silvestres y transfectados. Resultados. Se observó una disminución de 40 y 70 % en el ARNm de SmB y SmD3, respectivamente. El crecimiento y el enquistamiento no se vieron afectados en estos parásitos. Conclusión. La disminución de SmB y SmD3 no afectó al parásito, lo que indica que el empalmosoma sigue siendo funcional, o que el empalme no es una función vital del parásito.

Introduction. Giardia intestinalis is an early divergent organism that was recently shown to have introns. The machinery responsible for the removal of introns in higher eukaryotes is the spliceosome, which consists of five ribonucleoproteins. Each of these ribonucleoproteins has a small nuclear RNA, a set of seven Sm proteins (B, D1, D2, D3, E, F and G) and several specific proteins. Some genes that encode spliceosome proteins have been bioinformatically identified in the parasite genome. Although it is assumed that the spliceosome is responsible for splicing in this parasite, biochemical characterization is lacking. Objective. To inhibit two G. intestinalis spliceosome protein genes in order to determine whether this inhibition affects parasite growth or encystation. Materials and methods. Antisense sequences of the genes encoding the spliceosomal parasite proteins SmB and SmD3 were cloned into a specific G. intestinalis vector. G. intestinalis individuals were subsequently transfected with the recombinant vectors and those parasites that incorporated the vector were selected. A decrease in mRNA levels by real-time PCR was confirmed and the growth and encystation in wild and transfected parasites was assessed. Results. A decrease of 40% and 70% of SmB and SmD3 mRNA levels, respectively, was observed. Growth and encystation in these parasites were not affected. Conclusion. Decrease of SmB and SmD3 mRNA levels does not affect the parasite, indicating that the spliceosome remains functional or that splicing is not essential for parasite viability.

Neurofibromatosis tipo I: Mutación de splicing detectada por MLPA y secuenciación en la Argentina

La neurofibromatosis tipo 1 (NF1) es un desorden genético autosómico dominante, con una prevalencia de 1 en 2500-3000 nacidos vivos. La dificultad diagnóstica se debe al tamaño extenso del gen NF1 con pocos sitios hot-spot, la ausencia de una clara relación genotipo-fenotipo y rasgos clínicos con un espectro muy heterogéneo. Un caso sospechoso de NF1 procedente de la provincia de Jujuy fue analizado por MLPA (multiplex ligation-dependent probe amplification) en nuestro laboratorio. Mujer, adolescente mestiza (Amerindia/Europea), con un osteoma maxilar, lordosis lumbar, neurofibromas cutáneos y manchas café con leche. Por MLPA se detectó una alteración en el exón 13 del gen NF1. Por secuenciación del exón 13 se identificó una mutación "missense" en la posición 1466 del ARNm (NM_000267.3:c.1466A>G) que introduce un sitio de splicing aberrante. La patogenicidad de la mutación fue corroborada en la base de datos de variantes clínicas del National Center for Biotechnology Information. En nuestro conocimiento, este es el primer registro de una mutación NF1 en un paciente proveniente de poblaciones mestizas del Noroeste Argentino. La alteración ha sido reportada en individuos de otras poblaciones de origen muy disímil al del caso presentado, como la europea, sugiriendo que el sitio podría considerarse un sitio hot-spot del gen. Donde exista baja disponibilidad de diagnósticos moleculares, como en nuestro caso, se puede aplicar un algoritmo que comience por el estudio del gen NF1 por MLPA, metodología relativamente sencilla y de costo accesible. Con ella se evita enviar muestras al extranjero para análisis genéticos.

Neurofibromatosis type 1 (NF1) is a dominant autosomic genetic disorder, with a birth incidence of 1 in 2500-3000. Diagnosis is difficult because of the size of gene NF1 that has few hot-spots sites, the absence of a clear genotype-phenotype relation, and a heterogeneous clinical manifestation. A NF1 suspected case from Jujuy province was analyzed by multiplex ligation-dependent probe amplification (MLPA). Mestizo female teenage (Amerindian/European), with a maxilar osteoma, lumbar lordosis, cutaneous neurofibromas and café au lait spots. MLPA detected an alteration in exon 13 of the NF1 gene. By sequencing of exon 13, a missense mutation (NM_000267.3:c.1466A>G) was found which introduces an aberrant splicing site and is registered as pathogenic in the clinical variants database of NCBI. As far as we are aware, this is the first report of a NF1 mutation in mestizo population of Northwest Argentina. 1466A>G has been described before in patients of European origin, suggesting that the affected site could be a hot-spot site of the gene. For countries as Argentina, with limited availability of molecular diagnostic methods, we propose a diagnosis algorithm by starting the mutational analysis of NF1 with MLPA. This methodology is relatively simple and of low cost, avoiding to send samples abroad for genetic analyses.

