Induction of two cytochrome P450 ...

Induction of two cytochrome P450 genes, Biologia, Kofeina

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Gene 377 (2006) 56
–
64
www.elsevier.com/locate/gene
Induction of two cytochrome P450 genes, Cyp6a2 and Cyp6a8,
of Drosophila melanogaster by caffeine
in adult flies and in cell culture
Srividya Bhaskara, Erika Danielle Dean, Vita Lam, Ranjan Ganguly
⁎
Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, United States
Received 8 December 2005; received in revised form 7 February 2006; accepted 28 February 2006
Available online 5 April 2006
Received by Igor B. Rogozin
Abstract
To examine whether caffeine, the most widely used xenobiotic compound, would induce insect cytochrome P450 or CYP gene expression,
upstream DNA fragments of Cyp6a2 (0.12, 0.26, 0.52 and 0.98-kb) and Cyp6a8 (0.06, 0.1, 0.2, 0.5 and 0.8-kb) genes of Drosophila melanogaster
were individually fused to the firefly luciferase (luc) reporter gene. Promoter activities of these constructs were examined in Drosophila SL-2 cells
using luciferase assays. Activity of 0.2- and 0.8-kb upstream DNA of Cyp6a8 was also measured in transgenic female flies. When these flies were
treated with 2 mMpure caffeine or Vivarin caffeine, both DNA fragments showed a 4
–
5-fold induction of promoter activity. Endogenous Cyp6a8 and
Cyp6a2 genes in these flies also showed caffeine-induced expression. In addition, both 0.2- and 0.8-kb DNAs showed differential basal and caffeine-
induced activity in head, ovaries, gut, cuticle plus fat body and malpighian tubules. However, in all tissues 0.8-kb DNA always showed higher basal
and caffeine-induced activities compared to the 0.2-kb DNA, suggesting that the additional DNA present in the 0.8-kb fragment has sequences that
enhance both activities. In SL-2 cells, all reporter constructs of each Cyp6 gene showed significantly higher basal activity than the empty vector.
Sequences that boost basal activity are located in
−
265/
−
129 and
−
983/
−
522 DNA of Cyp6a2, and
−
199/
−
109 and
−
491/
−
Keywords: Vivarin; Reporter gene assay; Transgenic flies; Cell transfection; Luciferase assay; Promoter assay; AP1 binding sites
1. Introduction
perspectives. One such perspective is the induction and
regulation of mammalian and human CYP genes, especially in
relation to the metabolism of different toxic chemicals,
carcinogenic compounds and drugs including barbiturate com-
pounds such as phenobarbital and barbital (
Wang and Negishi,
2003; Mandal, 2005
). Interestingly, many of these xenobiotic
compounds have been used as tools to identify the cis-regulatory
elements, receptors and other factors that regulate CYP gene
expression in mammals (
Sueyoshi and Negishi, 2001; Hankin-
son, 2005
).
Besides drugs and environmental toxicants, caffeine is
probably another xenobiotic compound to which most people
are exposed everyday. Caffeine is naturally found in berries,
seeds and leaves of many plants including coffee, cocoa and tea.
Cytochrome P450 monooxygenases or CYPs comprise a
superfamily of enzymes that are involved in the biosynthesis of
many biologically important compounds and metabolism of a
variety of xenobiotic (foreign) chemicals to which living organ-
isms are exposed to on a daily basis (
Guengerich, 2004
). CYPs
have been studied in various organisms from different biological
Abbreviations: bp, base pair(s); CYP, cytochrome P450 monooxygenase;
Cyp (or CYP), CYP-encoding gene; kb, kilobase(s) or 1000 bp; LUC,
Luciferase; luc, LUC-encoding gene; nt, nucleotide(s).
⁎
Corresponding author. Tel.: +1 865 974 5148; fax: +1 865 974 6306.
E-mail address:
(R. Ganguly).
0378-1119/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:
199 DNA of Cyp6a8
genes. While the 0.12- and 0.1-kb upstream DNAs of Cyp6a2 and Cyp6a8 genes respectively did not show caffeine-inducibility in SL-2 cells, the
longest upstream DNA of each gene gave the highest level of induction. Caffeine-responsive sequences are not clustered at one place; they appear to
be dispersed in
−
983/
−
126 and
−
761/
−
109 regions of Cyp6a2 and Cyp6a8 genes which also contain many binding sites for activator protein 1 (AP1)
and cyclic AMP response element binding protein (CRE-BP). Significance of these binding sites in caffeine-inducibility has been discussed.
