scientific research. Longevity is widely accepted as a complex trait whose manifestation is determined by genes, environment, and chance.1 Family studies esti- mate that heritability in human longevity is generally in the range of 20% to 30%,2 which leads to the possibil- ity that several genes with smaller effects account for this complex trait. To search for genes that influence longev- ity is a major avenue to reveal its essence and predict the people accordingly. Recently, Christensen et al3 have written an excellent review on the genetic determinants of human longevity and pointed out that variants in the apolipoprotein E gene had been found to be consistently associated with survival and longevity. However, these variants with a modest effect on lifespan cannot fully ac- count for the longevity prediction, which suggests that some biologic mechanisms implicated in longevity are still unknown. The physiologic features of aging, such as a progressive accumulation of molecular damage and an attenuation of the cellular defense mechanisms, have now shifted the focus of longevity association study to the pathways of stress response genes, espe- cially the heat shock protein 70 (Hsp70) family genes.4

METHODS

Study population. This study was reviewed and ap- proved by the local bioethical committee, and informed written consent was obtained from each subject. A total of 191 unrelated naturally long-lived Uygur subjects (131 men and 60 women) was recruited in this study, in which 52 individuals were more than 100 years old and 139 individuals were within 90–99 years old. Details of this study population have been published previ- ously.12 In brief, the age of the long-lived people was determined based on the following 5 standards:

pany.

The

generations kin relation as learned through interviewing villages and families one by one.

Their experience of the

great historical events, including the first pandemic plague in

, the sec- ond occurrence of the plague during

–

, the war against Hui ethnicity, and the land reform in

We established their ages on the following hypotheses: Generally, a person had not any mem- ory about those events before

years of age; he/ she could remember any of them after

years of age; his/her age at the time of marriage$

years; and the age of joining the army$

years. In this way, a deduction would be made by contrasting

proteins whose expression is triggered when organisms are exposed to stress or injury, which include infections, mechanical stress, oxidants, and cytokine stimulation.5 In response, the arterial wall cells produce high levels of heat shock proteins to protect themselves against the unfavorable conditions.6 The Hsp70 family serves as a dominant chaperone and plays an important role in

such primary knowledge with the census register in

, with an error#

years.

No contradiction in medical records. Fact was veri- fied by consulting different doctors for

times.

The error between actual age and self-reported age#

years. The reference formula is as follows: actual age

(self-reported age)

In ad- dition,

short-lived people (

men and

women) were collected without consanguinity at enrollment. They all naturally died within

–

years, and none in their family lived longer than

years within the past

generations. We started investigating on this population

years ago, col- lected their blood samples, and confirmed their cases through many years of follow up. Short-lived people were randomly chosen and matched in terms of age, gender, and resident area.

MgCl , 2.5 mmol/L KCl, and 0.5 units Taq DNA poly- 2 merase. The restriction enzyme NcoI (Promega, Beijing, China) recognized the amplified fragment (627 bp) and digested it into 354 bp and 273 bp when 2437 T was present. After restriction of enzyme treatment, the reac- tion mixture was separated on 2% agarose gels. The primers and amplification conditions for the other 2 var- iants were according to a report of Wu et al.13 Moreover, the accuracy of our screening method was confirmed by direct sequencing of amplified DNA from randomly selected samples (10%), and no difference in results was found between the 2 methods (data not shown).

activity of HSPA1A, the promoter fragment (488 bp) encompassing G190C variant was cloned into the pMD19-T Easy vector (TaKaRa) with the forward primer: 5’-CGG GGT ACC CGC CAT GGA GAC CAA CAC CC (contained KpnI restriction site) and 5’- CCG CTC GAG CCG GTT CCC TGC TCT CTG TC. The confirmation for construction was done by restric- tion analysis as well as DNA sequencing (BGI LifeTech Co., Ltd., Fremont, Calif). The fragment was then inserted upstream of the firefly luciferase reporter gene at the EcoRI site added between the KpnI/XhoI sites of the pGL3-basic vector (Promega Corporation, Madison, Wisc). The insert orientation in the HSPA1A-pGL3 con- struct was confirmed by restriction assay. Human lung carcinoma A549 cells cultivated in FK12 medium con- taining 10% fetal calf serum were plated into 6-well dishes (5 3 105/well), grown for 20 h, and transfected by the lipofectin method (New England Biolabs, Ips- wich, Mass). The test plasmid (HSPA1A-pGL3 4.0 mg) and internal Renilla luciferase control plasmid (phRL- TK, 1.0 mg) were cotransfected into cells, which were grown for 48 h. The cells were incubated at 42(cid:2)C for 1 h and harvested 5 h after the heat treatment. Cell lysates were prepared, and luciferase activity was measured with a luninometer by using a dual luciferase assay sys- tem. Each promoter activity was measured directly by the ratio of the firefly luciferase level to the Renilla lucif- erase level. For each reporter construct, 5 independent

DNA extraction and phenotype measurements. Blood