Obtaining Immunization Record In Ontario

Introduction

elucidate the molecular events triggered in response to changes in nutrient availability, analyzing gene regulation processes in model organism Saccharomyces cerevisiae

Any cell

different types of nutrients needed for growth. Cells are able to sense changes in nutrient growth medium and adapt to them. Thus, the processes that mediate various cellular responses occur sequentially and are: reception, transduction and response. The reception involves the sensing of different nutrients and proteins in the plasma membrane of cells. Transduction is the mechanism by which signals are sent or within cells generate a cellular response, which may be the activation of enzymes of a particular metabolic pathway and / or activate the transcription of specific genes. Each cell
has within it a nucleus that contains, among other things, the information (DNA) to enable it to function properly. This information in turn, is divided into small segments, in packages that are called genes. Each gene, after being transcribed and translated, produces a special protein, ie each gene has enough information to synthesize a protein.

As experimental model, cells will be used yeast Saccharomyces cerevisiae is a unicellular eukaryotic organism non-pathogenic, easy handling, rapid growth in defined culture media and whose complete genome sequence is known. Taking into account the evolutionary conservation of the signaling pathways triggered in response to nutrient medium, the use of S. cerevisiae as a model for the study of signal transduction will clarify the major challenge is to understand how signals produced by nutrients present in the extracellular environment are integrated and coordinated by the cells.

In this picture you can see a yeast cell gemando (reproducing asexually).

can also observe the nucleus and vacuole.

In the laboratory studying the regulation of gene UGA4, which is an excellent model for studying gene response to changes in the extracellular environment and is subject to complex regulation and also some of the key proteins involved in this regulation are conserved from yeast to man. This is the case, for example, transcription factors GATA family, many of which are important in regulating UGA4, and also the Tor1 protein, kinase that has a central role in controlling growth response to nutrient availability in both flies and yeast as cells mamíferos1, 2.
UGA4 is the gene that codes for Uga4 protein. This protein is a permease located in the plasma membrane and allows the incorporation of d-aminolevúlico acid (ALA) 3.4 and y-aminobutyric acid (GABA) 5.6.

ALA has no biological function that is essential for the cell, is incorporated by this because of its structure , which is similar to that of GABA.
GABA can be used by the yeast cells as a source of nitrogen, but is a poor source in compared to rich sources of nitrogen and ammonia. Factors influencing the suppression or induction of gene expression, depending on the situation in which the cell is located. The gene to be expressed only in the absence of a source of nitrogen (as ammonium) in the absence of such a source, the cell triggers mechanisms to survive in such conditions. In the promoter sequence of the gene UGA4 two important sites known as GABA and UAS-UAS-GATA. In each of these factors influence the sticking repress or induce its expression. UGA4 gene expression depends on the presence of an inducer, GABA5, 7. Induction requires the action of two factors transcription, a specific Uga3 and other pleiotropic, Uga35, which act through activating sequence rich in GC nucleotides (UASGABA) 8.9. The UGA4 gene regulatory region also contains four adjacent repeats heptanucleótido 5'-CGAT (A / T) AG-3 'which is an UASGATA. On the latter acting GATA factors can act as positive and negative factors. The negative (and Gzf3 Uga43) compete with the positive (Gln3 and GAT1) and that "wins" sticks to the gene promoter. When there is a rich source of nitrogen, the cell does not waste energy synthesizing proteins carrying GABA8, 10.11, positive factors. Gln3 and GAT1, was found in the cytoplasm bound to the protein Ure2 from acting on the UAS-GATA element, and therefore, the negative factors "win", which leads to repression of the expression of UGA4. In this case the negative factors bind to the UAS-GATA site.

