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Department of Small Animal Clinical Studies, University of Glasgow Veterinary School, Glasgow, UK and * Waltham Centre for Pet Nutrition, Leicestershire, UK
3To whom correspondence should be addressed. E-mail: t.mckevitt{at}vet.gla.ac.uk.
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ABSTRACT |
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KEY WORDS: • telomere • dog • age
Telomeres are specialized nucleoprotein structures that cap the ends of all eukaryotic chromosomes. Their structure is highly conserved across species boundaries and consists of a large number of tandem repeats of short G-rich sequences and associated proteins. In vertebrates the telomere repeat sequence is TTAGGG (1).
Although the structure of the telomeric repeat sequence is highly conserved, the number of repeats within telomeres varies widely; for example, canine telomeres have been shown to range from 10 to 23 kbp (2) compared to the much larger 10 to 60 kbp found in mice (3). Aside from this interspecies variation, telomere lengths have been found to differ widely within the same species. This variation may be associated with the age of the individuals concerned; for example, Harley et al. (4) demonstrated a reduction in the mean length of human fibroblast telomeres with increasing age. Variation in telomere length may also be found within cell lines taken from a single individual and even among the chromosomes of a single cell (4). The in vitro studies have been paralleled by in vivo studies showing loss of telomeric repeats with age in humans (5). Telomeric attrition is ascribed to the end replication problem, a phenomenon that is responsible for the loss of 50–100 bp of telomeric DNA per cell division. It is primarily the result of the inability of DNA polymerases to replicate the extreme 5' end of linear DNA molecules during double-stranded (ds) DNA replication(6).
With regard to function, telomeres act to protect the ends of chromosomes from degradation, rearrangements, loss of genetic information and from the ligation of DNA ends by repair enzymes (1). Telomeres may also act as so-called generation counters, keeping track of the number of divisions a cell has undergone and then triggering cellular senescence when a critical number of divisions has occurred(7). Hayflick et al. (8) over 30 y ago equated cellular senescence with normal cellular aging, and further evidence of the link between the two was established by telomere studies in patients suffering from premature aging syndromes (9,10). This is interesting in the context of canine breeds, given that we know differing dog breeds have relatively wide variations in natural life span.
Telomere length may be assessed by measuring the length of telomere restriction fragments (TRFs) in the cell population. TRFs are a measure of telomeric DNA and include a small portion of subtelomeric DNA resulting from digestion of high-molecular-weight genomic DNA with frequently cutting restriction enzymes that do not have recognition sites within the telomere sequence (11). The aim of this study was to investigate whether the reduction in TRFs with age predicted by the end replication problem is demonstrable in dogs from a number of different breeds and to evaluate whether interbreed differences exist in TRFs.
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MATERIALS AND METHODS |
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Peripheral blood mononuclear cell (PBMC) (3) derived DNA was obtained from EDTA and heparinized 5-mL blood samples taken from canine subjects using standard protocols. A total of 47 dogs were studied, ranging in age from <1 y to 13 y of age from three different breeds: Labrador Retriever (n = 22), Miniature Schnauzer (n = 17) and Beagle (n = 8). The protocol used for blood sampling the subjects complied with Home Office regulations for the Care and Use of Laboratory Animals.
Telomeric restriction fragment analysis
TRF analysis was performed by Southern blot hybridization using the Telomere Length Kit (BD PharMingen, UK). Briefly, high-molecular-weight genomic DNA was isolated from blood samples using the QIAamp DNA Blood Maxi Kit (Qiagen, UK) and verified by gel electrophoresis. DNA (3 µg per sample) was digested with an equal mixture of RsaI and HinfI enzymes at a concentration of 4 U enzyme mix/µg of DNA at 37°C for 16 h. DNA fragments were separated by 0.6% agarose gel electrophoresis at 160 V for between 2 and 3 h. Digested DNA was then transferred to a Hybond N+ nylon membrane (Amersham, Buckinghamshire, UK) and hybridized with a biotinylated (TTAGGG)7 probe at 55°C for 16 h. Membranes were washed in 2x SSC/0.01% SDS and subjected to chemiluminescence detection and exposed to autoradiography film for between 30 s and 10 min. Biotinylated DNA ladders and digested DNA with known mean telomeric repeat lengths were used as size markers. The mean TRF length was calculated by integrating the signal intensity above background over the entire TRF distribution as a function of TRF length using the formula: L = 
(OD1L1)/ (OD1), where OD1 and L1 are the signal intensity and TRF length, respectively, at position 1 on the gel image.
