Identification of CSN1S1 polimorphisms in Malagueña goat stallions to improve the dairy production

Motivation: Alpha-S1-casein is one of the most abundant proteins in milk of ruminants, and the extensive polymorphisms at CNS1S1 locus not only affect the quantity, but also the quality of milk (Martin et al. 2002). Up to 18 polymorphisms have been identified at CNS1S1 locus and have been classified into 4 groups based on milk production (high, medium, low, null) (Caravaca et al. 2008). Currently, there is a program to improve the Malagueña Goat breed, whose purpose is the selection of individuals based on different criteria, including the optimization of dairy production (Serradilla, 2012). One of the most important characteristic of Malagueña goat is the high milk production, both in quantity and quality; thus, our objective is to identify individuals with a favorable allelic combination to select those stallions with a value suitable for reproduction. Genotyping of 77 stallions will provide advice on best individuals for insemination in the improvement program.Methods: Genomic DNA is obtained from blood samples. After DNA extraction, three levels of diagnostic PCRs will be performed in a progressive order based on the results obtained in each level. The results of the diagnostic PCRs will be analyzed by gel electrophoresis, obtaining the final results according to the size of fragment expected for each polymorphism (Ramuno et al. 2000; Cosenza et al. 2003; Caravaca et al. 2008)

Crispr-cpf1 edition in the fission yeast to characterize the regulatory function of 3´UTR RNA loops in transcription termination

The wos2 gene encodes for the protein p23, which acts as a co-chaperone in the presence of the protein Hsp90. Their function is to fold proteins during a heat shock stress. This process is very similar both in human and the fission yeast S.pombe, our model organism in this project a. It was previously discovered that there exist three mRNA sizes depending on the 3’UTR length. Interestingly, the abundance of each depends on growth temperature. We then hypothesized that the secondary structure of the 3´UTR RNA could dictate the termination site depending on temperature. We are trying to demonstrate this assumption as well as to develop a temperature sensitive switch to control gene expression by inserting the wos2 3’UTR in the 5’UTR of rpl42S59Q gene marker. This allele is resistant to cycloheximide whilst the deletion is not. This way we may easily asses gene expression. To this end, we are using the CRISPR/Cpf1 technology, to engineer the genome region of interest. We are in the process of checking engineered candidates

Improve the production and quality of milk malagueña goat by the identification and validation of genetic markers (QTLs)

Motivation:Malagueña goat is one of the most dairy goat breeds in the world with an average production of 500 liters of milk, per female, per year; with approximately 300,000 animals.Currently, the Malagueña goat breed improvement program, is based on the selection of the best individuals based on morphologic criteria. So that, a greater number of morphological skills exist in a specimen, better genetic evaluation is assigned.On the other hand, for years it has been studying the association of molecular genetic markers with cheese production. Specifically, they have been described in several global locations even 18 alleles of the caseins directly related to the total protein content. There are described alleles of high, medium, low and zero protein production.In this paper we perform a genetic identification by genotyping of these alleles to validate the program to improve the Malaga goat breed.Methods:The DNA is obtained by extraction from blood and/or hair for later identification of each copy of haplotype.To this end, we perform 4 levels of diagnostic PCRs that allows us to separate high production alleles from others.Comparing the allelic frequencies obtained in a selected group as producers, with a low production control group we can validate the polymorphism of α-S1-casein protein as a marker of individual milk production.Results:Assuming that the in process method will be validated, the results will be applied to design a controlled crosses program aimed to improve the cheese degree apply

New connection between transcriptional regulation and genomic instability

The genes involved in cell and nuclear division ensure an equitable distribution of genetic material between daughter cells. Defects in their functioning cause what is known as genomic instability, a molecular origin in the development of cancer and other genetic diseases.Among these proteins are crucial those that produce the sister chromatid cohesion and release cycles. This is achieved by the antagonistic action between the loading and unloading pathways of the cohesin complex, which are activated or inhibited in coordination with the progress of the cell cycle, keeping the sister chromatids together until the moment of their separation in anaphase. This regulation is evolutionarily very well conserved and is very similar between multicellular eukaryotes and the fission yeast, Squizosaccharomyces pombe, the model organism in this work. In previous work, the conditional mutant cwf15.d53 was isolated, which has a deletion of the last 53 amino acids that confers sensitivity to formamide and shows an abnormal pattern of mitotic segregation associated with a quantitative and qualitative increase in chromosome-loaded cohesin.The aim of the project is twofold: on the one hand, to characterise a new thermosensitive allele of cwf15 (cwf15.ts4) and, on the other hand, to determine if this is the molecular cause of this segregation defect. The approximation we follow in the first case is to reveal the cellular defects caused under restrictive conditions, by fluorescence microscopy in live and fixed cells. To deal with the second objective, we expect to over-express one of the proteins involved in cohesin unloading, Wpl.To do this, its promoter at the native locus will be replaced by a powerful regulatable promoter (nmt promoter). Its expression is regulated by the amount of thiamine present in the medium, so that in the absence of thiamine it is highly expressed. In this situation, we can check if the cellular defect is reversed and monitor the amount of cohesin throughout the cell cycle by biochemical assays and fluorescent markers in live cells

Phosphorylation-mediated regulation of the gamma tubulin complex.

