the induction of dna puff bhc4-1 gene is a late response to the

The induction of DNA puff BhC4-1 gene is a late response to the increasein 20-hydroxyecdysone titers in last instar dipteran larvae

L.R. Basso Jr.a, C. Vasconcelosa, A.M. Fontesa,1, K. Hartfelderb, J.A. Silva Jr.a,P.S.R. Coelhoa, N. Monesic, M.L. Paco-Larsona,*

aDepartamento de Biologia Celular, Molecular e Bioagentes Patogenicos, Faculdade de Medicina de Ribeirao Preto,

Universidade de Sao Paulo, Av. Bandeirantes 3.900, Ribeirao Preto 14049-900, SP, BrazilbDepartamento de Biologia, Faculdade de Filosofia Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, Brazil

cDepartamento de Analises Clınicas, Toxicologicas e Bromatologicas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto,

Universidade de Sao Paulo, Ribeirao Preto 14040-903, SP, Brazil

Received 16 July 2001; received in revised form 24 September 2001; accepted 4 October 2001

Abstract

The characterization of DNA puff BhC4-1 expression was extended and its response to 20-hydroxyecdysone investigated in Bradysia

hygida and in transgenic Drosophila carrying the BhC4-1 gene. In both organisms the activation of BhC4-1 in salivary glands occurs at the

end of the larval stage coinciding with the peak in ecdysone titers which induces metamorphosis. Injections of 20-hydroxyecdysone into mid-

fourth instar larvae of B. hygida show that the induction of BhC4-1 expression, as well as amplification and puff C4 expansion, are late events

induced by the hormone. This late response of BhC4-1 expression was also observed in transgenic salivary glands cultivated in the presence

of 20-hydroxyecdysone. In vitro studies using transgenic Drosophila indicate that both repressor and activator factors regulate the timing of

BhC4-1 expression in salivary glands. q 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Ecdysone response; Late gene; DNA puff gene promoter; Transgenic Drosophila

1. Introduction

The opening and closing of RNA puffs in salivary gland

polytene chromosomes of dipteran larvae permit the direct

visualization of molting hormone-induced changes in

patterns of gene expression. In addition to these common

RNA puffs, salivary gland polytene chromosomes of sciarid

flies exhibit a second type of puffs, DNA puffs, which are

sites of amplification and transcription triggered by the

molting hormone ecdysone (Lara et al., 1991; Gerbi et al.,

1993; Stocker et al., 1996). Earlier cytological studies have

shown that DNA puffs fulfill the requirements for classifica-

tion as ecdysone late induced puffs. The injection of 20-

hydroxyecdysone (hereafter referred to as ecdysone) in

mid-fourth instar larvae of different sciarid species induces,

after a delay of several hours, generalized DNA synthesis

followed by the formation of DNA puffs (Crouse, 1968;

Stocker and Pavan, 1974; Amabis and Amabis, 1984a).

Ligature experiments result in the inhibition of DNA puff

formation (Amabis and Amabis, 1984b). Moreover, DNA

puff induction requires RNA (Berendes and Lara, 1975) and

protein synthesis (Amabis and Amabis, 1984a), since its

induction is blocked by actinomycin D and cycloheximide

treatment, respectively.

Attempts to reproduce the DNA puffing pattern in sciarid

salivary glands cultured in vitro have had only limited

success. The B2 DNA puff of Rhynchosciara americana

was fully induced in vitro and shown to be active in RNA

synthesis when the proximal region of glands from larvae

initiating cocoon spinning were incubated in a media speci-

fic for this organism which contained hemolymph from

young larvae and ecdysone (Alvarenga, 1980). A series of

DNA puffs was induced in glands of Trichosia pubescens

cultured in modified Cannon’s medium supplemented with

bovine serum albumin and ecdysone shortly before the time

that these puffs would normally appear, but DNA puffs

could not be induced in cultured glands from younger larvae

(Ferreira, 1981). In no case, has the entire DNA puffing

cycle been induced in vitro in glands of late fourth instar,

feeding sciarid larvae.

Although DNA puff formation is induced by the injection

Mechanisms of Development 110 (2002) 15–26

0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0925-4773(01)00589-5

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* Corresponding author. Tel.: 155-16-6023347; fax: 155-16-6331786.

E-mail address: [email protected] (M.L. Paco-Larson).1 Present adress: Fundacao Hemocentro Ribeirao Preto, R. Tenente Catao

Roxo, 2501, Ribeirao Preto 14051-140, SP, Brazil.

of exogenous hormone the activity of different DNA puffs

seems to be differentially controlled by ecdysone. While

high levels of ecdysone are necessary to maintain in vitro

the synthesis of a set of polypeptides related to a group of B.

hygida DNA puffs, the presence of the hormone had an

inhibitory effect on the synthesis of polypeptides related

to another group of DNA puffs that expands later (Carvalho

et al., 2000). In this context, studies on specific DNA puff

genes are needed to understand the mechanisms underlying

the ecdysone response of these genes.

During the last decade, the use of transgenic Drosophila

has been employed to extend the characterization of DNA

puff gene regulation at the molecular level (Bienz-Tadmor

et al., 1991; Monesi et al., 1998, 2001). Two of the genes

analyzed were able to drive transcription in prepupal sali-

vary glands in a temporally regulated manner, suggesting

that factors necessary for tissue-specific and developmen-

tally regulated expression of these sciarid genes may be

conserved in Drosophila.

The mechanisms by which ecdysone regulates RNA puff

genes in Drosophila has been extensively studied. The

entire sequence of puffing in third instar larvae can be repro-

duced in vitro when salivary glands are cultured in the

presence or absence of ecdysone. The model proposed by

Ashburner et al. (1974), based on a series of in vitro experi-

ments, predicted the existence of a hormone receptor

complex which would directly activate a set of early

genes, visualized as a set of early puffs. The product of

the early genes would repress their own transcription and

induce another set of puffs, the so-called late puffs.

