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Molecular and Cellular Biology, November 1998, p. 6737-6744, Vol. 18, No. 11
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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

Masatomo Kobayashi,1,2 Noriko Aita,1,2 Shigeo Hayashi,1,3 Kohichi Okada,1,2 Tsutomu Ohta,2,dagger and Susumu Hirose1,2,*

The Graduate University for Advanced Studies,1 Department of Developmental Genetics,2 and Genetic Stock Research Center,3 National Institute of Genetics, Mishima, Shizuoka-ken 411-8540, Japan

Received 14 May 1998/Returned for modification 14 July 1998/Accepted 14 August 1998

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.

* Corresponding author. Mailing address: Department of Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka-ken 411-8540, Japan. Phone: 81-559-81-6771. Fax: 81-559-81-6776. E-mail: shirose{at}lab.nig.ac.jp.

dagger Present address: Department of Cell Genetics, National Institute of Genetics, Mishima, Shizuoka-ken 411-8540, Japan.

Molecular and Cellular Biology, November 1998, p. 6737-6744, Vol. 18, No. 11
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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