Gene expression and nuclear architecture during development and differentiation

Development requires a precise program of gene expression to be carried out. Much work has focussed on the regulatory networks that control gene expression, for example in response to external cues. However, it is important to recognize that these regulatory events take place within the physical context of the nucleus, and that the physical position of a gene within the nuclear volume can have strong influences on its regulation and interactions. The first part of this review will summarize what is currently known about nuclear architecture, that is, the large-scale three-dimensional arrangement of chromosome loci within the nucleus. The remainder of the review will examine developmental processes from the point of view of the nucleus.     

Identification of a signal for nuclear targeting in platelet-derived-growth-factor-related molecules.

The v-vis gene encodes p28sis, the transforming protein of simian sarcoma virus. This gene resulted from a fusion of the env gene of simian sarcoma-associated virus and the woolly monkey gene for the B chain of platelet-derived growth factor (PDGF). Previous work has shown that the v-sis gene product undergoes signal sequence cleavage, glycosylation, dimerization, and proteolytic processing to yield a secreted form of the protein. It transport across the endoplasmic reticulum is blocked by the introduction of a charged amino acid residue within the signal sequence, the protein does not dimerize, is not secreted, and is no longer transforming as assayed by focus-forming ability in NIH 3T3 cells. Instead, this mutant protein localizes to the nucleus as demonstrated by both indirect immunofluorescence and cell fractionation. Using a series of deletion mutations, we delimited an amino acid sequence within this protein which is responsible for nuclear localization. This region is completely conserved in the predicted human c-sis protein, although it lies outside of regions required for transformation by the v-sis gene product. This nuclear transport signal is contained within amino acid residues 237 to 255, RVTIRTVRVRRPPKGKHRK. An amino acid sequence containing these residues is capable of directing cytoplasmic v-sis mutant proteins to the nucleus. This sequence is also capable of directing less efficient nuclear transport of a normally cytoplasmic protein, pyruvate kinase. Pulse-chase experiments indicate that the half-lives of nuclear and cytoplasmic v-sis mutant proteins are approximately 35 min. Using the heat-inducible hsp70 promoter from Drosophila melanogaster, we showed that the nuclear v-sis protein accumulates in the nucleus within 30 min of induction. The identification of a nuclear transport signal in the v-sis gene product raises interesting questions regarding the possibility of some function for PDGF or PDGF-related molecules in the nucleus.     

Nuclear DNA content and chromatin pattern of rat rhabdomyosarcoma cell sublines with different metastatic potentials.

There is a constant need of features able to characterize potentially metastatic cells among the heterogeneous cell subpopulations which constitute a tumor. Image cytometry of metastatic tumor cells give rise to variable results, partly because of a heterogeneous origin of cells, or potential drug effects. The aim of this work was to characterize nuclear changes observed in metastatic cell clones issued in vitro from the same parental cell population The nuclear phenotypes of 6 cell sublines isolated from a rat rhabdomyosarcoma cell line and differing in their metastatic ability were evaluated by image cytometry on Feulgen-stained preparations. Densitometric [5], geometric [3] and textural [9] features were computed from each nuclear image. For each cell subline, a metastatic score, ranging from 0 to 10, was calculated on the basis of in vivo invasivity data, by measuring the number of pulmonary metastases observed after s.c. graft of tumor cells in rats. Data obtained were compared to karyotype, growth characteristics, and oncogene expressions of cell lines. The nuclear DNA content, the chromosome numbers, the cell sublines doubling times, and the distribution of cells within the cell cycle appear unrelated with this score. On the contrary, increase in metastatic ability is accompanied by changes in chromatin pattern as assessed by textural features. Progressive increase in chromatin condensation can be observed in cell sublines with increasing metastatic score. These results were confirmed by an unsupervised multivariate partitioning of rhabdomyosarcoma cells which identified two separate subsets whose distributions within the analyzed cell lines correlate with their metastatic ability. These data suggest that, in rat rhabdomyosarcoma cell sublines, metastatic ability could be associated with nuclear morphological changes at the level of chromatin texture.     

