Analysis Of Mitotic Chromosomes Research Articles

Triploidy in tambaqui Colossoma macropomum identified by chromosomes of fish larvae

Triploid fish are excellent models for use in commercial scale because they can show higher rates of growth, carcass yield and meat quality. If sterile, tambaqui (Colosso mamacropomum) triploids can be used to avoid the problems related to early puberty, especially the negative impacts of male sexual maturation on harvest weight. In the present study, we evaluated the triploidy induction efficiency (thermal shocks) in the fish tambaqui Colossoma macropomum, the most important Neotropical species used for aquaculture. To verify the result of the triploidy experiments, we used a protocol of ploidy identification through analysis of mitotic chromosomes of early life stages (larvae cytogenetics). We obtained better results of hatching and triploid rates through temperature shock at 41°C for 2 min (heat shock) than cold shock. In conclusion, additional treatments will allow to refine the protocols to obtain 100% of triploidy in tambaqui, particularly those from heat shocks. These methodologies can also be applied to other Neotropical fish species, as alternative methods for breeding programs and to delineate conservation plans for the proper management of the stocks.

Cytogenetic characterization of the Antarctic silverfish Pleuragramma antarctica (Boulenger 1902) through analysis of mitotic chromosomes from early larvae

This paper describes the cytogenetic features of the Antarctic silverfish Pleuragramma antarctica (Boulenger 1902), a keystone species of the Antarctic coastal marine ecosystem. Conventional cytogenetic analyses and physical mapping of repetitive DNA sequences were performed on metaphase plates obtained through direct chromosome preparation from P. antarctica early larvae.The Antarctic silverfish have a diploid number (2n) = 48, and a karyotype made up of a majority of two-armed chromosomes (karyotype formula36m/sm + 10st + 2a, fundamental number = 94). Major ribosomal gene repeats were detected on three chromosome pairs (20, 21, and 23), in correspondence of dim DAPI stained regions. Long Interspersed Nuclear Elements (LINEs) were abundant and wide spread over all chromosomes.Overall, the cytogenetic data presented herein are consistent with a long independent cytogenetic and evolutionary history for the species. The large number of two-armed chromosomes, indicative of highly-rearranged karyotype, coupled with a diploid number of 48, a presumed primitive character for this fish group, and the spread of the major ribosomal genes on three chromosome pairs, make the Antarctic silverfish distinct from all other notothenioid species.

Extraordinary centromeres: differences in the meiotic chromosomes of two rock lizards species Darevskia portschinskii and Darevskia raddei.

According to the synthesis of 30 years of multidisciplinary studies, parthenogenetic species of rock lizards of genus Darevskia were formed as a result of different combination patterns of interspecific hybridization of the four bisexual parental species: Darevskia raddei, D. mixta, D. valentini, and D. portschinskii. In particular, D. portschinskii and D. raddei are considered as the parental species for the parthenogenetic species D. rostombekowi. Here for the first time, we present the result of comparative immunocytochemical study of primary spermatocyte nuclei spreads from the leptotene to diplotene stages of meiotic prophase I in two species: D. portschinskii and D. raddei. We observed similar chromosome lengths for both synaptonemal complex (SC) karyotypes as well as a similar number of crossing over sites. However, unexpected differences in the number and distribution of anti-centromere antibody (ACA) foci were detected in the SC structure of bivalents of the two species. In all examined D. portschinskii spermatocyte nuclei, one immunostained centromere focus was detected per SC bivalent. In contrast, in almost every studied D. raddei nuclei we identified three to nine SCs with additional immunostained ACA foci per SC bivalent. Thus, the obtained results allow us to identify species-specific karyotype features, previously not been detected using conventional mitotic chromosome analysis. Presumably the additional centromere foci are result of epigenetic chromatin modifications. We assume that this characteristic of the D. raddei karyotype could represent useful marker for the future studies of parthenogenetic species hybrid karyotypes related to D. raddei.

Flow Sitometri ile Bazı Ispanak Aksesyonlarının Çekirdek DNA İçeriklerinin Belirlenmesi

The aim of this research is to determine ploidy levels of 53 spinach (Spinacia oleracea L.) accessions obtained from abroad sources by using flow cytometry. According to the results of the statistical analysis, the differences among DNA content of accessions were statistically important. The average DNA content of spinach accessions used in the study ranged between 2.225 pg/2C (Esfenaj accession) and 2.059 pg/2C (Matador and Godir accessions). The mean nuclear DNA of accessions shown to be relatively stable due to low standard deviation (0.003-0.096). Based on the mitotic chromosome analyses of the some plants with different DNA contents, mitotic chromosome number of the all plants analyzed in the study were determined as 2n=12. These results indicate that all the accessions used in the study are diploid although their DNA content is significantly vary. According to the clustering analysis of the 2C DNA contents results, it was observed that eight sub clusters were formed under two main clusters. The clustering of axioms was performed in a similar manner to the multiple comparison test. As a result, the data will save time and labor convenience in a further breeding studies deal with the same 53 spinach accessions of our study.

