Alternativa para el tratamiento de la atrofia severa de maxilar superior: casos clínicos de implantes cigomáticos / Zygomatic implants for the management of severe alveolar atrophy: clinical cases

La rehabilitación implanto-protética de maxilares con atrofia severa, presenta un desafío en la odontología actual. La falta de tejido óseo para la colocación de implantes estánda- res, conlleva tener que decidir una alternativa de tratamiento para el paciente. El objetivo de esta publicación es presentar dos casos clínicos del tratamiento de la atrofia de maxi- lar superior a través de implantes cigomáticos. Se presentan dos casos clínicos operados mediante la metodología descrita por Branemark, debatiendo las alternativas quirúrgicas actuales. Una paciente presentó dolor peri-orbitario, que fue resuelto inmediatamente. No se presentaron otras complicaciones post quirúrgicas. Se discuten alternativas pro- téticas de la resolución de estos casos. Transcurridos 12 y 18 meses de la rehabilitación implanto protética, no se presentaron otras complicaciones quirúrgicas ni protéticas en ambos pacientes. Se concluye que la rehabilitación con implantes cigomáticos es una alternativa válida para rehabilitar los maxilares superiores atróficos, de corto tiempo, baja morbilidad, y que presenta tasas de éxito similares a los implantes convencionales.

Implant-prosthetic rehabilitation of severe atrophic maxilla is a challenge for clinicians. Decision making when patients lack of bone density or quantity is usually difficult. The aim of this study is to report two cases of severe atrophic maxilla treated through zygo- matic implants. Two clinical reports using the Branemark methodology are presented, in which surgical alternatives are discussed. One patient had post-surgical pain in the peri orbital area and could be solved immediately. No other surgical complications were observed. Prosthetic alternatives are also discussed. After 12 and 18 months of functional loading, neither surgical nor prosthetic problems occurred. In conclusion, zygomatic implants are a viable, short time, low morbidity treatment in patients with an atrophic maxilla and present success rates similar to conventional implants.

Distinct routes to metastasis: plasticity-dependent and plasticity-independent pathways.

The cascade that culminates in macrometastases is thought to be mediated by phenotypic plasticity, including epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT and MET). Although there is substantial support for the role of EMT in driving cancer cell invasion and dissemination, much less is known about the importance of MET in the later steps of metastatic colonization. We created novel reporters, which integrate transcriptional and post-transcriptional regulation, to test whether MET is required for metastasis in multiple in vivo cancer models. In a model of carcinosarcoma, metastasis occurred via an MET-dependent pathway; however, in two prostate carcinoma models, metastatic colonization was MET independent. Our results provide evidence for both MET-dependent and MET-independent metastatic pathways.

Complicaciones intra y postquirúrgicas de exodoncias en un programa de atención comunitaria de la FOUBA en Junín de los Andes / Intra and postsurgical tooth extraction complications at a FOUBA community dentistry program in Junin de los Andes

El objetivo de este estudio fue analizar la incidencia de complicaciones intra y postquirúrgicas de extracciones simples en 5 días de atención comunitaria en Junín de los Andes, provincia de Neuquén, Argentina. Se realizaron 109 extracciones dentales en 74 de los pacientes que concurrieron al centro único de atención, entre el 27 y 31 de octubre de 2014, presentando piezas dentarias con indicación de extracción. Las complicaciones más frecuentemente encontradas fueron la fractura de la tabla ósea vestibular, la fractura de la corona de la pieza dentaria y el dolor posoperatorio. Complicaciones de frecuencia intermedia fueron el trismus, la alveolitis, la laceración de los tejidos blandos y la fractura de la raíz de la pieza dentaria. Se registró un caso de hemorragia primaria y una inyección accidental dentro de un vaso sanguíneo. Cuando se comparó estadísticamente la presencia de complicaciones pre y posquirúrgicas entre cirugías que duraron menos de 30 minutos y 30 minutos más, se encontró una asociación significativa; para ambas complicaciones, el porcentaje fue mayor en el segundo grupo. La práctica de extracciones simples ocasionalmente conlleva tener que manejar complicaciones. Es importante que el odontólogo general sea capaz de prevenirlas, diagnosticarlas y tratarlas. Reducir los tiempos operatorios parecería ser una medida clave para disminuir las probabilidades de aparición de complicaciones intra y posquirúrgicas.

