Telomere analysis of platyhelminths and acanthocephalans by FISH and Southern hybridization

Genome ◽  
2009 ◽  
Vol 52 (11) ◽  
pp. 897-903 ◽  
Author(s):  
Marta Bombarová ◽  
Magda Vítková ◽  
Marta Špakulová ◽  
Božena Koubková
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Southern Hybridization ◽  
Telomeric Repeat ◽  
Telomere Maintenance ◽  
Repeat Motif ◽  
Pomphorhynchus Laevis ◽  
Nippotaenia Mogurndae ◽  
Parasitic Worms

We examined the composition of telomeres in chromosomes of parasitic worms, representatives of the flatworm groups Monogenea and Cestoda (Platyhelminthes), and thorny-headed worms (Syndermata: Acanthocephala) by fluorescence in situ hybridization (FISH) with different telomeric repeat probes. Our results show that the (TTAGGG)n sequence, supposed to be the ancestral telomeric repeat motif of Metazoa, is conserved in Monogenea ( Paradiplozoon homoion ) and Cestoda ( Caryophyllaeus laticeps , Caryophyllaeides fennica , and Nippotaenia mogurndae ) but not in Acanthocephala ( Pomphorhynchus laevis and Pomphorhynchus tereticollis ). In the Pomphorhynchus species, no hybridization signals were obtained with the “nematode” (TTAGGC)n, “arthropod” (TTAGG)n, and bdelloid (TGTGGG)n telomeric probes using FISH with their chromosomes and Southern hybridization with P. laevis DNA. Therefore, we suggest that parasitic Acanthocephala have evolved yet unknown telomeric repeat motifs or different mechanisms of telomere maintenance.

HER-2/neu gene amplification characterized by fluorescence in situ hybridization: poor prognosis in node-negative breast carcinomas.

1997 ◽  
Vol 15 (8) ◽  
pp. 2894-2904 ◽  
Author(s):  
M F Press ◽  
L Bernstein ◽  
P A Thomas ◽  
L F Meisner ◽  
J Y Zhou ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Gene Amplification ◽  
Poor Prognosis ◽  
Southern Hybridization ◽  
Recurrent Disease ◽  
Breast Cancers ◽  
Node Negative ◽  
Her 2

PURPOSE The HER-2/neu gene codes for a membrane receptor protein that is homologous, but distinct from the epidermal growth factor receptor. This investigation was performed to validate fluorescence in situ hybridization (FISH) as a sensitive and specific method for assessing HER-2/neu gene amplification in archival tissue and to test whether this alteration is associated with poor prognosis. MATERIALS AND METHODS HER-2/neu gene amplification was determined by FISH in 140 archival breast cancers, previously characterized for gene amplification by Southern hybridization or dot-blot hybridization, and for gene expression by Northern hybridization, Western immunoblot, or immunohistochemistry. A separate cohort of 324 node-negative breast cancers was assessed for amplification by FISH to determine the utility of HER-2/neu gene amplification. RESULTS Relative to solid-matrix blotting procedures, FISH analysis of HER-2/neu gene amplification showed a sensitivity of 98% and a specificity of 100% in 140 breast cancers. Among patients treated by surgery only, the relative risks (relative hazard) of early recurrence (recurrent disease within 24 months of diagnosis), recurrent disease (at any time), and disease-related death were statistically significantly associated with amplification. The prognostic information contributed by HER-2/neu amplification was independent of the other markers studied. CONCLUSION FISH was an alternative technique for determining gene amplification and had some distinct advantages over Southern hybridization. Our results demonstrate that HER-2/neu gene amplification in the absence of adjuvant therapy is an independent predictor of poor clinical outcome and is a stronger discriminant than tumor size. Women with small tumors that had gene amplification were at increased risk of recurrence and disease-related death.

scholarly journals Very short telomere length by flow fluorescence in situ hybridization identifies patients with dyskeratosis congenita

Blood ◽  
2007 ◽  
Vol 110 (5) ◽  
pp. 1439-1447 ◽  
Author(s):  
Blanche P. Alter ◽  
Gabriela M. Baerlocher ◽  
Sharon A. Savage ◽  
Stephen J. Chanock ◽  
Babette B. Weksler ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Telomere Length ◽  
Bone Marrow Failure ◽  
Correct Diagnosis ◽  
Dyskeratosis Congenita ◽  
Telomere Maintenance

