From the Department of Neuroscience and Cell Biology,
Robert Wood Johnson Medical School/University of Medicine and Dentistry
of New Jersey, Piscataway, New Jersey 08854 and § National
Centre for Biological Sciences, TIFR Centre, IISc Campus, P. O. Box 1234, Bangalore 560012, India
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ABSTRACT |
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Biotransformation enzymes have been found in the
olfactory epithelium of vertebrates. We now show that in
Drosophila melanogaster, a UDP-glycosyltransferase (UGT),
as well as a short chain dehydrogenase/reductase and a cytochrome P450
are expressed specifically or preferentially in the olfactory organs,
the antennae. The evolutionarily conserved expression of
biotransformation enzymes in olfactory organs suggests that they play
an important role in olfaction. In addition, we describe five
Drosophila UGTs belonging to two families. All five UGTs
contain a putative transmembrane domain at their C terminus as is the
case for vertebrate UGTs where it is required for enzymatic activity.
The primary sequence of the C terminus, including part of the
transmembrane domain, differs between the two families but is highly
conserved not only within each Drosophila family, but also
between the members of one of the Drosophila families and
vertebrate UGTs. The partial overlap of the conserved primary sequence
with the transmembrane domain suggests that this part of the protein is
involved in specific interactions occurring at the membrane surface.
The presence of different C termini in the two Drosophila
families suggests that they interact with different targets, one of
which is conserved between Drosophila and vertebrates.
All organisms live in environments that contain potentially
harmful chemicals, both natural and man-made. Extensive studies of
detoxification in the vertebrate liver provide a framework to the study
of detoxification mechanisms in other systems (1-3). Detoxification
often occurs in two phases; in phase I, the initial compound is
transformed into a more reactive species. A variety of different
chemical transformations are involved, including redox reactions
catalyzed by enzymes of the cytochrome P450 superfamily (4, 5) and
members of the short chain dehydrogenase/reductase (SDR)1 family (6). Phase II
reactions consist in the addition, either to a product of a phase I
reaction or directly to many toxic chemicals, of a highly polar group
such as UDP-glucuronosyl (catalyzed by UDP-glucuronosyltransferases)
(2) or glutathione (catalyzed by glutathione S-transferases
(7). Products of phase II reactions are hydrophilic; they can no longer
cross membranes and are eliminated by secretion. In addition to the
elimination of environmental toxins, phase I and II biotransformation
enzymes participate in the removal of toxic side products of normal
metabolism (e.g. bile acids), participate in drug clearance,
and play an important role in the synthesis of hormones such as
prostaglandins and some steroids (3). Finally, the involvement of these
enzymes in the production of carcinogens (8), drug clearance, and some hereditary diseases (9) makes an understanding of their function important for human health.
Biotransformation enzymes related to those found in vertebrates have
also been found in insects and are likely to play equally important
roles. Cytochrome P450s and glutathione S-transferases in
particular have been implicated in insect resistance to pesticides (10). UDP-glucuronosyltransferases are part of a superfamily of
UDP-glycosyltransferases (UGTs) present in plants, animals, and
bacteria (11). These enzymes transfer the sugar moiety of UDP-glucose,
UDP-glucuronic acid, UDP-galactose, or UDP-xylose to a variety of
hydrophobic substrates (11). Insects contain UGT activities that can
use UDP-glucose but not UDP-glucuronic acid as a glycosyl donor
(12-14). Although no molecular information on any insect UGT was
available until this work, baculoviruses infecting several species of
moths have been shown to encode ecdysteroid UDP-glucosyltransferases
(15, 16). These viral enzymes specifically inactivate ecdysteroids, the
molting hormones of the infected hosts, and thus prolong the larval
stage permissive to viral replication. ecdysteroid
UDP-glucosyltransferases lack a C-terminal transmembrane domain and are
secreted in the hemolymph where ecdysone is present (15).
Here we report that, in Drosophila, several phase I and II
biotransformation enzymes are expressed preferentially in the olfactory organs, the antennae. This observation is reminiscent of the
preferential or exclusive expression of a cytochrome P450, UGT, and
glutathione S-transferase in the vertebrate olfactory
epithelium (17-20). The presence of these enzymes in the olfactory
organs of such evolutionarily distant organisms supports the notion
that they play an important role in olfaction. In addition, the
availability of the first sequences of UGTs from insects sheds light on
the structure and function of the C-terminal domain of vertebrate UGTs.
