A myosin heavy-chain-like polypeptide is associated with the nuclear envelope in higher eukaryotic cells.

A high molecular weight polypeptide, identified as an ATPase subunit by direct ultraviolet photoaffinity labeling, has been shown to be a component of nuclear envelope-enriched fractions prepared from a variety of higher eukaryotes (Berrios, M., G. Blobel, and P. A. Fisher, 1983, J. Biol. Chem., 258:4548-4555). In rat liver as well as Drosophila melanogaster embryos, this polypeptide appears to be a form of myosin heavy chain. This conclusion is based on both immunochemical and immunocytochemical data, as well as on the results of CNBr and chymotryptic peptide map analyses. In Drosophila, the identification of this myosin heavy chain-like polypeptide as a nuclear envelope component has been corroborated in situ by indirect immunofluorescence analyses using permeabilized whole cells, mechanically extruded nuclei, and cryosections obtained from a number of larval tissues. Localization appears to be restricted to the nuclear periphery in a manner similar to that observed for the nuclear lamins and the pore complex glycoprotein. Antibodies directed against the Drosophila nuclear envelope ATPase have also been shown to decorate mammalian and higher plant cell nuclei in situ. Implications for intracellular nuclear mobility and for nucleocytoplasmic exchange of macromolecules in vivo are discussed.

An RNase-sensitive particle containing Drosophila melanogaster DNA topoisomerase II.

Most DNA topoisomerase II (topo II) in cell-free extracts of 0-2-h old Drosophila embryos appears to be nonnuclear and remains in the supernatant after low-speed centrifugation (10,000 g). Virtually all of this apparently soluble topo II is particulate with a sedimentation coefficient of 67 S. Similar topo II-containing particles were detected in Drosophila Kc tissue culture cells, 16-19-h old embryos and extracts of progesterone-matured oocytes from Xenopus. Drosophila topo II-containing particles were insensitive to EDTA, Triton X-100 and DNase I, but could be disrupted by incubation with 0.3 M NaCl or RNase A. After either disruptive treatment, topo II sedimented at 9 S. topo II-containing particles were also sensitive to micrococcal nuclease. Results of chemical cross-linking corroborated those obtained by centrifugation. Immunoblot analyses demonstrated that topo II-containing particles lacked significant amounts of lamin, nuclear pore complex protein gp210, proliferating cell nuclear antigen, RNA polymerase II subunits, histones, coilin, and nucleolin. Northern blot analyses demonstrated that topo II-containing particles lacked U RNA. Thus, current data support the notion that nonnuclear Drosophila topo II-containing particles are composed largely of topo II and an unknown RNA molecule(s).

Interconversion of Drosophila nuclear lamin isoforms during oogenesis, early embryogenesis, and upon entry of cultured cells into mitosis.

Two isoforms of a single nuclear lamin, distinguishable on one-dimensional SDS-polyacrylamide gels, have previously been identified in Drosophila nuclei during interphase. A third species, designated lamin Dmmit, has now been identified as soluble in extracts of Drosophila tissue culture cells blocked in mitosis by drugs. An apparently identical form is the only lamin species detectable in late-stage egg chambers and early embryos. Phosphoamino acid analyses suggest that the conversion of lamins Dm1 and Dm2 to lamin Dmmit is brought about by a specific rearrangement of phosphate groups rather than by dramatic net changes in the levels of lamin phosphorylation. The residues involved in these phosphorylation/dephosphorylation reactions have been tentatively mapped to a 17.8-kD cyanogen bromide fragment containing amino acids 385-547. This represents a potential "hinge" domain in the lamin structure between the end of coil 2 and the globular COOH terminus. These results have implications for understanding the regulation of nuclear envelope breakdown during mitosis and karyoskeletal dynamics during oogenesis and early embryogenesis.

Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots.

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 matrix-pore 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.

Isolation and characterization of a proteinaceous subnuclear fraction composed of nuclear matrix, peripheral lamina, and nuclear pore complexes from embryos of Drosophila melanogaster.

