Expressed sequence tag analysis of expression profiles of zebrafish testis and ovary☆
Introduction
The expressed sequence tag (EST) approach, first demonstrated in the human genome project (Adams et al., 1991), is powerful in massive cloning of cDNAs as well as in large scale characterization of cDNA sequences for deciphering genome sequence. This approach is also valuable in studies of mRNA expression profiles at a single gene level from unbiased cDNA libraries. In general, two types of information can be obtained by this approach: the composition of expressed transcripts and the relative abundance of these transcripts. Both types of information are important to the understanding of molecular composition and function of source tissues and cells.
The zebrafish, Danio rerio, is currently the most popular fish model in genetic and developmental analyses. In the past few years, tremendous research infrastructure for genetic analysis has been established for this species. For example, large scale mutagenesis has been carried out both by a chemical mutagen and by transgenic insertion. Many of the mutated genes have been cloned either by a candidate gene approach or by chromosomal walking; many of the cloned genes have implications in human diseases (for review, see Dooley and Zon, 2000). Several approaches have been established for zebrafish genome mapping, including randomly amplified polymorphic DNA (RAPD) (Postlethwait et al., 1994), simple sequence length polymorphism (SSLP) (Knapik et al., 1998), single strand conformation polymorphism (SSCP) (Postlethwait et al., 1998), and radiation hybrid panels (Geisler et al., 1999). Several zebrafish EST projects have been carried out in small research laboratories as well as in a large sequencing center (Gong et al., 1997, Ton et al., 2000, Clark et al., 2001). Finally, a zebrafish genome project has been initiated and the genome is expected to be completed by the end of 2003 (http://www.sanger.ac.uk/Projects/Drerio/).
So far, over 170,000 zebrafish EST sequences are available in GenBank (December, 2001), largely due to the contribution by the Washington University group. These EST clones are generated from several zebrafish cDNA libraries, mainly from different embryonic stages and whole adult. So far, only a few tissue-specific cDNA libraries have been used to generate zebrafish EST clones, including heart, brain, kidney, etc. Compared to the comprehensive cDNA libraries such as whole-adult and embryonic cDNA libraries, the expression profile of tissue-specific cDNA libraries can provide new and different information that sometimes presents more functional meaning. In addition, tissue-specific and rarely expressed genes are more likely to be obtained from these tissue-specific cDNA libraries than from a whole-adult or embryonic cDNA library, in which these genes may represent a very small proportion in the transcription profile. Furthermore, characterization of gonad cDNAs will be helpful to identify gonad molecular markers for developmental analyses.
In the present studies, we report a total of 1025 zebrafish EST sequences from two zebrafish gonad cDNA libraries: testis and ovary. In order to find suitable molecular markers for zebrafish gonads, some of the EST clones were selected to analyze the tissue distribution of their mRNAs and their expression in the testis and ovary.
Section snippets
Construction of two zebrafish gonad cDNA libraries
Zebrafish were purchased from a local ornamental fish farm. Two zebrafish gonad cDNA libraries from the testis and ovary were constructed in the present study. PolyA+ RNA was extracted using the Micro-fast Track™ Kit (Invitrogen). For construction of the testis library, ∼6 μg of polyA+ RNA was extracted from a pool of testes collected from 31 male zebrafish of 4–5 months old. For the ovary library, ∼5 μg of polyA+ RNA was purified from the ovaries of two female fish of 4–5 months old. cDNA
Summary of EST clones in zebrafish testis and ovary cDNA libraries
A total of 1025 random clones from the two zebrafish gonad cDNA libraries were partially sequenced, including 501 clones from the testis library and 524 from the ovary library (Table 1). The cDNA identification rates are 60% (301 clones) for the testis clones and 65% (340 clones) for the ovary clones. In total, 641 clones were identified, representing at least 478 different zebrafish genes, of which only 61 have been previously reported in the public DNA databases. Thus, 417 identified
Acknowledgements
Our research was supported by the Academic Research Fund from National University of Singapore (NUS). S.Z. is a recipient of a NUS postgraduate scholarship. We thank Dr Mingru Chen for contribution of eight testis EST clones and help in analysis of in situ hybridization results. The EST sequences reported here have been submitted to GenBank and the Accession numbers are BM141139–BM141637 for T1 to T501 (excluding T59, BM485237; and T200, BM485238), and BM140615–BM141138 for O1 to O524. The list
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A list of identified zebrafish cDNA clones from this study is available at the following website: http://www.dbs.nus.edu.sg/Staff/gong.html