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PubMed2003

Gradual and quantum genome size shifts in the hystricognath rodents.

We assessed genome size variation by flow cytometry within and among 31 species of nine families of African and South American hystricognath rodents. Interspecific variation was extensive and genome size was relatively high among the South American radiation whereas only moderate variation and smaller estimates of genome size were observed in the African counterparts. The largest genome size, indicating tetraploidy was recorded in the South American octodontid, Tympanoctomys barrerae (16.8 pg DNA). This quantum shift in DNA content represents a novel mechanism of genome evolution in mammals. As expected in polyploid organisms, varying nucleotypic effects were observed in the dimensions of the sperm cells and lymphocytes of T. barrerae. The role of control mechanisms that influence cell dimensions in polyploid organisms is discussed.

Africa↗
PubMed2002

Delinking of S phase and cytokinesis in the protozoan parasite Entamoeba histolytica.

The alternation of DNA replication in S phase and chromosome segregation in M phase is a hallmark in the cell cycle of most well-studied eukaryotes and ensures that the progeny do not have more than the normal complement of genes and chromosomes. An exception to this rule has been described in cancer cells that occasionally become polyploid as a result of failure to restrain S phase despite the failure to undergo complete mitosis. Here, we describe the cell division cycle of the human pathogen, Entamoeba histolytica, which routinely accumulates polyploid cells. We have studied DNA synthesis in freshly subcultured cells and show that, unlike most eukaryotes, Entamoeba cells reduplicate their genome several times before cell division occurs. Furthermore, polyploidy may occur without nuclear division so that single nuclei may contain 1-10 times or more genome contents. Multinucleated cells may also accumulate several genome contents in each nuclei of one cell. Thus, checkpoints that normally prevent DNA reduplication until after cytokinesis in most eukaryotes are not observed in E. histolytica.

Animals↗
PubMed2002

Severe intestinal bleeding caused by intestinal metastases of a primary angiosarcome of the thyroid gland.

A 75 year old male presented with gastrointestinal bleeding after resection of both upper lobes of the lungs because of metastases. One year ago an angiosarcoma was the reason for a complete removal of the thyroid gland. In esophago-gastro-duodenoscopy we found multiple hemorrhagically stained polyploids lesions in the postbulbar duodenum and jejunum. Colonoscopy showed isolated polyploid lesions of the right flexura. Because of persistent gastrointestinal bleeding a diagnostic laparotomy was done. Intraoperative intestinoscopy demonstrated multiple bleeding metastasis. To remove many of the bleeding lesions two longer intestinal segments of the jejunum and ileum were resected. The histology of the metastases showed arrangements of polygonal cells with prominent nucleoli and atypical mitosis. Immunohistochemistry identified CD 31, vimentin and factor VIII associated antigen. There was an erosion of the superficial intestinal mucosal cells with resulting hemorrhage; same histology had been found in the thyroid gland and the right upper lobe of lung. Eight days after surgery the patient died because of respiratory and circulatory insufficiency.

Aged↗
PubMed2004

Cytogenetic evidence for asexual evolution of bdelloid rotifers.

DNA sequencing has shown individual bdelloid rotifer genomes to contain two or more diverged copies of every gene examined and has revealed no closely similar copies. These and other findings are consistent with long-term asexual evolution of bdelloids. It is not entirely ruled out, however, that bdelloid genomes consist of previously undetected pairs of sequences so similar as to be identical over the regions sequenced, as might result if bdelloids were highly inbred sexual diploids or polyploids. Here, we employ fluorescent in situ hybridization with cosmid probes to determine the copy number and chromosomal distribution of the heat shock gene hsp82 and adjacent sequences in the bdelloid Philodina roseola. We conclude that the four copies identified by sequencing are the only ones present and that each is on a separate chromosome. Bdelloids therefore are not highly homozygous sexually reproducing diploids or polyploids.

Animals↗
PubMed2006

Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium-legume symbiosis.