Motivation: Microtubules perform vital functions such as chromosome segregation, nuclear positioning, vesicular transport and determination of cell polarity and morphogenesis. The gamma tubulin complex is an essential element responsible for microtubule nucleation and is highly conserved from yeast to human and the core of the complex is basically composed of three esential proteins (Alp4/GCP2, Alp6/GCP3 and Gtb1/GTB). Its function is subject to a multiple regulation processes both temporally and spatially, and phosphorilation is one of the main postranslational modifications to regulate protein function. The fission yeast Schizosaccharomyces pombe is a potent model for studying microtubules (MT) nucleation because of its simple MT cytoskeleton, genetic tractability, limited number of regulators, and amenability to assays of individual nucleation events. In this work, we use S. pombe to determine the role of different putative phosphorylation sites to understand the regulation of this complex in different stages of the cell cycle. Methods: In our laboratory, a collection of mutants of Alp4 and Alp6 genes was generated. These mutants consisted of the modification of the phosphorylation sites described in massive phosphoproteomes. Usind directed mutagenesis we generated non-phosphorylatable and phosphomimetic alleles (changins Serines to Alanine or Aspartic acid residues respectively. In this project, a phenotypic analysis of the different mutants from this collection has been performed using different approaches such as spot tests, western blots, fluorescence microscopy with live cells, and genetic interaction analysis techniques. Results: We found that different mutants display phenotypic differences under the conditions analysed. The most representative phenotypes are related to delayed and aberrant mitotic spindle formation, no mitotic spindle formation and the appearance of stress granules at high temperatures. This establishes a connection between the phosphorylation state of Alp4 and Alp6 with microtubule formation, and its implication in the biology of microtubules in the cell. Conclusions: Our data suggest that phosphorylation of components of the gamma tubulin complex is involved in the regulation of microtubule nucleation in different stages of the cell cycle and cell phisiology. This shed light in new molecular mechanisms not been previously described

Functional characterization of splicing factor Cwf15 in the maintenance of genomic stability

Motivation: The accumulation of mutations and rearrangements, or the asymmetrical distribution of genetic material during the mitotic process lead to cellular defects known generically as genomic instability. Previously, we isolated a truncated version of cwf15 lacking it last 53 aa (cwf15.d53) which presents two clearly deleterious phenotypes associated with genomic instability: readthrough and aberrant distribution of the cohesin loaded in the chromosomes. These data show that, in addition to its function in splicing, cwf15 plays a fundamental role in the end of transcription and in the charge/discharge cycle of cohesin. The specific objectives of this project are: Expression in trans of the 53 amino acid that lacks the mutant to evaluate self-contained function. Testing in vivo a “proof of concept” design to follow specifically the loaded cohesin in the chromosomes. Finding extragenic suppressors of cwf15 defective function. Methods:  First, we characterized the chromatin segregation defects of cwf15 and compared quantitative-and-quantitatively to other splicing mutant to show that there exist functional differences. Second, we have engineered, and inserted into the genome, different control and experimental expression constructs to assess functional complementation of Cwf15 c-terminal domain in trans. Third, we have designed and constructed a novel system by bi-molecular complementation to monitor cohesin specifically loaded onto the chromosomes in living cells. Fourth, we have generated genetic combinations of cwf15 deletion and truncation with very recently published mutations in transcription termination genes (yth1 and seb1) to show that cwf15 is involved in a RNA processing different from splicing. Results and conclusions: We show that defective cwf15 function phenotype is different from other characterized splicing factors and that a specific gain-of-function mutation in transcription termination seb1 is able to suppress growth defects of cwf15.d53 mutant. Furthermore, a missense mutation in the RNA polII pausing factor yth1 also rescues lethality of cwf15 whole deletion. On the other hand, preliminary experiments suggest that expression in trans of the last 53 amino acids does not reverse the phenotype of cwf15.d53 although it accumulates in the nucleolus

Estrategia cooperativa para la maximización de recursos en proyectos fin de grado

En el marco actual de la limitación de los recursos docentes por la situación económica que atraviesa nuestra sociedad, se hace esencial desarrollar estrategias para maximizar los recursos educativos en asignaturas eminentemente prácticas como los Proyectos Fin de Grado. En este proyecto se pretende maximizar la dedicación del tutor multiplicando por cuatro las horas que cada estudiante puede pasar en el laboratorio con una supervisión y entrenamiento experto. Para ello Se han tutorizado 4 Proyectos Fin de Grado íntegramente de laboratorio de manera simultánea. Los proyectos son paralelos, con un organismo modelo, temática, dinámica y objetivos comunes pero manteniendo elementos diferenciadores para mantener la individualidad en la adquisición de competencias. Cada sesión de laboratorio, se realiza sin excepción con los 4 estudiantes, de modo que el contacto que cada uno de ellos tiene con el tutor es 4 veces mayor que si fuera individual. Sin embargo, cada uno de ellos realizó de modo individual todas y cada una de las tareas necesarias tanto de diseño como de ejecución experimental. Al ser proyectos diseñados con coincidencias conceptuales y desarrollo de competencias comunes, las dudas resueltas en grupo así como las discusiones y análisis de los resultados de forma colectiva beneficia a todos y cada uno de los estudiantes y el tiempo que se ahorra en procesos comunes se puede invertir en más trabajo de laboratorio. Los resultados han sido óptimos tanto por los resultados experimentales como por la opinión de los estudiantes implicados