The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains.

CP190, a protein of 1,096 amino acids from Drosophila melanogaster, oscillates in a cell cycle-specific manner between the nucleus during interphase, and the centrosome during mitosis. To characterize the regions of CP190 responsible for its dynamic behavior, we injected rhodamine-labeled fusion proteins spanning most of CP190 into early Drosophila embryos, where their localizations were characterized using time-lapse fluorescence confocal microscopy. A single bipartite 19-amino acid nuclear localization signal was detected that causes nuclear localization. Robust centrosomal localization is conferred by a separate region of 124 amino acids; two adjacent, nonoverlapping fusion proteins containing distinct portions of this region show weaker centrosomal localization. Fusion proteins that contain both nuclear and centrosomal localization sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion proteins containing only the centrosome localization sequence are found at centrosomes throughout the cell cycle, suggesting that CP190 is actively recruited away from the centrosome by its movement into the nucleus during interphase. Both native and bacterially expressed CP190 cosediment with microtubules in vitro. Tests with fusion proteins show that the domain responsible for microtubule binding overlaps the domain required for centrosomal localization. CP60, a protein identified by its association with CP190, also localizes to centrosomes and to nuclei in a cell cycle-dependent manner. Experiments in which colchicine is used to depolymerize microtubules in the early Drosophila embryo demonstrate that both CP190 and CP60 are able to attain and maintain their centrosomal localization in the absence of microtubules.

Horizontal transfer of Wolbachia between phylogenetically distant insect species by a naturally occurring mechanism.

Wolbachia is a genus of alpha-proteobacteria found in obligate intracellular association with a wide variety of arthropods, including an estimated 10-20% of all insect species [1]. Wolbachia represents one of a number of recently identified 'reproductive parasites' [2] which manipulate the reproduction of their hosts in ways that enhance their own transmission [3] [4] [5] [6] [7] [8] [9]. The influence of Wolbachia infection on the dynamics of host populations has focused considerable interest on its possible role in speciation through reproductive isolation [3] [10] [11] and as an agent of biological control [2] [12] [13]. Although Wolbachia normally undergoes vertical transmission through the maternal line of its host population [14], there is compelling evidence from molecular phylogenies that extensive horizontal (intertaxon) transmission must have occurred [1] [9] [15] [16] [17]. Some of the best candidate vectors for the horizontal transmission of Wolbachia are insect parasitoids [15], which comprise around 25% of all insect species and attack arthropods from an enormous range of taxa [18]. In this study, we used both fluorescence microscopy and PCR amplification with Wolbachia-specific primers to show that Wolbachia can be transmitted to a parasitic wasp (Leptopilina boulardi) from its infected host (Drosophila simulans) and subsequently undergo diminishing vertical transmission in this novel host species. These results are, to our knowledge, the first to reveal a natural horizontal transfer route for Wolbachia between phylogenetically distant insect species.

Assembly of the zygotic centrosome in the fertilized Drosophila egg.

Zygotic centrosome assembly in fertilized Drosophila eggs was analyzed with the aid of an antiserum Rb188, previously shown to be specific for CP190, a 190 kDa centrosome-associated protein (Whitfield et al. (1988) J. Cell Sci. 89, 467-480; Whitfield et al. (1995) J. Cell Sci. 108, 3377-3387). The CP190 protein was detected in two discrete spots, associated with the anterior and posterior ends of the elongating nucleus of Drosophila spermatids. As the spermatids matured, this labelling gradually disappeared and was no longer visible in sperm dissected from spermathecae and ventral receptacles. gamma-Tubulin was also found in association with the posterior end of the sperm nucleus during spermiogenesis, but was not detected in mature sperm. This suggests that CP190 and gamma-tubulin are not present in detectable quantities in fertilizing sperm. CP190 was not detected in association with the sperm nucleus of newly fertilized eggs removed from the uterus, whereas many CP190-positive particles were associated with microtubules of the sperm aster from anaphase I to anaphase II. These particles disappeared during early telophase II and only one pair of CP190-positive spots remained visible at the microtubule focus of the sperm aster. These spots were associated with one aster through telophase, and then moved away to form two smaller asters from which the first mitotic spindle was organized. Colchicine treatment suggested that at least some CP190 protein is an integral part of the centrosome rather than merely being transported along microtubules. Centrosomal localization of the CP190 antigen was prevented by incubation of the permeabilized zygote in 20 mM EDTA.

