Telomeric Probes Research Articles

Genome content in the non-model ciliate Chilodonella uncinata: insights into nuclear architecture, gene-sized chromosomes among the total DNA in their somatic macronuclei during their development.

Ciliates are a model lineage for studies of genome architecture given their unusual genome structures. All ciliates have both somatic macronuclei (MAC) and germline micronuclei (MIC), both of which develop from a zygotic nucleus following sex (i.e., conjugation). Nuclear developmental stages are not as well explored among non-model ciliate genera, including Chilodonella uncinata (Class- Phyllopharyngea), the focus of our work. Here, we characterize nuclear architecture and genome dynamics in C. uncinata by combining DAPI (4',6-diamidino-2-phenylindole) staining and fluorescence in situ hybridization (FISH) techniques with confocal microscopy. We developed a telomere probe for staining alongside DAPI, which allows for the identification of fragmented somatic chromosomes among the total DNA in the nuclei. We quantify both total DNA and telomere-bound signals to explore changes in DNA content and chromosome maturation across Chilodonella's nuclear life cycle. Specifically, we find that MAC developmental stages in the ciliate C. uncinata are different than the data reported from other ciliate species. These data provide insights into nuclear dynamics during nuclear development and enrich our understanding of genome evolution in non-model ciliates.

Comparative molecular and conventional cytogenetic analyses of three species of Rhinella (Anura; Bufonidae).

The genus Rhinella corresponds to a group of anurans characterized by numerous taxonomic and systemic challenges, leading to their organization into species complexes. Cytogenetic data for this genus thus far are limited to the diploid number and chromosome morphology, which remain highly conserved among the species. In this study, we analyse the karyotypes of three species of the genus Rhinella (Rhinella granulosa, Rhinella margaritifera, and Rhinella marina) using both classical (conventional staining and C-banding) and molecular (FISH-fluorescence in situ hybridization with 18S rDNA, telomeric sequences, and microsatellite probes) cytogenetic approaches. The aim of this study is to provide data that can reveal variations in the distribution of repetitive sequences that can contribute to understanding karyotypic diversification in these species. The results revealed a conserved karyotype across the species, with 2n = 22 and FN = 44, with metacentric and submetacentric chromosomes. C-banding revealed heterochromatic blocks in the pericentromeric region for all species, with a proximal block on the long arms of pairs 3 and 6 in R. marina and on the short arms of pairs 4 and 6 in R. margaritifera. Additionally, 18S rDNA probes hybridized to pair 5 in R. granulosa, to pair 7 in R. marina, and to pair 10 in R. margaritifera. Telomeric sequence probes displayed signals exclusively in the distal region of the chromosomes, while microsatellite DNA probes showed species-specific patterns. These findings indicate that despite a conserved karyotypical macrostructure, chromosomal differences exist among the species due to the accumulation of repetitive sequences. This variation may be attributed to chromosome rearrangements or differential accumulation of these sequences, highlighting the dynamic role of repetitive sequences in the chromosomal evolution of Rhinella species. Ultimately, this study emphasizes the importance of the role of repetitive DNAs in chromosomal rearrangements to elucidate the evolutionary mechanisms leading to independent diversification in the distinct phylogenetic groups of Rhinella.

Mapping of Repetitive Sequences in Brachyhypopomus brevirostris (Hypopomidae, Gymnotiformes) from the Brazilian Amazon.