The objective of this study was to analyze the incidence of intra and postoperative complications of simple extractions in 5 days of community care in Junín de los Andes, province of Neuquén, Argentina. A total of 109 dental extractions were performed in 74 patients who attended the single care center between October 27 and 31, 2014, presenting teeth with indication of extraction. The most frequent complications were the fracture of the buccal bone table, the fracture of the crown of the tooth and the postoperative pain. Complications of intermediate frequency were trismus, alveolitis, laceration of the soft tissues and fracture of the root of the tooth. There was a case of primary haemorrhage and an accidental injection into a blood vessel. When statistically comparing the presence of pre and postsurgical complications between surgeries that lasted less than 30 minutes and 30 minutes more, a significant association was found; for both complications, the percentage was higher in the second group. The practice of simple extractions occasionally entails having to handle complications. It is important that the general dentist be able to prevent, diagnose and treat them. Reducing operative times seems to be a key measure to decrease the chances of intraoperative and postoperative complications.

Development of an autoimmune syndrome affecting the skin and internal organs in P-selectin glycoprotein ligand 1 leukocyte receptor-deficient mice.

OBJECTIVE: To define and characterize the progression of the spontaneous autoimmune disease that develops in mice in the absence of the leukocyte adhesion receptor P-selectin glycoprotein ligand 1 (PSGL-1). METHODS: Skin-resident immune cells from PSGL-1-deficient mice and C57BL/6 control mice of different ages were isolated and analyzed by flow cytometry. Biochemical parameters were analyzed in mouse serum and urine, and the presence of serum autoantibodies was investigated. Skin and internal organs were extracted, and their structure was analyzed histologically. RESULTS: Skin-resident innate and adaptive immune cells from PSGL-1(-/-) mice had a proinflammatory phenotype with an imbalanced T effector cell:Treg cell ratio. Sera from PSGL-1(-/-) mice had circulating autoantibodies commonly detected in connective tissue-related human autoimmune diseases. Biochemical and histologic analysis of skin and internal organs revealed skin fibrosis and structural and functional abnormalities in the lungs and kidneys. Furthermore, PSGL-1(-/-) mice exhibited vascular alterations, showing loss of dermal vessels, small vessel medial layer remodeling in the lungs and kidneys, and ischemic processes in the kidney that promote renal infarcts. CONCLUSION: Our study demonstrates that immune system overactivation due to PSGL-1 deficiency triggers an autoimmune syndrome with characteristics similar to systemic sclerosis, including skin fibrosis, vascular alterations, and systemic organ involvement. These results suggest that PSGL-1 expression contributes to the maintenance of the homeostasis of the immune system and could act as a barrier for autoimmunity in mice.

Puede el paciente advertir cercanía al nervio dentario inferior bajo anestesia infiltrativa?: presentación de un caso clínico / Can the patient notice the proximity to mental nerve under local anesthesia?: report of a clinical case

Cuando se colocan implantes dentales en el sector posterior de la mandíbula, el profesional debe decidir si utilizar la técnica troncular o la infiltrativa. Algunos estudios afirmaron que bajo anestesia infiltrativa el paciente podría advertir al profesional a través de su dolor, la cercanía del fresado o la colocación del implante al nervio dentario inferior. En contra posición, el objetivo de esta presentación de un caso clínico es evidenciar a través de tomografías pre y post quirúrgicas,que bajo anestesia infiltrativa la paciente no refirió dolor a pesar que el implante fue colocado íntimamente en relación al conducto dentario inferior.(AU)

Revisión bibliográfica de la anatomía del nervio dentario inferior y su implicancia en la cirugía implantológica de los sectores posteriores mandibulares / A review of the literature on inferior alveolar nerve anatomy and its involvement with implant surgery of the posterior mandibular sectors

El nervio dentario inferior es una de las estructuras anatómicas más importantes a considerar previo a la colocación de implantes dentales en la mandíbula. La lesión de este nervio es una situación temida por los pacientes y los profesionales por la posibilidad de daño permanente en su conducción nerviosa. Es fundamental para todos los odontólogos que realicen maniobras quirúrgicas próximas a la entidad nerviosa, conocer ampliamente su recorrido, distribución y características normales, para prevenir la injuria del mismo y sus indeseables consecuencias. El objetivo de esta publicación es brindar información actualizada del conocimiento del nervio dentario inferior y su relación con la implantología oral...

