 |
 Previous Article | Next Article 
Volume 16, Number 12,
Issue of June 15, 1996
pp. 3900-3911
Copyright ©1996 Society for Neuroscience
Characterization of Drosophila Tyramine  -Hydroxylase
Gene and Isolation of Mutant Flies Lacking Octopamine
Maria Monastirioti1,
Charles E. Linn, Jr.2, and
Kalpana White1
1 Biology Department and Volen National Center for
Complex Systems, Brandeis University, Waltham, Massachusetts 02254, and
2 Entomology Department, New York State Agricultural
Experiment Station, Cornell University, Geneva, New York 14456
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Octopamine is likely to be an important neuroactive molecule in
invertebrates. Here we report the molecular cloning of the
Drosophila melanogaster gene, which encodes tyramine
 -hydroxylase (TBH), the enzyme that catalyzes the last step in
octopamine biosynthesis. The deduced amino acid sequence of the encoded
protein exhibits 39% identity to the evolutionarily related mammalian
dopamine -hydroxylase enzyme. We generated a polyclonal antibody
against the protein product of Th gene, and we
demonstrate that the TBH expression pattern is remarkably similar to
the previously described octopamine immunoreactivity in
Drosophila. We further report the creation of null mutations
at the Th locus, which result in complete absence of TBH
protein and blockage of the octopamine biosynthesis.
Th-null flies are octopamine-less but survive to
adulthood. They are normal in external morphology, but the females are
sterile, because although they mate, they retain fully developed eggs.
Finally, we demonstrate that this defect in egg laying is associated
with the octopamine deficit, because females that have retained eggs
initiate egg laying when transferred onto octopamine-supplemented
food.
Key words:
cloning of Tyramine -hydroxylase;
TBH-immunocytochemistry;
Dopamine -hydroxylase;
octopamine-null mutant;
egg-laying behavior;
Drosophila
neurogenetics
INTRODUCTION
Octopamine is likely to be an important
neuroactive molecule in many invertebrates, because physiological
studies, carried out primarily in arthropods, have provided
considerable evidence about its role as a neurotransmitter, a
neuromodulator, or a neurohormone (for review, see Evans, 1980 , 1985,
1992; David and Coulon, 1985). Octopamine appears to regulate diverse
physiological functions in different organisms such as neuromuscular
transmission in locusts (Hoyle, 1984; Malamud et al., 1988), light
production in fireflies (Nathanson, 1979), production of submissive
postures in lobsters (Livingstone et al., 1980), feeding and sting
response in honey bees (Braun and Bicker, 1992) (for review, see Bicker
and Menzel, 1989; Burrell and Smith, 1995), and induction of lipid and
carbohydrate metabolism at times of stress (for review, see Orchard et
al., 1993). Because of the structural similarities between
noradrenaline and octopamine and an absence of noradrenaline from
several arthropod species, octopamine is at times considered to be
the arthropod noradrenaline. Although studied primarily in
invertebrates, octopamine also is found in vertebrates, but its
functions there have not been well studied (for review, see David and
Coulon, 1985).
We were interested in developing molecular-genetic tools to study the
role of octopamine in the fruit fly Drosophila melanogaster,
because one could conceive strategies to manipulate genetically the
level of octopamine and study its effects on specific behaviors of the
organism. In principle, it should be possible to create flies that are
congenitally devoid of any octopamine or flies in which the presence of
octopamine is conditional. Drosophila is known to contain
octopamine (O'Dell et al., 1987) and recently, octopamine
immunoreactivity has been described in specific neurons and
neuropil areas of the nervous system and also in neuronal terminals
innervating the larval body wall muscles (Monastirioti et al.,
1995). Pharmacological studies in Drosophila have
suggested involvement of octopamine in behaviors such as phototaxis and
learning as well as in certain aspects of development (Dudai et al.,
1987). High-affinity octopamine binding sites have been described
in studies with Drosophila head homogenates (Dudai and Zvi,
1984), and a gene encoding a putative octopamine/tyramine receptor
has been cloned and its pharmacological properties have been
investigated (Arakawa et al., 1990; Saudou et al., 1990; Robb et al.,
1994). Mutation in the inactive locus causes hypoactivity,
and the mutant flies are reported to have 15% of normal octopamine
(O'Dell et al., 1987; O'Dell and Burnett, 1988). Despite all of these
studies, at present there is no well defined function assigned to
octopamine in Drosophila.
To manipulate the levels of octopamine genetically, we decided to first
identify a gene encoding one of the enzymes in the biosynthetic pathway
of octopamine. Molecular identification of such a gene allows
determination of the cytogenetic location of the gene on salivary
chromosomes, which can facilitate isolation of mutations in that
genetic locus. Octopamine biosynthesis requires tyrosine decarboxylase
activity to convert tyrosine to tyramine and tyramine -hydroxylase
(TBH) activity to convert tyramine to octopamine. Livingstone and
Tempel (1983) have demonstrated that these two enzymatic activities
exist in Drosophila melanogaster and that the tyrosine
decarboxylase activity is distinct from the Ddc-encoded dopa
decarboxylase activity. No other information existed on the two enzymes
in Drosophila. However, studies on biochemical properties of
a partially purified TBH from the thoracic nervous system of lobster
Homarus americanus had demonstrated that it was remarkably
similar to dopamine -hydroxylase (DBH), which converts dopamine to
noradrenaline in mammals (Wallace, 1976). The salient features of this
similarity are (1) both enzymes recognize either tyramine or dopamine
(the precursors of octopamine and noradrenaline, respectively) as
substrates, although with different affinities, (2) both enzymes
require oxygen and ascorbic acid as cosubstrates, and (3) both enzymes
bind copper and are inhibited by metal chelators (Wallace, 1976). These
similar properties make it highly likely that the enzymes TBH and DBH,
which catalyze the final hydroxylation step in the production of
octopamine and noradrenaline, are functionally homologous (Wallace,
1976) and are probably related evolutionarily. Because DBH amino acid
sequences from several mammalian species were available (Lamouroux et
al., 1987; McCafferty and Angeletti, 1987; McMahon et al., 1990), we
decided to attempt to clone the Drosophila Tyramine
-hydroxylase (Th) gene by using the possible
evolutionary homology between the TBH and DBH amino acid sequences.
In this study, we describe cloning of a Drosophila gene that
has homology to DBH, and we provide immunocytochemical,
biochemical-genetic, and behavioral-genetic evidence confirming this
gene as the Drosophila melanogaster Th gene. We report on
creation of null mutations at the Th locus and analysis
of the phenotype associated with the octopamine deficit. We show that
these octopamine-less flies survive to adulthood and are normal in
external morphology and that the males are fertile. Interestingly,
mutant females are sterile, because although they mate, they retain
fully developed normal oocytes. Finally, we demonstrate that the
egg-retention defect is associated with the octopamine deficit, because
mated gravid females that have retained oocytes initiate egg
laying when transferred onto octopamine-supplemented food.
MATERIALS AND METHODS
PCR. Degenerate oligonucleotides were designed for
the regions of homology among the three mammalian DBH proteins shown in
Figure 1. The primer sequences were:
Fig. 1.
Alignment using LINEUP program of a portion of DBH
amino acid sequence from three mammalian species (bovine, human, rat)
in the histidine-rich regions. The numbers to the right
correspond to amino acid coordinates of the rat DBH.
Asterisks indicate the paired histidines or the H-X-H
residues; dot indicates Tyr236 of the
rat sequence, conserved in all three species. The regions selected for
primer design are underlined with arrows
indicating the 5  3 direction of the primer.
[View Larger Version of this Image (57K GIF file)]
5-ACNACNTAT/CTGGTGT/CTAT/CAT-3,
5-GGA/GTTA/GTGA/GTAA/GTGNACT/CTC-3,
5-GGNGAA/GATGGAA/GAAT/CGC-3,
5-AAN-ACC/TTCCATA/GTGA/GTGNAC-3,
where n = A/G/C/T. PCR reactions were done in
a total volume of 20 µl, and the incubation mixture contained 1 µg
genomic DNA as template, 5 µM of each primer,
1.5 mM MgCl2, 200 µM each of deoxynucleotidyl triphosphates, and
2 U of Taq polymerase (Perkin-Elmer Cetus, Emeryville, CA).
For reamplification reactions, 0.5 µl of the first PCR reaction was
used as template. Conditions for the PCR amplification were as follows:
initial denaturation at 94°C for 2 min followed by 5 cycles at 94°C
for 1 min, 42°C for 2 min, and 72°C for 2 min, and 25 cycles at
94°C for 1 min, 50°C for 2 min, and 72°C for 2 min.
PCR products were gel purified, treated with the Klenow fragment of DNA
polymerase I, and then phosphorylated with T4 kinase (enzymes were
purchased from New England Biolabs, Beverly, MA). The products then
were subcloned into the SmaI site of BlueScript-KS
(Stratagene, La Jolla, CA), and their sequences were determined.
Screening of cDNA libraries and sequencing. cDNA clones were
isolated by standard methods from a  gt11 recombinant fly head cDNA
library (Itoh et al., 1985). The plasmid clone containing the 120 bp
PCR product was used as a hybridization probe labeled by the random
primer method. The entire insert from the positive clones was subcloned
into the EcoRI site of Bluescript-SK (pDmDBH). DNA fragments
from pDmDBH then were subcloned into polylinker sites of
pBluescript-SK, and sequencing of both strands was performed. The
sequence also was confirmed from double-stranded overlapping deletions
covering the entire 3 kb length of pDmDBH.
RNA isolation and Northern analysis. Total RNA was isolated
from adult heads and bodies of Canton-S flies using the guanidium
hydrochloride method (Davis et al., 1986); 12 µg from each sample RNA
was separated on 1.2% formaldehyde/agarose gel and transferred to
Biotrans nylon membrane. Antisense RNA probe was synthesized with T7
polymerase from a subclone (pPXDBH) carrying a 0.87 kb
Pst-Xho fragment from the cDNA clone (Fig.
2B). The filter was hybridized overnight at
65°C under conditions described in Yao et al. (1992). Subsequently,
the filter was hybridized with a random-primed DNA probe of pRP49
(Kongsuwan et al., 1985) as a control.
Fig. 2.
A, Nucleotide and deduced amino acid
sequences of Drosophila Th cDNA (EMBL Data Library number
Z70316[GenBank]). Amino acid residues in bold indicate paired
histidines or the H-X-H sites. Residues in bold with an
asterisk correspond to those amino acids of the bovine DBH
protein modified by mechanism-based inhibitors (see text, Fig. 3).
Residues in bold italics correspond to consensus for
potential N-glycosylation sites, and underlined tetrads of
amino acids correspond to potential phosphorylation sites. The stop
codon is underlined. B, Restriction map of the
Drosophila Th cDNA. Restriction sites: H,
HindIII; P, PstI; R,
EcoRI; S, SalI; X,
XhoI. The open reading frame region of the cDNA is indicated
by the arrow below the restriction map. The fragments
used for the RNA in situ probe and the fusion protein
construct are represented with solid lines above and
below the restriction map, respectively. The first and last
amino acid included in the fusion protein construct are indicated at
the end of the corresponding line.
[View Larger Version of this Image (50K GIF file)]
RNA in situ hybridization. In situ
detection of RNA in whole-mount larval CNS was as described in Ebens et
al. (1993) using digoxygenin-labeled DNA probe synthesized for the
Pst-Xho fragment of the cDNA clone (Fig.
2B) according to the manufacturer's protocols (Boehringer
Mannheim, Indianapolis, IN).
Protein purification and preparation of antibodies. The 1.2 kb Sal-Xho fragment of the DmDBH cDNA (Fig.
2B) was inserted into the XhoI site of the
bacterial expression vector pET15b (Novagen, Madison, WI) in frame with
the His6 tag and under the bacteriophage T7
promoter present in the vector. The resulting plasmid was introduced
into the Escherichia coli BL21(DE3) strain (Studier and
Moffatt, 1986); cultures of the transformed cells were grown to
OD600 = 0.5, induced with 1 mM isopropyl
-D-thiogalactopyranoside, then grown for 3 more hr and harvested. Pellets were resuspended in 8 M urea, 0.1 M
NaH2PO4, 0.01 M Tris, pH 8, 7.5 mM
-mercaptoethanol, and cells were lysed completely by 2 × 30 sec
sonication. After 30 min centrifugation at 10,000 rpm, the lysate was
adsorbed to a nickel chelate affinity resin (Qiagen, Chatsworth, CA)
column, and the protein was eluted with a pH gradient in the above
buffer. The purified protein was renatured at 4°C by dialysis against
a buffer containing (in mM): NaCl 500, Tris 20, pH 7.4, PMSF 0.5, containing 6, 4, 2, 1, 0.5, and 0 M urea, and it was stored at 70°C. Rats were
immunized subcutaneously at multiple sites with 25 µg of purified
protein in incomplete Freund's adjuvant every 2 weeks.
For specific antibody purification, 60 µg of purified protein was
subjected to Western blotting; the nitrocellulose band was fragmented,
washed, and incubated in a 1:5 dilution of anti-TBH serum for 48 hr at
4°C. The specific immunoglobulins were eluted from the fragments at
4°C with 500 µl of elution buffer (100 mM
glycine/HCl, pH 2.6, 0.2% Tween 20, 100 mM NaCl,
and 100 µg/ml BSA) for 10 min. The eluate was neutralized immediately
with 25 µl of 2 M Tris/HCl, pH 8, and was kept
aliquoted at 70°C.
Immunoblot analysis of wild-type and Th mutants.
Crude homogenates of head and body tissue were used for the
immunoblots. Purified anti-TBH IgG was used at 1:30 dilution for the
primary antibody incubations. The enhanced chemiluminescence system
(Amersham, Arlington Heights, IL) was used for detection of the primary
antibody according to the manufacturer's instructions. Horseradish
peroxidase-conjugated goat anti-rat secondary antibody was used at a
final concentration of 1:1500.
Immunohistochemistry. Third instar larval brains were chosen
for these studies for the following reasons: (1) the staining can be
done in whole mounts; (2) the pattern of immunoreactivity is simple,
which makes comparisons possible; and (3) the staining was
reproducible. In contrast, the adult staining had to be done in
sections and was variable in our hands, and thus, comparisons were
difficult.
Third instar larval brains were dissected in PBS containing (in
mM): NaCl 137, KCl 3, KH2PO4 1.8, Na2HPO4 10, pH 7.5, and
fixed in ice-cold Bouin's fixative containing (in ml): saturated
aqueous picric acid solution 75, formaldehyde 25, glacial acetic acid 5 for 1 hr at room temperature. After fixation, samples were washed in
PBS (three times, 15 min each) and in PBT (PBS containing 0.3% Triton
X-100 and 0.1% BSA) (two times, 15 min each). Washes were followed by
preincubation with 5% normal goat serum for 1 hr at room temperature
and overnight incubation with purified anti-TBH antibody (1:25 in PBT)
at 4°C. The samples then were washed in PBT (six times, 15 min each)
and incubated overnight at 4°C in goat anti-rat FITC-conjugated
secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA)
diluted at 1:100 in PBT. After several washes in PBT and a final wash
in PBS, the samples were mounted in mounting media (80% glycerol, 2%
N-propylgalate in PBS). For each genotype, a minimum of four
samples were immunoprocessed together; the staining intensity was
similar between samples of the same genotype in a given batch. Each
genotype was examined at least three times. Samples were observed on a
Bio-Rad MRC600 confocal microscope (Hercules, CA). Samples were viewed
under identical conditions with respect to laser level and gain. Images
were photographed from the computer screen using similar exposures.