This model has been confirmed at the molecular level.

Ecdysone binds a heterodimer formed by two nuclear recep-

tors, EcR and USP, and this complex directly induces the

transcription of early puff genes (Koelle et al., 1991; Yao et

al., 1992, 1993; Talbot et al., 1993; Thomas et al., 1993).

The molecular characterization of three early puffs (2B5,

74EF and 75B) resulted in the isolation of the Broad-

Complex (Br-C), E74 and E75 genes, which consist of tran-

scription factors that serve to transmit the ecdysone signal to

downstream target genes (Burtis et al., 1990; Segraves and

Hogness, 1990; Thummel et al., 1990; DiBello et al., 1991;

Bayer et al., 1996a). More recently two other early puffs

have been characterized at the molecular level. The 23E puff

contains the E23 gene, encoding an ecdysone-induced ABC

transporter, whose expression is correlated with the ecdy-

sone pulses. This transporter probably acts as a negative

regulator of ecdysone-mediated signaling (Hock et al.,

2000). The 63 F early puff contains two divergently tran-

scribed genes, E63-1, a member of the EF-hand family of

Ca21 binding proteins and E63-2, which has no apparent

open reading frame (Vaskova et al., 2000).

The late puffs, which are more numerous, are less well

characterized. The late genes are expected to be the targets

of early gene products and to encode effector molecules

with specific roles during metamorphosis. The 71E late

puff contains a cluster of six salivary gland specific genes,

which appear to encode antimicrobial peptides which may

protect metamorphosing tissues (Restifo and Guild, 1986;

Wright et al., 1996). The study of L71 regulation has

provided evidence for direct regulation of late genes by

early genes (Fletcher and Thummel, 1995; Crossgrove et

al., 1996). Another late puff is 4F, which contains a single

transcription unit (Wolfner, 1980). The characterization of

late puffs was recently extended by the molecular and

genetic characterization of the 63E and 82F late puffs.

The L63 and L82 genes are essential, comprise up to 85

kb of genomic DNA and encode multiple RNA isoforms

displaying complex developmental patterns. L63 shows

strong homology to the cyclin dependent kinase protein

family and the L82 has homology to a novel gene family

(Stowers et al., 1999, 2000). At present, the mechanisms by

which ecdysone regulates the late genes are poorly under-

stood. The understanding of these mechanisms is necessary

to comprehend how the hormonal signal is correctly trans-

duced in each target tissue.

In this report we focus on a special class of late response

puffs, namely the DNA puffs of sciarid flies. In particular,

we extend the characterization of DNA puff BhC4-1 gene by

analyzing its expression during development and investigat-

ing the response of this gene to ecdysone in Bradysia hygida

and in a Drosophila melanogaster line transformed with the

BhC4-1 gene. Our results indicate that the induction of

BhC4-1 expression in B. hygida and in Drosophila, as

well as amplification and puff C4 expansion in B. hygida,

are secondary events induced by the increase of ecdysone

titers that occur at the end of the last larval instar. Several

hours after the injection of ecdysone into B. hygida mid-

fourth instar larvae, the expression of BhC4-1 mRNA and

protein, as well as BhC4-1 amplification and puff C4 expan-

sion are induced. In vitro experiments in transgenic Droso-

phila show that the BhC4-1 mRNA is induced after 6 h of

incubation in the presence of ecdysone. Interestingly, in the

absence of exogenous hormone, but in the presence of

cycloheximide, the BhC4-1 mRNA is also detected,

suggesting that the temporal regulation of BhC4-1 requires

a repressor which prevents its transcription in the salivary

gland before the hormone titers increase at the end of the last

larval instar.

2. Results

2.1. The BhC4-1 expression in B. hygida

Previous studies have shown that the BhC4-1 gene is

amplified and abundantly expressed in fourth instar salivary

glands at the time when DNA puff C4 expands (Paco-Larson

et al., 1992). In order to verify if BhC4-1 expression occurs

in other developmental stages, Northern blots containing

about 10 mg of total RNA from whole animals were hybri-

dized with a 1.2 kb BhC4-1 cDNA fragment (Fig. 1).

Confirming previous data, the 1.4 kb BhC4-1 mRNA is

L.R. Basso Jr. et al. / Mechanisms of Development 110 (2002) 15–2616

detected at late fourth instar stages (E7 and E7 1 4 h) at the

time DNA puff C4 is formed. Lower levels of BhC4-1

mRNA are still detected in early pupae (P), when DNA

puff C4 has already regressed, whereas no expression was

observed in embryos (E), whole second and third instar

larvae (2/3), adult males (M) or adult females (F). These

results indicate that BhC4-1 expression is restricted to the

end of the fourth larval instar at the time of DNA puff C4

formation. Furthermore, by Northern blot analysis the

BhC4-1 mRNA is detected in late fourth instar salivary

glands but not in the respective carcasses, suggesting that

high levels of BhC4-1 expression only occur in the larval

salivary glands (Fig. 1).

The characterization of BhC4-1 mRNA expression in the

salivary gland during larval-pupal transition was further

investigated through RT-PCR experiments. One point five

micrograms of total RNA extracted from salivary glands at

different times at the end of the fourth larval instar and

beginning of pupal life, and ten fold serial dilutions, were

employed as the starting point in each reverse transcriptase

reaction. PCR reactions were performed using primers

specific for the BhC4-1 gene (Fig. 2A) and 16S mitochon-

drial RNA (employed as an internal control for the cDNA

amount), which resulted in the amplification of 362 and 580

bp PCR products, respectively. BhC4-1 mRNA is initially

detected at stage E3. It reaches its maximum levels at stage

E7 and decreases during the beginning of the pupal stage

(P). This analysis indicates that between E3 and E7 there is

about a 10,000 fold increase in the amounts of BhC4-1

mRNA in the gland (Fig. 2B).

