Non-syndromic Hirschsprung's disease as a result of a RET gene variant. / Enfermedad de Hirschsprung no sindrómica por variante en el gen RET.

INTRODUCTION: Hirschsprung's disease (HD) is characterized by the absence of ganglion cells in the submucosal and myenteric plexuses of the colon as a result of disorders in the migration and differentiation of enteric neural crest cells during embryogenesis. It is a cross-factor condition, with more than 11 genes identified in its pathogenesis, including the RET proto-onco gene. CASE REPORTS: We present the case of two siblings with total colon HD where a potentially pathogenic variant of the RET gene was found. Their father also had this condition. DISCUSSION: Prenatal diagnosis through genetic testing allows for informed decisions and care planning for the newborn, thus reducing delayed diagnosis and treatment, and minimizing long-term complications. Mutations such as the RET gene variant highlight the importance of the genetic approach in understanding and managing HD.

INTRODUCCION: La enfermedad de Hirschsprung (EH) se caracteriza por la ausencia de células ganglionares en los plexos submucoso y mientérico del intestino grueso, resultante de deficiencias en la migración y diferenciación de las células de la cresta neural entérica durante la embriogénesis. Es una condición multifactorial, con más de 11 genes identificados en su patogénesis, incluyendo el protooncogén RET. CASO CLINICO: Se presenta el caso de dos hermanos con EH de colon total, cuyo padre también padeció la enfermedad, y en quien se encontró una variante potencialmente patogénica en el gen RET. COMENTARIOS: El diagnóstico prenatal mediante pruebas genéticas permite decisiones informadas y la planificación de cuidados para el neonato afectado, reduciendo demoras en el diagnóstico y tratamiento, y minimizando las complicaciones a largo plazo. La identificación de mutaciones como la variante en el gen RET destaca la importancia del enfoque genético en la comprensión y manejo de la EH.

Yeast transformation by the LiAc/SS carrier DNA/PEG method.

Efficient transformation of yeast has been the corner stone of molecular studies in yeast for numerous years. It is essential for techniques such as the yeast two-hybrid system as well as genome modification. Here I present protocols for a number of different transformation applications. A quick and easy protocol is for situations in which large numbers of transformants are not required. The high-efficiency protocol generates large numbers of transformants from small amounts of DNA that can be scaled up for the library transformation protocol. A transformation protocol for a microtiter plate format is also included. Finally, a protocol for the production of frozen competent yeast cells is included, which allows cells to be taken from the freezer at a moment's notice and transformed with high efficiency.

Yeast transformation by the LiAc/SS carrier DNA/PEG method.

Transformation is essential to many molecular and genetic investigations in the yeast Saccharomyces cerevisiae. Yeast transformation protocols utilizing the LiAc/ssDNA/PEG method are presented. Protocols for various applications are listed including a method for transformation in 96-well microtiter plate format and another for the production of frozen competent yeast cells that can be used at a moment's notice.

Yeast two-hybrid liquid screening.

Yeast two-hybrid (YTH) method consists of a genetic trap that selects for "prey" cDNA products within a library that interact with a "bait" protein of interest. Here, we provide a protocol for YTH screening using a liquid medium screening method, which improves the sensitivity of this technique and streamlines the laborious classic screening in solid medium plates. The method uses a simple series of dilutions with established yeast strains transformed with diverse baits and complex cDNA libraries. This allows for prompt detection of positive clones revealed by liquid growth, due to activation of HIS3 reporter gene. Activation of a second reporter gene and reconstruction of the YTH interaction is highly reproducible using this system. This approach can either be performed using culture flasks or deep-well 96-well plates and the number of interactions obtained is similar, when compared to the classic method. In addition, the liquid screening method is faster and more economical for YTH screening and has the added benefit of automation if 96-well plates are used.

Chemical reactivity and microbicidal action of bethoxazin.

Bethoxazin is a new broad spectrum industrial microbicide with applications in material and coating preservation. However, little is known of its reactivity profile and mechanism of action. In this study, we examined the reactivity of bethoxazin toward biologically important nucleophilic groups using UV-vis spectroscopy and LC-MS/MS techniques and found the molecule to be highly electrophilic. Bethoxazin reacted with molecules containing free sulfhydryl groups such as GSH and human serum albumin to form covalent adducts that were detectable by MS, but did not react with amino, carboxylic, phenolic, amino oxo, alcoholic, and phosphate functional groups. Bethoxazin potently inhibited the catalytic activity of yeast DNA topoisomerase II and the growth of yeast BY4742 cells at low micromolar concentrations. However, the reduced form of bethoxazin and GSH-treated bethoxazin were both inactive in these assays. The experimentally determined relative reactivity of bethoxazin and its reduced form analog correlated with their biological activities as well as their quantum-mechanically calculated electrophilicity properties. Taken together, the results suggest that bethoxazin may exert its microbicidal action by reacting with sensitive endogenous sulfhydryl biomolecules of microbial cells. Consistent with this view, the inhibitory activity of bethoxazin on topoisomerase II may be due to its ability to react with critical free cysteine sulfhydryl groups on the enzyme. Our studies have provided for the first time a better understanding of the reactivity of bethoxazin, as well as some insights into the mechanism by which the compound exerts its microbicidal action.

Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method.

Here we describe a protocol for the production of frozen competent yeast cells that can be transformed with high efficiency using the lithium acetate/single-stranded carrier DNA/PEG method. This protocol allows the production of highly competent yeast cells that can be frozen and used at a later date and is especially useful for laboratories using one or two strains repeatedly. The production of yeast cells for freezing takes only approximately 30 min, once the yeast culture has grown up. Transformation with frozen competent yeast cells will take approximately 30 min, depending on the heat shock used.

Microtiter plate transformation using the LiAc/SS carrier DNA/PEG method.

Here, we describe a protocol that has been adapted for the transformation of yeast cells in 96-well microtiter plates. This protocol can be tailored for multiple applications and is suitable for high-throughput applications. It can be completed in 2-3 h, once the yeast cells have been grown depending on the heat shock used.

High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method.

Here we describe a high-efficiency version of the lithium acetate/single-stranded carrier DNA/PEG method of transformation of Saccharomyces cerevisiae. This method currently gives the highest efficiency and yield of transformants, although a faster protocol is available for small number of transformations. The procedure takes up to 1.5 h, depending on the length of heat shock, once the yeast culture has been grown. This method is useful for most transformation requirements.

Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method.

Here, we describe a quick and easy version of the lithium acetate/single-stranded carrier DNA/PEG method of transformation for Saccharomyces cerevisiae. This method can be performed when only a few transformants are needed. The procedure can take less than an hour, depending on the duration of the heat shock. It can be used to transform yeast cells from various stages of growth and storage. Cells can be transformed from freshly grown cells as well as cells stored on a plate at room temperature or in a refrigerator.

Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method.

Here, we describe a Library screen transformation protocol using the lithium acetate/single-stranded carrier DNA/PEG method of transformation for Saccharomyces cerevisiae. This method is suitable for screening complex plasmid libraries such as those used for yeast two-hybrid analysis. This procedure takes up to 2.5 h to complete once the yeast culture has been grown.

Yeast transformation by the LiAc/SS Carrier DNA/PEG method.

The technique for the transformation of Saccharomyces cerevisiae using the LiAc/SS Carrier DNA/PEG method is described. We describe a rapid method, for use when large numbers of transformants are not necessary. A high-efficiency method for the generation of large numbers of transformants is also given. A method for the transformation of plasmid libraries, which includes yeast two-hybrid applications, also is listed to aid the reader in generating transformants to effectively cover the library complexity. Finally, a protocol for transformation using a 96-well format is included for transformation applications that require it.

Yeast two-hybrid system screening.

The yeast two-hybrid system is a powerful molecular genetic tool conceived by Fields and Song. The article is a comprehensive set of methods designed to take the reader through a yeast two-hybrid analysis of your favorite gene (YFG). This article details the preparation for a screen, the screen itself, as well as the analysis of the positives identified. Using these methods, the readers should be able to successfully negotiate a yeast two-hybrid screen using the Fields' or related systems.

Psoriasin interacts with Jab1 and influences breast cancer progression.

Psoriasin (S100A7) is expressed at low levels in normal breast epithelial cells but is highly expressed in preinvasive ductal carcinoma in situ. Persistent psoriasin expression occurs in some invasive carcinomas and is associated with poor prognostic factors. Whereas there is evidence that secreted psoriasin can act as a chemotactic factor for CD-4-positive lymphocytes in psoriatic skin lesions, an intracellular biological function is unknown. We have found that psoriasin physically interacts with Jab1 (c-jun activation-domain binding protein 1) in the yeast two-hybrid assay and confirmed this by coimmunoprecipitation assay in breast cancer cells. Psoriasin-transfected breast cancer cells showed increased nuclear Jab1 and demonstrated several features consistent with an alteration in Jab1 activity including an increase in activator protein-1 (AP-1) activity, increased expression of AP-1 and HIF-1-dependent genes, and reduced expression of the cell-cycle inhibitor p27(Kip1). Psoriasin overexpression was also associated with alteration of cellular functions that are associated with increased malignancy, including increased growth, decreased adhesion, and increased invasiveness in vitro, as well as increased tumorigenicity in vivo in nude mice. We conclude that intracellular psoriasin influences breast cancer progression and that this may occur through stimulation of Jab1 activity.

