Cytogenetic analysis of two related Deltochilum (Coleoptera,Scarabaeidae) species: Diploid number reduction,extensive heterochromatin addition and differentiation

Male mitotic and meiotic chromosomes of two species of the genus Deltochilum (Scarabaeidae) were analyzed through conventional staining, C-banding, base-specific fluorochromes, silver nitrate staining (AgNO₃) and FISH (45S rDNA). The two species possessed karyotypes with 2n = 14, neo-XY and meta-submetacentric chromosomes. The analysis of constitutive heterochromatin (CH) revealed mainly diphasic chromosomes in the two species, showing heterochromatic long arms. Silver nitrate staining labeled the blocks corresponding to CH in D. (Deltohyboma) aff morbillosum while in D. (Deltohyboma) calcaratum, AgNO₃ staining revealed only the CH blocks of the diphasic autosomes. The fluorochrome staining revealed in D. (D.) calcaratum the diphasic autosomes and the sex chromosomes with CMA₃⁺ blocks, and in D. (D.) aff morbillosum, the GC-rich sequences were restricted to the terminal regions of the long arms of the pairs 1 and 2 and the X. The FISH revealed 45S rDNA sites in two autosomic pairs and in the X chromosome. The analyses performed allowed for the identification of cytogenetic markers and the discussion of possible chromosome rearrangements that have been involved in the karyotypic differentiation of these species mainly related to the repetitive genome.     

First description of B chromosomes in the family Auchenipteridae,Parauchenipterus galeatus (Siluriformes) of the São Francisco River basin (MG,Brazil)

B chromosomes are considered additional and non-essential; they likely originate from A chromosomes and follow a distinct evolution. In fish, approximately half of the Neotropical species with B chromosomes are Characiformes and 35% are Siluriformes. There has been no report of B chromosomes in Auchenipteridae until this moment. B chromosomes found in a population of Parauchenipterus galeatus from the São Francisco River basin in the state of Minas Gerais (Brazil) were small, metacentric, totally heterochromatic and exhibited intra-individual and inter-individual variation. The diploid number was 58 chromosomes (22 metacentric, 16 submetacentric, 12 subtelocentric and 8 acrocentric). The nucleolar organizing regions were simple and the heterochromatin intercalated in the ribosomal sites, characterized by CMA₃ and DAPI fluorochromes, was of a GC-rich constitution. The 5S rDNA genes were located in an intercalary position in only one chromosome pair. An hypothesis about the origin of the B chromosomes in P. galeatus and a review on B chromosomes in catfish are also presented in this study.     

Chromatin condensation and sensitivity of DNA in situ to denaturation during cell cycle and apoptosis--a confocal microscopy study

The goal of this study was to construct high resolution 3D confocal images of regions of condensed and extended chromatin in cell nuclei and individual chromosomes. It has been shown previously that sensitivity of DNA in situ to denaturation correlates with chromatin condensation and varies during cell cycle and apoptosis. Thus, detection of DNA which was partially denatured in situ provided a means to image areas of condensed chromatin. DNA denaturation was detected using a metachromatic dye acridine orange (AO) which differentially stains single stranded (ss) and double-stranded (ds) DNA sections. Early studies of denaturability of cellular DNA utilized flow cytometry and standard fluorescence microscopy. These techniques could not reveal small local differences in DNA denaturability within cell nucleus or in individual chromosomes. For instance, it was not possible to detect the initial points of chromosome condensation in G2-phase of the division cycle or in apoptosis. In order to achieve this goal we have recently extended these studies by applying confocal microscopy. We investigated DNA denaturability in normal human fibroblasts and HL-60 leukemic cells, at different stages of cell cycle and apoptosis. Following removal of RNA and partial denaturation of DNA with acid cells were stained with AO. Green (530 nm) and red (640 nm) fluorescence (exc. 457 nm) of non-denatured and denatured DNA was imaged by confocal microscopy. Blind deconvolution was used to further improve the quality of 3D images. Photobleaching of AO fluorescence was minimized and a correction for chromatic aberration and register shift was implemented. Nuclei of interphase cells exhibited predominantly green fluorescence representing AO binding to ds DNA. Punctuate areas of red fluorescence representing AO binding to denatured DNA and most likely associated with local regions of condensed chromatin were also present in all interphase nuclei. The proportion of denatured DNA increased in cells entering mitosis. In prophase individual condensing chromosomes exhibited varied proportions of green and red fluorescence indicating different content of denatured chromatin. In some chromosomes bands of denatured and denaturation-resistant chromatin were clearly resolved. In metaphase and anaphase chromosomes exhibited red fluorescence along all length of their arms indicating the highest and uniform susceptibility to denaturation. In telophase chromosomes contained predominantly denaturation-resistant DNA again and denaturated regions were significantly less abundant. At cytokinesis some decondensing chromosomes were still resolved. At this stage almost all regions of denatured DNA were located close to nuclear envelope. These regions may correspond to pockets of heterochromatin reforming at nuclear periphery. In early apoptosis condensation of chromatin appeared to commence in several distinct regions within nucleus. Some apoptotic bodies contained condensed chromatin surrounding central regions of extended chromatin. At late stages of apoptosis the whole volume of apoptotic bodies was occupied by condensed chromatin.     

