Patterns of E74A RNA and protein expression at the onset of metamorphosis in Drosophila Page: 3 of 19
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peripodial membranes of the imaginal discs, and
proliferation centers of the brain, where relatively high
levels of E74A RNA persist in 1.5 h prepupae. The
distribution of nuclear E74A protein correlates with the
E74A RNA distribution with one exception: we find no
evidence for E74A protein in the proliferation centers
of the brain. Studies of the timing of E74A expression
revealed that the broad 8-10 h expression of E74A
mRNA at the end of larval development is refined into
a 5-7 h burst of E74A protein, with the peaks offset by
-2h. This delay in E74A protein accumulation is not
evident 10h later, in response to the next pulse of
ecdysone, suggesting that it may be of regulatory
significance and may contribute toward the timing of
subsequent steps in the regulatory hierarchy.
Materials and methods
Developmental staging of animals
Larvae were grown on food containing 0.05 % bromophenol
blue (Sigma). As the larvae begin wandering the blue dye
gradually clears from their intestine indicating the time since
the cessation of feeding (Maroni and Stamey, 1983). This
technique can be used to stage late-third instar larvae with an
error of approximately 2-3 h at 250C (L. B. and A. Andres,
unpublished results). Larvae whose guts were completely blue
were greater than 12 h away from puparium formation; these
we designate as early wandering larvae. Larvae whose guts
were cleared of dye were -3 h from puparium formation
( 1.8h); these we designate as late wandering larvae.
Prepupae were synchronized at the white prepupal stage (0Oh)
and allowed to develop on moistened filter paper at 25 C for
the desired time. This staging is highly reproducible since the
prepupae begin to tan approximately 15-20 min after pupari-
ation.
In situ hybridization
Stationary late-third instar larvae, white prepupae, and 1.5 h
prepupae were frozen, sectioned with a cryostat (8pm
sections), and mounted on subbed slides as described (Hafen
and Levine, 1986). High specific-activity 35S-labeled RNA
probes were prepared by in vitro transcription of a cloned
E74A cDNA containing the first three exons. This probe is
specific for E74A transcripts and will not cross-react with
E74B. Probe synthesis and in situ hybridization were
performed essentially as described by Ingham et a. (1985),
following a protocol provided by C. Rushlow and M. Levine.
We did not acetylate and pronase digest the sections before
hybridization. After coating with film emulsion, the slides
were exposed for 2-3 weeks.
In situ hybridization to RNA in intact organs was
performed according to Mlodzik et al. (1990), with minor
modifications. The first fixation was supplemented with 0.1 %
deoxycholate. Larval and prepupal organs were dissected in a
9-well Corning dish and all subsequent incubations were done
in such a dish placed on a rotating platform. The probe for
whole-mount in situ hybridization was made by digoxygenin
labeling an antisense oligonucleotide from nucleotides no.
409-461 of the E74A cDNA (Burtis et al. 1990). The labeling
reaction consisted of 1.5 mm CoCl2, 10 M dATP, 200 M
digoxigenin-dUTP (Boehringer-Mannheim), 1 M E74A oli-
gonucleotide, and 75units terminal transferase (Boehringer-
Mannheim). The reaction was incubated at 37 C for 20 minE74A RNA and protein expression patterns 983
after which the labelled oligonucleotide was ethanol precipi-
tated and resuspended in 40 pl TE. Approximately 5 pl of
probe was used for each hybridization (5 pl probe in -95 p1
hybridization solution). Anti-digoxigenin antibodies (Boeh-
ringer-Mannheim) were preabsorbed to dissected and fixed
larval organs. Alkaline phosphatase-conjugated antibodies
were used at a 1:200 final dilution in PBT. Incubation with the
antibody ranged from 3 h at room temperature to overnight at
4 C. The alkaline phosphatase stained organs were mounted
in Mowiol mounting solution (Harlow and Lane, 1988) and
photographed with Kodacolor Gold 200 film on a Zeiss
Axiophot photomicroscope equipped with differential inter-
ference contrast.
For confocal microscopy, E74A RNA was identified with an
anti-digoxigenin-fluorescein antibody (Boehringer-Mann-
heim, preabsorbed and then diluted 1:10 in PBT) and the
chromosomes were detected with a monoclonal antibody
supernatant (8C5: a gift from S. Benzer) which was added at a
1:4 dilution with the anti-digoxigenin antibody. This was
followed by three 10 min washes and a 1.5 h incubation with a
goat anti-mouse Texas Red antibody (Jackson ImmunoRe-
search Laboratories) diluted 1:100 in PBT. After further
washing, the organs were mounted in Mowiol mounting
solution which was supplemented with 2mg ml-1 phenylene-
diamine (Sigma). The fluorescent stains were imaged on a
BioRad confocal laser scanning microscope (MRC 600).
Images were generated from Kalman averaging of -20 scans.
Each specimen was scanned with dual detector channels for
fluorescein and Texas Red emission wavelengths. The dual
images were merged with the 'alternate pixel' command.
RNA isolation and northern blots
RNA was isolated from 30-50 staged larvae or prepupae
placed in a 7 ml homogenizer with 200 pl RNA lysis buffer
(100 mM Tris-HCl pH 7.8, 0.3M sodium acetate, 20 mM
EDTA, 1% sarkosyl) and homogenized with five strokes of a
B pestle. Immediately, 200 pl phenol was added followed by
five more strokes with the B pestle. The mixture was
centrifuged and the aqueous portion was extracted with
phenol two more times. Then, 400 pl ethanol was added and
allowed to sit for 5 min at room temperature. The RNA was
pelleted, resuspended in 30-50 pl water, and stored at -80 C.
Approximately 20 pg of total RNA from each time point
was electrophoresed on 1 % formaldehyde agarose gels and
blotted onto Nytran. Northern blot hybridizations were
performed as described (Thummel et a. 1990). Blots were
probed with a 32P-labelled single-stranded cDNA probe,
derived from the first 3 exons of E74A, synthesized as
described (Burtis et al. 1990).
Antibodies
An E74A/#-galactosidase fusion protein was constructed by
inserting the entire E74A ORF into pWR-590-1 (Guo et al.
1984). The E74A C-terminal coding region was then removed
as a PstI restriction fragment. The protein encoded by this
plasmid contains most of /3-galactosidase fused to 111
N-terminal amino acids of E74A, including 19 amino acids of
sequence common to E74A and E74B. The fusion protein was
expressed in E. coli DH5 and purified from inclusion bodies
essentially as described (Rio et al. 1986), except that we used
25 mm or 250 mm Hepes pH 7.5 in place of 50 mm and 500 mM
Tris-HCl. After urea extraction, the protein was dialyzed
overnight in 0.1 M Hepes pH 8 and centrifuged to removeinsoluble material. The fusion protein was further purified by
precipitation with 30 % ammonium sulfate, after which the
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Boyd, Lynn; O'Toole, Erin & Thummel, Carl S. Patterns of E74A RNA and protein expression at the onset of metamorphosis in Drosophila, article, August 1, 1991; [Cambridge, England]. (https://digital.library.unt.edu/ark:/67531/metadc674070/m1/3/: accessed March 29, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .