Pràctiques de Bioquímica

El document forma part dels materials docents programats mitjançant l'ajut del Servei de Política Lingüística de la Universitat de ValènciaDocument PDF amb diverses Pràctiques de Laboratori de Bioquímica General per a un Grau de Biologia, però també utilitzable en Graus afins com Bioquímica, Biomedicina i Biotecnologia. Conté les descripcions de 5 sessions pràctiques on es fan servir les tècniques bàsiques de bioquímica: valoració d’àcids febles i tampons, aïllament, purificació i caracterització de proteïnes i d’àcids nucleics, experiències amb cadena fotosintètica de cloroplasts i del metabolisme d’hidrats de carboni (fermentació i glicogen).PDF document with several Practices of Laboratory of General Biochemistry for a Degree of Biology, but also usable in Degrees related like Biochemistry, Biomedicine and Biotechnology. It contains the descriptions of 5 practical sessions where the basic techniques of biochemistry are used: assessment of weak acids and buffers, isolation, purification and characterization of proteins and nucleic acids, experiences with photosynthetic chain of chloroplasts and metabolism of carbohydrates (fermentation and glycogen)

Protein interactions within the Set1 complex and their roles in the regulation of histone 3 lysine 4 methylation

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5′ end of active coding regions but a decrease of H3K4 dimethylation at the 3′ end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3′ end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.Acciones Integradas Hispano-Francesas HF2003-0170Ministerio de Educación y Ciencia BFU2005-02603Ministerio de Ciencia y Tecnología BMC2003- 07072-C03-0

Autoavaluacions Bioquímica

Document PDF d’Autoavaluació sobre Bioquímica General per a un Grau de Biologia, però també utilitzable en Graus afins com Bioquímica, Biomedicina i Biotecnologia. Conté tant preguntes tipus test, com a textos amb buits per omplir amb paraules des d’un llistat i també conceptes a definir. Fan referencia a: proteïnes, enzimologia, àcids nucleic, bioenergètica i metabolisme.PDF document of Self-assessment on General Biochemistry for a Degree in Biology, but also usable in related Degrees such as Biochemistry, Biomedicine and Biotechnology. It contains both test-type questions, as well as texts with empty spaces to fill with words from a list and also concepts to be defined. They refer to: proteins, enzymology, nucleic acids, bioenergetics and metabolism

Material Docent: Bioquímica. Problemes i Qüestions. Grau Biologia

Document PDF amb Problemes i Qüestions de Bioquímica General per a un Grau de Biologia, però també utilitzable en Graus afins com Bioquímica, Biomedicina i Biotecnologia. Conté tant problemes com a qüestions de: solucions amortidores de pH, proteïnes, enzimologia, àcids nucleic, bioenergètica i metabolisme. Els problemes venen amb les solucions numèriques. Inclou annexes amb explicacions que faciliten la resolució de problemes de dissolucions amortidores de pH i també de enzimologia. Alguns problemes d’aquests amb el desenrotllament de la seua resolució.PDF document with Problems and Questions about General Biochemistry for a Degree in Biology, but also usable in associated Degrees such as Biochemistry, Biomedicine and Biotechnology. It contains both problems and questions: about buffers and pH, proteins, enzymology, nucleic acids, bioenergetics and metabolism. The problems come with the numerical solutions. It includes annexes with explanations that facilitate the resolution of problems about buffer solutions and also about enzymology. Some of these problems contain the development of its resolution

Data for the identification of proteins and post-translational modifications of proteins associated to histones H3 and H4 in S. cerevisiae, using tandem affinity purification coupled with mass spectrometry

Tandem affinity purification method (TAP) allows the efficient purification of native protein complexes which incorporate a target protein fused with the TAP tag. Purified multiprotein complexes can then be subjected to diverse types of proteomic analyses. Here we describe the data acquired after applying the TAP strategy on histones H3 and H4 coupled with mass spectrometry to identify associated proteins and protein post-translational modifications in the budding yeast, Saccharomyces cerevisiae. The mass spectrometry dataset described here consists of 14 files generated from four different analyses in a 5600 Triple TOF (Sciex) by information‐dependent acquisition (IDA) LC–MS/MS. The above files contain information about protein identification, protein relative abundance, and PTMs identification. The instrumental raw data from these files has been also uploaded to the ProteomeXchange Consortium via the PRIDE partner repository, with the dataset identifier PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002671 and http://dx.doi.org/10.6019/PXD002671. These data are discussed and interpreted in http://dx.doi.org/10.1016/j.jprot.2016.01.004. Valero et al. (2016) [1]. Keywords: Chromatin, Histones, Post-translational modifications, Proteomics, Tandem affinity purification, Yeas

Metabolisme i Regulació: protocols per al laboratori experimental

El document forma part del materials docents de l'assignatura obligatòria "Metabolisme i regulació" del Grau en Biotecnologia i han estat objecte de revisió mitjançant un ajut del Servei de Política Lingüística de la Universitat de València.L'assignatura del Grau en Biotecnologia de la Universitat de València "Metabolisme i regulació" inclou un part de pràctiques de laboratori. Aquest text inclou una introducció general amb protocols detallats de les pràctiques així com els qüestionaris que cal resoldre abans i després de realitzar els experiments.The subject of the Degree in Biotechnology of the University of Valencia "Metabolism and regulation" includes a part of laboratory practices. This text includes a general introduction with detailed protocols of the practices as well as the questionnaires that must be solved before and after the experiments