Caracterización clínica y genético-molecular de un paciente con enfermedad granulomatosa crónica ligada al X: reporte de una nueva mutación asociada al splicing: caso clínico / Clinical, genetic and molecular characterization of the X-linked chronic granulomatous disease: a case report of a new splicing mutation

Introducción: La Enfermedad Granulomatosa Crónica (EGC) se presenta como consecuencia de mutaciones en los genes que codifican 5 de las subunidades del sistema NADPH oxidasa humano. Su forma más común es causada por cambios en el gen CYBB que codifica gp91 phox. Objetivo: Identificar el defecto molecular que lleva a la presentación de la EGC. Caso clínico: Paciente de sexo masculino con antecedentes de enfermedad diarreica aguda y abscesos perianales recurrentes desde los 2 meses. A los 6 meses, presentó una inflamación crónica del colon y colitis bacteriana. A los 3 años tenía infecciones en las vías respiratorias inferiores y perianales. Estudio compatible con EGC. El análisis del ADNc identificó expresión anormal del ARNm, lo cual se confirmó al realizar la secuenciación. Específicamente se observó la ausencia del exón 2. Adicionalmente, los datos de la secuenciación del ADNg identificaron una alteración en el sitio aceptor de "splicing" del intrón 1, que incluye una deleción seguida de la inserción de 3 nucleótidos (c.46-14_-11delTTCT insGAA). Conclusiones: Se presenta el primer estudio molecular de un paciente con EGC por defecto de "splicing" reportado en Colombia. La definición de la mutación y su correlación con el fenotipo es importante para proveer una apropiada consejería genética al paciente y su familia.

Chronic granulomatous disease (CGD) is caused by mutations in the genes that encode five of the subunits of the human NADPH oxidase. The most common form is caused by mutations in CYBB, the human gene encoding gp 91 phox. Objective: To identify the molecular defects causing CGD. Case report: A male patient with a history of acute diarrhea and recurrent perianal abscess since two months old. At 6 months, the patient presented a chronic inflammatory disease of the colon and bacterial colitis. After three years, he developed infections in the lower and perianal respiratory tract. The cDNA analysis identified abnormal mRNA expression, which was confirmed by sequencing. Specifically the exclusion of exon 2 was observed. Additionally, gDNA sequencing identified an alteration in the acceptor splice site of intron 1, including a deletion followed by insertion of three nucleotides (c.46-14_-11delTTCT insGAA). Conclusions: The first molecular study of a patient with CGD due to splicing pattern change, reported in Colombia, is presented. The definition of the mutation and its correlation with the phenotype is essential to provide appropriate genetic counseling to patients and their families.

Investigação da diversidade proteica, transcricional e genômica de TP53 e seu impacto em adenocarcinomas colorretais / Investigation of protein, transcriptional and genomic diversity of TP53 and its impact on colorectal adenocarcinomas

