Multicolor Fluorescence In Situ Hybridization Research Articles

Neo-XY body: an analysis of XY1Y2 meiotic behavior in Carollia (Chiroptera, Phyllostomidae) by chromosome painting

Classical and molecular cytogenetic analyses of mitotic and meiotic cells were performed on two species of Carollia from the family Phyllostomidae (Chiroptera), which have an XX/XY₁Y₂ sex determination system. Our results show that the species Carollia perspicillata and Carollia brevicauda have the same Xq-autosome translocation (neo-X). Using multicolor FISH we observed different levels of condensation of the original X and Y chromosomes when compared to the translocated autosomal segment, a likely consequence of the nucleolar organizer region blocking spreading of inactivation to the autosomal region of the neo-X. The use of chromosome painting showed the behavior of the sex chromosome trivalent – here called the ‘neo-XY body’ – in meiosis. We compared the variation between the condensation of the original X and Y and the autosome-sex chromosome axis and described the pairing between the original X-Y segments (pseudoautosomal region) and the XY₂ homologous segments, suggesting genetic activity of the latter during meiosis.

Reprobing Multicolor Fish Preparations in Lepidopteran Chromosome

Multicolor fluorescence in-situ hybridization (FISH) and subsequent reprobing of chromosome preparations increase the number of chromosomes and/or anchor loci on the chromosomes simultaneously identified. Reprobing techniques have been widely applied to chromosomes of vertebrates and plants. We have developed a novel reprobing protocol that utilizes multicolor FISH and bacterial artificial chromosome (BAC) probes to examine chromosome preparations in a model lepidopteran species, the silkworm, Bombyx mori. With standard two-color BAC-FISH, routinely used to map genes on B. mori chromosomes, we could localize only two probes on one preparation, whereas our new protocol combining five-color BAC-FISH and preparation reprobing enabled us to simultaneously map 10 probes, as demonstrated with the Bombyx Z chromosome. The improved BAC-FISH technique will facilitate karyotyping and synteny analysis in Lepidoptera.

Is it Williams syndrome?GTF2IRD1implicated in visual–spatial construction andGTF2Iin sociability revealed by high resolution arrays

Genetic contributions to human cognition and behavior are clear but difficult to define. Williams syndrome (WS) provides a unique model for relating single genes to visual-spatial cognition and social behavior. We defined a approximately 1.5 Mb region of approximately 25 genes deleted in >98% of typical WS and then rare small deletions, showing that visual-spatial construction (VSC) in WS was associated with the genes GTF2IRD1 and GTF2I. To distinguish the roles of GTF2IRD1 and GTF2I in VSC and social behavior, we utilized multiple genomic methods (custom high resolution oligonucleotide microarray, multicolor FISH and somatic cell hybrids analyzed by PCR) to identify individuals deleted for either gene but not both. We analyzed genetic, cognitive and social behavior in a unique individual with WS features (heart defects, small size, facies), but with an atypical deletion of a set of genes that includes GTF2IRD1, but not GTF2I. The centromeric breakpoint localized to the region 72.32-72.38 Mb and the telomeric breakpoint to 72.66 Mb, 10 kb downstream of GTF2IRD1. Cognitive testing (WPPSI-R, K-BIT, and PLS-3) demonstrated striking deficits in VSC (Block Design, Object Assembly) but overall performance 1.5-3 SD above WS means. We have now integrated the genetic, clinical and cognitive data with previous reports of social behavior in this subject. These results combine with previous data from small deletions to suggest the gene GTF2IRD1 is associated with WS facies and VSC, and that GTF2I may contribute to WS social behaviors including increased gaze and attention to strangers.