Brachyhypopomus (Hypopomidae, Gymnotiformes) is a monophyletic genus consisting of 28 formally described species. Karyotypic data are available for 12 species. The same karyotype is described for two species (B. brevirostris and B. hamiltoni), as well as different karyotypes for the same species from distinct locations (B. brevirostris). In this context, B. brevirostris may constitute a cryptic species complex. Thus, in the present study, we analyzed the karyotype of B. brevirostris, from Santarém, Pará, and Tefé, Amazonas, using classical cytogenetics (conventional staining and C-banding) and molecular techniques (fluorescence in situ hybridization using 18S rDNA, 5S rDNA, U2 snRNA, and telomeric probes). The results show that samples from both locations present 2n = 38, with all chromosomes being acrocentric (FC = 38a). In both populations, 18S rDNA sequences are present on only one pair of homologous chromosomes and telomeric sequences occur only at the ends of the chromosomes. In the Tefé sample, the 5S rDNA occurs in two pairs, and the U2 snRNA in three pairs. These results are the first descriptions of these sequences for B. brevirostris samples from the Tefé locality, as well as the first karyotypic description for the Santarém locality. Future cytotaxonomic studies of this genus can benefit from these results.

3D Super-Resolution Nuclear Q-FISH Imaging Reveals Cell-Cycle-Related Telomere Changes.

We present novel workflows for Q-FISH nanoscopy with the potential for prognostic applications and resolving novel chromatin compaction changes. DNA-fluorescence in situ hybridization (DNA-FISH) is a routine application to visualize telomeres, repetitive terminal DNA sequences, in cells and tissues. Telomere attrition is associated with inherited and acquired diseases, including cancer and cardiomyopathies, and is frequently analyzed by quantitative (Q)-FISH microscopy. Recently, nanoscopic imaging techniques have resolved individual telomere dimensions and their compaction as a prognostic marker, in part leading to conflicting conclusions still unresolved to date. Here, we developed a comprehensive Q-FISH nanoscopy workflow to assess telomeres with PNA telomere probes and 3D-Stimulated Emission Depletion (STED) microscopy combined with Dynamic Intensity Minimum (DyMIN) scanning. We achieved single-telomere resolution at high, unprecedented telomere coverage. Importantly, our approach revealed a decrease in telomere signal density during mitotic cell division compared to interphase. Innovatively expanding FISH-STED applications, we conducted double FISH targeting of both telomere- and chromosome-specific sub-telomeric regions and accomplished FISH-STED in human cardiac biopsies. In summary, this work further advanced Q-FISH nanoscopy, detected a new aspect of telomere compaction related to the cell cycle, and laid the groundwork for future applications in complex cell types such as post-mitotic neurons and muscle cells.

A Nested PCR Telomere Fusion Assay Highlights the Widespread End-Capping Protection of Arabidopsis CTC1.

Telomeres protect the ends of linear eukaryotic chromosomes from being recognized as DNA double-strand breaks. Two major protein complexes are involved in the protection of telomeres: shelterin and CST. The dysfunction of these complexes can challenge the function of telomeres and lead to telomere fusions, breakage-fusion-bridge cycles, and cell death. Therefore, monitoring telomere fusions helps to understand telomeres biology. Telomere fusions are often analyzed by Fluorescent In Situ Hybridization (FISH) or PCR. Usually, both methods involve hybridization with a telomeric probe, which allows the detection of fusions containing telomeric sequences, but not of those lacking them. With the aim of detecting both types of fusion events, we have developed a nested PCR method to analyze telomere fusions in Arabidopsis thaliana. This method is simple, accurate, and does not require hybridization. We have used it to analyze telomere fusions in wild-type and mutant plants altered in CTC1, one of the three components of the Arabidopsis CST telomere capping complex. Our results show that null ctc1-2 mutant plants display fusions between all telomeric regions present in Arabidopsis chromosomes 1, 3 and 5, thus highlighting the widespread end-capping protection achieved by CTC1.

