Architecture and evolution of dinoflagellate chromosomes: an enigmatic origin.

Dinoflagellates are a highly diversified group of unicellular protists that present fascinating nuclear features which have intrigued researchers for many years. As examples, a dense nuclear matrix accommodates permanently condensed chromosomes that are composed of fibers organized without histones and nucleosomes in stacked rows of parallel nested arches. The macromolecular chromosome structure corresponds to cholesteric liquid crystals with a constant left-handed twist. RNA acts to maintain the chromosome structure. Whole mounted chromosomes have a left-handed screw-like configuration with coils which progressively increase their pitch. This helical arrangement seems to be the result of a couple of narrow strands coiling together. Chromosomes do not show Q, G and C banding patterns. However, a roughly spherical differentiated upper end (primitive kinetochore?) and two differentiated coiling regions, the upper one composed of two to three coils where a couple of sister strands run together and parallel to each other, and the lower one where sister strands run out of phase by 180 degrees angular difference along the immediate next turns, can be distinguished. The chromosome segregation into two daughter chromatids begins at the telomere that attaches to the nuclear envelope, follows along the chromosome axis constituting first a Y-shaped and afterwards a V-shaped chromosome, which packs the newly synthesized DNA inside the "old" chromosome. Dividing chromosomes remain highly condensed, and the diameters of the new chromatids and the undivided chromosome are similar, but the number of arches is twice as large in G1 as in G2. The nuclear envelope remains through the cell cycle and shows spindle fibers, which penetrate intranuclear cytoplasmic channels during mitosis constituting an extra nuclear spindle. These and other cytogenetic features suggest that dinoflagellates are a group of enigmatic protists, unique and different from the usual eukaryotes. In contrast, DNA sequence studies propose that dinoflagellates are true eukaryotes, closely related to Apicomplexa, and ciliates (Alveolata), suggesting that the unusual features of chromosome and nuclear organization are not primitive but derived characters. Nevertheless, dinoflagellates have reached enigmatic specific nuclear and chromosome solutions, extremely far from those of other living beings.

FISHing in the microwave: the easy way to preserve proteins. I. Colocalization of DNA probes and surface antigens in human leukocytes.

Most of the techniques for simultaneous visualization of proteic cell components and DNA probes are difficult to carry out. We have developed an alternative protocol for simultaneous visualization of DNA probes and cell surface antigens in human cells. The method exploits microwave energy to denature DNA and to anneal the probe after antigen fluorescent detection of unfixed cells previously embedded in a microgel. CD3 antigens and whole genome DNA probes or specific repetitive DNA sequences were colocalized in peripheral blood samples. The results show a strong, specific and consistent hybridization pattern in each cell that allowed correlation between cell subtype (PROTEINS) and nuclear phenotype (DNA).

[Anterior sacral meningocele with Currarino's syndrome: report of two cases]. / Meningocele sacro anterior en pacientes con síndrome de Currarino: aportación de dos casos.

The anterior sacral meningocele is the congenital hernia of the thecal sac into the pelvis through a bone defect of the anterior wall of the sacrum. The association of anorectal anomaly, anterior sacral defect and presacral mass, known as Currarino's triad, can be diagnosed during the first decade of life in the eighty per cent of the cases However, in the incomplete syndrome, the symptoms appear in adults. In order to avoid late complications and due to the clear genetic origin of this uncommon pathology, the investigation of the family is of paramount importance for the early diagnosis and treatment. We review the literature and add two cases of anterior sacral meningocele with rectal fistula and incomplete Currarino's syndrome. In one of the cases the pathology is also present in another member of the same family and is complicated with meningitis. The importance of early diagnosis and treatment of these malformations and the need for fammily genetic investigation are discussed.

Meningocele sacro anterior en pacientes con síndrome de Currarino: aportación de dos casos / Anterior sacral meningocele with Currarino´s syndrome: Report of two cases

El meningocele sacro anterior es la hernia congénita del saco tecal en la pelvis a través de un defecto óseo en la pared anterior del sacro. La asociación de anomalía anorectal, defecto sacro anterior y masa presacra, conocida como triada de Currarino, puede ser diagnosticada durante la primera década de la vida, en el ochenta por cien de los casos, sin embargo, en el del síndrome incompleto, los síntomas aparecen en el adulto. Para evitar complicaciones tardías y debido a que esta rara patología tiene un claro origen genético, la investigación de la familia es de vital importancia en el diagnóstico precoz y tratamiento. Revisamos la literatura y aportamos dos casos de meningocele sacro anterior con fístula rectal y síndrome incompleto de Currarino, uno de ellos complicado con meningitis y con esta anomalía en un miembro de su familia. Se discute la importancia del diagnóstico precoz y tratamiento de estas malformaciones y la necesidad de una investigación genética familiar (AU)

High frequency of constitutive alkali-labile sites in mouse major satellite DNA, detected by DNA breakage detection-fluorescence in situ hybridization.

