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Patterning through differential endoreduplication in epithelial organogenesis of the chordate, Oikopleura dioica.

The contributions that control of cell proliferation and cell growth make to developmental regulation of organ and body size remain poorly explored, particularly with respect to endocycles in polyploid tissues. The epithelium of the marine chordate Oikopleura dioica is composed of a fixed number of cells grouped in territories according to gene-specific expression and nuclear sizes and shapes. As the animal grows 10-fold during the life cycle, epithelial cells increase in size differentially as a function of their spatial position. We show that this cellular pattern reflected differences in ploidy levels ranging from 34 to 1,300 C. The diverse ploidy levels in defined cellular fields resulted both from different timing of entry into endocycles and from cell-specific regulation of endocycle lengths. Throughout the life cycle, differential cell size and ploidy increases were accompanied by field-specific profiles of progressive reductions in G-phase duration. Endocycles were asynchronous among cells of a given epithelial territory, but at the resolution of individual cells, both DNA replication timing and ploidy levels were bilaterally symmetric. The transparent, accessible, oikoplastic epithelium is a model of choice for the study of endoreduplication in the context of pattern formation and growth.

Animals↗

Chromatin bridges between sister chromatids induced in late G2 mitosis in CHO cells by trimethylpsoralen + UVA.

A hypothesis is proposed that sister chromatid pairing is due, at least in part, to the pairing between DNA strands belonging to each of the two sister chromatids (conservative pairing). To test this hypothesis interstrand DNA cross-links were induced in late G2-mitosis in CHO cells in order to bind covalently paired DNA strands eventually coming from both sister chromatids and detect the consequent chromatin bridges between sister chromatids (SCCBs). Therefore cells were treated with trimethylpsoralen (TMP) + UVA (365 or 405 nm). Chromatin bridges in ana-telophase were induced by an UVA irradiation at 365 nm, which gives rise to both monoadducts and cross-links, but not by a 405-nm irradiation, which gives rise only to monoadducts. An analysis of colchicine-induced c-anaphases demonstrated that such chromatin bridges were really SCCBs and that terminal regions of chromosomes were particularly involved. The evolution of SCCBs was studied to rule out that they were masked isochromatid exchanges. So TMP + UVA-treated cells were induced to polyploidize with colchicine and labeled with 5-bromodeoxyuridine. Cells treated with TMP + UVA in G2-mitosis appeared as M1 tetraploid c-metaphases; in such cell populations there was not an increase in isodicentric chromosomes, which are derived from isochromatid exchanges. The present data, as a whole, support the hypothesis that a "conservative pairing" between DNA strands of sister chromatids can be present in mitosis.

Animals↗

DNA synthesis and Fos and Jun protein expression in mitotic and postmitotic WI-38 fibroblasts in vitro.

Normal human embryonic lung fibroblasts WI-38 differentiate spontaneously along the cell lineage mitotic fibroblasts (MF) I, II, and III and postmitotic fibroblasts (PMF) IV, V, VI, and VII in the fibroblast stem cell system in vitro, when appropriate methods are applied. The mitotic fibroblasts can be induced to shift to postmitotic fibroblasts by two treatments with mitomycin C (2 x MMC) in a short period of time compared to spontaneous development. Mitotic and postmitotic fibroblast cell types have specific morphological and biochemical properties, e.g., [35S]methionine polypeptide markers in 2D PAGE. Spontaneously arisen and experimentally induced (2 x MMC) PMF have the same morphological and biochemical characteristics. Mitotic fibroblasts have 2n DNA and undergo DNA synthesis for reduplication. Postmitotic cells undergo, on average, two rounds of DNA synthesis for endoreduplication (polyploidization). Spontaneously arisen and experimentally induced postmitotic populations are composed of postmitotic fibroblasts PMF IV, V, and VI with 2n, 4n, and 8n DNA. DNA synthesis of mitotic and postmitotic WI-38 cell populations may be regulated by the expression of Fos and Jun proteins. The Fos level of MFs was higher by a factor of 15-24 and the Jun level of MFs by a factor of 4.2-6.3 than those of spontaneously arisen PMFs. In 2 x MMC-induced PMFs, the Fos level was about 4.4-7.5 times higher and the Jun level 1.7-3.3 times higher than that of spontaneously arisen PMFs. The down-regulation of these two parameters is a normal event in the development of mitotic to postmitotic WI-38 fibroblasts in the fibroblast stem cell system and is not related to cellular aging.

