Abstract

By screening Drosophila mutants that are potentially defective in synaptic transmission between photoreceptors and their target laminar neurons, L1/L2, (lack of electroretinogram on/off transients), we identified ort as a candidate gene encoding a histamine receptor subunit on L1/L2. We provide evidence that the ort gene corresponds to CG7411 (referred to as hclA), identified in the Drosophila genome data base, by P-element-mediated germ line rescue of the ort phenotype using cloned hclA cDNA and by showing that several ort mutants exhibit alterations in hclA regulatory or coding sequences and/or allele-dependent reductions in hclA transcript levels. Other workers have shown that hclA, when expressed in Xenopus oocytes, forms histamine-sensitive chloride channels. However, the connection between these chloride channels and photoreceptor synaptic transmission was not established. We show unequivocally that hclA-encoded channels are the channels required in photoreceptor synaptic transmission by 1) establishing the identity between hclA and ort and 2) showing that ort mutants are defective in photoreceptor synaptic transmission. Moreover, the present work shows that this function of the HCLA (ORT) protein is its native function in vivo.

Highlights

  • Arthropod photoreceptors release histamine as their major neurotransmitter [1]

  • Zheng et al [8] and Gisselmann et al [9] showed that two of the genes identified in the Drosophila Genome Project data base encode chloride channel subunits that are sensitive to histamine when expressed in oocytes

  • We show that mutants of a single complementation group, named ort [20], have defective responses of L1/L2 and that the complementation group corresponds to the gene, CG7411, identified in the Drosophila Genome Project data base

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Summary

EXPERIMENTAL PROCEDURES

Fly Stocks—There are seven known alleles of ort in our collection: ortP306, ortP364, ortUS2515, ortUS6096, ortUS1471, ortUS1606, and oraJK84. Germ Line Rescue by P Element-mediated Transformation—To generate transgenic flies carrying hclA cDNA in an ort background, 2.2-kb hclA cDNA was cloned into the NotI site of the CaSpeR-hs transformation vector. This vector contains the promoter of the heat shock gene, hsp, to allow heat-induced, ubiquitous expression of inserted cDNA. The 147-bp stretch was subcloned into the pSPT18 vector, the DNA template was linearized with restriction enzymes downstream of the cloned insert, and the sense and antisense RNA probes were synthesized and labeled with digoxigeninUTP by in vitro transcription with SP6 and T7 RNA polymerases, respectively. Slides containing tissue sections hybridized with sense and antisense RNA probes were processed in parallel

RESULTS
Normalized off transient amplitude
DISCUSSION

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