5) BLAST can be used to determine which amino acids have mutated between these t

Question

5) BLAST can be used to determine which amino acids have mutated between these two strains of the virus. Perform another BLAST, but this time select “align two or more sequences.” Use the original GP protein ID “AHX24649.2” and compare it to the homologous sequence found in question 4. Look at the amino acid alignment: which amino acid(s) were mutated in the new virus isolate? If any are present, you will see blank spots or mismatches in the overlaying sequence alignments. (5 points)

6) Uniprot is an extremely useful protein analysis database. Search Uniprot for “Ebolavirus glycoprotein” and click on the top link that matches the amino acid size you determined in question 3. What is the role of this protein in Ebola infection? Based on your findings, which cells are infected by Ebola? (5 points)

7) On the same page, scroll down to “Structure” and under “3D Structure Databases” select “RCSB PDB” then click “3CSY” to the right. On the next page, click “3D view.” Alternatively, use this link: (http://www.pdb.org/pdb/explore/explore.do?pdbId=3CSY). You may have to update or enable Java to view the model. What is this super cool 3D model you are viewing? (10 points)

8) In order to study the cellular entry mechanism of Ebola in your lab, you wish to clone the native sequence of the GP gene found in question #2 into the plasmid pUC19. In order to clone the gene into pUC19, you decide to use PCR to add restriction enzyme sites at its 5’ and 3’ ends. Use NEB cutter version 2 (http://tools.neb.com/NEBcutter2/) and look under “standard sequences” to view available restriction enzyme sites in pUC19. Select “linear display” to make it easier to see all of the restriction sites. Find the section of pUC19 with a large number of restriction sites – this is the polylinker region specially designed to have many restriction sites for cloning.

You can also view a pUC19 map here: http://bio.classes.ucsc.edu/bio20L/info/content/molbio2/puc19.gif

Select two enzymes that you will use to clone your fragment. Why did you choose these enzymes? (10 points)

9) What would the size of the recombinant plasmid be that you will obtain if you did successfully clone the nucleotide sequence of the Ebola glycoprotein from question 2 into pUC19? Explain. (10 points)

10) Using NEB cutter, do a custom digest of pUC19 with enzymes 1 and 2 (from question 8). Copy and paste the virtual map into your PLQ (10 points) as well as the in silico gel electrophoresis results (10 points) which includes the sizes of the fragments.

Explanation / Answer

1. The organism is Zaire Ebola Virus, The genome size is 18,959 bp cRNA

The protein is minor nucleoprotein, The gene symbol is VP30.

It is isolated from blood of rhesus monkey, reported to cause infection

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