Chromatography. Brains of adult male flies were dissected
and fixed immediately and homogenized in ice-cold 0.1 M perchloric acid solution. Samples were
centrifuged for 15 min in an Eppendorf at 4°C, and supernatants were
kept at 70°C until used for the HPLC analysis. Chromatographic
separations were achieved as in Linn et al. (1994), using a Vydac
reverse-phase C-18 HS-54-15 HPLC column (15 cm × 4.6 mm inner
diameter, 3 µm particles) (Hesperia, CA). The mobile phase contained
70 mM monobasic sodium phosphate, 0.5 µM EDTA, 0.1 mM
l-octanesulfonate (sodium salt), 8% methanol, and 2% acetonitrile,
and was adjusted to pH 5.5. The mobile phase was run isocratic at 0.85 ml/min. The first electrode was set at a potential of 0.38 V and the
second electrode at 0.73 V. Using these potential settings, dopamine
and serotonin were detected selectively on channel 1, whereas
octopamine, N-acetyl octopamine, and tyramine were detected
on channel 2. Identification of compounds was based on comparison of
retention times with external standards, which were run at the
beginning and end of each daily series. Standards were prepared in 0.1 N perchloric acid solution daily. Peak identification also was
determined on the basis of changes in retention time or peak area as a
function of systematic change in chromatographic conditions, including
pH, percentage organics, or applied channel voltage (Linn et al.,
1994). Chemicals were purchased from Sigma (St. Louis, MO) except
N-acetyloctopamine, which was provided by Research
Biochemicals (Natick, MA) incorporated as a part of the Chemical
Synthesis Program of the National Institute of Mental Health Contract
278-90-0007.
Fly stocks and genetics. The following strains were obtained
from the Bloomington, Indiana, Drosophila stock center: (1)
p845 (sn[Pw+-lacW], w1118), (2)
Df(1)snC128/FM6, (3)
Df(1)snC128/C(1)DX y f; Dp
(1;2)sn+72d/bwD, (4) y2 sc w
snx2 B/Df(1)sxl, (5) Cy/Sp; ry506 Sb
P[ry+ 2-3]/TM6 Ubx (see Lindsley and Zimm, 1992). The genetic
schemes used to generate local transpositions took advantage of the
p845 element, which is a transposition in the sn locus.
Briefly, virgin p845 sn females were crossed to P
transposase-bearing males. Male or virgin female progeny were crossed
en masse to females or males carrying X chromosome balancer
y2 sc w snx2 Bar. Progeny were
screened for female or male flies (depending on the F1 cross), which
were revertant for the singed phenotype. To establish individual lines,
sn revertants were mated singly to balancer-carrying flies.
Lines that carry the P insertion in the X chromosome were determined
based on the cosegregation of the yellow and white markers, and they
were maintained. Mutations at the Th locus that disrupt
the gene were found based on loss of TBH protein band in immunoblot
analysis of the X-linked lines.
Excisions of the MF372 transposon were done as follows: virgin females
from the insertion line were crossed en masse to males of the stock
carrying the P transposase. Male progeny of this cross were mated
individually to females from the X balancer stock, FM7a, and
male progeny of the F1 cross were used in immunoblot analysis as
above.
Fertility assay at 25°C. Single virgin females, 1-2
d old, were mated to three Canton-S males in unyeasted test tubes
containing standard food, and they were allowed to lay eggs for 6 d. At
day 6, parents were discarded and progeny were counted at day 17. The
number of progeny per female was calculated by dividing the total
number of progeny produced by the total number of females assayed
(including those that laid no eggs).
Octopamine feeding. Single THM18
females were mated with three Canton-S males in unyeasted test
tubes containing standard food. After 5-6 d, the mated females were
transferred to food supplemented with different concentrations of
octopamine or other biogenic amines used in this study. For initial
tests, instant fly food (Carolina Biochemicals) was used; in subsequent
tests, standard sucrose-inactivated yeast/agar fly food was used.
Initial tests were done with 50, 25, 10, and 4 mg/ml of octopamine to
determine the optimal concentration. In all subsequent tests, a
concentration of 10 mg/ml was used. Dopamine, norepinephrine, and
tyramine were used to supplement the food at concentrations of 20 and
10 mg/ml. At a minimum, 10 flies were tested for all the drugs used.
Flies were removed on day 6 after transfer and progeny counted at day
17 after transfer. The number of progeny produced was divided by the
total number of females tested.
RESULTS
Cloning and sequencing of a Dopamine -hydroxylase
(DH)-like Drosophila gene
As a first step toward identifying the Tyramine
-hydroxylase gene of Drosophila, we cloned
Drosophila sequences with homology to mammalian
Dopamine -hydroxylase genes using PCR technology. We used
degenerate oligonucleotide primers designed for the amino acid regions
implicated in the function of the protein such as copper-binding sites
and the putative active center (discussed in McMahon et al., 1990). In
these regions, the amino acid sequences of the human, rat, and bovine
DBH are 100% identical (Fig. 1). A low-stringency PCR reaction using
genomic DNA as a template and primers 1 and 2 yielded an amplified 328 bp product. When the 328 bp product was used as a template along with
primers 1 and 4, a 120 bp band was amplified. This was the expected
size band, assuming spacing conservation between the corresponding
amino acid residues of primers 1 and 4 in mammalian and
Drosophila proteins and absence of intronic sequence in the
Drosophila genomic DNA. A 120 bp band also was produced when
we used primers 3 and 4 in the initial PCR reaction and in a follow-up
reaction, primers 1 and 4. The 120 bp product was subcloned, and 10 independent clones were sequenced. Only two of these, which contained
the same insert in opposite orientations, were flanked by sequences
corresponding to primers 1 and 4 at either ends, and these also
included an open reading frame of about 40 amino acids. A protein data
bank search revealed these to be most homologous to a 40 amino acid
region of known mammalian DBH sequences.
Armed with this 120 bp probe, we screened a Drosophila head
cDNA library (Itoh et al., 1985) and isolated one cDNA clone out of 2 × 105 clones. This cDNA was sequenced completely
from both strands (Fig. 2) and found to be 2895 nucleotides in length
and with a long open reading frame (ORF) from nucleotide 172 to 2193. The first ATG codon of the ORF at position 214 is likely to encode the
initiator methionine, because the sequence preceding it exhibits a
perfect match with the Drosophila initiation codon consensus
C/A A A A/C (ATG) (Cavener, 1987). The stop codon at position
2194-2196 is followed by stop codons in all three frames, and a 702 nucleotide long 3 untranslated region, yet polyA tail is not included.
The deduced translation product of this ORF, assuming translational
initiation at ATG214, is a 660 amino acid
polypeptide, with a predicted molecular mass of ~76,000.
Two potential N-glycosylation sites, Asn-X-Ser/Thr (Hubbard and Ivatt,
1981) (Fig. 2A) were found at Asn227
and Asn604. In addition, four potential
calmodulin-dependent protein kinase phosphorylation sites,
Arg-X-Y-Ser/Thr (Pearson et al., 1985) (Fig. 2A) were found
at Ser77, Thr58,
Thr214, and Thr462.
