〃′

″″″α
s′ ゴア 2イ ′-250`ノ 992)

A difFerence in the proteins found in young adults of inbred

strains of D″ θsθ ′あilα ]“ ιJα agα s′ ι″Which correlates with
genetically‐ deterⅡ lined,long“or short life span

ISAMU YONEMURAl,AKIRA OKAN02,YOSHIHARU SHIMIZU3,HAYATO HASEKURAl

and BARRY BOETTCHER4
lD`ρ α″ ″′ぼ Lagα ′几イ′訪ι″ ttα 助 ルα 1/77Jυ sJrl Sc力 οοノげ ν メ タ″り ο ,

`777ι `,7b/clο `″ `r77`ル

7ο /cl・ ο, ffJ Jcρ αれ
2D"α ″′777θ rげ 二 ′
ψ グ β′ り ,772S″ た び Cα r″ ουαSε ″/r7rDお θαsc,動 ノ
ο 777お ′ ′s力 び″′ υ′rs′ rl
“ `ル “
S`力 ο
οノげ ノ レグ
12″ ′,ン ′αrs“ ο′ο,J9θ ノ ″α
`ク
η
3D"α ″′ θ″ `加Plrb′ ′εル α “′
′力,渋 力οο′ぼ んイ たαノ伽 ノ υα ク ,3ο S′ ′
″ノ ,政 力α″α sノ
`訪 `加 `,動
,

“ `グ
Kα″αgα ″α,259-Fゴ J￠ α″ ρ
4D9,α ″′ οgた α′Sι Jθ ″ε′s,「 /7′
了0/Bノ ο′ υ′″ Иイ
α々s,2θ θθ И″s″ α″α
711`″ C/77′ sノ
ヶ 9/N`lllε αsr々 ,Ⅳ ン
`ll Sο `力

YoNEMURA,I,OKANO,A,SHIMIzu,Y,HASEKURA,H and BoETTCHER.B 1992 A di∬ crcncc in thc

protcins found in young adults of inbrcd strains of D″ ο5ο ρ /α 771θ /α ″r7gas′
/1′ 、
VhiCh cOrrclatcs 、
vith
genetically― detcrmincd,long or short lifc span ―
―〃′
″′′ α ′′7:241-250 Lund,S、`/ vedcn ISSN 0018-0661
7′

`
Rcccivcd January 30, 1992 Acccptcd July 3, 1992

0nc― dimcnsiOnal clcctrophorcsis was pcrformed on extracts of lies collected flom acrOss an agcs Protcin
gcl pattcrns wcrc comparcd for two strains of D″ ο,ο ′力〃αj77θ た″Ogα ,′ ′ r with distinctly long and short aduit
life spans that rcsult from diよ 、rcnt allclcs of 10ngcvity gcncs An inter‐ strain difFerence、 vas observed in the
changes in protcin pattcrn in thc 77 kDa rcgion in pcriod of day O、 5a■ cr cmcrging We propose that the
protcin involvcd is a prOduct of autosoma1 longcvity allclcs И′and И2 at thc■、?И locus and is rclatcd to
dcvclopmcnt o「 longcvity potcntials in the preimaginal stage

おα777″ y。 ″ε r″ ,Dψ α″7pld″ ′9/L′ gα /″ θ

グ′ ο
たJο ttα S力 ,た α ιう
,ル ″
● みοο′9/″ ′
清 ″ε,′ エイ5
力711α ,3″““ `171a,「 `iSて `′
ylr、 ′ ″ゎ ο.ル ● ο,′ ノ ′力ρα″

