BIO 224 Laboratory
CSU, Sacramento
October 18 & 20, 2010
Lab Assignment 7
(due Monday, October 27th)
1) Using your assigned human protein/mRNA sequence, find one other
mammalian sequence in addition to your mouse & rat sequences (one outside of
the rodent family) and also two sequences from non-mammalian species. List all
the genes and accession numbers for both the mRNA (only mammals) and
protein sequence in a table (human, mouse, rat and the other three species).
2), Use the Clustal W (http://www.ebi.ac.uk/clustalw/ ) program to align the
mammalian mRNA and then their protein sequences separately (use the default
values). You will need to input each sequence as a fasta file and in consecutive
order. You will need to change the title of each sequence to one that you will
recognize in the output (meaning, don't use the NM_ ## or NP_## but rather a
short descriptive title like human ALB).
A) Cut and paste the protein alignment into this document (note: to get the
sequences to align properly, you need to change the text to Courier and use a
smaller font such as 8 or 9).
B) What was the best alignment score for the nucleotide and protein
alignments and between which sequences?
C) Which alignment do think more accurately reflects the proper sequence
alignment and why? (discuss both of the resulting alignments in your answer).
3) Now that you have observed the alignment of the mammalian homologues,
include the non-mammalian homologues and perform a ClustalW alignment
using the protein sequences.
A) Cut and paste this alignment into this document.
B) Compare the two alignments (protein alignment from question 2 and
3).
i)
Did the alignment change much with respect to divergent
and conserved regions? How so? (If not, then why do you
think it didn’t change much?)
ii)
Examine the gaps and aligned residues. For the gaps,
discuss why those gaps were likely placed there. For the
aligned residues, discuss the conserved and hypervariable
regions found within the alignment.
C) Copy your dendrogram for the guide tree for the protein alignment in
question 3 into this document.(this is the .dnd file or also at the bottom
of your alignment file you will see the guide tree and a button for "View
DND File) .
BIO 224 Laboratory
CSU, Sacramento
October 18 & 20, 2010
Then go to the following website:
(http://www.trex.uqam.ca/index.php?action=newick&project=trex) and
copy your dendrogram into the input box provided and click "View Tree".
Then format the tree by checking the box for "Proportional edge lengths".
Then right click on the figure and "Save Picture As" a bmp file extension
and import into this word document.
D) Interpret the guide tree and dendrogram. Describe the guide tree in
terms of the relationships of the genes. Does the guide tree reflect the
alignment order? (e.g. which genes are most closely related down to
the most divergent). Explain the dendrogram with respect to
branching order and lengths of branches (what do the numbers refer
to?). .
4) Reformat your ClustalW alignment generated in question 3 using the
Boxshade program (“Pretty Printing and Shading of Multiple-Alignment files”
Follow the steps below.
a)
First, go back and redo the alignment within the ClustalW
program but this time change the Output format for the
alignment to be “aln wo/ numbers” (means align without
numbers, this is so that the boxshade program can read the
alignment properly). Save the output alignment file (make sure it
has a “.aln” file extension)
b)
Open the Boxshade program
(http://www.ch.embnet.org/software/BOX_form.html ). Enter the
alignment into the program (make sure you copy the whole file
into the input box including the header). Change the input
sequence format to “ALN” rather than the MSF default. Chose
which type of output format you want (postscript is for adobe
acrobat or reader ; RTF or rich text format works well for word
documents). Run the program.
c)
Save the shaded output and paste it into the document.
d)
Explain the output with regards to the shading (discuss
conserved and hypervariable regions as discussed in question
3B above).