three atpl genes are present on chromosome ii in

J. Biochem. 118, 607-613 (1995)

Three ATPl Genes Are Present on Chromosome II in Saccharomycescerevisiae

Masaharu Takeda,1 Takahito Okushiba, Takaaki Satoh, Shinren Kuniyoshi,Chika Morishita, and Yoshinobu Ichimura

Department of Applied Microbial Technology, Kumamoto Institute of Technology, Kumamoto, Kumamoto 860

Received for publication, May 15, 1995

Chromosome fragmentation, ATPl disruption, and Southern blot analyses of total DNAsand prime clones of chromosome II showed that three identical ATPls are present, directingfrom the telomere to the centromere on the 36-55 kb far from the left telomere sequence ofchromosome II. That is, the coding and 5'-, 3'-non-coding regions of ATPl are repeated 3times at approximately 7 kb intervals. These three ATPls are expressed, and one and two^TPls-disrupted strains respectively, showed ca. 70 and 40% decreases in their ATPaseactivities and a subunit contents, compared to those of the wild-type, DC-5 or W303-1Astrain, but could grow on glycerol.

Key words: ATP1, chromosome II, FjFo-ATPase, gene organization, Saccharomycescerevisiae.

The FiF0-ATPase complex is found in all prokaryotes andeukaryotes. The complex, which is located in mitochondria,chloroplasts, and bacteria, consists of three sectors, Fj(catalytic portion), Fo (proton channel embedded in themembrane), and FA (associated components between F, andFo). Though in prokaryotes all the subunit genes arepolycistronically organized {1-3), in eukaryotes the geneorganization is quite different (4, 5). Five subunits consist-ing of the Ft-sector are encoded in the nuclear genome,except for the a subunit of plant mitochondria! F^o-ATPase, which is synthesized within mitochondia (6, 7).However, the subunit organization of F] is generallyctifhyde, and the structure and function of the complex areessentially similar in all organisms (8). Here we show thegene organization of the a subunit gene (ATP1) (9) of thecomplex in Saccharomyces cerevisiae.

MATERIALS AND METHODS

Strains, Vectors, and Media—The yeast strains used inthis study were YNN290 (a/a, ura3-52/ura3-52, ade2-101/ade2-101, lys2-801/lys2-801, trpl-A 1/trpl-A 1),DC5 (a, leu2-3, Ieu2-112, his3, canl-11), W303-1A (a,leu2-3, Ieu2-112, his3-ll, his3-15, trpl-1, ura3-l, ade2-l,canl-100), and SEY2102 (a, leu2-3, ku2-112, ura3-52,his4-519, gal2, suc2-/]9). The construction of chromosomefragmentation centromeric vectors for ATPl, pTOCF41and pTOCF41' was performed as described (4, 10). For aHIS3 chromosome fragmentation vector, a 1.7 kb BarriH.1fragment of YCp403 (gift from Dr. G.R. Fink) was insertedinto the BaniH.1 site of centromeric vector YCF4 (gift fromDr. P. Hieter) as described (4, 10). The preparation ofselective (SD) and rich (YPD and YPGE) media (11), andyeast transformation (12) were performed as described.

1 To whom correspondence should be addressed. E-mail: takeda©kumamoto-it.ac.jp

Pulsed Field Gel Electrophoresis—The preparation ofchromosomes and pulsed field gel electrophoresis wereperformed as described (4).

Miscellaneous—Yeast prime clones (23) were purchasedfrom ATCC. Total DNAs were prepared by a publishedmethod. (14). The preparation of mitochondria (15) andWestern blotting were carried out as described (16).ATPase activity was performed by a published procedure(17).

RESULTS AND DISCUSSION

ATPl, encoding the yeast F^ATPase a subunit, wasmapped on the far left end of chromosome II in Sacchar-omyces cerevisiae (4). Chromosome fragmentation ana-lyses (4, 10), using the centromeric vector, pT0CF41,showed that three different proximal fragments, 43 (strainTSY413), 50 (strain TSY412), and 57 kb (strain TSY411),were detected, and hybridized with ATPl (Fig. IB). In thecontrol, pTOCF41' (opposite strand ATPl), no chromo-some fragmentation occurred. PKC1, which is located atthe left end of chromosome II (18), hybridized with thesame fragments (Fig. 1C). The sizes of the chromosome IIfragments were calculated considering the size (ca. 8 kb) ofthe vector, YCF4, used (4, 10). The results indicated thatthree ATPl proximal fragments, 35, 42, and 49 kb, werederived from chromosome II and ATPl located on thecentromere-side of PKC1, and the ATP1(B) was directedfrom the telomere to the centromere.

