A highly conserved homologue of bovine neurocalcin in Drosophila melanogaster is a Ca(2+)-binding protein expressed in neuronal tissues.
Polymerase chain reaction was used to search for genes encoding recoverin-like proteins in Drosophila melanogaster. We identified a gene that codes for a cognate of bovine neurocalcin; hence, we have named it neurocalcin (nca). A cDNA of nca was isolated and sequenced. The deduced polypeptide product of the cDNA is 22 kDa in size, and its amino acid sequence is 88% identical to that of bovine neurocalcin. This deduced Drosophila neurocalcin (DrosNCa) protein has three putative EF-hands and has a sequence in its NH2 terminus required for fatty acylation. DrosNCa was expressed in Escherichia coli and subsequently purified by phenyl-Sepharose chromatography and Mono Q anion exchange fast protein liquid chromatography. This recombinant protein was capable of binding 45Ca2+ and exhibited Ca(2+)-dependent mobility shifts in both SDS-polyacrylamide gel electrophoresis and native gel electrophoresis. DrosNCa was tritiated when it was coexpressed in E. coli with N-myristoyl transferase in the presence of [3H]myristic acid. The nca transcript was approximately 1 kilobase long, and tissue in situ hybridization showed that this message was present in the brain of adult flies. Antibodies raised against recombinant DrosNCa cross-reacted with rat hippocalcin on an immunoblot but not with bovine recoverin. When immunohistochemical analysis was performed, staining was observed throughout the central nervous system of adult flies, particularly in the neuropil, where neurons synapse. The nca locus maps to or near 76F on the Drosophila third chromosome.
Teng
D H
DH
Howard Hughes Medical Institute, University of Washington, Seattle 98195.
Chen
C K
CK
Hurley
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004331:Drosophila melanogaster / Q000235:genetics*; D005136:Eye Proteins; D015870:Gene Expression; D017344:Genes, Insect; D051597:Hippocalcin; D017403:In Situ Hybridization; D008074:Lipoproteins; D008969:Molecular Sequence Data; D009226:Myristates / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D051596:Neurocalcin; D012333:RNA, Messenger / Q000235:genetics; D044169:Receptors, Calcium-Sensing; D051603:Recoverin; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D005136:Eye Proteins; D008074:Lipoproteins; D009226:Myristates; D009419:Nerve Tissue Proteins; D051596:Neurocalcin; D012333:RNA, Messenger; D044169:Receptors, Calcium-Sensing; D051603:Recoverin; D051597:Hippocalcin; D002118:Calcium