Symbiosis between legumes and Rhizobium bacteria leads to the formation of root nodules where bacteria in the infected plant cells are converted into nitrogen-fixing bacteroids. Nodules with a persistent meristem are indeterminate, whereas nodules without meristem are determinate. The symbiotic plant cells in both nodule types are polyploid because of several cycles of endoreduplication (genome replication without mitosis and cytokinesis) and grow consequently to extreme sizes. Here we demonstrate that differentiation of bacteroids in indeterminate nodules of Medicago and related legumes from the galegoid clade shows remarkable similarity to host cell differentiation. During bacteroid maturation, repeated DNA replication without cytokinesis results in extensive amplification of the entire bacterial genome and elongation of bacteria. This finding reveals a positive correlation in prokaryotes between DNA content and cell size, similar to that in eukaryotes. These polyploid bacteroids are metabolically functional but display increased membrane permeability and are nonviable, because they lose their ability to resume growth. In contrast, bacteroids in determinate nodules of the nongalegoid legumes lotus and bean are comparable to free-living bacteria in their genomic DNA content, cell size, and viability. Using recombinant Rhizobium strains nodulating both legume types, we show that bacteroid differentiation is controlled by the host plant. Plant factors present in nodules of galegoid legumes but absent from nodules of nongalegoid legumes block bacterial cell division and trigger endoreduplication cycles, thereby forcing the endosymbionts toward a terminally differentiated state. Hence, Medicago and related legumes have evolved a mechanism to dominate the symbiosis.

Cell Cycle↗
PubMed2026

N6-methyladenine DNA modification modulates pathogen virulence in nematodes.

Understanding the global regulatory mechanisms that control pathogen virulence gene expression is essential for elucidating the molecular basis of pathogenicity. N6-methyladenine (6 mA) plays a crucial role in regulating gene expression in response to various environmental stresses; however, its role in pathogen virulence remains largely unexplored. Here, we report the widespread occurrence of 6 mA across 17 nematode isolates and map its genomic landscape in six notorious agriculturally important pathogen root-knot nematodes (RKNs). We demonstrated that 6 mA is characterized by a conserved GAG motif across nematodes, but exhibits species-specific distribution patterns and distinct effects on gene expression. In particular, its enrichment in transposable elements (TEs) differs between polyploid and diploid nematodes, suggesting lineage-specific epigenetic regulation potentially associated with polyploidy. We further identified two functional 6 mA demethylases, MiNMAD-1 and MiNMAD-2, and confirmed their catalytic activity and active sites. Host-induced gene silencing (HIGS) of minmad-1 significantly increased plant resistance to three polyploid RKN species. A detailed functional analysis revealed that minmad-1 knockdown broadly affected gene expression during the parasitic stage, including genes involved in virulence, thereby reducing nematode infectivity. Together, our findings suggest 6 mA demethylase as a key epigenetic regulator of RKNs' virulence, providing new insights into nematode biology and offering promising targets for the development of sustainable control strategies.

Animals↗
PubMed1981

Smooth muscle cell hypertrophy versus hyperplasia in hypertension.

Arteries of hypertensive animals have a greater mass of smooth muscle than those of normotensive controls. We examined the contribution of smooth muscle cell hypertrophy and hyperplasia to this increase in mass. Cell size measurements obtained by (i) image analysis of enzyme-dispersed cells, (ii) morphometric evaluation of tissue sections, and (iii) biochemical measures of protein/cell and actin/cell ratios on isolated cells showed that average cell size was greater in spontaneously hypertensive rats than in normotensive Wistar-Kyoto and Sprague-Dawley controls. Average DNA/cell ratios were also increased in spontaneously hypertensive rats while protein/DNA ratios were not different. Analysis of nuclear DNA content of individual cells by flow microfluorimetry and Feulgen-DNA microdensitometry measurements showed that greater than 20% of spontaneously hypertensive rats cells were polyploid while less than 10% of control cells were polyploid. Estimates of cell number per centimeter of aortic length, based on ploidy and DNA content, show no difference between control and hypertensive rats. Thus, smooth muscle hypertrophy alone accounts for the increased mass of smooth muscle in aortas of spontaneously hypertensive rats. Furthermore, this cellular hypertrophy is accompanied by a change in nuclear ploidy. This nuclear response in hypertension may represent a fixed change related to the establishment of a chronic hypertensive state.