Characterization of a family of nuclear and chromosomal proteins identified by a monoclonal antibody.

A monoclonal antibody (3C5) isolated from a mouse immunized with human chromatin stained the nuclei of all cultured cell types tested by indirect immunofluorescence. Experiments with HeLa and PtK1 cells demonstrated striking cell-cycle-related changes in the staining properties of the target antigen. A rapid increase in nuclear fluorescence was seen in prophase, with antigen located between the condensing chromosomes. In metaphase and anaphase cells antigen was present throughout the cytoplasm with the chromosomes apparently unstained. However, isolated metaphase chromosomes showed intense, peripheral staining. In telophase cells immunofluorescent staining was most intense among the decondensing chromosomes and by early G1 staining was predominantly nuclear. Nuclear fluorescence faded as cells progressed through interphase. By protein blotting and immunostaining, 3C5 recognized protein bands with subunit molecular weights of 130, 73, 50, 38, 32 and 22 to 25 kDa. These bands were present in all human and rodent cultured cell types tested. All bands were extracted by 6 M urea or 1% sodium dodecyl sulfate (SDS) but not by Triton X-100. Our results provide evidence against the involvement of a common carbohydrate moiety, in vitro proteolysis or non-specific cross reaction in this multi-banded pattern. The same family of proteins was detected in mitotic and interphase cells, suggesting that the changes in immunofluorescent staining through mitosis are due to changes in antigen accessibility. Subcellular fractionation experiments showed that all major bands were present in the nuclear fraction. Only two (50 and 32 kDa) were detected also in the post-nuclear membrane fraction and none were present in the soluble cytoplasmic fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody identifies a 215 000-dalton nuclear envelope protein restricted to certain cell types.

The monoclonal antibody, AGF2.3, was isolated from mice immunised with the human promyeloid cell line HL60. By immunofluorescence and immunoelectron microscopy the antibody was shown to bind to the nuclear envelope in uninduced HL60 cells. Immunofluorescent staining was reduced to very low levels in HL60 cells induced to mature to monocytes or neutrophils by addition of 12-0-tetradecanoylphorbol-13-acetate or dimethyl sulfoxide respectively. Blood neutrophils did not express the antigen. Weak immunofluorescent staining of cell nuclei was observed in peripheral blood lymphocytes and in sections of normal human kidney, tonsil and skin epithelium. The AGF2.3 antigen was strongly expressed on the nuclei of 21/21 haemopoietic cell lines and 21/25 permanent non-haemopoietic cell lines representing various cell types. In contrast, the antigen was not expressed by any of six primary (untransformed) cell cultures. These included fibroblasts, endothelial cells and keratinocytes. The antigen was expressed in the Q10 SV-40 transformed cell line derived from a non-expressing primary fibroblast culture. AGF2.3 antibody precipitated a protein with an apparent subunit molecular weight of approximately 215 kDa from Triton X-100 extracts of HL60 and HeLa cells labelled with 35S-methionine. This protein was not detectable in extracts of primary skin fibroblasts prepared in parallel. We conclude that AGF2.3 antibody recognises a previously undescribed protein associated with the nuclear envelope which is expressed at high levels in most transformed cell lines but which is weakly expressed or absent in normal tissues and primary cell cultures.

Evidence for the direct involvement of lamins in the assembly of a replication competent nucleus.