Base Sequence↗

Uncoupling of S-phase and mitosis by recombinant cytotoxic necrotizing factor 2 (CNF2).

Cytotoxic necrotizing factor 2 (CNF2) is an exotoxin identified from virulent clinical isolates of Escherichia coli. It has been characterized in adherent cell lines as an inducer of cellular death, hyperploidy (multinucleation), and cytoskeletal reorganization. The molecular mechanism of these actions is unclear. Two cellular mechanisms can be hypothesized to explain the DNA content increase (hyperploidy) induced by the toxin. The first is that the toxin interferes with cytoplasmic division without interfering with normal nuclear cycling, such that DNA is replicated in the absence of cell division. The second is that the toxin drives the nuclear machinery to replicate the DNA multiple times within one cell cycle, without interfering with cytoplasmic division. In order to investigate these phenomena, we have constructed a recombinant CNF2 gene that expresses a toxin with both an epitope tag and a polyhistidine tag. Extracts made from E. coli that express this gene have a high multinucleating activity that colocalizes with the recombinant 115-kDa protein. To distinguish between these hypotheses, we used recombinant CNF2 and several growth conditions (time, partial differentiation, and stage of growth) to establish a relationship between cellular divisions and generation of hyperploidy. It was also determined that the toxin had no effect upon in vitro DNA replication using a Xenopus egg extract system. In aggregate, these data are consistent with the hypothesis that CNF2 is affecting cytoplasmic division and thereby removing the requirement for a completed mitosis before the initiation of another S-phase. These data are discussed in relation to the generation of polyploid cells during megakaryopoeisis and the generation of aneuploid cells during tumorigenesis.

3T3 Cells↗

p53-independent apoptosis and p53-dependent block of DNA rereplication following mitotic spindle inhibition in human cells.

We have studied the response of human transformed cells to mitotic spindle inhibition. Two paired cell lines, K562 and its parvovirus-resistant KS derivative clone, respectively nonexpressing and expressing p53, were continuously exposed to nocodazole. Apoptotic cells were observed in both lines, indicating that mitotic spindle impairment induced p53-independent apoptosis. After a transient mitotic delay, both cell lines exited mitosis, as revealed by flow-cytometric determination of MPM2 antigen and cyclin B1 expression, coupled to cytogenetic analysis of sister centromere separation. Both cell lines exited mitosis without chromatid segregation. K562 p53-deficient cells further resumed DNA synthesis, giving rise to cells with a DNA content above 4C, and reentered a polyploid cycle. In contrast, KS cells underwent a subsequent G1 arrest in the tetraploid state. Thus, G1 arrest in tetraploid cells requires p53 function in the rereplication checkpoint which prevents the G1/S transition following aberrant mitosis; in contrast, p53 expression is dispensable for triggering the apoptotic response in the absence of mitotic spindle.

Apoptosis↗

Identification of PML oncogenic domains (PODs) in human megakaryocytes.

Megakaryocytes (Mks) are unique cells in the human body in that they carry a single and polyploid nucleus. It is therefore of interest to understand their nuclear ultrastructure. PML oncogenic domains (PODs) were described in several types of eukaryotic cells using human autoantibodies which recognize nuclear antigens with a specific speckled pattern (dots) in indirect immunofluorescence (IF). Two main antigens, PML and Sp 100, usually colocalize and concentrate in these nuclear subdomains. We investigated the presence of PODs using IF and immunoelectron microscopy (IEM) in cells from megakaryocytic lineage: the HEL cell line and human cultured Mks. Antibodies against PML, Sp100, and anti-nuclear dots were used in single and double labeling. PODs were identified in HEL cells and in human Mks, and their ultrastructure was characterized. We then used IF to quantify PODs within Mks and showed that their number increased proportionally to nuclear lobularity. In summary, we report the identification of PODs in human Mks at an ultrastructural level and an increase in PODs number in parallel with Mk ploidy. We show that endomitosis not only leads to DNA increase but also to the multiplication of at least one of the associated nuclear structures.

Antigens, Nuclear↗

Hypotetraploidy in a patient with small cell carcinoma.

