ABSTRACT
Gonadal development usually involves alternative splicing of sex-related genes. Vasa, a highly conserved ATP-dependent RNA helicase present mainly in germ cells, has an important function in gonadal development. As an important sex-related gene, recent evidence indicates that different splice variants of vasa exist in many species. In this study, there was identification of two types of vasa splice variants in the Chinese mitten crab Eriocheir sinensis, termed Esvasa-l and Esvasa-s, respectively. Furthermore, splice variants of Esvasa-s were sub-divided into Esvasa-s1, Esvasa-s2, Esvasa-s3, Esvasa-s4, and Esvasa-s5, based on differing numbers of TGG repeats. Results from genomic structure analyses indicated that these forms are alternatively spliced transcripts from a single vasa gene. Results from tissue distribution assessments indicate the vasa splice variants were exclusively expressed in the gonads of male and female adult crabs. In situ hybridization results indicate Esvasa mRNA was mainly present in the cytoplasm of previtellogenic oocytes. As oocyte size increased, relative abundance of Esvasa mRNA decreased and became distributed near the cellular membrane. The Esvasa mRNA was not detectable in mature oocytes. In testis, Esvasa mRNA was detected in spermatids and spermatozoa, but not in spermatogonia and spermatocytes. Notably, results from qPCR analysis of Esvasa-l and Esvasa-s indicate there are different relative proportions during gametogenesis, implying that splice variants of the Esvasa gene may have different biological functions during crab gonadal development.
Subject(s)
Alternative Splicing , Brachyura/genetics , DEAD-box RNA Helicases/metabolism , Gonads/growth & development , RNA, Messenger/metabolism , Amino Acid Sequence , Animals , Base Sequence , DEAD-box RNA Helicases/genetics , Gene Expression Regulation, Developmental/physiology , Gene Expression Regulation, Enzymologic/physiology , RNA, Messenger/genetics , Sexual MaturationABSTRACT
OBJECTIVE: To develop a method for detection of coxsackie B virus type 1-6 by RT-PCR. METHODS: A pair of primers were designed to amplify all types of coxsackie B virus 1-6 efficiently. The PCR product was hybridized in micro-wells in which 6 type specific oligonucleotide probes had been coated respectively, colorimetric detection was performed to discriminate the types of coxsackie B virus. RESULTS: This method was shown to be concordant with the IgM ELISA, 71.7% of anti-coxsackie B positive cases could be detected by RT-PCR. CONCLUSION: The RT-PCR method can type coxsackie B virus efficiently and provides a tool for clinical diagnosis and epidemiological investigation.