Triploid Production from Interspecific Crosses of Two Diploid Perennial Helianthus with Diploid Cultivated Sunflower (Helianthus annuus L.).

Wild Helianthus species are a valuable genetic resource for the improvement of cultivated sunflower. We report the discovery and characterization of a unique high frequency production of triploids when cultivated sunflower was pollinated by specific accessions of diploid Helianthus nuttallii T. & G. and H. maximiliani Schr. Genomic in situ hybridization (GISH) analyses indicated that the triploid F1s had two genomes from the wild pollen sources and one from the cultivated line. Mitotic chromosome analyses indicated that the frequency of triploid progenies from the crosses of cultivated lines × H. nuttallii accession 102 (N102) was significantly higher than those of unexpected polyploid progenies from the crosses of wild perennial species × N102, and no unexpected polyploids were obtained from the reverse crosses. Pollen stainability analysis suggested the existence of a low percentage of unreduced (2n) male gametes in some accessions, especially N102 and H. maximiliani accession 1113 (M1113), which were generated at the telophase II and tetrad stages of meiosis. The triploid F1s could be the results of preferred fertilization of the low frequency of 2n male gametes with the female gametes of the cultivated sunflower, due to the dosage factors related to recognition and rejection of foreign pollen during fertilization. The triploids have been used to produce amphiploids and aneuploids. Future studies of the male gametes’ fate from pollination through fertilization will further uncover the mechanism of this whole genome transmission. Studies of the genetic control of this trait will facilitate research on sunflower polyploidy speciation and evolution, and the utilization of this trait in sunflower breeding.

SUMOylation Regulates Polo-like Kinase 1-interacting Checkpoint Helicase (PICH) during Mitosis

Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres.

Direct preparation protocol to obtain mitotic chromosomes from canine mammary tumors.

Currently, mammary neoplasms in female canines are a serious problem in veterinary clinics. In addition, the canine species is an excellent disease model for human oncology because of the biological and genetic similarities between the species. Cytogenetics has allowed further study of the characterization of neoplasms in canines. We hypothesized that the use of a direct preparation protocol for mitotic chromosome analysis would provide a simple and low cost protocol for use in all laboratories. The objective of this method is to display in a few hours of dividing cells just like the time of collection since cell division in tissue can be obtained. Ten female canines with the spontaneous occurrence of mammary neoplasia were used to test a pioneering direct preparation protocol to obtain mitotic chromosomes. The excised breast tumor tissue fragments were subjected to the protocol consisting of treatment with colchicine, treatment with hypotonic solution, and fixation. Mitotic chromosomes were absent in cell suspensions of only two samples among the 10 materials analyzed, based on the analysis of five blades for each preparation obtained. So, the cell suspension obtained allowed for the observation of eight tissue samples viable for cytogenetic analysis, five of which had excellent numbers of mitotic chromosomes. However, the technique was unsuccessful in producing high-quality cell suspensions because of inadequate condensation and scattering of chromosomes. While adjustments to methodological procedures are needed, this protocol represents a low cost and simplified method to study the cytogenetics of canine tumors.

Interference in DNA Replication Can Cause Mitotic Chromosomal Breakage Unassociated with Double-Strand Breaks

Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs). We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea) in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways). Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54−/−/KU70−/− DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54−/−/LIG4−/− Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.

