AT-specific Fluorochrome Research Articles

Small genome size variation across the range of European beech (Fagus sylvatica)

Interpopulation variation of relative and absolute genome size was studied in Fagus sylvatica subsp. sylvatica and subsp. orientalis. The study included 18 populations of beech planted in a common-garden experiment in central Slovakia and three additional populations from the Caucasus. Nuclear DNA content was determined by means of flow cytometry using the AT-specific fluorochrome 4′,6-diamidino-2-phenylindole and non-specific propidium iodide, and its associations with climate, growth, phenology and physiological traits were assessed. The approximate average nuclear DNA content (2C) across all samples was 1.178 ± 0.020 pg. The lowest mean relative genome sizes were observed in the Alpine range, whereas they increased toward the range margins; no clear trend was observed for 2C values. Temperature seasonality and temperature annual range were found to be negatively associated with genome size. Among phenotypic traits, the maximum chlorophyll a fluorescence yield (Fv/Fm) was found to be negatively correlated with relative genome size, whereas phenology and some photosynthetic parameters were correlated with the 2C values.

Heterochromatin patterns and ribosomal DNA loci distribution in diploid and polyploid Crotalaria species (Leguminosae, Papilionoideae), and inferences on karyotype evolution

Most Crotalaria species display a symmetric karyotype with 2n = 16, but 2n = 14 is found in Chrysocalycinae subsection Incanae and 2n = 32 in American species of the section Calycinae. Seven species of the sections Chrysocalycinae, Calycinae, and Crotalaria were analyzed for the identification of heterochromatin types with GC- and AT-specific fluorochromes and chromosomal location of ribosomal DNA loci using fluorescent in situ hybridization (FISH). A major 45S rDNA locus was observed on chromosome 1 in all the species, and a variable number of minor ones were revealed. Only one 5S rDNA locus was observed in the species investigated. Chromomycin A(3) (CMA) revealed CMA(+) bands colocalized with most rDNA loci, small bands unrelated to ribosomal DNA on two chromosome pairs in Crotalaria incana, and CMA(+) centromeric bands that were quenched by distamycin A were detected in species of Calycinae and Crotalaria sections. DAPI(+) bands were detected in C. incana. The results support the species relationships based on flower specialization and were useful for providing insight into mechanisms of karyotype evolution. The heterochromatin types revealed by fluorochromes suggest the occurrence of rearrangements in repetitive DNA families in these heterochromatic blocks during species diversification. This DNA sequence turnover and the variability in number/position of rDNA sites could be interpreted as resulting from unequal crossing over and (or) transposition events. The occurrence of only one 5S rDNA locus and the smaller chromosome size in the polyploids suggest that DNA sequence losses took place following polyploidization events.

Conserved Cytogenetic Features in the Amazonian Arapaima, Arapaima gigas (Schinz 1822) from Jamari River, Rondônia–Brazil

Specimens of Arapaima gigas from Jamari River (RO) were cytogenetically analyzed. A diploid number of 2n=56 chromosomes was found (28m-sm + 28st-a). Secondary constrictions were observed on the short arms of chromosome 3. Nucleolar Organizer Regions (NORs) were detected at the subterminal region on short arms of the third chromosomal pair by both silver nitrate staining and FISH with 45S rDNA probe, being equivalent to secondary constrictions. The ribosomal sites were also characterized by size heteromorphism and presence of CMA3+/DAPI- blocks. The constitutive heterochromatin was located at pericentromeric region of some chromosomes. After sequential Cbanding and base-specific fluorochromes staining, most of the heterochromatins proved to be neutral, i.e., with similar amounts of AT and GC bases. Nonetheless, some heterochromatic regions were marked by GC-specific fluorochromes in one chromosomal pair and by AT-specific fluorochrome staining on two pairs. The present data are in agreement with previous reports in populations from Araguaya River, indicating that conserved cytogenetic features are present in this important fish species.

Multiple malformation of bovine spermatozoa with special reference to their lightmicroscopic fluorescing pattern and electron-microscopic structure.

