Diploid Chromosome Set Research Articles

Epigenetic Control May Explain Large Within-Plant Heterogeneity of Meiotic Behavior in Telocentric Trisomics of Rye

In telocentric trisomics (telotrisomics) of organisms in which the chromosomes normally have two distinct arms, a single chromosome arm with a centromere is present in addition to a complete diploid set of chromosomes. It is the simplest form of polysomy and suitable for analyzing meiotic pairing and recombination patterns in situations where chromosomes compete for pairing. When no suitable meiotic chromosome markers are available, four metaphase I configurations can be distinguished. Their relative frequencies are indicative of the pairing and recombination patterns. In short arm (1RS) telotrisomics of chromosome 1R of rye (Secale cereale) we observed great differences in pairing and recombination patterns among spikes from different tillers and clones of the same plants. Anthers within spikes were only very rarely different. We analyzed a large number of genotypes, including inbreds as well as hybrids. The effects of genetic and environmental conditions on heterogeneity, if any, were limited. Considering that the reproductive tissue of a spike is derived from one primordial cell, it seems that at the start of sexual differentiation there was variation among cells in chromosomal control, which at meiosis determines pairing and crossing-over competence. We suggest that it is an epigenetic system that rigidly maintains this pattern through generative differentiation. In competitive situations the combination most competent for pairing will pair preferentially, forming specific meiotic configurations with different frequencies for different spikes of the same plant. This would explain the heterogeneity between spikes and the homogeneity within spikes. The epigenetic system could involve chromatin conformation or DNA methylation. There were no signs of heterochromatinization.

Cytogenetic variability of cell lines of Ungernia victoris grown on nutrient media of different compositions

Cell lines of U. victoris obtained from a single bulb and grown over lengthy periods of time in agarsolidified and liquid media differed significantly in terms of chromosome number. No effect of the components of the nutrient media, including phytohormones, on the evolution of the governing ratios between cells of different ploidy was found. An increase in the proportion of metaphases with the diploid chromosome set when the callus was transferred into a liquid medium was also established.

Expression of somatic DNA repair genes in human testes

Meiosis is the key process for recombination and reduction of the diploid chromosome set to a haploid one. Many genes that have been found in yeast or mouse models to play a role in meiosis are also important for the repair of DNA damage in somatic cells. To study the DNA repair gene transcriptome during male germ cell development, we have developed a specialized cDNA microarray with 181 human genes which are involved in different somatic DNA repair pathways and/or cell cycle control and 45 control house-keeping genes. This DNA repair gene chip was used to quantify the mRNA expression levels in three human testes samples versus a fibroblast RNA pool. Two hundred twenty genes on the chip (including house-keeping genes) showed detectable expression levels in adult testes. Sixty-four DNA repair- and cell cycle-associated genes showed higher expression levels in testicular cells than in mitotically dividing fibroblasts and, therefore, are likely to be implicated in meiosis. The microarray results of 17 genes with increased expression levels were validated with reverse Northern blots or real-time quantitative RT PCR. Systematic analyses of the meiotic DNA repair gene transcriptome may provide new insights into the genetics of male (in)fertility.

HPLC analysis of leaf surface flavonoids for the preliminary classification of birch species

Flavonoid aglycones found on the surfaces of birch (Betula spp.) leaves may constitute up to 10% of the dry weight of the leaf. A facile extraction and HPLC procedure has been developed that can be used for the preliminary classification of birch species according to the patterns of their leaf surface flavonoids. The procedure involves no complex sample preparation steps, and is able to provide HPLC chromatograms from fresh leaves in less than 30 min. If necessary, leaves do not even need to be removed from the tree. Since the genus Betula is taxonomically complex and separation of different birch species can be problematic, the developed method was applied to 15 Betula species and four sub-species of Betula pendula Seven of the studied species were classified as B. pubescens and eight as B. pendula-type birches. The remaining four species did not belong to either of these two classes on account of their unique pattern of external flavonoids. The difference between the leaf surface flavonoid composition of B. pubescens and B. pendula type birch species was unambiguously clear, and the developed method could reliably distinguish between the two species. Whilst leaf surface flavonoids can be valuable chemotaxonomic markers, they classify birch species differently from morphological markers. Birch species with diploid chromosome sets did not contain any of the flavanones that were present in the leaves of other species. The close relationship between the occurrence of some flavonoid aglycones and the ploidy level of Betula species suggests that these chemotaxonomic markers may be useful both in taxonomic and phylogenetic analyses.

