Characterisation of two genes for guanylate cyclase activator protein (GCAP1 and GCAP2) in the Japanese pufferfish, Fugu rubripes.
cDNA and genomic clones encoding guanylate cyclase activating proteins (GCAP1 and GCAP2) in the Japanese puffer fish (Fugu rubripes) were identified by probing, respectively, a retinal cDNA library and a whole genomic cosmid library with human GCAP1 and GCAP2 cDNA probes. Clones were identified as GCAP1 and GCAP2 on the basis of amino acid identity with the equivalent frog sequences and their placement into GCAP1 and GCAP2 clades within a GCAP phylogenetic tree. The Fugu genes have an identical four exon/three intron structure to GCAP1 and GCAP2 genes from other vertebrates but the introns are smaller, with the result that the four exons spread over approximately 1 kb of DNA in each case. The two genes are separated on to separate cosmids. However, the results of Southern analysis of the cosmids and of genomic DNA are consistent with a tail-to-tail gene arrangement, as in other species, but with a surprisingly large intergenic separation of around 18.7 kb. Recombinant Fugu GCAP1 failed to activate human retinal guanylate cyclase (retGC) in vitro although CD spectroscopy shows that the protein is folded with a similar secondary structure to that of human GCAP1. The failure to activate may be due therefore to a lack of molecular compatibility in this heterologous assay system.
Wilkie
Susan E
SE
Division of Molecular Genetics, Institute of Ophthalmology, University College London, Bath Street, London EC1V 9EL, UK.
Stinton
Inez
I
Cottrill
Phillippa
P
Deery
Evelyne
E
Newbold
Richard
R
Warren
Martin J
MJ
Bhattacharya
Shomi S
SS
Hunt
David M
DM
D000595:Amino Acid Sequence; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D005796:Genes; D051604:Guanylate Cyclase-Activating Proteins; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D012160:Retina / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D023741:Takifugu / Q000235:genetics*
D002135:Calcium-Binding Proteins; C496736:GUCA1A protein, human; C496739:GUCA1B protein, human; D051604:Guanylate Cyclase-Activating Proteins; D011994:Recombinant Proteins