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The authors established a cartilaginous fish cell line [Squalus acanthias embryo cell …


Biology Articles » Zoology » Ichthyology » RNA expression in a cartilaginous fish cell line reveals ancient 3′ noncoding regions highly conserved in vertebrates

Abstract
- RNA expression in a cartilaginous fish cell line reveals ancient 3′ noncoding regions highly conserved in vertebrates

RNA expression in a cartilaginous fish cell line reveals ancient 3′ noncoding regions highly conserved in vertebrates

  1. David Forest,
  2. Ryuhei Nishikawa,
  3. Hiroshi Kobayashi,
  4. Angela Parton,
  5. Christopher J. Bayne, and
  6. David W. Barnes*

+Author Affiliations

  1. Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672
  1. Communicated by Gordon H. Sato, Ministry of Fisheries, Massawa, Eritrea, November 21, 2006 (received for review October 12, 2006)

 
An Open Access article from PNAS January 23, 2007 vol. 104 no. 4 1224-1229.
 

Abstract

We have established a cartilaginous fish cell line [Squalus acanthias embryo cell line (SAE)], a mesenchymal stem cell line derived from the embryo of an elasmobranch, the spiny dogfish shark S. acanthias. Elasmobranchs (sharks and rays) first appeared >400 million years ago, and existing species provide useful models for comparative vertebrate cell biology, physiology, and genomics. Comparative vertebrate genomics among evolutionarily distant organisms can provide sequence conservation information that facilitates identification of critical coding and noncoding regions. Although these genomic analyses are informative, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. Using ESTs defining mRNAs derived from the SAE cell line, we identified lengthy and highly conserved gene-specific nucleotide sequences in the noncoding 3′ UTRs of eight genes involved in the regulation of cell growth and proliferation. Conserved noncoding 3′ mRNA regions detected by using the shark nucleotide sequences as a starting point were found in a range of other vertebrate orders, including bony fish, birds, amphibians, and mammals. Nucleotide identity of shark and human in these regions was remarkably well conserved. Our results indicate that highly conserved gene sequences dating from the appearance of jawed vertebrates and representing potential cis-regulatory elements can be identified through the use of cartilaginous fish as a baseline. Because the expression of genes in the SAE cell line was prerequisite for their identification, this cartilaginous fish culture system also provides a physiologically valid tool to test functional hypotheses on the role of these ancient conserved sequences in comparative cell biology.

  • noncoding sequence
  • sequence conservation
  • Squalus acanthias

 

Efforts toward a comprehensive compilation of cell biological, physiological, and genomic information from a variety of vertebrate species are providing a rich source of comparative data representing critical points in evolutionary divergence (1). Although available information is rapidly expanding for teleosts (bony fishes; ref. 2), few cell biological or genomic data are available for the most primitive jawed vertebrates, the cartilaginous fish, class Chondrichthyes, largely represented by the order Elasmobranchii (3, 4). This information would extend comparative data through >450 million years of evolution. We have derived an embryonic mesenchymal stem cell line [Squalus acanthias embryo cell line (SAE)] from the elasmobranch S. acanthias, the spiny dogfish shark (5). This cell line from a cartilaginous fish provides a means to study the cell biology, physiology, and genomics of Chondrichthyes. Recently, the National Human Genome Research Institute targeted for comprehensive genomic sequencing Leucoraja erinacia, an elasmobranch of the superorder Squaliformes, related to S. acanthias, underscoring the value of Chondrichthyes for comparative biology (6)

In efforts to explore the cell biology and genomics of cartilaginous fish, we used the SAE shark cell line to prepare a normalized cDNA library primed from the 3′ end of the mRNA. A database of >5,000 EST sequences representative of gene expression in the cell line was generated by sequencing from the 5′ end of the ESTs. Additional sequencing of a portion of these ESTs from the 3′ end allowed an analysis of 3′ UTRs of mRNA transcripts from well defined genes. Among these, we identified eight with remarkably long and evolutionarily well conserved gene-specific noncoding nucleotide sequences. These phylogenetic footprints were found in the 3′ UTRs of shark-homologous genes in a variety of vertebrate orders, from cartilaginous fish to mammals, suggesting functional roles. Interestingly, all eight genes are involved in the regulation of cell growth and proliferation. Our data demonstrate the utility of elasmobranch genomics as a tool for comparative vertebrate biology and also point toward the use of the SAE cartilaginous fish cell line as a tool for functional verification at the cellular level of molecular genomic predictions and hypotheses.


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