Puede el paciente advertir cercanía al nervio dentario inferior bajo anestesia infiltrativa?: presentación de un caso clínico / Can the patient notice the proximity to mental nerve under local anesthesia?: report of a clinical case

Cuando se colocan implantes dentales en el sector posterior de la mandíbula, el profesional debe decidir si utilizar la técnica troncular o la infiltrativa. Algunos estudios afirmaron que bajo anestesia infiltrativa el paciente podría advertir al profesional a través de su dolor, la cercanía del fresado o la colocación del implante al nervio dentario inferior. En contra posición, el objetivo de esta presentación de un caso clínico es evidenciar a través de tomografías pre y post quirúrgicas,que bajo anestesia infiltrativa la paciente no refirió dolor a pesar que el implante fue colocado íntimamente en relación al conducto dentario inferior.

Implant prosthetic rehabilitation with a free fibula flap and interpositional bone grafting after a mandibulectomy: a clinical report.

This clinical report describes the multidisciplinary treatment of a 16-year-old girl diagnosed with cemento-ossifying fibroma in the mandible. The resection of the lesion and reconstruction with a free osseous fibula flap with microvascular anastomosis was performed. Four months later, interpositional bone grafting of iliac spongy bone was used to gain bone height at the treated site. Twenty-four months later, 5 dental implants were placed. After a 6-month osseointegration period, a partial screw-retained fixed dental prosthesis was fabricated. Prosthodontic planning and treatment considerations are discussed.

A functional alternative splicing mutation in human tryptophan hydroxylase-2.

The brain serotonergic system has an essential role in the physiological functions of the central nervous system and dysregulation of serotonin (5-HT) homeostasis has been implicated in many neuropsychiatric disorders. The tryptophan hydroxylase-2 (TPH2) gene is the rate-limiting enzyme in brain 5-HT synthesis, and thus is an ideal candidate gene for understanding the role of dysregulation of brain serotonergic homeostasis. Here, we characterized a common, but functional single-nucleotide polymorphism (SNP rs1386493) in the TPH2 gene, which decreases efficiency of normal RNA splicing, resulting in a truncated TPH2 protein (TPH2-TR) by alternative splicing. TPH2-TR, which lacks TPH2 enzyme activity, dominant-negatively affects full-length TPH2 function, causing reduced 5-HT production. The predicted mRNA for TPH2-TR is present in postmortem brain of rs1386493 carriers. The rs13864923 variant does not appear to be overrepresented in either global or multiplex depression cohorts. However, in combination with other gene variants linked to 5-HT homeostasis, this variant may exhibit important epistatic influences.

[Total homocysteine levels in children with diabetes type 1. Conditional factors]. / Valores plasmáticos de homocisteína total en niños con diabetes mellitus tipo 1. Factores condicionantes.

OBJECTIVES: To measure the plasma levels of total homocysteine (tHcy) in children with type I diabetes mellitus and their relationship with the control of the disease. MATERIAL AND METHODS: We studied a total of 46 patients with ages between 4 and 19 years. The analyzed variables were: sex, age, puberty stage by Tanner, BMI, years of evolution of the illness, self-monitoring, associated diseases, tHcy, folic acid, vitamin B12, glycosylated haemoglobin (HbA1c), lipid profile and renal function. RESULTS: The mean tHcy was of 5.48 +/- 1,64 microm/l, similar to that in our control population. There was a positive correlation with tHcy when analyzing the puberty stage by the Tanner scale. The years of evolution of diabetes varied between 0.4 and 15, with a mean of 5.77 +/- 3.69, with no correlation with tHcy. The glycosylated haemoglobin mean was 7.35 %, with no correlation with tHcy. The levels of folic acid and vitamin B12 were similar to the control population. The lipid profile of our patients was normal, with no association with tHcy levels. There was no correlation between GFR and tHcy. CONCLUSIONS: A clinically correct control of children with diabetes mellitus type 1, appears to ensure a normal total homocysteinemia, with no significant differences with the healthy individuals of the same age and social environment.

Valores plasmáticos de homocisteína total en niños con diabetes mellitus tipo 1. Factores condicionantes / Total homocysteine levels in children with diabetes type 1. Conditional factors

