Two luciferase reporter plasmids encompassing of ATM promoter were constructed. DNA samples from human homozygous with either C > T or T > C were PCR amplified and cloned into pGL3-basci vector (Promega). Then, Hela cells were seeded at 8   103 per well in 24-well plates and cotransfected with 0.6 mg reporter construct and 15 ng hRL-TK renilla luciferase plasmid using 3.3 ml of ExGene500reagentin500 ml media.All transfectionswereconductedintriplicate. After 24 h of transfection, luciferase activity was analyzed according to manufacturer’s protocol for Dual Luciferase1 Assay (Promega). The pGL3-Basic was used as controls, and Renilla luciferase was used as an internal control to normalizethefireflyluciferaseactivity.Theexperimentwasperformedintriplicate.

2.2. Denaturing high-performance liquid chromatography (DHPLC)

Three pairs of PCR primers were used to create three overlapping products (226 bp, 240 bp or 287 bp fragment) for DHPLC analysis. PCR reaction was carried out in 20 ml reaction volumes containing 200 ng of genomic DNA, 5 pmol of each primer,1(cid:4)reactionbufferwithoutMgCl ,2.5 mMMgCl ,0.2 mMofeachdNTP,and 2 2 1.25 U Taq DNA polymerase. The PCR conditions were as follows: an initial denaturation step at 94 8C for 3 min, followed by 32 cycles of denaturation at 94 8C for 30 s, annealing for 30 s at 56–58 8C according to the primers used (Table 1), and elongation for 30 s at 72 8C, with a final extension of 5 min at 72 8C.

2.8. Statistical analysis

x2orFisher’sexacttest(whereanexpectedcellcountwas<5)wasconductedto compare the genotypic and allelic frequency between LLIs and controls, and also to assess deviation of the genotypic frequencies from those expected under Hardy– Weinbergequilibrium.One-wayANOVAwascarriedouttoexaminethedifferences in ATM mRNA expression. Student’s test was used to examine the difference in luciferase reporter gene expression. All statistical analysis was done by using SPSS software (version 11.5, SPSS, Inc., Chicago, IL, USA). P < 0.05 was used as the criterion of statistical significance. All statistical tests were two sided.

Detection of DNA sequence variation with DHPLC was carried out on a Transgenomic Wave DNA Fragment Analysis System (Transgenomic Inc., Omaha, NE, USA). The DNA molecules eluted from the column were detected by scanning with a UV detector at 260 nm. Prior to DHPLC analysis, PCR products were denatured by heating at 96 8C for 5 min and then gradually cooling down to 25 8C over a period of 30 min. Then, aliquots of 5 ml PCR product were injected into the mobile phase (Buffer A, 0.1 M TEAA, pH 7.0; Buffer B, 0.1 M TEAA/25% acetonitrile) using a flow rate of 0.9 ml/min. The start- and end-points of the gradient were adjusted according to the size of the PCR products. The melting points were 62.5 8C, 65 8C and 64.1 8C for 226 bp, 240 bp and 287 bp respectively.

3. Results

To maximize the opportunity to screen genetic variations in promoter of ATM, we deployed denaturing high-performance liquid chromatography (DHPLC), a highly sensitive method for detecting DNA sequence variants (Liu et al., 1998; Wolford et al., 2000), to analyze PCR product of genomic DNA of ATM promoter from 5 long-lived individuals with familial history of longevity and 5 matched controls. Three pairs of primer were used to amplify three overlapping DNA fragments from sequences of the known ATM promoter for DHPLC analysis (Fig. 1A). Except for the third amplicon of the ATM promoter, both amplicons 1 and 2 did not show distinct elution profiles between LLIs and controls (Fig. 1B), so those results suggested that genetic variations might exist in the third amplicon. Then, we examined the distribution of variance in both LLIs and controls (156 LLIs and 53 controls) using DHPLC. The long-lived individuals (LLIs) displayed higher frequency of variance than the controls (51.9% versus 24.5%, data not shown). To validate genetic variations, we randomly selected 41 DNA samples from 29 variant and 12 invariant subjects for sequencing.

2.3. Sequencing

DNA fragments amplified from LLIs and controls were sequenced in both the forward and reverse directions using an ABI PRISM 3730 automatic sequencer.

2.4. Restriction fragment length polymorphism (RFLP)

The 5 ml PCR products of the targeted DNA fragments were digested at 37 8C for at least 3 h with Sac II (New England Biotechnologies, USA), then electrophoresed onan8%polyacrylamidegelandstainedwithsilvernitrate.Theconcurrencerateof direct sequencing and Sac II restriction digestion was 100%, and for quality control, the positive and negative controls (with no DNA) were used.

2.5. RT-PCR

Total RNA was isolated from peripheral blood mononuclear cells of 42 old individuals (33 men and 9 women, from 70 to 80 years, mean age 77.3, SD   3.72) using the QIAamp RNA Blood Mini kit. One microgram of total RNA per sample was reversely transcribed into cDNA according to the manufacturer’s instructions (PrimeScriptTM RT-PCR Kit TaKaRa Japan). cDNA was PCR amplified using the forward, 50-GCT CCT AAT CCA CCT CAT-30, and reverse, 50-CTC CTC CTA AGC CAC TTT-30. The amplified product was 256 bp. PCR cycles were 94 8C for 3 min, 30 cycles at 94 8C for 30 s, 51 8C for 30 s and 72 8C for 30 s, and the final extension was 72 8C for 5 min. PCR products were analyzed on 2% agarose gel and visualized by staining with ethidium bromide. Semi-quantitative PCR wasperformed asa limited dilutionoftemplates, and theanalysis ofamplified products wasperformedbyanimage analyzer(ChemiDocTM XRS, BIO-RAD, USA). b-Actin was used as internal control (Giacconi et al., 2004).

To validate genetic variations, we randomly selected 41 DNA samples from 29 variant and 12 invariant subjects for sequencing. One single nucleotide polymorphism (SNP, rs189037) in the third amplicon of the ATM promoter (Fig. 1C) was identified. The SNP contains CC, CT, and TT genotypes in the tested human individuals.

2.6. Electrophoretic mobility shift assay (EMSA)