Terminal Restriction Fragment Length Research Articles

Variations in T-RFLP profiles with differing chemistries of fluorescent dyes used for labeling the PCR primers

Culture independent molecular methods have emerged as indispensable tools for studying microbial community structure and dynamics in natural habitats, since they allow a closer look at microbial diversity that is not reflected by culturing techniques. Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis is one of the informative and widely used techniques for such studies. However, the method has a few limitations to predict microbial community structure with significant accuracy. One of the major limitations is variation in real Terminal Restriction Fragment (TRF) length and observed TRF length. In the present study we report the generation of TRF length variations using different fluorescent dyes to label the PCR primers. T-RFLP profiles generated from primers labeled with different dyes varied significantly and led to inconsistent microbial species identification. Occurrence of such variations can have serious consequences on interpretation of the T-RFLP profiles from environmental samples representing complex microbial community. Therefore, in a T-RFLP study, the primers and labeling dye system should be carefully evaluated and optimized for an individual community under investigation. Further, it would be recommended to establish a target gene library in parallel with T-RFLP analysis to facilitate the accurate prediction of microbial community structure.

Arterial baroreflex function does not influence telomere length in kidney of rats

To investigate the relationship between arterial baroreflex (ABR) function and telomere length in kidney of rats. Stroke-prone spontaneously hypertensive rats (SHR-SP) and sinoaortic denervated rats (SAD) were used as models with depressed arterial baroreflex. In the first experiments, SHR-SP rats were examined at the age of 24 weeks for both sexes and 40 weeks for female rats. In the second experiments, SAD rats were studied 4 and 35 weeks after SAD operation. Blood pressure was continuously recorded for 4 h in a conscious state. After the determination of baroreflex sensitivity (BRS), the terminal restriction fragment (TRF) of rat kidney was analyzed using Southern blot. The TRF length was found shorter in: a) male SHR-SP compared with age-matched female SHR-SP; b) female SHR-SP 40 weeks of age compared with 24 weeks of age; c) in rats 35 weeks after operation compared with rats 4 weeks post operation in both sham-operated and SAD rats. In SHR-SP, the TRF length did not correlate with BRS. In addition, SAD did not affect TRF length at either 4 or 35 weeks post-surgery. It may be concluded that baroreflex function does not influence the terminal restriction fragment (TRF) length in rats.

Leukocyte Telomere Length and Cardiovascular Disease in the Cardiovascular Health Study

The telomere length of replicating somatic cells is inversely correlated with age and has been reported to be associated cross-sectionally with cardiovascular disease (CVD). Leukocyte telomere length, as expressed by mean terminal restriction fragment (TRF) length, was measured in 419 randomly selected participants from the Cardiovascular Health Study, comprising a community-dwelling cohort recruited in four US communities. The authors investigated associations between TRF length and selected measures of subclinical CVD/risk factors for CVD (data were collected at the 1992/1993 clinic visit) and incident CVD (ascertained through June 2002). In these participants (average age = 74.2 years (standard deviation, 5.2)), mean TRF length was 6.3 kilobase pairs (standard deviation, 0.62). Significant or borderline inverse associations were found between TRF length and diabetes, glucose, insulin, diastolic blood pressure, carotid intima-media thickness, and interleukin-6. Associations with body size and C-reactive protein were modified by gender and age, occurring only in men and in participants aged 73 years or younger. In younger (but not older) participants, each shortened kilobase pair of TRF corresponded with a threefold increased risk of myocardial infarction (hazard ratio = 3.08, 95% confidence interval: 1.22, 7.73) and stroke (hazard ratio = 3.22, 95% confidence interval: 1.29, 8.02). These results support the hypotheses that telomere attrition may be related to diseases of aging through mechanisms involving oxidative stress, inflammation, and progression to CVD.