Germinação das sementes e análise citogenética em duas espécies de palmeiras

<p><em>Euterpe edulis </em>e <em>Archontophoenix alexandrae </em>são palmeiras usadas para ornamentação e extração do palmito. Foram avaliados a germinação e o IVG das sementes, o comportamento dos cromossomos mitóticos e meióticos e a quantificação de pólen viável. Para cada espécie, em cada localidade e em cada substrato, foram usadas 60 sementes. As análises dos cromossomos mitóticos e a identificação diferencial dos nucléolos foram feitas na zona meristemática da raiz primária. Os corantes usados foram giemsa 2% (m/v) e AgNO3 50% (m/v), respectivamente. A análise dos cromossomos na microsporogênese e a viabilidade do pólen foram feitas em anteras de botões florais, coradas com carmin propiônico 2% (m/v). A maior porcentagem de germinação e IVG das sementes, nas duas espécies, foi observada nas amostras provenientes de Maquiné em substrato contendo vermiculita. <em>A. alexandrae </em>é um diploide com 2n=2x=32 cromossomos com fórmula cariotípica 14m+12sm+4st+2t. <em>E. edulis </em>é um diploide com 2n=2x=36 cromossomos com fórmula cariotípica 16m+12sm+8t. Ambas possuem um par de cromossomos submetacêntricos com constrição secundária intermediária, contendo nucléolos com genes ribossomais ativos. Nas duas espécies, análises da microsporogênese e pólen mostraram alto percentual de células normais em todas as fases da meiose I e II, resultando elevados índice meiótico (tétrades normais) e viabilidade do pólen, indicando que estas são espécies nas quais os genes se expressam normalmente nas condições ambientais em que vivem e não apresentam problemas na formação de células reprodutoras masculinas.</p>

Cytogenetic evidence of mixed disomic and polysomic inheritance in an allotetraploid (AABB) Musa genotype

Edible bananas originated mainly from two wild species, Musa acuminata Colla (AA) and Musa balbisiana Colla (BB), and triploid cultivars with an AAA, AAB or ABB genome are the most widely used. In the present study, chromosome pairing affinities are investigated in a sterile AB Indian variety and in its fertile colchicine-induced allotetraploid (AABB) derivative to determine the inheritance pattern of the tetraploid genotype. The potential implications of interspecific recombination and chromosomal composition of diploid gametes for Musa improvement are presented. The pairing of different chromosome sets at diploid and tetraploid levels was investigated through a combination of conventional cytogenetic and genomic in-situ hybridization (GISH) analyses of meiotic chromosomes, leading to a likelihood model of the pairing behaviour. GISH analysis of mitotic chromosomes was also conducted to reveal the chromosome constitution of hybrids derived from crosses involving the allotetraploid genotype. Analysis of chromosome associations at both ploidy levels suggested that the newly formed allotetraploid behaves as a 'segmental allotetraploid' with three chromosome sets in a tetrasomic pattern, three sets in a likely disomic pattern and the five remaining sets in an intermediate pattern. Balanced and unbalanced diploid gametes were detected in progenies, with the chromosome constitution appearing to be more homogenous in pollen than in ovules. Colchicine-induced allotetraploids in Musa provide access to the genetic background of natural AB varieties. The segmental inheritance pattern exhibited by the AABB allotetraploid genotype implies chromosome exchanges between M. acuminata and M. balbisiana species and opens new horizons for reciprocal transfer of valuable alleles.

Introgression of Swertia mussotii gene into Bupleurum scorzonerifolium via somatic hybridization.

BackgroundThe wild herb Swertia mussotii is a source of the anti-hepatitis compounds swertiamarin, mangiferin and gentiopicroside. Its over-exploitation has raised the priority of producing these compounds heterologously. Somatic hybridization represents a novel approach for introgressing Swertia mussotii genes into a less endangered species.ResultsProtoplasts derived from calli of Bupleurum scorzonerifolium and S. mussotii were fused to produce 194 putative hybrid cell lines, of which three (all derived from fusions where the S. mussotii protoplasts were pre-treated for 30 s with UV light) later differentiated into green plants. The hybridity of the calli was confirmed by a combination of isozyme, RAPD and chromosomal analysis. The hybrid calli genomes were predominantly B. scorzonerifolium. GISH analysis of mitotic chromosomes confirmed that the irradiation of donor protoplasts increased the frequency of chromosome elimination and fragmentation. RFLP analysis of organellar DNA revealed that mitochondrial and chloroplast DNA of both parents coexisted and recombined in some hybrid cell lines. Some of the hybrid calli contained SmG10H from donor, and produced swertiamarin, mangiferin and certain volatile compounds characteristic of S. mussotii. The expression of SmG10H (geraniol 10-hydroxylase) was associated with the heterologous accumulation of swertiamarin.ConclusionsSomatic hybrids between B. scorzonerifolium and S. mussotii were obtained, hybrids selected all contained introgressed nuclear and cytoplasmic DNA from S. mussotii; and some produced more mangiferin than the donor itself. The introgression of SmG10H was necessary for the accumulation of swertiamarin.