Of the lambda prime clones containing left end areas ofchromosome II (13), three on the filter were hybridizedwith ATPl. Clones 70511 and 70113, and 70113 and 70804overlapped each other at 4.4, and 9.3 kb long regions,respectively. But clones 70511 and 70804 did not overlap.Southern hybridization analyses of these DNAs clones werecarried out using ATPl (2.9 kb EcoRl-Sphl fragmentincluding the coding, 5'- and 3'-noncoding regions contain -

Vol. 118, No. 3, 1995 607

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608 M. Takeda et al.

(A) (B) (C)

A 1 2 3 4 1 2 3 4 1 2 3 4

220-I600kb -• 1*1

23kb- -»•

lelomere(Y') SUP11 URA3pTOCF41 ••••HIM

(10.1kb)

CKN4 ATPl

H I

telomere(Y') SUP11 UKA3 CKN4 ATPlpTOCF41'

(10.7kb) J-

Fig. 1. Chromosome fragmentation wascarried out according to published methods(4, 10). Each centromeric plasmid, pTOCF41or 41', alone was introduced to diploid strainYNN290 {4, 10), and then stable light-pinkcolonies were selected. The fragmented chro-mosomes from the transformants were appliedto a CHEF apparatus (A), blotted onto a hybondN+ nylon membrane, and then hybridized withATPl (B, 1st Southern hybridization) andPKC1 (C, after 1st Southern hybridization, there-probed membrane was used for the 2ndSouthern hybridization). The proximal frag-ments of chromosome II were separated in a 1%agarose gel on an alternating CHEF gel appara-tus. Electrophoresis was carried out for 20 h in0.5 XTBE buffer at 200 V and 14'C with a 2.8-3.4 s linear gradient in order to separate theproximal fragments. Under the conditionsused, all chromosomes, larger than 200 kb,could not be separated. Lane 1, SH964; lane 2,TSY411; lane 3, TSY412; lane 4, TSY413.

I 2 3 4 5 6 7 8 9 1 0 1 1 1 2

9.42 kb

4.36

2.32

2.02

0.56

Fig. 2. Southern hybridization of membranes forSaccharomyces cerevisiae clones. Southern hybridiza-tion of purified DNAs from prime clones, 70511, 70113,and 70804, using DIG-labeled ATPl [EcoRl-Sphl 2.9 kbfragment of pBR12-5 carrying the ATPl coding (1.6 kb),5'- (0.6 kb), and 3'-non-coding region (0.7 kb) containing0.2 kb pBR322 DNA] as a probe. Lanes 1, 4, 7, and 10,EcoRl/HindUl; lanes 2, 5, 8, and 11, BamUl/HinAUl;lanes 3, 6, 9, and 12, Hindm/Sall; P, Pstl; H, Hin6Ill;S, Sail; B, SomHl; E, EcoRl.

Clone \

70511

701 13

70804

Fragment

ue E/ll

1.7

2.01.7

2.01.7

hybrldized(kb)

HfM

2.4

3.12.4

4.33.12,4,

S/II

2.70.6

3.12.70.6

4.33.12.70.6

Chromosome I I left arm

II, S , D, E, S^i!2 F.j/Pi H3 S j B ^ j S j l l j Ej /P 2 >l5 S s B j E 5 S , H , E , / P 3I i l l I ' I , I i I I ' I I I, i I I I I I I I

clone 70511 (I8.028M clone 70804 (16,033bp)

clone 70113 (15,398bp)

_L25 30 35 40 45

Distance from (elomtre (kb)

50 55

E; EcoRl, B; Boml l l , II; I l indl l l ,S; Sai l , P; Prtl.