Avian chromosomes can be examined by injection of erythrocyte nuclei into mouse oocytes.

Avian chromosomes can be examined by injection of erythrocyte nuclei into mouse oocytes.

Chromosome abnormalities in secondary pig oocytes matured in vitro

Abnormalities of chromosome segregation during in vitro maturation of oocytes cause failure of in vitro fertilization. Oocytes collected from pig ovaries after slaughter were matured in vitro (IVM) for 30–48 h. In total, 1144 secondary oocytes were studied cytogenetically. An unreduced (diploid) chromosome set was identified in 146 spreads (12.8%). A higher proportion of diploidy was noticed in secondary oocytes matured for 40 h and longer (15.0%) than in the groups matured for 30 and 36 h (9.0%). Among 998 secondary oocytes with the reduced chromosome number, 612 could be analyzed in detail. Hypohaploidy ( n=19−1) was identified in 22 cells (3.59%) and a hyperhaploid ( n=19+1) set of chromosomes was identified in 15 cells (2.45%). The rate of aneuploidy, estimated by doubling the rate of hyperhaploidy was 4.9%. It was also found that aneuploid spreads occurred more frequently in the group of oocytes matured for 40 h and longer. Small acrocentrics were mostly found as an extra chromosome in the hyperhaploid spreads. Our study indicates that to avoid an excess of chromosomally abnormal secondary oocytes, IVM duration of pig oocytes should not exceed 40 h.

The genome composition of hexaploid Psammopyrum athericum and octoploid Psammopyrum pungens (Poaceae: Triticeae)

The genomic constitution of two species in the genus Psammopyrum, i.e., Ps. athericum (2n = 6x = 42) and Ps. pungens (2n = 8x = 56), was studied by genomic in situ hybridization (GISH). In Ps. athericum, one diploid chromosome set hybridized to a genomic probe from Pseudoroegneria ferganensis (St genome), one diploid set to a probe from Agropyron cristatum (P genome), and one diploid set to a probe from Thinopyrum junceiforme (EbEe genomes) or Th. bessarabicum (Eb genome). Substituting the St-genome probe with an L-genome probe from Festucopsis serpentinii resulted in exactly the same hybridization pattern, suggesting a genomic constitution of EStP or ELP for Ps. athericum. The same probes used on Ps. pungens showed two diploid sets of chromosomes hybridizing to the St-genome probe, one diploid set hybridizing to the P-genome probe, and one diploid set hybridizing to the EbEe-genome probe. The L-genome probe hybridized to approximately 14 of the chromosomes that were labeled by the St-genome probe. Hence the genomic constitution for Ps. pungens is proposed to be EStStP or EStLP.

Identifying the chromosomes of the A- and C-genome diploid Brassica species B. rapa (syn. campestris) and B. oleracea in their amphidiploid B. napus