O câncer colorretal (CCR) é resultado da interação de fatores ambientais com múltiplos fatores genéticos. O gene TP53 regula muitas funções celulares e apresenta-se mutado na maioria dos casos de CCR. Mutações, polimorfismos e variações de expressão gênica fornecem a base genética para a diversidade humana e podem ter um papel crucial na variabilidade fenotípica e susceptibilidade às doenças. O estudo analisou as alterações em TP53 encontradas e buscou associações entre os achados com a expressão imunoistoquímica e os dados clinicopatológicos. Foram realizadas análises da proteína, do cDNA e gDNA de TP53 em 101 pacientes com CCR submetidos à cirurgia no Hospital AC Camargo, no período de 2000-2006. Foram encontradas mutações em TP53 em 54,5% dos tumores. As mutações foram mais frequentes no grupo de pacientes que apresentaram reação imunoistoquímica positiva para p53 (48,5%) do que no grupo negativo (51,5%) (p < 0,001). Foram encontradas três (3%) mutações sinônimas, o que corresponde a 8,8% das mutações pontuais; uma (33,4%) delas levando à ruptura do sítio de splicing. Foram encontradas oito inserções/deleções (7,9%), quatro (50%) delas descritas pela primeira vez e três (37,5%) delas levando à ruptura do sítio de splicing. O polimorfismo PIN2 (c.74+38C>G) foi encontrado em 89,1% dos pacientes. A frequência do alelo G foi de 0,72. O polimorfismo PIN3 (c.96+41_96+56del16) foi encontrado em 21,8% dos pacientes. A frequência do alelo A2 foi de 0,12. O polimorfismo 72Pro>Arg foi encontrado em 85,4% dos pacientes. A frequência do alelo Pro72 foi de 0,26 para o cDNA e 0,23 para o gDNA, diferença encontrada devido a expressão preferencial de um dos alelos em 40,7% dos heterozigotos. A expressão de p53, a mutação de TP53, a presença do alelo A2 e expressão do alelo Pro72 foram associadas significativamente às características clinicopatológicas que conferem pior comportamento tumoral e prognóstico adverso. Assim, a análise do cDNA de TP53 foi importante para determinar seu padrão de alteração e a contribuição específica do alelo expresso. Os dados obtidos foram congruentes com a forte associação que existe entre a mutação de TP53 e o seu acúmulo proteico, ainda que este acúmulo tenha sido verificado na minoria das amostras. Existe envolvimento de mutação sinônima com o mecanismo de splicing. As associações com os dados clinicopatológicos indicam que os polimorfismos em TP53 podem ter um papel na agressividade do CCR

Colorectal cancer (CRC) is the result of the interaction of environmental factors with multiple genetic factors. The TP53 gene regulates many cellular functions and is presented mutated in the majority of CRC cases. The mutations, polymorphisms and variations in gene expression provide the genetic basis for human diversity and may have a crucial role in the phenotypic variability and disease susceptibility. The study examined the TP53 alterations and sought associations between the findings with the immunohistochemical expression and clinicopathological data. We studied 101 patients, who underwent surgery at AC Camargo Hospital in the 2000-2006 period, through analysis of the protein, cDNA and gDNA of TP53. We found 54.5% of tumors with mutations in TP53. The mutations were significantly more frequent in the group of patients with positive immunohistochemistry reactions for p53 than in the negative group (p < 0.001). Synonymous mutations were found in three tumors, one of them leading to disruption of the splicing site. We found eight insertions/deletions, four of them described for the first time and three of them leading to the rupture of the splicing site. The PIN2 (c.74+38C>G) polymorphism was found in 89.1% of patients. The frequency of the G allele was 0.72. PIN3 (c.96+41_96+56del16) polymorphism was found in 21.8% of patients. The frequency of the A2 allele was 0.12. The polymorphism 72Pro>Arg was found in 85.4% of patients. The frequency of allele Pro72 was 0.26 for the cDNA and 0.23 for the gDNA. This difference was found because it was noticed the preferential expression of one allele in 40.7% of heterozygotes.The expression of p53, the TP53 mutation, the presence of the A2 allele and the allele Pro72 expression were significantly associated with tumor characteristics that confer worse tumor behavior and unfavorable prognosis. The analysis of TP53 cDNA is important to determine the pattern of alteration and the specific contribution of the allele expressed. This approach is consistent with the strong association that exists between TP53 mutation and its protein accumulation. Synonymous mutations are involved with splicing. The associations with linocophatological characteristics indicate that TP53 polymorphisms may play a role in the aggressiveness of CRC.

A preferable approach to clone hLIF cDNA from the genomic DNA

Complementary DNA (cDNA) is valuable for investigating protein structure and function in the research of life science, but it is difficult to obtain by traditional reverse transcription. In this study, we employed a novel strategy to clone the human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA directly isolated from the mucous membrane of mouth. The hLIF sequence can be acquired within a few hours by means of amplification of each exon and splicing using overlap-PCR. Thus, the new approach developed in this study is simple, time- and cost-effective, and it is not limited to particular gene expression levels of each tissue.