Monoclonal antibodies linked to paramagnetic immunobeads (Dynabeads) have been used to investigate the distribution of lamin B3 in fractions of Xenopus egg extracts. Lamin B3 behaved as if it were completely soluble and did not co-precipitate with membrane fractions. Sperm pronuclei assembled in lamin depleted egg extracts were compared to pronuclei assembled in mock depleted extracts by field emission in-lens electron scanning microscopy (FEISEM). This technique revealed that the surface structures of the nuclear envelopes, including nuclear pores, appeared to be identical, indicating that lamin depletion does not affect nuclear envelope assembly. One-dimensional and two-dimensional gel electrophoresis was used to analyze soluble proteins co-precipitated with lamin B3 on Dynabeads. Our results indicate that two major species (molecular mass: 105 kDa and 57 kDa) specifically co-precipitate with lamin B3 as well as several minor species. At least three proteins which co-precipitate with lamin B3 were identified as nuclear matrix proteins. Lamin B3 was separated from these proteins and re-inoculated into lamin depleted extracts. This resulted in partial rescue of both lamina assembly and DNA replication. These results imply that lamin B3 is directly involved in the assembly of structures required for the initiation of DNA replication.

Single-locus complementary sex determination in Diadegma chrysostictos (Gmelin) (Hymenoptera: Ichneumonidae).

Following the establishment of isofemale lines and subsequent inbreeding, the ichneumonid parasitoid wasp Diadegma chrysostictos (Gmelin) was shown by segregation of polymorphic alloenzyme loci to have single-locus complementary sex determination (sl-CSD). This and the biparental nature of diploid males was confirmed using two independent Mendelian recessive phenotypic markers. The existence of diploid males, sl-CSD, and the abrogation of diploid males following outbreeding was further confirmed by flow cytometry, a potentially general method that is independent of the maternal sex allocation or the need for genetic markers. Estimates of the number of sex alleles in several British populations demonstrated 17-19 alleles in Britain, with a decline toward the northerly limit of the parasitoid's range, varying from 16 in the south of England to 4-5 in central Scotland, in broad agreement with the rate of attainment of a male-biased sex ratio when used to establish en masse laboratory cultures. These data represent the second confirmation of the existence of sl-CSD in the Ichneumonidae (and the first in the Campopleginae subfamily), lending further support to the notion that sl-CSD was the ancestral condition in the Aculeata/Ichneumonoidea clade (Cook 1993a; Periquet et al. 1993).

Centriole and centrosome dynamics during the embryonic cell cycles that follow the formation of the cellular blastoderm in Drosophila.

We have used immunofluorescence and electron microscopy to examine centrosome dynamics during the first postblastodermic mitoses in the Drosophila embryo. The centrosomal material, as recognized by antibodies against CP190 and gamma-tubulin, does not show the typical shape changes observed in syncytial embryos, but remains compact throughout mitosis. Centrioles, however, behave as during the syncytial mitoses, with each daughter cell inheriting two separated centrioles at the end of telophase. During interphase in epithelial cells that have a distinct G1 phase, two isolated centrioles are found, suggesting that the separation of sister centrioles is tightly coupled to a mitotic oscillator in both the "abbreviated" and the "complete" embryonic division cycles. The centrioles of the Drosophila embryo sharply differed from the sperm basal body, having a cartwheel structure with nine microtubular doublets and a central tubule. This "immature" centriolar morphology was shown to persist throughout embryonic development, clearly demonstrating that these centrioles are able to replicate despite their apparently neotenic structure.

Characterization of monoclonal antibodies to histone 2B. Localization of epitopes and analysis of binding to chromatin.

Two mouse monoclonal IgM antibodies have been isolated which bind to histone 2B (H2B), as shown by protein blotting and immunostaining and by solid-phase radioimmunoassay (RIA). One of these (HBC-7) was specific for H2B by both techniques whereas the other (2F8) cross-reacted with histone H1 by RIA. Both antibodies failed to recognize H2B limit peptides from trypsin-digested chromatin and did not bind to Drosophila H2B, which differs extensively from vertebrate H2B only in the N-terminal region. These findings indicate that both antibodies recognize epitopes within the trypsin-sensitive, N-terminal region comprising residues 1-20. Binding of antibody HBC-7 was inhibited by in vitro ADP-ribosylation of H2B at glutamic acid residue 2. This strongly suggests that the epitope recognized by HBC-7 is located at the N-terminus of H2B, probably between residues 1 and 8. We have used solid-phase radioimmunoassay to investigate factors which influence the accessibility of this epitope in chromatin. Removal of H1 ('stripping') from high-molecular-mass chromatin had no effect on HBC-7 binding, nor was any difference observed between binding to stripped chromatin and to 146-base-pair (bp) core particles derived from it by nuclease digestion. These results suggest that accessibility of the N-terminal region of H2B is not influenced by H1 itself or by the size or conformation of linker DNA. In contrast, binding of antibody HBC-7 to 146-bp core particles derived from unstripped chromatin was reduced by up to 70%. Binding was restored by exposure of these core particles to the conditions used for stripping. Analysis of the protein content of core particle preparations from stripped and unstripped chromatin suggests that these findings may be attributable to redistribution of non-histone proteins during nuclease digestion. Pre-treatment of high-molecular-mass chromatin or 146-bp core particles with the intercalating dye ethidium bromide resulted in a severalfold increase in binding of HBC-7. The major changes in nucleosome morphology induced by ethidium are therefore accompanied by an increase in accessibility of the N-terminal region of H2B, possibly as a direct result of changes in the spatial relationship between H2B and core DNA.

