DNA Supercoiling Factor Localizes to Puffs on Polytene Chromosomes in Drosophila melanogaster

ABSTRACT

DNA supercoiling factor (SCF) was first identified in silkworm as a protein that generates negative supercoils in DNA in conjunction with eukaryotic topoisomerase II. To analyze the in vivo role of the factor, we cloned a cDNA encoding Drosophila melanogaster SCF. Northern analysis revealed 1.6- and 1.8-kb mRNAs throughout development. The longer mRNA contains an open reading frame that shares homology with mouse reticulocalbin whereas the shorter one encodes a truncated version lacking the N-terminal signal peptide-like sequence. An antibody against SCF detected a 45-kDa protein in the cytoplasmic fraction and a 30-kDa protein in the nuclear fraction of embryonic extracts. Immunoprecipitation suggests that the 30-kDa protein interacts with topoisomerase II in the nucleus, and hence that it is a functional form of SCF. Immunostaining of blastoderm embryos showed that SCF is present in nuclei during interphase but is excluded from mitotic chromosomes. In larvae, the antibody stained the nuclei of several tissues including a posterior part of the salivary gland. This latter staining was associated with natural or ecdysteroid-induced puffs on polytene chromosomes. Upon heat treatment of larvae, the staining on the endogenous puffs disappeared, and strong staining appeared on heat shock puffs. These results implicate SCF in gene expression.

ACKNOWLEDGMENTS

We thank J. Tamkun, M. Scott, and Y.-N. Jan forDrosophila libraries, K. Tamai and A. Kikuchi for antibodies, M. Yamamoto for assignment of the chromosomal sites, M. Jindra and H. Ueda for critical reading of the manuscript, and M. Yanagida for discussion.

This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan to S. Hirose and S. Hayashi.

M.K. and N.A. contributed equally to this work.