When there is a rich source but there are GABA in the medium, the cell adapts to change and active all the machinery to synthesize the protein Uga4, in other words, GABA induces gene expression UGA45, 7.9 . Positive factors dissociate from Ure2 and translocate to the nucleus where they move to the negative factors binding to the promoter and allowing expression gen9, 12. The interaction between the positive factors with GAT1 Gln3 and Ure2 is modulated by the phosphorylation status of these factors which in turn depends on the kinase activity Tor13-15.
The main objective of this internship is to familiarize with different molecular biology techniques through participation in a project that studies the signaling cascade that leads to the regulation of gene expression UGA4 by extracellular amino acids. Molecular mechanisms signaling cascade that is not fully understood but it is known that the SPS sensor complex is involved.
This complex is known as SPS16-18 for its three protein components (Ssy1p, Ptr3p and Ssy5p) and activated after the sensing of extracellular amino acids and possibly intracelulares19. Ssy1 is a membrane protein that resembles an amino acid permease but functions only as a sensor. Ssy5 ptr3 and interact with Ssy1 form a dynamic protein complex. It has been determined a large number of genes regulated by the activity of this sensor and many of these genes encode proteins involved in amino acid transport. Among them, AGP1 BAP2 and respond to amino acids via the SPS through Stp1 transcription factors, and Uga3520 STP2.

Stp1 and STP2 factors act in response to extracellular amino acid sensing on UASA elements present in promoters of permeases, such as BAP2 and BAP3. Importantly, the large sequence similarity between elements and UASGABA21 UASA, 22. STP2 Stp1 and are synthesized as latent factors and remain trapped in the cytoplasm through the amino terminal regulatory domain. Also three proteins in the inner nuclear membrane, ASI1, Asi3 ASI2 and participate in retaining and STP2 Stp1 latent in citoplasma23, 24. In response to amino acids, active Ssy1 Ssy5 protease that in turn cleaves the regulatory domain and STP2 Stp1, leading to activation of these factors. Their processed forms are transported to the nucleus where genes regulated transactivator SPS20 ,25-27.