Finally, among the 31 histidine residues found in the deduced amino
acid sequence, there are two paired histidines at positions 288/289 and
304/305 and one His-X-His at position 452-454 (Fig. 2A).
Such closely spaced histidines have been reported in binding sites for
copper in copper-binding proteins (Sigel, 1981), and they also have
been found in the mammalian DBH proteins (Lamouroux et al., 1987;
McMahon et al., 1990).
Sequence comparisons
Comparison of the deduced amino acid sequence with protein
sequences in data bases using the GENIFRO experimental BLAST Network
Service (Altschul and Lipman, 1990) revealed highest score of homology
to the bovine, human, and rat DBH proteins. Figure 3
shows alignment of the Drosophila amino acid sequence with
mammalian sequences using multiple sequence alignment program, LINEUP.
The overall identity between the Drosophila protein and the
mammalian DBH proteins is 39%, and the similarity is 59% with
inserted gaps. However, certain regions exhibit greater conservation;
e.g., residues 404-519 and 325-392 (Drosophila protein
coordinates) show 55% and 47% identity and 68% and 63% similarity,
respectively.
Fig. 3.
Alignment using LINEUP program of
Drosophila TBH protein sequence to the DBH protein from
three mammalian species. The top line corresponds to the
Drosophila TBH amino acids 1-660. Only the identical amino
acids are shown, whereas gaps are marked with dots and
nonconserved residues are marked with dashed lines.
Asterisks indicate conserved His-His or His-X-His residues,
and # indicates an His-X-His site of the mammalian DBH not
conserved in the Drosophila TBH. The residues
Tyr273 and His452 (TBH
coordinates), which correspond to those modified by mechanism-based
inhibitors (DeWolf et al., 1988, 1989), are indicated with a dot
below them.
[View Larger Version of this Image (54K GIF file)]
Several amino acid residues have been reported to be important for the
function of DBH protein. These residues are conserved between the
mammalian and the Drosophila proteins, indicating an
evolutionary relationship. One stretch of six conserved amino acids
(270-275) and another of seven (448-454) include
Tyr273 and His452,
respectively. These are residues that have been identified as putative
active sites for the bovine DBH using mechanism-based inhibitors
(DeWolf et al., 1988, 1989). In addition, the two paired histidines
(288-289 and 306-307) and the His-X-His residue (453-455), which are
likely to be involved in copper binding, also are conserved. However, a
second His-X-His residue present in the mammalian proteins is not
conserved in the Drosophila protein (374-376). It also
should be pointed out that the spacing between the above regions is
almost the same in the Drosophila and the mammalian
proteins. Finally, the two glycosylation sites also are conserved.
Northern analysis and RNA in situ
Total RNA from adult head and body tissues was hybridized with a
riboprobe created from the antisense strand of the 870 bp
Pst-Xho fragment of the cDNA (Fig.
2B). A single transcript of ~3.4 kb, which appeared to be
head enriched, was detected in the adult heads (Fig.
4A). The absence of signal in the body
does not imply that there is no transcript in the body, but just that
it is at a much lower concentration than in the head.
Fig. 4.
Th transcript analysis. A, Total
Canton-S RNA (12 µg/lane) from adult head and body tissue was
hybridized with a 32P-labeled RNA probe
corresponding to the Pst-Xho fragment of
Th cDNA and subsequently with a
32P-labeled DNA probe of RP49. The size of the
transcript was determined relative to RNA markers (RBL) shown on the
right. B, Cellular distribution of the
Th transcript. Confocal image of whole-mount third instar
larval CNS hybridized with a digoxigenin-labeled DNA probe
corresponding to the Pst-Xho fragment of the
Th cDNA. Anterior is to the top. The CNS is composed of
the paired brain lobes and a fused ventral ganglion composed of the
subesophageal ganglion, thoracic ganglion, and abdominal ganglion (see
legend to Fig. 6C). The in situ hybridization
signal as detected with an alkaline phosphatase-conjugated
antidigoxigenin antibody was present in the ventral ganglion in a
cluster of cells in the subesophageal ganglion (white arrow)
and in cells along the midline of the thoracic and abdominal ganglia.
Scale bar, 50 µm.
[View Larger Version of this Image (95K GIF file)]
We studied the in situ distribution of the transcript in
whole-mount third instar larval CNSs, using a digoxigenin-labeled DNA
probe synthesized for the Pst-Xho fragment (Fig.
2B). A discrete expression pattern consisting of a small
population of the larval CNS cells localized exclusively in the midline
of the ventral ganglion was revealed (Fig. 4B). The pattern
comprises a large cluster of cells at the midline of the subesophageal
ganglion and unpaired cells in the midline of the thoracic and
abdominal ganglia. In contrast to the ventral ganglion, the brain lobes
were devoid of any signal. In the larval CNS, cell-specific transcript
localization pattern shows a remarkable correlation with the previously
described octopamine-immunoreactive neuronal pattern (Monastirioti et
al., 1995). This strongly supports the identification of the gene we
have isolated as the gene that encodes TBH. We will, therefore, refer
to this gene as Th and its protein product as TBH.
TBH immunoreactivity resembles octopamine immunoreactivity
Polyclonal anti-TBH serum was raised in rats against a bacterially
expressed purified internal part of the protein
(Sal-Xho fragment) (Fig. 2B) and was
affinity purified as described in Materials and Methods. In
immunoblots, the affinity-purified antibody revealed a single band
corresponding to the predicted 76 kDa protein. Comparison of protein
extracts from adult heads and bodies shows that TBH is enriched in the
head, but it is also present in the body at lower levels (Fig.
5A).
Fig. 5.
Immunoblot analysis of wild-type and mutant lines.
Affinity-purified anti-TBH antiserum was used as the primary antibody;
signal was visualized by chemiluminescent detection. A,
Immunoblot analysis of head and body homogenates from Canton-S male and
female flies. Protein (30 µg) from each sample was analyzed by
SDS-PAGE on a 7.5% gel. B, TBH immunodetection in a sample
of lines produced by the P element mutagenesis. Protein, equivalent of
one head homogenate, from the original p845 line (insert in the
sn locus) and five independent new insertion lines was
loaded. To control for sample loading, the blot was stripped and
reprobed using an anti-Tubulin antibody. A total of 250 lines were
screened by this method. Note the lack of signal in MF372 lane.
C, TBH immunodetection in a sample of lines in which the
transposon in MF372 was excised. The procedure was the same as in
B. A total of 67 lines were screened. Note the lack of
signal in M18 and MF372 lanes and normal signal in M6 and M11
lanes.
[View Larger Version of this Image (29K GIF file)]
Immunocytochemical analysis of the larval CNS using the
affinity-purified antibody showed that the protein is detected in cell
bodies of the ventral ganglion in a pattern that correlates with the
RNA expression pattern. A large group of intensely stained cells is
detected in the ventral midline of the subesophageal ganglion, and two
to three cells are stained in the midline of each of the thoracic and
abdominal neuromeres (Fig. 6A). In
addition, pairs of paramedial cells (arrows in Fig.
6A) were detected in the three thoracic neuromeres
and in the first abdominal neuromere. Intense staining also was
detected in the neuropil. Immunoreactive fibers travel along both sides
of the midline and extend from the ventral ganglion to the central
brain lobes. Transverse fibers also are detected between the brain
hemispheres extending to the center of each lobe where they form an
immunoreactive focus (Fig. 6A).
Fig. 6.