Two distinct mechanisms localise cyclin B transcripts in syncytial Drosophila embryos.

We demonstrate that two independent mechanisms act on maternally derived cyclin B transcripts to concentrate the transcripts at the posterior pole of the Drosophila oocyte and at the cortex of the syncytial embryo. The cortical accumulation occurs because the cyclin B transcript is concentrated around nuclei and comigrates with them to the cortex. The perinuclear localisation of the transcript is blocked by inhibitors of microtubule polymerisation and the transcript colocalises with microtubular structures during the cell cycle, suggesting that the transcript is associated either directly or indirectly with microtubules. Neither microtubules nor actin filaments are required to maintain the posterior concentration of cyclin B transcripts. Instead, this seems to depend on the association of the transcripts with a component of the posterior cytoplasm. The distribution pattern of the transcript at the posterior pole throughout embryogenesis and in a variety of mutant embryos suggests that this component is associated with polar granules.

Microsatellite frequency and size variation in the parthenogenetic parasitic wasp Venturia canescens (Gravenhorst) (Hymenoptera: Ichneumonidae).

Nine genomic libraries of the parthenogenetic wasp Venturia canescens were screened for microsatellite loci. In contrast to other Hymenoptera (GT)n and not (CT)n, was the predominant repeat category found with 14 kb and 28 kb genomic DNA between loci, respectively. Mono- and trinucleotide microsatellites were rarer, occurring at frequencies between 231 kb and 589 kb of genome, whilst tetranucleotide repeats are scarce, with (ATTC)n and (CCGG)n loci occurring every 692 kb and 983 kb, respectively, and only one small imperfect (GATA)n locus and no (GACA)n loci were revealed. Over 70% of the dinucleotide, and all the trinucleotide microsatellites were small (less than eleven repeats), whilst 60% to 80% of loci were imperfect. Moreover, very few compound microsatellites and only a single association between different microsatellites were observed.

The 190 kDa centrosome-associated protein of Drosophila melanogaster contains four zinc finger motifs and binds to specific sites on polytene chromosomes.

Microinjection of a bacterially expressed, TRITC labelled fragment of the centrosome-associated protein CP190 of Drosophila melanogaster, into syncytial Drosophila embryos, shows it to associate with the centrosomes during mitosis, and to relocate to chromatin during interphase. Indirect immunofluorescence staining of salivary gland chromosomes of third instar Drosophila larvae, with antibodies specific to CP190, indicate that the protein is associated with a large number of loci on these interphase polytene chromosomes. The 190 kDa CP190 protein is encoded by a 4.1 kb transcript with a single, long open reading frame specifying a polypeptide of 1,096 amino acids, with a molecular mass of 120 kDa, and an isoelectric point of 4.5. The central region of the predicted amino acid sequence of the CP190 protein contains four CysX2CysX12HisX4His zinc-finger motifs which are similar to those described for several well characterised DNA binding proteins. The data suggest that the function of CP190 involves cell cycle dependent associations with both the centrosome, and with specific chromosomal loci.

Xenopus lamin B3 has a direct role in the assembly of a replication competent nucleus: evidence from cell-free egg extracts.