Increased genomic alteration complexity and telomere shortening in B-CLL cells resistant to radiation-induced apoptosis

B-cell chronic lymphocytic leukemia (B-CLL) results in an accumulation of mature CD5⁺/CD23⁺ B cells due to an uncharacterized defect in apoptotic cell death. B-CLL is not characterized by a unique recurrent genomic alteration but rather by genomic instability giving rise frequently to several chromosomal aberrations. Besides we reported that ∼15% of B-CLL patients present malignant B-cells resistant to irradiation-induced apoptosis, contrary to ∼85% of patients and normal human lymphocytes. Telomere length shortening is observed in radioresistant B-CLL cells. Using fluorescence in situ hybridization (FISH) and multicolour FISH, we tested whether specific chromosomal aberrations might be associated with the radioresistance of a subset of B-CLL cells and whether they are correlated with telomere shortening. In a cohort of 30 B-CLL patients, all of the radioresistant B-CLL cell samples exhibited homozygous or heterozygous deletion of 13q14.3 in contrast to 52% of the radiosensitive samples. In addition to the 13q14.3 deletion, ten out of the 11 radioresistant B-cell samples had another clonal genomic alteration such as trisomy 12, deletion 17p13.1, mutation of the p53 gene or translocations in contrast to only three out of 19 radiosensitive samples. Telomere fusions and non-reciprocal translocations, hallmarks of telomere dysfunction, are not increased in radioresistant B-CLL cells. These findings suggest (i) that the 13q14.3 deletion accompanied by another chromosomal aberration is associated with radioresistance of B-CLL cells and (ii) that telomere shortening is not causative of increased clonal chromosomal aberrations in radioresistant B-CLL cells.

Short Telomeres Initiate Telomere Recombination in Primary and Tumor Cells

Human tumors that lack telomerase maintain telomeres by alternative lengthening mechanisms. Tumors can also form in telomerase-deficient mice; however, the genetic mechanism responsible for tumor growth without telomerase is unknown. In yeast, several different recombination pathways maintain telomeres in the absence of telomerase—some result in telomere maintenance with minimal effects on telomere length. To examine non-telomerase mechanisms for telomere maintenance in mammalian cells, we used primary cells and lymphomas from telomerase-deficient mice (mTR−/− and Eμmyc+mTR−/−) and CAST/EiJ mouse embryonic fibroblast cells. These cells were analyzed using pq-ratio analysis, telomere length distribution outliers, CO-FISH, Q-FISH, and multicolor FISH to detect subtelomeric recombination. Telomere length was maintained during long-term growth in vivo and in vitro. Long telomeres, characteristic of human ALT cells, were not observed in either late passage or mTR−/− tumor cells; instead, we observed only minimal changes in telomere length. Telomere length variation and subtelomeric recombination were frequent in cells with short telomeres, indicating that length maintenance is due to telomeric recombination. We also detected telomere length changes in primary mTR−/− cells that had short telomeres. Using mouse mTR+/− and human hTERT+/− primary cells with short telomeres, we found frequent length changes indicative of recombination. We conclude that telomere maintenance by non-telomerase mechanisms, including recombination, occurs in primary cells and is initiated by short telomeres, even in the presence of telomerase. Most intriguing, our data indicate that some non-telomerase telomere maintenance mechanisms occur without a significant increase in telomere length.

Color Compensation of Multicolor FISH Images

Multicolor fluorescence in situ hybridization (M-FISH) techniques provide color karyotyping that allows simultaneous analysis of numerical and structural abnormalities of whole human chromosomes. Chromosomes are stained combinatorially in M-FISH. By analyzing the intensity combinations of each pixel, all chromosome pixels in an image are classified. Due to the overlap of excitation and emission spectra and the broad sensitivity of image sensors, the obtained images contain crosstalk between the color channels. The crosstalk complicates both visual and automatic image analysis and may eventually affect the classification accuracy in M-FISH. The removal of crosstalk is possible by finding the color compensation matrix, which quantifies the color spillover between channels. However, there exists no simple method of finding the color compensation matrix from multichannel fluorescence images whose specimens are combinatorially hybridized. In this paper, we present a method of calculating the color compensation matrix for multichannel fluorescence images whose specimens are combinatorially stained.