TBH immunoreactivity in representative samples of
larval CNSs from wild-type (Canton-S) and Th mutant
strains. Third instar larval CNSs were immunoreacted with
affinity-purified anti-TBH antiserum primary antibody and an anti-rat
FITC-conjugated secondary antibody. These images were collected as Z
series at the same laser level and gain settings. Care was taken to
photograph and develop the negatives using similar conditions, but the
images from the mutant brains were developed longer to show the
residual signal. Each image represents a superimposition of 8 to 10 confocal sections taken at a Z step of 2.16 µm. In all images, the
anterior is to the top. A, Confocal image of wild-type
larval CNS. Note the intense immunoreactivity in the neuropil and the
characteristic foci (white arrows), one in each brain lobe.
In the ventral ganglion, note the cells in the subesophageal region
(black arrow) and along the midline in the thoracic and
abdominal ganglia and the paramedial cells (black
arrowheads). B, Confocal image of a larval CNS of the
ThMF372 mutant. Note the near absence of the
neuropil staining and the reduced signal in the TBH-immunoreactive
cells compared with the wild type. (Because of the low TBH signal, the
background signal is high). C, Confocal image of a larval
CNS from the ThnM18 excision mutant. Note the
complete absence of immunoreactivity from both the neuropil and the
TBH-expressing cells. Black arrow points to the
subesophageal ganglion; black arrowheads delineate thoracic
ganglia. (We believe that the dotted pattern along the midline of the
ventral ganglion is caused by nonspecific staining, because we have
observed such staining in dorsal midline using other antibodies,
especially when the antibody concentration is high. This pattern is
confined to the dorsal-most sections, which are not included in the
wild-type CNS representation in A. Br, Brain
lobes; sb, subesophageal ganglion; th: thoracic
ganglion; ab: abdominal ganglion. Scale bar, 50 µm in
A-C.
[View Larger Version of this Image (104K GIF file)]
The overall cellular and neuropil expression of TBH in the larval CNS
shows a striking similarity to the octopamine immunoreactivity pattern
(Monastirioti et al., 1995, their Fig. 1). However, more cell bodies in
the ventral midline of the abdominal neuromeres seem to be
TBH-immunoreactive than were observed to be octopamine-immunoreactive.
It is possible that octopamine levels in the extra cells were below
detectable levels or that the TBH is expressed in additional cells.
Because of specific and incompatible fixation conditions necessary for
each antigen [formaldehyde for TBH, high percentage (6.25%) of
glutaraldehyde for octopamine], we have been unable to detect both
octopamine and TBH antigens simultaneously.
Generation of hypomorphic and null Th mutants
In situ hybridization of the cDNA clone to third instar
larval polytene chromosomes and preliminary molecular characterization
of the genomic DNA (data not shown) localize the gene to 7D1-2 region
on the X chromosome between the sn and l(1)mys
loci (Fig. 7).
Fig. 7.
Genetic map of the 7D1-2 region of the X
chromosome. Four known loci besides Th are represented by
boxes. The signs below each box indicate
the loci uncovered by Df(1)snC128 () or
covered by the Dp(1;2)sn+72d (+), two of the
chromosomal rearrangements corresponding to the area. Arrows
point to the position of p845 and MF372 transposon insertions.
olfE, Olfactory E; sn, singed; Th,
Tyramine -hydroxylase; fs(1)h, female sterile
homeotic; l(1)mys, lethal (1) myospheroid.
[View Larger Version of this Image (9K GIF file)]
Our strategy to generate Th mutants did not presuppose
any specific phenotypic consequence. We used P element-associated
``local transposon jumps'' methodology (Tower et al., 1993; Zhang and
Spradling, 1993) as a mutagen and screened the putatively mutagenized
chromosomes by assaying for TBH in immunoblots (Dolph et al., 1993).
When P elements are mobilized, there is a high incidence of local
transposon hops that can create insertions and possible disruption in
the neighboring genes (Cooley et al., 1988). A fertile P element
insertion line, p845, which carries an insertion of the synthetic
transposon PlacW (Bier et al., 1989) in the 5 end of the sn
gene, causing a singed phenotype, was available (K. O'Hare, personal
communication) (Fig. 7). We mobilized this P element using the 2-3
transposase and isolated new insertions that are accompanied by
reversion at the sn locus (details of the mutagenesis to be
described elsewhere). Approximately 250 lines carrying new insertions
on the X chromosome were screened for TBH protein in immunoblots. A
representative immunoblot (Fig. 5B) presents the results
from six lines, including line MF372 in which TBH protein was not
detected. Southern analysis showed that the P transposon has been
inserted in the genomic region of the Th gene, 3 to a
genomic EcoRI fragment containing the ATG (data not
shown).
TBH immunoreactivity in the third instar larval MF372 CNS was much
reduced in the cell bodies, although the pattern was similar to the
wild type, and neuropil was devoid of any signal (Fig. 6B).
This suggested that MF372 transposon insertion in the Th
locus has caused a hypomorphic mutation,
ThMF372.
To generate null Th mutants, excisions of the P
transposon insertion in ThMF372 were created
by using the 2-3 transposase. Individual lines from 62 independent
excision chromosomes were established and screened for disruption of
the Th locus by assaying protein levels in immunoblots.
Excision events can be grouped in three classes with a distinct
phenotypic outcome in each case. First, many excisions are precise and
restore the gene and the phenotype. In these cases, wild-type TBH
signal is observed (51/67), as in lines M6 and M11 (Fig.
5C). Second, in some excision events, the element is excised
only partially, which may result either in the same phenotype or in one
distinct from the original insertion. Examples are lines M22 and M25,
which show reduced TBH signal (Fig. 5C). Finally, some
imprecise excisions also delete flanking genomic sequences resulting in
disruption of the gene, creating protein nulls such as line M18 (Fig.
5C). Larval CNSs from lines M18, the putative protein null,
and M6, the revertant to wild type, then were checked
immunocytochemically. In contrast to the initial MF372 insertion line,
TBH immunoreactivity was not detected in the larval CNS of the M18 line
(Fig. 6C), whereas staining in the M6 line was
indistinguishable from the wild type (data not shown). These data
suggest that M18 represents a null mutation,
ThnM18, and that M6 is a revertant
(ThrM6) of the ThMF372
hypomorphic mutation.
Th-null mutants are devoid of octopamine and
accumulate tyramine
Null mutations of the Th gene should block
conversion of tyramine to octopamine and, thus, eliminate octopamine
from the fly brain while perhaps accumulating tyramine. To assess
levels of octopamine, its precursor tyramine, and its metabolite
N-acetyloctopamine, brain extracts of mutants, null
ThnM18, hypomorph
ThMF372 and control flies, Canton-S, and
revertants ThrM6 and
ThrM11 were analyzed by HPLC with
electrochemical detection (HPLC-ECD) (Table 1).
For comparison, we also measured dopamine and
serotonin in the same extracts. In the wild-type (Canton-S) brains, the
amount of octopamine measured was ~263 pg/brain; the values in the
revertants were comparable at 275 and 150 pg/brain. However, an
octopamine peak was not detected in the null mutant
ThnM18, and very little octopamine; ~7.6
pg/brain was found in the hypomorph ThMF372.
Interestingly, a six- to eightfold increase in tyramine was observed in
the null ThnM18 and the hypomorph
ThMF372 (Table 1) compared with the normal
level of ~8 pg/brain. In addition, the metabolite
N-acetyloctopamine was not detected (<0.5 pg) in the mutant
lines. No significant differences in dopamine and serotonin levels were
detected. Norepinephrine was not detected in either wild-type or mutant
extracts (data not shown).