Generation of an Appendage cDNA Library, Cloning, and
Sequencing--
Partial cDNA clones for AntP450,
AntDH, and DmeUgt35b were initially found through
random sequencing of clones in an antennae-minus-heads subtracted
cDNA library that was described previously (21). All cDNA
sequences discussed in this paper were obtained from full-length
cDNA clones isolated by using the partial clones as probes to
screen an appendage cDNA library in Lambda-ZAP (Stratagene). Appendage RNA was generated from poly(A)+ RNA isolated from an appendage fraction (see below and Ref. 22).
Analysis of Gene Expression--
Total RNA for Northern blots
was isolated either from hand-dissected antennae or legs or from
mass-produced body fractions generated as follows
(22). Frozen flies are vortexed, and the
resulting body parts are then sieved to yield three fractions: appendages (antennae, legs, and wings), heads (without antennae), and
bodies (abdomen and thorax, decapitated and without legs or wings).
Because all the proteins under study belong to multigene families, the
probes used were first tested on Southern blots under identical
conditions to ensure that there was no detectable cross-reactivity to
related genes (not shown). The probes used are indicated in the legend
to Fig. 1.
Sequence Analysis--
Sequences were assembled and analyzed
using Wisconsin Package Version 9.1, Genetics Computer Group, Madison,
WI. Data base searches were performed using BLAST (23) both on the
Berkeley Drosophila Genome Project and National Center for
Biotechnology Information www servers (http://www.fruitfly.org/blast/
and
http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast?Jform=0, respectively).
The central portion of the DmeUgt35a cDNA is represented
by nucleotides number 1-324 of P1 clone DS07339 that has been mapped by the BDGP to 86D5-10. Similarly, the 3' end of DmeUgt35b
is partially represented in preliminary sequence (nucleotides 1-326) of DS08785 mapped to 86D1-D2. AntP450 is a partial cDNA
sequence identical to EST number GH06928 and with significant sequence similarity to the C terminus of the cyp6 family of
cytochrome P450s (24).
The three members of the DmeUgt37 family result from
conceptual translation of sequences from the Drosophila
Genome Project. The sequences encoding the three open reading frames
(ORFs) are present at nucleotides number 68193 to 66570 (reverse
strand) of P1 clone DS51087 for DmeUgt37a1; positions
22203-22207 of DS00108 for DmeUgt37b1 and nucleotides
111614 to 113188 of DS07321 for DmeUgt37c1. To generate the
DmeUgt37a1 ORF, we removed a likely intron at
positions 67516-67464. In the case of DmeUgt37b1*, deletion of a single T in a stretch of 5 Ts at positions 22203-22207 results in
the creation of an ORF with high similarity to the other
Drosophila UGT37 ORFs (see text). The frameshift in the
database sequence could be caused by a sequencing error or a recent
mutation resulting in a pseudogene. In either case, conceptual
translation of the "corrected" DmeUgt37b1* sequence
represents a UGT with high similarity to the other UGT37 proteins
throughout its open reading frame and therefore likely represents a
real UGT, even if it no longer exists in present day laboratory canton
S strains. For the purpose of this publication we will keep the
asterisk to denote the ambiguity. Note that the inclusion of the
DmeUgt37b1* ORF is not necessary to reach the conclusions
about the domain structure of Drosophila UGTs that are
discussed in the text. The sequences of the three novel cDNAs
discussed here have been deposited in the GenBankTM data
base and their accession numbers are as follows: AntDH, AF116553; DmeUgt35a, AF116555; and Dme
Ugt35b, AF116554. Non-Drosophila UGTs are designated
according to the names given by the UGT Nomenclature Committee and
accession numbers are given in the figures.