Oilseed rape ( Brassica napus L.) is an amphidiploid species that originated from a spontaneous hybridisation of Brassica rapa L. (syn. campestris) and Brassica oleracea L., and contains the complete diploid chromosome sets of both parental genomes. The metaphase chromosomes of the highly homoeologous A genome of B. rapa and the C genome of B. oleracea cannot be reliably distinguished in B. napus because of their morphological similarity. Fluorescence in situ hybridisation (FISH) with 5S and 25S ribosomal DNA probes to prometaphase chromosomes, in combination with DAPI staining, allows more dependable identification of Brassica chromosomes. By comparing rDNA hybridisation and DAPI staining patterns from B. rapa and B. oleracea prometaphase chromosomes with those from B. napus, we were able to identify the putative homologues of B. napus chromosomes in the diploid chromosome sets of B. rapa and B. oleracea, respectively. In some cases, differences were observed between the rDNA hybridisation patterns of chromosomes in the diploid species and their putative homologue in B. napus, indicating locus losses or alterations in rDNA copy number. The ability to reliably identify A and C genome chromosomes in B. napus is discussed with respect to evolutionary and breeding aspects.

Predictive value of progression-associated chromosomal aberrations for the prognosis of meningiomas: a retrospective study of 198 cases.

The goal of this study was to determine whether in meningiomas cytogenetic findings are suitable as a predictive parameter relevant to prognosis. Between 1992 and 1998 at the Department of Neurosurgery, Saarland University, 198 patients underwent surgery to resect meningiomas. The meningiomas were investigated cytogenetically and the patients were followed up for a mean period of 33 months. On the basis of the cytogenetic findings, the meningiomas were subdivided into four groups: Group 0 meningiomas displayed a normal diploid chromosome set; Group 1 tumors were found to have monosomy 22 as the sole cytogenetic aberration; Group 2 tumors were markedly hypodiploid meningiomas with loss of additional autosomes in addition to monosomy 22; and Group 3 meningiomas had deletions of the short arm of a chromosome 1, as well as additional chromosomal aberrations including loss of one chromosome 22. One hundred ninety-eight patients in whom tumor resections were determined to be Simpson Grade I or II could be followed up after complete tumor extirpation. In 20 patients, one or several recurrences were documented during the period of observation. The tumors were classified according to their different, but mostly uniform chromosomal aberrations. Recurrences were found in six (4.3%) of 139 tumors in Groups 0 and 1 and in two (10.5%) of 19 tumors in Group 2; the highest rate of recurrence was found in 12 (30%) of 40 tumors in Group 3. This supports the notion that the deletion of the short arm of one chromosome 1 is an important prognostic factor in meningiomas. The results of this study document a significant correlation between histological grade (p < 0.0001), location (p < 0.0001), and recurrences of meningiomas (p < 0.0001) (significance determined using chi-square tests). The cytogenetic classification of meningiomas provides a significant contribution to the predictability of tumor recurrence and is, therefore, a valuable criterion for the neurosurgeon's postoperative management protocol.

Effect of activation with Ca ionophore A23187 and puromycin on the development of human oocytes that failed to fertilize after intracytoplasmic sperm injection

Objective: To determine the effect of sequential oocyte activation with calcium ionophore A23187 (A23187) and puromycin on oocytes that were unfertilized after intracytoplasmic sperm injection (ICSI). Design: Laboratory examination. Setting: The in vitro fertilization/embryo transfer unit in Tokushima University Hospital. Patient(s): Discarded unfertilized oocytes following ICSI. Intervention(s): All 172 oocytes that showed no evidence of normal fertilization 18 hours after ICSI were exposed to A23187 (5 μM) for 5 minutes and consequently were treated with puromycin (10 μg/mL) for 5 hours. Main Outcome Measure(s): The activation rate, proportion of oocytes that showed two pronuclei with the second polar body (2PN2PB), and cleavage rate were calculated. Chromosomal analysis of the oocytes with 2PN2PB was also performed. Result(s): The treatment activated 146 out of 172 oocytes (84.9%) and 30.1% of the activated oocytes showed 2PN2PB. Sixteen of 25 oocytes with 2PN2PB (64.0%) cleaved. There was no difference in the activation rate, proportion of activated oocytes with 2PN2PB, or cleavage rate between oocytes that were injected with a motile spermatozoon and those injected with an immotile spermatozoon. Chromosomal analysis showed a normal diploid set of chromosomes (n = 46) in four of five analyzable oocytes. Conclusion(s): The sequential treatment of calcium ionophore A23187 and puromycin activates unfertilized oocytes after ICSI. The resultant oocytes with 2PN2PB can cleave.