Actins↗
PubMed1989

New World tetraploid cottons contain Old World cytoplasm.

New World tetraploid cottons (Gossypium spp.) originated through hybridization of ancestral diploid species that presently have allopatric ranges in Asia-Africa (the A genome) and the New World tropics and subtropics (the D genome). Despite intensive study, the identity of the parental diploids and the antiquity of polyploidization remain unresolved. In this study, variation in the maternally inherited chloroplast genome was assessed among species representing both of the parental genomes and the tetraploids. Approximately 560 restriction sites were assayed in each accession, representing sequence information for about 3200 nucleotides. The resulting maternal phylogeny has no convergent restriction site mutations and demonstrates that the cytoplasm donor for all tetraploid species was an A genome diploid with a chloroplast genome that is similar to Gossypium arboreum and Gossypium herbaceum. No mutational differences were detected between these two species, and few mutations distinguish the chloroplast genomes of A genome diploids from those of tetraploid taxa. In contrast to expectations based on extensive taxonomic, geographic, and genetic diversity, a surprisingly low level of sequence divergence has accumulated subsequent to polyploidization. Chloroplast genomes of tetraploid species are distinguished from each other by between one and six apparent point mutations. The data suggest that tetraploid cotton originated relatively recently, perhaps within the last 1-2 million years, with subsequent rapid evolution and diversification throughout the New World tropics.

Journal Article↗
PubMed1990

Defective chlorophyll a/b-binding protein genes in the genome of a homosporous fern.

The majority of homosporous ferns have a chromosome number that is severalfold greater than that of diploid seed plants. These fern species have therefore been generally considered to be of polyploid origin. Enzyme electrophoretic investigations have demonstrated, however, that within fern genera, species having the lowest chromosome numbers (n = 27-52) have the number of isozymes typical of diploid seed plants; there is no isozyme evidence for polyploidy of these plants. We have constructed a genomic DNA library from Polystichum munitum (n = 41), a homosporous fern, and have screened the library for sequences homologous to the chlorophyll a/b-binding (CAB) protein genes of higher plants. The majority of the sequences isolated and characterized by nucleotide sequence determination represent defective CAB genes. This result is in contrast to the situation in the genomes of diploid angiosperms, where most, and sometimes all, copies of the CAB gene family represent functional members. Several hypotheses could explain the existence of multiple defective CAB genes in P. munitum. (i) The defective CAB genes are the result of "gene silencing" following polyploidy. (ii) P. munitum has not gone through a polyploidization event, but several, and perhaps the majority, of its CAB genes have mutated to a nonfunctional state (a phenomenon not yet observed in any of the genomes of non-fern plants so far examined). (iii) Some defective CAB genes have been specifically amplified in the genome of P. munitum.

Amino Acid Sequence↗
PubMed1991

Genetic diversity and adaptedness in tetraploid Avena barbata and its diploid ancestors Avena hirtula and Avena wiestii.

Avena barbata, a tetraploid grass, is much more widely adapted and successful in forming dense stands than its diploid ancestors. The success of such polyploids has often been attributed to heterosis associated with ability to breed true for a highly heterozygous state in which allelic differences between the parents are fixed in the polyploid by chromosome doubling. We have examined the relationship between genetic diversity and adaptedness for 14 allozyme loci in A. barbata and its diploid ancestors in samples collected from diverse habitats in Israel and Spain. The relationship varied from locus to locus: superior adaptedness was associated with genetic uniformity for five loci, in part with genetic uniformity and in part with genetic diversity (monomorphism for a single heteroallelic quadriplex) for one locus, and with allelic diversity in the form of heteroallelic quadriplexes combined with genotypic diversity in the form of complex polymorphisms among different homoallelic and/or heteroallelic quadriplexes for the eight remaining loci. These results indicate that allelic diversity fixed in nonsegregating form through chromosome doubling was an important factor in the evolution of adaptedness in A. barbata. However, it is unlikely that heterosis associated with heterozygosity contributed significantly to superior adaptedness in either the diploids or the tetraploid because virtually all loci (approximately 99%) were homozygous in the Avena diploids and tetraploid.