Several Biodegradation Enzymes Are Expressed Preferentially in the
Antennae of Drosophila--
We have previously described a subtracted
cDNA library (antennae-minus-heads) enriched in cDNAs expressed
specifically or preferentially in the antennae of Drosophila
melanogaster. Analysis of a number of those cDNAs led to the
discovery of several putative odorant-binding proteins with
distinct expression patterns on the surface of the antennae, suggesting
a role for odorant-binding proteins in olfactory discrimination (21).
Here we report that, in addition to odorant-binding proteins, sampling
of our library has led to the discovery of cDNAs coding for a
cytochrome P450 (AntP450, see "Experimental
Procedures"), a UGT (Dme Ugt35b, see below for
explanation of the nomenclature), and a short chain dehydrogenase/reductase (AntDH). The proteins encoded by
these three cDNAs are related to vertebrate enzymes involved in
detoxification. In addition to DmeUgt35b isolated from our
subtractive library, a second UGT-encoding cDNA
(DmeUgt35a) was isolated from an appendage cDNA library
by cross-hybridization to DmeUgt35b (see below).
Analysis of expression patterns was performed by Northern blots using
probes specific for DmeUgt35a, DmeUgt35b,
AntDH, and AntP450. In every case the probes were
generated from sequences that show little similarity with other genes
of the same family and the lack of cross-hybridization was verified on
Southern blots performed under identical hybridization conditions (data
not shown). The Northern blot shown in Fig.
1 analyzes RNAs prepared from two types
of samples as indicated above the lanes. First, fly parts were
separated into three fractions (see "Experimental Procedures"): appendages (third antennal segments, legs, and wings), heads (without third antennal segments), and bodies (decapitated and without legs).
Second, to differentiate between different appendages, we separately
hand-collected third antennal segments and legs from approximately 200 flies. Expression of AntDH is restricted to appendages, and
within appendages it is much higher in third antennal segments than in
legs. AntP450 and DmeUgt35b are more ubiquitous
because they are detected in heads and bodies albeit at slightly lower
levels than in appendages. Nevertheless, both genes are expressed at
highest levels in the third antennal segment. In contrast to these
three genes, DmeUgt35a shows lower expression in the third
antennal segment than in legs. Although most sensilla involved in taste
can be found on the legs, head, and wings of Drosophila, the
third antennal segment contains the vast majority of olfactory sensilla
(25). The preferential expression of AntDH, DmeUgt35b, and AntP450 in the third antennal
segment is therefore suggestive of a role in olfaction.
To further delineate expression patterns we used in situ
hybridization on cryosections of heads and antennae (Fig.
2). Of the genes discussed here, only
AntDH expression was detected by this method, most likely
because of its higher expression levels (data not shown). Consistent
with the Northern blot analysis, in situ hybridization to
AntDH mRNA is restricted to the third antennal segment;
no expression is detected in the head (Fig. 2) or in the second
antennal segment (not shown). This observation further supports a role
for AntDH in olfaction, because the second antennal segment
does not contain chemosensory hairs (25). Within the third antennal
segment however, AntDH expression appears uniformly distributed (Fig. 2 and data not shown), in contrast with several odorant-binding proteins, each of which is restricted to a single morphological type of sensillum with a nonuniform distribution on the
antennal surface (21, 26,
27).2
AntDH Is a Short Chain Dehydrogenase/Reductase Specifically
Expressed in Third Antennal Segments--
The initial AntDH
cDNA clone was used as a probe to isolate a full-length cDNA
clone from an appendage cDNA library (see "Experimental Procedures"). A single ORF has sequence similarity to the members of
a large family of SDRs (Fig. 3) (28)
found in organisms ranging from prokaryotes and plants to humans (28).