Objetivos: Conocer las concentraciones plasmáticas de homocisteína total en niños afectados de diabetes mellitus tipo 1 y su relación con el control de la enfermedad. Material y métodos: Estudiamos un total de 46 pacientes con edades comprendidas entre los 4 y los 19 años. Las variables analizadas fueron: sexo, edad, estadio puberal de Tanner, índice de masa corporal, años de evolución de la enfermedad, autocontrol, patologías asociadas, homocisteína total (tHcy), ácido fólico, vitamina B12, hemoglobina glucosilada (HbA1c), perfil lipídico y función renal. Resultados: La homocisteína (Hcy) media fue de 5,48 ± 1,64 μm/l, similar a la de nuestra población control. Analizando el estadio puberal mediante la escala Tanner encontramos una correlación positiva con la Hcy. Los años de evolución de la diabetes oscilaban entre 0,4 y 15, con una media de 5,77 ± 3,69, sin correlación con la Hcy. La HbA1c media era del 7,35 %, sin correlación con la Hcy. Las concentraciones de ácido fólico y vitamina B12 fueron similares a la población control. El lipidograma de nuestros pacientes fue normal, sin relación con las cifras de Hcy. No hallamos correlación entre el índice de filtrado glomerular (GFR) y la Hcy. Conclusiones: Un correcto control clínico de los niños afectados de diabetes mellitus tipo 1 parece garantizar una homocisteinemia total normal, sin diferencias significativas con los individuos sanos de su misma edad y ambiente social (AU)

Objectives: To measure the plasma levels of total homocysteine (tHcy) in children with type I diabetes mellitus and their relationship with the control of the disease. Material and methods: We studied a total of 46 patients with ages between 4 and 19 years. The analyzed variables were: sex, age, puberty stage by Tanner, BMI, years of evolution of the illness, self-momitoring, associated diseases, tHcy, folic acid, vitamin B12, glycosylated haemoglobin (HbA1c), lipid profile and renal function. Results: The mean tHcy was of 5.48 ± 1,64 μm/l, similar to that in our control population. There was a positive correlation with tHcy when analyzing the puberty stage by the Tanner scale. The years of evolution of diabetes varied between 0.4 and 15, with a mean of 5.77 ± 3.69, with no correlation with tHcy. The glycosylated haemoglobin mean was 7.35 %, with no correlation with tHcy. The levels of folic acid and vitamin B12 were similar to the control population. The lipid profile of our patients was normal, with no association with tHcy levels. There was no correlation between GFR and tHcy. Conclusions: A clinically correct control of children with diabetes mellitus type 1, appears to ensure a normal total homocysteinemia, with no significant differences with the healthy individuals of the same age and social environment (AU)

Valores plasmáticos de homocisteína total en el recién nacido normal en el pretérmino. Factores condicionantes / Serum total homocysteine levels in normal and preterm newborn infants: conditioning factors

La hiperhomocisteinemia se ha involucrado en las enfermedades propias del embarazo y el parto, así como en las alteraciones fetales y del recién nacido. Para conocer el alcance real de este hecho y prevenir estas alteraciones, debemos conocer los valores normales de homocisteína total (tHcy) en los recién nacidos, tanto pretérmino como nacidos a término, y su relación con diversas variables que podrían influir en sus concentraciones. En este artículo mostramos que los niveles de tHcy están relacionados directamente con la edad gestacional y el peso de los recién nacidos

Hyperhomocysteinemis has been involved in health concerns associated with pregnancy and delivery and in fetal and neonatal anomalies. In order to determine the real implication of this fact and be able to prevent these alterations, we should know the normal values of serum total homocysteine (tHcy) in preterm and tern newborns and their relationship to different variables that could influence their concentrations. In this article, we show how tHcy levels are directly related to the gestational age and birthweight of infants

Tratamiento de la hiperfenilalaninemia por déficit de fenilalanina hidroxilasa con tetrahidrobiopterina. ¿Cuándo y cómo? / Tetrahydrobiopterin therapy for hyperphenylalaninemia due to phenylalanine hydroxylase deficiency. When and how?

Introducción: Algunos pacientes afectados de hiperfenilalaninemia por déficit de fenilalanina hidroxilasa responden con un descenso variable de las concentraciones plasmáticas de fenilalanina, a la administración por vía oral de tetrahidrobiopterina (BH4), de tal modo que pueden liberalizar o incluso abandonar la dieta con ingesta limitada de fenilalanina. Habitualmente, la identificación de los pacientes sensibles a BH4 se realiza mediante el test de sobrecarga con BH4, pero no existe acuerdo unánime con relación a su metodología e interpretación de los resultados. Desde esta perspectiva, es importante disponer de una herramienta que nos ayude a identificar del modo más sencillo posible a los pacientes tributarios de este tipo de tratamiento y conocer cuál es su evolución a largo plazo. Material y métodos: Se ha practicado el test de sobrecarga combinado de fenilalanina (100 mg/kg) y BH4 (20 mg/kg) en 20 pacientes con hiperfenilalaninemia sometidos a dieta limitada en fenilalanina. Resultados: Con independencia de su genotipo, la respuesta al test fue positiva en los 9 pacientes cuyo máximo nivel de fenilalanina al diagnóstico fue menor de 815 nmol/ml. Todos están en tratamiento con dosis de BH4 entre 7 y 15 mg/kg/día y en todos los casos ha sido posible aumentar notablemente la ingesta diaria de fenilalanina. En este momento 6 están con dieta libre, y los otros muy cerca de alcanzar este objetivo. Ninguno de los pacientes con un nivel de fenilalanina máximo al diagnóstico de más de 938 nmol/ml ha respondido al test de sobrecarga. Conclusiones: El nivel máximo de fenilalanina en el momento del diagnóstico, parece ser un método sencillo y fiable para predecir la respuesta al tratamiento con BH4. En los pacientes respondedores, el tratamiento continuado con BH4 permite la eliminación de la dieta limitada en fenilalanina en un alto porcentaje de casos

