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

<p>Abstract</p> <p>Background</p> <p>Physiological processes occur in many species for which there is yet no sequenced genome and for which we would like to identify the genetic basis. For example, some species increase their vascular network to minimise the effects of reduced oxygen diffusion and increased blood viscosity associated with low temperatures. Since many angiogenic and endothelial genes have been discovered in man, functional homolog relationships between carp, zebrafish and human were used to predict the genetic basis of cold-induced angiogenesis in <it>Cyprinus Carpio </it>(carp). In this work, carp sequences were collected and built into contigs. Human-carp functional homolog relationships were derived via zebrafish using a new Conditional Stepped Reciprocal Best Hit (CSRBH) protocol. Data sources including publications, Gene Ontology and cDNA libraries were then used to predict the identity of known or potential angiogenic genes. Finally, re-analyses of cold carp microarray data identified carp genes up-regulated in response to low temperatures in heart and muscle.</p> <p>Results</p> <p>The CSRBH approach outperformed all other methods and attained 8,726 carp to human functional homolog relationships for 16,650 contiguous sequences. This represented 3,762 non-redundant genes and 908 of them were predicted to have a role in angiogenesis. The total number of up-regulated differentially expressed genes was 698 and 171 of them were putatively angiogenic. Of these, 5 genes representing the functional homologs NCL, RHOA, MMP9, GRN and MAPK1 are angiogenesis-related genes expressed in response to low temperature.</p> <p>Conclusion</p> <p>We show that CSRBH functional homologs relationships and re-analyses of gene expression data can be combined in a non-model species to predict genes of biological interest before a genome sequence is fully available. Programs to run these analyses locally are available from <url>http://www.cbrg.ox.ac.uk/~jherbert/</url>.</p

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

Abstract Background Physiological processes occur in many species for which there is yet no sequenced genome and for which we would like to identify the genetic basis. For example, some species increase their vascular network to minimise the effects of reduced oxygen diffusion and increased blood viscosity associated with low temperatures. Since many angiogenic and endothelial genes have been discovered in man, functional homolog relationships between carp, zebrafish and human were used to predict the genetic basis of cold-induced angiogenesis in Cyprinus Carpio (carp). In this work, carp sequences were collected and built into contigs. Human-carp functional homolog relationships were derived via zebrafish using a new Conditional Stepped Reciprocal Best Hit (CSRBH) protocol. Data sources including publications, Gene Ontology and cDNA libraries were then used to predict the identity of known or potential angiogenic genes. Finally, re-analyses of cold carp microarray data identified carp genes up-regulated in response to low temperatures in heart and muscle. Results The CSRBH approach outperformed all other methods and attained 8,726 carp to human functional homolog relationships for 16,650 contiguous sequences. This represented 3,762 non-redundant genes and 908 of them were predicted to have a role in angiogenesis. The total number of up-regulated differentially expressed genes was 698 and 171 of them were putatively angiogenic. Of these, 5 genes representing the functional homologs NCL, RHOA, MMP9, GRN and MAPK1 are angiogenesis-related genes expressed in response to low temperature. Conclusion We show that CSRBH functional homologs relationships and re-analyses of gene expression data can be combined in a non-model species to predict genes of biological interest before a genome sequence is fully available. Programs to run these analyses locally are available from http://www.cbrg.ox.ac.uk/~jherbert/.</p

Association of Csk to VE-cadherin and inhibition of cell proliferation

Vascular endothelial cadherin (VE-cadherin) mediates contact inhibition of cell growth in quiescent endothelial cell layers. Searching for proteins that could be involved in VE-cadherin signaling, we found the cytosolic C-terminal Src kinase (Csk), a negative regulator of Src family kinases. We show that Csk binds via its SH2 domain to the phosphorylated tyrosine 685 of VE-cadherin. VE-cadherin recruits Csk to cell contacts and both proteins can be co-precipitated from cell lysates of transfected cells and endothelial cells. Association of VE-cadherin and Csk in endothelial cells increased with increasing cell density. CHO cells expressing the tyrosine replacement mutant VE-cadherin-Y685F grow to higher cell densities than cells expressing wild-type VE-cadherin. Overexpression of Csk in these cells under an inducible promoter inhibits cell proliferation in the presence and absence of VE-cadherin, but not in the presence of VE-cadherin-Y685F. Reduction of Csk expression by RNA interference enhances endothelial cell proliferation. Our results suggest that the phosphorylated tyrosine residue 685 of VE-cadherin and probably the binding of Csk to this site are involved in inhibition of cell growth triggered by cell density