Physical Mapping of Ribosomal DNA Genes on <i>Jatropha curcas</i> Chromosomes by Multicolor FISH

Jatropha curcas L. belonging to the Euphorbiaceae is becoming a significant crop in tropical areas nowadays. The genetic diversity among 1 African and 5 Asian varieties were analyzed by multicolor fluorescence in situ hybridization (McFISH) using 5S and 45S ribosomal RNA genes (rDNAs). One locus of 5S rDNA and 2 loci for 45S rDNA were detected at specific regions of the chromosomes in all the materials. Also, telomeric repeats (TTTAGGG)n were localized on the terminal regions of all chromosomes. The present results confirm the stability of these major repeated sequences among Jatropha lines.

Molecular Cytogenetic Mapping of Chromosomal Fragments and Immunostaining of Kinetochore Proteins in Beta

By comparative multicolor FISH, we have physically mapped small chromosome fragments in the sugar beet addition lines PRO1 and PAT2 and analyzed the distribution of repetitive DNA families in species of the section Procumbentes of the genus Beta. Six repetitive probes were applied, including genotype-specific probes—satellites pTS4.1, pTS5, and pRp34 and a dispersed repeat pAp4, the telomere (TTTAGGG)n, and the conserved 18S-5.8S-25S rRNA genes. Pachytene-FISH analysis of the native centromere organization allowed proposing the origin of PRO1 and PAT2 fragments. Comparative analysis of the repetitive DNA distribution and organization in the wild beet and in the addition lines allowed the development of a physical model of the chromosomal fragments. Immunostaining revealed that the PRO1 chromosome fragment binds α-tubulin and the serine 10-phosphorylated histone H3 specific for the active centromere. This is the first experimental detection of the kinetochore proteins in Beta showing their active involvement in chromosome segregation in mitosis.

Human Erythroblasts Generated in Vitro Remain Functional with a Normal Karyotype 8 Years after Cryopreservation: Implications for Ex Vivo Generated Erythroid Transfusion Products.

The safety and adequacy of the blood supply is threatened by natural disasters, social and political events, epidemics, and emerging infections. During shortages, frozen blood is used to supplement the blood supply. Current regulations allow red blood cells to be stored frozen up to ten years; however, the shelf-life of such products is limited once blood is thawed. Cultured human erythroid cells derived in vitro from either fresh or cryopreserved CD34+ cells or peripheral blood mononuclear cells potentially represent an alternative source of erythrocytes for transfusion. However, it is unknown if normal erythroid cells undergoing ex-vivo expansion with growth factors will remain functional or develop genetic rearrangements in culture making them unsuitable for transfusion. We have compared the proliferative and differentiation potential of human erythroblasts obtained in culture from the peripheral blood mononuclear cells (PBMC) of adult donors. This analysis included freshly expanded erythroblasts as well as erythroblasts cryopreserved and stored for short (1 month) and long (8 years) periods.PBMC from four volunteer blood donors were prepared using gradient-density centrifugation and cryopreserved in DMSO in June 2000. One months later, 2x107 PBMC from one of the donors were thawed and cultured under conditions that allow massive ex vivo generation of erythroblasts (HEMA culture, Migliaccio et al Blood Cells Mol Dis 2002; 28:169-80). These cultures were stimulated with recombinant hSCF (50ng/mL), hGM-CSF (1ng/ml), hIL3 (1U/mL), hEPO (1U/mL) and contained dexamethasone and estradiol (each 10−6 M). Twenty million PBMC from the three additional donors were thawed and cultured under HEMA conditions in 2008. In all the three cases, the day 9 cultures contained an average of 10x107 cells, 95% of which were erythroid by CD36 and CD235a staining. These day 9 cells were either cultured for 4 additional days or cryopreserved (>10 individual vials per donor containing 5x106 each). Cells were subcultured and maintained either under HEMA conditions (to assess their proliferation ability) or stimulated with EPO alone (5U/ mL) (to assess maturation). In May 2008, aliquots of the erythroblasts obtained from all donors were thawed and cultured again and amplification and differentiation potential of the freshly expanded and thawed cells were compared. Cells thawed after few months or 8 years of cryopreservation gave similar results and the data were pooled. The viability of the erythroblasts after thawing was 60–70%. After 4 days under HEMA conditions, both freshly expanded and cryopreserved erythroblasts doubled in numbers and retained an immature erythroid phenotype (CD36highCD235alow). On the other hand, in cultures containing EPO alone, the erythroblasts remained constant in number but progressed to a mature CD36posCD235ahigh phenotype. The results are summarized in the following table:Proliferation and Maturation Profile of Fresh and Cryopreserved Human ErythroblastsFold IncreasePhenotypeCD36highCD235alowCD36highCD235ahighFresh cellsHEMA culture253%40%EPO alone115%80%Thawed CellsHEMA culture246%36%EPO alone15%90%The eight-years cryopreserved erythroblasts expanded in culture were also cytogenetically evaluated. Karyotype and multicolor FISH analyses demonstrated a normal 46,XY karyotype with no obvious genomic rearrangements. To determine whether cells carried any known in utero leukemic genomic rearrangements, interphase FISH studies were performed for TEL/ETV6-AML1, MLL, 5q31 (EGR1) and 7q31 loci. In 800 evaluated interphase nuclei, all loci were present in disomy. This data indicates that human erythroblasts obtained in culture can be efficiently cryopreserved, remain functional in culture and do not acquire chromosomal abnormalities detectable by multicolor FISH analysis. These observations suggest that cultured erythroblasts should be further evaluated to determine if they represent a more suitable long term storage product than cryopreserved mature red blood cells.

