The Compatibility Between Biomphalaria glabrata Snails and Schistosoma mansoni: An Increasingly Complex Puzzle.

This review reexamines the results obtained in recent decades regarding the compatibility polymorphism between the snail, Biomphalaria glabrata, and the pathogen, Schistosoma mansoni, which is one of the agents responsible for human schistosomiasis. Some results point to the snail's resistance as explaining the incompatibility, while others support a "matching hypothesis" between the snail's immune receptors and the schistosome's antigens. We propose here that the two hypotheses are not exclusive, and that the compatible/incompatible status of a particular host/parasite couple probably reflects the balance of multiple molecular determinants that support one hypothesis or the other. Because these genes are involved in a coevolutionary arms race, we also propose that the underlying mechanisms can vary. Finally, some recent results show that environmental factors could influence compatibility. Together, these results make the compatibility between B. glabrata and S. mansoni an increasingly complex puzzle. We need to develop more integrative approaches in order to find targets that could potentially be manipulated to control the transmission of schistosomiasis.

Chromosome positioning is largely unaffected in lymphoblastoid cell lines containing emerin or A-type lamin mutations.

Gene-poor human chromosomes are reproducibly found at the nuclear periphery in proliferating cells. There are a number of inner nuclear envelope proteins that may have roles in chromosome location and anchorage, e.g. emerin and A-type lamins. In the last decade, a number of diseases associated with tissue degeneration and premature aging have been linked with mutations in lamin A or emerin. These are termed laminopathies, with mutations in emerin causing Emery-Dreifuss muscular dystrophy. Despite highly aberrant nuclear distributions of A-type lamins and emerin in lymphoblastoid cell lines derived from patients with emerin or lamin A mutations, little or no change in chromosome location was detected.

Both emerin and lamin C depend on lamin A for localization at the nuclear envelope.

Physical interactions between lamins and emerin were investigated by co-immunoprecipitation of in vitro translated proteins. Emerin interacted with in vitro translated lamins A, B1 and C in co-immunprecipitation reactions. Competition reactions revealed a clear preference for interactions between emerin and lamin C. Structural associations between lamins and emerin were investigated in four human cell lines displaying abnormal expression and/or localisation of lamins A and C. In each cell line absence of lamins A and C from the nuclear envelope (NE) was correlated with mis-localisation of endogenous and exogenous emerin to the ER. In two cell lines that did not express lamin A but did express lamin C, lamin C as well as emerin was mis-localised. When GFP-lamin A was expressed in SW13 cells (which normally express only very low levels of endogenous lamin A and mis-localise endogenous emerin and lamin C), all three proteins became associated with the NE. When GFP-lamin C was expressed in SW13 cells neither the endogenous nor the exogenous lamin C was localised to the NE and emerin remained in the ER. Finally, lamins A and C were selectively eliminated from the NE of HeLa cells using a dominant negative mutant of lamin B1. Elimination of these lamins from the lamina led to the accumulation of emerin as aggregates within the ER. Our data suggest that lamin A is essential for anchorage of emerin to the inner nuclear membrane and of lamin C to the lamina.

Human acrocentric chromosomes with transcriptionally silent nucleolar organizer regions associate with nucleoli.

Human ribosomal gene repeats are distributed among five nucleolar organizer regions (NORs) on the p arms of acrocentric chromosomes. On exit from mitosis, nucleoli form around individual active NORs. As cells progress through the cycle, these mini-nucleoli fuse to form large nucleoli incorporating multiple NORs. It is generally assumed that nucleolar incorporation of individual NORs is dependent on ribosomal gene transcription. To test this assumption, we determined the nuclear location of individual human acrocentric chromosomes, and their associated NORs, in mouse> human cell hybrids. Human ribosomal genes are transcriptionally silent in this context. Combined immunofluorescence and in situ hybridization (immuno-FISH) on three-dimensional preserved nuclei showed that human acrocentric chromosomes associate with hybrid cell nucleoli. Analysis of purified nucleoli demonstrated that human and mouse NORs are equally likely to be within a hybrid cell nucleolus. This is supported further by the observation that murine upstream binding factor can associate with human NORs. Incorporation of silent NORs into mature nucleoli raises interesting issues concerning the maintenance of the activity status of individual NORs.

