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PubMed2001

Zona pellucida solubility and cortical granule complements in human oocytes following assisted reproductive techniques.

In this study the solubility to alpha-chymotrypsin of the zona pellucida (ZP) of human oocytes and polyploid embryos obtained during various clinical procedures of assisted fertilisation (IVF, ICSI, cyropreservation) was evaluated. The aim of the study was to determine whether changes in ZP solubility occur during such procedures and whether abnormal solubility could be likened to fertilisation failure. Correlation between ZP solubility and cortical granule (CG) density was also studied. The results showed that ZP solubility varied considerably among germinal vesicle or metaphase oocytes obtained from different subjects, but was essentially identical for the oocyte cohort obtained from individual women. On the basis of ZP solubility metaphase oocytes were subdivided into two classes: class I, average ZP dissolution time +/- SE = 24.1+/-0.9 min, n = 28; and class II, 46.7+/-2.0 min, n = 13. Prolonged ZP dissolution times of metaphase oocytes were significantly correlated with a low in vitro fertilisation rate in sibling oocytes. The zonae of fertilised eggs (polyploid embryos) showed long solubilisation times (IVF: 45.3+/-3.4 min, n = 18; ICSI: 48.9+/-2.7 min, n = 19). ZP solubility of oocytes that failed to fertilise was intermediate between that of class I metaphase oocytes and embryos (unfertilised IVF: 33.0+/-2.7 min, n = 13; unfertilised ICSI: 43.0+/-2.4 min, n = 9). A moderate spontaneous ZP hardening occurred when metaphase oocytes were cultured for 24 h. Finally, cryopreservation of unfertilised oocytes caused hardening of their ZP, with dissolution times that were comparable to those found in fertilised eggs (49.5+/-2.3 min, n = 10). In most cases, an inverse correlation was found between ZP dissolution time and CG density (longer solubilisation times corresponding to lower CG density). ZP hardening caused by cryopreservation, however, was not associated with a significant reduction in CG density in most of the oocytes examined.

Adult↗
PubMed2000

Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates.

Increasing evidence supports the idea that various transgene silencing phenomena reflect the activity of diverse host defense responses that act ordinarily on natural foreign or parasitic sequences such as transposable elements, viroids, RNA and DNA viruses, and bacterial DNA. Transgenes or their transcripts can resemble these cellular invaders in a number of ways, thus making them targets of host protective reactions. At least two distinct host defense systems operate to silence transgenes. One acts at the genome level and is associated with de novo DNA methylation. A second line of defense operates post-transcriptionally and involves sequence-specific RNA degradation in the cytoplasm. Transgenes that are silenced as a consequence of the genome defense are revealing that de novo methylation can be cued by DNA-DNA or RNA-DNA interactions. These methylation signals can be interpreted in the context of transposable elements or their transcripts. During evolution, as transposable elements accumulated in plant and vertebrate genomes and as they invaded flanking regions of genes, the genome defense was possibly recruited to establish global epigenetic mechanisms to regulate gene expression. Transposons integrated into promoters of host genes could conceivably change expression patterns and attract methylation, thus imposing on endogenous genes the type of epigenetic regulation associated with the genome defense. This recruitment process might have been particularly effective in the polyploid genomes of plants and early vertebrates. Duplication of the entire genome in polyploids buffers against insertional mutagenesis by transposable elements and permits their infiltration into individual copies of duplicated genes.

Animals↗
PubMed2001

Evolution in action through hybridisation and polyploidy in an Iberian freshwater fish: a genetic review.

