cut-1-like genes are present in the filarial nematodes, Brugia pahangi and Brugia malayi, and, as in other nematodes, code for components of the cuticle.
A fragment of a cut-1 like gene from the filarial nematode Brugia pahangi (designated Bp-cut-1) was isolated by PCR from genomic DNA. The sequence was used to design primers for use in RT-PCR and resulted in the isolation of a cDNA fragment from larvae in the process of the L3-L4 moult. Screening of a B. malayi genomic library identified a single clone, Bm-cut-1. Using primers designed from the Brugia sequences, semi-quantitative RT-PCR was carried out on 11 different life cycle stages chosen to cover periods around the moult and inter-moult periods. This analysis demonstrated that the cut-1 mRNA was most abundant preceding the moult, consistent with its function as a cuticular protein. Immuno-gold electron microscopy using an affinity purified antiserum raised to the highly conserved region of Ascaris CUT-1 confirmed that the protein was restricted to a tight band in the median layer of the cuticle. Despite the fact that no transcripts could be detected in mature adult worms by RT-PCR, immuno-gold microscopy revealed staining of the microfilarial cuticle within the uterus of the adult female worm, suggesting that other cut-1-like genes are present in Brugia.
Department of Veterinary Parasitology, University of Glasgow, UK.
E
E
Lewis
S J
SJ
Hunter
L
L
Tetley
C P
CP
Nunes
P
P
Bazzicalupo
E
E
Devaney
D000595:Amino Acid Sequence; D000818:Animals; D017178:Brugia malayi / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development; D017179:Brugia pahangi / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development; D029742:Caenorhabditis elegans Proteins; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D015723:Gene Library; D017238:Genes, Helminth; D015801:Helminth Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D007150:Immunohistochemistry; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D029742:Caenorhabditis elegans Proteins; D018076:DNA, Complementary; D015801:Helminth Proteins; D012333:RNA, Messenger; C070207:CUT-1 protein, C elegans