Comprehensive analysis of interacting proteins and genome-wide location studies of the Sas3-dependent NuA3 histone acetyltransferase complex

Histone acetylation affects several aspects of gene regulation, from chromatin remodelling to gene expression, by modulating the interplay between chromatin and key transcriptional regulators. The exact molecular mechanism underlying acetylation patterns and crosstalk with other epigenetic modifications requires further investigation. In budding yeast, these epigenetic markers are produced partly by histone acetyltransferase enzymes, which act as multi-protein complexes. The Sas3-dependent NuA3 complex has received less attention than other histone acetyltransferases (HAT), such as Gcn5-dependent complexes. Here, we report our analysis of Sas3p-interacting proteins using tandem affinity purification (TAP), coupled with mass spectrometry. This analysis revealed Pdp3p, a recently described component of NuA3, to be one of the most abundant Sas3p-interacting proteins. The PDP3 gene, was TAP-tagged and protein complex purification confirmed that Pdp3p co-purified with the NuA3 protein complex, histones, and several transcription-related and chromatin remodelling proteins. Our results also revealed that the protein complexes associated with Sas3p presented HAT activity even in the absence of Gcn5p and vice versa. We also provide evidence that Sas3p cannot substitute Gcn5p in acetylation of lysine 9 in histone H3 in vivo. Genome-wide occupancy of Sas3p using ChIP-on-chip tiled microarrays showed that Sas3p was located preferentially within the 5′-half of the coding regions of target genes, indicating its probable involvement in the transcriptional elongation process. Hence, this work further characterises the function and regulation of the NuA3 complex by identifying novel post-translational modifications in Pdp3p, additional Pdp3p-co-purifying chromatin regulatory proteins involved in chromatin-modifying complex dynamics and gene regulation, and a subset of genes whose transcriptional elongation is controlled by this complex

Unveiling novel interactions of histone chaperone Asf1 linked to TREX-2 factors Sus1 and Thp1

13 páginas, 7 figuras, 2 yablasAnti-silencing function 1 (Asf1) is a conserved key eukaryotic histone H3/H4 chaperone that participates in a variety of DNA and chromatin-related processes. These include the assembly and disassembly of histones H3 and H4 from chromatin during replication, transcription, and DNA repair. In addition, Asf1 is required for H3K56 acetylation activity dependent on histone acetyltransferase Rtt109. Thus, Asf1 impacts on many aspects of DNA metabolism. To gain insights into the functional links of Asf1 with other cellular machineries, we employed mass spectrometry coupled to tandem affinity purification (TAP) to investigate novel physical interactions of Asf1. Under different TAP-MS analysis conditions, we describe a new repertoire of Asf1 physical interactions and novel Asf1 post-translational modifications as ubiquitination, methylation and acetylation that open up new ways to regulate Asf1 functions. Asf1 co-purifies with several subunits of the TREX-2, SAGA complexes, and with nucleoporins Nup2, Nup60, and Nup57, which are all involved in transcription coupled to mRNA export in eukaryotes. Reciprocally, Thp1 and Sus1 interact with Asf1. Albeit mRNA export and GAL1 transcription are not affected in asf1Δ a strong genetic interaction exists between ASF1 and SUS1. Notably, supporting a functional link between Asf1 and TREX-2, both Sus1 and Thp1 affect the levels of Asf1-dependent histone H3K56 acetylation and histone H3 and H4 incorporation onto chromatin. Additionally, we provide evidence for a role of Asf1 in histone H2B ubiquitination. This work proposes a functional link between Asf1 and TREX-2 components in histone metabolism at the vicinity of the nuclear pore complex.This work has been supported by MINECO, Spain (BFU2011-23418) and by the GV (PROMETEO/2013/061 Valencian Regional Government) grants to S.R.-N. M.P. is funded by MICINN, Spain (BFU2008-01976), and the GV (ACOMP2011/057 Valencian Regional Government). P.O.-C. and E.G.-O. are holders of a MINECO FPI grant and CIPF PhD grant respectively.Peer reviewe

Structural Characterization of Set1 RNA Recognition Motifs and their Role in Histone H3 Lysine 4 Methylation

International audienceThe yeast Set1 histone H3 lysine 4 (H3K4) methyltransferase contains, in addition to its catalytic SET domain, a conserved RNA recognition motif (RRM1). We present here the crystal structure and the secondary structure assignment in solution of the Set1 RRM1. Although RRM1 has the expected betaalphabetabetaalphabeta RRM-fold, it lacks the typical RNA-binding features of these modules. RRM1 is not able to bind RNA by itself in vitro, but a construct combining RRM1 with a newly identified downstream RRM2 specifically binds RNA. In vivo, H3K4 methylation is not affected by a point mutation in RRM2 that preserves Set1 stability but affects RNA binding in vitro. In contrast mutating RRM1 destabilizes Set1 and leads to an increase of dimethylation of H3K4 at the 5'-coding region of active genes at the expense of trimethylation, whereas both, dimethylation decreases at the 3'-coding region. Taken together, our results suggest that Set1 RRMs bind RNA, but Set1 RNA-binding activity is not linked to H3K4 methylation