Morphologically intact nuclei have been prepared from embryos of Drosophila melanogaster by a simple and rapid procedure. These nuclei have been further treated with high concentrations of DNase I and RNase A followed by sequential extraction with 2% Triton X-100 and 1 M NaCl to produce a structurally and biochemically distinct preparation designated Drosophila subnuclear fraction I (DSNF-I). As seen by phase-contrast microscopy, DSNF-I is composed of material which closely resembles unfractionated nuclei; residual internal nuclear structures including nucleolar remnants are clearly visible. By transmission electron microscopy, nuclear lamina, pore complexes, and a nuclear matrix are similarly identified. Biochemically, DSNF-I is composed almost entirely of protein (greater than 93%). SDS PAGE analysis reveals several major polypeptides; species at 174,000, 74,000, and 42,000 predominate. A polypeptide coincident with the Coomassie Blue-stainable 174-kdalton band has been shown by a novel technique of lectin affinity labeling to be a glycoprotein; a glycoprotein of similar or identical molecular weight has been found to be a component of nuclear envelope fractions isolated from the livers of rats, guinea pigs, opossums, and chickens. Antisera against several of the polypeptides in DSNF-I have been obtained from rabbits, and all of them show only little or no cross-reactivity with Drosophila cytoplasmic fractions. Initial results of immunocytochemical studies, while failing to positively localize either the 174- or 16-kdalton polypeptides, demonstrate a nuclear localization of the 74-kdalton antigen in all of several interphase cell types obtained from both Drosophila embryos and third-instar larvae.

Developmental regulation of Drosophila DNA topoisomerase II.

Affinity-purified polyclonal antibodies were used to quantitate steady-state levels of DNA topoisomerase II (topo II) throughout Drosophila development. Although wide fluctuations were recorded at different stages, these fluctuations were paralleled by changes in levels of the nuclear lamin, a nuclear structural protein used as an internal standard. The exception to this was adult males where lamin levels were significantly elevated relative to topo II. Northern blot analyses of topo II and lamin mRNA, performed in conjunction with immunoblot analyses of protein revealed fluctuations in levels of the two different messages that paralleled changes in each other and in their respective translation products. Biochemical and immunochemical analyses were complemented by indirect immunofluorescence and immunoperoxidase experiments performed in situ. topo II was found distributed throughout nuclei in most but not all cell types examined. These results for Drosophila topo II are apparently at odds with those obtained by others working in vertebrate systems (see, for example, Heck, M. M. S. and W. C. Earnshaw. 1986. J. Cell Biol. 103:2569-2581; Heck, M. M. S., W. N. Hittelman, and W. C. Earnshaw. 1988. Proc. Natl. Acad. Sci. USA. 85:1086-1090) and suggest that in Drosophila, topo II may not be a useful marker for the proliferative state.

Biosynthesis and interconversion of Drosophila nuclear lamin isoforms during normal growth and in response to heat shock.

Two major immunocross-reactive polypeptides of the Drosophila nuclear envelope, distinguishable in interphase cells on the basis of one-dimensional SDS-PAGE mobility, have been localized to the nuclear lamina by immunoelectron microscopy. These have been designated lamins Dm1 and Dm2. Both lamins are apparently derived posttranslationally from a single, primary translation product, lamin Dm0. A pathway has been established whereby lamin Dm0 is processed almost immediately upon synthesis in the cytoplasm to lamin Dm1. Processing occurs posttranslationally, is apparently proteolytic, and has been reconstituted from cell-free extracts in vitro. Processing in vitro is ATP dependent. Once assembled into the nuclear envelope, a portion of lamin Dm1 is converted into lamin Dm2 by differential phosphorylation. Throughout most stages of development and in Schneider 2 tissue culture cells, both lamin isoforms are present in approximately equal abundance. However, during heat shock, lamin Dm2 is converted nearly quantitatively into lamin Dm1. Implications for understanding the regulation of nuclear lamina plasticity through normal growth and in response to heat shock are discussed.

Heat shock-induced changes in the structural stability of proteinaceous karyoskeletal elements in vitro and morphological effects in situ.

Karyoskeletal protein fractions prepared from Drosophila melanogaster embryos contain morphologically identifiable remnants of nuclear pore complexes and peripheral lamina as well as what appears to be an internal nuclear "matrix" (Fisher, P. A., M. Berrios, and G. Blobel, 1982, J. Cell Biol., 92: 674-686). Structural stability of these proteinaceous assemblies is dependent on thermal incubation in vitro (37 degrees C, 15 min) before subfractionation of nuclei. In the absence of such incubation, greater than 90% of the total karyoskeletal protein including major polypeptide components of internal "matrix," pore complexes, and the peripheral lamina, is solubilized by 1 M NaCl. In vivo heat shock induces karyoskeletal stabilization resembling that resulting from thermal incubation in vitro. Immunocytochemical studies have been used to establish the effects of heat shock on the organization and distribution of major karyoskeletal marker proteins in situ. Taken together, these results are consistent with the notion that in vivo, regulation of karyoskeletal plasticity (and perhaps form) may be a functionally significant component of the Drosophila heat shock response. They also have broad practical implications for studies pertaining to the structure and function of karyoskeletal protein (nuclear "matrix") fractions isolated from higher eukaryotic cells.

Drosophila nuclear lamin precursor Dm0 is translated from either of two developmentally regulated mRNA species apparently encoded by a single gene.

