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.

FIG. 1

Nucleotide sequence of Drosophila SCF. Complete coding sequence and the 5′- and 3′-noncoding sequences are shown. The first 369 nucleotides represent the genomic sequence, and the following region was derived from cDNA. Comparison of the genomic and cDNA sequences revealed a 118-bp intron between nucleotide positions 877 and 878 (marked by a filled triangle). The predicted amino acid sequence of the entire ORF is shown in the single-letter code. The asterisk represents the stop codon. The boldfaced A residues and double underlining show the presumptive initiation sites for the 1.6- and 1.8-kb mRNA and the first in-frame methionine in the 1.6-kb mRNA, respectively. The single underlining represents the poly (A) tail.

FIG. 2

Sequence comparison of Drosophila SCF, silkworm SCF (22), and mouse reticulocalbin (23) deduced proteins. Amino acids identical between Drosophila and silkworm proteins are shaded, and those identical in all three proteins are boxed. Identity: Drosophila versus silkworm, 56%; Drosophila versus mouse, 43%; silkworm versus mouse, 45%. I to V represent loops of the EF-Hand domains. The presumptive signal peptides are underlined.

FIG. 3

Supercoiling activity of recombinant Drosophila SCF. ³²P-labeled relaxed closed circular DNA of pHSAR was incubated with Drosophila topoisomerase II (topo II) and/or bacterially expressed and purified his-tag SCFf, and analyzed by two-dimensional agarose gel electrophoresis. The first dimension was normal gel electrophoresis from the top to the bottom, while the second dimension was in the presence of 5 ng of ethidium bromide/ml from the left to the right. nc, nicked circular DNA; SC, supercoiled circular DNA with several negative superhelical turns; 1, linear DNA. Supercoiled DNA with 10 or more negative superhelical turns migrated faster than linear DNA in the first dimension (19).

FIG. 4

Transcripts from the SCF locus. (A) Northern blot hybridization with region-specific probes. A blot of total cellular RNA from prepupae was successively probed with the indicated DNA fragments (probes 1-3) and a full-length cDNA (probe 4). Size marker RNAs were run in a parallel lane on the same gel, and their positions are indicated. N and C represent the N and C termini of the full-length protein. Signal peptide denotes the signal-peptide-like sequence, and I to V represent the loops of the EF-Hand domains. (B) Mapping of the 5′ end of each mRNA by 5′ RACE. Underlining represents sequences of the longest PCR products from 1.6 and 1.8 kb mRNAs. INIT consensus, initiator consensus sequence; DPE consensus, downstream promoter element consensus. The filled triangles show the presumptive initiation sites for each mRNA.

FIG. 5

Identification of SCF. (A) Western blot analyses with region-specific antibodies. The proteins in 10 μl of the cytoplasmic (lanes 2 and 5) or 5 μl of the nuclear (lanes 3 and 6) fraction from 0- to 22-h-old embryos or 2.5 ng each of the recombinant proteins used for production of αNT and αCT (αNT-Ag and αCT-Ag, lanes 1 and 4) were resolved by SDS–10% PAGE. After blotting, the membrane was probed with αNT (lanes 1 to 3) or αCT (lanes 4 to 6). Positions of size marker proteins are indicated. Symbols are the same as those in Fig. 4A. (B) Coimmunoprecipitation of SCF and topoisomerase II. Upper panels: Western blots of nuclear proteins from the 0- to 22-h-old embryos incubated with αCT (middle) or the preimmune serum (left) were probed with antitopoisomerase II monoclonal antibody (α-topoII MAb). Lower panels: Western blots of the same nuclear proteins incubated with α-topoII monoclonal antibody (middle), or anti-hemagglutinin monoclonal antibody (αHA MAb, left) were probed with αCT. Western blots of input proteins are shown in the right column. Only regions of the blots containing the relevant bands are shown because each antibody detected only SCF (see panel A, lane 6) or topoisomerase II (data not shown) among the proteins in the nuclear fraction. (C) Scheme of expression of SCF and DCB-45. ER, endoplasmic reticulum. Other abbreviations are as described in the legend for Fig. 4A.

FIG. 6

Immunostaining of embryos. (A) Blastoderm embryo stained with αNT. (B) Another blastoderm embryo processed as just described but without primary antibody. (C) Part of a blastoderm embryo stained with DAPI. (D) The same embryo as in panel C but stained with αCT. (E) Part of a blastoderm embryo in metaphase stained with DAPI. (F) The same embryo as in panel E but stained with αCT. (G) Late embryo stained with αCT. A similar staining pattern was observed with αNT (data not shown). (H) Late embryo stained with αNT. Similar staining was obtained with αCT (data not shown). In panels C through F, parts of the embryos are shown at higher magnifications to show the shape of nuclei. In panels C to F and in panel H, the focus was on the upper surface of the embryos, while in panels A, B, and G, it was on the inside. All embryos are oriented anterior to the left.

FIG. 7

Immunostaining of the salivary gland. (A) Anterior parts of salivary glands from a third instar wandering larva stained with αNT (brown). Note that posterior cells with large nuclei were not stained with αNT but were positive for the PAS staining (purple). (B) Posterior part of a salivary gland from a third instar wandering larva stained with DAPI. (C) The same gland as in panel B but stained with αCT. a and p indicate the anterior and posterior parts of the salivary gland, respectively.

FIG. 8

Immunostaining of SCF on salivary gland polytene chromosomes. (A) Polytene chromosomes from a third instar wandering larva stained with αCT (red) and DAPI (blue). (B) Part of the second chromosome stained with αCT and DAPI. (C) Polytene chromosomes from an ecdysteroid-treated gland stained with αCT and DAPI. (D) Polytene chromosomes as in panel A but stained with the antitopoisomerase II monoclonal antibody (red) and DAPI (blue). Most regions of the chromosomes appear purple due to overlapping staining. (E) Polytene chromosomes from a heat-treated wandering larva stained with αCT and DAPI. The numbers in panels B, C, and E represent the chromosomal sites.