An affinity-purification method has been developed for the rapid, efficient, and precise elution of antibodies specifically bound to antigens immobilized on nitrocellulose after blot transfer from SDS polyacrylamide gels. The applicability of this technology has been demonstrated using antisera raised against the nuclear matrix-pore complex-lamina fraction prepared from Drosophila melanogaster embryos . In so doing, we have established the existence in whole embryo lysates, of two nearly identical forms of the predominant 74-kilodalton polypeptide previously identified in lower resolution studies of the nuclear matrixpore complex-lamina fraction . These species, distinguishable on the basis of a slight difference in SDS PAGE mobilities on low concentration polyacrylamide gels, are immunochemically cross-reactive and have been localized exclusively to the nuclear periphery (nuclear envelope) by indirect immunofluorescence analyses of cryosections . The steady-state levels of these two polypeptides have been examined in total embryo lysates both as a function of embryogenesis and in response to heat shock . The larger species is not detectable in early embryos but approaches levels approximately equal to that of the smaller form by about the temporal midpoint of embryonic development . In response to heat shock, this larger form appears to be converted nearly quantitatively into the lower molecular weight polypeptide . These results, as well as the general reliability of the nitrocellulose blot immunoaffinity-purification methodology, have been substantiated through the use of monoclonal antibodies.A method for the affinity purification of antibodies from diazotized paper blots ofSDS polyacrylamide gels has recently been published by Olmsted (1) . Although a detailed quantitative assessment of the efficiency of this technique was not presented in her report, Olmsted did note that in order to achieve signals with the affinity-purified IgG that were of comparable intensity to those seen with the unfractionated serum, she eluted antibodies from approximately 10 times the amount ofpaper used to characterize those eluates in the reprobe . This, in conjunction with data showing significant residual signal remaining on the original blot after elution, suggested a relatively poor recovery of affinity-purified IgG. Further limitations of the procedure reported by Olmsted include the selection ofdiazotized paper for the original blotnitrocellulose is easier to use, gives higher resolution, and is hence preferred for routine blotting applications-as well as the use of a radiolabeled protein A probe to detect the first antibody bound to the blot. This detection method necessi-

DNA topoisomerase II has been immunochemically identified on protein blots as a major polypeptide component of the Drosophila nuclear matrix-pore complexlamina fraction. Indirect immunofluorescence analyses of larval cryosections have confirmed the nuclear localization of topoisomerase II in situ. Although apparently excluded from the nucleolus, the topoisomerase protein is otherwise distributed throughout the interior of interphase nuclei. Similar immunocytochemical studies performed with permeabilized whole giant cells from third-instar larval salivary glands have shown topoisomerase II to be largely restricted to the polytene chromosomes. Upon nuclear disassembly during mitosis, the topoisomerase polypeptide appears to redistribute diffusely throughout the cell. Faint immunofluorescent staining of mitotic chromosomes is also observed.The Drosophila melanogaster embryo nuclear matrix-pore complex-lamina (NMPCL) fraction is operationally defined as that material which remains insoluble (pellets at 10,000 x g) during sequential treatments with RNase, DNase, 2% Triton X-100, and 1 M NaCl (1). By phase-contrast microscopy, NMPCL components resemble unfractionated nuclei. However, they are almost entirely devoid of DNA, RNA, phospholipid, and histones. By transmission electron microscopy, these nuclear remnants appear as ovoid or spherical structures surrounded by a limiting layer of fibrous material presumably derived from the nuclear lamina. They also contain an internal meshwork termed the nuclear matrix. Nucleolar remnants can be readily identified within the matrix, while higher-magnification examination of the NMPCL periphery reveals the presence of nuclear pore complexes apparently attached to the lamina. The Drosophila NMPCL is morphologically indistinguishable from fractions similarly derived from a variety of vertebrate nuclei (2-5). It is also similar to the rat liver nuclear pore complex-lamina fraction with regard to elements of the nuclear periphery (6-8).When analyzed by one-dimensional NaDodSO4/PAGE, the Drosophila NMPCL is heterogeneous (1). Two major polypeptides, migrating as a closely spaced doublet in the 74-to 76-kDa region of the gel, have been shown to be antigenically related and to be specifically localized to the nuclear envelope in situ (9). These polypeptides are apparently homologous to the three vertebrate lamins, A, B, and C (10, 11). A third major NMPCL band, migrating in the 170-to 190-kDa region of the NaDodSO4/polyacrylamide gel, has been shown to be composed of two immunochemically distinct polypeptides of nearly identical molecular weight (12 (14) and appears to be homologous to the 190-kDa rat liver nuclear pore complex glycoprotein (15). To date, no quantitatively major NMPCL polypeptide has been definitively localized to the nuclear interior in situ.Recent work in our laboratory on NMPCL ATPases (12, 13) has led to the consideration of DNA topoisomerase II.Type II DNA topoisomerases catalyze the relaxation of superhelical turns in topologically constrained DNA by a d...

Barrier-to-autointegration factor (BAF) is potentially a DNA-bridging protein, which directly associates with inner nuclear membrane proteins carrying LEM domains. These features point to a key role in regulation of nuclear function and organization, dependent on interactions between the nuclear envelope and chromatin. To understand the functions of BAF in vivo, Drosophila baf null mutants generated by P-element-mediated imprecise excision were analyzed. Homozygous null mutants showed a typical mitotic mutant phenotype: lethality at the larval-pupal transition with small brains and missing imaginal discs. Mitotic figures were decreased but a defined anaphase defect as reported for C. elegans RNAi experiments was not observed in these small brains, suggesting a different phase or phases of cell cycle arrest. Specific abnormalities in interphase nuclear structure were frequently found upon electron microscopic examination of baf null mutants, with partial clumping of chromatin and convolution of nuclear shape. At the light microscopic level, grossly aberrant nuclear lamina structure and B-type lamin distribution correlated well with the loss of detectable amounts of BAF protein from nuclei. Together, these data represent evidence of BAF's anticipated function in mediating interactions between the nuclear envelope and interphase chromosomes. We thus conclude that BAF plays essential roles in nuclear organization and that these BAF functions are required in both M phase and interphase of the cell cycle.