Interstitial telomere sequences in Original Article chromosomes of Baikal planarians

The presence of internal telomeric sequences (ITSs) in chromosomes typically indicates instances of genome reorganization. Changes in morphology and chromosome number can be sources of intraspecific polymorphism and also lead to speciation. Both variants are found in flatworms, but ITSs are rare in chromosomes, as is common in other invertebrate animals. Out of 23 flatworm species ITSs has been identified in only three parasitic species. Using FISH with telomeric probes, we found that ITSs are also present in the chromosomes of the endemic Baikal planarians Baikalobia Kenk, 1930 (Tricladida, Continenticola, Dendrocoelidae). This is the first time that ITSs has been identified in free-living flatworms. Like Shistosoma Weinland, 1858, the appearance of ITSs in the Baikal planarians could be associated with the process of speciation. There is no data yet on ITSs in other dendrocoelids, and the question remains whether ITSs are a specific feature of Baikal planarians or a special feature of all dendrocoelids.

Genetic diversity analysis in the Brazilian Amazon reveals a new evolutionary lineage and new karyotype for the genus Mesomys (Rodentia, Echimyidae, Eumysopinae).

Morphological, molecular and chromosomal studies in the genera Lonchothrix and Mesomys have contributed to a better understanding of taxonomic design, phylogenetic relationships and karyotypic patterns. Recent molecular investigations have shown a yet undescribed diversity, suggesting that these taxa are even more diverse than previously assumed. Furthermore, some authors have questioned the limits of geographic distribution in the Amazon region for the species M. hispidus and M. stimulax. In this sense, the current study sought to understand the karyotypic evolution and geographic limits of the genus Mesomys, based on classical (G- and C-banding) and molecular cytogenetic analysis (FISH using rDNA 18S and telomeric probes) and through the sequencing of mitochondrial genes Cytochrome b (Cytb) and Cytochrome Oxidase-Subunit I (CO using phylogeny, species delimitation and time of divergence, from samples of different locations in the Brazilian Amazon. The species M. stimulax and Mesomys sp. presented 2n = 60/FN = 110, while M. hispidus presented 2n = 60/FN = 112, hitherto unpublished. Molecular dating showed that Mesomys diversification occurred during the Plio-Pleistocene period, with M. occultus diverging at around 5.1 Ma, followed by Mesomys sp. (4.1 Ma) and, more recently, the separation between M. hispidus and M. stimulax (3.5 Ma). The ABGD and ASAP species delimiters support the formation of 7 and 8 potential species of the genus Mesomys, respectively. Furthermore, in both analyzes Mesomys sp. was recovered as a valid species. Our multidisciplinary approach involving karyotypic, molecular and biogeographic analysis is the first performed in Mesomys, with the description of a new karyotype for M. hispidus, a new independent lineage for the genus and new distribution data for M. hispidus and M. stimulax.

Chromosome Painting in Cercopithecus petaurista (Schreber, 1774) Compared to Other Monkeys of the Cercopithecini Tribe (Catarrhini, Primates).

The Cercopithecini tribe includes terrestrial and arboreal clades whose relationships are controversial, with a high level of chromosome rearrangements. In order to provide new insights on the tribe's phylogeny, chromosome painting, using the complete set of human syntenic probes, was performed in Cercopithecus petaurista, a representative species of the Cercopithecini tribe. The results show C. petaurista with a highly rearranged karyotype characterized by the fission of human chromosomes 1, 2, 3, 5, 6, 8, 11, and 12. These results compared with the literature data permit us to confirm the monophyly of the Cercopithecini tribe (fissions of chromosomes 5 and 6), as previously proposed by chromosomal and molecular data. Furthermore, we support the monophyly of the strictly arboreal Cercopithecus clade, previously proposed by the molecular approach, identifying chromosomal synapomorphies (fissions of chromosomes 1, 2, 3, 11, 12). We also add additional markers that can be useful for deciphering arboreal Cercopithecini phylogeny. For example, the fission of chromosome 8 is synapomorphy linking C. petaurista, C. erythrogaster, and C. nictitans among the arboreal species. Finally, a telomeric sequence probe was mapped on C. petaurista, showing only classic telomeric signals and giving no support to a previous hypothesis regarding a link between interspersed telomeric sequences in high rearranged genomes.

Karyotypic and Molecular Analysis of Pterygoplichthys pardalis (Castelnau 1855) from the Lower Amazon River.

