Bioinformatics Workshop - Assignment Example

Bioinformatics Workshop Question TH Write a short paragraph about the protein P53-human cellular tumor antigen andits function TP53 is known to be in increased amount in several transformed cells and it is frequently inactivated or mutated in approximately sixty percent of cancers. As a trans-activator, it plays a role in cell cycle regulation whereby it functions by negatively regulating cell division by controlling the required genes associated with cell division. Cyclin-dependent kinases as an inhibitor are known to be one of the activated genes. The expression of FAS and BAX antigens Stimulation mediates the apoptosis induction.LincRNA-p21 is known to be involved in TP53-dependent- transcriptional repression and this results to apoptosis as well as effecting regulation of cell –cycle.p53 is seen to be kept inactive in unstressed cells and in this cases its acted on by ubiquitin ligase whereby the transcription activity of p53 is inhibited as well as the its degradation is promoted by ubiquitinates p53.In human cancer the p53 activity is lost either through cell signaling loss or mutations in the p53 gene it self 2) GAPDH, ACTB, ACTA2, ALB, KRT16, CFTR genes in three different cell types (k562 Erythroid cells, HepG2 Liver cells and NHEK epidermal Keratinocytes (skin cells) you have to show for each gene where its made, what kind of protein encode and in which cell its active and where its repressive by reading the (H3 K4 me1, H3 K4 me2, H3 K4me3, H3 K9Ac, H3 K9me3, H3K27AC) GAPDH The gene encodes a member of glyceraldehydes-3-phosphate dehydrogenase protein family It is a membrane protein found in several intracellular locations and it’s active in all cell types (it is a house keeping gene) ACTA2 Chromosomal location: 10q23.31 It encodes actin, aortic smooth muscle Sub cellular location: cytoskeleton, cytoplasm Function: Actin are known to be highly conserved proteins involved in cell motility as well as ubiquitously expressed in all cells of eukaryotes ALB Gene Found in the liver cells It encodes a soluble monomeric protein referred to as albumin Function: The key plasma protein, it regulates the colloidal osmotic blood pressure. Sub-cellular location: it is secreted Disease: Dysalbuminemic hyperthyroxinemia (DH) KRT16 It encodes Keratin which is type I cytoskeletal Function: Intermediate filaments that are responsible for epithelial cells structural intergrety as well as are subdivided into hair keratins and cytokeratins Tissue specificity: They are known to be expressed in nail bed, hair follicle as well as in mucosal stratified squamous epithelia. They are as well in sweat luminal cells and the ducts of mammary glands Disease associated to the gene: Unilateral palmoplantar verrucous nervus, Keratoderma CFTR It encodes a member of ATP-binding cassette transporter super family Function: the channel of chloride as well as it controls the transport pathways regulation. Mutation leads to autosomal recessive known as cystic fibrosis. Sub cellular location: Cell membrane. Multi-pass membrane protein Tissue specificity: Located on the epithelial surfaces which line the lungs as well as other organs. Most of the above genes are not expressed in all tissues of the body apart from the GAPDH which is known to be a house keeping gene, hence expressed in all types of tissues. These genes that are not expressed in other types of tissues might have been modified through processes such as silencing. For instance, ALB gene is expressed in the liver cells but it’s not expressed in other cells. These modifications occur during cell differentiation 3) Comment on the different modifications found at each gene in the different cell types and suggest how these might relate to their expression status Differences between cells in eukaryotes are determined by expression of genes that are of different sets. For example, an eggs which is fertilized and undifferentiated appears and behaves in a different way when compared to the neuron, skin or a muscle cell (Callinan and Feinberg, 2006).This is due to different expression by the genes. This occurrence can be utilized in diagnosis as well as selecting appropriate treatment for cancer. It is well known that some genes are silenced in other tissues hence not expressed. There are several processes that are involved here such as imprinting that involves eukaryotic gene regulation. Here, there is silencing of the genes alleles for the rest of the life. This process affects few genes but there are several critical growth regulators involved. There is always silencing of mother copy in some genes .On the other hand, the paternal copy is ever silenced for various genes (Callinan