Abstract Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome in which the known susceptibility genes (DKC1, TERC, and TERT) belong to the telomere maintenance pathway; patients with DC have very short telomeres. We used multicolor flow fluorescence in situ hybridization analysis of median telomere length in total blood leukocytes, granulocytes, lymphocytes, and several lymphocyte subsets to confirm the diagnosis of DC, distinguish patients with DC from unaffected family members, identify clinically silent DC carriers, and discriminate between patients with DC and those with other bone marrow failure disorders. We defined “very short” telomeres as below the first percentile measured among 400 healthy control subjects over the entire age range. Diagnostic sensitivity and specificity of very short telomeres for DC were more than 90% for total lymphocytes, CD45RA+/CD20− naive T cells, and CD20+ B cells. Granulocyte and total leukocyte assays were not specific; CD45RA− memory T cells and CD57+ NK/NKT were not sensitive. We observed very short telomeres in a clinically normal family member who subsequently developed DC. We propose adding leukocyte subset flow fluorescence in situ hybridization telomere length measurement to the evaluation of patients and families suspected to have DC, because the correct diagnosis will substantially affect patient management.

scholarly journals Quantification of telomere features in tumor tissue sections by an automated 3D imaging-based workflow

2016 ◽  
Author(s):  
Manuel Gunkel ◽  
Inn Chung ◽  
Stefan Wörz ◽  
Katharina I. Deeg ◽  
Ronald Simon ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence Microscopy ◽  
Fluorescence In Situ Hybridization ◽  
Length Distribution ◽  
Nuclear Bodies ◽  
Telomere Maintenance ◽  
Proliferative Potential ◽  
Tissue Sections ◽  
Pml Nuclear Bodies

AbstractThe microscopic analysis of telomere features provides a wealth of information on the mechanism by which tumor cells maintain their unlimited proliferative potential. Accordingly, the analysis of telomeres in tissue sections of patient tumor samples provides can be exploited to obtain diagnostic information and to define tumor subgroups. In many instances, however, analysis of the image data is conducted by manual inspection of 2D images at relatively low resolution for only a small part of the sample. As the telomere feature signal distribution is frequently heterogeneous, this approach is prone to a biased selection of the information present in the image and lacks subcellular details. Here we address these issues by using an automated high-resolution imaging and analysis workflow that quantifies individual telomere features on tissue sections for a large number of cells. The approach is particularly suited to assess telomere heterogeneity and low abundant cellular sub-populations with distinct telomere characteristics in a reproducible manner. It comprises the integration of multi-color fluorescence in situ hybridization, immunofluorescence and DNA staining with targeted automated 3D fluorescence microscopy and image analysis. We apply our method to telomeres in glioblastoma and prostate cancer samples, and describe how the imaging data can be used to derive statistically reliable information on telomere length distribution or colocalization with PML nuclear bodies. We anticipate that relating this approach to clinical outcome data will prove to be valuable for pretherapeutic patient stratification.Abbreviations3D-TIM3D targeted imagingALTalternative lengthening of telomeresAPBALT-associated PML-NBCLSMconfocal laser scanning fluorescence microscopyECTRextrachromosomal telomeric repeatFFPEformalin-fixed, paraffin-embeddedFISHfluorescence in situ hybridizationIFImmunofluorescencepedGBMpediatric glioblastomaPMLpromyelocytic leukemiaPML-NBPML nuclear bodyPNApeptide nucleic acidROIregion of interestTMAtissue microarrayTMMtelomere maintenance mechanismSMLMsingle molecule localization microscopy

scholarly journals Cell Type–Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry

2018 ◽  
Vol 66 (7) ◽  
pp. 485-495 ◽  
Author(s):  
Aernoud A. van Batenburg ◽  
Karin M. Kazemier ◽  
Ton Peeters ◽  
Matthijs F. M. van Oosterhout ◽  
Joanne J. van der Vis ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Telomere Length ◽  
Dna Sequences ◽  
Double Strand Breaks ◽  
Telomere Maintenance ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Telomeres are small repetitive DNA sequences at the ends of chromosomes which act as a buffer in age-dependent DNA shortening. Insufficient telomere repeats will be recognized as double-strand breaks. Presently, it is becoming more evident that telomere attrition, whether or not caused by mutations in telomere maintenance genes, plays an important role in many inflammatory and age-associated diseases. In this report, a method to (semi)quantitatively assess telomere length and DNA double-strand breaks in formalin-fixed paraffin-embedded (FFPE) tissue is described. Therefore, a novel combination of quantitative fluorescence in situ hybridization, tissue elution, and immunofluorescence staining techniques was developed. Caveolin-1 (type 1 pneumocytes), pro-surfactant protein C (type 2 pneumocytes), club cell-10 (club cells), and alpha smooth muscle actin (smooth muscle cells) markers were used to identify cell types. To visualize all the different probes, restaining the tissue by heat-mediated slide elution is essential. Fluorescent signals of telomeres and DNA double-strand breaks were quantified using the Telometer plugin of ImageJ. As example, we analyzed lung tissue from a familial pulmonary fibrosis patient with a mutation in the telomere-associated gene poly(A)-specific ribonuclease ( PARN). The protocol displays a novel opportunity to directly quantitatively link DNA double-strand breaks to telomere length in specific FFPE cells.

