Materials and Methods 5

Phylogeny

1PUJA FASTA sequence was obtained from NCBI Entrez Protein. This was used as a query to BLASTP the non-redundant protein database. Sequence matches were selected for multiple-sequence alignment (MSA) on the basis of presence in model organisms representative of the three major kingdoms of life (bacteria, archaea and eukaryotic) (Hedges, S. 2002). MSA was performed using CLUSTAL X (version 1.83), sequences were removed if they did not contain the N-terminal NKxD motif. N-terminal and C-terminal ends of sequences were trimmed downed so as only regions of conservation were aligned. The phylogenetic tree was constructed using the PROT-DIST (distance matrix construction), NEIGHBOUR (neighbour-joining method of tree construction), and CONSENSE programs of the PHYLIP package. Confidence in tree branches was determined by bootstrapping (100 resamplings of the PHYLIP analysis).

Function

To determine the function of 1pujA a variety of data-mining strategies and tools were utilized. First a literature search was conducted using Google Scholar and PubMed to familiarize ourselves with our protein and to find any knowledge or additional information already in databases and published literature. From this point on we predicted a possible function for our protein and using proceeded to test our hypothesis. This was done using the ProKnow and the Profunc web servers. ProKnow is a relatively simple database which simply determines the likely molecular function and biological process of a sequence. ProFunc is a server which was developed to help identify the likely function of a protein from its three-dimensional structure. It uses both sequence and structure based methods to try to provide clues as to the protein's likely or possible function (REF= http://www.ebi.ac.uk/thornton-srv/databases/ProFunc/). Of all the ProFunc searches Interpro, PDB, SSM and DALI and 3D functional template searches all returned useful data however the most promising results came from UniProt. UniProt is a tool for performing a multiple sequence alignment on a submitted sequence. It was also important to investigate the function of any orthologs of our protein. Mouse and human sequences were analysed with a server called SymAtlas, useful for determining the expression levels of a sequence in different tissues.

To determine the function of 1pujA (and related YlqF proteins) a variety of data-mining strategies and computational tools were utilized. First a literature search was conducted using Google Scholar and PubMed to familiarize ourselves with YlqF proteins and to find any knowledge or additional information already in databases and published literature. From this point on we predicted a possible function for our protein and using this proceeded to test our hypothesis. This was done using the ProKnow and the Profunc web servers. ProKnow is a relatively simple database which determines the likely molecular function and biological process of a queried sequence. ProFunc is a server which was developed to help identify the likely function of a protein from its three-dimensional structure. It uses both sequence and structure based methods to try to provide evidence as to a protein's likely or possible function (REF= http://www.ebi.ac.uk/thornton-srv/databases/ProFunc/). UniProt is a tool for performing a multiple sequence alignment on a submitted sequence. It was also important to investigate the function of any suspected orthologs or homologs of our protein. Mouse and human sequences were analysed with a server called SymAtlas, useful for determining the expression levels of a sequence in different tissues.