The spatial organization of human chromosomes within the nuclei of normal and emerin-mutant cells.

To fully understand genome function, the linear genome map must be integrated with a spatial map of chromosomes in the nucleus. Distinct nuclear addresses for a few human chromosomes have been described. Previously we have demonstrated that the gene-rich human chromosome 19 is located in a more central position in the nucleus than the similarly sized, but gene-poor, chromosome 18. To determine whether these two chromosomes are a paradigm for the organization of chromatin in the human nucleus, we have now analysed the nuclear organization of every human chromosome in diploid lymphoblasts and primary fibroblasts. We find that the most gene-rich chromosomes concentrate at the centre of the nucleus, whereas the more gene-poor chromosomes are located towards the nuclear periphery. In contrast, we find no significant relationship between chromosome size and position within the nucleus. Proteins of the nuclear membrane or lamina are candidates for molecules that might anchor regions of the genome at the nuclear periphery and it has been suggested that disruption of this organization may play a role in some disease pathologies. We show that the intranuclear organization of chromosomes is not altered in cells that lack the integral nuclear membrane protein emerin, from an individual with X-linked Emery--Dreifuss muscular dystrophy. This suggests that emerin is not necessary for localizing chromosomes at the nuclear periphery and that the muscular dystrophy phenotype in such individuals is not due to grossly altered nuclear organization of chromatin.

Re-modelling of nuclear architecture in quiescent and senescent human fibroblasts.

Spatial organisation of the genome within the nucleus can play a role in maintaining the expressed or silent state of some genes [1]. There are distinct addresses for specific chromosomes, which have different functional characteristics, within the nuclei of dividing populations of human cells [2]. Here, we demonstrate that this level of nuclear architecture is altered in cells that have become either quiescent or senescent. Upon cell cycle exit, a gene-poor human chromosome moves from a location at the nuclear periphery to a more internal site in the nucleus, and changes its associations with nuclear substructures. The chromosome moves back toward the edge of the nucleus at a distinctive time after re-entry into the cell cycle. There is a 2-4 hour period at the beginning of G1 when the spatial organisation of these human chromosomes is established. Lastly, these experiments provide evidence that temporal control of DNA replication can be independent of spatial chromosome organisation. We conclude that the sub-nuclear organisation of chromosomes in quiescent or senescent mammalian somatic cells is fundamentally different from that in proliferating cells and that the spatial organisation of the genome is plastic.

Differences in the localization and morphology of chromosomes in the human nucleus.

Using fluorescence in situ hybridization we show striking differences in nuclear position, chromosome morphology, and interactions with nuclear substructure for human chromosomes 18 and 19. Human chromosome 19 is shown to adopt a more internal position in the nucleus than chromosome 18 and to be more extensively associated with the nuclear matrix. The more peripheral localization of chromosome 18 is established early in the cell cycle and is maintained thereafter. We show that the preferential localization of chromosomes 18 and 19 in the nucleus is reflected in the orientation of translocation chromosomes in the nucleus. Lastly, we show that the inhibition of transcription can have gross, but reversible, effects on chromosome architecture. Our data demonstrate that the distribution of genomic sequences between chromosomes has implications for nuclear structure and we discuss our findings in relation to a model of the human nucleus that is functionally compartmentalized.

Putting the genome on the map.

The first complete genomic sequence of a eukaryote (Saccharomyces cerevisiae) has already been accomplished. It is estimated that the sequence of the human genome will be known early in the next millennium. Yet it is already apparent that, despite their immense length, these linear primary sequence maps will be inadequate descriptions of the eukaryotic genome, be it of a budding yeast or a human. To reflect our growing awareness of the importance of spatial context in chromosome function and in gene expression we argue that a more complete map of the genome should seek to embody the richness of information that we expect of the maps we use to navigate our way around the outside world.

The extended packaging sequence of MoMLV contains a constitutive mRNA nuclear export function.