Pterygoplichthys pardalis is an armored catfish native to South America and an important resource for the ornamental fish industry. Recently, several exotic populations have been introduced into rivers on five continents. Despite its commercial and environmental importance, P. pardalis is poorly studied from a genetic perspective. In this study, we analyzed the karyotype of P. pardalis from the Amazon River and molecular variations in the mitochondrial gene Cytochrome oxidase I (COI) between native and exotic populations. The karyotype presented diploid number 2n = 52 and NF = 100 without cytogenetic variation between males and females. Nucleolus organizer regions (Ag-NOR) in the distal region of the long arm of pair 12 coincided with the 18S hybridization signal, whereas 5S was syntenic to this chromosome but localized in the short arm. The constitutive heterochromatin was restricted in the distal regions of pairs 4, 12, 25, and 26. Telomeric probes showed only distal hybridization signals. The karyotype of P. pardalis diverged from that of its congeners, and COI molecular variation revealed four haplotypes. The Philippine population revealed the greatest diversity with three haplotypes, while haplotype H1 was the most abundant and observed in both native and exotic populations. This new genetic data contributes to species management and provides useful information from an aquaculture perspective.

Supernumerary Marker Chromosome Identified in Asian Elephant (Elephas maximus).

We identified a small, supernumerary marker chromosome (sSMC) in two phenotypically normal Asian elephants (Elephas maximus): a female (2n = 57,XX,+mar) and her male offspring (2n = 57,XY,+mar). sSMCs are defined as structurally abnormal chromosomes that cannot be identified by conventional banding analysis since they are usually small and often lack distinct banding patterns. Although current molecular techniques can reveal their origin, the mechanism of their formation is not yet fully understood. We determined the origin of the marker using a suite of conventional and molecular cytogenetic approaches that included (a) G- and C-banding, (b) AgNOR staining, (c) preparation of a DNA clone using laser microdissection of the marker chromosome, (d) FISH with commercially available human painting and telomeric probes, and (e) FISH with centromeric DNA derived from the centromeric regions of a marker-free Asian elephant. Moreover, we present new information on the location and number of NORs in Asian and savanna elephants. We show that the metacentric marker was composed of heterochromatin with NORs at the terminal ends, originating most likely from the heterochromatic region of chromosome 27. In this context, we discuss the possible mechanism of marker formation. We also discuss the similarities between sSMCs and B chromosomes and whether the marker chromosome presented here could evolve into a B chromosome in the future.

Contributions of species Rineloricaria pentamaculata (Loricariidae:Loricariinae) in a karyoevolutionary context

Species of Rineloricaria demonstrate an interesting evolutionary history from a cytogenetic point of view, due to the occurrence of extensive variation in diploid number (2n=36 -70 chromosomes), with Robertsonian rearrangements mostly responsible for this karyotypic diversity. In this study we present the karyotypic data for a population of Rineloricaria pentamaculata, collected in the Itiz stream, a tributary of the Paraná River Basin (Paraná, Brazil), which exhibited 2n=56 chromosomes distributed in 8m/sm+48st/a (number fundamental equal to 64) and simple NOR system revealed by fluorescent in situ hybridization (FISH) with 18S rDNA probe, silver nitrate and positive C band, located on the first submetacentric chromosome pair (pair 5). In addition, the NOR pair showed a size heteromorphism for this region, rich in GC composition (positive CMA3). Clusters of 5S rDNA were located in 14 chromosomes and the FISH with a telomeric probe was used to map possible evidence of chromosomal fusions, however, it showed only telomeric sites. These results corroborate the data for the species R. pentamaculata and the genus Rineloricaria, showing that they are similar to most of the populations analyzed. About the cytogenetic data of R. pentamaculata, we reaffirm that most populations were conserved, but in those with derived characteristics, Robertsonian chromosomal rearrangements probably contributed to the karyotypic evolution of the group.