The effects of social status on biological aging as measured by white‐blood‐cell telomere length

Low socio-economic status (SES) is associated with a shortened life expectancy, but its effect on aging is unknown. The rate of white-blood-cell (WBC) telomere attrition may be a biological indicator of human aging. We tested the hypothesis that SES is associated with telomere attrition independent of known risk factors influencing the aging process. We studied 1552 female twins. A venous blood sample was taken from each twin and isolated WBCs used for extraction of DNA. Terminal restriction fragment length (TRFL) was measured. Questionnaire data were collected on occupation, education, income, smoking, exercise, height and weight. Standard multiple linear regression and multivariate analyses of variance tested for associations between SES and TRFL, adjusting for covariates. A discordant twin analysis was conducted on a subset to verify findings. WBC telomere length was highly variable but significantly shorter in lower SES groups. The mean difference in TRFL between nonmanual and manual SES groups was 163.2 base pairs (bp) of which 22.9 bp (approximately 14%) was accounted for by body mass index, smoking and exercise. Comparison of TRFL in the 17 most discordant SES twin pairs confirmed this difference. Low SES, in addition to the harmful effects of smoking, obesity and lack of exercise, appears to have an impact on telomere length.

Insulin resistance, oxidative stress, hypertension, and leukocyte telomere length in men from the Framingham Heart Study

Insulin resistance and oxidative stress are associated with accelerated telomere attrition in leukocytes. Both are also implicated in the biology of aging and in aging-related disorders, including hypertension. We explored the relations of leukocyte telomere length, expressed by terminal restriction fragment (TRF) length, with insulin resistance, oxidative stress and hypertension. We measured leukocyte TRF length in 327 Caucasian men with a mean age of 62.2 years (range 40-89 years) from the Offspring cohort of the Framingham Heart Study. TRF length was inversely correlated with age (r = -0.41, P < 0.0001) and age-adjusted TRF length was inversely correlated with the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) (r =-0.16, P = 0.007) and urinary 8-epi-PGF(2alpha) (r = -0.16, P = 0.005) - an index of systemic oxidative stress. Compared with their normotensive peers, hypertensive subjects exhibited shorter age-adjusted TRF length (hypertensives = 5.93 +/- 0.042 kb, normotensives = 6.07 +/- 0.040 kb, P = 0.025). Collectively, these observations suggest that hypertension, increased insulin resistance and oxidative stress are associated with shorter leukocyte telomere length and that shorter leukocyte telomere length in hypertensives is largely due to insulin resistance.

Ets2 binding site single nucleotide polymorphism at the hTERT gene promoter – effect on telomerase expression and telomere length maintenance in non-small cell lung cancer

The aim of this study was to elucidate the occurrence of DNA sequence changes in the promoter region of hTERT gene, and its effect on telomerase expression and telomere length maintenance in non-small cell lung cancer (NSCLC). Between January 2002 and December 2003, 66 NSCLC patients were studied. The expression of hTERT, telomerase activity (TA), and c-Myc were examined, and the terminal restriction fragment length (TRFL) was measured. A t/n-TRFLR was obtained by dividing the TRFL of the tumour tissue by TRFL of the paired normal tissue. PCR products were sequenced and compared with known hTERT gene promoter sequence for a length of 716 bp upstream of the transcription starting code. The changes of any known sequence and/or c-Myc expression with their impact on telomerase activity and TRFL maintenance were measured. Positive hTERT, TA and c-Myc expression was observed in 43 (65.2%), 39 (59.1%) and 59 (89.4%) of the tumour tissue samples, respectively. Except for one patient who had C/C (in normal tissue) homozygotes to T/C (in tumour tissue) heterozygotes point mutation, a novel single nucleotide polymorphism (SNP) −245kb upstream ( Ets2 binding site) of the hTERT gene was observed in all normal and tumour tissues, including C/C in 9, T/C in 35, and T/T in 22 of the tumour tissues. The TA of C/C homozygotes was lower than that of T/T homozygotes (P = 0.0331), while the t/n-TRFLR of C/C homozygotes was higher than that of T/T homozygotes (P = 0.0621). The latter was even more obvious when c-Myc were positive (P = 0.0185). Our data shows that T/T homozygotes have a lower t/n-TRFLR, but a stronger TA expression, suggesting that the studied Ets2 binding site is a positive regulator of hTERT gene. SNP may interfere with Ets2 binding and lower TA expression in T/C heterozygotes and C/C homozygotes.