The HPLC results are consistent with the biochemical expectation that
loss of tyramine hydroxylation in Th-null animals will
result in absence of octopamine and accumulation of tyramine.
Th-null females retain eggs
Mutant Th flies survive to adulthood; their external
appearance is normal, and they do not exhibit any obvious defects.
Under optimal growing conditions, their viability is comparable to
their heterozygous siblings, but under unfavorable, crowded conditions,
their viability is reduced. Mutant males are fertile, but the mutant
females are sterile. These females appear to mate normally; they
produce fully developed ovarioles that become abnormally large within a
few days of eclosion as eggs are retained.
To further investigate the sterility and egg retention observed in the
mutant females, and to ascertain that the defects were a direct
consequence of the genetic lesion at the Th locus,
individual females of different genotypes were assayed for number of
progeny produced over a defined time. The data are tabulated in Table
2, the genetic limits of Deficiency
(Df(1)snC128) and Duplication
(Dp(1;2)sn72d+) chromosomes that uncover and
cover Th are explained in Figure 7. The following
observations support association of the egg-retention defect with the
lesion at the Th locus. (1) During a 6 d assay period,
ThnM18-null mutant females or
ThnM18/Deficiency females did not produce any
progeny. (2) Flies of the genotype
ThnM18/Deficiency/Duplication were fully
fertile. (3) Females homozygous for the insertion chromosome
ThMF372 showed near normal fertility, but
ThMF372/Deficiency females showed very low
fecundity. The decrease in fertility of the
ThMF372/Deficiency-bearing females suggests a
gene-dosage effect on the expression of the phenotype. (4) Females
carrying the revertant chromosomes ThrM6 and
ThrM11 are fully fertile. These revertants
are important controls, because they have wild-type TBH protein levels
and normal fertility.
Octopamine feeding induces egg-laying in Th
mutant females
To check whether the egg-retention defect is caused by
octopamine deficit, 6-d-old mated mutant
ThnM18 females were transferred to food
supplemented with different concentrations of octopamine and allowed to
lay eggs for 6 d, and progeny counted on day 17 from the transfer.
Females transferred to 4-10 mg/ml octopamine produced low but
significant numbers of progeny (average 12 progeny per fly). Higher
concentrations (25-50 mg/ml) of octopamine induced egg-laying, but the
mutant females died within a few hours. No progeny was produced when
food was supplemented with 10 mg/ml of tyramine or dopamine. However,
norepinephrine had the same effect as octopamine at 10 mg/ml. Thus, we
can conclude that the sterility of Th mutant females is a
direct consequence of octopamine deficit resulting from the disruption
of the Th locus.
DISCUSSION
The experiments described in this paper represent the initial step
toward our ultimate goal to understand the role of octopamine in insect
behavior and physiology. We undertook the molecular identification of
the Drosophila gene that encodes TBH, the enzyme that
catalyzes the final step in the synthesis of octopamine, using an
approach based on the supposition that TBH is related evolutionarily to
mammalian DBH. TBH, the protein product of the gene we identified,
showed ~39% overall identity to the mammalian DBH protein. The
functional similarity between these two proteins was underscored
further by the high conservation around Tyr273
and His452 (TBH sequence), the two residues that
have been identified as putative active sites of the bovine DBH (DeWolf
et al., 1988, 1989), and by the conservation of the paired histidine
residues that are important for copper binding. An antibody generated
against TBH demonstrated that TBH expression in the nervous system
closely resembles the octopamine immunoreactivity (Monastirioti et al.,
1995). Thus, the immunocytochemical localization was consistent with
the notion that TBH, as part of the octopamine biosynthetic machinery,
should be localized in the same cells as octopamine. TBH
immunoreactivity is detected in both neuronal cell bodies and in
neuronal processes.
A genetic strategy was developed to create mutations at the
Th locus that did not depend on ``a priori'' knowledge
of the phenotype other than a decrease or absence of TBH protein. A P
transposon insert in the Th locus was recovered initially
(ThMF372) using local transposon hops (Tower
et al., 1993; Zhang and Spradling, 1993), and precise and imprecise
excisions of this insert were subsequently induced. Based on immunoblot
and immunocytochemical analysis, we had the following genotypes on the
same parental chromosome: (1) ThMF372, a
hypomorphic mutation that reduces TBH level dramatically, (2)
ThrM6, a revertant that restores the TBH
levels completely, and (3) ThnM18, a null
mutation that results in flies devoid of any TBH protein.
We measured the levels of octopamine, tyramine,
N-acetyloctopamine, dopamine, and serotonin in brain
extracts of mutant and wild-type flies. Several conclusions regarding
the Th-null flies can be drawn from this analysis. First,
Th-null flies have octopamine levels below detection
capability, but the relatively low wild-type levels of tyramine are
increased by approximately eightfold. Second,
N-acetyloctopamine, a metabolite of octopamine, also is
below the level of detection. Third, dopamine and serotonin are not
affected significantly. Therefore, the two major factors that could
impact the behavior and physiology of Th-null mutants are
the absence of octopamine and increased levels of tyramine. Although
low levels of tyramine are present in insect nervous systems, at
present its function is not known. However, pharmacological studies
using crude membrane preparations from Drosophila have
suggested tyramine as a partial agonist for octopamine receptors (Uzzan
and Dudai, 1982). In fact, in the case of a cloned
Drosophila octopamine/tyramine receptor, tyramine has been
shown to be more potent than octopamine when assayed in binding studies
or as an inhibitor of adenylate cyclase activity in stably transfected
mammalian cells (Saudou et al., 1990; Robb et al., 1994). Although the
endogenous ligand for this receptor in Drosophila has not
been identified, its existence and pharmacological properties suggest
that a severalfold increase in the tyramine level could have a
physiological effect. Moreover, because TBH is transported in neuronal
processes, in the null mutants, TBH-containing vesicles must accumulate
tyramine, which in all likelihood will be released at the synapse.
Flies devoid of octopamine eclose, and the eclosed adults, in general,
appear normal. They are able to walk, fly, and mate. The viability of
these flies suggests that octopamine is not essential in any vital
physiological function. However, we cannot rule out that an increased
level of tyramine may substitute functionally for octopamine in this
genotype. The reduced viability of Th nulls under crowded
conditions suggests that in the wild, these flies would be severely
handicapped.
Homozygous Th-null females retain fully developed eggs.
This functional deficit is correlated directly with the null mutation
at the Th locus for the following reasons. (1) Both
homozygous females carrying the null mutation
ThnM18, and hemizygous females
ThnM18/Df(1)snC128 produce no
progeny, whereas 100% of the examined females having the above
hemizygous phenotype as well as the duplication chromosome
Dp(1;2)sn72d produce progeny. (2) Revertant
females, ThrM6 and
ThrM11, produce normal numbers of progeny.
(3) Homozygous females carrying the hypomorphic mutation
ThMF372 do produce progeny, but hemizygous
ThMF372/Df(1)snC128 females show
impaired fertility, because they produce reduced numbers of
progeny.