© 2006 Elsevier B.V. All rights reserved.
S. Bhaskara et al. / Gene 377 (2006) 56
–
64
57
A variety of commercial food and drinks also contain a high
quantity of caffeine. Because caffeine is consumed heavily and
it is chemically very similar to other purines, extensive studies
have been done on the effect of caffeine on various biological
processes in humans and other mammals such as brain motor
activity, cognitive functions, sleep and hypertension (
Fredholm
et al., 1999; Lorist and Tops, 2003
). However, not much work
has been done on the effect of caffeine on gene expression
except for three studies. One study showed that caffeine
treatment increases CYP1A1 and CYP1A2 mRNA levels in rat
liver and kidney (
Goasduff et al., 1996
). In the other two studies
caffeine was found to increase the levels of c-fos, c-jun, and
junB mRNAs in rat striatum (
Svenningsson et al., 1995
), and
sonic hedgehog mRNA in primary murine neuronal and
astroglial cells in culture (
Sahir et al., 2004
).
In insects, CYPs are known to be involved in conferring
insecticide resistance, and insecticide resistance-associated
overexpression of one or more CYP genes has been observed
in many insect species (see
Scott, 1999
for review). In
Drosophila also, at least five Cyp genes, including Cyp6a2
and Cyp6a8, show overexpression in DDT resistant strains
(
Maitra et al., 1996; Dombrowski et al., 1998; Daborn et al.,
2001;Brandteta ,2002
). However, the mechanism of
overexpression or the regulation of insect CYP genes in general
is not well-understood. Since xenobiotic compounds have been
successfully used to understand mammalian CYP gene regula-
tion, we surmised whether caffeine could also be used to study
insect CYP gene regulation. Therefore, in the present study we
used Drosophila as a model insect and examined the effect of
caffeine on the Cyp6a2 and Cyp6a8 gene expression in adult fly
and also in Drosophila cells in culture. The results showed that
both genes are induced by caffeine, and Cyp6a8 shows
differential constitutive and caffeine-induced expression in
different tissues of the adult flies.
ry
506
host strain were used: 0.2-luc30-4 (also referred as 0.2-luc4),
0.8-luc110, 0.8-luc14, 0.8-luc121 and 3.1-luc2.Eachofthese
transgenic lines has a single reporter transgene with a firefly
luciferase (luc) reporter gene under the control of 0.2- (
−
11/
3100) upstreamDNA of
the Cyp6a8 gene. The chromosomal locations of the transgene in
different lines have been described earlier (
Maitra et al., 2002
). The
0.2-luc4 and 0.8-luc110 strains were made homozygous for their
2nd chromosome-linked transgene by using appropriate balancer
stocks (
Maitra et al., 2000
) and two genetic crossing schemes. One
scheme gave 0.2-luc4H and 0.8-luc110H stocks. Genotypes of
these flies are +/+; 0.2-luc/0.2-luc; +/+ and +/+; 0.8-luc/0.8-luc;+/
+, respectively. The transgene carrying 2nd chromosomes of these
stocks are from the ry
506
strain that was used for transformation.
The other scheme produced 0.2-luc4H-ry and 0.8-luc110H-ry
stocks. Besides the transgene carrying 2nd chromosomes, the X
and 3rd chromosomes in these stocks are also from the ry
506
strain
(indicated with r). Genotypes of these stocks are X
r
/X
r
; 0.2-luc/
0.2-luc; 3
r
/3
r
and X
r
/X
r
; 0.8-luc/0.8-luc; 3
r
/3
r
, respectively. The
source of the 4th chromosome in any of these stocks is not known.
Other reporter transgenic lines, 0.8-luc14, 0.8-luc121 and 3.1-
luc2, carrying 3rd chromosome-linked reporter transgenes are the
original transgenic lines made in the ry
506
host strain. These strains
have been maintained by crossing ry
+
transgenic males and
females in every generation. Flies were reared at 25 °C on standard
cornmeal-agar-molasses Drosophila medium.
Schneider line 2 or SL-2 cells of D. melanogaster (
Schneider,
1972
), obtained from Invitrogen (Carlsbad, CA), were used for
promoter assays. Cells were maintained at 25 °C in Schneider's
Drosophila medium (Gibco-BRL) supplemented with 10% heat-
inactivated bovine calf serum and 0.01% penicillin
199), 0.8- (
−
11/
−
761) or 3.1-kb (
−
11/
−
streptomy-
cin (Sigma). Every fourth day the cells were transferred to fresh
media.