Soil bacterial diversity in a loblolly pine plantation: influence of ectomycorrhizas and fertilization

We studied the effect of ectomycorrhizas and fertilization on soil microbial communities associated with roots of 10-year-old loblolly pine. Ectomycorrhizas were identified using a combination of community terminal restriction fragment profiling and matching of individual terminal restriction fragments to those produced from ectomycorrhizal clones and sequences recovered from roots and sporocarps. Differences between bacterial communities were initially determined using cluster analysis on community terminal restriction fragment profiles and through subsequent recovery of 16S rDNA clones. Analysis of bacterial clones revealed that terminal restriction fragment length was often shared between taxonomically dissimilar bacterial types. Consequently, we could not reliably infer the identity of peaks in the bacterial community profile with some exceptions, notably chloroplast rDNA that generated an approximate peak size of 80.2 bp. Fertilization increased the frequency of a Piloderma-like ectomycorrhiza. However, we did not detect clear effects of fertilization or the presence of viable ectomycorrhizas on bacterial communities. Bacterial communities seemed to be determined largely by the carbon and nitrogen content of soil. These results suggest that important soil microbial groups respond differently to soil conditions and management practices, with ectomycorrhizal communities reflecting past nutrient conditions and bacterial communities reflecting current environmental conditions of soil microsites.

Aldosterone and Telomere Length in White Blood Cells

Aldosterone accelerates cardiovascular aging by mechanisms that generate reactive oxygen species. Telomere length in white blood cells (WBCs) may be a bioindicator that registers the accruing burden of systemic oxidative stress. The aim of the present study was, therefore, to examine the relationship between plasma aldosterone and telomere length in WBCs. We studied 75 normotensive and never-treated mildly hypertensive men whose blood was drawn for the measurements of plasma aldosterone concentration and the terminal restriction fragment (TRF) length in WBCs. The slope of the TRF-age relationship in the entire cohort showed a decrease in telomere length of 26 +/- 5 base pairs per year (r = -0.46, p <.001). Age-adjusted TRF length was the longest in the lowest aldosterone quartile (6.74 +/- 0.12 kb) and shortest in the highest aldosterone quartile (6.36 +/- 0.11 kb), with intermediate TRF lengths in the second and third aldosterone quartiles (analysis of variance [ANOVA] trend test, p =.025). In telomeric attrition equivalence, participants in the upper aldosterone quartile were 15 years older than their peers in the lowest quartile. The inverse relationship between aldosterone and WBC telomere length suggests not only that aldosterone is pro-oxidant but that elevated concentrations of this hormone might be linked to a higher rate of telomere attrition and perhaps increased biological aging in humans.

Chromosomal telomere shortening of kidney cells in IgA nephropathy by the measurement of DNA in urinary sediment

The histology and function of the kidney deteriorates with age and progressive renal failure, but the mechanisms involved in renal ageing are not known. In vitro studies suggest that telomere shortening is important in replicative senescence, and is accelerated by stress factors that increase replication. We investigated whether IgA nephropathy, a prototype chronic kidney disease, is associated with localized intrarenal cellular ageing. We studied the mean length of terminal restriction fragments (TRF), a measure of average telomere size, in the DNA of peripheral blood mononuclear cells and urinary sediment of 15 patients with IgA nephropathy. The mean TRF lengths in peripheral blood is 7043.8 +/- 1 182.8 base pairs, and in urinary sediment is 6 749.7 +/- 636.5 base pairs. The mean TRF lengths of urinary DNA significantly correlate with the serum creatinine (r = -0.525, p = 0.044) and estimated glomerular filtration rate (GFR) (r = 0.651, p = 0.009). The mean TRF lengths of urinary DNA had an insignificant inverse correlation with patient age (r = -0.364, p = 0.2), and do not correlate with the degree of glomerulosclerosis (r = 0.004, p = 0.9) or tubulointerstitial scarring in renal biopsy (r =-0.032, p = 0.9). After 30 months of follow-up, the rate of decline of estimated GFR has an inverse correlation with the mean TRF lengths of urinary DNA (r = -0.699, p = 0.004). The TRF lengths of peripheral blood DNA do not correlate with any clinical or histological parameter or the rate of renal function decline. Although this is a pilot study, our observation indicates that the TRF lengths of genomic DNA extracted from urinary sediment is related to the degree of renal impairment. However, a long telomere length of genomic DNA in urinary sediment is associated with a more rapid decline of renal function. Our findings might be relevant to the pathogenesis of progressive renal failure.