Gcnctic thcorics of agcing may gcncra‖
classincd into cithcr thc somatic mutation thcory
or thc progranl thcory(HAYFLICK 1985) Both
groups of thcorics postulatc somc participation of
gcncs 、 vhich 、vould producc changcs in protcin
synthcsis as scncsccncc procccds Ho、 vcvCr,Studies
rcportcd to datc havc not found signincant changcs
in protcin during thc coursc of ageing(VAUGHAN
and CALVIN 1977:WILSON Ct a1 1978:PARKER Ct
al 1981:FLEMING Ct a1 1986)
Rcccntly, adult lirc span in inbrcd strains of
D″ οsο ρカメ
ル 771`/77″ οgα sr`″ (D777)haS bCCn bund to
bc contrOncd by autosomal and X― chromosomal
mttOr gCncs(YoNEMURA ct a1 1989,1990)ThcSC
longcvity gcncs wcrc namcd thc J/11(Ju―
tCmS J772 genes have bccn shown to amcct thc
dcvclopmental speed of D 777 in the prё linaginal
stagc,indicating that thcy havc a function around,
or bcforc,thc cmcrging pcriod(YoNEMURA Ct al
1991)
Thcsc indings suggcst that protcin changcs duc
to Stt gcncs,if any,would occur at an carlicr stagc
y bc than thosc prcsumcd by othcr rcscarchcrs Consc―
qucntly,wc cxalllincd prOtcins in D 771 0f diffcrcnt
agcs,varying from young, rcccntly cmcrgcd adults
to thosc rcccntly dcad ol‐ old agc(thC dCad on thc
modal days of mortality pcaks)ProtCins fl・ Om two
inbrcd strains,kno、 vn to posscss diffcrcnt J777 gcnCS
vcrc comparcd and a dimcrcncc、 vas obscrvcd in a
、
protcin in thc youngcst adult nics This dimcrcncc
appcars to bc rclatcd to thc intcr― strain dittcrcncc
of lifc span Thc modc of inhcritancc of this
protcin has also bccn invcstigatcd
PIIaterials and lncthods
myO)SyS―
D 797′ あ″οgα sr′ ″ ― TwO highly inbrcd strains of
D777 with diffcrcnt liた spans wcrc uscd hcrc as in
prcvious studies(YoNEMURA Ct a1 1989;YoNE―
MURA Ct al 1990)ThC ShOrt-l cd strain was
tcrmcd Pl and thc long― livcd, P2 ThCy wcrc brcd
at 27・ Con food containing 8シ6 dry ycast Thcir
lifc spans arc controllcd by two pairs of maJOr
242 1 YONEMURA ET AL
gcncs at autosomal, J47И , and X― chromosomal,
J777二 10Ci,Which intcract with additivity(YoNE
MURA ct a1 1989;YoNEMURA ct a1 1990)ThC J/27
gcnotypcs for Pl arc И′И′χf in malcs, and
И/И ′ X■χr in fcmalcs; and for P2, И2И 2Й 12 in
malcs,and/2A2λ「2Й f2 in fcmalcs И2 or X2 givc
longcr lifc span than cither И′ Or Й´ F and arc
dominant Thc χf anclc sho、vs a dosagc c∬ ect,
giving longcr lifc to hOmozygous fcmalcs than to
hcnlizygous malcs X2 has no dOsagc cmcct as P2
shows nO scx dimcrcncc in lifc span
Mα′
ノ72g θ 7'7`77′ ―
κ′ ′ ―Rcciprocal mating Of Pl
`″
malcs and P2 fCmalcs produccd thc Fl l gcncration
and that of Pl fcmalcs and P2 malCS prOduccd thc
F12 gcnCratiOn Notation for gencrations f0110、 vcd
that of our prcviOus rcport(YoNEMURA ct al
1989) In gCncral, thc Fl gcncrations havc gcnO_
typcs which includc thc И′И2Й「2 allclcs, and arc
long‐ livcd HOwcvcr, only thc F12 malCS havc
ИfИ Ⅸ ■ (with nO X2 allclc)and havc a lifc span
intcrmcdiatc bctwccn thcir 10ng― livcd and shOrt―
livcd parcnts(YoNEMURA ct al 1989,1990)
Thc Bl gcncration v′ as obtaincd by backcrossing
Fll malcs to Pl fcmalcs This gcncration scgrcgatcs
two life spans in thc rati0 0f l:1, short livcd
(И ゴ
И′)and 10ng― livcd(И ノ
И_71,rcnecting thc di5cr―
cncc of J711И allclcs both in malcs and in fcmalcs
(YoNEMURA ct a1 1991)
Prο ttθ lll α″αJl・ sな ――Adult nics wcrc Obtaincd at
diffcrcnt stagcs of lit,■ om s00n a■ cr cmcrgcncc
to thc modal days of inOrtality Thrcc individuals
of thc samc agc wcrc poOlcd to obtain avcragc
data, with thc cxccption of thc cxpcrilncnt t0 0b―
scrvc individual dimcrcnccs Thc bOdics、 vcre storcd
at-20° C
Protcins werc cxtractcd for elcctrophOrcsis as
follo、 vsi nics wcrc hOmOgcnizcd in 0 5 n1l of chloro―
form― mcthanol(2/1,v/V)pcr indi duaL lipids wcrc
rcmovcd 、
vith a nitcr papcri thc rcsiduc was
lyophilizcdi 25 μl of samplc bumcr(o125 M Tris,
pH 68: 2シ 6 SDS: 150。 glyccrol: 5シ 6 2-lllcrCap_
tocthanol:0001%brOmphcn01 bluc)Was addcd tO
thc lyophilizatc;thc samplc was hcatcd for 3 11ain in
a bath of bOiling watcr and was ccntrifugcd at
10,000× G for 10 1nin PrOtcins、 /crc analyzcd by
sodiun dodccyl sulphatc polyacrylanlidc gcl clcc_
trophorcsis(SDS― PAGE)(LAEMML1 1970):10 μ
ofthc supcrnatant iuid was loadcd on cach lane of
10%or 15° /O acrylamidc gcl(80× 70× lmm)
and was clcctrophOrcscd at roOln tcmpcraturc at
25 111A fOr 90 1nin Thc clcctrophorcscd protcin
bands wcrc staincd with O.005シ t Coomassic bril―
〃ピ
だ′″α
ざ′′
7(′ 9,2,
liant bluc R-250(acctiC acid/isOprOpano1/Watcr,
10/25/65, v/v/V)
Results
Protcin gcl pattcrns obtaincd frOlla 1 5シ 6 polyacryl―
amide gcl clcctrOphorcsis(15%SDS― PAGE)wcrc
comparcd on poolcd samplcs fronl thrcc individu‐
als cach of thc shOrt_livcd(Pl)and thC 10ng― livcd
(P2)Strains c011cctcd on days O(S00n aftcr cmcr―
gcncc),5,10,20 and 30;also tcstcd wcrc samplcs
of thc 10ng― livcd strain cOncctcd on days 40, 50,
and samplcs of rcccntly dcad individuals on thc
modal days of bOth strains
lntcr― strain dimcrcnccs、 vcrC ObScrvcd soon aftcr
cmcrgcncc in thrcc bands of 77 kDa, 45 kDa and
20 kDa Espccially, thc diffcrencc of a 77 kDa
prOtcin band bctwccn thc two strains、 vas rcmark―
ablc, bcing thick in thc 10ng‐ livcd (P2)and VCry
thin in thc short― livcd(Pl)(Fig l)Thrcc individ―
uals wcrc uscd for cach samplc ElcctrophOrcsis
was pcrformcd in duplicatc、 vith thc samc samplcs,
and idcntical pattcrns、 vcrc obtaincd Thcsc rcsults
appcar tO rcncct thc intcr― strain diScrcncc of lifc
span cOntro‖ cd genctically
Fig 2 shows gcl pattcrns of samplcs f10m malcs
Thc lifc span(thc mOdal day ofthc mOrtality pcak)
is 36 days for Pl males and 56 days for P2 malCS:
nics which dicd On thc modal day wcrc takcn as
scnilc dcad bodics Markcd difFcrcnccs,both quali―
tativc and quantitativc,wcrc obscrvcd bct、 vccn gcl
pattcrns of day O and day 5,although comparisons
bct、 vccn lancs is difncult in Fig 2 as it is cOmposcd
of scvcral gcls Thc most distinct dintrcncc bc_
t、vccn thc strains vas a band of apprOxilnatcly
、
77 kDa(arrOWCd a in Fig 2);it appcarcd thickcr
in P2 0n day O and bccamc thin frOm day 5 0n ln
Pl this band was thin frOm day O on Thc intcr_
strain dimcrcncc notcd in Fig l in t、 vo protcin
bands,45 kDa and 20 kDa,was not obscrvcd hcrc
From day 5 to thc modal day of mortality, no
markcd changc in prOtcin pattcrns 、vas obscrvcd
among thc dimcrcnt lifc stagcs and bct、 /ccn thc
strains, cxccpt twO protcin bands labcncd b
(95 kDa)and C(88 kDa)in Fig 2 Band b bccamc
distinct aftcr day 20 and disappcarcd in scnilc dcad
l bodics in bOth Pl and P2 gcnCratiOns Band c
appcarcd aftcr day 10 and continucd to dcath on
thc modal days, incrcasing with agc Changcs
of thcsc t、 vo bands、 vcrc silllilar in thc t、 vo strains
AlthOugh no quantitativc mcasurcmcnt with a
dcnsitOmctcr was pcrformcd, bands b and c did
″ ″″
″α5′ ′
7`′ 997, A PROTEIN DIFFERENCE AND LONGEVITY IN DROSO′ ″′
ιИ 243
“