New insights into trypanosomatid U5 small nuclear ribonucleoproteins

Several protozoan parasites exist in the Trypanosomatidae family, including various agents of human diseases. Multiple lines of evidence suggest that important differences are present between the translational and mRNA processing (trans splicing) systems of trypanosomatids and other eukaryotes. In this context, certain small complexes of RNA and protein, which are named small nuclear ribonucleoproteins (U snRNPs), have an essential role in pre-mRNA processing, mainly during splicing. Even though they are well defined in mammals, snRNPs are still not well characterized in trypanosomatids. This study shows that a U5-15K protein is highly conserved among various trypanosomatid species. Tandem affinity pull-down assays revealed that this protein interacts with a novel U5-102K protein, which suggests the presence of a sub-complex that is potentially involved in the assembly of U4/U6-U5 tri-snRNPs. Functional analyses showed that U5-15K is essential for cell viability and is somehow involved with the trans and cis splicing machinery. Similar tandem affinity experiments with a trypanonosomatid U5-Cwc21 protein led to the purification of four U5 snRNP specific proteins and a Sm core, suggesting U5-Cwc-21 participation in the 35S U5 snRNP particle. Of these proteins, U5-200K was molecularly characterized. U5-200K has conserved domains, such as the DEAD/DEAH box helicase and Sec63 domains and displays a strong interaction with U5 snRNA.

Splicing variants impact in thyroid normal physiology and pathological conditions / Variantes de splicing e seu impacto em condições fisiológicas e patológicas da tireoide

RNA splicing is an essential, precisely regulated process that occurs after gene transcription and before mRNA translation, in which introns may be removed and exons, retained. Variability in splicing patterns is a major source of protein diversity from the genome and function to generate a tremendously diverse proteome from a relatively small number of genes. Changes in splice site choice can determine different effects on the encoded protein. Small changes in peptide sequence can alter ligand binding, enzymatic activity, allosteric regulation, or protein localization. Errors in splicing regulation have been implicated in a number of different disease states. This study reviewed the mechanisms of splicing and their repercussion in endocrinology, emphasizing its importance in some thyroid physiological and pathological conditions.

Após a transcrição genética e antes da tradução do mRNA, ocorre o splicing do RNA, que consiste em um processo essencial, precisamente regulado, por meio do qual podem ocorrer excisões de íntrons e retenções de éxons. A variabilidade dos padrões de splicing é a principal fonte de diversidade de proteínas geradas por um pequeno número de genes. Alterações na escolha do sítio de splicing podem determinar efeitos diferentes nas proteínas codificadas. Pequenas alterações na sequência peptídica podem alterar o seu sítio de ligação de substratos, sua atividade enzimática, a regulação alostérica ou a localização proteica. Erros na regulação do splicing têm sido implicados em grande número de doenças. Nessa revisão, foram descritos os mecanismos de splicing enfatizando sua importância em algumas condições fisiológicas e patológicas envolvendo a tireoide.

Analysis of the intronic single nucleotide polymorphism rs#466452 of the nephrin gene in patients with diabetic nephropathy

We present the analysis of an intronic polymorphism of the nephrin gene and its relationship to the development of diabetic nephropathy in a study of diabetes type 1 and type 2 patients. The frequency of the single nucleotide polymorphism rs#466452 in the nephrin gene was determined in 231 patients and control subjects. The C/T status of the polymorphism was assessed using restriction enzyme digestions and the nephrin transcript from a kidney biopsy was examined. Association between the polymorphism and clinical parameters was evaluated using multivaríate correspondence analysis. A bioinformatics analysis of the single nucleotide polymorphism rs#466452 suggested the appearance of a splicing enhancer sequence in intron 24 of the nephrin gene and a modification of proteins that bind to this sequence. However, no change in the splicing of a nephrin transcript from a renal biopsy was found. No association was found between the polymorphism and diabetes or degree of renal damage in diabetes type 1 or 2 patients. The single nucleotide polymorphism rs#466452 of the nephrin gene seems to be neutral in relation to diabetes and the development of diabetic nephropathy, and does not affect the splicing of a nephrin transcript, in spite of a splicing enhancer site.

