Genomic organization of an avermectin receptor subunit from Haemonchus contortus and expression of its putative promoter region in Caenorhabditis elegans.
Avermectins and milbemycins are believed to exert their anthelmintic effects by binding to glutamate-gated chloride channels (GluCls). Two GluCl subunits have been localized in the pharynx in Caenorhabditis elegans, and the pharynx has been implicated as a major target for avermectins in C. elegans. However, in parasitic nematodes, the pharyngeal localization of the GluCl subunits needs to be determined. The HcGluCla gene encoding an alpha-type GluCl subunit has been cloned from Haemonchus contortus previously. To investigate the expression site of the HcGluCla gene we have isolated a 1439bp 5'-flanking region and determined the genomic organization of this gene. The HcGluCla gene is composed of 12 exons separated by 11 introns and spans approximately 7.3kb of genomic DNA. Analysis of the 1439bp 5'-flanking region of the HcGluCla gene revealed that it contained TATA, CCAAT boxes, and several other consensus transcriptional factor recognition sequences. The 1439bp 5'-flanking region and the first exon and intron and part of the second exon of the HcGluCla gene were fused to green fluorescence protein (GFP) reporter gene and microinjected into the gonads of C. elegans. After microinjection of the construct into C. elegans, four stable transformed lines were established and assayed for GFP expression. The transformed animals exhibited fluorescence in the two pairs of MC and M2 pharyngeal neurons, but no expression was detected in the muscle cells. Expression of HcGluCla in pharyngeal neurons suggests a mechanism for the effects of avermectins and milbemycins on pharyngeal function in parasitic nematodes.
Liu
Jie
J
Institute of Parasitology, McGill University, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Que., Canada H9X 3V9.
Dent
Joseph A
JA
Beech
Robin N
RN
Prichard
Roger K
RK
D020413:3' Untranslated Regions; D024506:5' Flanking Region / Q000235:genetics; D020121:5' Untranslated Regions; D000818:Animals; D030801:Animals, Genetically Modified; D000871:Anthelmintics / Q000378:metabolism / Q000494:pharmacology; D019677:Artificial Gene Fusion; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003062:Codon; D018509:DNA, Helminth / Q000737:chemistry / Q000302:isolation & purification; D005091:Exons; D017238:Genes, Helminth; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006189:Haemonchus / Q000235:genetics*; D007438:Introns; D007559:Ivermectin / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D016385:TATA Box / Q000235:genetics
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000871:Anthelmintics; D018118:Chloride Channels; D003062:Codon; D018509:DNA, Helminth; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; C109777:glutamate-gated chloride channels; D049452:Green Fluorescent Proteins; D007559:Ivermectin; C019264:avermectin