BIOL2609_L20_2007 - Molecular Ecology ECOL 2007 Lecture 20:...

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Unformatted text preview: Molecular Ecology ECOL 2007 Lecture 20: Population structure: Telomeres as age markers Age structure of populations • As organisms age, their fertility and survival is affected • A knowledge of age structure in a population is therefore important, particularly in understanding: – Conservation implications – Effects of exposure to pollutants But…. • Field ecologists often work with organisms for which there is no prior history • It is sometimes difficult to estimate age from phenotype, especially with ‘remote’ observations Telomeres • Telomeres are short tandem repeat sequences found at the linear ends of all eukaryotic chromosomes • They comprise repeated units of: TTAGGG (CCCTAA) • This sequence is highly conserved among vertebrates, in which most studies of telomeres to date have been carried out • Telomeres function to stabilize the integrity of chromosome ends: – During duplication – Broken chromosomes (inhibit fusion) • In germ cells and carcinomas telomere length is maintained by an enzyme called telomerase – a ribonuclear protein that synthesizes telomere sequences to replace lost bases • In somatic cells, telomeres shorten by 50200 nucleotides per cell division • This is because DNA polymerase cannot completely replicate the 3’ end of linear DNA • As a result telomeres shorten with each cell division, and this is linked to cellular ageing • It is therefore suggested that telomere length can serve as an age marker – ‘biological clock’ hypothesis • This would be potentially advantagous over traditional methods – Ie: dentition, otoliths etc • Telomere assays can be performed on tissue or blood samples taken non-destructively • High-throughput analysis would allow large numbers of individuals to be studied How do we measure telomeres? • The most widely used technique is based upon hybridization of an oligonucleotide probe (TTAGGG)4 to restriction digested genomic DNA • The hybridized fragments are then visualized by electrophoresis – Telomere length – estimated by comparison to a size marker – Telomere amount – estimated from band intensity • This can be performed by membrane hybridization, or solution hybridization • We will refer to all telomere assays as TALA (telomere amount and length assays) – Nb: this term was originally used to describe only the solution-hybridization method (Gan et al 2001) – Here the term includes membrane and solution hybridization, FISH and Q-PCR methods Non-destructive sampling Genomic DNA extraction Southern blot, membranehybridization Solutionhybridization, electrophoresis Restriction-digest (make sure no telomere recognition sites) A) Solutionhybridization B) Membranehybridization M = marker, 1-4 = human cancer cell lines • The digested and hybridized genomic DNA appears as a broad smear – why? – Different chromosomes have different restriction sites relative to the telomeric end Mean telomere length = 7495 +/-563 bp - mean telomere length = position where 50% of signal occurs - determined by image analysis Telomere amount -estimated by comparison of band intensity to that of the highest DNA concentration used - this gives a linear relationship • So how do you know your oligonucleotide has bound to chromosome-end telomeres and not some interstitial sequence? – Remove the G-strand overhang of telomeres in your template (using a nuclease enzyme) – (CCCTAA)4 oligo’s will only bind to the terminal G overhang – The telomeres are then ‘blunt’ ended – Hybridization should not be observed Lanes 2-4, 5-7, 8-10 are triplicates of three individuals The 3rd lane for each individual (4,7,10) contain samples pre-treated with mung bean nuclease to remove the G overhang (samples = zebra finch, Haussmann & Vleck 2002) Does it work? Case study – zebra finches Haussmann & Vleck, 2002 • Captive populations of the zebra finch (Taeniopygia guttata) – Known age structure • nondestructive blood samples from: – Juvenile (n = 11) < 4 months – Young (n = 9) 11-15 months – Old (n = 11) >18 months • Lifespan = 5-7 years Southern blot (membrane hybridization) of zebra finch individuals Lanes 1,12,24 = standards Specificity of oligo’s tested with nucleasetreated controls • Mean telomere length of age-classes: – Juvenile 8,656 (+/-104) – Young 8,448 (+/-115) – Old 7,979 (+/-104) • Mean telomere length decreases with age – Rate = ~516 (+/-94) yr-1 Linear relationship in age range 424 months No sex-related differences • The study included individuals up to age 24 months, but zebra finches can live up to 7 years • So, we do not know if telomere-age relationship is linear over the whole life span • Also unknown is if telomeres from cells in different tissues age at different rates Alternative TALA appoaches Alternative methods: • Flourescent in situ hybridization (FISH) – Hybridization of oligo’s to intact chromosomes – Visualization by flourescence microscopy – Area of stain vs area of chromosome • Real time quantitative PCR – PCR primers specific to telomeres – Compare PCR copy number to that dilution series of a known standard FISH using telomere-specific oligo’s TRF = terminal restriction fragment, measured by TALA T/S ratio = telomere:single copy (control) gene ratio Telomere measurement by Q-PCR Summary & directed reading • Telomeres have been demonstrated to show a linear relationship to age for certain cell types within certain age ranges of some organisms • More work is needed to develop standards for telomere measurement and to establish the validity of the relationship • Direcetd reading: Oecologia 130: 325-328 (2002) ...
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This note was uploaded on 03/19/2010 for the course DEB BIOL2609 taught by Professor Drstephenb.pointing during the Spring '10 term at HKU.

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