Telomere Length Among the Elderly and Oldest-Old

Human chromosomes terminate in a number of repeats of the sequence TTAGGG. At birth, each chromosome end is equipped with approximately 15 kb of telomere sequence, but this sequence is shortened during each cell division. In cell cultures telomere shortening is associated with senescence, a phenomenon that has also been observed in normal adult tissues, indicating that telomere loss is associated with organismal ageing. Previous work has established that the rate of telomere loss in humans is age dependent, and recent work shows a sex-specific difference in telomere length and shortening in individuals over the age span of 20 to 75 years. Here, terminal restriction fragment lengths on DNA purified from whole blood were measured to examine the mean telomere length in a cross-sectional cohort of 816 Danish individuals of age 73 to 101 years. In this age group, females show a linear correlation between telomere length and age, whereas the pattern tends to be nonlinear (quadratic in age) for males. This difference in telomere length dynamics between the 2 sexes may be caused by several different mechanisms, including differences in selection by mortality, differences in leukocyte population or different telomerase expression pattern.

Telomere Length Among the Elderly and Oldest-Old

AbstractHuman chromosomes terminate in a number of repeats of the sequence TTAGGG. At birth, each chromosome end is equipped with approximately 15 kb of telomere sequence, but this sequence is shortened during each cell division. In cell cultures telomere shortening is associated with senescence, a phenomenon that has also been observed in normal adult tissues, indicating that telomere loss is associated with organismal ageing. Previous work has established that the rate of telomere loss in humans is age dependent, and recent work shows a sex-specific difference in telomere length and shortening in individuals over the age span of 20 to 75 years. Here, terminal restriction fragment lengths on DNA purified from whole blood were measured to examine the mean telomere length in a cross-sectional cohort of 816 Danish individuals of age 73 to 101 years. In this age group, females show a linear correlation between telomere length and age, whereas the pattern tends to be nonlinear (quadratic in age) for males. This difference in telomere length dynamics between the 2 sexes may be caused by several different mechanisms, including differences in selection by mortality, differences in leukocyte population or different telomerase expression pattern.

Telomere shortening occurs in Asian Indian Type 2 diabetic patients

Telomere shortening has been reported in several diseases including atherosclerosis and Type 1 diabetes. Asian Indians have an increased predilection for Type 2 diabetes and premature coronary artery disease. The aim of this study was to determine whether telomeric shortening occurs in Asian Indian Type 2 diabetic patients. Using Southern-blot analysis we determined mean terminal restriction fragment (TRF) length, a measure of average telomere size, in leucocyte DNA. Type 2 diabetic patients without any diabetes-related complications (n = 40) and age- and sex-matched control non-diabetic subjects (n = 40) were selected from the Chennai Urban Rural Epidemiology Study (CURES). Plasma level of malondialdehyde (MDA), a marker of lipid peroxidation, was measured by TBARS (thiobarbituric acid reactive substances) using a fluorescence method. Mean (+/- SE) TRF lengths of the Type 2 diabetic patients (6.01 +/- 0.2 kb) were significantly shorter than those of the control subjects (9.11 +/- 0.6 kb) (P = 0.0001). Among the biochemical parameters, only levels of TBARS showed a negative correlation with shortened telomeres in the diabetic subjects (r = -0.36; P = 0.02). However, telomere lengths were negatively correlated with insulin resistance (HOMA-IR) (r = -0.4; P = 0.01) and age (r = -0.3; P = 0.058) and positively correlated with HDL levels (r = 0.4; P = 0.01) in the control subjects. Multiple linear regression (MLR) analysis revealed diabetes to be significantly (P < 0.0001) associated with shortening of TRF lengths. Telomere shortening occurs in Asian Indian Type 2 diabetic patients.

Sequence-specific DNA damage by reactive oxygen species: Implications for carcinogenesis and aging

