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Replication timing of human telomeric DNA and other repetitive sequences analyzed by fluorescence in situ hybridization and flow cytometry
Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
Department of Immunocytochemistry, DAKO A/S, Glostrup, Denmark.
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2001 (Engelska)Ingår i: Experimental Cell Research, ISSN 0014-4827, Vol. 271, s. 223-229Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The replication timing of telomeres seems to differ between species. Yeast telomeres are late replicating, whereas limited data from very few human cell lines have indicated telomere replication throughout S phase. In the present study a series of permanent cell lines and patient samples was investigated using a flow cytometric approach for telomere length determination based on in situ hybridization using peptide nucleic acid probes and DNA staining. This method permits selective analysis of cells in specific phases of the cell cycle without perturbation of the cell cycle machinery. The timing of replication of telomeric C(3)TA(2) and T(2)AG(3) repeats was found to differ between individual samples and could precede or be concomitant with the replication of bulk DNA. Replication of the T(2)AG(3) strand seemed to occur somewhat later than that of the C(3)TA(2) strand in some samples. (GTG)(n) and other repetitive sequences generally showed a replication pattern similar to that of the bulk of DNA with slightly individual differences, whereas centromeric DNA repeats consistently replicated within a short time frame in late S phase. The apparent variability in replication timing seen for telomeric DNA might suggest individual differences in firing of replication origins.

Ort, förlag, år, upplaga, sidor
Elsevier, 2001. Vol. 271, s. 223-229
Nyckelord [en]
telomere, centromere, repetitive sequence, fluorescence in situ hybridization, flow cytometry, replication, timing
Nationell ämneskategori
Cancer och onkologi Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Identifikatorer
URN: urn:nbn:se:umu:diva-5077DOI: 10.1006/excr.2001.5391ISI: 000172749500003PubMedID: 11716534Scopus ID: 2-s2.0-0035842522OAI: oai:DiVA.org:umu-5077DiVA, id: diva2:144444
Tillgänglig från: 2003-06-26 Skapad: 2003-06-26 Senast uppdaterad: 2023-03-24Bibliografiskt granskad
Ingår i avhandling
1. Telomere analysis of normal and neoplastic hematopoietic cells: studies focusing on fluorescence in situ hybridization and flow cytometry
Öppna denna publikation i ny flik eller fönster >>Telomere analysis of normal and neoplastic hematopoietic cells: studies focusing on fluorescence in situ hybridization and flow cytometry
2003 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

The telomeres are specialized structures at the end of the chromosomes composed of the repeated DNA sequence (TTAGGG)n and specific proteins bound to the DNA. The telomeres protect the chromosomes from degradation and end to end fusions. Due to the end-replication problem, the telomeric DNA shortens every cell division, forcing the cells into senescence at a critical telomere length. This process can be counteracted by activating a specialized enzyme, telomerase, which adds telomeric repeats to the chromosome ends leading to an extended or infinite cellular life span. Telomerase activity is absent in most somatic tissues but is found in germ cells, stem cells, activated lymphocytes and the vast majority of tumor cells and permanent cell lines. Hence, telomerase has been suggested as a target for cancer treatment as malignant cells almost exclusively express the enzyme and in that context telomere length measurements will be of great importance.

Telomere length is traditionally measured with a Southern blot based technique. A new method for telomere analysis of cells in suspension, called flow-FISH, was developed based on fluorescence in situ hybridization using a telomeric peptide nucleic acid (PNA) probe,

DNA staining with propidium iodide and quantification by flow cytometry. Flow-FISH had high reproducibility and the telomere length measurements showed good correlation with Southern blotting results. The flow-FISH technique also allows studies of cells in specific phases of the cell cycle and the replication timing of telomeric, centromeric and other repetitive sequences were analyzed in a number of cells. Like previous studies, centromeres were shown to replicate late in S phase while the telomere repeats were found to replicate early in S phase or concomitant with the bulk DNA, which is opposite to the patterns described in yeast.

In benign immunopurified lymphocytes from tonsils, high telomerase activity was found in germinal center (GC) B cells. This population also had high hTERT mRNA levels and displayed a telomere elongation as shown by flow-FISH and Southern blotting. Combined immunophenotyping and flow-FISH on unpurified tonsil cells confirmed the results.

Chronic lymphocytic leukemia (CLL), the most common leukemia in adults, can be divided into pre-GC CLL, characterized by unmutated immunoglobulin VH genes and worse prognosis, and post-GC CLL, with mutated VH genes and better prognosis. In 61 cases of CLL, telomere length was measured with Southern blotting and VH gene mutation status was analyzed. A new association was found between VH mutation status and telomere length, where cases with longer telomeres and mutated VH genes (post-GC CLL) had better prognosis

than CLL with short telomeres and unmutated VH genes (pre-GC CLL). A larger study of 112 CLL cases was performed using flow-FISH. The same correlation between telomere length and VH mutation status was found but gender seemed to be of importance as telomere length was a significant prognostic factor for the male CLL patients but not in the female group. Age of the patients and spread of disease seemed to affect the prognostic value of VH gene mutation status.

Ort, förlag, år, upplaga, sidor
Umeå: Medicinsk biovetenskap, 2003. s. 66
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 840
Nyckelord
Biomedicine, telomere, telomerase, fluorescence in situ hybridization, flow cytometry, flow-FISH, replication timing, chronic lymphocytic leukemia, immunoglobulin gene, prognosis, Biomedicin
Nationell ämneskategori
Mikrobiologi inom det medicinska området
Forskningsämne
medicinsk cellbiologi
Identifikatorer
urn:nbn:se:umu:diva-76 (URN)91-7305-444-5 (ISBN)
Disputation
2003-05-23, Betula, 6M, Umeå, 09:00
Tillgänglig från: 2003-06-26 Skapad: 2003-06-26 Senast uppdaterad: 2018-01-13Bibliografiskt granskad

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Hultdin, MagnusGrönlund, ElisabethNorrback, Karl-FredrikRoos, Göran

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Hultdin, MagnusGrönlund, ElisabethNorrback, Karl-FredrikRoos, Göran
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Cancer och onkologiMedicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)

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