1C1524 生きた細胞の分裂期染色体の動的なクロマチン構造(核酸,第49回日本生物物理学会年会)

1C1524 生きた細胞の分裂期染色体の動的なクロマチン構造(核酸,第49回日本生物物理学会年会)

Lampbrush chromosomes of the Japanese quail (Coturnix coturnix japonica): A new version of cytogenetic maps

Avian oocyte chromosomes are transfomed into giant transcriptionally active lampbrush chromosomes (LBCs) at meiosis 1 diplotene. These chromosomes are a convenient tool for high-resulution cytogenetic analysis. Using differential staining with fluorochromes DAPI and CMA3, we have constructed detailed cytological maps for lampbrush macrochromosomes 1-5 and ZW of the Japanese quail Coturnix coturnix japonica. We also performed a comparative analysis ofmitotic chromosomes and LBCs corresponding to them. We estimated the decondensation coefficient during LBC formation and determined the centromere indices for mitotic and diplotene chromosomes and thus found that different chromosomes and chromosomal regions demonstrate unequal degrees of decondensation.

Chromatin differentiation between Theobroma cacao L. and T. grandiflorum Schum

A comparative analysis of mitotic chromosomes of Theobroma cacao (cacao) and T. grandiflorum (cupuaçu) was performed aiming to identify cytological differences between the two most important species of this genus. Both species have symmetric karyotypes, with 2n = 20 metacentric chromosomes ranging in size from 2.00 to 1.19 μm (cacao) and from 2.21 to 1.15 μm (cupuaçu). The interphase nuclei of both species were of the arreticulate type, displaying up to 20 chromocentres, which were more regularly shaped in cacao than in cupuaçu. Prophase chromosomes of both species were more condensed in the proximal region, sometimes including the whole short arm. Both species exhibited only one pair of terminal heterochromatic bands, positively stained with chromomycin A 3 , which co-localized with the single 45S rDNA site. Each karyotype displayed a single 5S rDNA site in the proximal region of another chromosome pair. Heterochromatic bands were also observed on the centromeric/pericentromeric regions of all 20 chromosomes of cacao after C-banding followed by Giemsa or DAPI staining, whereas in cupuaçu they were never detected. These data suggest that the chromosomes of both species have been largely conserved and their pericentromeric chromatin is the only citologically differentiated region.

In vitro root induction in weedy amaranthus species to obtain mitotic chromosomes

Mitotic chromosome analysis has proven to be an important tool in monitoring the potential for genetic exchange among related plant species. One major obstacle to using mitotic chromosome analysis in any species is obtaining large numbers of clear, well-spread metaphase chromosomes necessary to perform cytological techniques such as chromosome banding and fluorescent in situ hybridization. The ability to obtain good chromosome spreads is in part determined by the number and morphology of the roots, which contain the metaphase tissue. Many Amaranthus species produce very thin, delicate roots. The technique used in the process described herein provides for much more substantial roots, allowing for higher probability of obtaining well-spread metaphase chromosomes. Seeds were planted in a soilless mixture, and then cuttings and leaves were taken from the plants. The cuttings were sterilized and placed in Murashige and Skoog (MS) media, while leaf tissue was analyzed by flow cytometry, both pre- and post-propagation, to obtain DNA contents. No changes in DNA content were observed. The in vitro procedure produced significantly larger roots than were produced in soilless mix. Furthermore, all of the in vitro roots observed had 32 chromosomes of normal morphology. In vitro root propagation allowed large numbers of roots to be obtained from a single plant, thereby resulting in increased probability of obtaining cells with metaphase chromosomes that reflected the original plants' chromosome numbers and therefore may be used for molecular cytogenetic analysis.

Sex-specific role of Drosophila melanogaster HP1 in regulating chromatin structure and gene transcription

Drosophila melanogaster heterochromatin protein 1 (HP1a or HP1) is believed to be involved in active transcription, transcriptional gene silencing and the formation of heterochromatin. But little is known about the function of HP1 during development. Using a Gal4-induced RNA interference system, we showed that conditional depletion of HP1 in transgenic flies resulted in preferential lethality in male flies. Cytological analysis of mitotic chromosomes showed that HP1 depletion caused sex-biased chromosomal defects, including telomere fusions. The global levels of specific histone modifications, particularly the hallmarks of active chromatin, were preferentially increased in males as well. Expression analysis showed that approximately twice as many genes were specifically regulated by HP1 in males than in females. Furthermore, HP1-regulated genes showed greater enrichment for HP1 binding in males. Taken together, these results indicate that HP1 modulates chromosomal integrity, histone modifications and transcription in a sex-specific manner.