Phylogenetic distribution of TTAGG telomeric repeats in insects

Genome ◽  
2004 ◽  
Vol 47 (1) ◽  
pp. 163-178 ◽  
Author(s):  
Radmila Frydrychová ◽  
Petr Grossmann ◽  
Pavel Trubac ◽  
Magda Vítková ◽  
František Marec
Keyword(s):  
In Situ Hybridization ◽  
Phylogenetic Tree ◽  
Fluorescence In Situ Hybridization ◽  
Southern Hybridization ◽  
Published Data ◽  
Phylogenetic Distribution ◽  
Telomeric Repeats ◽  
Insect Evolution ◽  
Single Primer

We examined the presence of TTAGG telomeric repeats in 22 species from 20 insect orders with no or inconclusive information on the telomere composition by single-primer polymerase chain reaction with (TTAGG)6 primers, Southern hybridization of genomic DNAs, and fluorescence in situ hybridization of chromosomes with (TTAGG)n probes. The (TTAGG)n sequence was present in 15 species and absent in 7 species. In a compilation of new and published data, we combined the distribution of (TTAGG)n telomere motif with the insect phylogenetic tree. The pattern of phylogenetic distribution of the TTAGG repeats clearly supported a hypothesis that the sequence was an ancestral motif of insect telomeres but was lost repeatedly during insect evolution. The motif was conserved in the "primitive" apterous insect orders, the Archaeognatha and Zygentoma, in the "lower" Neoptera (Plecoptera, Phasmida, Orthoptera, Blattaria, Mantodea, and Isoptera) with the exception of Dermaptera, and in Paraneoptera (Psocoptera, Thysanoptera, Auchenorrhyncha, and Sternorrhyncha) with the exception of Heteroptera. Surprisingly, the (TTAGG)n motif was not found in the "primitive" pterygotes, the Palaeoptera (Ephemeroptera and Odonata). The Endopterygota were heterogeneous for the occurrence of TTAGG repeats. The motif was conserved in Hymenoptera, Lepidoptera, and Trichoptera but was lost in one clade formed by Diptera, Siphonaptera, and Mecoptera. It was also lost in Raphidioptera, whereas it was present in Megaloptera. In contrast with previous authors, we did not find the motif in Neuroptera. Finally, both TTAGG-positive and TTAGG-negative species were reported in Coleoptera. The repeated losses of TTAGG in different branches of the insect phylogenetic tree and, in particular, in the most successful lineage of insect evolution, the Endopterygota, suggest a backup mechanism in the genome of insects that enabled them frequent evolutionary changes in telomere composition.Key words: chromosomes, fluorescence in situ hybridization, FISH, insects, phylogeny, single primer PCR, Southern hybridization, telomere, telomeric repeats.

890: Multiple Fluorescence in Situ Hybridization (FISH) Probes Increase Sensitivity for Detection of Bladder Cancer

2006 ◽  
Vol 175 (4S) ◽  
pp. 287-288 ◽  
Author(s):  
Juliann M. Dziubinski ◽  
Michael F. Sarosdy ◽  
Paul R. Kahn ◽  
Mark D. Ziffer ◽  
William R. Love ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization

588: Detection of Chromosome 8 Alterations in Paired Needle Biopsy and Prostatectomy Specimens Using Fluorescence In-Situ Hybridization

2004 ◽  
Vol 171 (4S) ◽  
pp. 156-156
Author(s):  
Chandler D. Dora ◽  
Yasushi Kondo ◽  
Fusheng X. Lan ◽  
Jeffrey M. Slezak ◽  
Erik J. Bergstralh ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Needle Biopsy ◽  
Chromosome 8
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