928 M.E. Kula, C.E. Rozek Insect Biochemistry and Molecular Biology 30 2000 927–935
2. Materials and methods
2.1. Nucleic acid manipulation, electrophoresis, and isolation
DNA was digested with appropriate restriction endonucleases according to manufacturer specifications
and size fractionated on agarose gels following standard methods Maniatis et al., 1982. DNA fragments were
isolated by electroelution into DEAE-ion exchange paper Dretzen et al., 1981. Vectors were linearized
with the appropriate restriction endonucleases and DNA fragments were subcloned following standard procedures
Maniatis et al., 1982.
An 800 bp EcoRI fragment representing the C-ter- minal half of the D. melanogaster cDNA sequence was
cloned into the trp expression vector pATH 3 Hardy and Strauss, 1988. Transformed cells were induced to
express a fusion protein by the addition of b-indoleac-
rylic acid. Following induction cells were pelleted and lysed for isolation of the insoluble fusion protein accord-
ing to the method of Hardy and Strauss 1988.
2.2. Polyclonal antibody production and immunoreactions
Fusion protein was mixed in a 1:1 emulsion with Freund’s complete adjuvant and injected into rabbits.
Antibodies were purified by the method of McKinney and Parkinson 1987.
Proteins immobilized on nitrocellulose were blocked with 5 non-fat dry milk in 5 BSA, 10 mM Tris-HCl
pH 8.0, 150 mM NaCl TBS. Primary antibody reac- tions were incubated overnight. Secondary antibodies
were peroxidase conjugated goat anti-rabbit IgG. Posi- tive reactions were visualized colormetrically.
2.3. Drosophila protein preparations and subcellular fractionation
Various protein preparations from Drosophila were used to identify and isolate the Cyt-b protein. The sub-
cellular fractionation procedure used in this communi- cation has been adapted from several methods which are
successful using insect models Gnagey et al., 1987; Alz- iari et al., 1985; Tupper and Tedeschi, 1969. Total fly
preparations consisted of whole adult D. melanogaster or D. virilis protein homogenized and boiled in 1X
Laemmli sample buffer. Large body parts wings, exo- skeleton, etc. and cellular debris were pelleted with two
10 min spins in a microcentrifuge and the supernatants were used as the sources of total proteins. Subcellular
fractionations were completed as an enriching protocol to localize the Cyt-b protein. Briefly, the subcellular
fractions were prepared in the following manner. Adult flies 2–5 g of 0–2 day old males and females were
homogenized at 0 °
C in 0.25 M sucrose, 50 mM sodium phosphate pH 7.5. Cellular debris was removed by cen-
trifugation at 1000g for 10 min at 4
° and the supernatant
was respun under the same conditions to further pellet eye pigments and nuclei. Mitochondria require higher
speeds and longer times of centrifugation, and were pel- leted from this supernatant at 15 000g for 1 h at 4
° . The
non-mitochondrial fraction supernatant was removed and centrifuged at 100 000g for 1 h at 4
° to pellet micro-
somes. The microsomal pellets were resuspended in 500 µ
l 50 mM NaPi buffer, pH 7.5. The mitochondrial pellets were fractionated as follows. Mitochondria were resus-
pended in 1 ml 50mM NaPi and were combined with an equal volume of 0.5 M NaCl and incubated at 0
° C for
1 h. The resuspended mitochondria were centrifuged at 100 000g for 45 min at 4
° . The final supernatant rep-
resented the high salt soluble peripheral mitochondrial proteins, and the final pellet, resuspended in NaPi, con-
tained the insoluble integral membrane mitochondrial proteins.
Aliquots of protein that were positive when tested with the anti-Cyt-b polyclonal were also tested in succi-
nate dehydrogenase assays to verify their mitochondrial origin. Spectrophotometric measurements were com-
pleted to monitor the presence of heme in these fractions. All fractionation pellets were used to test specificity of
antibody. We did not detect cytochrome b in blots of cytoplasmic, microsomal and nuclear proteins.
2.4. Electrophoresis of proteins and western blotting Proteins were separated on SDS-polyacrylamide dena-
turing gels Laemmli, 1970. After electrophoresis, the proteins were transferred to nitrocellulose by electroblot-
ting. The protein blots were blocked for 3 h to overnight in 5 BSA dissolved in 10 mM Tris pH 8.0, 150 mM
NaCl, 0.05 Tween-20 TBST. Primary antibodies were diluted in TBST containing 0.1 BSA and incu-
bated with the blots for 3 h at room temperature. After three washes in TBST, the secondary antibody was
added 1:7500 dilution of horseradish peroxidase conju- gated goat anti-rabbit or anti-mouse Cappel in TBST
containing 0.1 BSA and incubated for 3 h. After three washes in TBST, the proteins cross-reacting with the
antibodies were detected with 3,3
9-diaminobenzidine DAB dissolved in 0.05 M Tris-HCl pH 7.6 final conc.
600 ugmL. 1 uL of 30 hydrogen peroxide was added per milliliter of substrate solution. Following the sub-
strate reaction, the blots were washed in distilled H
2
O. 2.5. In vitro transcriptiontranslation of Cyt-b mRNA
The full length D. melanogaster Cyt-b cDNA Levin et al., 1989 was cloned into the transcription vector
pGEM-3Z Promega. Transcription of Cyt-b RNA was carried out at 37
° for 2 h with the addition of rATP,
929 M.E. Kula, C.E. Rozek Insect Biochemistry and Molecular Biology 30 2000 927–935
rCTP, rGTP,
rUTP and
T7 RNA
polymerase Boehringer Mannheim. The DNA template was
removed with DNase I Boehringer Mannheim and the reaction mix was extracted and precipitated. The in vitro
transcribed Cyt-b RNA was resuspended in 30 µ
l sterile ddH
2
O. The in vitro translation IVT of Cyt-b was car- ried out in rabbit reticulocyte lysate with or without the
addition of canine pancreatic microsomal membranes according to the manufacturer Promega at 30
° for 1 h.
2.6. Immunocytochemical localization of Cyt-b Pupal cases from 75 h old pupae were removed and
the thoraces
were dissected
and placed
in 4
formaldehydePBS for overnight fixation. The tissues were washed in PBS for 2 h following fixation. Adult
thoraces were then dehydrated through a graded series of ethanol solutions 25, 50, 75, 90, 95, and
100, embedded in LR White resin Polysciences, Inc., Warrington, Pennsylvania and sectioned. The thoraces
were blocked in filtered 5 normal goat serumTBS for 15 min at room temperature. The sections were then
washed in TBS and incubated with a 1:1000 dilution of the anti-Cyt-b polyclonal antibody made in a 1 normal
goat serumTBS solution overnight at 4
° . The adult sec-
tions were washed in TBS and incubated with gold con- jugated anti-rabbit antibody at 1:20 dilution for 4 h at
room temperature. All antibody incubations were carried out in a humidified chamber. All solutions were filter
sterilized before use, and all antibody sources were spun in a microcentrifuge for 5 min prior to use. Sections were
counterstained with 2 uranyl acetate and lead citrate.
3. Results