Reactive oxygen species (ROS) generated by environmental chemicals can cause sequence-specific DNA damage, which may lead to carcinogenesis and aging. We investigated the mechanism of DNA damage by environmental chemicals (catechol, propyl gallate and bisphenol-A), homocysteine and UVA radiation using human cultured cell lines and(32)P-labeled DNA fragments. Carcinogenic catechol induced piperidine-labile sites frequently at thymine residues in the presence of Cu(II) and NADH. Furthermore, catechol increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60, but not in HP100, a hydrogen peroxide (H(2)O(2))-resistant cell line derived from HL-60. Thus, it is concluded that oxidative DNA damage through generation of H(2)O(2) plays an important role in the carcinogenic process of catechol. In addition, an environmental factor, bisphenol-A, and a dietary factor, propyl, gallate, also induced sequence-specific DNA damage via ROS generation.UVA, as well as UVB, contributes to photoaging. In humans, telomere shortening is believed to be associated with cell senescence. In this study, we investigated the shortening rate of telomeres in human WI-38 fibroblasts exposed to UVA irradiation. The telomere length (as measured by terminal restriction fragment length) in WI-38 fibroblasts irradiated with UVA decreased with increasing the irradiation dose. UVA irradiation with riboflavin caused damage specifically at the GGG sequence in the DNA fragments containing telomere sequence (TTAGGG)(4). We concluded that the GGG-specific damage in telomere sequence induced by UVA irradiation participates in the increase of the telomere shortening rate.In this report, we show our experimental results and discuss the mechanisms of sequence-specific DNA damage in relation to carcinogenesis and aging.

Clinical significance of telomerase and its associate genes expression in the maintenance of telomere length in squamous cell carcinoma of the esophagus

To observe the interaction between the expression of telomerase activity (TA) and its associate genes in regulation of the terminal restriction fragment length (TRFL) in esophageal squamous cell carcinoma (SCC). Seventy-four specimens of esophageal SCC were examined. The TA was measured by telomeric repeat amplification protocol (TRAP) assay, and the associated genes [human telomerase-specific reverse transcriptase (hTERT), hTERC, TP1, c-Myc, TRF1, and TRF2] were detected using RT-PCR method. The TRFL was measured by Telomere Length Assay Kit and Southern blotting. The correlations between the expression of telomerase and its associated genes with the TRFL and survivals were examined. Expressions of the TA, hTERT, hTERC, TP1, c-Myc, TRF1, and TRF2 genes were observed in 85.1%, 64.9%, 79.7%, 100.0%, 94.6%, 82.4%, and 91.9% of the tumor tissues, respectively. The TRFL of the tumor and normal esophageal tissues were 2.70+/-1.42 and 4.93+/-1.74 kb, respectively (P<0.0001). The TRFL of the telomerase positive and telomerase negative tumor tissues were 2.72+/-1.44 and 2.58+/-1.32 kb, respectively (P = 0.767). The TRFL ratios (TRFLR) of the telomerase positive and telomerase negative tumor tissues were 0.55+/-0.22 and 0.59+/-0.41, respectively (P = 0.742). The expression rates of h-TERT (P = 0.0002), hTERC (P<0.0001), and TRF1 (P = 0.002) in the tumor tissues are higher than those of the normal paired tissues. Though TA is markedly activated in tumor tissues (P<0.0001), its expression is not related to clinicopathological parameters including gender, tumor differentiation, and TNM stages. The cumulative 4-year survival rates of telomerase positive and telomerase negative cases were 35.86% and 31.2%, respectively (P = 0.8442). The cumulative 4-year survival rates of patients with their TRFLR <or=85% and >85% were 38.7% and 15.7%, respectively (P = 0.1307). Though telomerase expression is not related to tumor stages and prognosis, our data support that the TA increased as the TRFL decreased, probably under the control of hTERT, hTERC, and TRF1. When telomerase expression was activated, only TRF2 overexpression persisted to stabilize T-loop formation. Furthermore, as the TRFLR decreased to 85%, a trend of better prognosis was observed. Cox model analysis indicates a higher t/n TRFLR and distant metastasis are independent poorer prognostic factors (P = 0.035 and P = 0.042, respectively).

Correlation between telomerase expression and terminal restriction fragment length ratio in non-small cell lung cancer--an adjusted measurement and its clinical significance.