Physical mapping by FISH and GISH of rDNA loci and discrimination of genomes A and B inScilla scilloides complex distributed in Korea

The chromosomal locations of the 18S-26S (45S) and 5S rDNA loci in cytotypes AA, BB, and AABB ofScilla scilloides Complex from Korea were physically mapped using multicolor fluorescencein situ hybridization (McFISH). Genomicin situ hybridization (GISH) was also performed to distinguish between the AA and BB genomes in allotetraploid AABB plants. One 18S-26S rDNA locus was detected in both AA (a2) and BB (b1 ); one locus also was found in the allopolyploid AABB (b1 ). This demon-strated the loss of that locus in genome A. GISH with biotin-labeled DNA from the BB genome and digoxigenin-labeled 18S-26S rDNA probes revealed that the 18S-26S rDNA in AABB plants was localized in the nucleolus organizer region (NOR) of genome B. One and two 5S rDNA loci were found in diploids AA and BB, respectively. As expected, all three 5S rDNA loci were detected in the AABB plants. The sequence identities of the 5S rDNA genes among cytotypes AA and BB, AA and AABB, and BB and AABB were 99%, 95%, and 95%, respectively.

MLL rearrangement with t(6;11)(q15;q23) as a sole abnormality in a patient with de novo acute myeloid leukemia: conventional cytogenetics, FISH, and multicolor FISH analyses for detection of rare MLL-related chromosome abnormalities

We report a rare case of acute myeloid leukemia (AML) with t(6;11)(q15;q23) in a 50-year-old female showing a poor prognosis. Bone marrow biopsy revealed markedly hypercellular marrow with infiltrates of myeloblasts, consistent with AML-M2 morphology. The karyotype of this patient was 46,XX,t(6;11)(q15;q23) in all analyzed cells, and the results of fluorescence in situ hybridization (FISH) and multi-color FISH analysis confirmed this unique MLL rearrangement as a sole abnormality. To our knowledge, t(6;11)(q13∼q15;q23) is the most rare type of MLL rearrangement involving the long arm of chromosome 6. Only two cases with t(6;11)(q13;q23) and three cases with t(6;11)(q15;q23) have been reported, but detailed clinical or laboratory data were not available. From this report, it is apparent that in a cytogenetic laboratory, the accurate detection of a rare type of MLL rearrangement is very important in the differential diagnosis, prompt treatment, and prediction of prognosis of leukemias.

