Multiple alignments and phylogeny

Re-examining Globin Gene Phylogeny

Print out your molecular phylogeny of [3-globin sequences as a phylogram and produce a similar one for the human globin genes, for which the file can also be found in the VLE Resources.

Explain in less than 500 words what the phylogenies tell us about the history of the gene cluster (you will need to do some research for this and include references).

Over twenty years ago, Thomas Kocher with colleagues in Alan Wilson's group published an important paper in the Proceedings of the National Academy of Sciences of the USA, entitled 'Dynamics of mitochondrial DNA evolution in animals: Amplification and sequencing with conserved primers' (Proc. Natl. Acad. Sci. USA 86: 6196¬6200, 1989). Go the PNAS website (http://www.pnas.org/), search for the paper, and download it.

Wilson's group was one of the pioneers of the polymerase chain reaction (PCR) and its use in evolutionary studies. This particular paper has been extremely useful for studies of animal evolution based on mitochondrial DNA (mtDNA).

The paper reports 15 mtDNA cytochrome b sequences. Retrieve these sequences using the NCBI database and align them with Clustal

Use Clustal to estimate a neighbour-joining (NJ) tree of the 15 sequences. Check the topology (that is, the branching structure) of the tree as a Cladogram then as a Phylogram. Print it out.

How is your tree similar or different from the three trees presented by Kocher et al. (1989)? If they differ at all, why might this be? (Check the legend to Figure 3.)

Retrieving the sequences

• Retrieve the sequences from the NCBI database: http://www.ncbi.nlm.nih.gov/

• Remember to search under 'nucleotide', and save them into a text file as FASTA format, i.e.: >DipodomysRltcagccctat ttctagctat acattataca ccagacaccc tcacggcatt ttcatcagtt... etc.

• Retrieve, in addition, a lamprey (Lampetra similis) cytochrome b sequence to use as an outgroup.

• Make one text file with all the FASTA sequences, separated by blank lines.

Gene Phylogeny Analysis of Tyrosine Hydroxylase

Tyrosine hydroxylase is the key enzyme for synthesis of dopamine and norepinephrine in neurones. Hence it is found in all animals with some form of brain. We will examine its phylogeny and investigate its orgins. Using NCBI, search the protein database for eukaryotic tyrosine hydroxylases. Make a list of 25 different species with a known tyrosine hydroxylase by reading through entries.

Select a set of ten from your search with as wide an evolutionary spectrum as possible based on the phylogeny shown next to the sequences (hint: right side of page). The spectrum should include mammals, mollusca, Arthropoda, and chordates.

Build a CLUSTAL alignment, find the optimal phylogenetic gene tree (which clustering method?) and print the dendogram. How does your tyrosine hydroxylase gene tree compare with published evolutionary trees for the included organisms found in the literature/textbooks?

Tyrosine hydroxylase is a member of a family of related proteins. Describe the family and it's characteristics. Download the protein sequences of members of this family from human and rat. Based on multiple alignments and phylogenetic analysis, describe the evolution of tyrosine hydroxylase. For example, you will need to determine whether there is stronger conservation between members of this family within an organism or within tyrosine hydroxylases. A few paragraphs are expected to explain and justify your results and their comparison to published literature. References should be cited. Further credit will be given for other appropriate choices of organisms to support your argument.

Attachment:- Assignment.zip