Potassium channels from NG108-15 neuroblastoma-glioma hybrid cells. Primary structure and functional expression from cDNAs

The complete amino acid sequences of two potassium channel proteins from NG108-15 neuroblastoma-glioma hybrid cells have been deduced by cloning and sequencing the cDNAs. One of these proteins (NGK2) is structurally more closely related to the Drosophila Shaw gene product than to the Shaker and Shab gene products, whereas the other (NGK1) is identical with a rat brain potassium channel protein (BK2) which is more closely related to the Drosophila Shaker gene product. mRNAs derived from both the cloned cDNAs, when injected into Xenopus oocytes, direct the formation of functional potassium channels with properties of delayed rectifiers.     

Structure of the voltage-dependent potassium channel is highly conserved from Drosophila to vertebrate central nervous systems.

Voltage-sensitive potassium channels are found in vertebrate and invertebrate central nervous systems. We have isolated a rat brain cDNA by cross-hybridization with a probe of the Drosophila Shaker gene complex. Structural conservation of domains of the deduced protein indicate that the rat brain cDNA encodes a voltage-sensitive potassium channel. Of the deduced amino acid sequence, 82% is homologous to the Drosophila Shaker protein indicating that voltage-sensitive potassium channels have been highly conserved during evolution. Selective pressure was highest on sequences facing the intracellular side and on proposed transmembrane segments S4-S6, suggesting that these domains are crucial for voltage-dependent potassium channel function. The corresponding rat mRNA apparently belongs to a family of mRNA molecules which are preferentially expressed in the central nervous system.     

Molecular cloning and characterization of the genes encoding the two subunits of Drosophila melanogaster calcineurin.

Genomic clones containing the full coding sequences of the two subunits of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin, were isolated from a Drosophila melanogaster genomic library using highly conserved human cDNA probes. Three clones encoded a 19.3-kDa protein whose sequence is 88% identical to that of human calcineurin B, the Ca(2+)-binding regulatory subunit of calcineurin. The coding sequences of the Drosophila and human calcineurin B genes are 69% identical. Drosophila calcineurin B is the product of a single intron-less gene located at position 4F on the X chromosome. Drosophila genomic clones encoding a highly conserved region of calcineurin A, the catalytic subunit of calcineurin, were used to locate the calcineurin A gene at position 21 EF on the second chromosome of Drosophila and to isolate calcineurin A cDNA clones from a Drosophila embryonic cDNA library. The structure of the calcineurin A gene was determined by comparison of the genomic and cDNA sequences. Twelve exons, spread over a total of 6.6 kilobases, were found to encode a 64.6-kDa protein 73% identical to either human calcineurin A alpha or beta. At the nucleotide level Drosophila calcineurin A cDNA is 67 and 65% identical to human calcineurin A alpha and beta cDNAs, respectively. Major differences between human and Drosophila calcineurins A are restricted to the amino and carboxyl termini, including two stretches of repetitive sequences in the carboxyl-terminal third of the Drosophila molecule. Motifs characteristic of the putative catalytic centers of protein phosphatase-1 and -2A and calcineurin are almost perfectly conserved. The calmodulin-binding and auto-inhibitory domains, characteristic of all mammalian calcineurins A, are also conserved. A remarkable feature of the calcineurin A gene is the location of the intron/exon junctions at the boundaries of the functional domains and the apparent conservation of the intron/exon junctions from Drosophila to man.     

Selection of Drosophila genes encoding secreted and membrane proteins

With the use of a yeast-based signal sequence trap (YSST) method, we screened a Drosophila cDNA library to isolate genes encoding secreted and membrane proteins. Of the 136 unique cDNA clones sequenced, 11 clones (8.1%) are identical to previously known Drosophila genes, 18 clones (13.2%) are homologous to other genes identified in various organisms, and 91 clones (66.9%) are novel. Most of these genes are secreted or membrane proteins, or appear to contain putative signal sequences at their amino termini. This indicates that YSST is an effective tool for the isolation and analysis of Drosophila genes that play roles in intercellular communication.     

Neuronal expression of a newly identified Drosophila melanogaster G protein alpha 0 subunit.

Guanine nucleotide-binding proteins (G proteins) mediate signals between activated cell-surface receptors and cellular effectors. A bovine G-protein alpha-subunit cDNA has been used to isolate similar sequences from Drosophila genomic and cDNA libraries. One class, which we call DG alpha 0, hybridized to position 47A on the second chromosome of Drosophila. The nucleotide sequence of the protein coding region of one cDNA has been determined, revealing an alpha subunit that is 81% identical with rat alpha 0. The cDNA hybridizes strongly to a 3.8 kb mRNA and weakly with a 5.3 kb message. Antibodies raised against a trp-E-DG alpha 0 fusion protein recognized a 39,000 Da protein in Drosophila extracts. In situ hybridization to adult Drosophila sections combined with immunohistochemical studies revealed expression throughout the optic lobes and central brain and in the thoracic and abdominal ganglia. DG alpha 0 message and protein were also detected in the antennae, oocytes, and ovarian nurse cells. The neuronal expression of this gene is similar to mammalian alpha 0, which is most abundantly expressed in the brain.