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Sequence, expression and genetic mapping of a rainbow trout retinoblastoma cDNA
1Department of Zoology, Washington State University, Pullman, WA 99164-4236, USA.
Abstract:
A full-length cDNA for retinoblastoma (RB1) has been cloned from a cDNA library prepared from 3-week-old rainbow trout (Oncorhynchus mykiss) eyed embryos. The trout RB1 cDNA encodes a predicted protein of 910 amino acids and is the most divergent cloned retinoblastoma gene sequence to date. RT-PCR studies reveal high levels of RB1 expression by the second week of embryogenesis, which remains uniformly expressed until hatching. Expression studies of adult fish tissues show the RB1 gene to be expressed in all tissues examined, including the oesophagus, eye, liver, intestine, posterior and anterior kidney, skin, stomach, muscle, spleen, gill, swim bladder, gonads and brain. The RB1 gene appears to be a single copy gene based on Southern analysis, and maps to linkage group XVI in the trout genome map. Polymorphisms in the RB1 gene and in closely linked markers should facilitate LOH analysis of RB1.
Insights
Researchers cloned the rainbow trout retinoblastoma (RB1) gene, finding it highly divergent and expressed throughout development and in adult tissues. This discovery aids future loss of heterozygosity (LOH) analysis in trout.
Area of Science:
- Molecular Biology
- Genetics
- Comparative Genomics
Background:
- The retinoblastoma (RB1) gene is a crucial tumor suppressor.
- Understanding RB1 gene evolution and expression across species provides insights into its function.
- Rainbow trout (Oncorhynchus mykiss) serves as a valuable model for fish genomics.
Purpose of the Study:
- To clone and characterize the full-length cDNA of the retinoblastoma (RB1) gene in rainbow trout.
- To investigate the expression patterns of the trout RB1 gene during embryogenesis and in adult tissues.
- To determine the gene copy number and map the RB1 gene within the trout genome.
Main Methods:
- Cloning of full-length cDNA from a rainbow trout embryo library.
- Bioinformatic analysis to predict protein sequence and assess divergence.
- Reverse transcription-polymerase chain reaction (RT-PCR) for expression studies.
- Southern blot analysis for gene copy number determination.
- Genome mapping to localize the RB1 gene.
Main Results:
- A full-length RB1 cDNA was successfully cloned, encoding a 910-amino acid protein, representing the most divergent RB1 sequence reported to date.
- High RB1 gene expression was observed from the second week of embryogenesis through hatching, with uniform expression in all examined adult tissues.
- Southern analysis indicated RB1 is a single-copy gene, mapped to linkage group XVI in the rainbow trout genome.
Conclusions:
- The cloned rainbow trout RB1 gene exhibits significant sequence divergence, suggesting evolutionary adaptation.
- Ubiquitous expression of RB1 in trout indicates a fundamental role in normal cellular processes across various tissues and developmental stages.
- The identification of polymorphisms in RB1 and linked markers will facilitate future loss of heterozygosity (LOH) studies for cancer research in this species.
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