With the aim of investigating a correlation between varia-

tions in the endogenous ecdysteroid titers and the induction

of BhC4-1 expression in the salivary gland, the hormone

titers in the hemolymph were measured by radio immuno

assay (RIA), at different time points during the fourth instar.

Two ecdysone peaks were detected, a small peak at stage

E1, which corresponds to a 15 fold increase in the ecdyster-

oid titer and a second and broader peak at stage E7, corre-

sponding to a 200 fold increase over basal hormone levels.

A decay in the ecdysone levels was observed before the

onset of pupation, after which the hormone levels start to

rise again (Fig. 2C). The BhC4-1 induction at stage E3

occurs at a time when the hormone levels are rising, whereas

the highest ecdysteroid titer in the hemolymph (2.3 £ 1027

M) was detected at stage E7, when BhC4-1 mRNA levels

was at maximum.

2.2. The formation of B. hygida DNA puff C4, and BhC4-1

expression and amplification are induced by ecdysone

DNA puffs of B. hygida are classified into two groups

based on the time of expansion. A first group is formed at

stage E7, and 8 h after E7 these DNA puffs regress. Sixteen

hours after E7, a second DNA puff group expands which

regresses 10 h later, just before the pupal molt (Sauaia et al.,

1971; Laicine et al., 1984).

The BhC4-1 gene is located at DNA puff C4, which is a

member of the first DNA puff group and its maximum

expression levels occur when the ecdysone titers in the

hemolymph are high. With the aim of investigating whether

high levels of ecdysone could induce the formation of DNA

puff C4 in B. hygida, fourth instar larvae at stage E1 (about

70 h before DNA puff C4 formation) were injected with 1.0

ml of 0.4 mM ecdysone and their salivary gland polytene

chromosomes were analyzed at different time points after

injection. Twenty hours after injection about 75% of the

analyzed chromosomes displayed the DNA puffs C4 and

C5 expanded and the DNA puff C7 regressed, a puffing

pattern characteristic of stage E7 1 2 h (Fig. 3). These

results show that the hormone treatment anticipates DNA

puff formation by 48 h. Analysis of control animals at differ-

ent times after injection reveals that the same puffing pattern

(E7 1 2 h) is only observed 60–70 h after ethanol injection

(data not shown). In addition, DNA puff anlagen, which are

characterized as an increase in the thickness of the chromo-

some bands involved in the formation of DNA puffs (Sauaia

et al., 1971), can also be observed at all major DNA puff

forming sites (Fig. 3).

In order to verify if BhC4-1 mRNA expression is also

induced by the hormone, larvae at stage E1 were injected

with ecdysone and analyzed by Northern blot at different

times after injection (1, 2, 4, 8, 16 and 24 h). The 1.4 kb

L.R. Basso Jr. et al. / Mechanisms of Development 110 (2002) 15–26 17

Fig. 1. The BhC4-1 expression is developmentally regulated and is salivary

gland specific. Upper autoradiograms: Northern blots containing 20 mg of

total RNA extracted from embryos (E), whole second and third instar larvae

(2/3), whole fourth instar larvae at stages E7 and E7 1 4 h, pupae (P), adult

males (M) and adult females (F) (left hand blot) or about 10 mg of total

RNA extracted from dissected carcasses (CS) and salivary glands (SG) of

fourth instar larvae at E7, (right hand blot), after hybridization with a 1.2 kb

BhC4-1 cDNA fragment (Monesi et al., 1995). The arrows point to the 1.4

kb BhC4-1 mRNA. Bottom autoradiograms: to demonstrate the loading of

the lanes, the blots were stripped, followed by hybridization with a rDNA

fragment from Rhynchosciara americana. (Zaha et al., 1982).

BhC4-1 mRNA is detectable in salivary glands 16 h after

hormone injection, and high levels of expression are

detected 8 h later (Fig. 4A). BhC4-1 mRNA expression is

not detected in salivary glands of control larvae, injected at

stage E1 with 1 ml of 5% ethanol and dissected 24 h later.

The induction of BhC4-1 protein expression by hormone

treatment was also investigated in immunoblots containing

salivary gland extracts from larvae dissected 16 or 24 h after

ecdysone injection. Using an affinity purified anti-BhC4-1

serum, the BhC4-1 43 kDa polypeptide is detected in sali-

vary glands 24 h after hormone injection, whereas no BhC4-

1 expression is verified in salivary glands of larvae injected

with 5% ethanol (Fig. 4B).

The DNA puff BhC4-1 gene is amplified about 21 fold in

the salivary gland at the end of the fourth larval instar (Paco-

Larson et al., 1992). In order to investigate if BhC4-1 ampli-

fication is induced by ecdysone, larvae at stage E1 were

hormone treated followed by Southern blot analysis. As

can be observed in Fig. 4C, salivary glands of ecdysone-

treated larvae exhibit a higher number of BhC4-1 copies 24

h after injection, when compared to control salivary glands

from larvae injected with 5% ethanol. BhC4-1 amplification

levels in the salivary gland reached peak levels 36 h after the

ecdysone injection. Taken together these results indicate

that DNA puff C4 formation, as well as BhC4-1 expression

and amplification are all induced by ecdysone treatment and

constitute a late response to elevated levels of this hormone.

2.3. The BhC4-1 response to ecdysone is conserved in

transgenic Drosophila

In a previous report we have shown that in transgenic

Drosophila the BhC4-1 gene is transcribed in salivary

glands of early-prepupae in a temporally regulated manner.


Fig. 2. Developmental pattern of BhC4-1 expression in the salivary gland of Bradysia hygida and titers of 20-hydroxyecdysone in the hemolymph at the end of

the fourth larval instar. (A) Diagram of the BhC4-1 gene. The open boxes represent the BhC4-1 exons. The position of the primers employed in the RT-PCR

experiments is indicated by arrowheads. Note that the use of this pair of primers results in a 362 bp fragment when the cDNA was amplified, and that a 429 bp

PCR product was amplified when genomic DNA was used as template. (B) BhC4-1 mRNA expression in the salivary gland at the end of the fourth instar.