The Iberian minnow Leuciscus alburnoides represents a complex of diploid and polyploid forms with altered modes of reproduction. In the present paper, we review the recent data on the origin, reproductive modes, and inter-relationships of the various forms of the complex, in order to predict its evolutionary potential. The complex follows the hybrid-origin model suggested for most other asexual vertebrates. Diploid and triploid females from the southern river basins exhibit reproductive modes that cannot be conveniently placed into the categories generally recognised for these vertebrate complexes, which imply continuous shifting between forms, where genomes derived from both parental ancestors are cyclically lost, gained or replaced. Replacement of nuclear genomes allow the introduction of novel genetic material, that may compensate for the disadvantages of asexual reproduction. Contrasting with most other vertebrate complexes, L. alburnoides males are fertile and play an important role in the dynamics of the complex. Moreover, diploid hybrid males may have initiated a tetraploidization process, when a diploid clonal sperm fertilised a diploid egg. This direct route to tetraploidy by originating fish with the right constitution for normal meiosis (symmetric), may eventually lead to a new sexually reproducing polyploid species. This case-study reinforces the significance of hybridisation and polyploidy in evolution and diversification of vertebrates.

Animals↗
PubMed2004

Laying the cytotaxonomic foundations of a new model grass, Brachypodium distachyon (L.) Beauv.

Brachypodium distachyon is a ubiquitous, temperate grass species which is being developed and exploited as an alternative model to rice, in order to gain access to important syntenic regions of the genomes of less tractable relatives such as wheat. As part of this initiative, this paper describes for the first time the cytotaxonomy of members of the polyploid series of this species, and challenges the assumption that the series evolved simply by chromosome doubling. In situ hybridization using genomic DNA probes and rDNA markers uncovers a hybrid origin of several of the polyploid ecotypes, and sheds light upon the complex evolution of this species and its close relatives.

Chromosome Painting↗
PubMed2002

Chromosome analysis in 31 cases of benign and malignant breast tumors: a study in Brazil.

Cultures of 31 breast tumors, being 20 carcinomas and 11 benign lesions, were cytogenetically analysed. Clonal chromosome aberrations were detected in 16 carcinomas and in 4 benign lesions. Nine carcinomas and 2 benign lesions had multiple cytogenetically unrelated and related clones, whereas a single abnormal clone was observed in 7 carcinomas and in 2 benign lesions. Polyploid clones were found in 7 carcinomas and in 2 benign lesions. The presence of clonal chromosome aberrations and polyploid cells was not associated with the clinicopathologic parameters tested. Carcinomas had more clonal changes than benign lesions (p = 0.031), showing that cytogenetic features are of diagnostic value and that different chromosome anomalies might have different pathogenetic and prognostic significance.

Adult↗
PubMed2001

The Ph1 locus is needed to ensure specific somatic and meiotic centromere association.

The correct pairing and segregation of chromosomes during meiosis is essential for genetic stability and subsequent fertility. This is more difficult to achieve in polyploid species, such as wheat, because they possess more than one diploid set of similar chromosomes. In wheat, the Ph1 locus ensures correct homologue pairing and recombination. Although clustering of telomeres into a bouquet early in meiosis has been suggested to facilitate homologue pairing, centromeres associate in pairs in polyploid cereals early during floral development. We can now extend this observation to root development. Here we show that the Ph1 locus acts both meiotically and somatically by reducing non-homologous centromere associations. This has the effect of promoting true homologous association when centromeres are induced to associate. In fact, non-homologously associated centromeres separate at the beginning of meiosis in the presence, but not the absence, of Ph1. This permits the correction of homologue association during the telomere-bouquet stage in meiosis. We conclude that the Ph1 locus is not responsible for the induction of centromere association, but rather for its specificity.

Binding Sites↗
PubMed2002

Adenovirus-mediated gene transfer of P16INK4/CDKN2 into bax-negative colon cancer cells induces apoptosis and tumor regression in vivo.