Telomere shortening in hypertensive heart disease depends on NOX2-mediated loss of PRDX1 and oxidative DNA damage

Abstract Background Heart failure (HF) coincides with cardiomyocyte telomere shortening. Arterial hypertension is the most prominent risk factor for HF. Both HF and arterial hypertension are associated with dysregulation of the neurohormonal axis and increased ROS. However, how neurohormonal activation is linked to telomere shortening in the pathogenesis of HF is incompletely understood. Further, recent findings suggest that peroxiredoxin 1 (PRDX1) can act as a telomere specific antioxidant. Methods To induce hypertensive HF, male C57BL/6J mice were subjected to AngII-infusion, uninephrectomy and high-salt (AngII++) for 5 weeks. Cardiac function was assessed by ultrasound. Mouse adult cardiomocytes (CMs) isolated from C57BL/6J mice and mice lacking the NADPH-oxidase (NOX) 2/gp91phox as well as rat ventricular CM-derived cells (H9C2) were stimulated with AngII. Telomere length was quantified by Q-FISH after staining with a C-rich telomere probe (TelC). DNA/RNA-damage was evaluated after staining for Oxo-8-Gua (8-oxo-7,8-dihydroguanine) and Oxo-8-G (8-oxo-7,8-dihydroguanosine). Superoxide (O2-) was quantified by 2-hydroxyethidium (2-HE) using HPLC analysis. Colocalization of Oxo-8-Gua and TelC was quantified by automated image analysis. (Sub)cellular and tissue expression of gp91phox/Nox2 and PRDX1 was evaluated by ICC/IHC. All image quantification was conducted semiautomatically. Results In mice subjected to hypertensive HF, CM telomere shortening correlated significantly with both left ventricular (LV) dilatation and impairment of LV systolic function, paralleled by a significant loss of myocardial PRDX1 and significantly increased myocardial DNA/RNA-damage. Similarly, CMs stimulated with AngII exhibited significant telomere shortening, significant loss of PRDX1 and significantly increased DNA/RNA-damage, together with significantly increased CM superoxide production and significantly increased expression of the superoxide generating enzyme gp91phox/NOX2. Correspondingly, deficiency of gp91phox/NOX2 significantly prevented AngII-induced CM telomere shortening, DNA/RNA-damage and PRDX1-depletion. A similar effect could be observed upon stimulation of CMs with the specific histone deacetylase (HDAC) 6-inhibitor tubastatin, which prevents deacetylation of PRDX1. Conclusion We could provide first evidence that in heart failure, ROS originating from increased NOX2-activity leads to depletion of the telomere-targeted antioxidant and repair-protein PRDX1, which results in damage to the telomeric DNA. Both depletion of NOX2 as well as harnessing the intrinsic antioxidant defense by stabilizing PRDX1 via HDAC6-inhibition prevented CM telomere shortening and could be further evaluated as potential therapeutic targets to address heart failure. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Federal Ministry for Education and Research

Comparative Cytogenetics of Lace Bugs (Tingidae, Heteroptera): New Data and a Brief Overview.