The cause of the reduced fertility appears to be retention of eggs in
the females, although some role in the late stage of egg maturation
cannot be ruled out. This retention could result from absence of
octopaminergic input in some process essential in transit of the egg
from the ovarioles into the ovipositor via the oviduct and/or extrusion
of the egg by the ovipositor. An obvious possibility is that
octopaminergic input is important in myogenic contractions; however,
that octopamine may have a role in some other as yet undefined
physiological process necessary for generating a viable fertilized egg
cannot be discounted. Studies in other insects, locusts for example,
have shown that myogenic contractions of the visceral muscles of the
oviduct and ovipositor, which appear to be under neurohormonal control,
are responsible for moving eggs along to the ovipositor in an orderly
fashion and for egg extrusion (Lange and Orchard, 1984; Lange et al.,
1984). Furthermore, it would appear that different regions of the
oviduct will exhibit distinct contractile patterns depending on whether
eggs are pushed, retained, or extruded. Octopamine has been implicated
in the modulation of oviductal visceral muscle by two lines of
evidence: innervation of the oviductal muscle by octopaminergic median
unpaired neurons (Kalogianni and Pflüger, 1992), and
physiological evidence that octopamine modulates activity of the
oviductal muscle (Kalogianni and Theophilidis, 1993). To our knowledge,
there is no physiological evidence of the effect of octopamine on the
ovipositor; however, modulation of the sting response by octopamine has
been demonstrated in the honey bee, Apis mellifera (Burrell
and Smith, 1995).
The egg retention observed in ThnM18 females
can be connected directly with the absence of octopamine, because
feeding octopamine to females is sufficient to induce egg deposition.
Because feeding tyramine has no effect, one can conclude that at least
the receptors involved in this behavior are octopamine-specific and
tyramine is not a potent agonist. On the other hand, noradrenaline is
an agonist, because it too can induce egg laying. Indeed, earlier
pharmacological studies support the idea that noradrenaline binds to
octopamine receptors and causes at least stimulatory effects in
adenylate cyclase activity, similar to that of octopamine (Uzzan and
Dudai, 1982). However, noradrenaline cannot be detected by HPLC either
in wild-type or Th mutant brain preparations (C.E. Linn,
unpublished observations). Additional analysis is necessary before we
know whether octopaminergic processes innervate the relevant muscle or
octopamine acts as a neurohormone.
Identification of the Drosophila Th gene and creation of
null mutations in the Th locus constitute an important
step in developing a molecular genetic approach to understand
octopamine-regulated processes. These approaches will allow
characterization of subtle mutant phenotypes and facilitate assignment
of defined neurons with specific behaviors, and are likely to help in
sorting out receptor functions. Progress in understanding
octopamine-mediated processes and the molecular reagents generated in
the fruit fly have potential to catalyze studies in other invertebrate
systems better suited for physiological studies. Moreover, biochemical
characterization of TBH may help develop strategies in insect pest
management.
FOOTNOTES
Received Nov. 14, 1995; revised March 27, 1996; accepted March 29, 1996.
This work was supported by National Institutes of Health Grant NS23510
and National Research Competitive Initiative Grants Program/U.S.
Department of Agriculture Grant 37302-1880 to K.W. Confocal microscopy
was made feasible by National Institutes of Health Shared
Instrumentation Grant RRO5615. We appreciate the excellent technical
assistance provided by Deborah Bordne in the genetic studies. We thank
Dr. Kevin O'Hare for information regarding the fly stock p845, Edward
Dougherty for photography and help with confocal microscopy, Patricia
Parmenter for careful reading of this manuscript, and members of the
White laboratory for helpful discussions. We gratefully acknowledge
critical comments on this manuscript from Leslie Griffith, Dimitris
Tzamarias, and Marshall Gorden.
Correspondence should be addressed to Kalpana White, Biology
Department, Brandeis University, Waltham, MA 02154.
Maria Monastirioti's present address: Institute of Molecular
Biology and Biotechnology, FORTH, P.O. Box 1527, 71110 Heraklion,
Crete, Greece.
REFERENCES
-
Altschul SF,
Lipman DJ
(1990)
Protein database searches
for multiple alignments.
Proc Natl Acad Sci USA
87:5509-5513.
[Abstract/Free Full Text]
-
Arakawa S,
Gocoyne JD,
McCombie WR,
Urquhart DA,
Hall LM,
Fraser CM,
Venter JC
(1990)
Cloning, localization, and permanent
expression of a Drosophila octopamine receptor.
Neuron
2:343-354.
-
Bicker G,
Menzel R
(1989)
Chemical codes for the control of
behaviour in arthropods.
Nature
337:33-39.
[Medline]
-
Bier E,
Vaessin H,
Shepherd S,
Lee K,
McCall K,
Barbel S,
Ackerman L,
Carretto R,
Uemura T,
Grell E,
Jan LY,
Jan YN
(1989)
Searching for pattern and mutation in the
Drosophila genome with a P-lacZ vector.
Genes Dev
3:1273-1287.
[Abstract/Free Full Text]
-
Braun G,
Bicker G
(1992)
Habituation of an appetitive reflex
in the honeybee.
J Neurophysiol
67:588-598.
[Abstract/Free Full Text]
-
Burrell BD,
Smith BH
(1995)
Modulation of the honey bee
(Apis mellifera) sting response by octopamine.
J Insect Physiol
41:671-680.
-
Cavener DR
(1987)
Comparison of the consensus sequence
flanking translational start sites in Drosophila and
vertebrates.
Nucleic Acids Res
15:1353-1361.
[Abstract/Free Full Text]
-
Cooley L,
Kelley R,
Spradling A
(1988)
Insertional
mutagenesis of the Drosophila genome with single P elements.
Science
239:1121-1128.
[Abstract/Free Full Text]
-
David JC,
Coulon JF
(1985)
Octopamine in invertebrates and
vertebrates: a review.
Prog Neurobiol
24:141-185.
[ISI][Medline]
-
Davis LG,
Dibner MD,
Battey JF
(1986)
Guanidine
isothiocyanate preparation of total RNA.
In: Basic methods in molecular biology,
, p. 129. New York: Elsevier.
-
DeWolf WE Jr,
Carr SA,
Varrichio A,
Goodhart PJ,
Mentzer MA,
Roberts GD,
Southan C,
Dolle RE,
Kruse LI
(1988)
Inactivation of
dopamine -hydroxylase by p-cresol: isolation and characterization of
covalently modified active site peptides.
Biochemistry
27:9093-9101.
[Medline]
-
DeWolf WE Jr,
Chambers PA,
Southan C,
Saunders D,
Kruse LI
(1989)
Inactivation of dopamine -hydroxylase by
-ethynyltyramine: kinetic characterization and covalent modification
of an active site peptide.
Biochemistry
28:3833-3842.
[Medline]
-
Dolph PJ,
Ranganathan R,
Colley NJ,
Hardy RW,
Socolick M,
Zucker CS
(1993)
Arrestin function in inactivation of a G
protein-coupled receptor rhodopsin in vivo.
Science
260:1910-1916.
[Abstract/Free Full Text]
-
Dudai Y,
Zvi S
(1984)
High affinity
[3H]octopamine-binding sites in D. melanogaster: interactions with ligands and relationship to
octopamine receptors.
Comp Biochem Physiol C
77:145-151.
-
Dudai Y,
Buxbaum J,
Corfas G,
Ofarim M
(1987)
Formamidines
interact with Drosophila octopamine receptors, alter the
flies' behavior and reduce their learning ability.
J Comp Physiol [A]
161:739-746.
-
Ebens AJ,
Garren H,
Cheyette BNR,
Zipursky SL
(1993)
The
Drosophila anachronism locus: a glycoprotein secreted
by glia inhibits neuroblast proliferation.
Cell
74:15-27.
[ISI][Medline]
-
Evans PD
(1980)
Biogenic amines in the insect nervous system.
Adv Insect Physiol
15:317-473.
-
Evans PD
(1985)
Octopamine.
In: Comprehensive insect physiology, biochemistry and pharmacology
(Kerknt, GA,
Gilbert, LI,
eds)
, p. 499. Oxford: Pergamon.
-
Evans PD
(1992)
Molecular studies on insect octopamine
receptors.