Introduction: Some patients with hyperphenylalaninemia due to phenylalanine hydroxylase deficiency respond with a variable decrease in plasma phenylalanine levels after oral tetrahydrobiopterin (BH4) administration and are then able to tolerate higher dietary phenylalanine intake or even to discontinue a phenylalanine-restricted diet. BH4-sensitive patients are usually identified by means of a BH4 loading test, but consensus on the methodology of this test and the interpretation of its results is lacking. Consequently, a simple tool to identify which patients are likely candidates for this treatment and how they will progress in the long-term is required. Material and methods: A combined oral BH4 loading test with phenylalanine (100 mg/kg) and BH4 (20 mg/kg) was performed in 20 patients with hyperphenylalaninemia under dietary phenylalanine restriction. Results: Independently of the genotype, the result was positive in all the 9 patients whose maximum phenylalanine level at diagnosis was below 815 nmol/ml. Currently, they are under treatment with tetrahydrobiopterin doses of 7-15 mg/kg/day. All these patients have been able to increase their oral phenylalanine intake. Six are currently following a normal diet and the remaining three are close to reaching this goal. None of the patients with a maximum phenylalanine level at diagnosis higher than 938 nmol/ml responded to the BH4 loading test. Conclusions: The maximum phenylalanine level at diagnosis seems to be a simple and reliable method to predict response to BH4 treatment. A high percentage of BH4-sensitive patients are able to discontinue a phenylalanine-restricted diet after long-term tetrahydrobiopterin treatment

[Severe fulminant form of neonatal citrullinemia. Report of a case]. / Forma grave fulminante de citrulinemia neonatal. Comunicación de un caso.

INTRODUCTION: Citrullinemia is an autosomal recessive disease, which is caused by a deficiency of the argininosuccinate synthetase. The neonatal forms are serious and many times are associated with a high level of mortality. CASE REPORT: A newborn that came in again on her third day of life due to a apneic episodes which required mechanical ventilation. Previously, she rejected feeding, had poor suction, lethargy and remarkable hypoactivity. During the following hours, she showed serious neurologycal deterioration with multifocal convulsions and coma, passing away 20 hours after admission due to endocraneal hypertension. The metabolic evaluation confirmed very significant hyperammonemia, with important increase of citrullin and glutamin, and arginine in the low limits of normality. She was treated with sodium benzoate and arginine and she also needed exanguinotransfusion. It was not possible to put her on hemodyalisis. The findings of the autopsy confirmed massive cerebral edema and characteristic hystological changes in the liver. The determination of the enzymatical activity in liver tissue showed a partial deficiency, with a residual activity of 25% of the average control. CONCLUSIONS: This is a case of fulminant neonatal citrullinemia that we considered of interest in order to draw the attention of the clinical on this type of diseases. The prognosis depends on early diagnosis, witch is based on clinical suspicion and analytical determination of ammonia in every newborn with unexplained vomiting, lethargy or other symptoms of encephalopathy.

The transcription elongation factor CA150 interacts with RNA polymerase II and the pre-mRNA splicing factor SF1.

