Drosophila melanogaster contains a single calmodulin gene. Further structure and expression studies.

We have previously characterized a calmodulin gene from the organism Drosophila melanogaster. In the higher vertebrates a multi-gene system for encoding calmodulin is present and, in at least one invertebrate species, genes encoding highly related calmodulin isotypes exist. We have therefore searched for additional calmodulin genes within D. melanogaster. Although our searches were sensitive enough to detect a relatively divergent gene encoding a calmodulin family protein, we were unable to detect any additional genes for calmodulin per se. Further studies of the structure and expression of the single calmodulin gene of D. melanogaster have established that the gene contains a tiny additional 5' exon encoding only 50 residues of the 5' leader. Sequencing at the 3' terminus has established that the two transcript size classes derived from the gene are produced as a result of alternative polyadenylation site usage. The relative abundance of the two size classes of mRNAs differs throughout the life cycle, indicating developmental regulation of polyadenylation site usage.

Structure and sequence of the Drosophila melanogaster calmodulin gene.

A series of phage clones overlapping the single calmodulin gene locus of Drosophila melanogaster has been isolated and the exons of the gene positioned and sequenced within these clones. A calmodulin cDNA clone of the electric eel was used to identify these clones and to position the two major protein-coding exons of the gene. cDNA clones for D. melanogaster calmodulin were then isolated, characterized and used to identify the remaining exons. The gene consists of four exons separated by three introns of 3400 to 4300 bases in length. Exon 1 consists of the 5' untranslated region and the initiator ATG; exon 2 encodes amino acid residues 1 to 58.3; exon 3 encodes residues 58.3 to 139.3; and exon 4 encodes residues 139.3 to 148 and the 3' untranslated region. From the sequence of the 3' untranslated region and the lengths of the cDNA clones, two or three polyadenylation sites are indicated. Sequences potentially involved in the control of transcription of the gene and splicing of the mRNA product have been identified. Comparison of the intron-exon structures of the D. melanogaster calmodulin gene, the chick calmodulin gene, and other genes of the troponin C superfamily reinforces previous hypotheses that these genes arose from a common progenitor and permits identification of four introns that were probably present in the progenitor gene structure. The D. melanogaster calmodulin gene contains three of these introns, and the chick gene contains all four. These gene comparisons also indicate that the region of these genes encoding Ca2+-binding loop 3 is highly variable in structure. The chick and D. melanogaster calmodulin genes differ in this region, the chick gene containing a fifth intron here that is absent from the D. melanogaster gene.

Tight genetic linkage between the kdr insecticide resistance trait and a voltage-sensitive sodium channel gene in the house fly.

The kdr insecticide resistance trait in the house fly, Musca domestica, confers resistance to the rapid paralysis (knockdown) and lethal effects of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and pyrethroids. Flies with the kdr trait exhibit reduced neuronal sensitivity to these compounds, which are known to act at voltage-sensitive sodium channels of nerve membranes. To test the hypothesis that a mutation in a voltage-sensitive sodium channel gene confers the kdr phenotype, we have cloned genomic DNA corresponding to a segment of the house fly homologue of the para sodium channel gene of Drosophila melanogaster, identified restriction-site polymorphisms within this segment between the kdr strain 538ge and an inbred insecticide-susceptible lab stain, and developed a sensitive polymerase chain reaction-based diagnostic procedure to determine the sodium channel genotype of individual flies. A genetic linkage analysis performed with these molecular markers shows that the kdr trait is tightly linked (within about 1 map unit) to the voltage-sensitive sodium channel gene segment exhibiting the DNA sequence polymorphism. These findings provide genetic evidence for a mutation at or near a voltage-sensitive sodium channel gene as the basis for kdr resistance.

Endothelial proliferation, migration, and differentiation are blunted by conditionally expressed protein kinase C pseudosubstrate peptides.

Peptides based on the pseudosubstrate (PS) sequence of conventional protein kinase C isoenzymes (alpha, beta, gamma) specifically inhibit PKC activity in permeabilized cells, but whether PS can be used to study the role of PKC in the proliferation or migration of intact endothelial cells (EC) and angiogenesis is unknown. Peptides based on the PKCeta pseudosubstrate (etaPS) sequence were 3.5- to 8-fold more potent in inhibiting the PKCalpha, delta, epsilon, or eta kinase activity than was the peptide based on the PKCalpha pseudosubstrate (alphaPS) sequence. Thus, etaPS was conditionally overexpressed in intact EC and compared to alphaPS. Serum-induced growth of EC expressing etaPS was significantly slower than that of control EC. etaPS EC demonstrated slower rate of serum stimulated migration than that of either control or alphaPS EC. Expression of either etaPS or alphaPS produced slower rates of PMA induced EC migration, as compared to control EC. In an in vitro three-dimensional assay in which EC organize into capillary tubules, the EC that expressed etaPS formed fewer such tubules. This study shows that pseudosubstrate inhibitors derived from PKCeta are more potent both in vitro and in vivo than one based on the conventional isoenzyme PKCalpha. These data further support a role for PKC in proliferation and migration of intact EC, and angiogenesis.