The tumor-suppressor gene p16INK4/CDKN2 (p16) is a cyclin-dependent kinase (cdk) inhibitor and important cell cycle regulator. Here, we show that adenovirus-mediated gene transfer of p16 (AdCMV.p16) into colon cancer cells induces uncoupling of S phase and mitosis and subsequently apoptosis. Flow cytometric analysis revealed that cells infected with AdCMV.p16 showed an initial G2-like arrest followed by S phase without intervening mitosis (DNA >4N). Using microscopic analysis, deformed polyploid cells were detectable only in cells infected with AdCMV.p16 but not in control-infected cells. Subsequently, AdCMV.p16-infected polyploid cells underwent apoptosis, as assessed by AnnexinV staining and DNA fragmentation, suggesting that cell cycle dysregulation is upstream of the onset of apoptosis. Treatment of mice with subcutaneously transplanted tumors of colorectal cancer cells with AdCMV.p16 but not AdCMV.p53 resulted in significantly reduced tumor volume and prolonged survival. Using an orthotopic model of liver metastasis, we observed both reduced local tumor growth and secondary intrahepatic metastasis after AdCMV.p16 treatment. Importantly, induction of apoptosis in vitro and reduction of tumor growth in vivo by p16 was p53- as well as bax-independent because identical results were obtained using cancer cells, either wild type or mutant for p53 or bax. The studies suggest that an AdCMV.p16-based treatment may be especially effective in patients with bax-negative colon cancer where overexpression of p53 appears not to be of therapeutic value.

Adenoviridae↗
PubMed1999

Deregulation of p53/p21Cip1/Waf1 pathway contributes to polyploidy and apoptosis of E1A+cHa-ras transformed cells after gamma-irradiation.

The p53/p21Cip1/Waf1-dependent checkpoint control of G1/S and G2/M phases of the cell cycle in response to DNA damage is an important mechanism of genome stability maintenance in normal cells. In many tumor cells, due to frequent point mutations and deletions of p53, the stringent control of the cell cycle and apoptosis is compromised. We have examined the cell cycle control and cell death of the rat embryo fibroblast cells (REF) transformed by E1A+cHa-ras oncogenes and expressing wild type p53. Gamma-irradiation at a dosage of 6 Gy has been used to analyse the p53-dependent trans-activation of the target p21cip1/waf1 gene and the levels of activity of cyclin-dependent kinases. Our results show that the cell cycle inhibitors p21Cip1/Waf1 and p27KIP accumulate in response to irradiation both in REF and E1A+cHa-ras cells. In contrast to normal REF cells, the accumulation of p21Cip1/Waf1 and p27KIP inhibitors, however, does not lead to inhibition of Cdk2 and cyclins E, A-associated kinase activities and to a G1/S block in E1A+cHa-ras cells. It is unlikely that the lack of inhibitory function of p21Cip1/Waf1 can be explained by its inability to bind Cdk2 and Cdk4 kinases or PCNA. Moreover, the p21Cip1/Waf1-associated kinase activity is increased upon gamma-irradiation of E1A+cHa-ras cells. We suggest that inactivation of p21Cip1/Waf1 may be accounted for by its interaction with E1A oncoproducts as the inhibitor is detected in immunoprecipitates using E1A-specific antibodies. During a temporary G2/M delay induced by gamma-irradiation, E1A+cHa-ras transformants continue DNA replication, which leads to accumulation of polyploid cells with lobulated nuclei and micronuclei. Thus, DNA damage of E1A+cHa-ras transformed cells, with a combination of functionally active wild type p53 and inactive p21Cip1/Waf1, contributes to formation of polyploid cells which then die due to apoptosis.

Adenovirus E1A Proteins↗
PubMed2000

Ectopic expression of cyclin E allows non-endomitotic megakaryoblastic K562 cells to establish re-replication cycles.

Megakaryocytes become polyploid by entering a truncated cell cycle, consisting of alternate S phases and abortive mitoses. We have investigated the regulation of the G1/S transition by comparing two megakaryoblastic cell lines, HEL and K562, which respectively do or do not become polyploid in response to phorbol esters. A pronounced downregulation of cyclin A, and to a lesser extent of cyclin E, occurred in K562 cells during the first 24 h after TPA treatment, in contrast with re-replicating HEL cells, in which both cyclins were present in individual G2/M cells. Transactivation experiments suggested that the absence of cyclin A in differentiated K562 cells could be due to a TPA-mediated inhibition of its transcription. To investigate the potential role of cyclin E in the establishment of re-replication cycles, we isolated K562 clones constitutively expressing cyclin E. The resulting clones, and also K562 cells transiently expressing cyclin E, entered re-replication cycles when treated with TPA. The transcriptional activity of the cyclin A promoter was not inhibited after TPA treatment, and although the levels of cyclin A fluctuated during further re-replication cycles, they never decreased below S phase levels. We conclude that the presence of cyclin E in megakaryoblastic G2/M cells determines cyclin A expression and allows the entrance into an extra S phase.

