Origin and culture conditions of cell lines used
For DNA end binding assays biopsies was taken from the skin of each of the following animals: mouse, Mexican Free Tailed bat, rabbit, Big Brown bat, cow and cat to produce primary fibroblast cultures. For the dog fibroblasts cultures were derived from a Beagle and a Rottweiler. For human, DNA binding activity was determined on a fibroblast cell line derived from a biopsy taken from an adult man but also on WI 38 lung embryo fibroblasts and on HeLa cells (a commonly used cell line derived from a cervical cancer). For Rhesus monkey, horse and gorilla skin fibroblast cultures were obtained from the Coriell Institute for Medical Research (Camden, NJ). For Chinese Hamster, we used CHO cells, a cell line derived from ovary cells.
For telomere length assays the following skin fibroblast cultures were used: little brown bat (2 cultures from 2 different wild caught individuals), mouse (2 cultures, one from a wild mouse from Pennsylvania and one from a wild mouse from Idaho), rat (2 cultures from 2 laboratory animals), rabbit (2 cultures from a laboratory animal), cat (1 line from a house cat), Rhesus monkey (2 cultures from the Coriell Institute), dog (2 cultures from a laboratory beagle), human (2 cultures from adult individuals), horse (2 cultures from the Coriell Institute), cow (2 cultures from a biopsy obtained from a New Jersey slaughter house).
The exact age of the majority of individuals used was known. It ranged between young and early middle age but it was generally young adult. The two mice and all the bats, which were all caught in the wild, were estimated to be young adults. Skin fibroblasts were grown according to our standard procedures (Cristofalo et al., 1980
) with the exception of the addition of antibiotics and antimycotic (100 IU/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin B). Bats skin samples were obtained from Dr Anja Brunet-Rossinni. Rottweiler skin samples were obtained from Dr David J. Waters and Deborah Schlittler (Purdue University, West Lafayette, IN).
Longevity and body mass data
Maximum longevity and adult body mass for the species analyzed were obtained from Dr Steven N. Austad’s personal database (The Sam and Ann Barshop Institute for Longevity and Aging Studies, San Antonio, TX) and from the online Longevity Records of the Max Planck Institute for Demographic Research (Rostock, Germany, http://www.demogr.mpg.de/
). Both databases are compiled from fully authenticated sources. Note that the reported maximum human longevity is 122.5 years. In this study, human longevity is adjusted to 90 years to account for the fact that, for the others species, only small cohorts are used to determine maximum longevity, and 90 years is an estimate for a similar sized random sample of humans.
Telomere restriction fragment (TRF) length analysis
TRF analysis was performed essentially as previously described (Steinert et al., 2002
). Briefly, 1 μg of genomic DNA was digested with a cocktail of AluI, HaeIII, HhaI, HinfI, MspI and RsaI, separated on a 0.5% agarose gel for 27 hours at 1 V/cm (or for PFGE at 6 v/cm with a ramped pulse from 1 to 15 secs for 20 hours), and the gel was then dried and probed using the 32
P-end labeled telomere repeat oligonucleotide (CCCTAA)4
. Complete DNA digestion was confirmed by ethidium staining of DNA run for four hours into the gel. The washed gel was visualized with a Molecular Dynamics Phosphoimager. Mean telomere length was calculated as the weighted average (ODi
), where ODi
is the background-corrected intensity of telomere signal in interval i and Li
is the average length of telomeres in interval i (each interval equal to a pixel), thus normalizing for the stronger signal emitted by longer telomeres. End-labeled full-length and HindIII-digested lambda DNA fragments were used as markers. Signals between 4 and 40 kb for standard gels (and between 5 and 65 kb for PFGE gels) were used for calculations. For little brown bat measurements, only telomere signals that were detected under non-denaturing conditions were used to estimate mean telomere length.
Fluorescence in situ hybridization
For telomere repeat fluorescence in situ
hybridization (FISH), a Cy3-conjugated peptide nucleic was used. Slide preparation, acid probe (CCCTAA)3
hybridization, and detection were performed as described in Unit 18.4, Current Protocols in Cell Biology Online, 2006 [www.interscience.wiley.com
DNA end binding activity assay
Nuclear protein extracts were prepared from nuclei using the method of (Dignam et al., 1983
) using 0.2% (v/v) NP-40 to lyse the cells. Extraction buffer contained 5mM MgCl2
to prevent clumping of the nuclei. Protein concentration of the nuclear extracts was determined using the Bradford method (Bio-Rad laboratories, Hercules, CA) and bovine serum albumin as a standard. DNA end binding activity was determined using our established protocol (Getts et al., 1994
), briefly, for each species increasing amounts of nuclear extracts (0.01–20 μg) were incubated with 1ng of a 144bp 32
P-labeled DNA probe for 15min at room temperature in the presence of 1μg of supercoiled circular pUC18 plasmid (1000 fold ratio of competitor to probe) in a final volume of 20μl in binding buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 150 mM NaCl, 5 mM DTT, 200μM PMSF, 2.5 mg/ml leupeptin, 1 mg/ml pepstatin A, and 10% (v/v) glycerol). The 144 bp probe was obtained from a Pvu II and Eco-RI digest of the pUC18 plasmid. Since the probe was derived from pUC18 plasmid, those proteins that bind to internal DNA sequences will be bound by the excess circular plasmid and will not bind to the probe. Also, since the plasmid does not contain DNA ends, only proteins that bind to DNA ends will bind to the probe. The reaction mixtures were electrophoretically separated in 5% polyacrylamide gels. The gels are then dried and the fraction of probe bound at each protein concentration is determined from phosphorimager scans of DNA end binding patterns. To estimate DNA binding activity, for each species we calculated the amount of protein required to bind to 50% of the probe.