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.

Karyological study of some Corvine birds (Corvidae, aves)

Karyotypes were studied in the hooded and carrion crows, their naturally occurred hybrids, the jungle crow, the azure-winged magpie (2n = 80 in all aforementioned birds), and the magpie (2n = 82). Corvine birds of Primorskii Krai were karyotyped for the first time. In addition to the similarity in the diploid chromosome sets, corvine birds were shown to have a similar structure of karyotype: in all studied birds, 14 macrochromosomes (Mchs) classified into three groups according to their size were detected. By karyotype structure, birds belonging to the same genus are similar. Some intergeneric differences are due to a change in the position of centromeres of the largest and sex chromosomes. Karyotypes of interspecific hybrids of crows are remarkable for the presence of heteromorphic (t/st) chromosome pair 2 in some individuals, which apparently does not affect their fecundity. Using differential C-banding, the sex chromosome W in female magpies was identified. In addition, heteromorphism was detected in C-bands of homologs of Mch pair 4 in the hooded crow. In the jungle crow, the azure-winged magpie, and the magpie, bright QH-bands and numerous G-bands were detected on Mchs and on some microchromosomes only. Active Ag-NOR-bands were detected on one macrochromosome pair in the magpie. In all, the karyotype structure of corvine birds is comparable to the basic structural scheme of the karyotype in the order Passeriformes, which confirms the concept of conservatism of the avian karyotype.

Polyploidy induces centromere association.

Many species exhibit polyploidy. The presence of more than one diploid set of similar chromosomes in polyploids can affect the assortment of homologous chromosomes, resulting in unbalanced gametes. Therefore, a mechanism is required to ensure the correct assortment and segregation of chromosomes for gamete formation. Ploidy has been shown to affect gene expression. We present in this study an example of a major effect on a phenotype induced by ploidy within the Triticeae. We demonstrate that centromeres associate early during anther development in polyploid species. In contrast, centromeres in diploid species only associate at the onset of meiotic prophase. We propose that this mechanism provides a potential route by which chromosomes can start to be sorted before meiosis in polyploids. This explains previous reports indicating that meiotic prophase is shorter in polyploids than in their diploid progenitors. Even artificial polyploids exhibit this phenotype, suggesting that the mechanism must be present in diploids, but only expressed in the presence of more than one diploid set of chromosomes.

Somatic embryogenesis and plant regeneration in myrtle (Myrtaceae)

Somatic embryos of myrtle (Myrtus communis L.) were induced from mature zygotic embryos cultured in MS medium supplemented with several concentrations of 2,4-D (2.26 μM – 18.98 μM) or Picloram (2.07 μM – 16.5 μM) combined with 0.087 M or 0.23 M sucrose. For all the concentrations of 2,4-D or Picloram tested, 0.087 M sucrose proved to be more effective than 0.23 M. The best frequencies of induction were obtained in a medium containing 2.26 μM 2,4-D in which 97.3% of the explants produced somatic embryos. Although most embryos were produced from the adaxial side of the cotyledons, some of them differentiated from the hypocotyl. Secondary somatic embryos were often seen arising from the periphery of the former somatic embryos. Somatic embryo development was not synchronous but practically all the embryos germinated well after being transferred to media containing GA3 (0.29, 0.58 and 1.44 μM) alone. When benzyladenine was combined with gibberellic acid, germinating somatic embryos produced adventitious shoot buds which contributed to an increase in plantlet regeneration. Histological observations suggested that somatic embryos arise from the upper surface of the cotyledons probably from peripheral cells. Polyphenol-rich cells were usually seen in association with meristematic-like cells from which somatic embryos originate or with earlier steps of somatic embryo differentiation. Regenerated plants were phenotypically normal, showing a diploid (2n = 22) set of chromosomes.

