Báo cáo khoa học: Điều hòa biểu hiện gene tiểu đơn vị a ATP synthase Drosophila melanogaster bởi yếu tố phiên mã chứa vị trí liên kết GAF và Adf-1

Expression of the

Drosophila melanogaster

ATP synthase asubunit

gene is regulated by a transcriptional element containing GAF

and Adf-1 binding sites

Ana Talamillo

*, Miguel Angel Ferna

´ndez-Moreno

, Francisco Martı

´nez-Azorı

´n

,Bele

´n Bornstein

1,2

Pilar Ochoa

and Rafael Garesse

Departamento de Bioquı´mica, Instituto de Investigaciones Biome

´dicas ‘Alberto Sols’, CSIC-UAM, Facultad de Medicina,

Universidad Auto

´noma de Madrid, Spain;

Servicio de Bioquı´mica, Hospital Severo Ochoa, Legane

´s, Madrid, Spain

Mitochondrial biogenesis is a complex and highly regulated

process that requires the controlled expression of hundreds

of genes encoded in two separated genomes, namely the

nuclear and mitochondrial genomes. To identify regulatory

proteins involved in the transcriptional control of key nuc-

lear-encoded mitochondrial genes, we have performed a

detailed analysis of the promoter region of the asubunit of

the Drosophila melanogaster F

ATP synthase complex.

Using transient transfection assays, we have identiﬁed a

56 bp cis-acting proximal regulatory region that contains

binding sites for the GAGA factor and the alcohol dehy-

drogenase distal factor 1. In vitro mutagenesis revealed that

both sites are functional, and phylogenetic footprinting

showed that they are conserved in other Drosophila species

and in Anopheles gambiae. The 56 bp region has regulatory

enhancer properties and strongly activates heterologous

promoters in an orientation-independent manner. In addi-

tion, Northern blot and RT-PCR analysis identiﬁed two

a-F1-ATPase mRNAs that diﬀer in the length of the 3¢

untranslated region due to the selection of alternative

polyadenylation sites.

Keywords: mitochondria; a-F

-ATPase; GAGA; Adf-1;

transcription regulation.

The bulk of cellular ATP is synthesized through oxidative

phosphorylation (OXPHOS) that takes place in the mito-

chondria. The OXPHOS system is composed of ﬁve

multisubunit complexes embedded in the inner mitochond-

rial membrane and two small electron carriers, ubiquinone

and cytochrome c[1]. The OXPHOS system is generated in a

unique manner. The majority of the more than 80 OXPHOS

subunits are encoded by genes in the nuclear DNA (n-DNA),

while 13 essential subunits are encoded in the mitochondrial

DNA (mtDNA), contributing to four out of the ﬁve

OXPHOS complexes. The mtDNA consists of a small,

double-stranded, circular DNA molecule that is transcribed

and translated within this organelle. However, all of the

components involved in the replication, maintenance and

expression of the mtDNA, as well as the factors that

participate in the assembly of the respiratory complexes, are

encoded in the nucleus. Therefore, correct OXPHOS func-

tion relies on the coordinated expression of numerous genes

encoded in two physically separated genetic systems [2,3].

The multisubunit enzyme ATP synthase (complex V of the

OXPHOS system) is present in the membranes of eubacteria,

mitochondria and chloroplasts. It synthesizes ATP by means

of a rotary mechanism coupled to the electrochemical

gradient generated by the electron transport chain [4]. The

mitochondrial ATP synthase of animals contains 16 subunits

and is responsible for the synthesis of the majority of cellular

ATP, thereby playing a crucial role in energy metabolism. It

is formed by an F

soluble complex containing ﬁve subunits

with a stoichiometry of a

dce, and a hydrophobic F

complex composed of 11 subunits that forms an H

channel

embedded in the inner mitochondrial membrane [1]. The F

subcomplex contains the three catalytic sites of the enzyme

located at the interfaces of the aand bsubunits where

nucleotide turnover takes place [4]. Two of the subunits are

encoded in the mtDNA; ATPase 6 (or a) and ATPase 8 (or

A6L). In contrast, the remainder are nuclear-encoded and

are translated by cytoplasmic ribosomes before being

imported to the mitochondria.