Multicolor fluorescence in situ hybridization and comparative genomic hybridization reveal molecular events in lung adenocarcinomas and squamous cell lung carcinomas

We have used the molecular cytogenetic techniques of multicolor fluorescence in situ hybridization (M-FISH) and comparative genomic hybridization (CGH) to analyze two established lung cancer cell lines (A549, H520), 80 primary lung adenocarcinoma samples and 80 squamous cell lung carcinoma samples in order to identify common chromosomal aberrations. M-FISH revealed numerous complex chromosomal rearrangements. Chromosomes 5, 6, 11, 12, and 17 were most frequently involved in interchromosomal translocations. CGH revealed regions on 1q, 2p, 3q, 5p, 5q, 7p, 8q, 11q, 12q, 14q, 16p, 17p, 19q, 20q, 21q and 22q to be commonly over-represented and regions on 2q, 3p, 4p, 5q, 7q, 8p, 9p, 13q, 14q, and 17p to be under-represented. In lung adenocarcinomas the most common gains were found in 16p13 (50%); while in squamous cell lung carcinomas the common gains were found in 17q21 (45%) and these alterations were observed to be associated with their specific pathological subtype. In conclusion, the present study contributes to the molecular biological characterization in lung adenocarcinomas and squamous cell lung carcinomas and through evaluation of molecular events to the recently emergent focus on novel markers for lung cancer treatment.

Metaphase Spreads and Spectral Karyotyping of Human Embryonic Stem Cells

INTRODUCTIONSpectral karyotyping is a powerful technique for enumeration of chromosomes in plant, animal, and human cells with high precision. It is also used as a tool for detecting and diagnosing abnormalities in individual chromosomes, such as deletions, duplications, and translocations. There are three basic steps to obtaining good spectral karyotypes: (1) making metaphase spreads, (2) hybridizing probes to the DNA, and (3) analyzing the spectral characteristics of the chromosomes. There are two techniques for spectral karyotyping that are widely used: spectral karyotype (SKY) and multicolor fluorescence in situ hybridization (M-FISH). Both techniques use multicolored fluorescent probes which emit at different wavelengths (200-700 nm) upon light excitation to hybridize to the target DNA sequences. The methods differ in the way in which light is captured and analyzed by the corresponding software. In this protocol, methods for making metaphase spreads from human embryonic stem cells (hESCs) for spectral karyotyping (SKY) using the spectral imaging system and software from Applied Spectral Imaging are described.

Structural analysis of interphase X-chromatin based on statistical shape theory

The 3D folding structure formed by different genomic regions of a chromosome is still poorly understood. So far, only relatively simple geometric features, like distances and angles between different genomic regions, have been evaluated. This work is concerned with more complex geometric properties, i.e., the complete shape formed by genomic regions. Our work is based on statistical shape theory and we use different approaches to analyze the considered structures, e.g., shape uniformity test, 3D point-based registration, Fisher distribution, and 3D non-rigid image registration for shape normalization. We have applied these approaches to analyze 3D microscopy images of the X-chromosome where four consecutive genomic regions (BACs) have been simultaneously labeled by multicolor FISH. We have acquired two sets of four consecutive genomic regions with an overlap of three regions. From the experimental results, it turned out that for all data sets the complete structure is non-random. In addition, we found that the shapes of active and inactive X-chromosomal genomic regions are statistically independent. Moreover, we reconstructed the average 3D structure of chromatin in a small genomic region (below 4 Mb) based on five BACs resulting from two overlapping four BAC regions. We found that geometric normalization with respect to the nucleus shape based on non-rigid image registration has a significant influence on the location of the genomic regions.