The light microscopic examinations of sperm samples from a sterile Fleckvieh-bull stained by the AT specific fluorochrome Hoechst 33258 showed beneath the constantly occurring paraxial tail numerous other malformations, which appeared under the electron microscope as multiple defects of essential cell organelles: defects of the acrosome as irregular thickening or forming of overturned folds "knobbed acrosome", sometimes with vesicles and inclusions inside, partial separation of the acrosome lamina from the nucleus. The defects of the nucleus were present as double-headed sperms, macro- and microcephalic forms besides bizarre shaped heads with gigantic cavities, often filled with membranes, cytoplasm and canaliculi even as heads with ridges which extended from the base to the apex. The main tail defect was a paraxial inserted tail, sometimes coiled, folded or bent down and combined with duplication or even multiplication of the tail with one or two implantation fossae. This multiplication was followed by extensive disturbances of the tail elements as lack or excessive numbers up to the isolation of tubules, fibers and mitochondria. Often there was no contact between tail and implantation fossa. Another defect was the existence of voluminous cytoplasmic droplets covering not only great parts of the tail but also the total head which included "arch like double membranes" and up to 15 tail segments. The persisting cytoplasmic droplet even as the generally occurring paraxial inserted tail are discussed in connexion with the existing hormonal imbalance as probably genetic damage leading to extensive defects during spermiogenesis.

Chromosome banding in Amphibia

The mitotic chromosomes of the Australian ground frogs Mixophyes fasciolatus and M. schevilli were analyzed by means of banding techniques and restriction endonuclease digestions. Chromosomal differentiation in these two species occurred exclusively by considerable changes in the amount of telomeric and centromeric heterochromatin, whereas the sizes and locations of interstitial heterochromatic regions, the sizes of all euchromatic segments as well as the positions of centromeres remained nearly identical during karyotype evolution. The major heterochromatic regions in the karyotypes of M. fasciolatus and M. schevilli amount to 30.2% and 20.7%, respectively. They consist of AT base pair-rich repetitive DNA sequences that are brightly labeled by AT-specific fluorochromes and display quenched fluorescence after staining with GC-specific fluorochromes. The heterochromatic regions can be differentiated by treatment of metaphase chromosomes and interphase cell nuclei with various restriction enzymes which either disclose the complete set of C-band patterns in the karyotypes of both species, or else reveal several subsets of these C-bands.

Measurement by flow cytometry of genomic AT/GC ratio and genome size.

Flow cytometry with the AT-specific fluorochrome Hoechst 33258 (HO) and the GC-specific fluorochrome olivomycin (OM) was used for measurement of base pair specific DNA content in 20 species of vertebrates. The results were found to be in good correlation with the biochemical literature on base pair frequencies (r = 0.972, P < 1 x 10(-8). This correlation allows one to determine the percent of GC/AT-pairs and genome size from flow cytometric data. The genome sizes obtained were compared with the literature data on flow cytometric genome size values determined with the use of propidium iodide (PI) that is usually believed to be non-base pair specific. The results were found to be in general agreement; however, the previously reported slight GC-preference of PI is confirmed. The optimal conditions for flow cytometry of AT/GC ratio and genome size with the use of OM and HO are discussed. The approach can be useful for research in ecology, fisheries science, species conservation, and other environmental studies as a tool for rapid survey of a vast array of specimens.

Fluorescent banding pattern analysis of eight taxa of Phaseolus and Vigna in relation to their phylogenetic relationships

Phylogenetic relationships among eight taxa of seven species of Phaseolus and Vigna (Phaseolus angularis, P. aureus, P. calcaratus, P. coccineus, P. vulgaris, Vigna sesquipedalis and V. sinensis; 2n = 22 each) were studied by the fluorescent chromosome banding technique. Preparations of somatic metaphase chromosomes of each taxon were sequentially stained with Giemsa, GC-specific fluorochrome chromomycin A3 (CMA) and AT-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). On the basis of the fluorescent banding patterns of the 22 chromosomes of each taxon, P. angularis, P. coccineus (from China and Korea) and P. vulgaris were grouped into one group ("Phaseolus group"), P. aureus and two Vigna species were grouped into another ("Vigna group") and P. calcaratus was grouped in an independent group.

