Materials and methods

Subjects and DNA samples

Centenarians were recruited with personal consent, through the “Chronos” Project at the Fondation Jean Dausset-Centre d’Etude du Polymorphisme Humain (CEPH), from all regions of France. They were Caucasians, born in France and were at least 99 years old on the day of blood collection. The mean age of the centenari- ans was 101 years. Controls were selected from unrelated Cauca- sian individuals who were born in France and matched with cente- narians for birthplace. The mean age of controls was 51 years. The proportion of women was 88.9% in the total sample tested (n=648) and 83.33% in the centenarian and control population re- spectively, reflecting the differential longevity of women and men in France. Peripheral blood leukocytes were routinely isolated by Ficoll gradient centrifugation, EBV-immortalised by standard methods and cryopreserved in liquid nitrogen until used. DNA was extracted from EBV-immortalised cell lines by standard meth- ods [30], resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH7.6) and kept frozen until used as described below.

Allele-specific amplification of the PARP-1 gene at the T1011C and T2444C loci

We analysed 648 DNA samples (324 centenarians and 324 con- trols), matched for birthplace and available in the DNA bank at Fondation Jean Dausset-CEPH, by allele-specific amplification of two regions comprising the T C and T C transitions, respec- 1011 2444 tively. Each site was amplified in the presence of three primers, two of which differed only in their 3′-most nucleotide (marked in bold). T C locus: “PexC” –GCT CCT CCA GGC CAA CGC; 2444 “PexT” –GCT CCT CCA GGC CAA CGT; “Pex19” –ACC CTG TTA CCT TAA TGT CAG TTT T; T C locus: “Pex6T” –TGC 1011 AGA TCT TGG ACC GAG TAG CT; “Pex6C” –TGC AGA TCT TGG ACC GAG TAG CC; and “P2cb” –GGT TGG GTG TCT GTG TCT TG. Each one of the two “competing”, allele-specific primers for the nucleotide variation locus (i.e. PexT/PexC and Pex6T/Pex6C for T C and T C respectively) was modified at 2444 1011 the 5′ end with a fluorescent dye (C6-FAM, TET; Applied Biosys- tems dye/Eurogentec), in such a way that genotypes could easily be distinguished. PCR conditions were the following: 40 ng ge- nomic DNA, 0.2 µM each primer, 4 mM MgCl , 10 mM Tris-HCl 2 pH8.3, 50 mM KCl, 200µM each dNTP and 2.5 U Stoffel frag- ment (Perkin Elmer) in a final volume of 50 µl. Samples were de- natured for 5 min at 97°C and subjected to 30 cycles consisting of 30 s at 95°C, 30 s at 64°C (T C) or 65°C (T C), 30 s at 72°C. 2444 1011 PCR products (expected sizes 148 bp and 159 bp for T C and 2444 T C, respectively) were analysed on an Applied Biosystems au- 1011 tomatic sequencer A377. Sizing of the products and collection of numbers of alleles were performed as described by the manufac- turer (Genescan User’s manual, Applied Biosystems). Statistical analysis of results was done with the Chi-square test.

Sequence analysis of PARP-1 cDNA

EBV-immortalised cell lines from 18 individuals (nine centenari- ans, nine controls) were selected according to the level of PARP activity previously measured [15] (i.e. either extremely high or low PARP activity, respectively). Polyadenylated RNA was ex- tracted from up to 107 cells with the QuickPrep micro mRNA puri- fication kit (Pharmacia Biotech), resuspended in RNAse-free wa- ter and used as a template for reverse transcription with the Ready-to-go T-primed first strand kit (Pharmacia Biotech). Eight pairs of primers were designed (see Table 1) allowing the PCR- based amplification of eight overlapping stretches of DNA (rang- ing in size from 311 bp to 564 bp) spanning the complete 3,320 bp PARP-1 cDNA sequence (GenBank accession number M32721). Amplification products were purified on a Microcon micro-con- centrator (Amicon) and resuspended in water. Both strands of each purified PCR product were sequenced with the Taq DyeDeoxy ter- minator cycle sequencing kit (Applied Biosystems). The same primers designed for PCR amplification of cDNAs were used for sequence reactions. Reactions consisted of 25 cycles under the fol- lowing conditions: 96°C for 10 s; 5 s at the hybridisation tempera-

are indicated. The 5′- and 3′-positions of the primer′′ were given with respect to the PARP-1 cDNA sequence described by Cherney et al. [32] (GenBank accession no. M32721).

Table 1 Oligonucleotides for PCR and sequence analysis of PARP-1 cDNA. Sequence, size, location on cDNA sequence, ex- pected sizes of amplification products and annealing temperatures

Sequence 5′ to 3′ Size (b) 5′-position 3′-position Product size Annealing on cDNA on cDNA (bp) temperature sequence sequence (°C) AAG CGT GTT TCT AGG TCG TG 20 101 120 564 60 CCT CCC TGT TCT TGA CAA AG 20 664 645 GCC ACA GCT AGG CAT GAT TG 20 603 622 311 52 GCT CGT CCT TGA TGT TCC AG 20 913 894 TGA GGT GGA TGG AGT GGA TG 20 792 811 356 58 GGTTGGGTGTCTGTGTCTTG 20 1147 1128 GAG CGA TGC CTA TTA CTG CAC 21 1074 1094 505 60 GAA CAA CTC CTG AAG GCT CTT G 22 1578 1557 GAG TTG TGT CTG AGG ACT TC 20 1514 1533 534 60 TTT TGG AGT GCC AAG CGT TC 20 2047 2028 TGG AAC AGA TGC CGT CCA AG 20 1961 1980 555 60 TGC TAT CAT CAG ACC CTC CC 20 2515 2496 CCT GAA CAA TGC AGA CAG TG 20 2412 2431 516 52 TCT CCC AAC AGG ATT AAG CC 20 2927 2908 CCA ACT ACT ACC ATA CGT CTC 21 2873 2893 547 52 CAA CCC CAA AAC AAC CCC TC 20 3419 3400