–
2. Materials and methods
2.2. Construction of reporter plasmids for Cyp6a2 and Cyp6a8
promoter assay in SL-2 cells
2.1. Fly strains and cell culture
Recombinant plasmids carrying 0.9-kb (
−
983/
−
1) and 0.8-
11) upstreamDNA of Cyp6a2-91R and Cyp6a8-91R
alleles of 91-R strain respectively (
Dombrowski et al., 1998;
Maitra et al., 2002
) were used as templates to amplify different
−
761/
−
The 91-C strain and the following five reporter transgenic lines
of Drosophila melanogaster created by
Maitra et al. (2002)
in the
Table 1
Sequences of the distal primers and PCR conditions used to amplify different regions of the Cyp6a2 and Cyp6a8 genes
Genes
Primer names
Regions amplified
Primer sequences and regions they are identical to
a
T
m
(°C)
Cyp6a2
b
A2-C
−
983/
−
1
5
′
-ctc
acgcgt
TTCATTCGTTTTATCGCCG-3
′
(
−
983/965)
61
A2R-1M
−
522/
−
1
5
′
-cc
acgcgt
CAAGTGGGATCGTCCTGTAC-3
′
(
−
522/
−
503)
53
A2R-2M
−
265/
−
1
5
′
-cc
acgcgt
GCAGTGAAGCGTATGAGTATC-3
′
(
−
265/
−
245)
52
A2R-3M
−
129/
−
1
5
′
-cc
acgcgt
GCTAGCTAGCTCACATGCTGTC-3
′
(
−
129/
−
112)
56
Cyp6a8
c
A8-5F
−
491/
−
11
5
′
-ggc
ctcgag
GTTGTGGTAGGTTAGTAGC-3
′
(
−
491/
−
473)
72.1
A8-1F
−
109/
−
11
5
′
-ggc
ctcgagC
TAGCGACTGAGAATGCATC-3
′
(
−
109/
−
90)
78
47) 78.6
a
Extra bases added to each primer are shown in lower case. Bases of the upstream DNA are in upper case. For each gene ATG is at +1. The MluI and XhoI sites
engineered respectively for Cyp6a2 and Cyp6a8 are underlined.
b
The same A2A proximal primer (5
′
-ctc
gtcgacT
TTGCGTAGCTGCTCCC-3
′
), complimentary to
−
1/
−
17 bases of the Cyp6a2 gene, was used with all distal
primers. Engineered bases are in lower case. Added SalI site is underlined. ATG is at +1. T
m
=63 °C.
c
G10 proximal primer (5
′
-cg
ttcgaa
ATGATCTTCGAATACG-3
′
), complimentary to
−
11/
−
26 bases of the Cyp6a8 gene, was used with all distal primers.
Engineered bases are in lower case. The added HindIII site is underlined. ATG is at +1. T
m
=70 °C.
A8-0.06F
−
67/
−
11
5
′
-ggc
ctcgagC
AGCGTTTAAAAGCAGTTTGC-3
′
−
67/
−
−
kb (
(
58
S. Bhaskara et al. / Gene 377 (2006) 56
–
64
regions of the upstream DNA of Cyp6a2 and Cyp6a8 genes via
PCR.