Heterochromatin and rDNA 5S and 45S sites as reliable cytogenetic markers for castor bean (Ricinus communis,Euphorbiaceae)

The increasing need for renewable energy resources has led to higher demands for biofuel, a scenario where the castor bean (Ricinus communis L.) seed oil represents a promising source of raw material. Despite that, information regarding the genome organization of R. communis is still scarce, impairing the application of modern biotechnological and breeding procedures. The present work brings the first evaluation of the mitotic chromosomes of this species, including 10 potentially interesting accessions for cultivation in semi-arid environments aiming at the biofuel production. The approach included standard staining, fluorochrome staining (CMA/DAPI), fluorescent in situ hybridization (FISH) with rDNA 5S and 45S, as well as silver impregnation. All accessions were diploid with 2n = 2x = 20, displaying mainly metacentric chromosomes, with CMA-positive bands (GC-rich) in all pairs of the complement. After silver impregnation, one to 14 nucleoli were observed, while the FISH with rDNA 45S revealed two large sites and a variety of minor dots, and the DNAr 5S hybridized in a single pair. The observed features were discussed and compared with literature data regarding pachytene bivalents.     

Cytogenetic analysis of two Coprophanaeus species (Scarabaeidae) revealing wide constitutive heterochromatin variability and the largest number of 45S rDNA sites among Coleoptera

The Coleopterans of Scarabaeinae clade presents Coprophanaeus (Megaphanaeus) ensifer and C. (Coprophanaeus) cyanescens (Scarabaeidae) when they are studied cytogenetically by different techniques. The species present symmetric karyotypes, diploid number of 2n = 20, and meta-submetacentric chromosomes. C. (M.) ensifer present an XY sex-determining mechanism and C. (C.) cyanescens an XY_p parachute mechanism. Analysis of constitutive heterochromatin (CH) in the two species revealed the presence of diphasic autosomes, with log arm heterochromatics. Moreover, an additional heterochromatic block in four autosomal bivalents were observed in C. (M.) ensifer. CMA₃/DA/DAPI fluorochrome staining detected CMA₃ positive heterochromatic blocks restricted to the sex chromosomes in C. (C.) cyanescens, whereas in C. (M.) ensifer CMA₃ positive pericentromeric blocks were present in all autosomes, in the Y chromosome and in the four additional heterochromatic blocks. DAPI staining was neutral in both species. Silver nitrate (AgNO₃) staining was inefficient for the detection of the nucleolar organizer region (NORs), but showed affinity for the heterochromatic regions. Fluorescence in situ hybridization (FISH) revealed the presence of 45S rDNA sites in the terminal region of the three autosomal bivalents of C. (C.) cyanescens and in seven bivalents and the Y chromosome of C. (M.) ensifer. These results contribute to a better understanding of chromosome evolution in the genus Coprophanaeus, and demonstrate a wide CH variability and the largest number of ribosomal sites among Coleoptera.     

Molecular cytogenetics of Salminus fish (Characiformes) based on 5S and 18S rRNA genes hybridization, fluorochrome staining and C-banding

Karyotype, mapping of nucleolar and 5S rRNA genes and distribution of constitutive heterochromatin supposedly AT-rich were characterized on two isolate populations of Salminus brasiliensis, the biggest characid fish, and three population of Salminus hilarii. The diploid number 2n=50 and the karyotype formulae (10M+20SM+20ST/A) were the same to Salminus species studied. The position of 18S rDNA cluster identified by FISH coincide with chromomycin A(3) labeling (CMA(+)) in the long arm telomeric portion of sixth pair. Subtle differences for the disposal of the 5S rRNA gene in the chromosome of the Salminus are presented. The distribution of the constitutive heterochromatins and DA/DAPI(+) bands are described.     

Cytogenetics of the land snails Cantareus aspersus and C. mazzullii (Mollusca: Gastropoda: Pulmonata)

A cytogenetic study was carried out on the chromosomes and nuclear DNA contents of the land snails Cantareus aspersus and C. mazzullii (Gastropoda: Pulmonata). Chromosomes were studied using Giemsa staining, banding methods and fluorescent in situ hybridization (FISH) with three repetitive DNA probes [18S rDNA, (GATA)(n) and (TTAGGG)(n)]. Results were very similar in the two species both showing (1) 54 bi-armed chromosomes [submetacentrics (SM) + metacentrics (M) + subtelocentrics (ST)]; (2) 10 terminal NORs after sequential application of rDNA FISH and silver staining; (3) uniform DNA fluorescence with CMA(3) and DAPI staining and (4) genomic composition considerably enriched both in highly- and moderately-repeated DNAs. The telomeric (TTAGGG)(n) sequence hybridized with the termini of all of the chromosomes in the two species. In spite of their apparent karyological uniformity, flow cytometry DNA assays showed that C. aspersus and C. mazzullii are characterized by different nuclear DNA content (C values are 3.58 and 3.08 pg, respectively) and slightly different base composition in their genomes. Present data on GS and AT% in C. mazzullii and C. aspersus confirm the trend toward high GS values and GC percentages among land snails.