15%SDS― PAGE Protcin samplcs(Cach poolcd

MW from three individuals)were takcn cvcry 4 hours
from soon aftcr cmcrgcnce to day 2 and, thcrc_
kDa aFtcr,atcvcry 24 hoursto day 5 Thc 77 kDa band
was prcscnt illlmcdiatcly aftcr cmcrgcnce,and was
奪
毅 a dcnsc band,in bOth strains lt bccamc thinncr in
thc PI strain fronl 12 to 24 hours aftcr cmcrgcncc,
7■ 0_ and thcn bccamc markcdly thinncr in both strains
66.2_ Elcctrophorcsis in 10シ 6 polyacrylanlidc gcl
(10% SDS― PAGE)was pCrぉ rmcd in Ordcr tO
obscrvc thc 77 kDa band mOrc prcciscly Fig 4
45.0- sho、 vs thc changcs of thc band in carly adult lifc
Thc 77 kDa protcin band 、
vas found tO bc scpa―
ratcd into t、 vO closcly associatcd uppcr and lowcr
bands by 10° 。 SDS‐ PAGE Thc lowcr band
showcd a strain‐ spccinc changc ln thc Pl gcncra‐
tion,it was distinctly thinncr than thc uppcr band
at 12 to 24 hours of agc and disappcarcd by 36
25.7_ hours ln P2, On thc cOntrary, both bands wcrc
about thc samc thickncss unti1 48 hours, and thc
lo、 vcr band disappcarcd aftcr 3 days Thcsc data