Adaptation, Physiological↗
PubMed1992

Chloroplast heteroplasmicity is stabilized by an amber-suppressor tryptophan tRNA(CUA).

Photosynthesis-deficient mutants of the green alga Chlamydomonas reinhardtii were previously shown to arise from nonsense mutations within the chloroplast rbcL gene, which encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Photosynthesis-competent revertants of these nonsense mutants have often been found to be stably heteroplasmic, giving rise to both mutant and revertant cells during mitotic or meiotic divisions under nonselective growth conditions. It was proposed that the stable heteroplasmicity might arise from a balanced polymorphism of suppressor and wild-type alleles of a tRNA gene maintained within the polyploid chloroplast genome. In the present study, we have focused on determining the molecular basis for the heteroplasmicity of one such revertant, named R13-3C, which was recovered from the 18-7G amber (UAG) mutant. Restriction-enzyme analysis and DNA sequencing showed that the amber mutation is still present in the rbcL gene of the revertant strain. In contrast, DNA sequencing of the suspected tRNA(Trp) gene of the revertant revealed a mutation that would change its CCA anticodon to amber-specific CUA. This mutation was found to be heteroplasmic, being present in only 70% of the tRNA(Trp) gene copies. Under nonselective conditions, the suppressor mutation was lost from cells that also lost the revertant phenotype. We conclude that stable heteroplasmicity can arise as a balanced polymorphism of organellar alleles. This observation suggests that additional tRNA suppressors may be identified due to their heteroplasmic nature within polyploid genomes.

Animals↗
PubMed2001

Gland formation in otitis media. An ultrastructural study in humans.

In an effort to evaluate mechanisms of new gland formation in otitis media, 14 human middle ear mucoperiostial tissue samples were obtained from 7 patients with a history of this disease. In areas of inflammatory reaction, especially in the promontory and anterior wall, the mucoperiosteum acquires polyploidal characteristics, with occasional epithelial breaks. The epithelium becomes thicker and the cells develop increased secretory activity. The invaginated portion of these polyploidal formations can be observed as pits lined by secretory epithelium. The cells in these invaginated areas: (i) fuse in areas of epithelial ruptures, leaving spaces into which cells secrete; (ii) develop dense cellular nests that bud off the epithelium, leaving empty spaces into which cells secrete. It is proposed that new gland formation can occur by means of fusion and formation of cellular nests that bud off the epithelium.

Chronic Disease↗
PubMed2001

In vitro expansion of human megakaryocytes as a tool for studying megakaryocytic development and function.

Research on normal human megakaryopoiesis has been limited by technical problems in obtaining megakaryocytic cells in sufficient quantities for experimental purposes. We describe here an ex vivo serum-free liquid culture system to expand normal human megakaryoblasts from purified bone marrow-, cord blood- or peripheral blood-derived CD34(+) cells. The early megakaryocytic cells are expanded in the presence of recombinant thrombopoietin (TpO) and interleukin-3 (IL-3), and if necessary further purified by employing anti-CD61 immunomagnetic beads. Our expansion system generates normal human megakaryoblasts in quantities sufficient to perform various functional studies on these cells as well as to isolate from them proteins and mRNA for molecular analysis. Megakaryocytic cells isolated from these cultures (i) express several markers characteristic of this lineage (CD41, CD61, CD62 P, CXCR-4, PAR-1, etc.), (ii) respond by calcium flux and phosphorylation of various intracellular proteins to stimulation by thrombin and (iii) adhere to fibrinogen and vitronectin. However, human megakaryoblasts derived from the cultures supplemented with TpO + IL-3, in contrast to murine megakaryocytic cells cultured under similar conditions, display poor polyploidization and do not release platelets. Since IL-3 has been reported to inhibit final maturation of megakaryocytic cells, we recently modified our expansion strategy. In this new approach CD34(+) cells are first expanded for 11 days in the presence of TpO + IL-3. Then megakaryoblasts derived are expanded for an additional 7 days supplemented with TpO only. We found that megakaryocytic cells expanded in this 'two step culture' model are more differentiated, are polyploid and release platelets. The model described here provides normal human megakaryoblasts in adequate numbers, to study megakaryopoiesis and megakaryocyte function.

