Materials and methods

sequencing in a 310 DNA sequencer with a BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems).

Population samples

VNTR polymorphism typing

the subjects lived in Calabria (southern Italy) and their T origin in the area was ascertained up to the grandparents generation (interview). The sample consisted of two subsamples: sample 1 was made up of 20- to 80-year-old subjects (median age 59 years); sample 2 was made up of 90- to 106-year-old subjects (median age 102 years). Sample 1 comprised 703 subjects (312 males and 391 females); sample 2 comprised 242 subjects (86 males and 156 females). For collecting sample 1, an appropriate campaign was addressed to Calabria University students and staff, as well as to people who attended the University for the Elderly or used local thermal baths. For collecting sample 2, subjects older than 90 years were identified by consulting the population registers of the Municipalities of Calabria, contacted by phone, and then visited.

Both Gene Scan DNA fragment analysis (TAMRA 500 internal size standard) in a 310 DNA sequencer (PE Applied Biosystems) and electrophoresis in a 1.5% agarose gel (Marker Step Ladder 50 bp, Sigma, as molecular weight marker) were used on PCR-amplified products for VNTR genotyping.

Allele 2 was analyzed by DNA sequencing in all the male carriers of the allele (10 homozygous plus 74 heterozygous subjects). After PCR amplification (see VNTR polymor- phism identification) and electrophoresis, the DNA fragment containing two repeats was recovered from agarose gel in TAE buffer 1(cid:1) (0.04 M Tris acetate, 0.001 M EDTA) by using the QIAquick gel extraction kit (Qiagen). The sequencing reactions were automatically carried out with the forward primer 5V-TCTTGCTGCATGTGGTTG-3V (5V position 12,275 nt).

After a detailed explanation of the aims of the research, the subjects who agreed to participate donated a blood sample for routine laboratory analyses and DNA prepara- tion; furthermore, people older than 60 years underwent a complete clinical and geriatric assessment. Subjects free of clinically overt pathologies and having blood and biochem- ical parameters in the normal age- and sex-specific range were enrolled in the study. In particular, the subjects enclosed in sample 2 belonged to health categories A and B previously described [16].

Plasmid construction

The pGL3 promoter vector (Promega) containing an SV40 promoter was used in the construction of reporter plasmids for the analysis of allele-specific regulatory functions. Preliminarily, to discriminate between alleles having the same length but different nucleotide sequence (T/C at position 63 in the second repeat) the common alleles (one to four repeats) were amplified and automati- cally sequenced from subjects who were homozygous for the 1–4 length-alleles. The forward primer 5V-CTCQ CCCGGGTGGGAACTCCCTGGAGGTT-3V (5V position 12,324 nt referring to www.ncbi.nlm.nih.gov/omim; MIM 604481, contig NT_ 035113) and the reverse primer 5V- CTCCCCGGGTGGCACCAGCCCTGGAAG-3Vwere used to have DNA fragments containing the VNTR sequence only (the italicized sequence was added to include the SmaI site for cloning). The PCR conditions were the same as described above. The PCR products of subjects having (CC) or (TT) homozygous genotype were purified by agarose gel in TAE buffer by using the QIAquick gel extraction kit (Qiagen) and digested with SmaI restriction enzyme. After purification by phenol extraction, the fragments were cloned into the SmaI-digested and dephos- phorylated pGL3 vector, upstream of the Luc-promoter transcriptional unit. The sequence of each insert (and its orientation in the reporter vector) was confirmed by restriction mapping and automated DNA sequencing. Cloned vectors were purified by Qiagen Plasmid Maxi Kit and used for subsequent experiments.

All the subjects analyzed in this study have given written informed consent for genetic studies on aging carried out by the present research group.

DNA samples

Six milliliters of blood was drawn from each subject. Plasma/sera were used for routine laboratory analyses, while DNA was extracted from blood buffy coats following standard procedures.

PCR amplification of genomic DNA was carried out in 25-Al final volume containing 100 ng of DNA; 0.08 AM each forward primer, (5V position 12,030 nt referring to www.ncbi.nlm.nih.gov/ _ omim; MIM 604481; contig NT 035113), and reverse primer, 5V-CATTCACCTTCCCAAAGTGG-3V; 200 AM dNTP; 0.3 U Finnzymes Taq DNA polymerase; 1.5 mM MgCl ; and 1(cid:1) thermophilic buffer. The following 2 thermal cycling conditions were used: 1 cycle at 958C for 45 s and 25 cycles of denaturation for 1 min at 958C, annealing for 1 min at 558C, and elongation for 1 min at 728C. An extra step was performed at 728C for 5 min. PCR-amplified fragments were analyzed by automated

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