suggcst that the protcin gcl pattcrns in young

6.5_ ￨￨￨￨￨￨￨￨￨￨￨￨
Fig.1. Protcin band pattcrns obtaincd frolal thc youngcst
malc■ ics of D 171′ /r7″ Ogα ,′ ′r soon aftcr cmcrgcncc,cxam―
incd by 15° 。SDS― PA(]E Protcin samplcs 針oln thrcc
individuals wcrc poolcd S=short lifё span strain L=
long lifc span strain
not appcar di∬ crcnt bct、 vccn thc strains Thc Only
intcr― strain
dimcrcncc which cOuld bc rclatcd to
digcrcnccs in lifc span Of thc gcnotypically di「
fcrcnt strains vas
thc density of band a on day
、 and fcmalcs)takcn 20 hours aftcr cmcrgcncc Thc
O, obscrvcd in thc protcins cxanlincd by 15シ 6
PAGE
Fig 3 shows a part of gcl pattcrns、 vhich dcmon―
stratc thc changcs of thc 77 kDa band(band a)by
adults from half a day to two days dimcrcntiatc Pl
and P2
Fcmalcs produccd rcsults all12ost idcntical to
thosc of malcs, cxCCpt lor an incrcasc of a 45 kDa
protcin band On,and aicr,day 2 or 3(ngurcs not
shown)This Was assumcd to bc a changc associ―
atcd with scxual ttaturity ofた malcs,and was not
invcstigatcd furthcr
ln an attcmpt to invcstigatc thc modc of inheri―
tancc of thc 77 kDa band, Pl and P2 Strains 、vcrc
rcciprocally matcd to producc thc hybrid gcncra―
tions Fll(Pl malcs× P2たmalCs)and F12(Pl
fcmalcs× P2malCS): 10%SDS― PAGE was pcr―
formcd On sarnplcs cxtractcd from 20 malcs and 20
fcmalcs at 20 hours aftcr cmcrgcncc All samplcs
cxhibitcd thc t、 v0 77 kDa sub― bands of about thc
samc dcnsity, that is, thc Fl sho、 vcd thc samc
protcin pattcrn as P2(Fig 5 shOws a part Of the
data) ThiS is consistcnt 、
vith control of thc trait
(that thC thick,lowcr band rcmains)by an autOso―
mal dolllinant allclc
To cxanlinc this proposition, thc protcin bands
wcrc also cxanlincd in thc BI(backcross to Pl)
gcncrations Fig 6 sho、 vs a part of thc clcc_
trophorcsis data of singlc■ y protcins scparatcd by
10%SDS― PAGE on 96 indi dual samplcs(malCS
long― livcd(L)andthe shOrt_livcd(S)nies scgrcgatc
l:l in thc Bl gcncrations Among thc pattcrns in
Fig 6, thosc with a singlc band, or with a thin
lo、 vcr band, 、
vcrc assigncd S, thc same typc as Pl,
 s′ 77`′ 992)

∽口 一”︼一∽ ｏ ′口 ０〓 一 口 ｏｏ ′一〇０ 口 “Ｑ ∽ ０﹂〓 ● 一 ｏＯ口ｏ■ＯＬ石Ｏ ｏ〓 一 一ｏＯｄ Ｏ﹄ ｏ 一 ● ｏ ｏ工一 ∽● ′ ＾口 一

 ０ ● ぅ ０﹂ ０ 口 につ ぁ 一 ｏ一ｏ ﹁。 “∩ ︼ いい に ＾ ● Ｏ Ｌ 、ｍ   ＯＯ● Ｏ﹄ｐに 一
０ ﹁¨ “ヽ︶∽︲﹁ｏ︺口 一
 ０ ● Ｏｏ ′ｏ 〓∽ ０ ０ ●に ０
， ， ， ，
∽０口 ヽｍ   ∽＾ ヽ０ ︻●つ ｏ 日 ｏ〓 口 〓 ］口 ● ● ｏ● ｏ ﹁ロロ ｏ０ ０ 口 “ ∽︵“０ ０ ︼ ︼ｏこ 一
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つ ∽０ ﹁ “０ ● ﹃Ｏ口Ｏ﹄Ｑ Ｏ ｏ、′ｏ﹁︼“ ０燎 一 や。 ＯＯＸＯ ● ｏｏ∽ ｏＬＯだ′ ∽０¨口ヽ轟Ｏ Ｏ︼一“一 ．
〓 ｏ い ぁ“０ 口︼ｏヽＬ   Ｏｏ＞〓︲¨ 口 〇 一 〇〓 一
 目 一 ﹁０︼ ｏ一一一 ．ハ一一
∽口ｏつ ｏ 一 〇〇Ｃ Ｏ“ＯＬ一
一 “﹁一∽︲﹄ｏ︸口 ︼ ● “ ００た′ｏ こ ∽ 〇 、“０ 口 ｏ ︵ヽ
０ ●¨
α
″c″ ″′

つ０ ′０■﹄“ ︶ ０ 口 ●Ｏ Ｌ 一ｏ一ＯＬＱ ●∩ 鱗 いい ● 一ｏＯ口 ｏ“コｏ日 ０ ﹁０と一 に い 、“Ｏ ｏ一 〇 ︵“Ｏ Ｆ ｏ 一﹂ ∽０ 口 ●０ 口 一ｏ︶ｏ﹄。 Ｃ ¨ Ｏ ｏ＞ヽｏ∽Ｏ ０ ０ＬＯ ′ ． ０＞“ “゛〓 ０ ●● げ ０ ● “ ｏ＞“ “一〓‘● σ 〓 一〇 ０ ＾∽０¨ 口”〓 〇︶ ｏ¨ヽ︺∽ 〓 ｏ“ｏ 一付
，
“

