Beginners Challenge

Answer all questions. Support your answers with screenshots and code where appropriate

  1. The process of glycolysis is important for generating the energy requirements of organisms.
    • Using bioinformatics pathway tools, display the full glycolytic pathway
    • What enzyme is responsible for phosphorylation of glucose to glucose-6-phosphate?
    • Identify the gene that codes for this enzyme, giving its alternate names
    • On which chromosome is the gene identified above found? Give its exact chromosomal location
    • Are there any SNPs associated with the gene? What Mendelian conditions, if any, can occur due to these SNPs?
    • Use NCBI tools to generate the fasta nucleotide sequence of the gene. How long is the nucleotide?
    • Use BLASTN to identify other hits similar to the gene. Why have we used BLASTN? What other types of BLAST tools do you know of? Comment on the E values obtained
    • Generate a fasta file containing the nucleotide sequence of our gene of interest and include sequences from the following organisms: Mus musculus, rattus norvegicus, bos Taurus, Pongo albeii, Neovisions visons, Xenopus laevis, Sus scrufa.
    • Perform a multiple sequence alignment using the fasta file above in CLUSTALW
    • Generate a phylogenetic tree of the aligned sequences using MEGA software and a suitable algorithm. Comment on the method used and the tree obtained, mentioning details about CpG islands, parsim informative sites, and branch lengths
    • Design a suitable primer for use in amplification of the enzyme. Comment on the suitability of this primer, mentioning the annealing temperature (Tm), GC clamps, GC content, and length in bp
    • The Wallace formula is used to compute primer Tm and is expressed as follows: Tm=2(A+T)+4(G+C) Write a web-based program containing a text-area and a submit button which allows a user to paste in a sequence and calculates the Tm. Use Perl and HTML.
    • Is the enzyme identified a suitable target for developing drugs against T. brucei? Give reasons
  2. Using NCBI tools, get the nucleotide sequence of the gene coding for the MnSOD protein in humans
    • What is the name of this gene?
    • How many transcript variants of the gene can you locate?
    • What are transcript variants and by which mechanism do they arise?
    • Comment on the length of the gene in bp
    • What are homologs, orthologs, and paralogs?
    • Download a fasta file containing 9 different homologs of the gene and name it mnsod.fasta
    • Using an appropriate phylogenetic tree, infer the evolutionary relatedness of the different organisms
    • Identify CpG islands and comment on methylation of the gene
  3. Using the GEO2R tool, identify at least 5 proteins that potentially interact with the MnSOD enzyme in humans
    • Validate your observations using the SMART Database
  4. Write a perl script that:
    • Determine the length of the human MnSOD ortholog
    • Specifies the start/stop sites
    • Prints out the length of the nucleotide sequence
    • Gives the complementary sequence of the nucleotide
    • Cuts the nucleotide sequence into two
    • Adds a CpG island at the end of any of the cut sequence
    • Translates any of the cut sequences into a protein
    • Locates promoter sequences
  5. Using a suitable perl command, read the data of the mnsod.fasta file onto your screen.
    • Create an array and insert the sequences in the fasta file into the array
    • Get the nucleotide sequence of Scedosporium boydii gene coding for the mnsod and append it at the end of the mnsod.fasta file using perl
    • Output the contents of the human MnSOD gene into a new text file using perl
    • Align the sequences in the fasta file
    • Using CLUSTALW, draw a phylogenetic tree and comment on the parsimony of the sequences.
  6. MnSOD catalyzes the dismutation of superoxide radical, a process that helps protect cells from cellular damage.
    • Upregulation of MnSOD expression is mediated by a number of transcription factors, most notably via the AMPK-CREB/Nrf2 pathway. What is the optimal solution for assessing the interaction between the transcription factors and SOD2 promoter sites in silico?
    • Is MnSOD a suitable target for anti-atherosclerotic drugs? Why or why not?
    • Cytoprotective drugs such as celecoxib may act in part by driving the nuclear translocation of Nrf2. Specify an in silico drug design strategy for the discovery of novel enhancers of Nrf2 nuclear translocation.
    • Using Perl, demonstrate the presence of EcoRII restriction sites, transposition sites, promoter sites, and start and stop codons in SOD2
    • Based on your findings above, is the Sleeping Beauty (SB) system a suitable vehicle for delivering therapeutic payloads to mutated SOD2?
  7. We want to clone the human hemeoxygenase enzyme and insert the sequence ACCGCTTA about a restriction site of your choice.
    • With the aid of a perl script, predict the feasibility of the protein encoded by the recombinant gene, mentioning its stability, ease of extraction, absorption profile in spectrophotometric analysis, among others.
  8. Is the sequence ACCTAGGT palindromic?
    • Justify your answer with the aid of a perl script, including the pseudocode for the script
    • Are there any restriction sites for Taq1 endonuclease in the human UCP2 gene? Justify your answer with the aid of a perl script
  9. Create an array containing orthologs of the TGF-β
    • Create another array containing orthologs of another anti-inflammatory cytokine
    • Create an array that contains elements of the two arrays above

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