The molecular basis underlying nucleo–mitochondrial

crosstalk is still poorly understood [3]. During the last few

years a number of processes have been shown to participate

in this process, including transcriptional and post-transcrip-

tional regulation of gene expression [5,6], changes in Ca

Correspondence to R. Garesse, Departamento de Bioquı

´mica, Insti-

tuto de Investigaciones Biome

´dicas ‘Alberto Sols’ CSIC-UAM, Fac-

ultad de Medicina, Universidad Auto

´noma de Madrid, C/Arzobispo

Morcillo 4, 28029 Madrid, Spain. Fax: +34 91 5854001,

Tel.: +34 91 4975453, E-mail: rafael.garesse@uam.es

Abbreviations: Adf-1, alcohol dehydrogenase distal factor; GAF,

GAGA factor; OXPHOS, oxidative phosphorylation; n-DNA, nuc-

lear DNA; mtDNA, mitochondrial DNA; NRF, nuclear respiratory

factor; RACE, rapid ampliﬁcation of cDNA ends; AEL, after egg

laying; UTR, untranslated region; DPE, downstream promoter

element.

*Present address: Departamento de Anatomı´a y Biologı´aCelular,

Facultad de Medicina, Universidad de Cantabria, Santander, Spain.

(Received 2 June 2004, revised 6 August 2004,

accepted 18 August 2004)

Eur. J. Biochem. 271, 4003–4013 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04336.x

concentration [7], control of the mitochondrial dNTP pool

[8,9], mitochondrial localization to speciﬁc cellular domains

[10], or changes in local ATP concentrations [11]. A

particularly fruitful experimental strategy to identify key

regulatory factors in mitochondrial biogenesis was pioneered

by Scarpulla’s group [5]. This involves characterization of the

promoter regions of mammalian nuclear-encoded mito-

chondrial genes, and has led to the identiﬁcation of several

transcription factors and coactivators that regulate the

expression of genes playing key roles in the biogenesis of

the OXPHOS system. In general, the transcription of nuclear

genes encoding proteins involved in OXPHOS biogenesis is

controlled by a combination of transcription factors that

are speciﬁc for each promoter [3,12]. However, one DNA

regulatory element that is more common in the 5¢upstream

regulatory region of respiratory genes is that recognized by

the constitutively expressed Sp1 factor [13]. Additionally,

two other transcription factors have been shown to play a

signiﬁcant role in mitochondrial biogenesis, the nuclear

respiratory factors (NRFs) 1 and 2 [5,14]. These factors are

likely to be involved in the integration of mitochondrial

biogenesis with other cellular processes related to cell growth

[15,16]. NRF-1 belongs to a novel class of regulatory

proteins, and it contains a DNA binding domain conserved

in two invertebrate developmental regulators, Erect Wing

and P3A2 [17]. Erect Wing is essential for the Drosophila

myogenesis and neurogenesis [18] while P3A2 regulates the

expression of several genes during sea urchin development

[19]. The transcription factor NRF-2 belongs to the ets family

and is the human homologue of the previously described

mouse transcription factor GABP [20]. Other DNA regula-

tory elements have been identiﬁed in the promoter of several

genes involved in mitochondrial biogenesis, such as

OXBOX/REBOX [21], Mts [22] or GRBOX [23]. However

the putative transcription factors that recognize these DNA

motifs remain to be identiﬁed.

In contrast, less is known about the mechanisms

controlling mitochondrial biogenesis in other animal sys-

tems or in invertebrates. We previously described how the

transcription of several Drosophila melanogaster genes

encoding components of the mtDNA replication machinery

was regulated. These included the mitochondrial single-

stranded binding protein (mtSSB), and the catalytic (a)and

accessory (b) subunits of the DNA polymerase c(Pol c)

[24,25]. Interestingly, the expression of the genes encoding

mtSSB and Pol c-bis transcriptionally regulated by the

DNA replication-related-element binding factor (DREF).

Indeed, in Drosophila this transcription factor regulates the

expression of genes that are essential for the cell-cycle and

for the nuclear DNA replication machinery [26], establish-

ing a link between mitochondrial and nuclear DNA

replication [24,25]. Here, we have identiﬁed essential

elements that participate in the transcriptional regulation

of the gene encoding the asubunit of the H

ATP synthase

(a-F1-ATPase)inD. melanogaster.

Materials and methods

Library screenings

We screened a D. melanogaster genomic library prepared in

the vector k-DASH using the previously described a-F1-

ATPase cDNA labelled with [

P]dCTP[aP] as a probe [27].