CA150 represses RNA polymerase II (RNAPII) transcription by inhibiting the elongation of transcripts. The FF repeat domains of CA150 bind directly to the phosphorylated carboxyl-terminal domain of the largest subunit of RNAPII. We determined that this interaction is required for efficient CA150-mediated repression of transcription from the alpha(4)-integrin promoter. Additional functional determinants, namely, the WW1 and WW2 domains of CA150, were also required for efficient repression. A protein that interacted directly with CA150 WW1 and WW2 was identified as the splicing-transcription factor SF1. Previous studies have demonstrated a role for SF1 in transcription repression, and we found that binding of the CA150 WW1 and WW2 domains to SF1 correlated exactly with the functional contribution of these domains for repression. The binding specificity of the CA150 WW domains was found to be unique in comparison to known classes of WW domains. Furthermore, the CA150 binding site, within the carboxyl-terminal half of SF1, contains a novel type of proline-rich motif that may be recognized by the CA150 WW1 and WW2 domains. These results support a model for the recruitment of CA150 to repress transcription elongation. In this model, CA150 binds to the phosphorylated CTD of elongating RNAPII and SF1 targets the nascent transcript.

Co-transcriptional splicing of pre-messenger RNAs: considerations for the mechanism of alternative splicing.

Nascent transcripts are the true substrates for many splicing events in mammalian cells. In this review we discuss transcription, splicing, and alternative splicing in the context of co-transcriptional processing of pre-mRNA. The realization that splicing occurs co-transcriptionally requires two important considerations: First, the cis-acting elements in the splicing substrate are synthesized at different times in a 5' to 3' direction. This dynamic view of the substrate implies that in a 100 kb intron the 5' splice site will be synthesized as much as an hour before the 3' splice site. Second, the transcription machinery and the splicing machinery, which are both complex and very large, are working in close proximity to each other. It is therefore likely that these two macromolecular machines interact, and recent data supporting this notion is discussed. We propose a model for co-transcriptional pre-mRNA processing that incorporates the concepts of splice site-tethering and dynamic exon definition. Also, we present a dynamic view of the alternative splicing of FGF-R2 and suggest that this view could be generally applicable to many regulated splicing events.

Messenger RNA repair and restoration of protein function by spliceosome-mediated RNA trans-splicing.

The functional repertoire of the human genome is amplified by the differential assortment of exons. Spliceosome-mediated RNA trans-splicing can mobilize these packets of genetic information to reprogram mRNAs. In principle, this process could repair defective transcripts in loss-of-function genetic disorders in humans. We developed a tractable lacZ repair system to serve as a model for these genetic disorders. Targeted pre-trans-splicing RNA molecules efficiently and specifically repaired mutated lacZ transcripts and restored enzymatic activity in human cells. The development of this model confirms the potential for spliceosome-mediated RNA trans-splicing in genetic repairs and provides a powerful tool for rational design and in vitro evolution of pre-trans-splicing molecules.

The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is critical for function.

In Saccharomyces cerevisiae, Prp17p is required for the efficient completion of the second step of pre-mRNA splicing. The function and interacting factors for this protein have not been elucidated. We have performed a mutational analysis of yPrp17p to identify protein domains critical for function. A series of deletions were made throughout the region spanning the N-terminal 158 amino acids of the protein, which do not contain any identified structural motifs. The C-terminal portion (amino acids 160-455) contains a WD domain containing seven WD repeats. We determined that a minimal functional Prp17p consists of the WD domain and 40 amino acids N-terminal to it. We generated a three-dimensional model of the WD repeats in Prp17p based on the crystal structure of the beta-transducin WD domain. This model was used to identify potentially important amino acids for in vivo functional characterization. Through analysis of mutations in four different loops of Prp17p that lie between beta strands in the WD repeats, we have identified four amino acids, 235TETG238, that are critical for function. These amino acids are predicted to be surface exposed and may be involved in interactions that are important for splicing. Temperature-sensitive prp17 alleles with mutations of these four amino acids are defective for the second step of splicing and are synthetically lethal with a U5 snRNA loop I mutation, which is also required for the second step of splicing. These data reinforce the functional significance of this region within the WD domain of Prp17p in the second step of splicing.

Coupled in vitro synthesis and splicing of RNA polymerase II transcripts.

Compelling in vivo studies suggest a tight functional linkage between RNA polymerase II transcription and premessenger RNA splicing. At present, the specific interactions involved in this coupling are poorly understood and deserve investigation. To this end, we developed an in vitro system that permits study of coupled transcription and splicing. Transcripts generated by RNA polymerase II were accurately and efficiently spliced under reaction conditions that permitted both transcription and splicing to occur simultaneously. The splicing of RNA-polymerase-II-driven transcripts was accelerated relative to that of the same transcripts driven by T7 RNA polymerase. Moreover, the product of exon ligation was found associated with the DNA template in reactions driven by RNA polymerase II. These two findings indicate that transcription and splicing were coupled in the in vitro system driven by RNA polymerase II, and suggest that this system will be useful for the biochemical study of this coupling.