Bone Marrow Cells↗
PubMed2005

New insights into cell cycle control from the Drosophila endocycle.

During metazoan development, the organization of the cell cycle is often modified in response to developmental signals. The endocycle provides a dramatic example of this phenomenon. In the endocycle, also referred to as the endoreplicative cycle, cells undergo successive rounds of DNA replication without an intervening mitosis. Often the endocycle is used to expand the genome of a group of specialized cells that are highly biosynthetically active. In these circumstances, large polyploid cells are produced in organisms that are primarily comprised of diploid cells. However, many organisms achieve growth by increasing cell size, rather than cell number. This strategy is more generally exploited in insects and plants. For instance, in the insect Drosophila melanogaster, the majority of the larval tissues, as well as many adult tissues, enter the endocycle and become polyploid. Therefore, Drosophila has been a rich source for studies on endocycle regulation. Recent work from Drosophila is beginning to reveal how developmental signals promote the transition from the mitotic cycle to the endocycle, as well as what drives endocycle progression. In addition, studies on the endocycle have provided insight into the regulatory principles underlying the once per cell cycle replication of the genome, as well as the relationship between S phase and mitosis.

Animals↗
PubMed1975

Relationship between enhanced turnover of phosphatidylinositol and lymphocyte activation by mitogens.

1. Various lectins [phaseolus vulgaris phytohaemagglutinin, Glycine max (soy-bean) agglutinin, Triticum vulgaris (wheat-germ) agglutinin and Axinella polyploides agglutinin] and antibodies to pig Ig (immunoglobulin) that are found by pig lymphocytes were assessed in terms of their capacities to stimulate lymphocyte transformation and to enhance phosphatidylinositol turnover. Transformation was measured after 45h of culture by incorporation of [6-(3)H]thymidine into DNA, whereas phosphatidylinositol metabolism was assessed after 1h of cultuis and G. max agglutinins and rabbit antibodies to pig Ig) increased phosphatidylinositol turnover, but non-transforming agents (T. vulgaris and A. polyploides agglutinins and Fab fragments of rabbit antibodies to pig Ig) failed to induce any significant enhancement. Subsequent cross-linkage of the bound, non-transforming Fab fragments with a goat antiserum to rabbit Ig stimulated transformation and phosphatidylinositol turnover. 3. Each transforming agent gave characteristic optimal dose responses that were similar for both phosphatidylinositol turnover and transformation. 4. The results indicate that activation of T- and B-lymphocytes is accompanied by enhanced phosphatidylinositol turnover and that in the case of B-cells this enhancement depends on the cross-linkage of surface receptors. They are consistent with the proposal that turnover represents an essential early step in the transformation process.

Animals↗
PubMed1997

Expression and function of receptors for extracellular matrix molecules in the differentiation of human megakaryocytes in vitro.