A cDNA clone encoding a portion of Drosophila nuclear lamins Dm1 and Dm2 has been identified by screening a lambda-gt11 cDNA expression library using Drosophila lamin-specific monoclonal antibodies. Two different developmentally regulated mRNA species were identified by Northern blot analysis using the initial cDNA as a probe, and full-length cDNA clones, apparently corresponding to each message, have been isolated. In vitro transcription of both full-length cDNA clones in a pT7 transcription vector followed by in vitro translation in wheat germ lysate suggests that both clones encode lamin Dm0, the polypeptide precursor of lamins Dm1 and Dm2. Nucleotide sequence analyses confirm the impression that both cDNA clones code for the identical polypeptide, which is highly homologous with human lamins A and C as well as with mammalian intermediate filament proteins. The two clones differ in their 3'-untranslated regions. In situ hybridization of lamin cDNA clones to Drosophila polytene chromosomes shows only a single locus of hybridization at or near position 25F on the left arm of chromosome 2. Southern blot analyses of genomic DNA are consistent with the notion that a single or only a few highly similar genes encoding Drosophila nuclear lamin Dm0 exist in the genome.

Spatial Organization and Time Dependence of Jupiter's Tropospheric Temperatures, 1980-1993.

The spatial organization and time dependence of Jupiter's temperatures near 250-millibar pressure were measured through a jovian year by imaging thermal emission at 18 micrometers. The temperature field is influenced by seasonal radiative forcing, and its banded organization is closely correlated with the visible cloud field. Evidence was found for a quasi-periodic oscillation of temperatures in the Equatorial Zone, a correlation between tropospheric and stratospheric waves in the North Equatorial Belt, and slowly moving thermal features in the North and South Equatorial Belts. There appears to be no common relation between temporal changes of temperature and changes in the visual albedo of the various axisymmetric bands.

Thermal stabilization of putative karyoskeletal protein-enriched fractions from Saccharomyces cerevisiae.

Elevated growth temperature (heat shock) promoted the structural stability of karyoskeletal protein-enriched fractions isolated from Saccharomyces cerevisiae. Similar stabilization could be induced by brief incubation of nuclei at 37 degrees C in vitro. These results are similar to those reported for higher eucaryotes and have practical implications for investigation of the karyoskeleton in S. cerevisiae.

p75, a polypeptide component of karyoskeletal protein-enriched fractions associated with transcriptionally active loci of Drosophila melanogaster polytene chromosomes.

A 75-kilodalton polypeptide has been identified which copurifies with karyoskeletal protein-enriched fractions prepared from Drosophila melanogaster embryos. Results of indirect immunofluorescence experiments suggest that this protein, here designated p75, is primarily associated with puffed regions of larval salivary gland polytene chromosomes. In nonpolytenized Schneider 2 tissue culture cells, p75 appeared to be localized throughout the nuclear interior during interphase. In mitotic cells, p75 was redistributed diffusely. A possible role for karyoskeletal elements in transcriptional regulation is discussed.

Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes.

Bacteriophage T7 RNA polymerase and derivatives that contain the nuclear localization signal (NLS) from simian virus 40 T antigen (J. J. Dunn, B. Krippl, K. Bernstein, H. Westphal, and F. W. Studier, Gene 68:259-266, 1988) were expressed in Saccharomyces cerevisiae under the control of the inducible GAL1 promoter. As determined by indirect immunofluorescence, T7 RNA polymerase lacking the NLS remained mostly in the cytoplasm, whereas the protein containing the NLS localized to the nucleus. T7 RNA polymerase containing a mutated NLS remained mostly cytoplasmic. Hybrid proteins containing the NLS near the amino terminus were enzymatically active in the yeast cell, initiating transcription selectively at a T7 promoter placed in yeast chromosomal or plasmid DNA and stopping at a specific T7 terminator. At limiting enzyme concentrations, 5 to 10 times as much target RNA was produced when the polymerase contained the NLS, presumably because more enzyme reached the nucleus. Although substantial amounts of intact mRNA accumulated, no translation of target mRNAs in yeast cells was detected.

Localization and posttranslational modifications of otefin, a protein required for vesicle attachment to chromatin, during Drosophila melanogaster development.

Otefin is a peripheral protein of the inner nuclear membrane in Drosophila melanogaster. Here we show that during nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. With the exception of sperm cells, otefin colocalizes with lamin Dm0 derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. In the egg chamber, otefin accumulates in the cytoplasm, in the nuclear periphery, and within the nucleoplasm of the oocyte, in a pattern similar to that of lamin Dm0 derivatives. There is a relatively large nonnuclear pool of otefin present from stages 6 to 7 of egg chamber maturation through 6 to 8 h of embryonic development at 25 degrees C. In this pool, otefin is peripherally associated with a fraction containing the membrane vesicles. This association is biochemically different from the association of otefin with the nuclear envelope. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. A major site for cdc2 kinase phosphorylation in vitro was mapped to serine 36 of otefin. Together, our data suggest an essential role for otefin in the assembly of the Drosophila nuclear envelope.