The overall degree of sequence identity is relatively low as is typical
in this family of proteins (the closest sequence we have found is
aldehyde reductase from the bacterium Streptomyces
clavuligerus, Cla9_Sc in Fig. 3, which has 36% identity with
AntDH). However, AntDH has all the residues that have been demonstrated
to be important for the function of SDRs (Fig. 3). In particular, the
GlyXXXGlyXGly motif close to the amino terminus
corresponds to a coenzyme binding pocket for either NAD or NADP. In
addition, the TyrXXXLys motif necessary for catalysis can be
found at positions 164 through 168 and Ser144 is the likely
homologue of the essential Ser139 of alcohol dehydrogenase
(29). Although most SDRs are cytoplasmic, some members of this family
are microsomal or even extracellular (30). Contrary to the case of the
membrane-associated mouse corticosteroid 11- Identification of Nine Putative Drosophila UGTs--
When probing
the appendage library with our partial Ugt clone we found
two classes of clones that hybridize at different intensities. Southern
blotting and sequence analysis shows that these phages correspond to
two different cDNAs each encoded by a separate gene, which we will
call DmeUgt35a and -b (see the last paragraph
under "Results" for a justification of this nomenclature). We have
mapped both sequences by in situ hybridization to
cytogenetic locations 86C-D in the Drosophila genome,
suggesting that these two genes have their origin in a relatively
recent duplication. More recently, the Berkeley and European
Drosophila genome projects have sequenced parts of both
DmeUgt35 genes, refining the mapping to 86D5-10 and 86D1-2
for the a and b genes, respectively.
We have also found that several other likely Ugt sequences
are present in the Drosophila genome project data. These
include three genomic DNA sequences containing full-length ORFs that
define three members of a second family of Drosophila UGTs,
UGT37: DmeUgt37a1, -b1 and -c1 (see below for an
explanation of the nomenclature). Five other likely Ugt
genes are represented by partial cDNA sequences or ESTs (EST
numbers GH06505, GH09393, GM04645, LD25345, and LD15335). The first
four are 5' sequences coding for NH2 termini (Fig.
4A), whereas the last one is a
3' sequence coding for a C terminus (Fig.
5). In all, we describe nine or ten
putative Drosophila UGTs (because each of the above ESTs has
only been sequenced from one end, LD15335 may be identical to one of
the other clones). The two DmeUgt35 cDNAs as well as the
three genomic DmeUgt37 sequences appear to represent
complete ORFs because they begin with ATG codons and end with stop
codons at positions that match closely those expected for this family
of genes (see "Experimental Procedures" for further discussion of
DNA sequence analysis). In contrast to the use of alternative splicing
for the generation of diversity, as is the case of the human
UGT1A1 gene (2), we have found no evidence of
alternative mRNA splicing, and an intron is present in only one of
the three genomic sequences (see "Experimental Procedures").
Some of the highest similarity between the five complete ORFs is
found near a sequence present in all known UGTs and defined by the
string:
[FVA]-[LIVMF]-[TS]-[HQ]-[SGAC]-G-X(2)-[STG]-X(2)-[DE]-X(6)-P-[LIVMFA]-[LIVMFA]-X(2)-P-[LMVFIQ]-X(2)-[DE]-Q, in which all amino acids that can occur at a given position are listed
inside brackets (11). The presence of this sequence strongly supports
the identification of these five proteins as UGTs (Fig. 4B).
In addition, the five complete ORFs contain C-terminal hydrophobic domains followed by several basic residues (see below and Fig. 5). In
the case of vertebrate UGTs, similar sequences have been identified as
a transmembrane domain and a positively charged "stop-transfer"
sequence that in combination are responsible for the anchoring of the
protein to the endoplasmic reticulum membrane (1) and are necessary for
enzymatic activity (31). Baculovirus ecdysteroid
UDP-glucosyltransferases, which are soluble and secreted in the
hemolymph of the hosts lack such C-terminal transmembrane domains
(15).
Finally, at least nine of the ten putative Drosophila UGTs
display a region of high similarity at their very amino terminus, immediately following the signal peptides (32, 33) (Fig.
4A). In the case of the tenth putative UGT: LD15335 only the
C-terminal sequence is presently known. In vertebrate UGTs this region
of the molecule is involved in the formation of dimers (34), which may
be the active form of the protein. In at least one case a heterodimer
has enzymatic activities that differ from those of either homodimer
(35), suggesting that the combinatorial association of different
subunits into heterodimers may provide added functional diversity.
UGT35a and -b Contain C-terminal Transmembrane Domains Similar to
Those of Vertebrate UGTs but Different from Those of the Drosophila
UGT37 Protein Family--
In consultation with the UGT nomenclature
committee (11), we have assigned the two cDNAs we have cloned to a
single family, Ugt35. The other three full-length sequences
found through the genome projects fall into a second family,
DmeUgt37. Both families fit the commonly accepted criteria
for protein families (more than 45% overall identity within a family
and less than 45% between different families, data not shown).