To establish a new model for analyzing the correlation between the terminal restriction fragment (TRF) length and telomerase activity in non-small cell lung cancer (NSCLC) patients due to inconsistent results in previous reports. Between January 1999 and December 1999, 79 NSCLC patients were studied. The activity of telomerase was measured by telomeric repeat amplification protocol, and the telomere restriction fragment (TRF) length was measured by Telomere Length Assay Kit and Southern blotting. The correlation between expression of telomerase activity and the TRF length ratio (TRFLR) using the tumor-to-normal TRFLR (t/n TRFLR) was examined. Positive telomerase activity was observed in 48 (60.8%) of the tumor tissue samples and in 5 (6.3%) of the normal tissue samples (P < 0.0001). The mean TRF lengths were 5.32+/-1.53 kb in normal tissue samples and 3.95+/-1.92 kb in tumor tissue samples, respectively (P = 0.0001). The 4-year cumulative survival rates of lower t/n TRFLR (< or = 75%) and higher t/n TRFLR (>75%) patients were 69.2 and 41.2%, respectively (P = 0.0227). Independent prognostic factors include t/n TRFLR (P = 0.014), T-status (P = 0.027), and N-status (P = 0.020) of the tumor. Our data suggest that there is a good correlation between the t/n TRFLR and expression of telomerase activity. A higher t/n TRFLR may indicate that the tumor regains its ability to repair the telomere lost and escapes the apoptosis scenario, which is subsequently reflected in poorer patient outcomes.

Telomere attrition and accumulation of senescent cells in cultured human endothelial cells.

The human umbilical vein endothelial cell (HUVEC) is an important model of the human endothelium that is widely used in vascular research. HUVECs and the adult endothelium share many characteristics including progression into senescence as the cells age. Despite this, the shortening of telomeres and its relationship to the progression into senescence are poorly defined in HUVECs. In this study of several HUVEC lines we show notable consistency in their growth curves. There is a steady decline in the growth rate of HUVECs grown continually in culture and we estimate complete cessation of growth after approximately 70 population doublings. The HUVECs lose telomeric DNA at a consistent rate of 90 base pairs/population doubling and show a progressive accumulation of shortened telomeres (below 5 kilobases). This telomeric loss correlates with the accumulation of senescent HUVECs in culture as assessed by staining for beta-galactosidase activity at pH 6. Although the telomere length of a large population of cells is a relatively crude measure, we suggest that in HUVECs a mean telomere length (as measured by terminal restriction fragment length) of 5 kilobases is associated with entry into senescence. These data demonstrate the strong relationship between telomere attrition and cell senescence in HUVECs. They suggest that DNA damage and subsequent telomere attrition are likely to be key mechanisms driving the development of endothelial senescence in the pathogenesis of vascular disease.

Rapid telomere attrition in cardiac tissue of the ageing Wistar rat

Many studies show an association between ageing and mean telomere length in DNA isolated from peripheral blood mononuclear cells, few studies have examined less accessible tissues. This study has two objectives: (i) to define the best method to prepare rodent DNA for telomere length measurement by Southern blotting and (ii) to determine whether there are differential rates of telomere attrition in different rodent tissues. We found that the use of agarose plugs for DNA isolation was essential for the accurate measurement of rodent telomere length. Tissue was collected from neonatal (3 days) or aged (18–24 months) male Wistar rats and terminal restriction fragment (TRF) length was measured by Southern blotting. Cardiac tissue from aged rats showed a 38% loss of TRF length compared with newborn animals ( p<0.001, n=13), this contrasts with much smaller reductions in brain (1.6%), liver (14.2%), kidney (8.9%) and lung (9.7%). This study demonstrates that the methods of DNA preparation are critical for accurate measurement of telomeres in rodent tissues. Moreover, we show differential rates of telomere attrition in rat tissues, the heart being most susceptible to telomere loss. These observations could have important implications for the study of age-specific changes in tissue function.

Telomere length and possible link to X chromosome

Because telomeres are eroded during mitosis, telomere length indicates the replicative history of human somatic cells. Clinical markers of ageing--such as pulse pressure and survival--are associated with telomere length. On the basis of findings of studies in twins, telomere length seems to be familial, but little is known about its mode of inheritance. We aimed to investigate the inheritance of telomere length. We measured terminal restriction fragment (TRF) length in white-blood-cell DNA taken from individuals from the family-based cohort of the Flemish Study on Environment, Genes, and Health Outcomes. We recorded no correlation in sex and age adjusted TRF length between spouses (r=-0.05; p=0.70) nor between fathers and sons (r=-0.16; p=0.35). By contrast, we noted robust correlations in TRF length between fathers and daughters (r=0.60; p<0.0001); between mothers and sons (r=0.41; p=0.0017) and daughters (r=0.59; p<0.0001); and among siblings (r> or =0.61; p< or =0.0004). X-linked inheritance of TRF length is the most probable explanation for our findings. Pending confirmation, our observations suggest that the process of ageing might be an X-linked trait.

