Integrative and Comparative Biology Advance Access published online on May 22, 2006
Integrative and Comparative Biology, doi:10.1093/icb/icl005
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David S. Durica 1 *,
Doris Kupfer 2,
Fares Najar 3,
Hongshing Lai 3,
Yuhong Tang 4,
Kathryn Griffin 5,
Penny M. Hopkins 5,
and
Bruce Roe 3
* To whom correspondence should be addressed. Synopsis We have constructed directional and randomly primed cDNA libraries from mRNAs isolated during progressive stages of fiddler crab (Uca pugilator) limb regeneration. Data from these libraries are being assembled into an on-line database (http://www.genome.ou.edu/crab.html) that is both BLAST and keyword searchable; the data set is also available through GenBank. The first characterized library was made from mRNA isolated 4 days post-autotomy, when the first sign of morphological differentiation, cuticle secretion, is observed. Analysis of 1698 cDNA clones led to assignment of 473 contigs and 417 singlets, for a total of 890 sequences. Of these,
Genomic and Proteomic Approaches in Crustacean Biology
EST library sequencing of genes expressed during early limb regeneration in the fiddler crab and transcriptional responses to ecdysteroid exposure in limb bud explants
1 Department of Zoology, University of Oklahoma, Norman, OK 73019, USA; Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA
2 Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA; Present address: DNA solutions, 840 Research Parkway, Oklahoma City, OK 73104, USA
3 Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA
4 Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA; Present address: Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Pky. Ardmore, OK 73401, USA
5 Department of Zoology, University of Oklahoma, Norman, OK 73019, USA
David S. Durica, E-mail: ddurica{at}ou.edu
Abstract 
86% showed no assignments to characterized genes on database searching, while 14% could be assigned to a known ortholog in the COG (Clusters of Orthologous Groups) database. BLAST searches to specific protein domains in the Gene Ontology database led to assignments for 40% of the assembled sequences. Sequence similarity searches of other crustacean EST databases produced hits to 13-30% of the Uca query sequences. The ESTs include several genes that may be potentially ecdysteroid-responsive, such as homologs to chaperone proteins and cuticle protein genes, as well as homologs to arthropod proteins involved in retinoid/terpenoid metabolism. We have tested 3 potential candidate genes for their ability to be induced by ecdysteroid in limb bud explants; an arthropodial cuticle protein gene, and the nuclear receptor genes EcR and RXR. A subset of early blastemal limb buds (8 days post autotomy) show a positive response to ecdysteroid by 1-1.5 h, followed by a decrease in transcript abundance at longer periods of sustained incubation. Later stage buds (12 days post autotomy-late premolt) show decreases in steady-state mRNA levels by 1.5 h, or are completely refractory to ecdysteroid exposure.
From the symposium "Genomic and Proteomic Approaches in Crustacean Biology" presented at the annual meeting of the Society for Integrative and Comparative Biology, January 4-8, 2006, at Orlando, Florida.
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