While numerical and structural chromosomal abnormalities characterize many hematopoietic and nonhematopoietic malignancies, the occurrence of polyploidy is by and large rare. We report here an interesting patient with small cell carcinoma (SCC) and hypotetraploidy initially referred to us because of a question of acute nonlymphocytic leukemia, M3 subtype, with a question of a 15;17 translocation characteristic of acute promyelocytic leukemia. However, the patient did not have a 15;17 translocation and the final hematopathologic analysis of the bone marrow aspirates and immunohistochemistry studies subsequently revealed the patient to have SCC. Small cell carcinoma is a highly malignant and a very aggressive neoplasm. A review of the literature, using Medline, Cancerlit, and the Science Citation Index, revealed that in most, if not all, reports, the presence of polyploidy is noted as a rare entity. In leukemia, reports of polyploidy point to a distinct category of patients with a poor risk for which more intensive treatment is needed. Limited information is currently available to assess the risk of polyploidy in small cell carcinoma. Our case is important not only because of the relative rarity of polyploidy, but also because insights gained from the study of this and other similar patients may help shed additional light on the mechanism of carcinogenesis, which is not fully known to date. As polyploidization is a manifestation of genetic instability and as genetic instability has been implicated in the genesis and progression of many cancers, it is perhaps not too surprising that polyploidy in our case was associated with a poor disease outcome. The patient has since expired.

Bone Marrow Neoplasms↗

Multiplex analysis of single-nucleotide extension products on a 16-capillary, denaturing, high-performance liquid chromatography array.

We constructed a high-performance liquid chromatography array consisting of 16 monolithic poly(styrene/divinylbenzene) capillaries for the parallel multiplex analysis of fluorescent dye-labeled single-nucleotide extension products. Because of the high chemical and physical robustness of the column bed that is covalently linked to the inner surface of the fused silica capillary, the array can be reused thousands of times without replenishment. The choice of fluorophore exerts a significant effect on resolution of the extension products. FAM, HEX, and TAMRA allowed complete resolution of all four possible allelic extension products not only from the extension primer but also from each other. The quantitative accuracy of the method enables the genetic typing of bi- and triallelic single-nucleotide polymorphisms in polyploid genomes and pooled samples.

Chromatography, High Pressure Liquid↗

A strong inhibitory element down-regulates SRE-stimulated transcription of the A3 cytoplasmic actin gene of Bombyx mori.

To identify the functional regulatory elements of the promoter of the cytoplasmic actin A3 gene in Bombyx mori, transient expression of A3-LacZ mutants was assayed in cultured Lepidoptera cells. This led to the recognition of two proximal and contiguous domains exerting strong negative and positive effects, respectively on promoter activity. The negative region contains a ten-base-pair sequence that binds Bombyx silk gland cell nuclear proteins in vitro. The positive regulatory element was identified as a serum response element (SRE) by its sequence, and its in vitro binding properties. Moreover, structural analysis of posterior and median silk gland cell chromatin by dimethyl sulfate-aided LMPCR revealed that SRE is bound to its cognate factor in situ, in most, if not all, the approximately 100,000 A3 copies of the polyploid DNA stock. The regulation of the A3 promoter in the silk gland would thus result from the combined action of these two antagonist factors.

Actins↗

Molecular cloning and expression of the Caenorhabditis elegans klp-3, an ortholog of C terminus motor kinesins Kar3 and ncd.

Common to all eukaryotes, kinesins are cytoskeletal motor proteins that mediate intracellular transport on microtubule tracks, using ATP hydrolysis. A Caenorhabditis elegans cDNA clone corresponding to the klp-3 gene, encoding a novel kinesin, was isolated, and mapped on LGII. Northern blot analysis using the klp-3 cDNA probe reveals a 1.9 kb mRNA that is transcribed at a low level during development. Temporal and spatial expression of the klp-3::lacZ fusion gene is limited to the marginal cells in the pharynx, and a group of muscle cells in the posterior gut region. The nucleotide sequence of klp-3 has been deduced from the cDNA and nematode genome sequencing consortium data. Conceptual translation of the klp-3 gene reveals a kinesin-like protein with its conserved motor domain containing the ATP binding and microtubule binding sites located in the C terminus. KLP-3 shares extensive homology with the yeast Kar3 and Drosophila ncd kinesins, which have previously been shown to mediate chromosomal movement and segregation during meiosis and mitosis. Overexpression of the klp-3 gene partially rescues the lethal phenotype of the maternal lethal him-14 ts(it44) mutants at non-permissive temperatures, and reduces the incidence of males caused by non-disjunction of the X-chromosome. Similarly, expression of a klp-3 antisense RNA, under the control of a heat shock promoter, causes embryonic arrest, dead eggs and polyploid cells in transgenic lines, suggesting a critical role for the klp-3 function in chromosome segregation. Further analysis of the klp-3 gene in C. elegans may elucidate diverse functions of the C terminus mitotic motor proteins during development.