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a new procedure for detecting and quantifying DNA breaks and alkali-labile sites in single cells. Cells trapped within an agarose matrix are deproteinized and treated with an alkaline unwinding solution that transforms DNA breaks and alkali-labile sites into single-strand DNA (ssDNA) motifs starting from the end of the break. These ssDNA motifs are susceptible to being hybridized with whole genome or specific DNA probes, and detected using current FISH procedures. As DNA breaks increase in a target region, more ssDNA is produced and more DNA probe hybridizes, thus increasing the FISH signal, which may be captured and analyzed using a digital image analysis system. This increase can be reflected in the surface area, mean and whole fluorescence intensity of the signal. When intact mouse splenocytes were processed with this technique using a whole genome probe, a very strong background signal was evident when compared with human blood leukocytes. In fact, when using 0.03M NaOH as the alkaline unwinding solution at 22 degrees C for 2.5min, the whole fluorescence intensity from mice cells was 50 times higher than that from human cells, thus suggesting the existence of a high frequency of constitutive alkali-labile sites in the DNA from mouse cells. Furthermore, when alkaline unwound mouse cells were simultaneously hybridized with the whole genome probe (FITC-revealed, green) and a major satellite DNA probe (Cy-3-labeled, red) both signals appeared co-localized. This result demonstrates that the high frequency of constitutive alkali-labile sites detected in the mouse genome is mainly located in the major satellite DNA sequences, resembling the findings from human 5bp classical satellite DNA sequences.

DBD-fish on neutral comets: simultaneous analysis of DNA single- and double-strand breaks in individual cells.

Humanblood leukocytes exposed to X-rays were immersed in an agarose microgel on a slide, extensively deproteinized, and electrophoresed under neutral conditions. Following this single-cell gel electrophoresis assay, characteristics of DNA migration (i.e., area of the comet) are related to the DNA double-strand breaks (dsbs) yield. After electrophoresis, comets were briefly incubated in an alkaline unwinding solution, transforming DNA breaks and alkali-labile sites into restricted single-stranded DNA (ssDNA) motifs. These motifs behave as target sites for hybridization with a whole genome probe, following the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure. As DNA breakage increases with dose, more ssDNA is produced in the comet by the alkali and more DNA probe hybridizes, resulting in an increase in the mean fluorescence intensity. Since radiation-induced DNA single-strand breaks (ssbs) are far more frequent than dsbs, the mean fluorescence intensity of the DBD-FISH signal from the comet is related to the ssb level, whereas the surface area of the same comet signal is indicative of the dsb yield. Thus, both DNA break types may be simultaneously analyzed in the same cell. This was confirmed in a repair assay performing the DBD-FISH on neutral comets from a human cell line defective in the repair of dsbs. Otherwise, treatment with hydrogen peroxide, a main inducer of ssbs, increased the mean fluorescence intensity, but not the surface, of X-ray-exposed human leukocytes.

Evidence of abundant constitutive alkali-labile sites in human 5 bp classical satellite DNA loci by DBD-FISH.

Human blood leukocytes within an agarose matrix were deproteinized and exposed to an alkaline denaturation that generates single-stranded DNA (ssDNA) starting from the ends of spontaneous basal DNA breaks and alkali-labile sites. Since the amount of ssDNA produced within a specific sequence area may be detected by hybridization with a specific probe, we quantified this in situ in different satellite DNA loci (DBD-FISH: DNA Breakage Detection FISH). The DBD-FISH signal, corrected for the respective FISH signals in metaphase, was remarkably strong in the 5bp classical satellite DNA domains analyzed (D1Z1, D9Z3, DYZ1), intermediate in the classical satellite 1 DNA sequences, and low in the alphoid satellite regions (D1Z5, DXZ1, all centromeres). This result is evidence of a high density of constitutive alkali-labile sites, probably abasic sites, within the 5bp satellite DNA sequences in human blood leukocytes. The presence and relative abundance of alkali-labile sites could explain the high frequency of spontaneous breakage and rearrangements in pericentromeric heterochromatin of chromosomes 1, 9, and 16, but not in Yqh, when this chromatin is undercondensed through spontaneous or induced demethylation, i.e. ICF syndrome or 5-azacytidine treatment.

DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features.

Non-irradiated and X-irradiated (80 Gy) human spermatozoa were processed for in situ DNA breakage detection-FISH (DBD-FISH) of the whole genome, following two alternative variations of the basic technique. In the first, cells were initially incubated in the alkaline unwinding solution for transformation of DNA breaks into single-stranded DNA (ssDNA) to be hybridized, followed by the lysing solutions for protein removal. In the second, incubation in the lysing solutions was carried out before the denaturation step. The first approach yielded two subpopulations. While most sperm nuclei were faintly labeled and had chromocenters, a small subpopulation was strongly and homogeneously labeled, due to extensive DNA breakage. X-ray exposure increased the surface and mean fluorescence intensity. Otherwise, when the denaturation step was performed after protein extraction, all sperm nuclei yielded strong and dispersed FISH signals. Protein removal allows access of the unwinding solution to the DNA, which has abundant alkali-labile sites, and thus gives rise to large areas of ssDNA that are labeled by FISH. X-ray exposure increased the dispersion of FISH signals but decreased their mean fluorescence intensity. A linear dose-response was generated using the second experimental variant, being 30 Gy the lowest dose for detecting induction of damage by X-rays in mature sperm chromatin. These results indicate that DBD-FISH is not only useful for in situ detection of DNA breakage but also for revealing structural features of chromatin.

Evidence of a differential organization of chromatin containing terminal or interstitial (TTAGGG)n repeats by in situ digestion with nucleases.

(TTAGGG)n sequence repeats in human telomeres and in Chinese hamster interstitial centromeric areas were digested in situ with exonuclease III (ExoIII) and exonuclease Bal 31. Incubation with AluI was performed beforehand to increase DNA breaks near telomere sequence areas. DNA removal at these specific regions was quantified by digital image analysis of the fluorescence in situ hybridization signal produced by a telomeric probe. Exonuclease III was 2.6 times more active in interstitial than in terminal telomeric sequence areas. Exonuclease Bal 31 was 2.3 times more effective in terminal than in interstitial telomeric sequence regions. These results support the hypothesis that chromatin is differentially organized in both telomeric sequence areas, despite their similar DNA composition.

Application of FISH for in situ detection and quantification of DNA breakage.

We describe a simple procedure that allows the use of fluorescence in situ hybridization (FISH) for in situ detection of DNA strand breaks in single cells (DBD-FISH: DNA Breakage Detection-FISH). After trapping within an agarose microgel, cells are incubated in an unwinding alkaline solution, deproteinized and dehydrated. Areas of single-stranded DNA are generated by the alkaline solution in proportion to the degree of DNA strand breakage. These then act as targets for FISH of whole genomic or region-specific probes (telomeric, human chromosome 8 painting, human alphoid DXZ1 locus, and human c-erbB-2 cosmid probes). Measurement of the amount and surface of FISH signals provides information on the breakage level in probed areas, permitting the assessment of possible intragenomic differences in sensitivity as well as intercellular heterogeneity in DNA damage induction or repair.

Image analysis of murine chromatin fiber structure after in situ digestion with restriction endonucleases.

OBJECTIVE: To determine and quantify the differences produced in chromatin structure after selective in situ DNA digestion with restriction endonuclease (RE). STUDY DESIGN: Chromatin fiber structure from a murine cell line was analyzed under light and electron microscopy before and after in situ digestion with AluI, HinfI and HaeIII using digital image analysis (DIA). The proposed DIA-based method entails the generation of a binary image and a skeleton characteristic of the chromatin fiber. Morphologic features (surface, perimeter, shape, number of triple points and Euler number) of the chromatin structure can then be quantified from the digitized images. The results of these experiments were compared with those obtained from direct digestion of naked DNA using the same endonucleases in an attempt to correlate the distribution of restriction sites, according to the DNA fragment size obtained, with the extent of chromatin disorganization after RE in situ digestion. RESULTS: Homologous chromatin fiber regions are differentially affected by the action of each RE, although they are indistinguishable under light microscopy. CONCLUSION: The proposed analytical routine constitutes a valuable tool for uncovering subtle alterations in the structure of chromatin fiber that has been modified under different experimental conditions.