Two genomic equivalents were transferred to Zeta-probe

ﬁlters (Bio-Rad), hybridized at 68 C in ZAP buffer [7%

(w/v) SDS, 0.25

phosphate buffer, pH 7.2], washed in

0.5% (w/v) SDS, 2·NaCl/Cit at 55 C (NaCl/Cit: 0.15

NaCl/0.015

sodium citrate) and visualized by autoradio-

graphy with intensifying screens at )70 C. Positive clones

were puriﬁed by two additional rounds of screening, and

positive phages were ampliﬁed using standard protocols,

and the inserts analysed by Southern blotting. The complete

sequence of the gene as well as 5¢upstream and 3¢

downstream regions (http://Flybase.bio.indiana.edu) were

included in two overlapping phages. Selected fragments

strongly hybridizing with the probe were subcloned into

pBluescript (Stratagene) and further characterized by

sequencing.

DNA sequencing

The nucleotide sequence of the genomic clones was

determined using the dideoxy chain-termination method

with Taq DNA polymerase and automatic sequencing (3T3

DNA sequencer, Applied Biosystems) following the manu-

facturer’s instructions. Both DNA strands were sequenced

in their entirety and the sequences were analysed using the

GCG

programme (University of Wisconsin) [28].

Mapping of transcriptional initiation sites

Identiﬁcation of the a-F1-ATPase transcription start site

was achieved by three different methods: primer extension,

high-resolution S1 mapping and rapid ampliﬁcation of

cDNA ends (RACE).

Primer extension analysis. Two different oligonucleotides

were used: a-PE1 (5¢-ACGGCCGGTCTCCTCCAGA

TC-3¢) from bp 216–195 and a-PE2 (5¢-GGACGC

CAGGCGGGCGGAAAAAATCG-3¢) from bp 30–4

from the ATG start codon in the a-F1-ATPase cDNA

sequence, respectively [27] (accession number Y07894). In

the assay, 50 pmol of the oligonucleotides were labelled with

50 lCi of [

P]ATP[cP] and polynucleotide kinase. Total

RNA (30 lg) from adults or from embryos obtained 0–18 h

after egg laying (AEL), and 8 lCi

1of

P-labelled primer

were used in each experiment. Annealing and reverse

transcription were carried out as described previously [29],

and the extended products were analysed in 8% (w/v)

polyacrylamide/7

urea gels. Sequencing reactions using

the same oligonucleotides were run in parallel.

S1 analysis. We PCR ampliﬁed a 506 bp fragment using

the forward primer 5¢-AGATGACCTGATTCCCTT

GG-3¢corresponding to bp )476 to )459 from the ATG

start codon in the genomic sequence (GenBank

2accession

number NT_033778) and the reverse primer 5¢-GGACGC

CAGGCGGGCGGAAAAAATCG-3¢corresponding to

bp 30–4 in the same sequence. The reverse oligonucleotide

was labelled at its 5¢end with 100 lCi of [

P]ATP[cP] using

T4 polynucleotode kinase under standard conditions. The

probe (3.2 lCi)

3was hybridized with 75 lgoftotalRNA

extracted from adult Drosophila, for 15 h in 80% (v/v)

formamide, 40 m

Pipes pH 6.4, 1 m

EDTA, 0.4

4004 A. Talamillo et al.(Eur. J. Biochem. 271)FEBS 2004

NaCl. After adding four volumes of S1 nuclease buffer

(40 m

sodium acetate, 250 m

NaCl, 4 m

ZnSO

), the

sample was incubated with 150 units of S1 nuclease

(Pharmacia) for 60 min. The reaction was stopped with

ammonium acetate and 0.1

EDTA, and the nucleic

acids extracted with phenol and precipitated with ethanol.

The pellet was resuspended in 75 m

NaOH, and after

incubating for 15 min at 90 C it was precipitated with

ethanol, resuspended in 98% (v/v) formamide, 25 m

EDTA, 0.02% (w/v) bromophenol blue, 0.02% (w/v)

xylene cyanol, and analysed in 8% (w/v) polyacrylamide/

urea gels. Sequencing reactions were run in parallel.

5¢RACE experiments. We used the RLM-RACE kit from

Ambion Inc. (cat. # 1700), following the manufacturer’s

instructions. We used a-PE1 as the outer primer oligo-

nucleotide for the a-F1-ATPase cDNA (see Primer exten-

sion analysis) and the inner primer was 5¢-TCTCCTCCA

GATCAGCCTTGGGGG-3¢.