Images of 2% agarose gels stained with ethidium bromide after electrophoresis of RT-PCR products. Salivary glands from animals at different stages (E1, E3,

E5, E7 and pupae) were dissected followed by total RNA extraction. 1.5 mg of total RNA and 10 fold serial dilutions (indicated on top of the lanes) were

employed to synthesize the cDNAs. The 362 and 580 bp PCR fragments were amplified using primers specific for the BhC4-1 gene and a fragment of 16 S

mitochondrial RNA, respectively. (C) Control PCR reaction in which no cDNA was added. (M) 100 bp DNA ladder (Gibco BRL). (C) Ecdysteroid titers in the

hemolymph of Bradysia hygida larvae determined from individual larvae at different stages using a highly sensitive RIA. Mean values and standard errors

represent measurements of at least four animals per developmental stage. Standard curves were generated using 20-hydroxyecdysone as the nonradioactive

ligand, and the results are consequently expressed as 20-hydroxyecdysone equivalents.

These results have indicated that the regulatory elements

involved in the expression of this gene are conserved

between D. melanogaster and B. hygida since Drosophila

transcription factors appear to recognize BhC4-1 regulatory

DNA sequences (Monesi et al., 1998). To precisely define

the stage during which BhC4-1 is activated in Drosophila,

we have analyzed its expression in transgenic salivary

glands using RT-PCR. Total RNA was extracted from single

salivary gland lobes dissected from animals at different

stages during the third larval instar and during the whole

prepupal stage. PCR reactions employed primers specific

for the BhC4-1 gene and for mitochondrial RNA, which

result in 362 and 580 bp PCR products, respectively. The

results show that BhC4-1 expression is initially detected at

stage PS9, about 90 min before pupariation. The levels of

BhC4-1 mRNA start to rise in 1 h prepupae, reach maximum

levels at 2–3 h prepupae, slowly decrease during the rest of

the prepupal stage, and are almost undetectable in 12 h

prepupae (Fig. 5).

The induction of BhC4-1 expression in transgenic Droso-

phila at stage PS9 could be a response to the presence of

high levels of ecdysone that occur in the larval-prepupal

transition in Drosophila (Bayer et al., 1996b). To investi-

gate this question, salivary glands at stage PS1 were

dissected, and one set of the sister salivary gland lobes

was cultured for defined times in Grace’s medium contain-

ing 2 £ 10 2 6 M ecdysone before being analyzed by RT-

PCR. The expression levels of BhC4-1, of 16S mitochon-

drial rRNA (internal control of cDNA amount), and of three

other Drosophila genes were simultaneously assessed in

aliquots of the total cDNA. The ecdysone response of the

EcR, E74A and E78B Drosophila genes had already been

characterized by RT-PCR (Huet et al., 1993, 1995). Accord-

ing to Huet et al. (1993) the EcR gene is an intermoult gene,

which is inhibited by high levels of the hormone, and it is

the only gene that should be active at stage PS1. The two

other Drosophila genes, E74A and E78B are representatives

of the early and early-late classes, respectively, and are

genes directly induced by the hormone. When E74A expres-

sion was detected in the set of sister salivary gland lobes


Fig. 3. The injection of 20-hydroxyecdysone induces DNA puff formation in the salivary gland chromosomes of Bradysia hygida. Photomicrographs of lactic-

acetic squashes of salivary glands. Larvae at stage E1 were injected with 5% ethanol (control group, left hand photographs) or 0.4 mM ecdysone (ecdysone

group, right hand photographs) and dissected 24 h latter followed by fixation. The four chromosomes (A, B, C and X) are shown. The arrows point to the

expanded DNA puffs C4 and C5. DNA puff C7, already regressed, is indicated by a star inside an oval. The stars indicate DNA puff anlagen, which were also

induced by the hormone treatment.

employed as control, which were not in vitro incubated with

the hormone, the whole experiment was not considered for

further analysis.

As shown in Fig. 6, BhC4-1 expression in transgenic

Drosophila is induced by ecdysone. Its mRNA is initially

detected in salivary glands after 6 h of incubation, indicating

that its induction occurs between 5 and 6 h after hormone

treatment. Higher levels of BhC4-1 mRNA are observed

after 8 h incubation, whereas shorter incubation times do

not result in BhC4-1 detection. The response of the EcR,

E74A and E78B Drosophila genes to the hormone is in

agreement with a previous report (Huet et al., 1995). The

EcR gene is repressed after 2 h incubation, and both E74A

and E78B are detected already after a 2 h incubation in the

presence of the hormone. Such responses are characteristic

of intermoult and early genes, respectively. In late third

instar larvae the late responding puffs show a delay in

their response to ecdysone by more than 4 h (Ashburner,

1972). Therefore, the BhC4-1 induction in Drosophila can

be classified as a late response to high levels of hormone.

We next investigated whether the induction of BhC4-1 by

the hormone was dependent on protein synthesis. Salivary

glands at stage PS1 were dissected and incubated for 6 or 8 h

in medium containing ecdysone (final concentration


Fig. 4. BhC4-1 expression and amplification are both induced by ecdysone. (A) Upper autoradiogram: Northern blot containing about 20 mg of total RNA

extracted from salivary glands of larvae injected at stage E1 with 0.4 mM ecdysone and dissected at different times after injection (1, 2, 4, 8, 16 and 24 h), up to