Table 1
shows the sequences of the distal and proximal
primers used for each Cyp6 gene. Appropriate restriction
enzyme sites that were added to these primers for cloning
purpose are detailed in
Table 1
. PCR amplifications and
purification of the amplified DNAs were done by using Easy-
Start PCR kit (Molecular Bioscience, Boulder) and kits from
Qiagen (CA), respectively. Four amplified DNA fragments
representing the
DNA mix. Cells to be transfected were resuspended in fresh
SL-2 medium at 1×10
6
cells/ml density. To each well of a
48-well microtiter plate, 500
μ
l of cells were dispensed and to
l of calcium phosphate-DNA mix was added
in a drop-wise manner. After 18 h incubation at 25 °C, the
medium was replaced with 500
μ
l of fresh medium and
incubated at 25 °C for 24 h. The cells were then spun down
and resuspended in 500
μ
l of fresh SL-2 medium containing
8 mM caffeine (Sigma, St. Louis). Following 24 h incubation
at 25 °C, cells were pelleted and 250
μ
1
regions (ATG at +1) of the Cyp6a2 gene were digested with MluI
and SalI, and cloned into MluI
−
983/
−
1,
−
522/
−
1,
−
265/
−
1 and
−
129/
−
l 1× passive lysis
buffer (Promega, WI) was added to each well. The plates
were agitated on a plate shaker for 15 min at room
temperature to lyse the cells and then centrifuged at
1500 rpm for 5 min at 4 °C. The clear supernatant was
collected from each well and assayed immediately for F-luc
and R-luc activities by using a dual luciferase assay kit
(Promega, WI). Briefly, to 20
μ
XhoI cut pGL3-basic vector
(Promega, WI). The resulting recombinant promoter-reporter
plasmids were named 0.98luc-A2, 0.52luc-A2, 0.26luc-A2 and
0.12luc-A2, respectively. In case of Cyp6a8 gene, three
amplified upstream DNAs, representing
–
−
491/
−
11,
−
109/
−
11
11 regions (ATG at +1) of Cyp6a8 were digested with
XhoIandHindIII and cloned into XhoI
−
67/
−
HindIII cut pGL2(N)
vector which is the pGL2-basic vector (Promega, WI) with SalI
site modified to NotI(
Maitra et al., 2002
). The recombinant
promoter-reporter plasmids were named 0.5luc-A8, 0.1luc-A8
and 0.06luc-A8, respectively. In addition to these plasmids,
0.8luc-A8 and 0.2luc-A8 plasmids carrying
–
l luciferase
assay reagent II was mixed, and placed in a luminometer
within 10 s to measure the firefly luciferase (F-luc) activity.
Immediately after recording the F-luc activity, 100
μ
l cell lysate, 100
μ
l Stop
and Glo reagent (Promega, WI) was added, vortexed gently
and the Renilla luciferase (R-luc) activity was measured
within 10 s. R-luc activity was used to normalize the data,
which were expressed as the ratio of F-luc to R-luc activity.
In each experiment, all reporter constructs of both Cyp6 genes
were used to transfect the same batch of cells. For each
reporter construct, triplicate transfection was done. Two such
experiments were done with two different isolates of each
reporter plasmid DNA to obtain the final mean F-luc/R-luc
ratio.
μ
−
766/
−
11 and
11 upstream DNA of Cyp6a8 (
Maitra et al., 2002
) were
also used in the present study. All PCR products were sequenced
and found to match with the reported sequence of Cyp6a2 and
Cyp6a8 upstreamDNAs (
Dombrowski et al., 1998; Maitra et al.,
2002
).
199/
−
2.3. Treatment of flies, fly extract preparation, and luciferase assay
4 days old, were allowed to
feed for 24 h on instant Drosophila food made with aqueous
solution of 2.0 mM Vivarin caffeine (obtained from local
store) or 16 mM pure caffeine (Sigma, St. Louis), a dose
found to be optimum for induction. Food made with water
was used as a control. After treatments, fly extracts were
prepared from groups of ten females for luciferase and
protein assays. Details of fly handling, extract preparation,
firefly luciferase and protein assays have been described
previously (
Maitra et al., 2002
). Luciferase activity and total
protein in the fly extract were determined by using a
luciferase assay reagent kit (Promega, Madison, WI) and
BCA protein assay kit (Pierce), respectively. Luciferase
activity in fly extracts was expressed as average Registered
Light Units per
Transgenic female flies, 2
–
2.5. Northern blot hybridization
The details of RNA blotting, probe synthesis and
hybridization have been described previously (
Maitra et al.,
2000
). Briefly, total RNA was isolated from caffeine-treated
and untreated adult female flies by using Tri-reagent (Sigma,
MO). For analysis, 20
μ
g of each RNA sample was dried,
l formaldehyde loading dye solution
(Ambion, Austin, TX) and denatured by incubating at
65 °C for 15 min. Samples were electrophoresed on a
2.2 M formaldehyde
μ
1.2% agarose gel and blotted onto
nylon membrane. The blots were cut into two pieces to probe
CYP RNA in the upper half and the control RP49 mRNA in
the lower half as described (
Maitra et al., 2000
). Upper
halves of three blots made from three independent RNA
samples and lower halves of these blots were prehybridized
in separate bottles for 2 h at 37 °C in Northern Max
Prehybridization/Hybridization buffer (Ambion, TX). After
prehybridization,
32
P-labeled CYP (5×10
9
cpm/
−
g). For each
treatment, extracts prepared from three independent groups of
flies were assayed.