and Feinberg, 2006). It is important to note that epigenetic research is very critical in terms of explaining the way cells that have identical DNA poses the capacity of differentiating into cells types that are different including the way the differentiated cellular state is maintained. Hence, epigenetic is considered to link phenotype and genotype. (Jaenisch, 2003).Epigenome is the term known to reflect the general cell’s epigenetic state. On the other hand, epigenetic is termed as the single genes or gene sets changes. Basing on these, it is very important to profile the Epigenome or modification patterns of epigenetic in a provided cell. In this case, the cell might be utilized as a biomarker of epigenetic for therapeutic development, diagnosis and clinical prediction DNA methylation is seen to be associated with transcriptional silencing, hence, its critical in the regulation of genes, tumorigenesis and development(Jaenisch, 20030).DNA methylation in mammals takes place in the cytosine ring at 5 prime position through methyl group addition. Cancer are known to have a characteristic that is unique whereby the chromatin structure is normally changed after methylation and this result to unexpected oncogenic activation as well as transposable elements. These modifications results to inactivation of the genes. Such epigenetic changes could act as oncogenic mechanism that is additional and it might contribute to tumor-genesis. Epigenetic Modifications that is as a result of dynamic process controlled by multiple processes as well as responsive to the stimuli of the environment could be inherited as well as remain stable. (Esteller, 2008).Thus, the complexity interaction among such markers is required to be made clear basing on varying conditions. The epigenetic modification distribution is not related o cell type or tissue specificity. Instead, they are as well linked to the areas where they are based. Hypomethylation of DNA occurs on the entire genome but on the other hand, Hypomethylation that is localized is seen to be on cancers cells TSGs (Feinberg and Tycko, 2004) Question 2): FS 1) About the gene a). From which species is the gene X sequence? The species is Mus musculus Evidence: Basing on the analysis, the sequence of gene x is 100% homology to the hit sequence of Mus musculus but for the other 4 species, their homology percentages are lower when compared to it. b). Which gene corresponds to gene X Sonic hedgehog gene c). Is there any evidence of alternative splicing for gene X? Yes there is evidence for alternative splicing basing on the above figure. It is observed that RNA gene that is involved in splicing is adjacent to gene X as indicated in the figure above. 2) About the evolution a) Diagram showing gene X flanked by other genes b) The gene arrangement is similar in humans and primates which are close relatives but not in some fish and invertebrates which are distant relatives. This means that arrangements of the genes on the genome differ relative to evolution of organisms. 3) About the gene family a) Can you identify the relative of gene x in the genome Indian hedgehog gene (Ihh) Desert hedgehog gene (Dhh) b) Can you find evidence from the neighboring genes to identify any of these relatives as true paraloguos of gene x The neighboring genes are not related to this relative genes to gene x and this confirms that the genes are true relative of gene x 4) About the regulation of the gene a) Where is the most conserved sequence around the transcription start of gene x located It is located at the transcription factor binding site downstream where transcription is initiated b) Which highly conserved transcription factor binding sites are found in that sequence? GFII CMAF CREB NFE2 NFKAPPAB50 c). Select one of the conserved binding sites and comment on the function of the transcription factors that may bind to it and thus may regulate the expression of gene X NFKAPPAB50 binding site NF-kB proteins consist of eukaryotic transcription factors that are related structurally and they are majorly involved in controlling of a large number of normal organisimal and cellular processes that include apoptosis, developmental processes, immune response, cellular growth etc (Beyaert , 2004). Additionally, the transcriptional factors are active persistently in several disease states that include arthritis, cancer, asthma, heart disease etc. NF-kB transcription factors consist of proteins that have conserved functions from fruit fly Drosophila melanogaster including human (Beyaert, 2004). Among the model organisms that are most studied, they are observed to be not present in yeast as well as nematode Caenorhabditis elegans (Beyaert , 2004). The