Xenopus egg extracts which assemble replication competent nuclei in vitro were depleted of lamin B3 using monoclonal antibody L6 5D5 linked to paramagnetic beads. After depletion, the extracts were still capable of assembling nuclei around demembranated sperm heads. Using field emission in lens scanning electron microscopy (FEISEM) we show that most nuclei assembled in lamin B3-depleted extracts have continuous nuclear envelopes and well formed nuclear pores. However, several consistent differences were observed. Most nuclei were small and only attained diameters which were half the size of controls. In a small number of nuclei, nuclear pore baskets, normally present on the inner aspect of the nuclear envelope, appeared on its outer surface. Finally, the assembly of nuclear pores was slower in lamin B3-depleted extracts, indicating a slower overall rate of nuclear envelope assembly. The results of FEISEM were confirmed using conventional TEM thin sections, where again the majority of nuclei assembled in lamin B3-depleted extracts had well formed double unit membranes containing a high density of nuclear pores. Since nuclear envelope assembly was mostly normal but slow in these nuclei, the lamin content of 'depleted' extracts was investigated. While lamin B3 was recovered efficiently from cytosolic and membrane fractions by our procedure, a second minor lamin isoform, which has characteristics similar to those of the somatic lamin B2, remained in the extract. Thus it is likely that this lamin is necessary for nuclear envelope assembly. However, while lamin B2 did not co-precipitate with lamin B3 during immunodepletion experiments, several protein species did specifically associate with lamin B3 on paramagnetic immunobeads. The major protein species associated with lamin B3 migrated with molecular masses of 102 kDa and 57 kDa, respectively, on one-dimensional polyacrylamide gels. On two-dimensional O'Farrell gels the mobility of the 102 kDa protein was identical to the mobility of a major nuclear matrix protein, indicating a specific association between lamin B3 and other nuclear matrix proteins. Nuclei assembled in lamin B3-depleted extracts did not assemble a lamina, judged by indirect immunofluorescence, and failed to initiate semi-conservative DNA replication. However, by reinoculating depleted extracts with purified lamin B3, nuclear lamina assembly and DNA replication could both be rescued. Thus it seems likely that the inability of lamin-depleted extracts to assemble a replication competent nucleus is a direct consequence of a failure to assemble a lamina.

GST-lamin fusion proteins act as dominant negative mutants in Xenopus egg extract and reveal the function of the lamina in DNA replication.

A cDNA encoding Xlamin B1 was cloned from a whole ovary mRNA by RT-PCR. GST-lamin fusion constructs were generated from this cDNA by first creating convenient restriction sites within the Xlamin B1 coding sequence, using PCR directed mutagenesis, and then sub-cloning relevant sequences into pGEX-4T-3. Two expression constructs were made, the first, termed delta 2+ lacked sequences encoding the amino-terminal 'head domain' of lamin B1 but included sequences encoding the nuclear localization signal sequence (NLS). The second expression construct, termed delta 2-, lacked sequences encoding the amino-terminal 'head domain' as well as sequences encoding the NLS. Purified fusion proteins expressed from these constructs, when added to egg extracts prior to sperm pronuclear assembly, formed hetero-oligomers with the endogenous lamin B3. The delta 2+ fusion protein prevented nuclear lamina assembly but not nuclear membrane assembly. The resulting nuclei were small (approximately 10 microns in diameter), did not assemble replication centers and failed to initiate DNA replication. When the delta 2- fusion protein was added to egg extracts prior to sperm pronuclear assembly, lamina assembly was delayed but not prevented. The resulting nuclei although small (approximately 12 microns), did form replication centers and initiated DNA replication. When added to egg extracts after sperm pronuclear assembly was completed delta 2+, but not delta 2-, entered the pre-formed nuclei causing lamina disassembly. However, the disassembly of the lamina by delta 2+ did not result in the disruption of replication centers and indeed these centres remained functional. These results are consistent with the hypothesis that lamina assembly precedes and is required for the formation of replication centers but does not support those centers directly.

Cloning of a gene encoding an antigen associated with the centrosome in Drosophila.