，
∽●﹄０一一“Ｑ ︻０“ ﹄“﹁口〓 ∽ 、′〇 一 ∽ ∽● ¨
に、一∽ 工一〇 ｍ   Ｏ ｏ一●０一つ 口一 ｏ■、 一ｏ¨ 〓 ｏ“ｏ ● ｏ ∽コ０脳 ︼“Ｃ 一長¨ 一 ∽ｏ 。 ｏ〓一 ＾
０ヽ′ ﹄●一● ｏ〇一● ●■ ﹂ｏ ∽口 ｏ ﹁ 一 ∽一〇ｍ ｏ一“﹄●ａ ｏ∽ ｏ＞に 日 ｏ﹁︺ ““ヽ０ ∽口 一 に一〓 Ｏ Ｏ Ｏヽ● ¨ 暉 ０工一 ＯＯ口 一
∽
，
︵■ ●つ ０３ ■ ︱ 場 目●謝 嘔 “ ¨目⊇ ＝ 口 ︵■ ヽつ ︼ ︶ Ｅ Ｉ 場 口“針 〓 ８ 楊 ＝ ∽
聖一
ＯＯＱ ●コ０‘ ∽一
Ｏｏ︻ ●●ｏ一
＞一 。口︼●∽ 口一
０●一ｏｏヽ長︸ ｃ口ｏと一∽ｏ一 ｏ一ｏ﹂﹄
面 〇 く Ｌあ ∩ ∽ ざ い一 ＾０
＾ 目ｏ﹁﹁ ＾
０ｏ〓■〓“Кｏ 辞●ｏＬ ︵〓一
●一﹄Ｏ日 ｏ〓一﹂ｏ ぁ“０ 一
“Ｏｏ日 ０〓一 ｏ一 〇〇●Ｏ¨﹂０日 ０ ■０こ ヽ 口Ｏｏ∽ ︻ ∽ｏ““ ︶口ｏ﹁ρこ一
ｏ ﹂ｏ ∽ｏ一
暉 、、ヽ一
．ヽ”ｏ、もヽもヽヽパ﹃ Я︻ “一つ０ ∽暉﹄ｏ一一”Ｑ Ｏ目●０ 口一
Ｏ﹁﹂ Ｏｏ●¨ ｏ一０﹄﹄ ．
Ｎ．¨︼﹄
鵞
饂 鷲

鰺
l

∞ａ︻
篠 一
〓ヽ■一
一
︱
徴ｌ 機蔭
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い０
〇．
いヾ
べつい
〇トト
に∩ 出
・
藍 筐 〓‡ 灘 群
1 YONEMURA ET AL

・
・ ・
・
，
∽ 日       ∽          口       ∽
０∽Ｑ ﹄Ｏ Ｏ
∽、ぉ０ ０い  ∽、に０ ０ヾ ∽ふに０ ０め ∽スにＯ ＯＮ ∽ぁに０ ０ 劇 ∽ぁ“０ い 酵Σ
ｏ〓 ● ０∽
244
 ｏＯ●０¨﹄ＯＦ一 に ∽﹄●０工 寸い ０一 ０ 一
 Я﹁ “﹄一∽ ∽
ｏ一﹂ 口一
∽■●ｏ工 ０［ ︼ｏこ一 工一 ｏ●﹄●ｏｏＯ 一一 一
●﹄︸∽ 〓一ｏ一 ●一 ﹄ｏＬ口¨
“ ∽口一 ｏ ﹂ｏこ一
A PROTEIN DIFFERENCE AND LONGEVITY INの ROSOP″ ′ιИ 245

●ｏＬ 。﹂。 ′ ∽一
ｏ一ｏ︼Ｑ “∩ 夕 いい 。エト  一。一 Ｑ日 “∽ 目 一
〓Ｏ“﹁ ∽ｏ︻
０●一 ｏｏ﹁〓一 ´ ｏ一。﹄Ｌ
〓一 ０日 ヽＯｏＯ 一一 一
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為を″ ′
で αs′ ′アィ rll′
246 1 YONEMURA ET AL ル″″
′′
α∫″7r′ 992)

Hours aftcr emcrging

S
MW

う′

う乙

И十
Standard 24 48 12

攣 t― ■1蝙 ■￨― ￨‐ ― ―― ヤ ￨￨￨￨■ r`￨■ ■11■ ド●摯゛￨14弯 や寧弯■■ ￨,「

kDa

770
662

450

Fig.4. Protcin band pattcrns,sho、 ving changcs in thc 77 kDa protcin band in short― livcd(S)and 10ng― livcd(L)strains
of 0 711 in thc carly adult stagc,up to 48 hours(10%SDS― PAGE)
Thc same samplcs asin Fig 3、 vere analyzcd by 10 0。 SDS‐ PAGE Thc 77 kDa protcin band、 vas found to scparatc
intO twO sub‐ bands,uppcr and lo、 ver ln thc S strain,thc lo、 ver band、 vas thinner than thc uppcr fron■ 12 to 24 hours
aftcr cmcrgcncc and disappcarcd aftcr 36 hoursi whilc in thc L strain, both bands retaincd about thc samc density,
cvcn at 48 hours aftcr cmcrgcncc