Amino Acid Sequence↗

Dominance--one hundred and fifteen years after Mendel's paper.

In The Molecular Basis of Dominance, Kacser & Burns (1981) demonstrated that dominance in diploids and polyploids, and pleiotropy in all organisms, were biochemical phenomena; they were the consequences of the response of a metabolic system to a genetically specified change in the activity of any one enzyme within the system. Epistasis was similarly explicable when each of at least two enzyme activities suffered a change. The significance of this achievement by Kacser & Burns (1981) for biochemistry, genetics, molecular biology, medicine and bio-technology is best seen against the background of 115 years of attempts to explain the origins of dominance.

Animals↗

Phylogeography of an asexual marine clam complex, Lasaea, in the northeastern Pacific based on cytochrome oxidase III sequence variation.

Northeastern Pacific populations of the intertidal marine clam Lasaea are exclusively composed of polyploid asexual clones that lack pelagic larval development. We investigated the phylogeographic structure of this clonal assemblage by assaying genetic divergence in 4 populations along 2000 km of the west coast of North America. In each population a 462-nt portion of the mitochondrial Cytochrome Oxidase III gene was amplified and sequenced for 30 individuals. A total of 21 haplotypes were detected and phylogenetic analyses resolved this variation into 5 main branches that differ in sequence composition by 8.9-14.5%. Two of the branches each encompass a single haplotype, the other 3 terminate in clades containing 3-11 lineages and within clade divergence ranges from 0.4 to 2.3%. Obvious geographic structuring was evident in that 19 of the 21 lineages were restricted in their distributions to single populations and 3 of the main phylogenetic branches were limited to specific marine biogeographic provinces. Two of the clades were encountered in multiple, geographically distant populations; however, the absence of shared haplotypes indicates that long distance gene flow is rare among these direct-developing populations. Northeastern Pacific Lasaea clones form a robust monophyletic grouping relative to other studied populations of the cosmopolitan genus. We estimate divergence times among northeastern Pacific clones to range from 3 to 6 myr. If the latest common ancestor of these exclusively asexual, genetically diverse, regionally monophyletic Lasaea lineages was also asexual, this represents a minimal age estimate for asexuality in the genus. The most plausible alternate hypothesis requires that at least 5 northeastern Pacific sexual parental species have recently become extinct.

Animals↗

Molecular evolution of the internal transcribed spacers (ITS1 and ITS2) and phylogenetic relationships among species of the family Cucurbitaceae.

Phylogenetic relationships of different members of the family Cucurbitaceae were estimated from sequences of the internal transcribed spacer (ITS1 and ITS2) regions of the nuclear ribosomal RNA genes. Twenty-six species of different genera belonging to different tribes and several subtribes were analyzed. The whole ITS regions were amplified by PCR technique and cloned, and three to five different clones of each species were sequenced; for some species PCR products were sequenced directly. ITS1 and ITS2 regions are slightly variable in length, with each length appearing genus-specific. A substitution rate of 3.62 x 10(-9) substitutions per site per year was calculated assuming 40 MYA separation time. Phylogenetic relationships inferred from ITS sequences of some species is in agreement with morphological data, but deviations to the taxonomic classification were also observed. A polyphyletic origin of the New World species must be considered. In the genus Cucurbita different "types" of ITS sequences within one species exist, possibly due to the high frequency of introgression during domestication or due to polyploidization events; in contrast, low intraspecific variability was detectable in the genus Cucumis, indicating different stages of speciation.

Base Composition↗

Gene duplication and the uniqueness of vertebrate genomes circa 1970-1999.

In this article I review research undertaken over the past 30 years into the role that gene duplication played in shaping vertebrate genomes. I discuss early karyotype studies that pointed to a relative stability of mammalian and avian genomes, the discovery and possible evolutionary significance of enormous genomes in urodele amphibians and lungfish, genome compaction in certain specialised bony fish, evidence for two rounds of total genome doubling in early vertebrate evolution and the fate of duplicated genes in polyploid fish.

Amphibians↗

Alterations in nuclear ploidy and cell phase distribution of rat liver cells in experimental alcoholic liver disease: relationship to antioxidant enzyme gene expression.