Multiple recurrent chromosomal breakpoints in mantle cell lymphoma revealed by a combination of molecular cytogenetic techniques

Mantle cell lymphoma (MCL) is genetically characterized by 11q13 translocations leading to the overexpression of CCND1, and additional secondary genomic alterations that may be important in the progression of this disease. We have analyzed 22 MCL cases and 10 MCL cell lines using multicolor fluorescence in situ hybridization (M-FISH), FISH, and comparative genomic hybridization (CGH). The 19 cases with abnormal karyotype showed the t(11;14)(q13;q32) translocation and, additionally, 89% of cases showed both numerical (n = 58) and structural (n = 77) aberrations. All but one MCL cell line showed t(11;14) and structural and numerical alterations in highly complex karyotypes. Besides 11 and 14, the most commonly rearranged chromosomes were 1, 8, and 10 in the tumors and 1, 8, and 9 in the cell lines. No recurrent translocations other than the t(11;14) were identified. However, we identified 17 recurrent breakpoints, the most frequent being 1p22 and 8p11, each observed in four cases and two cell lines. Interestingly, five tumors and four cell lines displayed a complex t(11;14), cryptic in one case and two cell lines, preferentially involving chromosome 8. In typical MCL, ATM gene deletions were significantly associated with a high number of structural and numerical alterations. In conclusion, MCL does not have recurrent translocations other than t(11;14), but shows recurrent chromosomal breakpoints. Furthermore, most MCL harbor complex karyotypes with a high number of both structural and numerical alterations affecting several common breakpoints, leading to various balanced and unbalanced translocations.

Multicolor fluorescence in situ hybridization and comparative genomic hybridization reveals molecular events in lung adenocarcinomas and squamous cell lung carcinomas

Objective To analyze the chromosomal aberrations and alterations of the genomes in lung adenocarcinomas and squamous cell lung carcinomas and thus to reveal the nature of the development of the two specific pathological subtypes of NSCLC and their's relationship with clinography.Methods We have used the molecular cytogenetic techniques of multicolor fluores- cence in situ hybridization (M-FISH) and comparative genomic hybridization (CGH) to analyze two established lung cancer cell lines (A549,H520),80 primary lung adenocarcinoma samples and 80 squamous cell lung carcinoma samples in order to identify common chromosomal aberrations.Results M-FISH revealed numerous complex chromosomal rearrangements.Chromosomes 5,6,11,12, 17 were most frequently involved in interchromosomal translocations.CGH revealed regions on 1 q, 2p,3q,5p,5q,7p,8q,11q,12q,14q,16p,17p,19q,20q,21q and 22q to be com- monly overrepresented and regions on 2 q,3 p,4 p,5 q,7 q,8 p,9 p,13 q,14 q,17 p to be un- derrepresented.In lung adenocarcinomas the most common gains were found in 16p13 (50%), while in squamous cell lung carcinomas the common gains were found in 17q21 (45%) and these al- terations were observed to be associated with their specific pathological subtype.Conclusions In conclusion,the present study contributes to the molecular biological characterization in lung adeno- carcinomas and squamous cell lung carcinomas and through evaluation of molecular events to the re- cently emergent focus on novel markers for lung cancer treatment.

Feature Normalization via Expectation Maximization and Unsupervised Nonparametric Classification For M-FISH Chromosome Images