and thosc 、
vith thc thick doublc bands wcrc as―
signcd L,thc samc typc as P2 and Fl Thc scgrcga―
tion ratio, accOrding to this classiflcation, was
48:48
From thc abovc data, thc pcriOd of disap―
pcarcncc of the 77 kDa protcin lowcr band dc―
tcctcd by 10シ 6 SDS― PAC」 E in D 777 WaS rCgardcd
as a quantitativc gcnetic trait, cOntrollcd by a
singlc mttor autOsomal gcnc,thc 10ng‐ lasting trait
bcing dOnlinant to thc carly― disappcaring Thcsc
charactcristics corrcspond to thc c∬ cct of J772И
autosoma1 longcvity gcncs cOntroling lifc spans.It
is prOposcd that thc 10、 vcr band of thc 77 kDa
protcin is a gcnc product Of J777И
E)iscuss10n
´ilanong thc many thcorics and mcchanisms to ex―
plain thc aging proccss, tWO main thcorics stand
outi thc thcOry of progralllincd aging,postulating
that gcncs control aging according to a prcdcter―
lllincd progranl:and thc lllutation thcory,clailning
that cclls arc dcstroycd whcn thc accumulation of
gcnc itturiCS rcachcs a thrcshold(aS rcvicwcd by
HAYFLICK 1985)Thc prOgram thcOry propOscs
that abnormal prOtcins arc produccd by scnility
gcncs, and thc mutatiOn thcory assumcs that in―
jurcd gcncs causc changcs in anlino acids and the
clcctric chargc of protcins Both Of thcsc thcOrics
,
prcdict that somc protein changcs(prOgrammed or
nOt)ヽ Vil1 0ccur during thc latcr period of lifc
Thcsc hypothcscs havc promptcd many expcri‐
mcnts to comparc protcins from individuals Of
di∬ crcnt agcs in various animals VAUGHAN and
CALVIN(1977)uscd Onc_dilalcnsional clcctrophorc―
sis to comparc band pattcrns and conccntrations Of
brain prOtcins frOnl lllicc of di∬ crcnt agcs Thcy
obscrved striking prOtcin changes during fctal and
iuVCnile stagcs,but found no changcs bctwcen thc
ル″
″ α,′ ′
″′ 7`′ ,9′ リ A PROTEIN DIFFERENCE AND LONGEVITY IN,ROSOP″ ′
ιИ 247

Fl l F12
MW
Standard
0 ? ?

‐鱚 瘍
,警 容ヾ,摯 零 fF,￨￨● :■ 1写

kDa

770

662

450

Fig. 5, Protcin band patterns, showing thc 77 kDa bands(indiCatcd)in thC tWO Fl gcncrations
(rCCiprOcal hybrids of S and L purcbrcd strains of D 771)at 20 hours aftcr emergcnce(10%
SDS‐ PAGE)
F !=Pl malcs× P2 たmalCs F12=Pl たmales× P2 malcs All individuals, irrcspective of scx,
rcvcalcd uppcr and lo、 vcr 77 kDa bands ofthc samc dcnsity,rcprcscnting thc P2 phcnOtypc,L strain
Conscqucntly,this trait,、 vith thc lowcr 77 kDa band rcmaining until thc latc stage oflifc,is dolllinant
and is controled by thc autosomal J771 gcnC

young(17-36 wccks of agc)and thC agcd(127-
134 wccks)WILSON et al(1978)comparcd the
ncrvc tissuc of young (2-14 months)and 01d
(21-24 months)ratS by two― dimcnsional clcc‐
trophorcsis of protcins labclcd with 14c_lccuinc,
and rcportcd that no diffcrcnccs 、 vCrC Sho、 vn in
protcin pattcrns or protcin amounts, and no no‐
ticcablc protcin synthcsis was dctcctcd in thc old
PARKER ct al(1981)comparcd protcins f10m thc
young(6 days post cclosion)and the。 ld(60 days)
D792 by t、 /o― dilllcnsional clcctrophorcsis, aftcr
fccding thcm 、
vith 35s_llacthionine, and failcd to
nnd any Signincant dimcrcnces
Bascd on thcsc rcsults,thc above rcscarchcrs arc
agrccd in thcir dissatisfaction 、 vith thc program
thcory, which assumcs scnility gcncs activatcd in
thc latcr stagcs Of life,and also against thc somatic
mutation thcory, which assumcs that mutations
vith agc Thcy cmphasizc
accumulate in protcins 、
thc nccd fOr a quitc dimcrcnt thcory on thc mccha‐
nism of scnility
Unlikc thc abOvc studics, cxpcrilllcntal rcsults
indicating signincant changc of a protcin during
lit,causing scnllity,wcrc repOrted by FLEMING Ct
al(1986)Thcy labclcd D 777 With 35s_mCthioninc
and comparcd protcins of flies from days 10, 28,
248 1 YONEMURA ET AL ″ご
″″″α
ざ′′
7`′ 992)