Simple SummaryTingidae, or lace bugs, is a family of herbivorous true bugs, with approximately 2600 identified species in 318 genera classified in two or sometimes three subfamilies, among which the largest subfamily Tinginae comprises about 2500 species. Here, an account is given of the karyotypes of 16 lace bug species studied using conventional chromosomal techniques and FISH with two repetitive DNA probes, 18S rDNA and (TTAGG)n. We also summarize and analyze all information accumulated to date on karyotypes of lace bugs. In general, such information is available for 60 species and 22 genera of Tinginae. We show that lace bugs are characterized by: (1) very conservative karyotypes, with six pairs of autosomes in a haploid set; (2) either an XY or X(0) sex chromosome system; (3) a conventional sequence of sex chromosome divisions in male meiosis; (4) absence of the (TTAGG)n mechanism for maintaining telomere integrity; (5) four discrete patterns of 18S rDNA chromosomal localization. We conclude that the search for chromosomal landmarks is of paramount importance for the characterization of the lace bug cytogenetics in more detail.The lace bug family Tingidae comprises more than 2600 described species in 318 genera that are classified into the subfamilies Tinginae (about 2500 species and 300 genera), Cantacaderinae, and Vianadinae. We provide data on karyotypes of 16 species belonging to 10 genera of the tribes Tingini and Acalyptaini (Tinginae) studied using conventional chromosome staining and FISH. The species of Tingini possess 2n = 12A + XY, whereas those of Acalyptaini have 2n = 12A + X(0). FISH for 18S rDNA revealed hybridization signals on one of the medium-sized bivalents in species of both tribes. FISH with a telomeric probe TTAGG produced no signals in any species. In addition, we provide a list of all data obtained to date on Tingidae karyotypes, which includes 60 species from 22 genera of Tinginae. The subfamily is highly conservative in relation to the number and size of autosomes, whereas it shows diversity in the number and chromosomal distribution of the rDNA arrays, which may be located either on a pair of autosomes (the predominant and supposedly ancestral pattern), on one or both sex chromosomes, or on an autosome pair and the X. The absence of the “insect” telomeric sequence TTAGG in all species implies that Tinginae have some other, yet unknown, telomere organization.

Bouquet Formation Failure in Meiosis of F1 Wheat-Rye Hybrids with Mitotic-Like Division.

Bouquet formation is believed to be involved in initiating homologous chromosome pairings in meiosis. A bouquet is also formed in the absence of chromosome pairing, such as in F1 wheat–rye hybrids. In some hybrids, meiosis is characterized by a single, mitotic-like division that leads to the formation of unreduced gametes. In this study, FISH with the telomere and centromere-specific probe, and immunoFISH with ASY1, CENH3 and rye subtelomere repeat pSc200 were employed to perform a comparative analysis of early meiotic prophase nuclei in four combinations of wheat–rye hybrids. One of these, with disomic rye chromosome 2R, is known to undergo normal meiosis, and here, 78.9% of the meiocytes formed a normal-appearing telomere bouquet and rye subtelomeres clustered in 83.2% of the meiocytes. In three combinations with disomic rye chromosomes 1R, 5R and 6R, known to undergo a single division of meiosis, telomeres clustered in 11.4%, 44.8% and 27.6% of the meiocytes, respectively. In hybrids with chromosome 1R, rye subtelomeres clustered in 12.19% of the meiocytes. In the remaining meiocytes, telomeres and subtelomeres were scattered along the nucleus circumference, forming large and small groups. We conclude that in wheat–rye hybrids with mitotic-like meiosis, chromosome behavior is altered already in the early prophase.

Fluorescent in-situ hybridization test performed on cell blocks is useful for establishing germ cell origin of metastatic tumors with unusual clinical presentation.

The majority of germ cell tumors (GCTs) are characterized by iso-chromosome 12p (i12p) abnormality. The aim of this study is to review the cytomorphologic features and analyze the utility of i12p fluorescent in-situ hybridization (FISH) test in diagnosing metastatic GCTs primarily evaluated by cytologic techniques in patients without prior history of GCTs. The laboratory information system was queried over a period of 10 years to search for cases where i12p FISH test was requested on cytology material. FISH test was performed using TelVysion 12p telomeric probe and CEP 12 centromere probe on cell-blocks. A ratio of 12ptel/CEP12 signal of 1.4 or greater was considered as positive. Patient demographics, clinical presentation, cytopathologic findings, and follow-up surgical resection data were reviewed and correlated. A total of three cases were identified, all men (age range 31-60 years). Cytologic diagnoses were favor metastatic embryonal carcinoma (Case 1, retroperitoneal fluid FNA), metastatic yolk sac tumor/YST (Case 2, lung mass FNA) and adenocarcinoma, likely representing a somatic-type malignancy (SM) arising from a preexisting GCT (Case 3, retroperitoneal mass FNA). This limited study demonstrated high sensitivity for detecting i12p abnormality by FISH test performed on cell blocks. The cytomorphology of extra-gonadal GCTs varies according to the histologic subtype. Sarcomatoid morphology of YST, SM or mixed GCTs further complicates cytology evaluation. FISH test for detection of i12p performed on cell-blocks is extremely useful in establishing germ cell origin of these metastatic GTCs with unusual cytomorphology and guides management in patients without prior history of GCTs.