The present report shows that incorporation of defined sequences from the Moloney murine leukaemia virus (MoMLV) into Rex dependent expression vectors based on the human T-cell leukaemia virus (HTLV-1) allows Rex independent gene expression. Deletion mutagenesis of the MoMLV derived sequences allowed this function to be localised to a 312 nt length sequence overlapping the MoMLV gag p15/p12 open reading frame. This 'extended packaging sequence' has been reported to markedly increase the titre of in vitro packaged retroviral vectors. Using fluorescent in situ hybridisation combined with confocal microscopy we show that the 312 nt element can replace Rex mediated nuclear export and expression of transcripts containing HTLV-1 cis acting repressive elements. Our observations are consistent with the extended packaging sequence of MoMLV exerting a constitutive mRNA nuclear export function.

Nucleocytoplasmic transport of HTLV-1 RNA is regulated by two independent LTR encoded nuclear retention elements.

Appropriate expression of HTLV-1 genes requires transcriptional transactivation by Tax and post-transcriptional regulation by Rex, both mediated by LTR encoded RNA sequences. Using a combination of deletion mutagenesis, Rex-reporter CAT assays, fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy it was established that in the absence of Rex, CAT mRNAs harboring HTLV-1 LTR sequences were unable to leave the nucleus. Deletion of the known U5 encoded cis-acting repressing sequence (CRS) led to a partial release of nuclear retention. A novel regulatory element overlapping the 3' Rex responsive element (RxRE) region was shown to prevent export and expression of these transcripts. Deletion of both the 5' LTR encoded CRS and 3' LTR encoded downstream repressive sequence (3' CRS) led to constitutive mRNA nuclear export and gene expression, independently of Rex. The locations of the two regulatory elements indicate that while the 5' CRS selectively acts to hinder export of unspliced transcripts, the 3' CRS has the capacity to induce nuclear retention of all HTLV-1 transcripts, and therefore could potentially contribute to viral latency in infected cells.

Identification of an interchromosomal compartment by polymerization of nuclear-targeted vimentin.

A number of structural and functional subnuclear compartments have been described, including regions exclusive of chromosomes previously hypothesized to form a reactive nuclear space. We have now explored this accessible nuclear space and interchromosomal nucleoplasmic domains experimentally using Xenopus vimentin engineered to contain a nuclear localization signal (NLS-vimentin). In stably transfected human cells incubated at 37 degrees C, the NLS-vimentin formed a restricted number of intranuclear speckles. At 28 degrees C, the optimal temperature for assembly of the amphibian protein, NLS-vimentin progressively extended with time out from the speckles into strictly orientated intranuclear filamentous arrays. This enabled us to observe the development of a system of interconnecting channel-like areas. Quantitative analysis based on 3-D imaging microscopy revealed that these arrays were localized almost exclusively outside of chromosome territories. During mitosis the filaments disassembled and dispersed throughout the cytoplasm, while in anaphase-telophase the vimentin was recruited back into the nucleus and reassembled into filaments at the chromosome surfaces, in distributions virtually identical to those observed in the previous interphase. The filaments also colocalized with specific nuclear RNAs, coiled bodies and PML bodies, all situated outside of chromosome territories, thereby interlinking these structures. This strongly implies that these nuclear entities coexist in the same interconnected nuclear compartment. The assembling NLS-vimentin is restricted to and can be used to delineate, at least in part, the formerly proposed reticular interchromosomal domain compartment (ICD). The properties of NLS-vimentin make it an excellent tool for performing structural and functional studies on this compartment.