Reappraisal of nuclear DNA content of rodlet cells compared to several cell types from some freshwater teleosts, using two methods of microdensitometry

Nuclear DNA contents of rodlet cells from Catostomus commersoni, Semotilus atromaculatus and Cyprinus carpio were compared with nuclear DNA of erythrocytes and larger cells of the same species, using scanning microdensitometry and averaging microdensitometry. This study reappraises the work of Barber & Westermann (1983), which employed averaging microdensitometry only, and compared rodlet cell nuclear DNA only with erythrocyte DNA. In addition, this work considers sources of error in both methods of microdensitometry, and comments upon the use of microdensitometry of either method as a mechanism for making distinctions among the DNA contents of cells of different types. The results of the present work consistently indiate no significant differences within species between nuclear DNA content of rodlet cells and larger teleost cells, using either method of microdensitometry. Because of the lack of statistically significant difference in DNA content between nuclei of rodlet cells and those of known teleost cells, it has been concluded that the rodlet cell itself is probably of teleost origin. However, the method indicates nothing about the origin of the rodlets, which have also been shown to contain DNA, but are Feulgen-negative.     

Nuclear magnetic resonance studies of flexible opiate conformations at monoclonal antibody binding sites. Quantitative interproton distances obtained from comparing theoretical and experimental transferred nuclear Overhauser enhancement: correlation with antibody sequence

A previous publication described the use of qualitative intramolecular 1H-transferred nuclear Overhauser effect measurements to determine the conformations of flexible ligands at monoclonal anti-opiate antibody binding sites. This paper concentrates on the quantitative interpretation of experiments of this type using the ligand nalorphine (N-allyl morphine) and a single anti-opiate monoclonal antibody. I compare the experimental unidimensional driven nuclear Overhauser effect buildup curves to theoretical curves derived with a knowledge of the fixed interproton distances in the ligand. The discussion covers the potential accuracies of derived distances and concentrates on two problem areas associated with determining structures from this type of experiment. The most serious one is the case where, because of particular multiproton spatial distributions, spin diffusion is so rapid that it cannot be determined experimentally and where numerical fits of theoretical calculations are misleading. The results show that, while intraligand spin diffusion complicates the interpretation for some proton pairs, with many others accuracies within about 0.3 A for interproton distances from 2 to 4 A are attainable. The results confirm the earlier report that the conformation of nalorphine in this antibody binding site differs from the major one present in solution or in the crystal. An important aspect of the work is that theoretical prediction of nuclear Overhauser effect time-dependence is an important practical tool for recognizing cases where interpretation of experiments will be difficult. Initial data on protein-to-ligand transferred nuclear Overhauser effect are presented, which show that at least one aromatic amino acid residue is closely involved in the binding of the ligand. The companion paper presents the primary sequences of the variable regions of the antibodies being used in our studies. In this paper, these and associated immunochemical studies are correlated with the nuclear magnetic resonance results. The combination of data presented in the two papers provides a basis for future work on protein-ligand interproton distances in the range 1 to 5 A using both transferred nuclear Overhauser effect (for rapidly exchanging ligands) and isotope-edited, indirectly detected nuclear Overhauser effect (for tightly bound ligands).     

A multi gene sequence-based phylogeny of the Musaceae (banana) family

Background  

The classification of the Musaceae (banana) family species and their phylogenetic inter-relationships remain controversial, in part due to limited nucleotide information to complement the morphological and physiological characters. In this work the evolutionary relationships within the Musaceae family were studied using 13 species and DNA sequences obtained from a set of 19 unlinked nuclear genes.     

Mutations in the nuclear lamin proteins resulting in their aberrant assembly in the cytoplasm.

We have constructed a series of mutations in the human A lamin cDNA to identify and alter the nuclear localization signal using an in vivo functional assay system. The nuclear localization signal in the lamin proteins has both structural and functional similarities with that of the SV40 large T-antigen. Mutations within this functional domain result in the assembly of cytoplasmic tubular structures, and the behavior of these mutants suggests a post-translational dimerization of the lamin proteins prior to their transport into the nucleus. In the course of this work other regions of the carboxy terminus of the A/C lamin proteins have been implicated in the proper assembly and structure of the nuclear envelope.     