AluI in situ digestion of human alphoid and classical satellite DNA regions: high-resolution digital image analysis of FISH signals from condensed and extended chromatin.

Human lymphocyte chromatin either extended or condensed in interphase nuclei and chromosomes was in situ digested by the restriction endonuclease AluI and then hybridized with alphoid probes specific for chromosome 1 (D1Z5 locus), for chromosome X (DXZ1 locus), and with a classical satellite DNA probe specific for chromosome 9 (D9Z1 locus). Fluorescent hybridization signals were quantified by digital analysis of high-resolution images obtained by a Photo-CD system in an attempt to analyze the differential DNA removal produced by AluI in specific repetitive DNA sequences with known restriction site frequency and distribution. The analysis of area and average pixel grey count of hybridization signals suggests that the greater the degree of chromatin stretching, the higher the accessibility of the probe and/or reporter molecules to the target. Nevertheless, this greater hybridization efficiency does not result in a higher fluorescence intensity due to dispersion of individual signals. Specific repetitive DNA at D9Z1 locus (classical) remained impervious to digestion, while that at DXZ1 (alphoid) was extensively removed, according to the frequency and distribution of restriction sites. Nevertheless, though the restriction sites were at least as frequent as at the DXZ1 locus, DNA at the D1Z5 locus (alphoid) was only partially removed. This indicates that chromatin organization within the C-band partially prevents extraction of alphoid sequences, supporting the hypothesis that alphoid DNA sequences are differentially organized among chromosomes. Overall, the same results were obtained from condensed and extended chromatin, suggesting that higher-order chromatin organization does not influence the in situ DNA cleavage and removal by AluI.

Differential sensitivity of some human alphoid and classical satellite DNA regions from lymphocyte chromosomes to in situ exonuclease III digestion.

Fluorescent in situ hybridization of alphoid and classical satellite III DNA sequences was performed on fixed chromosomes from human lymphocytes that were previously digested in situ with exonuclease III to produce single-stranded DNA motifs. Digital image analysis showed that while labeled alphoid satellite DNAs produced signals of similar strength to thermally denatured chromosomes, those of classical satellite III DNAs of chromosomes 9 and Yq were around 50% weaker. This result shows a differential sensitivity of these satellite DNA regions to in situ exonuclease III digestion and suggests structural differences in the higher-order organization of both subchromosomal constitutive heterochromatic regions.

Digital image analysis of chromatin fibre phenotype after "in situ' digestion with restriction endonucleases.

Restriction Endonucleases (REs) may recognize, cleave and remove DNA from fixed chromatin producing specific chromosome banding patterns. However, the modifications produced in the chromatin fibre are not easy to evaluate and compare. The aim of the present investigation was to visualize differences resulting in the texture of the chromatin fibre from metaphase chromosomes after each digestion using digital image analysis (DIA) facilities. To this purpose, metaphase chromosomes derived from a L-929 mouse cell line were digested with different REs (AluI, HpaII and HaeIII). Since light microscopy does not permit the observation of the chromatin fibre, DIA was performed on digitalized images of metaphase chromosomes under electron microscopy. The application of a LUT (Look Up Table) within the DIA software assigns a colour to each grey level of a digital image. The results obtained using a particular LUT, which permits the discrimination of specific chromatin fibre phenotypes resulting from each digestion, are reported and compared with those obtained under the light microscope.

Quantification of C-ERB-B2 gene amplification in breast cancer cells using fluorescence in situ hybridization and digital image analysis.

Fluorescence in situ hybridization (FISH) allows detection of the intercellular heterogeneity of C-ERB-B2 gene amplification in uncultured breast cancer cells. Nevertheless, because high levels of amplification result in coalescence of signals, direct microscopy quantification is restricted to cells wih low levels of amplification or with dispersed signals. A methodology of digital image analysis, using surface and grey-level FISH signals as parameters that permit a rapid, objective, and accurate estimation of gene copy number, is presented. This procedure is independent of the signal overlapping and results in a more accurate quantification and characterization of tumor cell heterogeneity.