g of total protein (RLU/
μ
2.4. SL-2 cell transfection, extract preparation and dual
luciferase assay
μ
g) and RP49
g) cDNA probes were added to the buffer in
respective bottles and hybridization was done at 37 °C for
22
μ
gofCyp6a2
or Cyp6a8 firefly luciferase (F-luc) reporter plasmid and
0.5
For transient transfection of SL-2 cells, 1.0
μ
24 h. The blots were washed under high stringent
conditions and scanned with a Packard Radioanalytical
Imager as described (
Dombrowski et al., 1998
). Data were
normalized using RP49 hybridization signal as the internal
control.
g of internal control Renilla luciferase (R-luc) plasmid
(Promega, Madison) were dissolved in sterile 0.1 M CaCl
2
(Invitrogen, CA) and mixed with equal volume of 2× HEPES
buffer (Invitrogen, CA) at pH 7.1 to make calcium phosphate-
μ
each well, 84
and
−
dissolved in 20
μ
(1.2×10
8
cpm/
–
S. Bhaskara et al. / Gene 377 (2006) 56
–
64
59
Fig. 1. Expression of luciferase reporter gene in 0.2-luc4H and 0.8-luc110H reporter transgenic lines. Adult females of each line were allowed to feed on
medium containing water or 2.0 mM caffeine (Vivarin) for 24 h. Extracts and RNA prepared from these flies were assayed for luciferase activity (A) and LUC
mRNA level (B) as described in the Materials and methods. The results represent the means ±S.D. of three independent experiments. ANOVA p
b
0.0001 for A
and B.
2.6. Statistics
from four- to six-fold (
Fig 2
). In another transgenic line (0.2-
luc7), the 0.2-luc transgene located at different chromosomal
location (
Maitra et al., 2002
) also showed about 5-fold induction
when treated with caffeine (data not shown). Since different
transgenic lines carrying reporter transgene at different chro-
mosomal position showed similar levels of induction, it may be
concluded that caffeine induction is not a result of chromosomal
position effect; it is the function of the Cyp6a8 upstream DNA
driving the luciferase reporter gene.
Data were analyzed using ANOVA program.
3. Results
3.1. Vivarin and caffeine induce Cyp6a8 promoter activity
To examine whether caffeine would induce Drosophila
Cyp6a8 gene, adult females of 0.2-luc4H and 0.8-luc110H
transgenic lines were exposed to instant Drosophila medium
made in aqueous solution of over-the-counter caffeine tablet
called Vivarin. The final concentration of caffeine in the me-
dium was 2.0 mM. The results (
Fig. 1
A) showed that in both
transgenic lines, luciferase activity was significantly higher in
Vivarin-treated flies compared to the water-treated controls.
Expression of the luc reporter gene was also examined by
Northern blot analysis. A 3-fold induction of LUC mRNA was
observed in both transgenic lines following caffeine treatment
(
Fig. 1
B), suggesting that Cyp6a8 promoter is induced by
caffeine. Results also showed that LUC enzyme activity and
LUC mRNA levels were significantly higher in the 0.8-luc line
compared to the 0.2-luc line. These data agree with our previous
observations that 0.8-kb upstream DNA of Cyp6a8 has higher
promoter activity than the 0.2-kb DNA (
Maitra et al., 2002
).
Vivarin tablet contains many other ingredients besides
caffeine. Therefore, to examine whether the effect of Vivarin
could be mimicked by caffeine, 0.2-luc4H, 0.8-luc110H, 0.8-
luc14 and 0.8-luc121 transgenic lines were treated with pure
caffeine. Since the latter two strains were not made homozygous
for the reporter transgene, all transgenic lines were crossed to the
ry
506
host strain used for germ line transformation, and the F1
ry
+
(transgene marker) females, heterozygous for the transgene,
were selected and treated with 16 mM caffeine, which was found
to be an optimum concentration in dose response experiments
(data not shown). While a six-fold induction was observed in
0.2-luc4 line, in three 0.8-luc lines the level of induction ranged
3.2. Caffeine induces the endogenous Cyp6a8 and Cyp6a2
genes in transgenic and wild type strains
To rule out the possibility that induction of the luc reporter
gene by caffeine is not due to some peculiarity of the reporter
Fig. 2. Effect of pure caffeine on the expression of luciferase reporter transgene
in different transgenic lines. Each transgenic line was crossed to the ry
506
host
strain, and the ry
+
(red-eyed) F1 females carrying the reporter transgene were
allowed to feed on water or 16 mM caffeine containing medium for 24 h.
Extracts prepared from these flies were assayed for luciferase activity. Data
represent mean±S.D. of three independent determinations. ANOVA p
b
0.0001.