Ultrastructural study of spermiogenesis and the spermatozoon of Microcotyle pancerii (Monogenea: Polyopisthocotylea: Microcotylidae), parasite of meagre Argyrosomus regius (Pisces: Teleostei)

The present work deals with the ultrastructure of spermiogenesis and the spermatozoon of Microcotyle pancerii, a gill parasite of meagre Argyrosomus regius collected in Corsican fish farms. Spermiogenesis was rather similar to that observed in other polyopisthocotylean Monogenea. The intercentriolar body was different from that described in digeneans. The nuclear condensation occurred in 2 successive stages. First, during the nuclear migration in the median cytoplasmic process, the nucleus developed a honeycomb-like appearance. Then, after the flagellar fusion, a discontinuous twisting of the chromatin appeared along the nucleus, with this process ending in total nuclear condensation. The structure of the spermatozoon is characterized by 2 axonemes (9 + "1" pattern), a single and continuous field of cortical microtubules, a mitochondrion, and a nucleus. Our findings were compared with various ultrastructural features in order to highlight variability within the group.     

Identification of a phosphoprotein in the nuclear matrix by monoclonal antibodies.

Previous work in this laboratory has established that a rat liver nuclear phosphoprotein (B2:Mr 68,000, pI 6.5-8.2) is associated with actively transcribed nucleosomes, as demonstrated by its preferential release after mild treatment with micrococcal nuclease. In the present report we provide further immunological evidence ('Western Blot' analysis, solid-phase radioimmunoassay and indirect immunofluorescence) that in addition establishes the presence of this phosphoprotein in the nuclear-matrix protein fraction. This paradoxical localization suggests that this phosphoprotein may function in two separate and distinct roles within the realm of nuclear organization.     

ALG11--a new variable DNA marker for sponge phylogeny: comparison of phylogenetic performances with the 18S rDNA and the COI gene

Phylogenetic relationships within sponge classes are highly debated. The low phylogenetic signal observed with some current molecular data can be attributed to the use of few markers, usually slowly-evolving, such as the nuclear rDNA genes and the mitochondrial COI gene. In this study, we conducted a bioinformatics search for a new molecular marker. We sought a marker that (1) is likely to have no paralogs; (2) evolves under a fast evolutionary rate; (3) is part of a continuous exonic region; and (4) is flanked by conserved regions. Our search suggested the nuclear ALG11 as a potential suitable marker. We next demonstrated that this marker can indeed be used for solving phylogenetic relationships within sponges. Specifically, we successfully amplified the ALG11 gene from DNA samples of representatives from all four sponge classes as well as from several cnidarian classes. We also amplified the 18S rDNA and the COI gene for these species. Finally, we analyzed the phylogenetic performance of ALG11 to solve sponge relationships compared to and in combination with the nuclear 18S rDNA and the COI mtDNA genes. Interestingly, the ALG11 marker seems to be superior to the widely-used COI marker. Our work thus indicates that the ALG11 marker is a relevant marker which can complement and corroborate the phylogenetic inferences observed with nuclear ribosomal genes. This marker is also expected to contribute to resolving evolutionary relationships of other apparently slow-evolving animal phyla, such as cnidarians.     

The road much traveled: trafficking in the cell nucleus

Trafficking of RNA molecules and proteins within the cell nucleus is central to genome function. Recent work has revealed the nature of RNA and protein motion within the nucleus and across the nuclear membrane. These studies have given insight into how molecules find their destinations within the nucleus and have uncovered some of the structural properties of the nuclear microenvironment. Control of RNA and protein trafficking is now emerging as a physiological regulatory mechanism in gene expression and nuclear function.     