Bone Marrow Cells↗
PubMed2003

Ancient allopolyploid speciation in Geinae (Rosaceae): evidence from nuclear granule-bound starch synthase (GBSSI) gene sequences.

A nuclear low-copy gene phylogeny provides strong evidence for the hybrid origin of seven polyploid species in Geinae (Rosaceae). In a gene tree, alleles at homologous loci in an allopolyploid species are expected to be sisters to orthologues in the ancestral taxa rather than to each other. Alleles at a duplicated locus in an autopolyploid, however, are expected to be more closely related to each other than they are to any orthologous copies in closely related species. We cloned and sequenced about 1.9 kilobases from the 5' end of the GBSSI-1 gene from two diploid, one tetraploid, and six hexaploid species. Each of the three loci in the hexaploid species forms a separate group, two of which are more closely related to copies in other species than they are to each other. This finding indicates that the hexaploid lineage evolved through two consecutive allopolyploidization events. Based on the GBSSI-1 gene tree, we hypothesized that there was an initial hybridization between a diploid species from the ancestral lineage of Coluria and Waldsteinia and an unknown diploid species to form the tetraploid Geum heterocarpum lineage. Backcrossing of G. heterocarpum with a representative of the unknown diploid lineage then resulted in a hexaploid lineage that has radiated considerably since its origin, comprising at least 40 extant species with various morphologies. A penalized likelihood analysis indicated that Geinae may be about 17 million years old, implying that the hypothesized allopolyploid speciation events are relatively ancient. Six of the 22 cloned Geinae GBSSI-1 copies in this study, which all are duplicate copies in polyploid taxa, may have become pseudogenes. We compared the GBSSI-1 phylogeny with one from chloroplast data and explored implications for the evolution of some fruit characters.

Base Sequence↗
PubMed1998

Incongruence between morphological and mitochondrial-DNA characters suggests hybrid origins of parthenogenetic weevil lineages (Genus aramigus).

An expanded matrix of morphological characters for the genus Aramigus (Coleoptera: Curculionidae), which includes numerous polyploid parthenogenetic lineages, was compared and combined with a published matrix of mitochondrial DNA (mtDNA) characters. The matrix of morphological characters provides little resolution of the A. tessellatus and A. uruguayensis species complexes but does resolve previously unresolved relationships among other morphologically defined species (A. globoculus + A. intermedius, A. curtulus + A. planioculus). The morphological and mtDNA characters are significantly incongruent (0.435 < or = IM < or = 0.463; IMF = 0.0735), according to the tests of Farris et al. (P = 0.010) and Templeton (P < 0.005), probably because of hybrid origins of polyploid parthenogenetic lineages. For the few sexual lineages included in both matrices, morphology and mtDNA provide congruent estimates of phylogeny. In spite of recent injunctions against combining data sets that are incongruent because of differing histories, the results of the combined analyses were used to select one of the most-parsimonious mtDNA trees as the best estimate of maternal-lineage genealogy and to reconstruct the evolution of parthenogenesis under the assumption that transitions from sexuality to parthenogenesis are irreversible. Where cytogenetically justified, as in weevils, the irreversibility assumption is useful for producing conservative estimates of the age of parthenogenetic lineages in spite of potential sampling bias against sexuals.