Analysis of telomerase activity and telomere function in cancer.

Telomeres are the structures at the ends of chromosomes, composed of repetitive sequences and associated proteins, which cap chromosome ends to maintain genomic stability. These structures are maintained by the enzyme complex telomerase in germ cells and some stem cells, but are absent in the majority of somatic cells. The consequence of this lack of telomerase in normal somatic cells is the shortening of the telomeric repeat, which results in a limited replicative life span. However, in cancer cells, which grow indefinitely, telomerase activity has been detected in a large number of different cancer cell types. This has lead to a great deal of interest in establishing techniques to measure telomerase activity, telomere length, and telomere function in both normal and cancer cells/tissue. Here we describe the TRAP (telomeric repeat amplification protocol) assay, a technique to measure telomerase activity in cells, TRF (terminal restriction fragment length) analysis to estimate telomere length, and both the anaphase bridge index and the frequency of dicentric chromosomes as indicators of telomere dysfunction.

24ASSESSMENT OF TELOMERE LENGTH IN NUCLEAR TRANSFER DERIVED SHEEP CLONES, THEIR OFFSPRING, AND CONTROL ANIMALS

The technique of somatic cell nuclear transfer (SCNT) involves the transplantation of a cultured somatic cell into an enucleated oocyte. Following activation, the nucleus of the somatic cell should be reprogrammed to a state of totipotency, which involves the resetting of epigenetic modifications of the somatic genome and also rebuilding of the chromosomal ends (telomeres). This study was carried out to investigate whether the nuclear transfer technique could rebuild the telomeres in sheep clones and their offspring when compared to age-matched control animals. Skin and blood samples were collected from three SCNT-derived sheep clones, and three of their offspring generated by natural mating. Control samples were collected from age-matched animals (n=17), spanning an age range from 1 month to 36 months of age. Ovine genomic DNA was extracted from samples using a Qiagen DNA extraction kit according to the manufacturer’s instructions. TeloTAGGG telomere length chemiluminescent assay kit (Roche Diagnostics) was used to determine the telomere length of all samples. Samples of ovine genomic DNA (2mg) were digested at 37°C for 2h using Hinf 1 and Rsa 1 endonuclease master-mix at the rate of 40 IU per sample. The resulted terminal restriction fragments (TRF) were separated on 1.2% agarose gel by pulsed field electrophoresis at 6V with 0.1s switch time for 4h in 0.5% TBE buffer at 14°C. The DNA fragments were southern-transferred to a nylon membrane and hybridized with telomere-specific (TTAGGG)n, digoxigenin (DIG)-labeled hybridization probe at 42°C. Following post-hybridization washes the membrane was incubated with DIG-specific antibody covalently coupled to alkaline phosphatase. The immobilized telomere probe was visualized by adding a chemiluminecence substrate to the membrane and exposed to X-ray film. Mean TRF length was determined by comparing the telomere densitometry signals to a molecular weight standard. High resolution bands resulting from pulsed field gel electrophoresis revealed that ovine TRF size distribution was in the range of 10–19kb. Linear regression analysis resulted in significant (t=25.84, P&amp;lt;0.05) decrease of mean TRF size with increasing age, at a mean rate of 0.08kb per every 6 months. Mean TRF length of SCNT-derived sheep clones, Matilda (28 months old), Macather (14 months old) and Frida (13 months old) were 11.12kb, 11.40kb, and 13.41kb, respectively. Their mean TRF values were significantly (P&amp;lt;0.05) shorter than those of their age-matched control animals: 16.95kb, 14.76kb, and 14.76kb, respectively (n=3 per group). The mean TRF length of the 3 offspring (21 months old) derived from natural mating was not significantly (P&amp;lt;0.05) different from age-matched controls (17.75±0.69kb v. 16.28±0.72kb). These results demonstrate that sheep clones derived from cultured somatic cells have shorter telomere lengths compared to age-matched controls, but this telomere loss is reset in the subsequent generation through natural breeding of the clones. (Funded by NSERC, OMAFRA and International Council for Canadian Studies.)