20 mg of total RNA extracted from salivary glands of larvae injected with 5% ethanol and dissected 24 h latter (Eth/24) and 20 mg of total RNA extracted from

salivary glands of larvae at stage E7 (E7) after hybridization with a 1.2 kb BhC4-1 cDNA fragment. The arrow points to the 1.4 kb BhC4-1 mRNA. Bottom

autoradiogram: to demonstrate the loading of the lanes, the blot was stripped, followed by hybridization with a 500 bp fragment from a cDNA encoding the

mitochondrial large rRNA subunit of Anopheles gambiae (Lemos et al., 1996). (B) Left hand picture, Coomassie Blue stained SDS-PAGE (9%) of total

salivary gland extracts of larvae injected at stage E1 with ecdysone (ecdysone lanes) or with 5% ethanol (ethanol lanes) and dissected (16) or (24) h latter and

total salivary gland extracts of larvae at stage (E7). Right hand picture, immunoblot of an identical gel after incubation with an affinity-purified anti-BhC4-1

serum (1:1000), followed by detection with the ECF western blotting kit (Amershan Pharmacia biotech). The 43 kDa polypeptide detected 24 h after hormone

treatment and present in salivary gland extracts of larvae at stage E7 is the BhC4-1 product (Silva Jr., 1997). The polypeptides detected in the 65-145 kDa

region in E7 salivary gland extracts consist of aggregates of the 43 kDa polypeptide. (C) Upper autoradiogram: Southern blot containing 10 mg of EcoRI

digested DNA extracted from salivary glands of larvae at stage E1 (prior to DNA puff C4 formation) and E7 (DNA puff C4 formation), 10 mg of EcoRI digested

DNA extracted from salivary glands of larvae injected at stage E1 with 0.4 mM ecdysone and dissected at different times after injection (14, 16, 24 and 36 h)

and 10 mg of Eco RI digested DNA extracted from salivary glands of larvae injected at stage E1 with 5% ethanol and dissected at different times after injection

(16, 24 and 36 h) after hybridization with a 1.2 kb BhC4-1 cDNA fragment. This cDNA fragment detects a 9 kb Eco RI genomic fragment. To demonstrate the

loading of the lanes, the blot was stripped, followed by hybridization with a cDNA fragment encoding the elongation factor 1a (EF-1a) of Rhynchosciara

(Graessmann et al., 1992).

2 £ 1026 M) plus cycloheximide (final concentration

1 2 1024 M) or cycloheximide alone (final concentration

1 £ 1024 M) or Grace’s medium alone. When E74 expres-

sion was detected in the control group (non-incubated sister

lobes) the result was not considered for further analysis. The

expression of BhC4-1, 16S mitochondrial rRNA, EcR, E74A

and E78B genes was assessed by PCR in aliquots of the total

cDNA obtained from each experimental group.

Unexpectedly, BhC4-1 expression is detected after incu-

bation in medium containing ecdysone plus cycloheximide,

as well as in medium containing only cycloheximide (Fig.

7). The incubation in Grace’s medium alone, without any

additives, does not lead to BhC4-1 expression, indicating

that its transcription is not inadvertently triggered by some

factor present in the medium. If the expression of BhC4-1

did not depend on protein synthesis, which is the case of E74

and E78 genes that are directly induced by the hormone,

higher levels of BhC4-1 expression should have been

detected in glands treated with ecdysone plus cyclohexi-

mide. In view of these results, the most likely explanation

for BhC4-1 expression in the absence of protein synthesis is

that the synthesis of a BhC4-1 repressor(s) is (are) being

inhibited by cycloheximide (Fig. 7).

3. Discussion

3.1. The induction of DNA puff BhC4-1 amplification and

expression is a late response to the increase of ecdysone

titers in the hemolymph of B. hygida late larvae.

Our results represent the first detailed characterization of

the ecdysone effect on the expression and amplification of

a particular DNA puff gene. BhC4-1 mRNA is initially

detected at stage E3, a time when extra copies of BhC4-1

gene are also initially detected in the gland (Paco-Larson et

al., 1992). The beginning of BhC4-1 amplification and

transcription coincides with the increase in the ecdysone


Fig. 6. Induction of BhC4-1 expression in transgenic salivary glands incu-

bated in the presence of ecdysone. Image of 2% agarose gels stained with

ethidium bromide after electrophoresis of RT-PCR products. Groups of

nine larvae at stage PS1 were collected and the salivary glands dissected.

One set of sister lobes was employed to analyze the pattern of gene expres-

sion at the time of dissection (0 h). The other set of sister lobes was

incubated for 2, 4, 5, 6 and 8 h, respectively, in Grace’s medium containing

2 £ 10 2 6 M ecdysone. Primers specific for each of the analyzed genes

were employed in the RT-PCR reactions. Of the total cDNA generated from

each set of nine lobes 8% was employed as a template to amplify the 16S

mitochondrial rRNA and 23% as a template for each the BhC4-1, EcR,

E74A and E78B genes.

Fig. 5. Pattern of BhC4-1 expression in the salivary gland of transgenic Drosophila. Images of 2% agarose gels stained with ethidium bromide after

electrophoresis of RT-PCR products. Salivary glands from animals at different larval stages (PS 1 to 11, left hand photograph) and from animals at different

times after 0 h prepupae (0–12 h, right hand photograph) were dissected followed by total RNA extraction from a single lobe. Analysis during the third instar

employed 100% of total RNA extracted from a single lobe in the reverse transcription reaction. From the total cDNA obtained, 80% was employed as a

template to amplify the BhC4-1 gene (362 bp) and 20% as a template for the 16 S mitochondrial RNA (580 b), respectively. Due to higher levels of BhC4-1

expression during the prepupal stage only 25% of total RNA extracted from a single lobe was employed in the reverse transcription reaction. Equal amounts

(50%) of the resulting cDNA were employed as templates to amplify both the BhC4-1 gene and the 16 S mitochondrial rRNA. (C) Negative control in which no

reverse transcriptase was added to the RT-PCR reaction.

titer that precedes the pupal molt, while puff C4 is maxi-

mally expanded 24 h later (stage E7), when the ecdysone

titers are highest. Therefore, these observations indicate

that BhC4-1 amplification and expression, as well as the

expansion of puff C4, depend on the presence of high

levels of ecdysone in the larval hemolymph. This interpre-

tation is in agreement with in vitro studies which have

shown that the presence of high levels of ecdysone are

necessary to maintain the synthesis of a set of polypeptides

characteristic of the period when the DNA puff C4 is active

(Carvalho et al., 2000).