The emergence of a new sex-system (XX/XY1Y2) suggests a species complex in the “monotypic” rodent Oecomys auyantepui (Rodentia, Sigmodontinae)

X-autosome translocation (XY1Y2) has been reported in distinct groups of vertebrates suggesting that the rise of a multiple sex system within a species may act as a reproductive barrier and lead to speciation. The viability of this system has been linked with repetitive sequences located between sex and autosomal portions of the translocation. Herein, we investigate Oecomys auyantepui, using chromosome banding and Fluorescence In Situ Hybridization with telomeric and Hylaeamys megacephalus whole-chromosome probes, and phylogenetic reconstruction using mtDNA and nuDNA sequences. We describe an amended karyotype for O. auyantepui (2n = 64♀65♂/FNa = 84) and report for the first time a multiple sex system (XX/XY1Y2) in Oryzomyini rodents. Molecular data recovered O. auyantepui as a monophyletic taxon with high support and cytogenetic data indicate that O. auyantepui may exist in two lineages recognized by distinct sex systems. The Neo-X exhibits repetitive sequences located between sex and autosomal portions, which would act as a boundary between these two segments. The G-banding comparisons of the Neo-X chromosomes of other Sigmodontinae taxa revealed a similar banding pattern, suggesting that the autosomal segment in the Neo-X can be shared among the Sigmodontinae lineages with a XY1Y2 sex system.

Molecular cytogenetics reveals an uncommon structural and numerical chromosomal heteromorphism in Zephyranthes brachyandra (Amaryllidaceae)

Background and aims: Zephyranthes brachyandra belongs to a tribe of ornamental Amaryllidaceae native of South America, whose genera circumscription and phylogenetic relationships are still unclear. Cytologically, Z. brachyandra is a tetraploid whose chromosomes are of similar size and morphology, hindering the identification of its 2n = 24 chromosomes. The aim of this study was to investigate the stability of the many CMA+ and DAPI+ bands and the occurrence of B chromosomes by a cytomolecular approach. M&M: For this investigation we conducted a cytomolecular analysis with CMA/DAPI staining and fluorescence in situ hybridization with 5S and 35S rDNA probes, and the TTTAGGG telomeric probe. Results: In the present work, a cytomolecular analysis of Z. brachyandra, revealed a large and variable number of CMA+ and DAPI+ heterochromatic bands and 5S and 35S rDNA sites, and a regular distribution of the TTTAGGG telomeric sequences. In addition, one individual was monotrisomic with 2n = 24, and another one had a B chromosome. Both numerical and structural chromosome alterations were clearly characterized by CMA/DAPI bands and rDNA sites. Conclusions: Comparing the present data with the cytological data for other species of Zephyranthes, it becomes clear that a cytomolecular approach is fundamental to the understanding of the chromosome variation and cytotaxonomy of the group.

Orofacial Muscle Weakening in Facioscapulohumeral Muscular Dystrophy (FSHD) Patients.