In: Comparative molecular neurobiology
(Pichon, Y,
eds)
, p. 286. Boston: BirkhauserVerlag.
-
Hoyle G
(1984)
Neuromuscular transmission in a primitive
insect: modulation by octopamine and catch-like tension.
Comp Biochem Physiol C
77:219-232.
-
Hubbard SC,
Ivatt RJ
(1981)
Synthesis and processing of
asparagine-linked oligosaccharides.
Annu Rev Biochem
50:555-583.
[ISI][Medline]
-
Itoh H,
Salvaterra P,
Itakura K
(1985)
Construction of an
adult Drosophila head cDNA expression library with lambda
gt11.
Dros Inf Serv
61:89.
-
Kalogianni E,
Pflüger HJ
(1992)
The identification of
motor and unpaired median neurones innervating the locust oviduct.
J Exp Biol
168:177-198.
[Abstract/Free Full Text]
-
Kalogianni E,
Theophilidis G
(1993)
Centrally generated
rhythmic activity and modulatory function of the oviductal dorsal
unpaired median (DUM) neurones in two orthopteran species
(Calliptamus SP. and Decticus albifrons).
J Exp Biol
174:123-138.
[Abstract]
-
Kongsuwan K,
Yu Q,
Vincent A,
Frisardi MC,
Roshbash M,
Lengyel JA,
Merriam J
(1985)
A Drosophila minute gene encodes
a ribosomal protein.
Nature
317:555-558.
[Medline]
-
Lamouroux A,
Vigny A,
Faucon Biguet N,
Darmon MC,
Frank R,
Henry JP,
Mallet J
(1987)
The primary structure of human dopamine-
hydroxylase and the relationship between the soluble and the
membrane-bound forms of the enzyme.
EMBO J
13:3931-3937.
-
Lange AB,
Orchard I
(1984)
Some pharmacological properties of
neuromuscular transmission in the oviduct of the locust, Locusta
migratoria.
Arch Insect Biochem Physiol
1:231-241.
-
Lange AB,
Orchard I,
Loughton BG
(1984)
Neural inhibition of
egg laying in the locust Locusta migratoria.
J Insect Physiol
30:271-278.
-
Lindsley DL,
Zimm GG
(1992)
The genome of Drosophila
melanogaster.
.
-
Linn CE Jr,
Poole KR,
Roelofs WL
(1994)
Studies on biogenic
amines and their metabolites in nervous tissue and hemolymph of adult
male cabbage looper moths. I. Quantitation of photoperiod changes.
Comp Biochem Physiol C
108:73-85.
-
Livingstone M,
Tempel B
(1983)
Genetic dissection of
monoamine neurotransmitter synthesis in Drosophila.
Nature
303:67-70.
[Medline]
-
Livingstone M,
Harris-Warrick RM,
Kravitz EA
(1980)
Serotonin
and octopamine produce opposite postures in lobsters.
Science II
208:76-79.
-
Malamud JG,
Mizisin AP,
Josephson RK
(1988)
The effects of
octopamine on contraction kinetics and power output of a locust flight
muscle.
J Comp Physiol [A]
162:827-835.
-
McCafferty B,
Angeletti RH
(1987)
Microsequencing of dopamine
beta-hydroxylase.
J Neurosci Res
18:289-292.
[ISI][Medline]
-
McMahon A,
Geertman R,
Sabban EL
(1990)
Rat dopamine
-hydroxylase: molecular cloning and characterization of the cDNA and
regulation of the mRNA by reserpine.
J Neurosci Res
25:395-404.
[ISI][Medline]
-
Monastirioti M,
Gorczyca M,
Rapus J,
Eckert M,
White K,
Budnik V
(1995)
Octopamine-immunoreactivity in the fruit fly
Drosophila melanogaster.
J Comp Neurol
356:275-287.
[ISI][Medline]
-
Nathanson JA
(1979)
Octopamine receptors, adenosine
3,5-monophosphate, and neural control of firefly flashing.
Science
203:65-68.
[Abstract/Free Full Text]
-
O'Dell K,
Burnet B
(1988)
The effects on locomotor activity
and reactivity of the hypoactive and inactive mutations of
Drosophila melanogaster.
Heredity
61:199-207.
-
O'Dell K,
Coulon JF,
David JC,
Papin C,
Fuzeau-Braesch S,
Jallon JM
(1987)
La mutation inactive produit une diminution marquee
d'octopamine dans le cerveau des Drosophiles.
C R Acad Sci III
305:199-202.
-
Orchard I,
Ramirez JM,
Lange AB
(1993)
A multifunctional role
for octopamine in locust flight.
Annu Rev Entomol
38:227-249.
[ISI]
-
Pearson RB,
Woodgett JR,
Cohen P,
Kemp BE
(1985)
Substrate
specificity of a multifunctional calmodulin-dependent protein kinase.
J Biol Chem
260:14471-14476.
[Abstract/Free Full Text]
-
Robb S,
Cheek TR,
Hannan FL,
Hall LM,
Midgley JM,
Evans PD
(1994)
Agonist-specific coupling of a cloned
Drosophila octopamine/tyramine receptor to multiple second
messenger systems.
EMBO J
13:1325-1330.
[ISI][Medline]
-
Saudou F,
Amlaiky N,
Plassat JL,
Borrelli E,
Hen R
(1990)
Cloning and characterization of a
Drosophila tyramine receptor.
EMBO J
9:3611-3617.
[ISI][Medline]
-
Sigel H
(1981)
Metal ions in biological systems, Vol 13.
.
-
Snodgrass RE
(1956)
Anatomy of the honey bee.
.
-
Studier FW,
Moffatt BA
(1986)
Use of bacteriophage T7 RNA
polymerase to direct selective high-level expression of cloned genes.
J Mol Biol
189:113-130.
[ISI][Medline]
-
Tower J,
Karpen GH,
Craig N,
Spradling AC
(1993)
Preferential
transposition of Drosophila P elements to nearby chromosomal
sites.
Genetics
133:347-359.
[Abstract]
-
Uzzan A,
Dudai Y
(1982)
Aminergic receptors in
Drosophila melanogaster: responsiveness of adenylate cyclase
to putative neurotransmitters.
J Neurochem
38:1542-1550.
[ISI][Medline]
-
Wallace BG
(1976)
The biosynthesis of
octopamine-characterization of a lobster tyramine -hydroxylase.
J Neurochem
26:761-770.
[ISI][Medline]
-
Yao KM,
Samson ML,
Reeves R,
White K
(1992)
Gene
Elav of Drosophila melanogaster: a prototype for
neuronal-specific RNA binding protein gene family that is conserved in
flies and humans.
J Neurobiol
24:723-739.
-
Zhang P,
Spradling AC
(1993)
Efficient and dispersed local P
element transposition from Drosophila females.
Genetics
133:361-373.
[Abstract]
This article has been cited by other articles:
|
|
|
|
 
A. Crocker and A. Sehgal
Octopamine Regulates Sleep in Drosophila through Protein Kinase A-Dependent Mechanisms
J. Neurosci.,
September 17, 2008;
28(38):
9377 - 9385.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Buhl, K. Schildberger, and P. A. Stevenson
A muscarinic cholinergic mechanism underlies activation of the central pattern generator for locust flight
J. Exp. Biol.,
July 15, 2008;
211(14):
2346 - 2357.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C.-h. Yang, P. Belawat, E. Hafen, L. Y. Jan, and Y.-N. Jan
Drosophila Egg-Laying Site Selection as a System to Study Simple Decision-Making Processes
Science,
March 21, 2008;
319(5870):
1679 - 1683.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