The monoclonal antibody Bx63 recognizes a centrosomal antigen of Drosophila melanogaster by indirect immunofluorescence and identifies two proteins, with apparent molecular weights of 185 x 10(3) and 66 x 10(3), on Western blots. We have used this antibody to isolate five clones (lambda cs1, -2, -3, -4 and lambda j63) from lambda gt11 expression libraries of Drosophila DNA. Using polyclonal anti-centrosomal sera raised against both immunoaffinity-purified Bx63 antigen and electrophoretically purified fusion protein from clone lambda cs3, we have demonstrated that the fusion proteins encoded by four of these clones (lambda cs1-4) share at least two epitopes with the 185 x 10(3) Mr centrosomal antigen. This indicates that clones lambda cs1-4 contain DNA from the gene coding for this protein. The four clones are independent isolates from a single chromosomal site, which we show by in situ hybridization to correspond with salivary gland chromosome region 88E 4-8. A low-abundance transcript of approximately 4.0 x 10(3) bases corresponding to the cloned gene is detected in all stages of the Drosophila life-cycle.

The A- and B-type cyclins of Drosophila are accumulated and destroyed in temporally distinct events that define separable phases of the G2-M transition.

We show that the sequence of Drosophila cyclin B has greater identity with B-type cyclins from other animal phyla than with Drosophila cyclin A, suggesting that the two cyclins have distinct roles that have been maintained in evolution. Cyclin A is not detectable in unfertilized eggs and is present at low levels prior to cellularization of the syncytial embryo. In contrast, the levels of cyclin B remain uniformly high throughout these developmental stages. In cells within cellularized embryos and the larval brain, cyclin A accumulates to peak levels in prophase and is degraded throughout the period in which chromosomes are becoming aligned on the metaphase plate. The degradation of cyclin B, on the other hand, does not occur until the metaphase-anaphase transition. In cells arrested at c-metaphase by treating with microtubule destabilizing drugs to prevent spindle formation, cyclin A has been degraded in the arrested cells, whereas cyclin B is maintained at high levels. These observations suggest that cyclin A has a role in the G2-M transition that is independent of spindle formation, and that entry into anaphase is a key requirement for the degradation of cyclin B.

Transcripts of one of two Drosophila cyclin genes become localized in pole cells during embryogenesis.

Cyclins, originally discovered in the eggs of marine invertebrates, are proteins which undergo dramatic cycles of synthesis followed by degradation at the metaphase-anaphase transition of cell division. That they participate in the G2-M transition is supported by the fact that when synthetic cyclin messenger RNAs from clam and sea urchin are microinjected into the G2-arrested oocytes of Xenopus, they induce maturation. The cyclin of fission yeast is the product of the cdc13 gene, which is known to interact with cdc2, a gene required for the entry into mitosis. We have cloned the genes that encode A-type and B-type cyclins from Drosophila melanogaster by virtue of their sequence similarity to oligonucleotides corresponding to conserved regions of the cyclin genes. We show that both genes encode abundant maternal mRNAs, but whereas the cyclin A mRNA is relatively uniformly distributed before cell formation, the cyclin B mRNA becomes localized to the developing pole cells. In larvae, cyclin A is expressed predominantly in brain and imaginal disks, whereas cyclin B transcripts are abundant in testes.

The Drosophila centrosome-associated protein CP190 is essential for viability but not for cell division.

The Drosophila CP190 and CP60 proteins interact with each other and shuttle between the nucleus in interphase and the centrosome in mitosis. Both proteins can bind directly to microtubules in vitro, and have been shown to associate with a specific pattern of loci on salivary gland polytene chromosomes, but their functions are unknown. Here we show that reducing the level of CP190 or CP60 by >90% in tissue culture cells does not significantly interfere with centrosome or microtubule organisation, with cell division, or with cell viability. However, CP190 is an essential protein, as flies homozygous for mutations in the Cp190 gene die at late pupal stages of development. In larval brains of Cp190 mutants, mitosis is not radically perturbed, and a mutated form of CP190 (CP190DeltaM), that cannot bind to microtubules or associate with centrosomes, can rescue the lethality associated with mutations in the Cp190 gene. Thus, CP190 plays an essential role in flies that is independent of its association with centrosomes or microtubules.