Interactions among Drosophila nuclear envelope proteins lamin, otefin, and YA.

The nuclear envelope plays many roles, including organizing nuclear structure and regulating nuclear events. Molecular associations of nuclear envelope proteins may contribute to the implementation of these functions. Lamin, otefin, and YA are the three Drosophila nuclear envelope proteins known in early embryos. We used the yeast two-hybrid system to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, lamin Dm, interacts with both otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina. In agreement with this interaction, lamin and otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among lamin, otefin, and YA. Lamin's rod domain interacts with the complete otefin protein, with otefin's hydrophilic NH2-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between lamin and YA showed that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA's COOH-terminal region is necessary and sufficient for interaction with lamin. Our results suggest that interactions with lamin might mediate or stabilize the localization of otefin and YA in the nuclear lamina. They also suggest that the need for both otefin and lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins.

Healthy ageing of cloned sheep.

The health of cloned animals generated by somatic-cell nuclear transfer (SCNT) has been of concern since its inception; however, there are no detailed assessments of late-onset, non-communicable diseases. Here we report that SCNT has no obvious detrimental long-term health effects in a cohort of 13 cloned sheep. We perform musculoskeletal assessments, metabolic tests and blood pressure measurements in 13 aged (7-9 years old) cloned sheep, including four derived from the cell line that gave rise to Dolly. We also perform radiological examinations of all main joints, including the knees, the joint most affected by osteoarthritis in Dolly, and compare all health parameters to groups of 5-and 6-year-old sheep, and published reference ranges. Despite their advanced age, these clones are euglycaemic, insulin sensitive and normotensive. Importantly, we observe no clinical signs of degenerative joint disease apart from mild, or in one case moderate, osteoarthritis in some animals. Our study is the first to assess the long-term health outcomes of SCNT in large animals.

[Specific DNA sequences are associated with nuclear envelopes of pseudonurse cells in Drosophila melanogaster otu 11].

Nuclei of ovarian pseudonurse cells from the mutant strain of Drosophila melanogaster otu 11 are suitable for mapping the attachment of chromosomes to the nuclear envelope (NE). Loci in contact with the NE included region 20CD of the X chromosome, region 41 of chromosome 2, the proximal end of region 81 of chromosome 3, and region 101 of chromosome 4. In situ hybridization revealed that all 4 regions contained sequences homologous to clone lambda20p1.4. DNA of clone lambda20p1.4 was previously found to bind specifically to purified D. melanogaster lamins. These results suggest that specific DNA sequences are involved in attachment of chromosomes to NE in vivo.

[A combined approach to mapping the proximal border of the right arm of polytene chromosome 2 in Drosophila melanogaster otu 11].

A combined approach based on cytological observations in situ hybridization, and qualitative Southern-blot analyses were used to localize the proximal border of the right arm of polytene chromosome 2 in Drosophila melanogaster otu 11 strain. A genetically functional chromosome 2 is bounded by "deletions" C', C, D, B, A and ms2-10. Using in situ hybridization in conjunction with comparative quantitative Southern-blot hybridization to deletions in centromeric heterochromatin, DNA of specific centromeric clone lambda20p1.4 was localized with respect to "deletions" and on otu 11 polytene chromosomes. Comparison of hybridization sites of lambda20p1.4 on polytene chromosomes, and its amount in mutant lines of D. melanogaster carrying known "deletions" in the centromeric heterochromatin enabled us to localize the proximal border of the right arm of chromosome 2 in D. melanogaster otu 11 strain between the 39/40 region and hybridization site of the k20p1.4 DNA fragment.

Drosophila gp210, an invertebrate nuclear pore complex glycoprotein.

Monoclonal antibodies were used to localize the putative Drosophila homolog of mammalian nuclear pore complex glycoprotein, gp210, to Drosophila nuclear pore complexes. Both immunofluorescence and immunoelectron microscopy were performed. To the best of our knowledge, this establishes Drosophila gp210 as the first invertebrate gp210 homolog. Results of developmental studies demonstrated that like nuclear lamin and DNA topoisomerase II, gp210 is found abundantly in nonnuclear form early in embryogenesis where it presumably fuels the rapid assembly of new nuclei. Unlike thse other two proteins, gp210 levels are maximal early after wich they decrease significantly; in addition, nonnuclear gp210 found in early Drosophila embryos is apparently associated with membrane vesicles. These results have implications for understanding the regulation of higher eukaryotic nuclear pore complex behavior through development as well as for determining gp210 function genetically.