Transcriptome analysis of channel catfish (Ictalurus punctatus): initial analysis of gene expression and microsatellite-containing cDNAs in the skin Attila Karsi, Dongfeng Cao, Ping Li, Andrea Patterson, Arif Kocabas, Jinian Feng, Zhenlin Ju, Kathryn D. Mickett, Zhanjiang Liu * The Fish Molecular Genetics and Biotechnology Laboratory, Department of Fisheries and Allied Aquacultures and Program of Cell and Molecular Biosciences, Auburn University, Auburn, AL 36849, USA Received 11 November 2001; received in revised form 20 December 2001; accepted 15 January 2002 Received by J.A. Engler Abstract Previous molecular genetic studies on channel catfish (Ictalurus punctatus) have focused on limited number of genes and gene products. Recent advancement of molecular techniques made high throughput analysis of transcriptomes possible. As part of our transcriptome analysis of channel catfish, we have analyzed 1909 expressed sequence tags (ESTs) derived from a skin library. Of the 1909 ESTs analyzed, 1376 (72.1%) ESTs representing 496 unique genes had homologies with other organisms while 478 (25.0%) ESTs had no significant homologies and were designated as unknown. The remaining 55 (2.9%) EST clones were eliminated because of their low quality or short sequences. Of the 496 unique genes, 327 (65.9%) genes were singletons while 169 (34.1%) genes represented by two or more ESTs. A total of 1007 (52.8%) ESTs representing 235 unique genes matched previously reported channel catfish ESTs while 847 (44.4%) ESTs representing 261 unique genes were newly identified from this research. Functional categorization of the channel catfish genes indicated that the largest group was ribosomal proteins with 65 unique genes represented by 500 clones. The most abundantly expressed gene, the calcium binding protein ictacalcin, accounted for almost 5% of overall expression, indicating its important function in the skin. Sequence analysis of ESTs revealed the presence of 89 microsatellite-containing genes that may be valuable for future mapping studies. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Fish; Skin; Expressed sequence tag; Marker; Simple sequence repeat 1. Introduction Genomic approaches provide alternatives for addressing the mechanistic matters of gene expression. Expressed sequence tags (ESTs) are particularly useful for the develop- ment of cDNA microarrays that allow differentially expressed genes to be determined in a systematic way (Schena et al., 1996; Wang et al., 1999). ESTs are single pass sequences generated from random sequencing of cDNA clones (Adams et al., 1991). Large scale EST analysis is also an efficient way for identification of genes and for analysis of their expression by means of expression profiling (Franco et al., 1995; Azam et al., 1996; Lee et al., 2000). It offers a rapid and valuable first look at genes expressed in specific tissue types, under specific physiological conditions, or during specific developmental stages. ESTs have also been great resources for genomic mapping (Boguski and Schuler, 1995; Hudson et al., 1995; Schuler et al., 1996). The potential use of ESTs for the discovery of new chan- nel catfish genes has previously been shown (Ju et al., 2000; Cao et al., 2001). In addition, EST analysis offers opportu- nities for rapid identification and analysis of genes involved in specific biological pathways. For instance, using a tran- scriptomic approach, we previously characterized all the 47 ribosomal protein genes in the 60S ribosome (Patterson et al., 2002) and all the 32 ribosomal protein genes in the 40S ribosome (Karsi et al., 2002), which could otherwise have required many years of labor-intensive and expensive analyzes. Such systematic EST analyzes allowed for the Gene 285 (2002) 157–168 0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S0378-1119(02)00414-6 www.elsevier.com/locate/gene Abbreviations: aa, amino acids(s); bp, base pair(s); cDNA, complemen- tary DNA; dNTP, deoxyribonucleotide triphosphate; ds, double stranded; EST, expressed sequence tag; kb, kilobase; LB, Luria-Bertani; NCBI, National Center for Biotechnology Information; PCR, polymerase chain reaction; ORF, open reading frame * Corresponding author. Tel.: 11-334-844-4054; fax: 11-334-844-9208. E-mail address: zliu@acesag.auburn.edu (Z. Liu).