[32]. We identified four new polymorphic loci in the PARP-1 coding sequence among this sample of 18 indi- viduals. These variants were all nucleotide transitions, namely C T (exon 2), T C (exon 7), G A (exon 8) 402 1011 1215 and T C (exon 17). We did not observe any deletion, 2444 insertion or duplication events. Genotype distributions and frequencies obtained after sequence comparisons are summarised in Table 2. In the sample collection tested (n=18), genotypes corresponding to the transitions C T, G A and T C were in total linkage disequi- 402 1215 2444 librium (data not shown), whereas analysis of the fourth transition, T C, showed genotype combinations totally 1011 independent of the previous ones, strongly suggesting two independent original events. The data presented here are definite proof that the bases at positions 402, 1,011, 1,215 and 2,444 of PARP-1 coding sequence are genetic polymorphisms. Moreover, as depicted in Fig. 1, we show that the first three nucleotide transitions located in the coding region of the DNA binding domain of PARP do not lead to amino acid exchanges, whereas the T C 2444 leads to a valine-to-alanine (V762A) change in codon 762. This amino acid position is located in the catalytic domain of the enzyme.

Results

In our previous study [15], EBV-immortalised cell lines from centenarian and control individuals have been char- acterised with respect to PARP activity. Here, we select- ed and analysed 18 of these samples (nine centenarians and nine controls) in order to search for polymorphisms in the PARP-1 coding region which might be responsible for the higher specific PARP activity we have previously observed among cell lines from centenarians. Polyade- nylated mRNA was prepared from these cell lines and reverse transcribed as described in Materials and meth- ods. The complete 3,320-bp sequence of PARP-1 cDNA was then PCR amplified with eight pairs of primer, re- sulting in eight overlapping PCR products. Primer pairs were designed to avoid any non-specific amplification, including amplification of the PARP pseudogene loci as- signed to 13q34 and 14q24 [31]. The PARP-1 coding se- quence defined by Cherney et al. [32] comprises nucleo- tide positions 160 to 3,204, and primers we designed spanned this region, from nucleotide position 101 to 3,419. Using the Sequence Navigator software (Applied Biosystems), we analysed each one of these fragments, then reconstructed the total cDNA sequence for each one of the 18 individuals and compared it to the PARP-1 cDNA sequence previously described by Cherney et al.

Having discovered these polymorphisms by systemat- ic sequencing of the complete PARP-1 open reading frame, we then studied the locus T C in a larger num- 2444 ber of individuals, to evaluate the potential impact of the V762A change on human longevity, as well as on the high inter-individual variability of specific PARP activi- ty that we had previously observed [15]. Furthermore, al- though T C is silent at the protein level, it appeared to 1011 be independent of the other mutational events and we therefore decided to analyse this locus as well. In order to do this we developed an allele-specific PCR amplifi- cation, in conjunction with a fluorescent read-out system (for details see Materials and methods). To illustrate the methodology used, Fig. 2 shows an example of electrop- herograms displaying curves for fluorescently labelled PCR products. In this approach, two homologous, allele- specific, labelled primers for the locus were used, to-

Table 2 Frequency distribution of genotypes at the PARP-1 loci C T, T C, G A and T C, in a sample of 18 individuals. 402 1011 1215 2444 Frequencies are expressed as a percentage in parentheses

Genotypes Loci C T T C G A T C 402 1011 1215 2444 TT 0 (0%) 5 (27.8%) – 11 (61.1%) CT 7 (38.9%) 10 (55.5%) – 7 (38.9%) CC 11 (61.1%) 3 (16.7%) – 0 (0%) GG – – 11 (61.1%) – GC – – 7 (38.9%) – CC – – 0 (0%) –

Exon 2 Exon 7 Exon 8Ben                De | Acres [ Kss2 Vre2A + >< ie > DNA binding domain Automodification Catalytic domaindomain

1800 2000 2200 180060 Heterozygous T/C0i) 7 B:17v7M1 / T GB vG:17v70M1/ c1200os Homozygous T/T0EM) 18 B:186F72M1 / T 18 G: 18°F72M1 / C42002100 Homozygous C/C(I 198: 19-v45m1 / T (BM) 9G: 19-va5m1 1c