An early stage of ZW/ZZ sex chromosome differentiation in Poecilia sphenops var. melanistica (Poeciliidae, Cyprinodontiformes)

The mitotic and meiotic chromosomes of the American cyprinodont fish Poecilia sphenops var. melanistica were analysed. All 46 chromosomes are telocentric. By specific staining of the constitutive heterochromatin with C-banding and various AT-specific fluorochromes, the homomorphic chromosome pair 1 could be identified as sex chromosomes of the ZW/ZZ type. All female animals exhibit a W chromosome with a large region of telomeric heterochromatin that is not present in the Z chromosome. These sex chromosomes cannot be distinguished by conventional staining; they represent the first demonstration of sex chromosomes in fishes in an early stage of morphological differentiation. The W heterochromatin and the telomeric heterochromatin in the two autosomes 18 show a very bright fluorescence when stained with AT-specific fluorochromes. This allows the direct identification of the chromosomal sex by examining the interphase nuclei: females exhibit three, males only two brightly fluorescent heterochromatic chromocenters in their nuclei. The significance of these ZW/ ZZ sex chromosomes and their specific DNA sequences, the dose compensation of the Z-linked genes, and the experimental possibilities using sex-reversed “ZW males” are discussed.

Characterization of heterochromatin in different species of the Scilla siberica group (Liliaceae) by in situ hybridization of satellite DNAs and fluorochrome banding

Satellite DNAs have been isolated from the monocotyledonous plants Scilla siberica, S. amoena, S. ingridae (all are highly GC-rich), and S. mischtschenkoana by using the Ag+ −Cs2SO4 density centrifugation technique. Hybridization in situ has been performed with 3H-cRNA to these satellite DNAs in all four species. In each species, the endogenous satellite DNA is located mainly in intercalary and major heterochromatin bands associated with terminal regions and nucleolar organizer regions (NORs) but not in centromeric regions. Patterns observed after cross-species hybridization show a high degree of satellite DNA homology between S. siberica, S. amoena, and S. ingridae. By contrast, satellite DNA of S. mischtschenkoana consists largely of different, non homologous DNA sequences, with two exceptions: (i) the NORs of all four species contain similar satellite sequences, and (ii) a strong homology exists between the satellite DNA of S. mischtschenkoana and centromeric DNA of S. siberica but not with those of S. amoena and S. ingridae. — Heterochromatin has also been characterized by the AT-specific fluorochromes quinacrine (Q) and DAPI and the GC-specific agent chromomycin A3 (CMA3), in combination with two counterstaining techniques. While CMA3-fluorescence is largely in agreement with data on base composition and location of the specific satellite DNAs, the results with Q and DAPI are conflicting. Prolonged fixation has been found to change the fluorescence character in certain instances, indicating that other factors than the base sequence of the DNA also play a role in fluorochrome staining of chromosomes. The results are discussed in relation to the taxonomy and phylogeny of the four species.

Modification of DAPI banding on human chromosomes by prestaining with a DNA-binding oligopeptide antibiotic, distamycin A

A DNA-binding AT-specific oligopeptide antibiotic, distamycin A, was used as non-fluorescent counterstain in conjunction with the DNA-binding AT-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) to investigate the effect of the antibiotic on DAPI fluorescent banding of human chromosomes. Distamycin A-pretreated metaphases and interphase nuclei exhibited a significantly lower overall fluorescence intensity than DAPI controls. Chromosome arms were pale and intercalary DAPI bands (Q bands) were obliterated, but some specific regions of constitutive heterochromatin remained brightly fluorescent. These were mainly the constrictions of chromosomes 1, 9 and 16, the short arm of chromosome 15, and the distal part of the Y. The distamycin A/DAPI banding pattern appears to be comparable to that reported for anti-5-methylcytosine binding [11]. The observations are discussed as they relate to the roles of chromosomal DNAs and proteins in chromosome banding.

Structure and variability of human chromosomes analyzed by recent techniques.

Besides the AT-specific fluorochromes, GC-specific fluorescent antibiotics are now available for chromosomal analysis. Chromosomal bands represent large accumulation of DNA sequences with similar AT:GC ratio. These uniform differences from the mean AT:GC ratio in the bands can be explained only by at least partial repetition of short DNA sequences in these regions. By comparison of various staining techniques more information also on the constitutive heterochromatin in man becomes available. The human NOR region exhibits a complex organization when studied by various base-specific fluorochromes and silver staining. The DNA-specific fluorochromes are also useful tools in cytophotometric DNA measurements.

Reverse fluorescent chromosome banding with chromomycin and DAPI.

Two DNA binding guanine-specific antibiotics, chromomycin A3 (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI.--In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI.--Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The cma-banding pattern appears to be similar to the pattern found by R-banding procedures.