The role of adhesive interactions with the extracellular matrix components of the bone marrow (BM) stroma has been widely studied in the differentiation of erythroid and myelomonocytic cells, but not in the megakaryocytic lineage. The development of efficient culture techniques for the production of megakaryocytes (Mks) from CD34+ purified BM cells, enables the study of the expression and function of adhesion receptors for collagen (VLA-2), fibronectin (VLA-4 and VLA-5) and laminin (VLA-6) during the maturation of Mks. We have shown that a significant percentage of CFU-MK (roughly 20%) adhere to fibronectin but not to collagen and laminin. The expression and adhesion of Mks developing in liquid culture from BM-CD34+ cells were tested at days 4, 7 and 10 of incubation. The expression of VLA-2, VLA-5 and VLA-6 on day 10 cultured Mks enabled purification of intermediate and large polyploid Mks by FACS sorting whereas VLA-4 appeared to label only immature Mks and myeloid cells. We observed that only a small proportion of mature Mks was able to adhere to collagen without spreading at day 10 of culture, whereas 30% of Mks adhered to fibronectin as early as day 4 of incubation, 40% of which also attached to laminin. Our data suggest that VLA-4 may be involved in the adhesion of CFU-MK and immature Mks on fibronectin, then replaced by VLA-5 in the final stages of maturation. The expression of VLA-6 and the number of adherent Mks on laminin increased sharply between day 7 and 10 of incubation. A number of mature polyploid Mks found in day 10 of culture exhibited characteristic features of intense spreading on laminin and fibronectin which were not observed on collagen.

Antigens, CD34↗
PubMed2000

Mating systems of diploid and allotetraploid populations of Tragopogon (Asteraceae). II. Artificial populations.

Polyploidization has long been recognized as an important force in the diversification of plants. Theoretical models predict that polyploids may be expected to exhibit higher rates of self-fertilization than do closely related diploid species. Wild populations of the neopolyploid Tragopogon mirus (4n) exhibited slightly higher rates of outcrossing than did populations of one of its progenitors, T. dubius (2n). In the current study, outcrossing rates in populations of T. dubius and T. mirus were estimated using artificial arrays constructed to maximize the chances of detecting outcrossing events. The artificial diploid population is more highly outcrossing (t=0.727; family-level estimates range from 0.00 to 1. 32) than the tetraploid population (t=0.591; family-level estimates range from 0.00 to 1.14), although the difference between them is not statistically significant. The results of this study, combined with those of the previous work on wild populations, suggest that mating systems in these species vary more among populations than between ploidal levels. This could be because of the relatively recent origins of the tetraploid species; there may have been insufficient time since the formations of the tetraploids for shifts in mating systems to occur.

Alleles↗
PubMed2000

Genetic structure and colonizing success of a clonal, weedy species, Pilosella officinarum (Asteraceae).

Introduced populations of weeds which are polyploid and reproduce primarily by apomixis are generally considered as having low levels of population genetic variation, highly differentiated populations and short evolutionary lifespans. Although polyploidy allows for habitat differentiation and colonization, lack of recombination because of apomixis means that long-term persistence is unlikely. However, variation can be introduced to a colonizing population by evolutionary changes in the mating system, or by somatic mutation and recombination. In this study hypersensitive genetic markers, inter-simple sequence repeats (ISSRs), were used to quantify genetic variation within Pilosella officinarum, a major weed of the New Zealand high country. Pilosella officinarum was introduced from Europe to New Zealand late in the 19th century and only polyploid, apomictic populations are thought to have survived. The combination of introduction history and breeding system has led to the assumption that New Zealand populations are necessarily genetically depauperate. However, our studies reveal variable levels of genetic variation and patterns of clonal distribution which indicate varying levels of sexual reproduction within New Zealand populations.

Asteraceae↗
PubMed2001

Haplotype variation of cpDNA in the agamic grass complex Pennisetum section Brevivalvula (Poaceae).

The Brevivalvula section of the grass polyploid complex Pennisetum shows various reproductive systems, apomixis being the most widespread. Haplotype variation of chloroplast DNA was studied in the six morphological taxa (species) of this section by using RFLP analysis in 54 plants corresponding to 14 elementary taxa, each characterized on the basis of morphology and ploidy level. Two additional species, Pennisetum glaucum and P. purpureum, which belong to another section of the same genus, were analysed for comparison. In Brevivalvula, chloroplast DNA size was estimated to range between 130 and 133 kb. Thirteen of 15 distinct haplotypes identified in the study were specific to the Brevivalvula section. They were unequally distributed among the morphotypes, the ploidy levels and sampling sites. Within the Brevivalvula section, plants of P. setosum, which are perennial and reproduce vegetatively or by agamospermy, possessed a single specific haplotype. This species differed clearly from the five other morphological species, which are known to be annual, to show either sexual or agamospermic reproduction and which shared most of the 12 other haplotypes observed in the section, suggesting the occurrence of multiple hybridization events between the taxa. Chloroplast DNA variation was highly geographically structured, suggesting low seed dispersal between sites, whereas the substantial haplotype diversity observed in the sites may indicate that agamic reproduction is responsible for the maintenance of distinct genetically isolated clones. Haplotype classification using Wagner's parsimony suggested the occurrence of bidirectional gene flow between the diploids and the polyploids, as reported already in other related apomictic complexes.