Cloning and molecular characterization of Trypanosoma cruzi U2, U4, U5, and U6 small nuclear RNAs

Small nuclear RNAs (snRNAs) are important factors in the functioning of eukaryotic cells that form several small complexes with proteins; these ribonucleoprotein particles (U snRNPs) have an essential role in the pre-mRNA processing, particularly in splicing, catalyzed by spliceosomes, large RNA-protein complexes composed of various snRNPs. Even though they are well defined in mammals, snRNPs are still not totally characterized in certain trypanosomatids as Trypanosoma cruzi. For this reason we subjected snRNAs (U2, U4, U5, and U6) from T. cruzi epimastigotes to molecular characterization by polymerase chain reaction (PCR) and reverse transcription-PCR. These amplified sequences were cloned, sequenced, and compared with those other of trypanosomatids. Among these snRNAs, U5 was less conserved and U6 the most conserved. Their respective secondary structures were predicted and compared with known T. brucei structures. In addition, the copy number of each snRNA in the T. cruzi genome was characterized by Southern blotting.

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs

No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 æM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.

Splice site prediction using stochastic regular grammars

This paper presents a novel approach to the problem of splice site prediction, by applying stochastic grammar inference. We used four grammar inference algorithms to infer 1465 grammars, and used 10-fold cross-validation to select the best grammar for each algorithm. The corresponding grammars were embedded into a classifier and used to run splice site prediction and compare the results with those of NNSPLICE, the predictor used by the Genie gene finder. We indicate possible paths to improve this performance by using Sakakibara’s windowing technique to find probability thresholds that will lower false-positive predictions.

Trypanosoma cruzi: establishment of permeable cells for RNA processing analysis with drugs

Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing which involves excision of introns and union of exons in two independent transcripts. For the first time, we present the standardization of Trypanosoma cruzi permeable cells (Y strain) as a model for trans-splicing study of mRNAs in trypanosomes, following by RNase protection reaction, which localizes the SL exon and intron. This trans-splicing reaction in vitro was also used to analyze the influence of NFOH-121, a nitrofurazone-derivative, on this mechanism. The results suggested that the prodrug affects the RNA processing in these parasites, but the trans-splicing reaction still occurred.

Mapeamento de bandas de Trans-splicing in vitro com extratos nucleares de Tryopanosoma cruzi / Band mapping of in vitro trans-splicing reaction using Trypanosoma cruzi nuclear extracts

A maturaçäo dos pré-mRNAs em tripanosomatídeos ocorre por meio de um mecanismo denominado trans-splicing, que envolve a excisäo dos íntrons e a uniäo dos éxonsde dois transcritos independentes. Um transcrito curto , splice leader (SL) RNA, é trans-spliceado a pré-mRNAs aceptores, originando o RNA maduro. O objetivo deste trabalho foi padronizar as condiçöes necessárias que favoreçam essa reaçäo in vitro, empregando, para este fim, extratos nucleares de formas epimastigotas de T. cruzi e pré-mRNA de alfa-tubulina de tripanosoma africano. Nas condiçöes experimentais para a reaçäo de trans-splicing, em que se utilizou extrato nuclear de cepa Bolíviae o pré-mRNA a-tubulina (aceptor) marcado radioativamente, na ausência do pré-mRNA SL exógeno, foi obtida uma banda acima do pré-mRNA de alfa-tubulina (possível produto), sugerindo que esta reagiu com o SL presente no extrato nuclear. A banda foi processada, mas näo se detectou a sequência do produto esperado(SL + éxon alfa-tubulina). Entretanto, o tamanho da banda na reaçäo de trans-splicing (acima do pré-mRNA) pode trtar-se do lariat, cuja estrutura molecular näo possibilitaria seu sequenciamento pela metodologia empregada. Interessante salientar que a banda somente aparece na reaçäo de trans-splicing contendo ATP, o que sugere tratar-se de uma reaçäo de transesterificaçäo.

Organización y expresión génica / Gene organization and expression

Este brevísimo resumen sobre la organización y la expresión génica tiene dos finalidades: introducir los conceptos más básicos sobre este tema de modo de ir familiarizando al lector sobre la terminología específica y segundo, la de hacer evidente hasta qué nivel de profundidad se conoce el mecanismo molecular de los procesos involucrados en la expresión génica. Todo el esclarecimiento de esta maravilha se hizo tan sólo en 20 años. No es de estrañar entonces la velocidad con que se van dilucidando actualmente las interrupciones en la normalidad de esta expresión génica como son las enfermedades genéticas, el cáncer, las infecciones virales, lo que hace tener esperanzas en el pronto hallazgo de soluciones para dichas enfermedades