J. Biochem.


Gene Organization of ATPl in Yeast 609

ing 0.2 kb pBR322 DNA) as a probe. Both DC5 and clone70113 contained 2.4 and 3.0 kb BamHl-Hinffll frag-ments, which were derived from the ATPl coding, 5'- and3'-noncoding regions. Whereas, on both BarriH.1-HindUland Sall-HindUl digestion, clone 70804 contained a 4.3 kbfragment, which might come from the ATPl coding regionand 3'-noncoding region carrying a part of a phage vectorDNA (A.MG3), because of deletion of the correspondingHindlU site (H7) in the clone DNA (Fig. 2), in addition tothe corresponding fragments of clone 70113. On the otherhand, clone 70511 contained only a 2.4 kb fragment, whichwas derived from the 5'-noncoding and a part of the codingregion of ATPl, because the clone DNA was devoid of thedown-stream region of the corresponding HindUl site (H2).In addition, both the 70113 and 70804 hybridization spotswere more intense than that of the 70511 clone (Table I).These results and other restriction patterns of the threeclones showed that clone 70511 contained one ATPl, andboth clones 70113 and 70804 contained two identicalATPls, as shown in Fig. 2.

The total DNAs from three different wild-type strains,DC5, W303-1A, and SEY2102, were digested, transferred,and then hybridized with ATPl (2.9 kb fragment) as aprobe. These DNAs gave the same hybridization patternswhen various restriction enzymes, EcoRl, BarnH.1, Hin-dni, Sail, and Pstl, were used (data not shown), and thePCR products (from - 2 0 bp up-stream of ATPl to 110 bpdown-stream of ATPl) from DC5 were the same (Mabuchi,

T. and Takeda, M., submitted for publication). Theseresults further support the conclusion that there are threeidentical ATPl genes 35-55 kb from the telomere of theleft-hand of chromosome II in each strain (designated asATPla, ATPlb, and ATPlc, respectively).

The ATPIB in two different wild-type strains, DC5 andW303-1A, were disrupted with the HIS3 or LEU2 gene, asdescribed previously {19). Approximately 20% of thetransformants were able to grow on glycerol. Then, totalDNA was isolated from each transformant and digestedwith EcoRl, followed by Southern hybridization withDIG-labeled ATPl (2.9 kb fragment) as a probe. As shownin Fig. 3, total DNAs from glycerol-grown transformantscontained the ATPl gene disrupted with HIS3 (4.9 kbEcoRl -fragment) and non-disrupted ATPl (3.7 kb EcoRl-fragment). In addition, in the case of the glycerol-grownHIS3-ATP1 transformants (ATPl disrupted with the

TABLE I. Hybridization analysis of a membrane containingDNA from physically mapped clones for the genome of Sacchar-omyces cerevisiae AB972 (ATCC 76269) purchased from ATCC(77284). DIG-labeled ATPl (EcoRl-Sphl 2.9 kb fragment) was usedas a probe for the hybridization. The intensities of the hybridizedspots were measured with a Densitograph AE-6900-M (Atto).

Clone Intensity (%)705117011370804

24.9138.5836.50

(A) (B)

1 2 3 4 5

4.9 kb3.7 kb

Strain

1.W303-1AfWT)

2. TKY4010(<dpl::MS3)

3. TKY4011(alpI::HIS3)

4. TKY4021(atpl::MS3)

5. TKY4031(atplr.HISJ)

native(3.7kb)

1

0

1

1

1

DNA •disrupted

(4.9kb)

(ratio

0

1

0.5

1.4

1.1

Chromosome **Ch.XV(1100kb)

of intensity)2

2

2"

i

i

Ch.n(830kb)

0

2.7

1.3

2.0

1.2

DNA fragment hybridized with ATPl. Ratio of disrupted ATPl (4.9kb)was calculated taking the native ATPl (3.7kb) intensity of each strain as 1.

Chromosome hybridized with HIS3. Ratio of HIS3 intensity in chromo-some II was calculated taking that in chromosome XV of each strain as 2.

•1100 kb (XV)

830 kb (II)

Fig. 3. Southern hybridization of total DNAs (A) andchromosomes (B) from ATPl disruptants. The inten-sities of the hybridized fragments were measured with aDensitograph (see Table I heading). Total DNAs wereisolated, digested with £coRl, and then subjected to 1%agarose gel electrophoresis. Chromosomes were isolatedand applied to a CHEF apparatus. Electrophoresis wascarried out for 34 h with a switching interval of 55 s for 17h and 90 s for 17 h. After the electrophoresis, Southernhybridization was carried out as described in the legend toFig. 1 using DIG-labeled ATPl and HIS3 as probes. Lane 1,W303-1A; lane 2, TKY4010; lane 3, TKY4011; lane 4,TKY4021; lane 5, TKY4031. The intensity of the hybrid-ized fragment was measured with a Densitograph AE-6900-M (Atto, Tokyo).