Multicolor fluorescence in situ hybridization (M-FISH) techniques provide color karyotyping that allows simultaneous analysis of numerical and structural abnormalities of whole human chromosomes. Chromosomes are stained combinatorially in M-FISH. By analyzing the intensity combinations of each pixel, all chromosome pixels in an image are classified. Often, the intensity distributions between different images are found to be considerably different and the difference becomes the source of misclassifications of the pixels. Improved pixel classification accuracy is the most important task to ensure the success of the M-FISH technique. In this paper, we introduce a new feature normalization method for M-FISH images that reduces the difference in the feature distributions among different images using the expectation maximization (EM) algorithm. We also introduce a new unsupervised, nonparametric classification method for M-FISH images. The performance of the classifier is as accurate as the maximum-likelihood classifier, whose accuracy also significantly improved after the EM normalization. We would expect that any classifier will likely produce an improved classification accuracy following the EM normalization. Since the developed classification method does not require training data, it is highly convenient when ground truth does not exist. A significant improvement was achieved on the pixel classification accuracy after the new feature normalization. Indeed, the overall pixel classification accuracy improved by 20% after EM normalization.

High‐resolution molecular cytogenetic analysis of Wilms tumors highlights diagnostic difficulties among small round cell kidney tumors

Many solid tumors exhibit characteristic gene fusions, which are reflected by balanced translocations at the cytogenetic level. These changes might be useful diagnostic and prognostic tools. In Wilms tumor (WT, nephroblastoma) no fusions genes or recurrent balanced translocations have been described thus far. To screen for cryptic balanced translocations, we have analyzed 17 renal neoplasms, histopathologically classified as WT, by a combination of G-banding, multicolor FISH, and subtelomeric FISH. This approach revealed several submicroscopic chromosomal aberrations and three different seemingly balanced translocations, resulting in a heterozygous deletion of HACE1, an EWSR1/ERG fusion, and an EWSR1/FLI1 fusion, respectively. As EWSR1 rearrangements are known to be a characteristic of Ewing tumors (ET), our findings illustrate the diagnostic problems regarding small cell kidney tumors and strongly argue for the need of adjuvant diagnostic techniques in this group of neoplasms. In summary, our genomic screening approach proved efficient in finding structural chromosomal aberrations. The fact that no recurrent translocations were found in the WTs of this study argues against the presence of a frequent pathognomonic translocation in this disease entity.

Genome-wide genetic characterization of an atypical meningioma by single-nucleotide polymorphism array–based mapping and classical cytogenetics

Most meningiomas, accounting for about 20% of intracranial tumors, can be cured by surgical removal. Yet, 8-22% of these tumors are classified as atypical or anaplastic (WHO grade II or III, respectively) presenting with a more aggressive behavior and a high relapse rate. We analyzed genomic alterations of an atypical meningioma using high-density single nucleotide polymorphism arrays (SNP-A) karyotyping combined with GTG-banding, multicolor fluorescence in situ hybridization (M-FISH), and locus-specific FISH. In accordance to recent studies applying SNP-A karyotyping in different malignancies we found that genomic lesions are present at a higher frequency than predicted by traditional cytogenetics. Most of these aberrations have not been described before. Additionally, we unveiled loss of heterozygosity (LOH) without copy number changes on chromosome regions 1p31.1, 2p16.1, 2q23.3, 6q14.1, 6q21, 9p21.1, 10q21.1, and 14q23.3, suggesting partial uniparental disomy (UPD). UPDs are currently considered to play an important role in the initiation and progression of different malignancies. Furthermore, we detected two de novo reciprocal translocations, t(8;19)(q24;q13) and t(10;16)(q22;q12.1). While GTG-banding and M-FISH data suggested balanced translocations, SNP-A analysis clearly demonstrated imbalances in the same region.

Multicolor FISH analysis of rDNA and telomere on spinach

In this study, multicolor fluorescence in situ hybridization (FISH) analysis on metaphase chromosomes of spinach with biotin-labeled 25S rDNA, DIG-labeled telomere sequences and biotin-labeled and DIG-labeled 5S rDNA was performed. There were six 25S rDNA loci located on the satellites of the third, the fifth and the sixth chromosomes, and four 5S rDNA loci located on the long arms of the third and the fifth chromosomes. The telomere loci were located on the end of the sixth chromosome and also on both the end and centromeric regions of other chromosomes. This study is an important complement to both traditional karyotype analysis and FISH karyotype analysis in spinach.