60
S. Bhaskara et al. / Gene 377 (2006) 56
–
64
Fig. 3. Northern blot analysis of endogenous Cyp6a8 and Cyp6a2 gene expression in 0.8-luc110H-ry reporter transgenic line and 91-C wild type strains of Drosophila.
Total RNAwas extracted from adult females of each strain, which were treated with water or 16 mM caffeine as described in
Fig. 2
. RNA samples were fractionated on
agarose gel, blotted and hybridized with Cyp6a8 (A) and Cyp6a2 (B) gene probes. The levels of RP-49 RNA in the RNA samples were used as internal control to
correct RNA loading discrepancy between lanes. The results represent the means±S.D. of three independent experiments done with three different RNA samples.
ANOVA p
b
0.0001 for A and B.
transgenes or the host ry
506
strain, RNA isolated from caffeine-
treated and untreated 0.8-luc110 (H-ry) transgenic flies
homozygous for the reporter transgene and a wild type strain
named 91-C were examined for endogenous Cyp6a8 gene
expression by Northern blot analysis. We also examined
Cyp6a2, another Cyp6 gene of Drosophila that appears to be
under the same gene regulatory mechanism that controls the
Cyp6a8 gene (
Maitra et al., 2000
). Results showed that in both
0.8-luc110 (H-ry) transgenic line and 91-C strain endogenous
Cyp6a8 and Cyp6a2 genes were induced by caffeine at the
steady-state mRNA level, although the level of induction of
these two genes varied between the two strains (
Fig. 3
).
line about a 12-fold induction of the reporter gene was observed
in caffeine-treated flies (
Fig. 5
A). In the gut (
Fig. 5
B), con-
stitutive expression of the reporter transgenes in the untreated
transgenic flies was very low; it was more than one order of
magnitude lower than that found in the ovaries. In fact the
specific activities of luciferase enzyme in treated and untreated
flies in the 0.2-luc4 (H-ry) line were too low to be considered as
an expression of the transgene. In the 0.8-luc110 (H-ry) line,
constitutive expression was also very low but, approximately a
10-fold induction was observed in the caffeine-treated flies
(
Fig. 5
B). In the cuticle plus fat body (
Fig. 6
A) and in the
malpighian tubules (
Fig. 6
B) of the untreated flies of both trans-
genic lines, luciferase activities were found to be several orders
magnitude higher than the activities observed in the ovary and
gut (
Fig. 5
). Both transgenes also showed a significant induction in
3.3. The 0.2- and 0.8-kb upstream DNAs of Cyp6a8 show
differential promoter activity in different tissues
To investigate the promoter activities of 0.2- and 0.8-kb
upstream DNAs of Cyp6a8 in different parts of the body,
luciferase activity in head, body (whole abdomen plus thorax)
and whole female transgenic flies were measured. Differential
expression of both transgenes was observed in the head and body
of the transgenic flies. Firstly, activities of both 0.2luc-A8 and
0.8luc-A8 transgenes were much higher in the heads than in the
bodies of the untreated control flies (
Fig. 4
). Secondly, in the
heads and bodies of the control flies, activity of the 0.8luc-A8
transgene was much higher than the activity of the 0.2luc-A8
transgene. Thirdly, both transgenes were induced by caffeine, but
the level of induced LUC activity was consistently higher in the
0.8-luc110 (H-ry) transgenic line than in the 0.2-luc4 (H-ry) line.
Expression of the 0.2luc-A8 and 0.8luc-A8 transgenes was
also examined in the ovary (
Fig. 5
A), gut (
Fig. 5
B), abdominal
cuticle plus fat body (
Fig. 6
A) and malpighian tubules (
Fig. 6
B)
of caffeine-treated and untreated adult female flies. In both
transgenic lines, expression of the reporter gene was very low in
the ovaries of the untreated flies (
Fig. 5
A) and caffeine treatment
did not induce the reporter gene in the 0.2-luc4 (H-ry) line
(p=0.34, Student's t-test). However, in the 0.8-luc110 (H-ry)
Fig. 4. Expression of the luciferase reporter transgene in different body parts of
caffeine-treated and untreated female flies of 0.2-luc4H-ry and 0.8-luc110H-ry
transgenic lines. Female flies of each transgenic line were fed with 16 mM
caffeine or water for 24 h and luciferase activity was measured as described. The
results shown are the means±S.D. of three independent experiments. ANOVA
p
0.0001.
b
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