Vol. 118, No. 3, 1995



HIS3 gene, designated as TKY4011), the intensity of the3.7 kb EcoRl-fragment was about twofold that of the 4.9kb EcoRl -fragment. On the other hand, the transformantsunable to grow on glycerol (TKY4010) contained onlyATPI disrupted with HIS3 (Fig. 3A). Hybridization analy-sis of chromosomes also indicated that the glycerol-growntransformants contained HJS3 in chromosome H in addi-tion to XV, where HIS3 is essentially present, and the bandintensity was almost the same. On the other hand, in theglycerol-nongrowth transformants, the intensity ratio ofchromosome II to XV was greater than those in theglycerol-growth transformants (Fig. 3B). Thus, the threeATPI genes were disrupted with the HIS3 gene in glycerol-nongrowth ATPI disruptant, TKY4010 (a, Ieu2, his3,ura3, trpl, ade2, atpla::HIS3, atplb::HIS3, atplc::HIS3). In the case of ATP 1 disrupted with the LEU2 gene,the same results were obtained (data not shown).

In the case of the introduction of the HIS3-ATP1 disrup-tion plasmid (pSK-4) into SKY2010 (a, Ieu2, his3, atpla::LEU2, atplb::LEU2, atplc::LEU2), all SD-plate (dex-trose) grown transformants could not grow on glycerol (Fig.

4). Southern blot analyses of chromosomes and total DNAsfrom these SD+/Gly- transformants (SKY2410) werecarried out using LEU2 (2.2 kb Hpal fragment), HIS3 (1.7kb BamHl fragment), and ATP1 (2.9 kb EcoRl-Sphlfragment, as described) as probes. Southern analyses ofchromosomes and total DNA from SKY2410 revealed thatthe three ATP1 genes in strain SKY2410 were disruptedwith both the LEU2 and HIS3 genes (Figs. 5 and 6). Thehybridization intensity of chromosomes hybridized withATP1, that is, the proportion of the hybridization of II tothat of XV (HIS3 probe), was greater than that of II to III(LEU2 probe) (Fig. 5). These results suggested that twoout of the three ATP1 genes were disrupted with HIS3, andthe other one, ATP1, was disrupted with the LEU2 gene.On Pstl digestion of total DNA from SKY2410, a 9.7 kbPstl fragment was detected (Fig. 6). This indicated thatL£[/2-disrupted ATP1 was the middle of the three identi-cal ATP1 genes, ATPlb. The results lead to the conclusionthat ATP la and ATPlc were disrupted with HIS3, andATPlb was disrupted with LEU2, respectively. That is,ATPla and ATPlc disrupted with LEU2 in SKY2010 were

Strain ATPI on chromosome II Glrctrol Selection

ATPl-a ATPl-c

DC-5

SXY2010

SKY2410

SKY2A11

SXY4A11

{ATP> grow SD-Heu+hii

not grow SD+hli

not grow SD

grow SD+bii

trow SD+leo

Construction oT ATPI dlsruntloti ol

V. Be II SphI

Hpal Hpal" 2 JKa | (<l«n'ed from YCp<03)

LEW(d«rirrt from YEpl3)

(KoBI-Sphl rntnot l «s<d for ATPI

Fig. 4. Reciprocal ATPI disruption. SKY2010 (a, Ieu2, his3, his3, atpla::HIS3, atplb::LEU2, atplc::HIS3,G\y-).SKY2410 wanatpla::LEU2, atplb::LEU2, atplc::LEU2, Gly") was transformed transformed with pBR12-5, native ATPI. Glycerol-grown colonieswith pSK-4, ATPI disruption plasmid with HIS3. SD-grown colonies were selected and restreaked on SD, SD-leu, and SD-his plates,that could not grow on glycerol were designated as SKY2410 (a, Ieu2,

(A) Probe : H1S3

2 3 4 5

(B) Probe : LEV2

1 2 3 4 5

(C) Probe : ATPI

1 2 3 4 5

Ch. XV(1100kb)

. Ch. II(830kb)

Ch. Ill1360kb)

Fig. 5. Southern hybridiza-tion of chromosomes isolatedfrom LEU2/HIS3 doubleATPI disruptants. Chromo-somes were isolated from cells.Hybridization involved thesame membrane, and was se-quentially carried out with DIG-labeled (A) HIS3, (B) LEU2,and (C) ATPI as probes, asdescribed in the legend to Fig. 1.Electrophoresis was carried outas described in the legend to Fig.3. Lane 1, DC5; lane 2, SKY-2010; lane 3, SKY2410; lane 4,SKY2A11; lane 5, SKY4A11.