The injection of high doses of ecdysone in B. hygida

larvae 96 h before the pupal molt anticipates the program

of DNA puff expansion by about 48 h. Twenty-four hours

after the hormone treatment the DNA puffs C5 and C4 are

expanded. These puffs are among the first DNA puffs to

form during B. hygida development (Laicine et al., 1984).

The fact that the induction of puffs C5 and C4 by ecdysone

requires a long time interval agrees with previous studies

showing that the expansion of DNA puffs is a late response

to the hormone. In Sciara coprophila (Crouse, 1968) and

Rhynchosciara hollaenderi (Stocker and Pavan, 1974) the

induction of the characteristic DNA puffs occurs within 24 h

after hormone injection, while in Trichosia pubescens

(Amabis and Amabis, 1984a) changes in DNA puff forming

sites are not observed until approximately 10 h after ecdy-

sone injection. Earlier autoradiographic studies have also

shown that 9–27 h after ecdysone injection the majority of

the salivary gland nuclei are engaged in DNA synthesis

(Crouse, 1968; Stocker and Pavan, 1974; Fresquez, 1979).

Although induction of DNA synthesis was observed all over

the chromosomes, the DNA puff loci present a much higher

rate of [3H] thymidine incorporation when compared with

the other chromosomal bands. This was interpreted as

evidence that DNA amplification was being induced by

the hormone (Crouse, 1968; Amabis and Amabis, 1984a).

The results presented here agree with those of earlier obser-

vations. Twenty-four hours after injection of the hormone,

DNA puff anlagen are clearly observed in all major DNA-

puff forming sites. DNA puff anlagen are characterized by

an increase in the thickness of the chromosome bands that

later will expand in DNA puffs, reflecting the presence of

the extra amounts of DNA which are synthesized at these

sites (Sauaia et al., 1971). Also in agreement with the cyto-

logical observations our studies on BhC4-1 gene shows that

the injection of ecdysone into larvae at an earlier stage leads

to early induction of BhC4-1 expression along with DNA

amplification and puff expansion. These events occur

around 16–24 h after ecdysone injection, indicating that

they constitute late responses to the hormone.


Fig. 7. The BhC4-1 expression in vitro is detected in the absence of protein synthesis. Images of 2% agarose gels stained with ethidium bromide after

electrophoresis of RT-PCR products. Groups of nine larvae at stage PS1 were collected and one set of salivary gland lobes was incubated during 6 or 8 h in

medium containing either 2 £ 1026 M ecdysone plus 1 £ 1024 M cycloheximide, or 1 £ 1024 M cycloheximide, or in Grace’s medium only. The other set of

lobes was employed to analyze the pattern of gene expression at the time of dissection (0 h). Of the total cDNA generated from each set of nine sister lobes 8%

were employed as a template to amplify the 16S mitochondrial rRNA and 23% as a template for each the BhC4-1, EcR, E74A and E78B genes. The slight

differences in the amount of BhC4-1 mRNA in glands incubated in the presence of ecdysone plus cycloheximide or cycloheximide alone are due to differences

in the loading of the gels.

3.2. The ecdysone response of BhC4-1 in transgenic

Drosophila reproduces the hormone effect in B. hygida

Our investigation of the ecdysone effect on BhC4-1

mRNA expression in transgenic Drosophila strengthens the

view that the regulatory mechanisms for BhC4-1 expression

are conserved between B. hygida and Drosophila.Previous

results have shown that a 3.6 kb fragment from the BhC4-1

gene 5 0 region is able to drive transcription in transgenic

Drosophila, specifically in salivary glands of prepupae

(Monesi et al., 1998). Here we extend the characterization

of BhC4-1 gene expression in the salivary glands of third

instar larvae and prepupae of a transgenic Drosophila line

carrying the BhC4-1 gene. The analysis by RT-PCR demon-

strates that BhC4-1mRNA is initially detected in the salivary

gland of late third instar larvae, reaches a peak in early prepu-

pae and is barely detectable at the end of the prepupal stage.

These results indicate that, similar to what is observed in B.

hygida, the induction of the BhC4-1 transgene coincides with

the increase in ecdysone titers in the late larval instar. When

less sensitive methods were employed (Monesi et al., 1998),

the BhC4-1 mRNA was mainly detected during prepupal

stage like what has been described for the mRNAs of Droso-

philalate puffs 71E and 4F (Andres et al., 1993), which are

induced in newly formed prepupae as a secondary response to

the metamorphic ecdysone peak. Consistent with a late gene

response is the observation that the addition of high amounts

of ecdysone to the medium of cultured transgenic salivary

glands induces BhC4-1 mRNA expression only after 6 h of

incubation. The ecdysone effect on BhC4-1 mRNA induction

in Drosophila is reminiscent of what we observe when inject-

ing ecdysone into mid-fourth instar larvae of B. hygida. This

same conclusion can not be drawn from the in vitro response

to ecdysone of a 719 bp promoter of DNA puff II/9-1 gene

from Sciara coprophila. In transgenic Drosophila the repor-

ter activity driven by the Sciara promoter was rapidly

induced by ecdysone (Bienz-Tadmor et al., 1991), different

than one would expected based on the cytological studies

which indicate that induction of S. coprophila DNA puffs

is a late ecdysone response (Crouse, 1968). This early

response of the S. coprophilapromoter to ecdysone could

be explained if the II/9-1 gene is, like BhC4-1, regulated by

a repressor. An early induction of the II/9-1 gene would occur

if the repressor binding site was missing in the 719 bp frag-

ment of II/9-1 promoter or if Drosophila lacked the repressor.