Chromosomal analysis of multipronuclear zygotes obtained after partial zona dissection of the oocytes.

We have attempted to analyse the chromosome constitution of multipronuclear 1-cell zygotes obtained after partial zona dissection (PZD) of the oocytes. Six cells with three pronuclei could not be evaluated whereas another one was characterized by the presence of a normal haploid and two uninterpretable metaphases. Complete karyotypes were established for 21 tripronuclear cells, taking the varying arrangement of the chromosome sets into consideration. Of the zygotes, 10 showed three separated haploid metaphases (distribution pattern n/n/n), eight zygotes had one haploid and one diploid chromosome set (n/2n) and in three cells the individual sets were not distinguishable (3n). The sex chromosome ratio XXX:XXY:XYY was 7:9:5. Chromosome abnormalities were found in eight of the completely or partially analysable tripronuclear zygotes (36.4%) and included numerical (4 cells), structural (2 cells) as well as combinations of numerical and structural alterations (2 cells). Three out of 11 zygotes with four pronuclei could not be evaluated at all. In three cases, only two chromosome sets were analysable and another cell displayed one uninterpretable set. Three out of eight completely or partially analysable zygotes with four pronuclei (37.5%) had chromosomal abnormalities. Excluding the four cells with one or two uninterpretable metaphases, the sex chromosome distribution XXXX:XXXY:XXYY:XYYY in the zygotes with four pronuclei was 0:1:1:2. Compared with previously analysed multipronuclear zygotes obtained after conventional in-vitro fertilization (IVF), the rate of aberrant zygotes as well as the incidence of aberrant (male + female) chromosome sets were not significantly changed after PZD.

Zytogenetische Analyse von Zygoten mit drei Vorkernen nach partieller Zona dissection (PZD) der Eizellen

Objective : Tripronuclear zygotes obtained after partial zona dissection (PZD) of the oocytes were analysed cytogenetically to determine the rate of chromosomal abnormalities and to allow a comparison with previously published data after conventional IVF. Material and Methods: ollowing incubation in hypotonic solution preparations were made from a total of 28 cells. Chromosomes were stained homogeneously with iemsa. Results: Six cells could not be evaluated whereas another one was characterised by the presence of a normal haploid and two non-interpretable metaphases. Complete karyotypes were established for 21 cells, taking the varying arrangement of the chromosome sets into consideration. Ten zygotes showed three separated haploid metaphases (distribution pattern n/n/n), eight zygotes had one haploid and one diploid chromosome set (n/ 2n) and in three cells the individual sets were not distinguishable (3n). The sex chromosome ratio XXX:XXY:XYY was 7:9:5. Cytogenetic abnormalities were found in eight zygotes including four with numerical and two with structural chromosome alterations. In two cells, aneuploidy and structural aberrations appeared simultaneously. Compared with previously analysed tripronuclear zygotes obtained after conventional IVF, the rate of aberrant zygotes (38.1% vs. 20%) as well as the incidence of aberrant (male + female) chromosome sets (14.1 % vs. 6.7%) were increased after PZD. However, these differences were not significant (probability of error=5%). Conclusion: The transmission of the abnormality could be ascribed to spermatozoa in two cases. The results indicate the need for further investigation of a possibly increased development of chromosomally aberrant zygotes after microinsemination. In addition, more information on the parental origin of the observed aberrations will be necessary.

A Hybrid Method for Parameter Estimation Concerning the Dynamic Modelling of Catabolic Pathways in Escherichia coli

Modelling catabolic pathways has become a very discussed area. Yet lacking enough reliable and consistent experimental and reproducible data that give hints at the intracellular state a method for parameter estimation was installed that allows modelling without the need of narrow preliminary assumptions for the parameters or the kinetics. From a population of individuals each provided with a freely chosen parameter set the one whose simulations fit best to experimental data is selected. Within some generations the model parameters are thus optimized. The genetic algorithm is featured with a diploid chromosome set. Having reached a high, i.e. 80%, fitness, classical parameter identification by the Neider Mead algorithm becomes possible. The results of these procedures are shown for a model of the glycolysis of Escherichia coli.