Nuclear extracts prepared as reported above were size-fractionated on 5–20% gradient SDS-polyacrylamide gels (Cambrex Biopharmaceuticals, Baltimore, MD) and electrotransferred on nitrocellulose membranes (Schleicher & Schuell BioScience Inc, Keene, NH) using a BioRad mini Protean electrophoresis system. Abundance of the proteins of interest was assayed using antibodies that react with sequences that are 100% conserved across mouse, cow and human: DNA dependent protein kinase catalytic subunit (cat# IMG-534) and Ku80 (cat# IMG-4174) antibodies were purchased from Imgenex (San Diego, Ca); they are both rabbit polyclonal antibody. For DNA-PKcs the antibody was raised against a synthetic peptide corresponding to amino acids 4118–4128 (GRTWEGWEPWM
) of human DNA-PKcs. For Ku80 the antibody was raised against a synthetic peptide corresponding to amino acids 323–338 (FSKVDEEQMKYKSEGK)
of the 80 kDa human Ku protein. The DNA Ligase IV antibody was made against a peptide (CELQEENQYLI)
at the carboxyl terminus of the Human DNA Ligase IV protein (Bryans et al., 1999
). Antibodies against serum response factor (SRF, G-20 cat# sc-335) and histone H3 (N-20 cat# sc-8653) were from Santa Cruz Biotechnology (Santa Cruz, CA). SRF G-20 is a polyclonal antibody raised against a peptide mapping within the C-terminus of SRF of human origin; SRF is more then 95% conserved across mouse, cow and human. Histone H3 N20 is an affinity purified goat polyclonal antibody raised against a peptide mapping at the N-terminus of histone H3 of human origin; the N-terminus of histone H3 is 100% conserved across mouse, cow and human. To control for loading, all membranes were stained with 0.5% w/v Ponceau S prior to antibody hybridization and gels were stained with Coomassie Brilliant Blue G-250 immediately after transfer.
Ten μg of the above cow and human nuclear protein extracts were separated in adjacent lanes on a 10% gel by SDS-PAGE. Proteins present in a series of horizontal 1mm wide gel samples across the sample lanes were digested with trypsin and the resulting peptides analyzed by high resolution tandem time of flight MALDI-(TOF/TOF) mass spectrometry. Peptides similarities were searched based on the human sequences information. Then identified proteins were arrayed in descending order of abundance based on their total peptide ion current. Thus the protein whose peptides produced the highest total ion current is number 1. Total ion current is a function of a protein’s abundance, the theoretical number of trypsin cleavable peptide fragments in its amino acid sequence, and their probability of cleavage. Relative abundance of human and cow DNA-PKcs, Ku80 and Ku70 was estimated by comparing total ion current and position in a list of proteins ordered by total ion current ().
DNA double strand break repair
Cells growing exponentially as monolayers in flasks were irradiated on ice using a Sheppard irradiator at approximately 12.1 Gy/min. and then incubated varying times at 37°C. Cells were removed from the flasks, washed with phosphate-buffered saline (PBS) at 0°C and resuspended at a density of 0.5–1.0 × 107
cells/ml in PBS containing 1.0% low melting point agarose (Incert, FMC), and agarose plugs were prepared by casing into 5 mm × 6mm × 1.0 mm inserts using a BRL mold cooled to 0–4°C. Cells in agarose plugs were placed in 50°C lysis solution containing; 0.5 M EDTA pH 7.9 (Sigma), 1.0% Sarkosyl (Sigma), 1.0 mg/ml proteinase K (Boeringer Manheim). After 18–24 h of digestion, the plugs were dialyzed twice against 10 volumes of TE (10 mM Tris, pH 7.8, 1 mM EDTA), and the RNA hydrolyzed by digestion in 1 volume of TE with 0.1 mg/ml RNAse A (Sigma) for 2 h at 37°C. The agarose plugs were subjected to AFIGE electrophoresis as described previously (Denko et al., 1989
; Stamato et al., 1993
; Stamato et al., 1990
), the gel stained with ethidium bromide, photographed, and agarose sections containing the DNA in the lane and the DNA in the plug were excised. The percentage of DNA released from the plug into the lane was determined by counting the radioactivity in agarose sections.
Statistical and phylogenetic analysis
Statistical analyses were performed using the software GraphPad InSat 3 and non linear regression analysis was performed with Graph Pad Prism 4 software (both from GraphPad Software, Inc. San Diego, CA). The phylogeny used for the phylogenetically independent comparisons was derived from Adkins and Murphy (Adkins et al., 2003
; Murphy et al., 2001
). Phylogenetic independent comparisons were performed using the “Comparative Analysis by Independent Contrasts” (CAIC) statistical program of Purvis and Rambault (Purvis et al., 1995