J. Biochem.


Gene Organization of ATP1 in Yeast 611

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

23.1kb6.564.36

2.32

0.56

Fig. 6. Total DNAs were isolated from transformants, digest-ed with restriction enzymes, and then subjected to Southernhybridization with ATP1 as described in the legend to Fig. 3.Lanes 1-3, DC5; lanes 4-6, SKY2010; lanes 7-9, SKY2410; lanes10-12, SKY2A11; lanes 13-15, SKY4A11; lanes 1, 4, 7,10, and 13,EcoRl; lanes 2, 6, 8, 11, and 14, Pstl; lanes 3, 6, 9, 12, and 15,Ecom/Pstl.

E, E,IPl E3

3.7kb , 4.3kbE4/P2 Es

3.7kb 4.3kb 3.7kbEJP,

DC 5

Fragment hybridizedwith ATP1 (2.9kb)

EcoRl Pstl

3.7kb 8.0kb

E/P

3.7kb

E, E,, E4/P2

2.4 , 3.0 . 4.3E3 E12, 2.4 , 3.0 4.3

E13 E4/P,

SKY2010 3 - °2.4

9.7 3.02.4

Pi: E6/P32 . 4 , 2 . 5 , 4 . 3 . 2 . 4 , 3 . 0 . 4 . 3 , 2 . 4 , 2 . 5

SKY2410 4.93.02.4

9.76.72.5

3.02.S2.4

4.3 , 2.4 , 3.0 . 4.3 . 3.7SKY2A11 3.7

3.02.4

9.78.0

3.73.02.4

2.4 I ,2.5 , 4.3 , 3.7SKY4A11 4.9

3.78.06.72.5

3.72.52.4

replaced to ATPla and ATPlc disrupted with MS3 (Fig.4).

In order to identify the disrupted ATP1 on chromosomeII, we performed two experiments. First, an ATP1 disrupt-ed strain, TKY4011 (a, Ieu2, his3, ura3, trpl, ade2, atpl::HIS3, Gly+), was transformed with a centromeric chromo-some fragmentation vector, pTS4-HIS31, and three stablepink colonies were selected. Chromosomes were isolatedfrom the transformant, followed by pulsed field gel electro-phoresis, and hybridization with HIS3 (1st) and PKCl(2nd) as probes, according to the legend of Fig. 1. The 57 kbDNA fragment was hybridized with HIS3 in the strainobtained on chromosome fragmentation of TKY4011, inwhich ATPlc was disrupted with HIS3, in addition tochromosome XV in which HIS3 is located (Fig. 7B). The 57kb fragment, which corresponds to 49 kb of chromosomeDNA as described above (Fig. 1), also hybridized withPKCl (Fig. 7C). These results indicated that A TPlc (49 kb

from the left telomere, see Fig. 2) on chromosome II instrain TKY4011 was disrupted with the HIS3 gene.Second, an EcoR.l-Sph.1 fragment of pBR12-5 carrying anative ATP1 was introduced into ATP1 triple disruptedstrain SKY2410 (a, Ieu2, his3, atpla::mS3, atplb::LEU2, atplc::HIS3, Gly~) as described (19). Two Gly+

transformants were isolated. One was selected on a SD-leuplate and the other on an SD-his plate (Fig. 4). Southernanalyses of the chromosomes and total DNAs from bothGly+ transformants (SKY4A11 and SKY2A11, respective-ly) are shown in Figs. 5 and 6. In strain SKY4A11,chromosome II was hybridized with HIS3 in addition toXV, in which HIS3 is essentially located, but was nothybridized with LEU2. On the other hand, in strainSKY2A11, chromosome II was hybridized with LEU2 inaddition to HI, in which LEU2 was originally present, butwas not hybridized with HIS3 (Fig. 5). Strain SKY2A11contained both the native ATP1 and L£U2-disrupted

Vol. 118, No. 3, 1995



Ch.I-XVI

57kb-50kb-43kb-

which were derived from YNN290(see Fig. 1), were used as controls.