Statistical methods
Analysis of covariance was carried out on the data set to establish whether age (covariate) or breed (fixed effect) had a significant effect on TRF. In all tests, the significance level was set at the 5% level. Statistical analysis was aided by use of the SAS statistics program (SAS Institute, Cary, NC).
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RESULTS |
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Previous work with canine subjects has not shown any significant difference between mean TRFs of different breeds. An analysis of covariance on the effect of breed on mean TRF for the data presented here has shown that breed differences do exist (P = 0.006). This is interesting because, although these are three breeds that are not recognized as having significantly different life spans, the results point to the possibility of large interbreed variations in mean TRFs.
It is possible that variation in the restriction-enzyme cutting sites between breeds may contribute to the results reported above. However, work carried out in human cell lines estimating telomere length by a modified fluorescence in situ hybridization (FISH) protocol (3), which does not include any subtelomeric DNA, correlated significantly with results obtained using restriction enzymes and Southern blot (14). This work showed that subtelomeric DNA is of a uniform length between human individuals and, given the similarities between human and canine telomere biology, the same situation is considered to exist in the dog. Development of a modified FISH protocol for analysis of canine telomeres will be necessary to confirm this.
Because cellular senescence has been linked to aging of the organism, it follows that the genetics of cellular senescence may be involved in determining the maximum life span of the organism (7). Such study in human subjects has been limited by the very small number of individuals whose natural life span has been significantly reduced by their genetic inheritance, such as Werner’s syndrome patients (9). Using mice as an animal model for human progeria syndromes is of limited use because of the significant differences between murine and human telomere biology (13). However, selective breeding of canines has produced a relatively large pool of animals whose genetic inheritance has included a reduced life span. Given that telomere biology in the dog has been shown to be a good model for human tissues (2), research into the genetics of cellular senescence in dogs may then be of relevance to human progeria studies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by a grant from Waltham Centre for Pet Nutrition.
4 Abbreviations used: FISH, fluorescence in situ hybridization; PBMC, peripheral blood mononuclear cell; TRF, terminal restriction fragment.
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LITERATURE CITED |
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1. Buys, C. H. (2000) Telomeres, telomerase, and cancer. N. Engl. J. Med. 342:1282-1283.
2. Nasir, L., Devlin, P., McKevitt, T., Rutteman, G. & Argyle, D. (2001) Telomere lengths and telomerase activity in dog tissues: a potential model system to study human telomere and telomerase biology. Neoplasia 3:351-359.[Medline]
3. Zijlmans, J. M., Martens, U. M., Poon, S. S., Raap, A. K., Tanke, H. J., Ward, R. K. & Lansdorp, P. M. (1997) Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats. Proc. Natl. Acad. Sci. U.S.A. 94:7423-7428.
4. Harley, C. B., Futcher, A. B. & Greider, C. W. (1990) Telomeres shorten during ageing of human fibroblasts. Nature (London) 345:458-460.[Medline]
5. Lindsey, J., McGill, N. I., Lindsey, L. A., Green, D. K. & Cooke, H. J. (1991) In vivo loss of telomeric repeats with age in humans. Mutat. Res. 256:45-48.[Medline]
6. Blackburn, E. H. (1991) Structure and function of telomeres. Nature (London) 350:569-573.[Medline]
7. Berube, N. G., Smith, J. R. & Pereira-Smith, O. M. (1998) The genetics of cellular senescence. Am. J. Hum. Genet. 62:1015-1019.[Medline]
8. Matsumura, T., Zerrudo, Z. & Hayflick, L. (1979) Senescent human diploid cells in culture: survival, DNA synthesis and morphology. J. Gerontol. 34:328-334.
9. Norwood, T. H., Hoehn, H., Salk, D. & Martin, G. M. (1979) Cellular aging in Werner’s syndrome: a unique phenotype?. J. Invest. Dermatol. 73:92-96.[Medline]
10. Kruk, P. A., Rampino, N. J. & Bohr, V. A. (1995) DNA damage and repair in telomeres: relation to aging. Proc. Natl. Acad. Sci. U.S.A. 92:258-262.
11. Mehle, C., Ljungberg, B. & Roos, G. (1994) Telomere shortening in renal cell carcinoma. Cancer Res 54:236-241.
12. Campisi, J. (1997) The biology of replicative senescence. Eur. J. Cancer 33:703-709.
13. Kipling, D. & Cooke, H. J. (1990) Hypervariable ultra-long telomeres in mice. Nature (London) 347:400-402.[Medline]
14. Hultdin, M., Gronlund, E., Norrback, K. F., Eriksson-Lindstrom, E. & Roos, G. (1998) Telomere analysis by fluorescence in situ hybridization and flow cytometry. Nucleic Acids Res 26:3651-3656.
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