reason why they are not present in yeast could be due to the transcription factors play a role controlling a wide range of physiological aspects of inflammatory and immune response. Primarily, NF-kB activity is seen to be regulated by the inhibitory IkB proteins and several of these proteins do exist and they varying affinities for NF-kB complexes (Gilmore, 2006). Question 3) : CS Annotated Diagram showing sequence of events that leads to RAR apha activation (Source; (Alsayed et al., 2001) The transcriptional trans-regulator, nuclear retinoic acid receptor alpha is known to control a subset of specific genes expression through binding at dynamic interactions and response elements with co-regulators that are ligand coordinated (Alsayed et al., 2001) The diagram (novel paradigm) above indicates the way RAR alpha target gene transcription is regulated through phosphorylation cascades that have been initiated through p38MAPK/MSK1 pathway activated rapidly by RA. It is seen that there is phosphorylation of RAR alpha by MSK1 at S369 found at the LBD. This enables the TFIIH to bind, hence, the N-terminal domain being phosphorylated at S77 by cdk7/cyclin H. The histon H3 is as well phosphorylated at S10.Then lastly; RAR alpha/TFIIH complex recruitment is controlled by MSK1 which initiates phosphorylation cascade. The p38MAPK that is deregulated can’t respond to RA, hence indicating the critical contribution of phosphorylation cascade that has been triggered in the RA signal by RA (Alsayed et al., 2001) Considering just RAR alpha, to what extent have the ligand binding domain phosphorylation and N-terminal domain phosphorylation sites been conserved during vertebrate evolution? It can be noted that the phosphorylation cascade is based on the domain flexibility increase involved in cyclin H binding (Bastien, and Rochette-Egly, 2004). This indicates a critical RAR alpha function in various species. In order to investigate if there is conservation of phosphorylation cascade, we need to consider both N-terminal domain and ligand binding domain (Altucci et al., 2005). For instance, a study was carried out in zebra fish (Danio rerio) to investigate if there was conserved phosphorylation cascade, whereby it expresses both RAR alpha A gene and RAR alpha B gene. The findings indicated that N terminal was conserved and phosphorylated while the ligand binding domain was absent for zebrafish RAR alphas (Bastien, and Rochette-Egly, 2004). After analyzing the conservation patterns of phosphorylation sites together with tracing back their evolution, it was observed that most ligand binding domain was frequently outside the RAR alpha of mammals and it emerges late during evolution of vertebrates (Altucci et al., 2005). On the other hand, N terminal domain was seen to be conserved, and this is an indication that functional site phosphorylation is considered to be under high selection constraint that is ancient. It can be suggested that various regulatory circuits do control the activity of RAR alpha during evolution (Bastien, and Rochette-Egly, 2004). Basing on the alignment results, they support the above study. The alignment indicated a conservation of RAR alpha sequence among all the species (vertebrates) compared. In this case, it implies that RAR alpha was conserved but the results never showed which domain was conserved. It can be concluded with support from the study that N terminal domain is highly conserved when compared to the ligand binding domain across the species Bibliography Alsayed Y, Uddin S, Mahmud N, Lekmine F, Kalvakolanu DV, Minucci S, Bokoch G, Platanias LC (2001) Activation of Rac1 and the p38 mitogen-activated protein kinase pathway in response to all-trans-retinoic acid. J Biol Chem 276: 4012–4019 Altucci L, Rossin A, Hirsch O, Nebbioso A, Vitoux D, Wilhelm E, Guidez F, De Simone M, Schiavone EM, Grimwade D, Zelent A, de The H, Gronemeyer H (2005) Rexinoid-triggered differentiation and tumor-selective apoptosis of acute myeloid leukemia by protein kinase A-mediated desubordination of retinoid X receptor. Cancer Res 65: 8754–8765 Bastien J, Rochette-Egly C (2004) Nuclear retinoid receptors and the transcription of retinoid-target genes. Gene 328: 1–16 Beyaert R (editor) (2004). Nuclear Factor-kappaB: Regulation and Role in Disease. Kluwer Academic Publishes, Dordrecht, The Netherlands. 426 pages Callinan P. and Feinberg A. P. (2006)Hum Mol Genet., 15, Spec No 1, R95-101 Esteller M. (2008) N Engl J Med, 358, 1148-59 Feinberg A. P. and Tycko B. (2004) Nat Rev Cancer, 4, 143-53 Gilmore TD (editor) (2006). NF-kB: from basic research to human disease. Oncogene (Reviews) 51: 6679-6899 Jaenisch R., Bird A. (2003)Nat Genet, 33 Suppl, 245-54 Read More