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A new procedure for determining the genetic basis of a physiological process in a non-model species, illustrated by cold induced angiogenesis in the carp PhD dinner seminars John Herbert
Why angiogenesis due to cold temperature? It is more difficult for muscles to utilise O 2 at lower temperatures
What Evidence? Cold induced angiogenesis has been shown in mammals and vertebrates 25 o C 5 o C
In the non-model species carp (no genome and little transcriptome ~20K EST sequences), ortholog relationships and microarray re-analyses were used to predict identity of cold induced angiogenic genes
Functionally linked together by orthologs Basic strategy overview What is an ortholog and how to create this link with 20K anonymous carp DNA sequences Model species Angiogenic genes Non-model species Cold induced genes
BLASTO illustrates limitations with public available software Zhou Y, Landweber LF: BLASTO: a tool for searching orthologous groups . Nucleic Acids Res 2007, 35 (Web Server issue):W678-682 Database from 2007 Singular, just one sequence Limited filtering options
Altenhoff AM, Dessimoz C: Phylogenetic and functional assessment of orthologs inference projects and methods . PLoS Comput Biol 2009, 5 (1):e1000262. bidirectional best-hits (BBH)
Another surprise is the good overall performance of the simple Bidirectional Best Hit approach. Although the method is restricted to 1:1 orthologs, the derived relations show good comparative accuracy in terms of Fitch's definition. Orthologs predicted by BBH also show close functional relatedness.
Reciprocal Best Hit (RBH): Ortholog assignment using BLAST Human Carp
How to improve on this? Zebrafish Another model species Genome is almost complete Currently 6 million ESTs 28 thousand Refseq proteins Carp……….only…. 20K ESTs Diverged 50 MYA Use zebrafish as stepping stone (SRBH)
Gracey et al. 2004: Coping with cold: An integrative, multitissue analysis of the transcriptome of a poikilothermic vertebrate. 17,825 sequences on chip This data set proved appropriate for meta-analysis to predict cold-induced angiogenic genes in skeletal and heart muscle tissues. Cold induced genes
Carp gene expression cooling regimen B) 57 genes up-regulated on initial cooling 567 total non-redundant genes up-regulated A) 547 persistently up-regulated genes
Identifying physiologically relevant genes in human
1) In-house method involved collecting genes based on the authors own angiogenesis research, combined with genes on the SABiosciences commercial angiogenic array and those listed in other literature.
2) Gene data was downloaded from Genbank: Perl programs, in conjunction with PubMed, were used to find article abstracts that contained angiogenesis keywords
3) Gene Ontology process data searched for the same keywords
4) Endothelial transcriptomes (Wallgard et al. 2008 and our list) were cross-referenced
From 3,762 genes found CSRBH, 908 were associated with endothelium or angiogenesis