Mre11 Deficiency in Arabidopsis Is Associated with Chromosomal Instability in Somatic Cells and Spo11-Dependent Genome Fragmentation during Meiosis

The Mre11/Rad50/Nbs1 complex is involved in many aspects of chromosome metabolism. Aberrant function of the complex is associated with defects in the DNA checkpoint, double-strand break repair, meiosis, and telomere maintenance. In this article, we report the consequences of Mre11 dysfunction for the stability of mitotic and meiotic chromosomes in Arabidopsis thaliana. Although plants homozygous for a T-DNA insertion in a conserved region of the MRE11 gene are viable, they exhibit growth defects and are infertile. Analysis of mitotic chromosomes prepared from the mutant plants revealed abundant dicentric chromosomes and chromosomal fragments. Fluorescence in situ hybridization showed that anaphase bridges are often formed by homologous chromosome arms. The frequency of chromosome fusions was not reduced in mre11 ku70 double mutants, suggesting that plants possess DNA end-joining activities independent of the Ku70/80 and Mre11 complexes. Cytogenetic examination of pollen mother cells revealed massive chromosome fragmentation and the absence of synapsis in the initial stages of meiosis. The fragmentation was substantially suppressed in mre11 spo11-1 double mutants, indicating that Mre11 is required for repair but not for the induction of Spo11-dependent meiotic DNA breaks in Arabidopsis.

Metaphase Karyotypes of Fruit Flies of Thailand. V. Cytotaxonomy of Ten Additional New Species of the Bactrocera dorsalis Complex.

Analysis of mitotic chromosomes, with special emphasis on the amount and distribution of constitutive heterochromatin, from larval samples of Thai populations of flies in the Bactrocera dorsalis complex has revealed 10 distinctive forms of metaphase karyotypes. This cytological evidence coupled with morphological observations of adults and host relationships with different host plant species have suggested the existence of 10 new cytotaxonomic species provisionally named species Q-Z belonging to the B. dorsalis complex. Metaphase karyotypes of Thai fruit flies were previously classified into 4 groups based on the quantitative differences in pericentric heterochromatin. Metaphase karyotypes of species Q, R, W, X, Y and Z are characterized by the presence of prominent blocks of pericentric heterochromatin in the X chromosomes and autosomes similar to the mitotic patterns found in B. kanchanaburi and B. raiensis. Hence, they are placed in Group 2 of this classification. Species U and V exhibit chromosome X patterns similar to that of B. carambolae and other related species. They are thus assigned to Group 4. In contrast, species S shows a minimum amount of pericentric heterochromatin in all autosomes, although the X chromosome follows the pattern of species Y. Interestingly, species T exhibits a unique pattern of euchromatin and heterochromatin in the X chromosome and conspicuous pericentric heterochromatin in all autosomes. Thus, species S and T are assigned to 2 new categories. Heterochromatin accumulations in the genome, as demonstrated in this study, appear to have played a significant role in chromosomal evolution. The different degree of variation in pericentric heterochromatin in mitotic chromosomes is, therefore, useful for cytotaxonomically distinguishing members of some cryptic species complexes of the dipteran insects.

A novel simple satellite DNA is colocalized with the Stalker retrotransposon in Drosophila melanogaster heterochromatin.

In the T(1:2)dor(var7) multibreak rearrangement the distal 1A-2B segment of the X chromosome of Drosophila melanogaster is juxtaposed to an inverted portion of the heterochromatin of chromosome 2. Analysis of mitotic chromosomes by a series of banding techniques has permitted us precisely to locate the heterochromatic breakpoint of this translocation in the h42 region of 2R. Cloning and sequencing of the eu-heterochromatic junction revealed that the translocated 1A-2B fragment is joined to (AACAC)n repeats, which represent a previously undescribed satellite DNA in D. melanogaster. These repeated sequences have been estimated to account for about 1 Mb of the D. melanogaster genome. The repeats are located mainly in the Y chromosome and in the heterochromatin of the right arm of chromosome 2 (2Rh), where they are colocalized with the Stalker retrotransposon.

The relationship between genetic and cytogenetic maps of pea. I. Standard and translocation karyotypes

A detailed cytogenetical study of inbred lines of pea and their F1 hybrids has been undertaken to study the relationship between the cytogenetic map and the molecular linkage map. The mitotic karyotypes of a standard pea line, JI15, a translocation line, JI61, and line JI281, a line used in the production of a mapping population, are given. A chromosome rearrangement detected by cytogenetic analysis of mitotic chromosomes has been further defined by synaptonemal complex (SC) analysis and the study of metaphase I chromosome behaviour. This meiotic analysis has allowed a comparison of SC physical lengths, observed chiasma frequencies, and recombination frequencies, as estimated from the genetic map, as a means of comparing physical and genetic distances.