title: Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells.
abstract: BACKGROUND
  Taxol(®) (paclitaxel) promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ) elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH) to identify genes involved in global pathway control.
  
  
  RESULTS
  Six separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation time points (6h, 18h and 5 day) to identify genes that are either induced or suppressed in response to MJ. Sequencing of 576 differentially screened clones from the SSH libraries resulted in 331 unigenes. Functional annotation and Gene Ontology (GO) analysis of up-regulated EST libraries showed enrichment of several known paclitaxel biosynthetic genes and novel transcripts that may be involved in MJ-signaling, taxane transport, or taxane degradation. Macroarray analysis of these identified genes unravelled global regulatory expression of these transcripts. Semi-quantitative RT-PCR analysis of a set of 12 candidate genes further confirmed the MJ-induced gene expression in a high paclitaxel accumulating Taxus cuspidata P93AF cell line.
  
  
  CONCLUSIONS
  This study elucidates the global temporal expression kinetics of MJ responsive genes in Taxus suspension cell culture. Functional characterization of the novel genes identified in this study will further enhance the understanding of paclitaxel biosynthesis, taxane transport and degradation.
authors.affiliation: Department of Biology, University of Massachusetts, Amherst, MA, 01003, USA.
authors.forename: Sangram K
authors.initials: SK
authors.lastname: Lenka
authors.forename: Nadia
authors.initials: N
authors.lastname: Boutaoui
authors.forename: Bibin
authors.initials: B
authors.lastname: Paulose
authors.forename: Kham
authors.initials: K
authors.lastname: Vongpaseuth
authors.forename: Jennifer
authors.initials: J
authors.lastname: Normanly
authors.forename: Susan C
authors.initials: SC
authors.lastname: Roberts
authors.forename: Elsbeth L
authors.initials: EL
authors.lastname: Walker
mesh_terms: D000085:Acetates / Q000494:pharmacology*; D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology*; D020224:Expressed Sequence Tags; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D054883:Oxylipins / Q000494:pharmacology*; D017239:Paclitaxel / Q000096:biosynthesis*; D010937:Plant Growth Regulators / Q000494:pharmacology; D020946:Taxus / Q000166:cytology / Q000235:genetics* / Q000378:metabolism
chemical_list: D000085:Acetates; D003517:Cyclopentanes; D054883:Oxylipins; D010937:Plant Growth Regulators; C072239:methyl jasmonate; D017239:Paclitaxel