Suppression subtractive hybridization PCR isolation of cDNAs from a Caribbean soft coral
نویسندگان
چکیده
Transcriptomic studies of marine organisms are still in their infancy. A partial, subtracted expressed sequence tag (EST) library of the Caribbean octocoral Erythropodium caribaeorum and the sea fan Gorgonia ventalina has been analyzed in order to find novel genes or differences in gene expression related to potential secondary metabolite production or symbioses. This approach entails enrichment for potential non-“housekeeping” genes using the suppression subtractive hybridization (SSH) polymerase chain reaction (PCR) method. More than 500 expressed sequence tags (ESTs) were generated after cloning SSH products, which yielded at least 53 orthologous groups of proteins (COGs) and Pfam clusters, including transcription factors (Drosophila Big Brother), catalases, reverse transcriptases, ferritins and various “hypothetical” protein sequences. A total of 591 EST sequences were deposited into GenBank [dbEST: FL512138 FL512331, GH611838, and HO061755-HO062154]. The results represent proof of concept for enrichment of unique transcripts over housekeeping genes, such as actin or ribosomal genes, which comprised approximately 17% of the total dataset. Due to the gene and sequence diversity of some ESTs, such sequences can find utility as molecular markers in current and future studies of this species and other soft coral biogeography, chemical ecology, phylogenetics, and evolution.
منابع مشابه
Suppression subtractive hybridization (SSH) combined with bioinformatics method: an integrated functional annotation approach for analysis of differentially expressed immune-genes in insects
The suppression subtractive hybridization (SSH) approach, a PCR based approach which amplifies differentially expressed cDNAs (complementary DNAs), while simultaneously suppressing amplification of common cDNAs, was employed to identify immuneinducible genes in insects. This technique has been used as a suitable tool for experimental identification of novel genes in eukaryotes as well as prokar...
متن کاملExpression of 35S::Pto globally activates defense-related genes in tomato plants.
The tomato (Lycopersicon esculentum) resistance gene Pto confers resistance to the bacterial pathogen Pseudomonas syringae pv tomato carrying the avirulent gene avrPto. Overexpressing Pto under the control of the cauliflower mosaic virus 35S promoter constitutively activates defense responses in the absence of pathogen infection and nonspecifically enhances disease resistance. To elucidate the ...
متن کاملDifferential screening of a subtracted cDNA library: a method to search for genes preferentially expressed in multiple tissues.
We have developed a new strategy for differential screening of genes that are expressed in two or more tissues, and have used it to identify genes that are preferentially expressed in both testis and ovary. In this approach, testis-specific cDNAs were first generated using the suppression subtractive hybridization technique, cloned and arrayed in microplates. The inserts from the subtracted tes...
متن کاملProfiling of differentially expressed genes in wound margin biopsies of horses using suppression subtractive hybridization.
Disturbed gene expression may disrupt the normal process of repair and lead to pathological situations resulting in excessive scarring. To prevent and treat impaired healing, it is necessary to first define baseline gene expression during normal repair. The objective of this study was to compare gene expression in normal intact skin (IS) and wound margin (WM) biopsies using suppression subtract...
متن کاملSuppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.
A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subt...
متن کامل