Association of pKi-67 with satellite DNA of the human genome in early G1 cells.

pKi-67 is a nucleolar antigen that provides a specific marker for proliferating cells. It has been shown previously that pKi-67's distribution varies in a cell cycle-dependent manner: it coats all chromosomes during mitosis, accumulates in nuclear foci during G1 phase (type I distribution) and localizes within nucleoli in late G1 S and G2 phase (type II distribution). Although no function has as yet been ascribed to pKi-67, it has been found associated with centromeres in G1. In the present study the distribution pattern of pKi-67 during G1 in human dermal fibroblasts (HDFs) was analysed in more detail. Synchronization experiments show that in very early G1 cells pKi-67 coincides with virtually all satellite regions analysed, i.e. with centromeric (alpha-satellite), telomeric (minisatellite) and heterochromatic blocks (satellite III) on chromosomes 1 and Y (type Ia distribution). In contrast, later in the G1 phase, a smaller fraction of satellite DNA regions are found collocalized with pKi-67 foci (type Ib distribution). When all pKi-67 becomes localized within nucleoli, even fewer satellite regions remain associated with the pKi-67 staining. However, all centromeric and short arm regions of the acrocentric chromosomes, which are in very close proximity to or even contain the rRNA genes, are collocalized with anti-pKi-67 staining throughout the remaining interphase of the cell cycle. Thus, our data demonstrate that during post-mitotic reformation and nucleogenesis there is a progressive decline in the fraction of specific satellite regions of DNA that remain associated with pKi-67. This may be relevant to nucleolar reformation following mitosis.

Centrosome amplification as a possible mechanism for numerical chromosome aberrations in cerebral primitive neuroectodermal tumors with TP53 mutations.

Although alterations in chromosome number have frequently been detected in human tumor cells and associated with tumor initiation and progression, the causal mechanisms are still not understood. One protein known to be involved in maintaining genetic stability is tumor suppressor p53. In mice, p53 has been implicated in the maintenance of diploidy (Cross et al., 1995) and the regulation of centrosome duplication (Fukasawa et al., 1996). Here we report on cerebral primitive neuroectodermal tumors that lacked the wild-type p53 gene (TP53) and showed multiple numerical chromosome aberrations, as detected by comparative genomic hybridization. In these tumors, the centrosome number was significantly higher than in a control tumor without a detected TP53 mutation and with few chromosomal imbalances. These findings indicate that abnormal centrosome amplification can occur in human tumors lacking wild-type TP53 and may be a mechanism by which numerical chromosome aberrations are generated.

Nuclear RNA accumulations contain released transcripts and exhibit specific distributions with respect to Sm antigen foci.

RNA polymerase II transcripts accumulate within mammalian nuclei at distinct sites and exhibit varying morphology. Certain RNA species are organized in elongated structures, whereas others appear as dot-like concentrations. To analyze the status of the RNA within these accumulations, we investigated the composition of accumulations derived from Epstein-Barr virus (EBV) genes, human papilloma virus 18 (HPV18) open reading frames E6 and E7, as well as heat shock protein 89a (hsp89alpha) and 89beta (hsp89beta) genes. No differential distribution of exon and intron sequences within concentrations of EBV RNA could be observed. Whereas accumulations of hsp89alpha and hsp89beta always coincided with Sm antigen foci, the RNA of EBV and HPV18 never co-localized with these foci. This excludes Sm antigen foci as the only sites of splicing and suggests gene-specific variation in the nuclear localization of transcripts. Two sets of experiments were performed to assess whether transcripts in the RNA accumulations are in statu nascendi or products released from a discrete gene locus. Because RNA transcripts derived from EBV genes, which are located on both ends of the genome, were all distributed along the entire length of the RNA signals, they cannot be derived from a highly decondensed genomic DNA extending throughout elongated RNA accumulations. Furthermore, removal of labeled RNA sequences and subsequent visualization of DNA confirmed the confinement of the genomic sequences to a small subregion of the area occupied by accumulated RNA. Therefore, this study supports the view of RNA accumulations as a stream of molecules that delineate a path from a dot-like gene locus toward the nuclear envelope for export into the cytoplasm.

Weaving a pattern from disparate threads: lamin function in nuclear assembly and DNA replication.