Fig. 7. Chromosome fragmentation of theHIS3-ATP1 disruptant with MS3. TKY-4011 (a, Ieu2, his3, ura3, trpl, ade2, atpl::HIS3, Gly+) was transformed with a centro-meric HIS3 chromosome fragmentation vector[pTS4-HIS31, which was constructed byHIS3 gene (1.7 kb BamHl fragment) inser-tion to the BamHl site of YCF4 (4, 10)].Electrophoresis of the chromosomes was car-ried out under the above (Fig. 1) conditions,blotted, and then hybridized with DIG-labeled(A) ATP1 (1st Southern hybridization), (B)HIS3 (1st Southern hybridization), and (C)PKC1 (2nd Southern hybridization) as probes.Lane 1, TSY411; lane 2, TSY412; lane 3,TSY413; lanes 4-6, strains obtained on chro-mosome fragmentation of TKY4011, in whichATPlc was disrupted with HIS3, with thepTS4-HIS31 vector. Strains TSY411-413,

transformed with pTOCF41, respectively, containing fragmented chromosome II, 57, 50, and 43 kb

TABLE n. Properties of ATP1 disruptants.

Strain F,-ATPase°(^mol/min/mg)

• subunit"(ratio)

Glycerol ATPrdisrupted

W303-1A (wt)TKY4010TKY4011TKY4021TKY4031DC5 (wt)SKY2010SKY2011SKY4010SKY4011SKY2410SKY2A11SKY4A11

1.65 (100%)01.07 (62.4%)0.89 (49.2%)0.92 (50.8%)1.39 (100%)01.07 (76.9%)00.55 (40.0%)01.25 (90%)0.83 (60%)

1.0000.880.280.301.0000.7000.400N.D.N.D.

+ no (0)ATPla,b,c (3)

+ ATPlc (1)+ N.D. (2)+ N.D. (2)+ no (0)

ATPla,b,c (3)+ N.D. (1)

ATPla,b,c (3)+ N.D. (2)

ATPla,b,c (3)+ ATPlb (1)+ ATPla.c (2)

"Percent calculated taking the activity of each wild-type as 100%.bRatio calculated taking the a subunit content of each wild-type as 1.'Number of disrupted ATP1 genes with the HIS3 or LEU2 gene inparentheses. N.D., not determined.

ATP1 genes, but not the HIS3-disrupted ATP1 one.Whereas, strain SKY4A11 contained both the native ATP1and HIS3-disrupted ATP1 genes but not the LEU2-disrupted A TP1 one (Fig. 6). Also,onPs£l digestion of totalDNA from 2A11, a 9.7 kb fragment was detected. There-fore, in SKY2A11, ATPlb was disrupted with LEU2 at thesame position as in SKY2410. Similarly, in SKY4A11, 6.7and 2.5 kb Pstl fragments were detected as well as those ofSKY2410 (Fig. 6). These results lead to the conclusion thatin SKY4A11, ATPla and ATPlc were disrupted withHIS3, and that in SKY2A11, ATPlb was disrupted withLEU2.

Mitochondria were isolated from the wild-type, and thevarious ATP1 disruptants described above, followed byWestern blotting using anti-a subunit antiserum andprotein A-gold (Bio-Rad, Richmond, CA). After enhance-ment, each band was scanned and quantitated with aShimadzu dual-wave length TLC scanner CS-900. Theamounts of the a subunit and Fi-ATPase activity inmitochondria were directly proportional to the amount ofATP1 (Table II). These results lead to the conclusion thatthe three equivalent ATPls present on chromosome II areexpressed. The physiological meaning of the three ATP1

genes in a cell and their participation in the molecularassembly for the F,F0-ATPase complex are still obscure.Studies on these points are currently in progress.

Quite recently, the DNA sequence of chromosome II(807,188 bp) was published, and one ATP1 gene presentwas directing from the telomere to the centromere on ca. 30kb far from the left telomere (20). The sequencing resultswere in accord with our previous ones (4, this study), butwere overlooked two other identical ATP1 genes whichwere maintained the same restriction sites, including both5'- and 3'-non-coding regions (ca. 7 kb) (Fig. 2).

We are especially grateful to Dr. Michael G. Douglas for criticalreading of the manuscript, providing us with the strains and antisera,and his encouragement. We also thank Messrs. Toshiharu Komataand Toshiaki Kodama for their technical assistance.

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Vol. 118, No. 3, 1995


three atpl genes are present on chromosome ii in

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