Background: Facioscapulohumeral muscular dystrophy is the third most commonly found type of muscular dystrophy. The aim of this study was to correlate the D4Z4 repeat array fragment size to the orofacial muscle weakening exhibited in a group of patients with a genetically supported diagnosis of FSHD. Methods: Molecular genetic analysis was performed for 52 patients (27 female and 25 male) from a group that consisted of 36 patients with autosomal dominant pedigrees and 16 patients with either sporadic or unknown family status. The patients were tested with the southern blotting technique, using EcoRI/Avrll double digestion, and fragments were detected by a p13E-11 telomeric probe. Spearman’s correlation was used to compare the fragment size with the degree of muscle weakening found in the forehead, periocular and perioral muscles. Results: A positive non-significant correlation between the DNA fragment size and severity of muscle weakness was found for the forehead (r = 0.27; p = 0187), the periocular (r = 0.24; p = 0.232) and the left and right perioral (r = 0.29; p = 0.122), (r = 0.32; p = 0.085) muscles. Conclusions: Although FSHD patients exhibited a decrease in muscular activity related to the forehead, perioral, and periocular muscles the genotype–phenotype associations confirmed a weak to moderate non-significant correlation between repeat size and the severity of muscle weakness. Orofacial muscle weakening and its association with a D4Z4 contraction alone may not have the significance to serve as a prognostic biomarker, due to the weak to moderate association. Further studies with larger sample sizes are needed to determine the degree of genetic involvement in the facial growth in FSHD patients.

Detection of TERRA R-Loops at Human Telomeres.

R-loops are three-stranded nucleic acid structures composed of a DNA-RNA hybrid and a displaced DNA strand. The long noncoding RNA TERRA forms R-loops at telomeres influencing the telomeric chromatin composition and impacting on telomere maintenance mechanisms by semiconservative DNA replication, homology directed DNA repair and telomerase. Here, we describe a method to detect R-loops at telomeres, which involves immunoprecipitation with the R-loop recognizing S9.6 antibody, followed by detection of telomeric DNA by either dot-blot hybridization with a radiolabeled telomeric probe, or qPCR using DNA primers that are specific for subtelomeric sequences.

Iso-chromosome 12p Analysis by Fluorescent In-Situ Hybridization: An Academic Institutional Experience

Abstract Introduction/Objective Chromosome 12 abnormalities like iso-chromosome 12p (i12p) and amplification of 12p are seen in majority (89%) of the primary and metastatic testicular germ cell tumors (TGCTs). i12p can be detected by karyotyping, fluorescent in-situ hybridization (FISH) or reverse transcriptase polymerase chain reaction. The aim of this study was to review i12p FISH data at our institution and assess the clinical utility. Methods/Case Report Laboratory information system was queried over a period of 15 years to search for cases where i12p FISH test was requested. FISH test was performed using TelVysion 12p telomeric probe and CEP 12 centromere probe on paraffin-embedded tissue or cell blocks. A ratio of 12ptel/CEP12 signal of 1.4 or greater was considered as positive. Patient demographics, clinical presentation, pathologic findings, and follow-up data were documented and correlated. Results (if a Case Study enter NA) Total 58 cases were identified with an age range of 14 to 76 years. Majority were male (M=52, F=6). Of these cases, 15 were testicular and 43 extra-testicular cases that included resection (n=35), biopsy (n=20) and cell-blocks (n=3). i12p was detected in 8 out of 15 testicular cases while i12p was detected in 16 out of the 43 extra-testicular cases. The extra- testicular cases included 17 retroperitoneal lesions, 8 lesions from the mediastinum, 6 lymph nodes from other sites and 12 miscellaneous lesions. Using pathology diagnosis with immunohistochemistry as gold standard, overall sensitivity was 60% and specificity was 86%. There were 3 false positive cases [Benign testicular parenchyma (n=1), suspicious for germ cell neoplasia in-situ (n=1) and undifferentiated epithelioid neoplasm (n=1)]. Conclusion Our results show that although the sensitivity was limited, FISH test for i12p demonstrated high specificity(86%) for diagnosis of primary or metastatic TGCTs. As an adjunct test, i12p FISH can help identify and further characterize a significant number of GCTs with unusual morphology or clinical presentation.