RanBPM interacts with psoriasin in vitro and their expression correlates with specific clinical features in vivo in breast cancer.

BACKGROUND: Psoriasin has been identified as a gene that is highly expressed in pre-invasive breast cancer, but is often downregulated with breast cancer progression. It is currently unknown whether psoriasin influences epithelial cell malignancy directly or by affecting the surrounding environment. However the protein is found in the nucleus, cytoplasm as well as extracellularly. In the present study we have sought to identify potential psoriasin-binding proteins and to describe their expression profile in breast tumors. METHODS: The yeast two-hybrid method was used to identify potential binding partners for psoriasin. The interaction of psoriasin with RanBPM was confirmed in-vitro by co-immunoprecipitation. The expression of RanBPM and psoriasin was measured by RT-PCR in a series of breast cell lines, breast tumors and primary lymphocytes. RESULTS: We have identified RanBPM as an interacting protein by the yeast two-hybrid assay and confirmed this interaction in-vitro by co-immunoprecipitation. RT-PCR analysis of RanBPM mRNA expression in cell lines (n = 13) shows that RanBPM is widely expressed in different cell types and that expression is higher in tumor than in normal breast epithelial cell lines. RanBPM expression can also be induced in peripheral blood mononuclear cells by treatment with PHA. RanBPM mRNA is also frequently expressed in invasive breast carcinomas (n = 64) and a higher psoriasin/RanBPM ratio is associated with both ER negative (p < 0.0001) and PR negative status (p < 0.001), and inflammatory cell infiltrates (p < 0.0001) within the tumor. CONCLUSIONS: These findings support the hypothesis that psoriasin may interact with RanBPM and this may influence both epithelial and stromal cells and thus contribute to breast tumor progression.

Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.

In this chapter we have provided instructions for transforming yeast by a number of variations of the LiAc/SS-DNA/PEG method for a number of different applications. The rapid transformation protocol is used when small numbers of transformants are required. The high efficiency transformation protocol is used to generate large numbers of transformants or to deliver DNA constructs or oligonucleotides into the yeast cell. The large-scale transformation protocol is primarily applicable to the analysis of complex plasmid DNA libraries, such as those required for the yeast two-hybrid system. The microtiter plate versions of the rapid and high efficiency transformation protocols can be applied to high-throughput screening technologies.

Genetic transformation of yeast.

Genetic transformation was first described by Griffith in 1928 and has since been demonstrated in a variety of organisms, including many species of fungi. This review focuses on the history and technology of the transformation of Saccharomyces cerevisiae. The application of protocols developed for S. cerevisiae to other important yeast species is discussed. The protocols for transformation by spheroplasting, LiAc/ssDNA/PEG, and electroporation are compared, and possible mechanisms for transformation are discussed.

Human growth factor receptor bound 14 binds the activated insulin receptor and alters the insulin-stimulated tyrosine phosphorylation levels of multiple proteins.

To identify proteins interacting in the insulin-signaling pathway that might define new pathways or regulate existing ones, we have employed the yeast two-hybrid system. In a two-hybrid screen of a human liver cDNA library, we identified the human growth factor receptor bound 14 (hGrb14) adaptor protein as a partner of the activated insulin receptor. Additional analysis of the insulin receptor--hGrb14 interaction in the yeast two-hybrid system revealed that the SH2 domain of hGrb14 was not the sole region involved in binding the activated insulin receptor. The insulin-stimulated interaction between hGrb14 and the insulin receptor was also observed in different mammalian cultured cell lines. This association was detected at 1 min of insulin stimulation and was maximal at 10 nM and greater concentrations of insulin. Chinese hamster ovary cells stably expressing the insulin receptor (CHO-IR) and hGrb14 were used to examine the effects of hGrb14 overexpression on insulin-stimulated tyrosine phosphorylation of proteins; in general, increasing levels of hGrb14 expression resulted in a reduction in tyrosine phosphorylation. This decrease was demonstrated for the specific proteins src homology-containing and collagen-related protein (Shc), insulin receptor substrate-1 (IRS-1), and Downstream of tyrosine Kinase (Dok). The broad effects of hGrb14 overexpression on insulin-stimulated tyrosine phosphorylation suggest that it acts early in the insulin-signaling pathway.