Recombinant D-AKAP2 Variant Construction and Functional Interac- tions with PKA Subunits. GST fusion constructs were made by fusing the 40 C-terminal amino acids of D-AKAP2 to the C terminus of GST as described by Huang et al. (13) and subcloned between the NdeI and BamHI sites of pRSET (Invitrogen). The constructs were transfected into BL21 cells and expressed for 6 h at 20°C. The cells were lysed in PBS with 5 mM BME and 0.1% Triton X-100. Three ¢ oat) pal microliters of supernatant were added to 200 l of this buffer and mea) pal MaatTa ( ane} Lg, 10 l of glutathione beads. After three washes, bovine RI (53 g, yp) pV AE.Ta ( wi |I ug, aeBM 20 M) and murine RII (2.4 g, 2 M) were added to the beads, m_pl. respectively, and the total volume was adjusted to 40 l. After incubating for 30 min at 4°C, the beads were washed three times, and separated in a 10% acrylamide gel.

which follows a standard normal distribution. Here pˆ and pˆ are Y O (cid:1)2 (cid:1)2 the allele frequency estimates and and are estimates of Y O measurement variability calculated from measurement replicates in the young and old pools, respectively. In this study, we made no attempt to correct for additional sources of variation or for multiple aaaient testing. Rather, SNPs were identified that had P values 0.05 among all measured SNPs, followed by a second, independent measurement of all significant SNPs based on three separate PCRs ofeachDNApool.Theresultsofthesecondroundofmeasurement were analyzed in a manner similar to the first round, and were compared for consistency. SNPs that showed statistically significant differences between young and old groups from pooled DNA analyses were individually genotyped for final validation.

Twenty-seven-residue peptides containing the two AKB domain variants of D-AKAP2 with a C-terminal cysteine were synthesized by SynPep (Dublin, CA). The peptides were HPLC purified and the molecular mass verified by mass spectrometry. Both peptides were labeled with tetramethylrhodamine-5-maleimide (Molecular Aimlaa ce Probes) at the cysteine residue and HPLC purified. RI and RII   “_1/3 were serially diluted into 10 mM Hepes 150 mM NaCl 3 mM wat\/0 EDTA 0.005% polysorbate 20 containing 10 nM fluorescently wowla t labeled peptide. For RII tests, 1 nM labeled peptide was used because the binding affinity to RII(cid:7) was greater than that to RI(cid:7)    and RII . The regulatory subunit concentrations were 1,000 0.5   vo) — ( UnIp, : pare a) — ( Met nM for RI , 500 0.1 nM for RII , and 100 0.02 nM for RII .

Estimates of allele frequencies using individual genotype were found by using the gene counting method. Comparisons allele frequencies as well as genotype frequencies between were carried out by using a (cid:6)2 test of independence.

The SNPs found to be associated with age were further analyzed for association with disease-related quantitative traits in the twin

Fluorescence anisotropy was monitored by using a Fluoromax-2 (Spex, Jobin Yvon Horiba) equipped with polarizers. The fluoro- phore was excited at 541 nm (5- to 10-nm bandpass) and emission was monitored at 575 nm (5- to 10-nm bandpass). For each protein, fluorescence anisotropy was monitored for three separate binding experiments. The data were averaged and fit to a 1:1 binding model by using the nonlinear regression application in GRAPHPAD PRISM version 3.00 (GraphPad Software).

AFYMYFOM OFHIAS

Subcellular Localization of D-AKAP2 Variants. The 30-aa mitochon- drial-anchoring domain of D-AKAP1 (14) was fused to the C- terminal 156 residues of mouse and human D-AKAP2, respectively, followed by a Flag-tag and subcloned into pcDNA4 (Invitrogen). n/d >/E The N terminus, including the dimerization docking (D D) do- p AADAE en  main of RI (residues 1–109) and full-length RII were each fused with GFP and cloned into pEGFPN1 (BD CLONTECH). Equal molar ratios of the Flag-tagged D-AKAP2 constructs and either aacTa-(   pr ACEy/I RII -GFP or RI -D D-GFP constructs were mixed and trans- —-1/2 fected into 10T(1 2) cells by using cytofectene (Bio-Rad). The AKB domain was detected by immuno-staining with monoclonal antibodies against the Flag-tag (Kodak) followed by rhodamine- conjugated secondary antibody (The Jackson Laboratory). The cells were imaged by using a Zeiss microscope equipped with a digital camera. Each channel was exposed for the same amount of time.

Fig. 1. Neighbor-joining tree of genetic distances among the four European- American discovery groups (YF, YM, OF, and OM) and DNA pools from three additionalethnicgroups.HI,HispanicAmerican;AF,AfricanAmerican;AS,Asian American;YF,youngfemale;YM,youngmale;OF,oldfemale;OM,oldmale.The lengths of branches are proportional to the pairwise genetic distances between pools.

Results

Identification of D-AKAP2 as a Candidate Gene. We identified 50 markers of the 6,500 tested markers that show a reproducibly significant allele frequency change between the two age groups in a<< at least one gender (P 0.05). The SNP that demonstrated the strongest association with age in both genders is located within the D-AKAP2 gene.

Genome-Wide SNP Analysis for Morbidity Gene Discovery. The