RT-PCR analysis of a

-F1-ATPase

mRNA-3¢ends

Total RNA from D. melanogaster embryos 0–18 h AEL or

adult ﬂies was extracted using Trizol (Gibco-BRL) and

treated for 30 min with RNAse-free DNAse I (1 unit per lg

of RNA). Reverse transcription was carried out following a

protocol described previously [24]. Ampliﬁcation of the 3¢

ends was performed with an oligo(dT) primer and one of

the two primers a-RT1 (5¢-TGCGCGGTCATCTGG

ACAA-3¢)anda-RT2 (5¢-ATCGCCAAGGACGGTGC

TA-3¢), at positions )184/)165

4and )83/)64 with reference

to the translation stop codon, respectively.

Promoter constructs

A909 bpDNAfragmentfromthe5¢region upstream of the

D. melanogaster a-F1-ATPase gene (from )914 to )5

considering +1 the ﬁrst nucleotide of the translation start

codon) was ampliﬁed by PCR from total DNA using the

primers pADm1 (forward; 5¢-AGCAGTCGACGA

AGCGACGAAGTGAAGCTGCGTGA-3¢) and pADm3

(reverse; 5¢-ATCCGTCGACATGCTTTTTAACTGTT

CG-3¢). After digestion with SalI (which recognizes the

sequence underlined in the oligonucleotides), the DNA

fragment was cloned into the pXp2 vector that contains the

luciferase reporter gene. The construct with the suitably

orientated insert was used as a parental DNA fragment for

the generation of a series of deletion constructs. These were

generated either by ExoIII digestion and blunt-end cloning,

by restriction endonuclease-based cloning, or by PCR

ampliﬁcation and cloning of selected DNA fragments.

Finally, we obtained the constructs shown below, where +1

represents the transcription start point according to the data

presented here.

Mutagenesis of the GAGA element was achieved by

PCR and subcloning of the ampliﬁed fragment. The

oligonucleotides used for PCR were the luciferase gene

internal primer 5¢-GGCGTCTTCCATTTTACC-3¢and

the oligonucleotide 5¢-CCGTCGACATTAATTTAATTT

ccccAATTATATTGCGTCG-3¢in which the SalI recogni-

tion site is in bold and the GAGA element is replaced by the

sequence underlined (lowercase letters show the nucleotide

changes). The )146/+79 construct was used as a template

and the ampliﬁed fragment was cloned into the pXp2

5vector.

This strategy was also used to mutate the alcohol dehy-

drogenase distal factor (Adf-1) element using the speciﬁc

primer 5¢-CCGTCGACATTAATTTGAGAAATTATAT

TGCGTCGCccgccggcCgcCacgGAGGGTGAC-3¢(again

the SalI recognition site is in bold, the location of the

Adf-1 element is underlined, and nucleotides in lowercase

have been changed). A similar strategy was carried out to

construct the GAGA or/and Adf-1 mutants in the hybrid

promoters (see Results).

Cell transfection assays

The pXp constructs (5 lg) were transiently transfected into

Schneider S2 cells (as described previously [25]) to assay

their promoter activity. To correct for variations in the

efﬁciency of transfection, we cotransfected the cells with the

plasmid pSV-bGAL and the quantiﬁcation of luciferase was

normalized to b-galactosidase activity. Luciferase activity

was determined using the Luciferase Assay System (Pro-

mega) according to manufacturer’s recommendations, and

b-galactosidase activity was measured as described previ-

ously [30].

Results

Transcriptional initiation sites of the

D. melanogaster

-F1-ATPase

gene

We have previously characterized a cDNA encoding the

D. melanogaster a-F1-ATPase subunit [27]. To isolate the

corresponding D. melanogaster a-F1-ATPase gene and

ﬂanking regions, we screened a genomic library using this

a-F1-ATPase cDNA as a probe. Two overlapping clones

containing the entire gene as well as 5¢upstream and 3¢

downstream sequences were selected for further analysis.

