Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths

Kimura, Masayuki; Stone, Rivka C.; Hunt, Steven C.; Skurnick, Joan; Lu, Xingrong; Cao, Xiaojian; Harley, Calvin B.; Aviv, Abraham

doi:10.1038/nprot.2010.124

Cited by 396 publications

(429 citation statements)

References 28 publications

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“…Serum insulin was analysed using a commercial time-resolved fluoroimmunoassay (Perkin-Elmer Life Sciences, Turku, Finland). Measurements of LTL were performed in duplicate on different gels by Southern blots as previously described [24]. The inter-assay coefficient of variation for the duplicate measures was 1.3%.…”

Section: Methodsmentioning

confidence: 99%

A short leucocyte telomere length is associated with development of insulin resistance

et al. 2016

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Aims/hypothesis A number of studies have shown that leucocyte telomere length (LTL) is inversely associated with insulin resistance and type 2 diabetes mellitus. The aim of the present longitudinal cohort study, utilising a twin design, was to assess whether shorter LTL predicts insulin resistance or is a consequence thereof. Methods Participants were recruited between 1997 and 2000 through the population-based national Danish Twin Registry to participate in the GEMINAKAR study, a longitudinal evaluation of metabolic disorders and cardiovascular risk factors. Baseline and follow-up measurements of LTL and insulin resistance over an average of 12 years were performed in a subset of the Registry consisting of 338 (184 monozygotic and 154 dizygotic) same-sex twin pairs. Results Age at baseline examination was 37.4± 9.6 (mean ± SD) years. Baseline insulin resistance was not associated with age-dependent changes in LTL (attrition) over the follow-up period, whereas baseline LTL was associated with changes in insulin resistance during this period. The shorter the LTL at baseline, the more pronounced was the increase in insulin resistance over the follow-up period (p < 0.001); this effect was additive to that of BMI. The co-twin with the shorter baseline LTL displayed higher insulin resistance at follow-up than the co-twin with the longer LTL. Conclusions/interpretation These findings suggest that individuals with short LTL are more likely to develop insulin resistance later in life. By contrast, presence of insulin resistance does not accelerate LTL attrition.

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Section: Methodsmentioning

confidence: 99%

A short leucocyte telomere length is associated with development of insulin resistance

et al. 2016

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“…Blots were visualized by autoradiography using a PhosphorImager (Typhoon Trio). Average telomere length was determined as described by Kimura et al (61) using Gel Analyzer software.…”

Section: Southern Blot Analysismentioning

confidence: 99%

Progerin-Induced Replication Stress Facilitates Premature Senescence in Hutchinson-Gilford Progeria Syndrome

Wheaton

Campuzano

et al. 2017

Molecular and Cellular Biology

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Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutation in LMNA that produces an aberrant lamin A protein, progerin. The accumulation of progerin in HGPS cells leads to an aberrant nuclear morphology, genetic instability, and p53-dependent premature senescence. How p53 is activated in response to progerin production is unknown. Here we show that young cycling HGPS fibroblasts exhibit chronic DNA damage, primarily in S phase, as well as delayed replication fork progression. We demonstrate that progerin binds to PCNA, altering its distribution away from replicating DNA in HGPS cells, leading to ␥H2AX formation, ATR activation, and RPA Ser33 phosphorylation. Unlike normal human cells that can be immortalized by enforced expression of telomerase alone, immortalization of HGPS cells requires telomerase expression and p53 repression. In addition, we show that the DNA damage response in HGPS cells does not originate from eroded telomeres. Together, these results establish that progerin interferes with the coordination of essential DNA replication factors, causing replication stress, and is the primary signal for p53 activation leading to premature senescence in HGPS. Furthermore, this damage response is shown to be independent of progerin farnesylation, implying that unprocessed lamin A alone causes replication stress.KEYWORDS HGPS, progerin, senescence, aging, p53, telomere T he study of children with rare accelerated aging (progeria) syndromes has provided insight into the normal process of aging. Hutchinson-Gilford progeria syndrome (HGPS) is caused by a heterozygous, autosomal dominant mutation in LMNA (1), the gene that encodes lamin A, a key structural protein in the nuclear lamina. The lamina, a protein network that lines the inner nuclear membrane, provides structural support for the nucleus and is important for chromatin attachment, DNA replication, nuclear organization, and gene transcription in ways that are not completely understood. Farnesylation of prelamin A at the C terminus allows targeting to the inner nuclear membrane, where it is subsequently cleaved by the zinc metalloproteinase Zmpste24 to release mature lamin A into the lamina and nucleoplasm. In HGPS, the most common LMNA mutation (G608G) activates a cryptic splice donor site in exon 11, resulting in prelamin A mRNA with a 150-bp internal deletion, leading to a 50-amino-acid truncation in a region that contains the Zmpste24 cleavage site (reviewed in reference 2). The mutant lamin, termed progerin, retains the farnesyl lipid anchor, interferes with the integrity of the nuclear lamina, and causes the formation of misshapen nuclei. The retention of progerin on the inner nuclear membrane interferes with the function and normal distribution of lamin A, causing a loss of peripheral heterochromatin, increased DNA damage, impaired recruitment of DNA repair proteins to sites of DNA damage (3), and persistent activation of DNA damage response proteins (4). In addition to its

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“…Telomere length assessment obtained using qPCR was verified by Southern blot (19), with certain modifications as previously described (20). Briefly, 1.5 µg DNA obtained from two samples was digested with the …”

Section: Southern Blot Analysismentioning

confidence: 99%

Telomere length assessment in leukocytes presents potential diagnostic value in patients with breast cancer

et al. 2016

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Abstract.Telomere shortening is associated with cancer development, primarily through the induction of genomic instability. The majority of studies have indicated that individuals with shorter blood telomeres may be at a higher risk of developing various types of cancer. There is increasing evidence that the study of the alterations in telomere length may improve cancer prognosis. The aim of the present study was to verify the use of telomere length parameters in the diagnostics of breast cancer stage. Telomere length was analyzed in the blood leukocytes of 52 patients with breast cancer relative to 47 control subjects using quantitative polymerase chain reaction. The effects of stage, grade, estrogen receptor, progesterone receptor and human epidermal growth factor 2 (HER2) status were assessed. The current study demonstrated that the average telomeric sequence length was significantly shorter in leukocytes from individuals diagnosed with a more severe stage of breast cancer (T2N1M0) than in leukocytes in the early stages of the disease (T1N0M0) (P=0.0207). Furthermore, the data indicated that telomeres in leukocytes derived from patients with HER2 + breast cancer were significantly longer compared with those with the HER2 -type (P=0.0347). These results suggest that the assessment of telomeres in blood leukocytes may, at least partially, correspond with breast cancer staging and HER2 receptor status.

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Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths

Cited by 396 publications

References 28 publications

A short leucocyte telomere length is associated with development of insulin resistance

A short leucocyte telomere length is associated with development of insulin resistance

Progerin-Induced Replication Stress Facilitates Premature Senescence in Hutchinson-Gilford Progeria Syndrome

Telomere length assessment in leukocytes presents potential diagnostic value in patients with breast cancer

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