Characterization of androcam: A novel calcium-binding protein and a close relative of calmodulin from Drosophila melanogaster
Beckingham, Kathleen M.
Doctor of Philosophy
The Androcam gene was originally isolated by Fyrberg et al. (1994), using reduced stringency cDNA library screening with a calmodulin (CaM) probe from our lab. The derived protein sequence of the new gene is 85% similar to CaM. Using Northern analysis, I established that, unlike CaM, transcripts from the new gene are not seen in all developmental stages of Drosophila melanogaster. I further determined that, the expression of this novel gene is confined to the testis. We thus named the gene Androcam. In situ hybridization with a RNA probe derived from the Androcam cDNA showed that within the testis, the transcripts are only present in germ-line cells from the polar spermatocyte stage onwards. Immunolocalization with Androcam antibodies further demonstrated that Androcam is first seen on the kl-3 Y loop in the polar spermatocytes. In the cytoplasm and nucleoplasm, the level of Androcam increases dramatically thereafter and remains at high levels in the elongating spermatids. Co-localization with a DNA dye and Androcam antibodies indicates that Androcam may be present in the head region of elongated spermatids. These data suggest that Androcam has roles during various stages of the spermatogenesis and possibly also in mature gametes. We, in collaboration with Dr. Peter Bayley's lab (NIMR, Mill Hill, U.K.) have examined the biophysical properties of Androcam. These biophysical results, accompanied with sequence comparison, suggested that Androcam is a new Ca2+-signaling protein. Our studies showed that Androcam is unlike CaM in that it has only three functional Ca 2+-binding sites. Two of these sites have very high affinity to Ca 2+ and may be in Ca2+- and target-bound form in the resting state, while the third site binds Ca2+ more weakly and is potentially the regulatory site. These Ca2+-binding properties are reminiscent of those of the cardiac/slow skeletal muscular troponin C, a relative of CaM, and a protein that is also involved in Ca 2+-signaling. The finding of Androcam in Drosophila melanogaster intrigued us to investigate whether a homolog exists in other organisms. Using the Androcam antisera as the main tools, I have attained evidence that Androcam homologs exist in other insect species and mammals.
Molecular biology; Cell biology