The a-F1-ATPase gene maps to the 2R arm of the

D. melanogaster polytene chromosomes and its structure

is shown schematically in Fig. 1. It contains four exons

separated by three 624, 92 and 113 bp introns. The ﬁrst

Fig. 1. Structure of the D. melanogaster a-F1-ATPase gene. Chromo-

somal location and structure of a-F1-ATPase. The gene maps to the

2R arm in D. melanogaster. In the schematic diagram of the gene

structure, white boxes represent introns, black boxes represent exons,

and the grey boxes represent UTRs. The line underneath the gene

shows the position of several restriction endonucleases. E: EcoRI; K:

KpnI; C: ClaI; Ev: EcoRV; S: SalI; P: PstI; B: BamHI.

FEBS 2004 Drosophila a-F

-ATPase gene expression (Eur. J. Biochem. 271) 4005

exon encodes the 5¢untranslated region (5¢-UTR) as well as

the ﬁrst 22 amino acids of the 23 residues which form the

targeting sequence. The last amino acid of the presequence,

the complete mature protein and the 3¢-UTR region are

encoded in exons 2–4. To determine the transcriptional

initiation sites of the a-F1-ATPase gene we ﬁrst carried out

primer extension analysis using total RNA extracted from

adults or embryos 0–18 h AEL, and two different

P-labelled oligonucleotide primers (a-PE1 and a-PE2).

Both primers produced identical results in embryos and

adults, three transcriptional initiation sites being detected at

positions )86, )91 (the majority) and )120, considering

position +1 as the ﬁrst nucleotide of the translation

initiation codon ATG (Fig. 2A,C).

This result was conﬁrmed by high resolution S1 mapping

using a 506 bp probe that extended from the coding region

(position +30) to 477 bp upstream of the ATG. In this

analysis, several DNA fragments were protected (Fig. 2B),

with the strongest signal corresponding to position )91, the

prominent position detected by primer extension. The

position )91 is 22 nucleotides upstream of the transcription

startpoint previously described for this gene [27] (GenBank

accession number Y07894). Additionally, more weakly

protected smaller fragments were detected, reﬂecting the

failure to precisely identify the initiation site typical in

housekeeping and TATA-less promoters. Finally, we per-

formed a RACE study on the 5¢end of the a-F1-ATPase

mRNA. All of the clones analysed end in the region )83 to

)115, most of them ending between )83 to )90 (Fig. 2C).

Interestingly, three clones identiﬁed in RACE experiments

detected the same nucleotides as the three weaker bands

shown by S1 mapping as the transcription start point.

Hence, we concluded that the D. melanogaster a-F1-ATP-

ase gene contains a heterogeneous region responsible for the

initiation of transcription although a common initiation site

was located at position )91.

As frequently observed in other housekeeping genes,

neither TATA nor CCAAT boxes were found in canon-

ical positions in the Drosophila a-F1-ATPase gene [31].

Moreover, in the 5¢upstream region of the a-F1-ATPase

gene we did not ﬁnd the TCAG/TTPy arthropod initiator

element [32]. Nevertheless, in the bovine and human a-F1-

ATPase genes, the transcriptional initiation sites located at

positions )91 and )120 lie within short conserved

sequences. Indeed, the sequence at the )91 transcriptional

start site, CCATCT, corresponds to a conserved

vertebrate initiator element (Inr; PyPyAT/APyPy), indica-

ting that it may be involved in tethering the basal

Fig. 2. Identification of the transcription start site of the D. melanogaster a-F1-ATPase gene. (A) Primer extension analysis. Transcripts from

a-F1-ATPase mRNA primers were obtained with both a-PE1 and a-PE2 primers, although because the results were identical only those with the

primer a-PE2 are shown. Sequencing was performed with the same primers using a genomic clone as the template. (B) High resolution S1 mapping.

Total RNA from D. melanogaster was hybridized with a 506 bp probe from )476 to +30, the ATG translation start codon being referred to as +1.

Bands that were protected from S1 nuclease were visualized in 8% (w/v) acrylamide/urea gels, close to the sequencing reactions. (C) The nucleotide

sequence of a-F1-ATPase 5¢upstream region. Black arrows show the transcription start points identiﬁed in the primer extension assays. White

arrows represent transcription start points from S1 mapping. The thickness of the black and white arrows is related to the intensity of the band.

Asterisks represent transcription start points from the 5¢end ampliﬁcation experiments (see Materials and methods). PCR products were cloned and

six of them were sequenced, identifying the nucleotide shown by asterisk as the 5¢end of a-F1-ATPase cDNA.

4006 A. Talamillo et al.(Eur. J. Biochem. 271)FEBS 2004

transcriptional apparatus to the a-F1-ATPase promoter.

