Kazuko Hanyu

Maternal Pumilio acts together with Nanos in germline development in Drosophila embryos

The maternal RNA-binding proteins Pumilio (Pum) and Nanos (Nos) act together to specify the abdomen in Drosophila embryos. Both proteins later accumulate in pole cells, the germline progenitors. Nos is required for pole cells to differentiate into functional germline. Here we show that Pum is also essential for germline development in embryos. First, a mutation in pum causes a defect in pole-cell migration into the gonads. Second, in such pole cells, the expression of a germline-specific marker (PZ198) is initiated prematurely. Finally, pum mutation causes premature mitosis in the migrating pole cells. We show that Pum inhibits pole-cell division by repressing translation of cyclin B messenger RNA. As these phenotypes are indistinguishable from those produced by nos mutation, we conclude that Pum acts together with Nos to regulate these germline-specific events.

Tudor protein is essential for the localization of mitochondrial RNAs in polar granules of Drosophila embryos

In Drosophila, polar plasm contains polar granules, which deposit the factors required for the formation of pole cells, germ line progenitors. Polar granules are tightly associated with mitochondria in early embryos, suggesting that mitochondria could contribute to pole cell formation. We have previously reported that mitochondrial large and small rRNAs (mtrRNAs) are transported from mitochondria to polar granules prior to pole cell formation and the large rRNA is essential for pole cell formation. Here we show that the localization of mtrRNAs is diminished in embryos laid by tudor mutant females, although the polar granules are maintained. We also found that Tud protein was colocalized with mtrRNAs at the boundaries between mitochondria and polar granules when the transport of mtrRNAs takes place. These observations suggest that Tud mediates the transport of mtrRNAs from mitochondria to polar granules.

Tetrahymena calcium-binding proteins, TCBP-25 and TCBP-23.

Publisher Summary Tetrahymena Ca 2+ -binding protein of 25 kDa (TCBP-25) and Tetrahymena Ca 2+ -binding protein of 23 kDa (TCBP-23) are calmodulin family proteins present in Tetrahymena . The analysis of cDNA suggests that TCBP-25 is composed of 218 amino acids and its molecular weight is estimated to be 24,702. On the other hand, TCBP-23 is composed of 207 residues and its molecular weight is estimated to be 23,413. Based on their deduced amino acid sequence, these proteins contain four EF-hand Ca 2+ -binding loops. Homology between TCBP-25 and TCBP-23 is 35%, but little sequence homology with other proteins is shown by a computer search. TCBP-25 and TCBP-23 are localized in the whole cell cortex except for the oral apparatus and the areas around the basal bodies. Furthermore, TCBP-25 is associated with both the migratory and stationary gametic pronuclei at the pronuclear exchange stage during conjugation. Therefore, TCBP-25 and TCBP-23 may play crucial roles in Ca 2+ -regulated processes in the cell cortex, and TCBP-25 may be involved in a Ca 2+ -dependent pronuclear exchange process during conjugation. This chapter presents the methods for preparation of recombinant TCBP-25 and TCBP-23, immunofluorescence staining of TCBP-25 and TCBP-23, and detection of TCBP-23-binding proteins.