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.Many biological processes that require unwinding or writhing of the DNA helix are thought to be facilitated by negative supercoiling of DNA. These processes include replication and transcription that require the unwinding of DNA and formation of nucleosomes and certain protein complexes on DNA that stabilize the writhing of DNA (37).Although the bulk of DNA in eukaryotic nuclei is not under superhelical tension (30), unconstrained supercoils occur locally in the chromatin DNA. Most of them appear to be produced by the tracking of processive enzyme complexes such as RNA polymerases along the DNA (38). However, recent studies have suggested the presence of unconstrained supercoils generated by mechanisms other than transcription (15,17). It is possible that an enzymatic activity similar to that of bacterial DNA gyrase may also exist in eukaryotes. In support of this idea, we detected and purified a novel supercoiling activity from the silkworm Bombyx mori (21). The activity consists of the DNA supercoiling factor (SCF) and topoisomerase II. Cloning and characterization of a cDNA encoding B. mori SCF revealed a distinctive Ca 2ϩ -binding protein and Ca 2ϩ -dependent activation of the supercoiling reaction (22).The silkworm B. mori is a useful organism for biochemical studies but it is far less suitable for the molecular genetic approach than the fly Drosophila melanogaster. To investigate the in vivo role of SCF, we initiated studies on a Drosophila homologue of the factor. We report here that Drosophila SCF interacts with topoisomerase II in nuclei and localizes to puffs on polytene chromosomes. These findings suggest a role of SCF in transcription on chromatin. MATERIALS AND METHODS...
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