Animals↗
PubMed1985

Nuclear ploidy of neonatal rat livers: effects of two hepatic carcinogens (mirex and dimethylnitrosamine).

The effect of two hepatic carcinogens, dimethylnitrosamine (DMN) (genotoxic) and mirex (epigenetic), on polyploidization in 12-d-old neonatal rats was investigated by Coulter counteranalysis and [3H] thymodine uptake in isolated hepatic nuclear classes. DMN disturbed the normal ploidy development in the neonatal liver and the proportion of nuclei in the ploidy classes by inducing the premature formation of a significant population of tetraploids with a concomitant reduction in diploids. A great proportion of the replicative activity was present in tetraploid nuclei as measured by the incorporation of [3H] thymidine. The labeling index and number of mitoses were also increased. In contrast to DMN, mirex had no influence on polyploidization. The neonatal rats used in these studies thus offer an opportunity to investigate in vivo the mode of action of genotoxic versus epigenetic compounds with reference to their effect on DNA.

Animals↗
PubMed2006

Separase: a universal trigger for sister chromatid disjunction but not chromosome cycle progression.

Separase is a protease whose liberation from its inhibitory chaperone Securin triggers sister chromatid disjunction at anaphase onset in yeast by cleaving cohesin's kleisin subunit. We have created conditional knockout alleles of the mouse Separase and Securin genes. Deletion of both copies of Separase but not Securin causes embryonic lethality. Loss of Securin reduces Separase activity because deletion of just one copy of the Separase gene is lethal to embryos lacking Securin. In embryonic fibroblasts, Separase depletion blocks sister chromatid separation but does not prevent other aspects of mitosis, cytokinesis, or chromosome replication. Thus, fibroblasts lacking Separase become highly polyploid. Hepatocytes stimulated to proliferate in vivo by hepatectomy also become unusually large and polyploid in the absence of Separase but are able to regenerate functional livers. Separase depletion in bone marrow causes aplasia and the presumed death of hematopoietic cells other than erythrocytes. Destruction of sister chromatid cohesion by Separase may be a universal feature of mitosis in eukaryotic cells.

Anaphase↗
PubMedDate not supplied

Thermal acclimation effects differ between voluntary, maximum, and critical swimming velocities in two cyprinid fishes.

Temperature acclimation may be a critical component of the locomotor physiology and ecology of ectothermic animals, particularly those living in eurythermal environments. Several studies of fish report striking acclimation of biochemical and kinetic properties in isolated muscle. However, the relatively few studies of whole-animal performance report variable acclimation responses. We test the hypothesis that different types of whole-animal locomotion will respond differently to temperature acclimation, probably due to divergent physiological bases of locomotion. We studied two cyprinid fishes, tinfoil barbs (Puntius schwanenfeldii) and river barbels (Barbus barbus). Study fish were acclimated to either cold or warm temperatures for at least 6 wk and then assayed at four test temperatures for three types of swimming performance. We measured voluntary swimming velocity to estimate routine locomotor behavior, maximum fast start velocity to estimate anaerobic capacity, and critical swimming velocity to estimate primarily aerobic capacity. All three performance measures showed some acute thermal dependence, generally a positive correlation between swimming speed and test temperature. However, each performance measure responded quite differently to acclimation. Critical speeds acclimated strongly, maximum speeds not at all, and voluntary speeds uniquely in each species. Thus we conclude that long-term temperature exposure can have very different consequences for different types of locomotion, consistent with our hypothesis. The data also address previous hypotheses that predict that polyploid and eurythermal fish will have greater acclimation abilities than other fish, due to increased genetic flexibility and ecological selection, respectively. Our results conflict with these predictions. River barbels are eurythermal polyploids and tinfoil barbs stenothermal diploids, yet voluntary swimming acclimated strongly in tinfoil barbs and minimally in river barbels, and acclimation was otherwise comparable.

Acclimatization↗