Bibliografía:
1. De Virgilio, C. & Loewith, R. Cell growth control: little eukaryotes make big contributions. Oncogene 25, 6392-415 (2006).
2. Beck, T. & Hall, M. N. The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature 402, 689-92 (1999).
3. Bermudez Moretti, M., Correa Garcia, S., Stella, C., Ramos, E. & Batlle, A. M. Delta-aminolevulinic acid transport in Saccharomyces cerevisiae. Int J Biochem 25, 1917-24 (1993).
4. Bermudez Moretti, M., Correa Garcia, S., Ramos, E. & Batlle, A. delta-Aminolevulinic acid uptake is mediated by the gamma-aminobutyric acid-specific permease UGA4. Cell Mol Biol (Noisy-le-grand) 42, 519-23 (1996).
5. Andre, B., Hein, C., Grenson, M. & Jauniaux, J. C. Cloning and expression of the UGA4 gene coding for the inducible GABA-specific transport protein of Saccharomyces cerevisiae. Mol Gen Genet 237, 17-25 (1993).
6. Uemura, T., Tomonari, Y., Kashiwagi, K. & Igarashi, K. Uptake of GABA and putrescine by UGA4 on the vacuolar membrane in Saccharomyces cerevisiae. Biochem Biophys Res Commun 315, 1082-7 (2004).
7. Andre, B. The UGA3 gene regulating the GABA catabolic pathway in Saccharomyces cerevisiae codes for a putative zinc-finger protein acting on RNA amount. Mol Gen Genet 220, 269-76 (1990).
8. Andre, B. et al. Two mutually exclusive regulatory systems inhibit UASGATA, a cluster of 5'-GAT(A/T)A-3' upstream from the UGA4 gene of Saccharomyces cerevisiae. Nucleic Acids Res 23, 558-64 (1995).
9. Talibi, D., Grenson, M. & Andre, B. Cis- and trans-acting elements determining induction of the genes of the gamma-aminobutyrate (GABA) utilization pathway in Saccharomyces cerevisiae. Nucleic Acids Res 23, 550-7 (1995).
10. Vissers, S., Andre, B., Muyldermans, F. & Grenson, M. Positive and negative regulatory elements control the expression of the UGA4 gene coding for the inducible 4-aminobutyric-acid-specific permease in Saccharomyces cerevisiae. Eur J Biochem 181, 357-61 (1989).
11. Grenson, M. 4-Aminobutyric acid (GABA) uptake in Baker's yeast Saccharomyces cerevisiae is mediated by the general amino acid permease, the proline permease and a GABA specific permease integrated into the GABA-catabolic pathway. Life Sci Adv Biochem 6, 35-39 (1987).
12. Idicula, A. M., Blatch, G. L., Cooper, T. G. & Dorrington, R. A. Binding and activation by the zinc cluster transcription factors of Saccharomyces cerevisiae. Redefining the UASGABA and its interaction with Uga3p. J Biol Chem 277, 45977-83 (2002).
13. Magasanik, B. & Kaiser, C. A. Nitrogen regulation in Saccharomyces cerevisiae. Gene 290, 1-18 (2002).
14. Kulkarni, A., Buford, T. D., Rai, R. & Cooper, T. G. Differing responses of Gat1 and Gln3 phosphorylation and localization to rapamycin and methionine sulfoximine treatment in Saccharomyces cerevisiae. FEMS Yeast Res 6, 218-29 (2006).
15. Bermudez Moretti, M. et al. Evidence that 4-aminobutyric acid and 5-aminolevulinic acid share a common transport system into Saccharomyces cerevisiae. Int J Biochem Cell Biol 27, 169-73 (1995).
16. Didion, T., Regenberg, B., Jorgensen, M. U., Kielland-Brandt, M. C. & Andersen, H. A. The permease homologue Ssy1p controls the expression of amino acid and peptide transporter genes in Saccharomyces cerevisiae. Mol Microbiol 27, 643-50 (1998).
17. Iraqui, I. et al. Amino acid signaling in Saccharomyces cerevisiae: a permease-like sensor of external amino acids and F-Box protein Grr1p are required for transcriptional induction of the AGP1 gene, which encodes a broad-specificity amino acid permease. Mol Cell Biol 19, 989-1001 (1999).
18. Klasson, H., Fink, G. R. & Ljungdahl, P. O. Ssy1p and Ptr3p are plasma membrane components of a yeast system that senses extracellular amino acids. Mol Cell Biol 19, 5405-16 (1999).
19. Wu, B. et al. Competitive intra- and extracellular nutrient sensing by the transporter homologue Ssy1p. J Cell Biol 173, 327-31 (2006).
20. Abdel-Sater, F., Iraqui, I., Urrestarazu, A. & Andre, B. The external amino acid signaling pathway promotes activation of Stp1 and Uga35/Dal81 transcription factors for induction of the AGP1 gene in Saccharomyces cerevisiae. Genetics 166, 1727-39 (2004).
21. de Boer, M. et al. Stp1p, Stp2p and Abf1p are involved in regulation of expression of the amino acid transporter gene BAP3 of Saccharomyces cerevisiae. Nucleic Acids Res 28, 974-81 (2000).
22. Nielsen, P. S. et al. Transcriptional regulation of the Saccharomyces cerevisiae amino acid permease gene BAP2. Mol Gen Genet 264, 613-22 (2001).
23. Boban, M. & Ljungdahl, P. O. Dal81 enhances Stp1- and Stp2-dependent transcription necessitating negative modulation by inner nuclear membrane protein Asi1 in Saccharomyces cerevisiae. Genetics 176, 2087-97 (2007).
24. Boban, M. et al. Asi1 is an inner nuclear membrane protein that restricts promoter access of two latent transcription factors. J Cell Biol 173, 695-707 (2006).
25. Abdel-Sater, F., El Bakkoury, M., Urrestarazu, A., Vissers, S. & Andre, B. Amino acid signaling in yeast: casein kinase I and the Ssy5 endoprotease are key determinants of endoproteolytic activation of the membrane-bound Stp1 transcription factor. Mol Cell Biol 24, 9771-85 (2004).
26. Andreasson, C., Heessen, S. & Ljungdahl, P. O. Regulation of transcription factor latency by receptor-activated proteolysis. Genes Dev 20, 1563-8 (2006).
27. Andreasson, C. & Ljungdahl, P. O. The N-terminal regulatory domain of Stp1p is modular and, fused to an artificial transcription factor, confers full Ssy1p-Ptr3p-Ssy5p sensor control. Mol Cell Biol 24, 7503-13 (2004).