Africa, Western↗
PubMed1999

Polymorphic chloroplast simple sequence repeat primers for systematic and population studies in the genus Hordeum

In this study we report the development of primers to amplify polymorphic chloroplast simple sequence repeats in the genus Hordeum, which includes cultivated barley (H. vulgare ssp. vulgare) and its wild progenitor H. vulgare ssp. spontaneum. Polymorphic products were amplified in a wide range of Hordeum spp. and intraspecific variation was detected in both cultivated and wild barley. A decrease in cytoplasmic diversity was observed between sspp. spontaneum and vulgare as well as between ssp. vulgare landraces and cultivars, which is characteristic of domestication processes in many corp species. We also observed possible evidence for reticulate evolution of H. brachyantherum polyploids, with apparent multiple cytoplasmic introgressions during successive polyploidization events.

Journal Article↗
PubMed2002

Polyploidy, phylogeography and Pleistocene refugia of the rockfern Asplenium ceterach: evidence from chloroplast DNA.

Chloroplast DNA sequences were obtained from 331 Asplenium ceterach plants representing 143 populations from throughout the range of the complex in Europe, plus outlying sites in North Africa and the near East. We identified nine distinct haplotypes from a 900 bp fragment of trnL-trnF gene. Tetraploid populations were encountered throughout Europe and further afield, whereas diploid populations were scarcer and predominated in the Pannonian-Balkan region. Hexaploids were encountered only in southern Mediterranean populations. Four haplotypes were found among diploid populations of the Pannonian-Balkans indicating that this region formed a northern Pleistocene refugium. A separate polyploid complex centred on Greece, comprises diploid, tetraploid and hexaploid populations with two endemic haplotypes and suggests long-term persistence of populations in the southern Mediterranean. Three chloroplast DNA (cpDNA) haplotypes were common among tetraploids in Spain and Italy, with diversity reducing northwards suggesting expansion from the south after the Pleistocene. Our cpDNA and ploidy data indicate at least six independent origins of polyploids.

Base Sequence↗
PubMed2002

Linkage relationships of genes coding for alpha2-macroglobulin, C3 and C4 in the zebrafish: implications for the evolution of the complement and Mhc systems.

The alpha2-macroglobulin (A2M) and the complement components C3 and C4 are related proteins derived from a common ancestor. Theoretically, this derivation could have occurred either by tandem duplications of their encoding genes or by polyploidization involving chromosomal segments, a chromosome or the whole genome. In tetrapods the A2M-, C3- and C4-encoding genes are generally each located on a different chromosome. This observation has been interpreted as supporting their origin by polyploidization. We identified and mapped (with the help of a radiation hybrid panel of cell lines) the A2M, C3 and C4 loci in the zebrafish, Danio rerio. Each of the three types of loci is present in the zebrafish in multiple copies, but all of the identified copies of a given type map to the same region in linkage groups 1 (C3) and 15 (A2M, C4). The A2M and C4 loci are mapped in the same region not linked to any of the class I or class II major histocompatibility complex (Mhc) loci. These observations are interpreted as supporting the origin of the A2M family of genes by tandem duplications, followed by the dispersal of the copies to different chromosomes. It is also argued that the association of C4 with the class I/II loci in tetrapods is accidental and without functional significance.

Animals↗