When protein synthesis was inhibited by the addition of

cycloheximide to the medium, BhC4-1 mRNA was detected

in transgenic salivary glands cultivated for 6 or 8 h even in

absence of exogenous hormone. The simplest explanation

for this result is that BhC4-1 regulation requires repressor

factor (s), which prevent(s) its transcription in the salivary

gland before the increase in endogenous hormone titers at

the onset of metamorphosis. Moreover, the time-course of

BhC4-1 hormonal induction indicates that the amount of

BhC4-1 mRNA in glands cultivated for 8 h in the presence

of the hormone is higher than the one observed after 6 h of

incubation. This increase in BhC4-1 mRNA, from 6 to 8 h of

incubation, does not seem to occur when protein synthesis is

inhibited by the addition of cycloheximide to the medium,

which agrees with the necessity of protein synthesis for the

induction of a late-responsive gene. The activation of the

BhC4-1 gene, specifically at the prepupal transition, might

involve stage- and tissue-specific regulatory proteins. Addi-

tionally, the timing of BhC4-1 expression in the salivary

gland could be determined by more ubiquitous trans-acting

regulators such as BR-C, as it was demonstrated for L71

(Crossgrove et al., 1996) and Fbp1 genes (Mugat et al.,

2000). The influence of the ecdysone receptor on BhC4-1

activity must also be considered. Using an antiserum against

a Chironomus tentans ecdysone receptor, imunofluores-

cence signals were observed at DNA puff sites of Trichosia

pubescens from the beginning of amplification/transcription

until puff regression (Stocker et al., 1997).

Taken together, our data provide clear evidence that an

increase in the endogenous ecdysone titer elicits an ecdy-

sone response cascade that results in DNA replication and

transcriptional activity of DNA puff BhC4-1 gene. Based on

time course characteristics and protein inhibitor experi-

ments, BhC4-1 mRNA expression can be classified as a

late response that requires removal of a repressor. This

information is valuable for future genetic and molecular

studies on the identification of factors that participate in

the hormonal regulation of tissue-specific late genes.

4. Experimental procedures

4.1. Bradysia hygida maintenance, life cycle and staging

Whole animals at different developmental stages or sali-

vary glands from fourth instar female larvae were obtained

from a laboratory culture kept at 208C (Sauaia et al., 1971).

At this temperature the life cycle lasts 36 days (Laicine et

al., 1984). Twelve days after hatching the larvae undergo the

third molt. On the 18th day after hatching (sixth day of the

fourth instar) the larval eyespots appear (E1). The eyespot

stage, E3 (eighth day of the fourth instar), is coincident with

the beginning of DNA puff anlage formation and also is the

stage during which the larvae abandon the food and start

spinning a cocoon. During the following 46 h there are

striking changes in the puffing pattern of the salivary

gland chromosomes. The eyespot stage, E7, is a reliable

indicator for predicting the puffing pattern. Staging of

older larvae is defined in hours after E7.

The salivary glands comprise three distinct morphologi-

cal regions, S1, S2 and S3, and there are four chromosomes,

A, B, C and X in the salivary gland cells. DNA puff C4 is

part of a DNA puff group that expands around E7, maxi-

mum DNA puff C4 expansion occurs at stage E7 1 4 h.

Sixteen hours after E7, when the first DNA puff group has

regressed, a second DNA puff group expands (Laicine et al.,

1984).


4.2. Drosophila maintenance and staging

Drosophila transgenic line P3A was described elsewhere

(Monesi et al., 1998). Stocks were kept at 258C in Caltech

medium (Elgin and Miller, 1980). The accurate staging of

larval stages (PS1–PS11) was performed through the cyto-

logical analysis of the pattern of RNA puffs in one of the

salivary gland lobes, according to Ashburner (1972). Prepu-

pal times were counted from the time of puparium forma-

tion. In order to stage larvae at PS1 the animals were reared

in Caltech medium containing 0.05% bromophenol blue and

staged following the methodology of Maroni and Stamey

(1983).

4.3. Nucleic acid isolation

Bradysia hygida RNA and DNA were extracted essen-

tially as described by Coelho et al. (1993). Drosophila sali-

vary gland RNA was extracted using Trizol LSe (Gibco-

BRL) according to the manufacturer’s instructions with the

following modifications: after dissection the salivary glands

were frozen in liquid nitrogen, Trizol LSe was added (75 ml

of Trizol LSe, per salivary gland lobe), followed by vortex-

ing during 5 min., at room temperature. After the addition of

chloroform (20% of the volume) followed by vortexing

during 3 min., and centrifuging at 12000 £ g, 15 min.,

48C, the upper aqueous phase was collected followed by

the addition of one volume of isopropanol and incubation

in a dry ice-ethanol bath during 45 min. Total RNA was

precipitated by centrifuging 12,000 £ g, 35 min., 48C,

followed by one wash in 75% ethanol. After drying, the

RNA was dissolved in water.

4.4. Northern and Southern blot hybridization

About 20 mg of total RNA from different developmental

times or 10 mg from dissected tissues were run in formalde-

hyde denaturing agarose gels (Sambrook et al., 1989). In

Southern blot experiments, about 10 mg of genomic DNA

extracted from salivary glands were digested with Eco RI

and run in 0.8 % agarose gels in 1 £ TAE buffer (Sambrook

et al., 1989). Northern and Southern blotting were

performed onto nylon membranes (Hybond N, Amershan).

Probes were labeled with a32P-dCTP by random primer

reaction. Hybridization and post-hybridization washes

were performed according to Sambrook et al. (1989).