Furthermore, several short sequences commonly found

downstream of transcriptional initiation sites in Drosophila

promoters include ACGT, ACAA, ACAG, and AACA

[32], and these were detected at )17, )18, )36 and )103

positions of the a-F1-ATPase gene (position relative to

ATG). However, the region did not contain a canonical

downstream promoter element (DPE), which is recognized

by the TAFII60 factor [33]. Indeed, the short elements

described above probably substitute for the DPE motif in

the Drosophila a-F1-ATPase promoter.

Functional analysis of the a-F1-ATPase promoter region

The function of the Drosophila a-F1-ATPase promoter

region was characterized by transient transfection into

Schneider SL2 cells. A series of deletions of the 5¢upstream

region of the gene were cloned in the pXp2 vector that

contains the luciferase reporter gene. A construct containing

the )823/+86 region (position +1 corresponds to the main

transcriptional initiation site located 91 nucleotides

upstream of the ATG initiation codon) promoted substan-

tial luciferase activity in Schneider cells, 2,300-fold higher

than the native pXP2 vector (Fig. 3). This activity was

orientation-dependent and indicates that the a-F1-ATPase

5¢proximal upstream region contains a strong promoter,

with similar activity in Schneider cells to the promoter of

the b-F1-ATPase gene [34] and 10-fold stronger than

the promoter of the gene encoding the catalytic subunit of

the mitochondrial DNA polymerase [25]. Similar luciferase

activity was maintained even when the upstream region was

reduced and contained only the )146/+86 region. In

contrast, a construct containing the )93/+86 region had

signiﬁcantly lower promoter activity, reaching only 13% of

the maximal activity, while the )61/+86 construct directed

similar levels of luciferase activity as the pXp2 vector

(Fig. 3).

These results indicated that although the 53 bp DNA

region located between nucleotides )146/)93 does not

itself have promoter activity, it contains DNA elements

critical for the activation of the a-F1-ATPase promoter.

Computer analysis revealed the presence of two DNA

sequence motifs in this region potentially recognized by

the GAGA factor (GAF) and the alcohol dehydrogenase

distal factor (Adf-1), respectively (Fig. 4A). GAF is a

Drosophila regulatory protein that overcomes transcrip-

tional repression produced by histones at the chromatin

level [35]. Adf-1 was initially identiﬁed as an activator of

the alcohol dehydrogenase (Adh) promoter and was

subsequently shown to control the expression of several

Drosophila genes [36,37].Interestingly, it has been shown

that GAF and Adf-1 act together to remodel nucleosome

structure and activate transcription both in vitro and

in vivo [38].

To analyse the involvement of the potential GAF and

Adf-1 binding sites in activating the a-F1-ATPase

promoter, we eliminated the target sequences by site-

directed mutagenesis and examined the activity of the

mutated constructs in cell transfection assays. Mutating

the GAGA or Adf-1 elements individually signiﬁcantly

reduced promoter activity by up to 40–60%, whereas

when both sites were abolished, the activity of the

promoter was reduced by 75% (Fig. 4B). In addition, we

carried out cotransfection studies in Schneider cells using

different a-F1-ATPase promoter constructs and a plasmid

that express GAF under the control of the actin 5C

promoter. The GAGA factor stimulated at least threefold

the activity of the promoter in constructs )397/+86 and

)146/+86, but had no effect on the activity of the

construct )93/+86, which does not contain the potential

GAF binding site (Fig. 4C).

The combination of GAF/Adf-1 has been shown to

activate transcription in a variety of promoter contexts.

Hence, we generated a construct containing a 56 bp

DNA fragment ()144/)89) that included the GAGA and

Adf-1 elements linked to the basal promoters of the

d-aminolevulinate synthase (ALAS)andb-F1-ATPase

genes [29,34]. In both constructs there was a substantial

Fig. 3. Functional analysis of the D. melanogaster a-F1-ATPase promoter. Scheme of the a-F1-ATPase promoter constructs used for transient

transfection assays in Schneider cells (see Materials and methods). The promoter regions are represented by solid lines and the luciferase reporter

gene is shown as a solid arrow. The numbers to the left of each construct indicate the limit of the promoter fragment with reference to the

transcription start point as established in this study. The relative promoter activities of the constructs measured in the luciferase assay are indicated

on the right by black boxes. The luciferase activity of the vector with no insert was deﬁned as being equal to one. Luciferase activity was normalized