Inching toward Armageddon: a psychiatric view

The existence of thermonuclear arsenals capable of destroying much of humanity takes its origins from, and has an influence on, processes that are largely psychological. The threat to use a single nuclear bomb to resolve contemporary conflict is an anathema in part because complex nation-states did not evolve with surviving a nuclear war in mind. The atomic arms race has proceeded apace because of maladaptive psychological mechanism including denial, distortion, projection, and, most relevantly, the need to enhance bonding within groups by creating stereotypes. One consequence of the arms race is a climate of fear and hopelessness, and especially destructive effect of which is seen in children. The physician's role in the prevention of nuclear war is critical, but divided between contradictory roles. On one hand, the physician is traditionally identified as a non-political advocate of the sick; on the other, as an advocate for the public health. It is this second model that enables physicians most legitimately to work for the prevention of nuclear war and to deal with the psychiatric concomitants of a planet drifting toward disaster.     

Examination of five nuclear markers for phylogenetic study of Hologalegina (Leguminosae)

Most legume phylogenies have relied heavily on plastid gene datasets, with or without nuclear ribosomal DNA ITS data, but the sequences of nuclear genes and gene-spanning regions offer certain advantages. We tested the phylogenetic utility of five nuclear loci across the species-rich legume clade Hologalegina: PGDH, TRPT, HRIP, RNAR, and CNGC4 (CNGC4-like protein). Our objective was to determine whether any of these nuclear loci could be beneficial at resolving lower-level phylogenetic relationships in this clade, with a particular interest in finding markers that might work at the species level. While the phylogenetic utility of these nuclear loci is unknown outside of Hologalegina, we determined that two of the loci, PGDH and TRPT, are useful for phylogenetic analyses within Hologalegina, depending upon the desired scale of resolution.     

Sampling Theory for Cytonuclear Disequilibria

We examine the statistical properties of cytonuclear disequilibria within a system including one diploid nuclear locus and one haploid cytoplasmic locus, each with two alleles. The results provide practical guidelines for the design and interpretation of cytonuclear surveys seeking to utilize the novel evolutionary information recorded in the observed pattern of cytonuclear associations. Important applications include population studies of nuclear allozymes in conjunction with genes from mitochondria, chloroplasts, or cytoplasmically inherited microorganisms. Our attention focuses on the allelic and genotypic disequilibria, which respectively measure the nonrandom associations between the cytotypes and the nuclear alleles and genotypes. We first derive the maximum likelihood estimators and their approximate large sample variances for each disequilibrium measure. These are each in turn used to set up an asymptotic test of the null hypothesis of no disequilibrium. We then calculate the minimum sample sizes required to detect the disequilibria under specified alternate hypotheses. The work also incorporates the deviation from Hardy-Weinberg equilibrium at the nuclear locus, which can significantly affect the results. The practical utility of this new sampling theory is illustrated through applications to two nuclear-mitochondrial data sets.     

Synthesis of nuclear acidic proteins in density-inhibited fibroblasts stimulated to proliferate

Previous work from this laboratory (Rovera and Baserga, 1971) has shown that, when density-inhibited WI-38 human diploid fibroblasts are stimulated to proliferate by a change of medium, the synthesis of nuclear acidic proteins increases within 30 minutes after stimulation; several hours before DNA synthesis begins to increase. Similar results have now been obtained with density-inhibited 3T6 mouse fibroblasts, also stimulated by a change of medium. Gel electrophoretic analysis of nuclear acidic proteins in both WI-38 human diploid fibroblasts and 3T6 mouse fibroblasts stimulated to proliferate indicates that the increased synthesis of nuclear acidic proteins is limited to certain classes of proteins while other classes are totally unaffected. The increase in nuclear acidic proteins synthesis is inhibited when WI-38 cells or 3T6 cells are stimulated in the presence of 5-azacytidine (10 μg/ml), a treatment which also inhibits the subsequent stimulation of DNA synthesis. These results, confirming and extending similar findings previously reported in other models of stimulated DNA synthesis, lend further support to the hypothesis that nuclear acidic proteins may play a critical role in the control of DNA synthesis and cell division in mammalian cells.