The chromosomal constitution of multipronuclear zygotes resulting from in-vitro fertilization.

We have attempted to analyse the chromosome constitution of 77 multipronuclear uncleaved zygotes obtained from our in-vitro fertilization programme. Complete karyotypes could be established for 51 tripronuclear cells and eight zygotes with four pronuclei. When compiling the results, the varying arrangement of the chromosome sets was taken into consideration. Eighteen tripronuclear zygotes showed three separate haploid metaphases (distribution pattern n/n/n), 16 cells had one haploid and one diploid chromosome set (n/2n), and in 15 zygotes the individual sets were not distinguishable (3n). Two zygotes were in fact tetraploid, the distribution of metaphases on the slide being n/3n and n/n/2n, respectively. In tripronuclear zygotes the sex chromosome ratio XXX:XXY:XYY was 14:16:18, excluding the two tetraploid cells and one zygote with a 23,X/23,X/22,-C or -Y karyotype. Chromosome abnormalities were found in 16 zygotes (31.4%) and included numerical (six cells), structural (four cells) as well as combinations of numerical and structural alterations (six cells). Four of the zygotes with four pronuclei (50%) had numerical and/or structural chromosome aberrations. Excluding two cells with one uninterpretable metaphase and a 22,-C or -Y karyotype, respectively, the sex chromosome distribution XXXX:XXXY:XXYY:XYYY was 1:1:2:1 in zygotes with four pronuclei. Another zygote was found to be pentaploid after fixation. These results suggest that analysis of multipronuclear zygotes yields valuable information about cytogenetic abnormalities occurring at the earliest stage of conception.

Production of Live Young by Serial Nuclear Transfer with Mitotic Stages of Donor Nuclei in Mice.

The developmental ability of reconstituted embryos that received nuclei from 2-, 4- and 8-cell embryos at M phase of the cell cycle was examined in vitro and in vivo. After fusion of M phase nuclei with enucleated oocytes, the reconstituted oocytes were artificially activated and cultured with cytochalasin B to achieve formation of two “pronuclei-like nuclei” (PN-like nuclei). To obtain embryos with a diploid set of chromosomes, nuclei from each reconstructed embryo were transferred individually into separate enucleated fertilized 1-cell embryos. These nuclear transplants developed to multicellular stages at higher rates and the number of reconstituted embryos doubled. Of the embryos reconstituted with 2-, 4-, and 8-cell nuclei at M phase, 26 (23/88), 40 (32/80) and 4% (5/135) developed to blastocysts, respectively. Live young were produced from blastocysts obtained from reconstituted embryos that received 2- and 4-cell nuclei at M phase, after transfer into recipient females.

Isolation and characterization of temperature-sensitive mammalian cell cycle mutants

Publisher Summary This chapter discusses the isolation and characterization of temperature-sensitive mammalian cell cycle mutants. Except for the sex chromosomes, mammalian cells have a diploid set of chromosomes. Therefore, it is difficult to isolate a recessive mutant from mammalian cells. Furthermore, mammalian cultured cell lines are an artificial biological material, since they are derived from mammalian tissues. When cells are cut out from mammalian tissues and plated on dishes, initially they grow well. Cell lines cultured presently have survived such a crisis and have acquired an ability to grow on dishes permanently, if materials necessary for cell proliferation are supplied. To characterize mammalian ts mutants at the molecular level, it is essential to identify the genes that cause the ts phenotype. Cell cycle analysis of mammalian ts mutants has been improved by combining the synchronization of cell proliferation with the expression pattern of cell cycle-specific genes.