The rad1 gene in Rainbow Trout (Oncorhynchus mykiss) is highly conserved and may express proteins from non-canonical spliced isoforms

• Published in: Comparative biochemistry and physiology. Toxicology & pharmacology 2013-01, Vol.157 (1), p.16-23
• Main Authors: Bozdarov, Johny, Sherry, James P., Duncker, Bernard P., Bols, Niels C., Dixon, Brian
• Format: Article
• Language: English
• Subjects: 9–1–1 complex; Alternate splicing; Alternative Splicing; Amino Acid Sequence; Animals; Antibody; Blotting, Western; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cell Line; Checkpoint protein; Cloning, Molecular; Conserved Sequence - genetics; Fish Proteins - classification; Fish Proteins - genetics; Fish Proteins - metabolism; Gene Expression Profiling; Gills - enzymology; Gills - metabolism; Molecular Sequence Data; Myocardium - enzymology; Myocardium - metabolism; Oncorhynchus mykiss - genetics; Oncorhynchus mykiss - metabolism; Phylogeny; Protein Isoforms - genetics; Protein Isoforms - metabolism; rad1; Rainbow trout; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Spleen - enzymology; Spleen - metabolism
• ISSN: 1532-0456; 1878-1659
• DOI: 10.1016/j.cbpc.2012.09.002

Cell-cycle checkpoint proteins maintain genomic integrity by sensing damaged DNA and initiating DNA repair or apoptosis. RAD1 is a checkpoint protein involved in the sensing of damaged DNA and is a part of the 9-1-1 complex. In this project rainbow trout rad1 (rtrad1) was cloned, sequenced, expressed as a recombinant protein and anti-rtRAD1 antibodies were developed. RAD1 protein levels were characterized in various rainbow trout tissues. It was determined that an 840bp open-reading frame encodes 279 aa with a predicted protein size of 31kDa. The rtRAD1 amino-acid sequence is highly conserved and contains conserved exonuclease and leucine zipper domains. RT-PCR was used to identify three non-canonical splice variants of rtrad1, two of which are capable of forming functional proteins. The rad1 splice variant that encodes an 18kDa protein appears to be abundant in rainbow trout spleen, heart and gill tissue and in the RTgill-W1 cell-line. Based on the genomic rtrad1 sequence the splice variants contain only partial exons which are consistent with the splicing of rad1 variants in mammals. This is the first time that rad1 has been fully characterized in a fish species.