MW
Standard

kDa

770
662

450

257

MW
Standard

kDa

770
662

450

Fig.6.Protcin band pattcrns obtaincd from a D 772 tcst cross progcny(10%SDS― PAGE)
Thc rcsults sho、 vcd thc thick,lo、 vcr 77 kDa band to scgregatc as a donlinant trait
Fll(Pi malcs× P2fCmalcs)malCS WCrc backcrosscd tO Pl fcmalcs to producc thc Bl
gcneration 20 hours aftcr cmcrgcncc, 96 individuals wcrc analyzcd fOr 77 kDa protcin
bands by 10° 。SDS― PAGE Thosc 、 vhO gave double bands Of thc same dcnsity 、 vcrc
groupcd L,thc long livcd as parcnt P2:thOSc whO had a singlc uppcr band or a doublc band
with thinncr 10、 ver band wcrc grouped S,thc shOrt_livcd as parcnt PI Thc distributiOns of
L and S individuals in Bl gencration、 vcrc 48:48, agreeing with thc cxpectatiOn
″crg671′ α
s′ ′
7`7992)
and 44, using two― dilncnsional clcctrophorcsis
Thcy found scvcn small polypcptidc spots on thc
protcin gcl pattcrn fronl thc day 28 samplc,、 vhich
wcrc abscnt in gcls fronl thc othcr days Thcy also
quantitatcd 100 polypcptidc spots and observed
scvcral changcs according to agc, and suggestcd
thc possibility that such changcs could bc thc causc
of sencsccncc
Thc abovc cxpCrimcnt of FLEMING Ct al(1986),
howcvcr, did not comparc multiplc strains 、vith
difFcrcnt lifc spans,and could not dcmonstratc that
stlch protcin changcs csscntially contributc to scnil‐
ity ln contrast, our prcscnt study comparcs t、 vo
strains with difFercnt lifc spans, and 、vc can con―
cludc that thc 77 kDa protcin(Fig l and 2)may
bc a causc of intcr― strain di∬ erence of lifc span,
although bands b or c in Fig 2 cannot,sincc thcy
show only changcs duc to tilne but not to intcr―
strain dimcrcnccs
On thc other hand,rcccnt cxpcrilncntal data on
D771 prOVidc cvidcncc for ncw conccpts in mccha‐
nisms of gcnctic control of longcvity
Wc havc suggcstcd that lifc span of D ηz is
controllcd by a fcw mttOr gcncs(YoNEMURA Ct al
1986)LucKINBILL Ct al(1988)havc rCportcd that
mttOr gcnes controlling lifc span of D″ arc lo‐
catcd on chromOsomes l and 3
Wc furthcr analyzed in detail the mode ofinher―
itancc of lifc span in D 777 and discovered an
autosomal and a scx chromosomal mttOr gene
controlling lifc span of D 721,acting in a Mcndclian
way, and nominatcd thcm J/77 gcncs Wc havc
dcnncd thc modal day ofthc mortality pcak as thc
phcnotypic valuc of J47 gcncs(YoNEMURA Ct al
1989)Thc dircct causc of dcath for cach individual
may bc classincd into diffcrcnt discascs, cg, can―
ccr,disordcr of hcart and othcr organs,or suffoca‐
tion by food, ctc, but any of thcm could bc
attributcd to thc rcsult of scnility Wc rcgardcd thc
dcad on thc modal days in our cxpcrilncnts as
scnilc dcaths(YoNEMURA Ct a1 1989,1990)Our
cxpcrilncntal work dcmonstratcd that 3憔 gcncs
also inttucncc thc prcilnaginal dcvclopmcntal spccd
in thc stagc from latc larva to cmcrgcncc,and that
thc gcnotypic valuc,or longcvity potcntial,is dctcr‐
mincd alrcady at thc cmcrgcncc stagc(YoNEMURA
ct al 1991)
Considcring thc pcriod of dittcrcntiation of
longcvity potcntial innucnccd by J7,2 gcncs,it sccms
likcly that Jη 2 gcncs、 vould lcavc somc tracc of thcir
function in thc youngcst adults soon aftcr emcr―
gcncc Thc prcscnt study has idcntincd a 77 kDa
protcin as a candidatc for a J777 gcnC product
A PROTEIN DIFFERENCE AND LONGEVlTY IN DROSOP″ ′
ιИ 249
This protcin is found in abundancc in adults
soon aftcr cmcrgcncc,and allnost disappcars aftcr
a fcw days Thc ratc of dccrcasc of this prOtcin is
slo、 vcr in thc long‐ livcd strain than in thc short―
livcd. This charactcr is shown by thc prcscnt
study to follow Mendelian inheritancc, thc slo、 v
ratc of disappcarancc bcing dominant There is a
difFerence in life span in Fl malcs Thc F12 1nalCS
(Pl females× P2malCS)uscd in thc prcscnt study
havc an intcrmcdiatc lifc span sincc thcy lack thc
J“ χ2allclc(gcnOtypc И FИ 2χ ′ ), whCrcas Fl_1
malcs havc thc ИfИ Ⅸ 2gcnotypc(YoNEMURA ct
al.1989, 1990).HoWCVCr, no rcmarkablc difFer―
cnce was observed among thc Fl progcnics in thc
inheritancc of thc 77 kDa protcin changcs Thcrc―
forc,if this protcin is a product of J773 genes,it is
not thc product of thc X‐ chromosomal Jη 2 gcncs,
but of thc autosomal J727 gcnCS(ノ И′ and J47
И “ morc abun‐
И2 allclc producing thc
O,thC J771
dant protein Our prclinlinary data sho、 v that this
protcin appcars in thc latc larval pcriod and dc‐
crcascs rapidly in thc latc pcriod of thc pupal
stagc This pcriod agrccs 、vith thc prcsumcd timc
、
vhcn di∬ crcntiation of longcvity potcntial occurs
(YoNEMURA ct al 1991)Detailcd data on thc
cmcct of this protcin on adult life span, on thc
changcs of this protcin in thc coursc of individual
dcvclopmcnt, and on its chcmical characteristics,
、vill bc publishcd clscv′ hcrc Thosc studics also
suggest that this protcin not only prolongs thc lifc
of the short― livcd strain but also that of thc long‐
livcd, if givcn in food as a supplcmcnt, whilc thc
othcr protcins dcvoid of thc 77 kDa protcin show
no cttcct on thc strains
D′ οsρ ρカノ″ larval serum protcin-1(LSP‐ 1)iS an
interesting protein for us in relation to thc 77 kDa
protcin Thcse two have some similaritics in thc
pcriod of appcarancc, thc molecular weight, or
localization Of their genes LSP-l is a polypcptidc
of 78-83 kDa which is found in hacmolymph of
thc third instar larvac in largc quantity during a
short pcriod bcforc pupation lt consists of thrcc
polypcptidc chains,α
,β ,and ν;it forms a hcxamcち
it contains no lipids or carbohydratcs(RoBERTS
and EvANS― RoBERTS 1979; SMITH ct al 1981)
Localization of its structural gcncs coding α
,β ,and
νpolypeptides is shown to be on chromosomcs l,2
and 3, respectively, by standard genctical tcch―
niqucs and in situ hybridization (RoBERTS and
EvANS‐ RoBERTS 1979; SMITH ct al 1981)Thc
function of this protein is not kno、 vn at all A
possibility is prcscntcd that it scrvcs as anlino acids
storagc br cuticlc synthcsis(SMITH Ct al 1981)
250 1 YONEMURA ET AL ル rピ ´″ド ″ 7`′ 9物

