The isolation of differentially expressed cDNA clones from the filarial nematode Brugia pahangi 1
A cDNA library constructed from 3 day post-infective L3 of the filarial nematode Brugia pahangi was screened by differential hybridization with cDNA probes prepared from different life-cycle stages. Five cDNA clones hybridizing selectively to the mosquito-derived L3 probe were isolated and characterized. Northern blot analysis of 4 of the clones confirmed that each was most highly expressed in the mosquito-derived L3. The expression of each mRNAduring parasite development in the mosquito vector was investigated using RT–PCR, and all were shown to be abundant in the immature L3. Four of the 5 cDNAs cloned coded for structural proteins: 2 cuticular collagens, and the muscle proteins tropomyosin and troponin. Further studies on troponin using an antiserum raised to the recombinant protein demonstrated that the protein, unlike the mRNA, was present in all life-cycle stages examined, while immunogold labelling demonstrated that it was localized to the muscle blocks.(Received August 25 1998)
(Revised December 21 1998)
(Revised February 11 1999)
(Accepted February 11 1999)
Key Words: Brugia pahangi; L3; differentially expressed genes; troponin; tropomyosin; cuticular collagens.
c1 Corresponding author: Department of Veterinary Parasitology, University of Glasgow, Bearsden Road, Glasgow G61 1QH. Tel: +0141 330 6925 (5751). Fax: +0141 330 5603. E-mail: firstname.lastname@example.org
1 Nucleotide sequence data reported in this paper are available in the EMBL and GenBankTM data bases under the accession numbers: SJ1 AJ224966; SJ5 AJ224967; clone A AJ130821; clone C AJ224968; clone F AJ224969; clone EE4 X91066.