4.5. Reverse transcriptase-polymerase chain reactions (RT-

PCR)

Total RNA samples were treated with 1 unit of RNase I

free-DNase I (Gibco-BRL) for 15 min. at 258C, followed by

the addition of 1 ml of 50 mM EDTA and incubation for 10

min. at 658C to inactivate DNase I. After the addition of 50

ng of hexamers, pd(N)6 (Pharmacia) and 50 ng of oligo dT

(Pharmacia), RNA samples were incubated at 708C for 10

min., after which the samples were kept in ice. Reverse

transcriptase reactions were performed in the presence of

1 £ SuperScript IIe buffer (Gibco-BRL), 5 mM DTT, 0.5

mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP

and 200 units of SuperScript IIe(Gibco-BRL), for 50 min.,

at 428C. SuperScript IIe was inactivated by incubation at

708C, for 15 min. PCR reactions were performed using

primers specific for each of the analyzed genes. Primer

sequences specific for the EcR, E74A and E78B genes

were described by Huet et al. (1993). Primer sequences

specific for the internal control (16S mitochondrial RNA)

were described by Simon et al. (1994). Primer sequences

specific for the BhC4-1 gene are as follows: 5 0CAGATTT-

GTTACTTACTTGC3 0 and 5 0CGGTGTTGATGTCTGT-

TTCC3 0. PCR reactions were performed separately for

each analyzed gene. Each PCR reaction contained 40

pmol of each primer, 1 £ Taq polymerase buffer (Gibco-

BRL), 1.5 mM MgCl2, 0.2 mM dATP, 0.2 mM dCTP, 0.2

mM dTTP, 0.2 mM dGTP and 2.5 units of Taq polymerase

(Gibco-BRL). Amplification reactions employed the

following cycles: 1 cycle at 948C, 5 min.; 30 cycles of

948C 1 min., 558C 2 min., 728C 3 min.; 1 cycle 728C, 10

min. PCR reactions in which the internal control fragment

was amplified employed 508C in the annealing step.

4.6. Radioimmunoassay (RIA)

Ecdysteroid contents of individual larvae at different

stages of development were determined by a highly sensi-

tive RIA, as previously described (Hartfelder et al., 2001).

Due to the small amount of hemolymph per larvae, the

animals were dissected and rinsed in 35 ml of Ringer solu-

tion, of which 30 ml were then transferred to an Eppendorff

tube. Ecdysteroids were extracted by adding 500 ml of

methanol (2208C), followed by a 1 h incubation at 48C

before quantification by RIA. Ecdysone concentration at

the major peak was measured by collecting 0.5 ml to 1.0

ml of hemolymph, with a microcapillary tube, from larvae at

stage E7, followed by extraction with 100-fold volume of

methanol. The antiserum employed in these measurements

was a gift of W. Bollenbacher (UNC Chapel Hill) and used

at a final concentration of 0.05%. Standard curves covering

the range of 25–5000 pg were generated using 20-hydro-

xyecdysone (Simes, Milan) as the nonradioactive ligand.

Results were therefore expressed as 20-hydroxyecdysone

(20E) equivalents.

4.7. Ecdysone injection and cytological technique

Bradysia hygida larvae at stage E1 were collected and

injected either with 0.5 ml of 0.4 mM 20-hydroxyecdysone

dissolved in 5% ethanol or with 5% ethanol (control

groups). At different times after the injection the larvae

were dissected, and the salivary glands were removed and

fixed in 7% perchloric acid. After 3 h fixation, the S1 region

of the glands was isolated and transferred to a drop of 50%

acetic acid, rinsed twice in 50% acetic acid, followed by

squashing in 87% lactic acid: acetic acid (1:1). Cytological


preparations were observed under phase contrast optics. The

photomicrographs were recorded in an Zeiss photomicro-

scope, using Technical Pan 2415 (Kodak), ASA 50.

4.8. BhC4-1 Protein analysis

A 1.2 kb cDNA fragment from the BhC4-1 gene was sub-

cloned in frame with the maltose binding protein (MBP)

into the expression vector pIH902 (New England Biolabs).

MBP-BhC4-1 fusion-protein was used to immunize rabbits.

Total serum was initially depleted from anti-MBP antibo-

dies through affinity purification. The serum depleted from

anti-MBP antibodies was incubated with a nitrocellulose

membrane to which MBP-BhC4-1 fusion protein had been

previously bound. After elution with triethylamine for 5

min., followed by neutralization with 1 M Tris–Cl, pH

8.0, the antibodies were microdialyzed against TBS (0.15

M NaCl; 0.1 M Tris, pH 8.0). For SDS-polyacrylamide gel

electrophoresis, salivary glands were resuspended in SDS-

PAGE loading buffer and run according to Laemmli (1970).

Gels were run in duplicate, where one gel was stained with

Coomassie Blue and the other was blotted onto nitrocellu-

lose membranes for Western blot analysis, according to

Towbin et al. (1979). Bound antibodies were detected

using the ECF Western blotting kit (Amersham Pharmacia

Biotech).

4.9. In vitro incubation of Drosophila salivary glands

Third instar larvae staged at PS1 were dissected in 1 £

Ringer solution and cleaned from adhering tissue. Each

incubation experiment employed groups of nine salivary

gland lobes. The other nine sister lobes were frozen in liquid

nitrogen for analysis of the pattern of gene expression at the

time of dissection. In each experiment, three lobes were co-

incubated in 75 ml of incubation medium (1 £ Grace’s

medium: distilled water: 2.5 % ethanol (50:9:1)) at 258C

during defined time intervals. Ecdysone was added to the

ethanol fraction (final concentration 2 £ 10 2 6 M) and

cycloheximide was added to the water fraction (final

concentration 1 £ 10 2 4 M).

Acknowledgements

We thank Cirlei A.V. Saraiva and Benedita O. de Souza

for their dedicated technical assistance, Valdir Mazzucatto

for the maintenance of the Drosophila room and Claudia

R.M. Faggion and Domingos S.S. Filho for maintenance of

the B. hygida culture. We also thank Dr Ann J. Stocker for

critical reading of the manuscript and suggestions. This

work has been supported by grants from FAPESP.

ML.P.L. is a CNPq fellow, N.M. is a research fellow of

F.A.P.E.S.P., J.A.S. Jr. was supported by a fellowship

from CNPq, and C.V., P.S.R.C. and L.R.B. Jr. received

fellowships from CAPES.

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the induction of dna puff bhc4-1 gene is a late response to the

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