RNA expression in a cartilaginous fish cell line reveals ancient 3′ noncoding regions highly conserved in vertebrates
- David Forest,
- Ryuhei Nishikawa,
- Hiroshi Kobayashi,
- Angela Parton,
- Christopher J. Bayne, and
- David W. Barnes*
+Author Affiliations
-
Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672
-
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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