Arylformamidase Sequence & Homology: Difference between revisions

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A similar BLAST search was performed using the human homologue to our query sequence. 126 of the top scoring matches were selected for a multiple sequence alignment. This was the minimum number of sequences which would also include the query sequence. The sequences were aligned, bootstrapped and a tree created as above. The tree revealed some questionable matches, joining humans with pufferfish for instance, which, whilst evolutionarily interesting poses more questions than answers.
A similar BLAST search was performed using the human homologue to our query sequence. 126 of the top scoring matches were selected for a multiple sequence alignment. This was the minimum number of sequences which would also include the query sequence. The sequences were aligned, bootstrapped and a tree created as above. The tree revealed some questionable matches, joining humans with pufferfish for instance, which, whilst evolutionarily interesting poses more questions than answers.
Top scoring sequences from the results of the BLAST search using the human homologue were appended to the original top scoring sequences of the results BLAST search on the bacterial query sequence.
As above, using CLUSTAL X, a multiple sequence alignment was generated, the data was then bootstrapped 1000 times and a phylogenetic tree generated using the neighbour-joining algorythm (Figure 2).
== 2. Results ==
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).




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Top scoring sequences from the results of the BLAST search using the human homologue were appended to the original top scoring sequences of the results BLAST search on the bacterial query sequence.  
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).


As above, using CLUSTAL X, a multiple sequence alignment was generated, the data was then bootstrapped 1000 times and a phylogenetic tree generated using the neighbour-joining algorythm (Figure 2).


'''Figure 2.'''
'''Figure 2.'''
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''Unrooted phylogenetic tree of highest scoring results from a BLAST search of bacterial sequences and highest scoring results of a BLAST search on a homologous human sequence. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).''
''Unrooted phylogenetic tree of highest scoring results from a BLAST search of bacterial sequences and highest scoring results of a BLAST search on a homologous human sequence. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).''


== 2. Results ==
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).


The alignment revealed several conserved regions accross all species, thereby indicating a high level of conservation from Bacteria through Eukaryota. Most significantly, the catalytic triad of 137S, 215E/D and 242H are conserved accross all species of prokaryotes and eukaryotes. These included vertebrates, invertebrates, yeasts, moulds and single-celled eukaryotes. The catalytic triad is thought to be involved in carboxylesterase activity.
The alignment revealed several conserved regions accross all species, thereby indicating a high level of conservation from Bacteria through Eukaryota. Most significantly, the catalytic triad of 137S, 215E/D and 242H are conserved accross all species of prokaryotes and eukaryotes. These included vertebrates, invertebrates, yeasts, moulds and single-celled eukaryotes. The catalytic triad is thought to be involved in carboxylesterase activity.

Revision as of 07:19, 3 June 2008

Our query sequence "Arylformamidase" is a putatuve thioesterase isloated from a Silicibacter sp. These organisms are best known for their ability to degrade sulfur compounds in the marine environment. The query sequence sequence Target 13, pdb:2pbl is 262 residues in length.

1. Method

Using the query sequence "Arylformamidase", a BLAST search was performed on the bacterial protein sequence Arylformamidase using a non-redundant database. The top scoring matches to an E-value of 3e-054, 35 sequences in total, were selected. Eukaryotic homologous sequences sequences were found using NCBI HomoloGene. These were appended to the list and a multple sequece alignment was performed using CLUSTAL X.

The data output from the multiple sequence alignment was bootstrapped 1000 times and a phylogenetic tree was created using the neighbour-joining algorythm. The program FigTree was used to create the visual representation of this tree(Figure 1).

A similar BLAST search was performed using the human homologue to our query sequence. 126 of the top scoring matches were selected for a multiple sequence alignment. This was the minimum number of sequences which would also include the query sequence. The sequences were aligned, bootstrapped and a tree created as above. The tree revealed some questionable matches, joining humans with pufferfish for instance, which, whilst evolutionarily interesting poses more questions than answers.

Top scoring sequences from the results of the BLAST search using the human homologue were appended to the original top scoring sequences of the results BLAST search on the bacterial query sequence.

As above, using CLUSTAL X, a multiple sequence alignment was generated, the data was then bootstrapped 1000 times and a phylogenetic tree generated using the neighbour-joining algorythm (Figure 2).


2. Results

Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).


Figure 1.

NewBOOT1000tree.png

Unrooted phylogenetic tree of highest scoring results from a BLAST search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from HOMOLOGENE. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).


To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).


Figure 2.

BacterANDhomoTREE.jpg

Unrooted phylogenetic tree of highest scoring results from a BLAST search of bacterial sequences and highest scoring results of a BLAST search on a homologous human sequence. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).


The alignment revealed several conserved regions accross all species, thereby indicating a high level of conservation from Bacteria through Eukaryota. Most significantly, the catalytic triad of 137S, 215E/D and 242H are conserved accross all species of prokaryotes and eukaryotes. These included vertebrates, invertebrates, yeasts, moulds and single-celled eukaryotes. The catalytic triad is thought to be involved in carboxylesterase activity.

Based on the phylogenetic information provided in Figure 2, we can see that the Silicibacter species from which our sequence originated is one of ancient lineage; the homologues of this protein are strongly conserved accross prokaryotic and eukaryotic species. The species coloured green within this tree are all gram-negative bacterial sequences which, by their nature may be pathogenic and partially responsible for horizontal transfer of the Arylformamidase gene.

References

NCBI HomoloGene: Arylformamidase

CLUSTAL Homepage

Fig Tree

MicrobeWiki: Silicibacter pomeroyi


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