Ho、 vcvcr, thc matcrial for the cuticlc should bc PARKER,J,FLANAGAN,J,MURPHY,J and CALLANT,J 1981
produccd mainly in thc pupal stagc whcn cuticlc 0n thc accuracy of protcin synthcsis in D″ α″ο―
οsο ′力〃α 792′ ′
″―-1イ ac力
gα s′ ′ Иgθ υ 761127-139
formation is most activc "gD′ RoBERTS,S 1979 The genctic and
RoBERTS,I) B and EvANS―
Although littlc attcntion has bccn paid to thc cytogenetic localizatiOn Of thc three structural gcncs cOding for
thC m● Or prOtan of D″ 。
soρ カカ hrval scrum― G´ ″′
:cs 93:
carly pcriod of lifc― tilllc in prcvious studics on 663-679
scncsccncc, thc rcsults Of thc prcscnt cxpcrilllcnts SMITH,D F,McCLELLAND,A,ヽ VHITE,B N,ADDISON,C F
indicatc that invcstigation of changcs in thc carlicst and GLOVER,D ヽ( 1981 The molecular cloning of a dis―
pcrscd sct of dcvclopmentally rcgulatcd gcncs、 vhich cncOde the
adult stagc is likcly to omcr impOrtant indings in malor larval scrum protcin of D ′′ gα s`′ r― ―Cθ 〃 2子
α″ο
thc study of incchanisms of senesccncc 441-449 “
VAUGHAN,｀ V J and CALVIN,M 1977 Elcctrophorctic analysis
of brain protcins「 lom young adult and agcd lanice― ― Gε ″
οη′
0/‐

og), 23: 110‐ 126

Иご
た″ο θ
771a″ お ― This study was supportcd by the Sllloking
WILSON,D L,HALL,M E and SToNE,G C 1978 Tcst o「
"′ “ `な
Rcscarch FOundation(Japan)
somc aging hypothcscs using t、 vo― dimcnsional protcin map‐
ping ―
― G″ ″
ο ο/Dgy
72′ 2′ :426-433
YoNEMURA,I,MOToYAMA,T and HASEKURA,H 1986 Evi
References dcncc Of maJOr gcnes controlling longcvity in D″ οsο ρ ′
α /1′

θ′
α″οgα ,`′ ――Prο εJの り α″И(″ ″ 62Sビ r β:41-44
FLEMING, J E, QuATTROCKI, E, LATTER, G, MIQuEL, J, YoNEMURA,I,MOToYAMA,T
“ and HASEKURA,H 1989 Modc
MARCUSON,R,ZucKERKANDL,E and BENSCH,K G 1986 of inhcritancc Of ma30r gcnes controning lifc span diミ 、rcnccs
Agc― dcpcndcnt changcs in protcins of D″ οs(〃 ,力 :￨● 7770′ α71ο ― bctwccn twO inbrcd strains of D″ ο
s(〃 ,力 〃α θ/rl″ ο
gα ピ
′― s′

″――Sc′ ′
gα s′ θ ′ご′237: 1157-1159 ″″´ ″′α,′ ′′:207-214 “
HAYFLiCK,L 1985 Thcorics Of bi010gical aging― Eχ,Gご ″ο″― YoNEMURA, I, ABE, M, IsHIDATE, R, IsHIYAMA. T, MO―
′ο′2θ : 145-159 TOYAMA,T,HASEKURA,H and BoETTCHER,3 1990 1nlu―
LAEM M LI,U K 1970 Clcavagc of structural proteins during thc ence of tcmpcraturc on thc inhcritancc Of adult life span in
asscmbly of thc hcad of bacteriophagc T4 -― ヽ_`“ ″ θ227: Drο sο ρ/1′ ′
α777ビ ′ gα s″ ″―
α″ο ― ″´ r´ r/1′ αs/′ 2:117-127
680-685 YoNEMURA, I, MOToYAMA, T, HASEKURA, H and BoETT―
Lじ cKINBILL,L S,GRAVES,J L,REED,A H and KoEST CHER, B 1991 Rclationship bct、 vccn gcnotypes of longcvity
AWANG, S 1988 Localizing gcncs that dclcr scncsccncc in gcncs and dcvclopmcntal spccd in D″ て
な カア ル″ ′ α″οgα ,′ ″― ―
∂″ Osο′力′
ル θ 物″οgα ,θ ″――〃σ ″ご′,ヶ :367-374 〃ο ″ο〃 ●66: 143-149
7′
`ψ “
“ `θ