The major residual structure that remains associated with the nuclear envelope following extraction of isolated nuclei or oocyte germinal vesicles with non-ionic detergents, nucleases and high salt is the lamina (Fawcett, 1966; Aaronson and Blobel, 1975; Dwyer and Blobel, 1976). The nuclear lamina is composed of intermediate filament proteins, termed lamins (Gerace and Blobel, 1980; Shelton et al., 1980), which polymerise to form a basket-weave lattice of fibrils, which covers the entire inner surface of the nuclear envelope and interlinks nuclear pores (Aebi et al., 1986; Stewart and Whytock, 1988; Goldberg and Allen, 1992). At mitosis, the nuclear envelope and the lamina both break down to allow chromosome segregation. As a consequence, each structure has to be rebuilt during anaphase and telophase, allowing cells an opportunity to reposition chromosomes (Heslop-Harrison and Bennett, 1990) and to reorganise looped chromatin domains (Franke, 1974; Franke et al., 1981; Hochstrasser et al., 1986), which may in turn control the use of subsets of genes. Because of the position that it occupies, its dynamics during mitosis and the fact that it is an essential component of proliferating cells, the lamina has been assigned a number of putative roles both in nuclear metabolism and in nuclear envelope assembly (Burke and Gerace, 1986; Nigg, 1989). However, to date there is little clear cut evidence that satisfactorily explains the function of the lamina in relation to its structure. In this Commentary we will describe some of the recent work that addresses this problem and attempt to provide a unified model for the role of lamins in nuclear envelope assembly and for the lamina in the initiation of DNA replication.

Internal lamin structures within G1 nuclei of human dermal fibroblasts.

The nuclear lamina is a mesh-like network of fibres subjacent to the inner nuclear membrane that is believed to be involved in the specific spatial reorganisation of chromatin after mitosis. To determine how the lamina might be involved in chromatin reorganisation, we have performed indirect immunofluorescence studies on quiescent and proliferating human dermal fibroblasts (HDF). Two monoclonal antibodies recognising human lamins A and C and three different fixation methods were employed. In indirect immunofluorescence studies, cultures of quiescent cells displayed a uniform perinuclear distribution of the antibodies. In proliferating cultures two distinct populations of cells were observed: one population displayed a typical perinuclear antibody distribution, while the second population displayed an unusual pattern consisting of a series of spots and fibres within the nucleus. By inducing cell-cycle synchrony in cultures we were able to determine that the unusual internal distribution of the lamin antibodies was restricted to cells in G1. Optical sectioning and 3-D reconstruction of the lamina structures in G1 nuclei was performed with a confocal laser scanning microscope (CLSM). This revealed that the internal lamin structures consisted of small foci and fibres proliferating throughout the nucleus. These structures were shown to be closely associated with areas of condensed chromatin but not nuclear membrane. As cells progress towards S phase the internal lamin foci disappear.

The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts.

The sites of nascent DNA synthesis were compared with the distribution of the proliferating cell nuclear antigen (PCNA) in S-phase nuclei of human diploid fibroblasts (HDF) by two in vitro techniques. Firstly, proliferating fibroblasts growing in culture that had been synchronised at S-phase were microinjected with the thymidine analogue biotin-11-dUTP. The sites of incorporation of biotin into injected cells were compared with the distribution of PCNA by indirect immunofluorescence microscopy and laser scanning confocal microscopy (LSCM). In common with other studies, a progression of patterns for both biotin incorporation and PCNA localisation was observed. However, we did not always observe coincidence in these patterns, the pattern of biotin incorporation often resembling the expected, preceding distribution of PCNA. In nuclei in which the pattern of biotin incorporation appeared to be identical to the distribution of PCNA, LSCM revealed that not all of the sites of PCNA immunofluorescence were incorporating biotin at the same time. Secondly, nuclei which had been isolated from quiescent cultures of HDF were innoculated into cell-free extracts of Xenopus eggs which support DNA replication in vitro. Following innoculation into these extracts DNA replication was initiated in each nucleus. The sites of DNA synthesis were detected by biotin-11-dUTP incorporation and compared with the distribution of PCNA by indirect immunofluorescence. Only a single pattern of biotin incorporation and PCNA distribution was observed. PCNA accumulated at multiple discrete spots some 15 min before any biotin incorporation was observed. When biotin incorporation did occur, LSCM revealed almost complete coincidence between the sites of DNA synthesis and the sites at which PCNA was localised.