Muscle-specific transcriptional regulation of the slowpoke Ca(2+)-activated K(+) channel gene.

Transcriptional regulation of the Drosophila slowpoke calcium-activated potassium channel gene is complex. To date, five transcriptional promoters have been identified, which are responsible for slowpoke expression in neurons, midgut cells, tracheal cells, and muscle fibers. The slowpoke promoter called Promoter C2 is active in muscles and tracheal cells. To identify sequences that activate Promoter C2 in specific cell types, we introduced small deletions into the slowpoke transcriptional control region. Using transformed flies, we asked how these deletions affected the in situ tissue-specific pattern of expression. Sequence comparisons between evolutionarily divergent species helped guide the placement of these deletions. A section of DNA important for expression in all cell types was subdivided and reintroduced into the mutated control region, a piece at a time, to identify which portion was required for promoter activity. We identified 55-, 214-, and 20-nucleotide sequences that control promoter activity. Different combinations of these elements activate the promoter in adult muscle, larval muscle, and tracheal cells.

Transcriptional control of Ca(2+)-activated K(+) channel expression: identification of a second, evolutionarily conserved, neuronal promoter.

Neuronal signaling properties are largely determined by the quantity and combination of ion channels expressed. The Drosophila slowpoke gene encodes a Ca(2+)-activated K(+) channel used throughout the nervous system. The slowpoke transcriptional control region is large and complex. To simplify the search for sequences responsible for tissue-specific expression, we relied on evolutionary conservation of functionally important sequences. A number of conserved segments were found between two Drosophila species. One led us to a new 5' exon and a new transcriptional promoter: Promoter C0. In larvae and adults, Promoter C0 was demonstrated to be neural-specific using flies transformed with reporter genes that either contain or lack the promoter. The transcription start site of Promoter C0 was mapped, and the exon it appends to the 5' end of the mRNA was sequenced. This is the second neural-specific slowpoke promoter to be identified, the first being Promoter C1. Promoter choice does not alter the encoded polypeptide sequence. RNAase protection assays indicate that Promoter C0 transcripts are approximately 12 times more abundant that Promoter C1 transcripts. Taken together, these facts suggest that promoter choice may be a means for cells to control channel density.

Behavioral and electrophysiological analysis of Ca-activated K-channel transgenes in Drosophila.

The slowpoke gene of Drosophila melanogaster encodes a Ca-activated K channel. This gene is expressed in neurons, muscles, tracheal cells, and the copper and iron cells of the midgut. The gene produces a large number of alternative products using tissue-specific transcriptional promoters and alternative mRNA splicing. We have described in great depth how transcription is regulated and are now cataloging the tissue-specificity of different splice variants. It is believed that the diversity of products serves to tailor channel attributes to the needs of specific tissues. Electrophysiological and behavioral assays indicate that at least some of these products produce channels with distinct properties.

Is there a Brachyury the Second? Analysis of a transgenic mutation involved in notochord maintenance in mice.

A new phenotype mapping to the t-complex, which is designated Brachyury the Second (T2), is characterized by a slightly shortened tail in heterozygotes and homozygous failure to form an organized notochord with subsequent abnormal development of posterior somites and neural tube. The phenotype of T2 superficially resembles that of Brachyury; however, there are several important differences. Brachyury homozygotes fail to make posterior somites, notochord, floor plate, and a placental connection, resulting in death by 10.5 days of development. In contrast, T2 homozygotes make posterior somites, scattered notochord cells, and floorplate and achieve an allantoic connection. However, despite making a maternal connection, T2 homozygotes cease development at E11.5 and die soon after. We have cloned and analyzed the transgene insertion site, which maps within 100 kb of the Brachyury gene, but does not seem to physically interrupt nor affect transcription from that locus. The existence of a second gene mapping near Brachyury and affecting the same developmental processes was alluded to over 50 years ago and has been debated ever since. An embryological description of T2 is presented, as is a discussion of the implications of a single, larger Brachyury locus versus two closely linked genes coordinately regulating axial development.