The ability of a cell to withstand oxidative stress has been hypothesized to be related to its ploidy status. We used the intragastric feeding rat model for alcoholic liver disease to evaluate the relationship between severity of liver injury, antioxidant mRNA levels, and DNA ploidy of liver cells. Rats were fed ethanol with different dietary fats (saturated fat, corn oil, and fish oil); pair-fed control animals received isocaloric amounts of dextrose. All animals were euthanized at 1 month and had evaluation of pathologic changes in the liver, DNA content by flow cytometry, and mRNA levels for catalase and glutathione peroxidase. The fish oil-ethanol group exhibited the most severe pathology, the corn oil-ethanol group had intermediate pathologic changes, and no pathologic changes were seen in the saturated fat-ethanol and dextrose-fed controls. Flow cytometric analysis of propidium iodide-stained nuclei revealed that saturated fat-dextrose and corn oil-dextrose groups had about 65% of cells with (diploid) G1 DNA content and about 30% of cells with tetraploid (4C) nuclei. The fish oil-dextrose had a significantly higher (p < 0.001) number of 4C cells (67.4 +/- 2.1%) compared to the other two dextrose-fed groups. In the animals showing pathologic liver injury, there was a higher percentage of cells with hypertetraploid nuclei. The highest percentage of these hypertetraploid cells was seen in the fish oil-ethanol group. Catalase and glutathione peroxidase mRNA levels correlated significantly with polyploidy. A significant correlation was seen between the number of cells in the greater than G2 + M phase and glutathione peroxidase mRNA levels (r = 0.91, p < 0.01) and catalase mRNA. The different slopes of correlation analysis between catalase mRNA and dietary fats show that the degree of saturation of fatty acids may influence catalase mRNA expression in cells with different ploidy states. We propose that polyploidization of liver cell nuclei may serve as a defense mechanism against ethanol-induced hepatotoxicity. This defense mechanism may also, in part, account for the antiregenerative effect of ethanol on hepatocytes.

Animals↗

Comparison of Sindbis virus-induced pathology in mosquito and vertebrate cell cultures.

We have compared Sindbis virus-induced cytopathology in vertebrate and mosquito (Aedes albopictus) cell cultures. It has been shown that vertebrate cells undergo apoptosis when infected by Sindbis virus and this was confirmed here using hamster cells (BHK). The occurrence of cell death in Sindbis virus-infected A. albopictus cells is a cell clone-specific phenomenon and, unlike in BHK cell cultures, mosquito cell death does not correlate with a large induction of apoptosis, as determined by assays testing for DNA fragmentation or reduced cellular DNA content. Cell cycle distribution changes were observed in Sindbis virus-infected BHK and C7-10 cell cultures, and the changes are distinct, both in the time of induction and the types of perturbations. In Sindbis virus-infected BHK cells, the major cell cycle profile change is the early accumulation of cells with sub-G1 DNA content and a corresponding reduction in the proportion of cells in G1 and G2/M. For Sindbis virus-infected C7-10 cells, the major perturbations are an increased proportion of cells showing G2/M or polyploid DNA content and a reduction in the proportion of G1 and S phase cells. These data suggest that the pathology induced in mosquito cell cultures by Sindbis virus infection may be distinct from the pathology which appears in vertebrate cell cultures.

Animals↗

Metabolic engineering of Saccharomyces cerevisiae for xylose utilization.

Metabolic engineering of Saccharomyces cerevisiae for ethanolic fermentation of xylose is summarized with emphasis on progress made during the last decade. Advances in xylose transport, initial xylose metabolism, selection of host strains, transformation and classical breeding techniques applied to industrial polyploid strains as well as modeling of xylose metabolism are discussed. The production and composition of the substrates--lignocellulosic hydrolysates--is briefly summarized. In a future outlook iterative strategies involving the techniques of classical breeding, quantitative physiology, proteomics, DNA micro arrays, and genetic engineering are proposed for the development of efficient xylose-fermenting recombinant strains of S. cerevisiae.

Biological Transport↗

Uses of wheat aneuploids.

There is available in wheat a unique series of aneuploids ranging from all 21 possible monosomics to complex types that are simultaneously deficient for one chromosome and duplicate for another. Furthermore, lines with chromosomes from related alien species either added to or substituted for wheat chromosomes are in common cytological use. This contribution condiders the use of this range of material in studies designed to elucidate the evolutionary relationships of the species, in investigations of the genetics of a polyploid with cytological diploidization, and in potential breeding manipulations.

Breeding↗