Finally, although the C-terminal sequence of LD15335 suggests that it
is a member of the DmeUgt37 family (Fig. 5), the definitive
assignment of the five UGTs presently only known as ESTs (Fig.
4A) to one of these two families, or yet new ones, will
require their complete sequences.
Amino acid residues present in all nine Drosophila proteins
occur in the first sixty residues (Fig. 4A) as well as in
the C-terminal half of the protein, particularly around the signature sequence (Fig. 4B). After the first sixty amino acids, the
amino-terminal halves of the proteins are highly divergent (not shown),
as is the case for vertebrate UGTs, perhaps corresponding to different substrate specificities.
Strikingly, although the C-terminal halves of all the
Drosophila proteins are closely related, there is a strong
discontinuity of this similarity at their very C termini. After a
highly conserved segment, the sequences of the two families diverge
abruptly, each encoding a different C-terminal domain containing
putative transmembrane stretches and stop-transfer sequences (Fig. 5).
Within each family, however, there is a high degree of sequence
conservation. Interestingly, the region of sequence similarity overlaps
with the likely transmembrane helix for members of the UGT35 (36) as
well as UGT37 families (Fig. 5). Five of the first six amino acids in
the putative transmembrane domain are identical between
Drosophila UGT35b and human UGT1A1, and conservation of
similarly located residues is apparent for the UGT37 family. This
pattern of conservation suggests specific and different roles for the
C-terminal domains of the two Drosophila families.
The Evolutionarily Conserved Presence of Biodegradation Enzymes
Argues for an Important Function in Olfaction--
The results
presented here suggest that, as in the olfactory epithelium of
vertebrates (17-20), several biodegradation enzymes are expressed
specifically or at higher levels in the antennae of
Drosophila. This conserved expression of biodegradation
enzymes in olfactory organs argues for an important function in
olfaction. Such a role is also consistent with the presence in the
antennae of an enzyme involved in cytochrome P450 activation, NADPH
P450 oxidoreductase (37).
In a highly specialized case of olfactory behavior, male moths are able
to find females located many miles away by rapidly alternating between
two types of behavior, upwind flight when inside a pheromone plume and
casting from side to side as soon as the pheromone is no longer
detected (38). The ability to monitor concentration changes without a
lag requires that the half-life of odorants inside the olfactory organs
be short relative to the time course of the outside fluctuations. Based
on these considerations, researchers have looked for and found enzymes that can specifically metabolize pheromones in the antennae of several
species of moths (39, 40). In vertebrate olfaction, a similar role has
been attributed to biotransformation enzymes that are better known for
their role in detoxification in the liver. The same sequence of events
that eliminates toxic chemicals may be involved in odorant degradation,
thereby preventing continuing stimulation of olfactory receptors.
Consistent with this hypothesis, an olfactory-specific UGT, UGTolf,
modifies odorants more efficiently than liver UGTs (18).
A second function for these enzymes might be in the protection of
olfactory organs from environmental toxins to which they are, by
necessity, preferentially exposed. Although odorant turnover and toxin
degradation are not mutually exclusive functions and any given protein
may be involved in both, the expression pattern of each gene may
suggest the relative contribution to either function. Because
detoxification occurs in many organs, proteins whose expression is
highly specific to the antennae, such as AntDH and UGT35b, are likely
to be involved in odorant turnover. On the other hand, proteins that
have more ubiquitous expression patterns, such as UGT35a, may
participate primarily in detoxification.
Parallel Conservation of Different Primary Sequences Suggests
Different Functions for the C-terminal Domains of the Two Drosophila
UGT Families--
We report here that the Drosophila genome
encodes at least nine different UGTs. The five genes for which complete
coding sequences are available contain the signature motif
characteristic of this superfamily and thus represent the first
reported UGTs from any insect other than ecdysteroid
UDP-glucosyltransferases from baculoviruses (11). In addition to the
overall similarity to UGTs in a variety of organisms, both
Drosophila and vertebrate proteins have at their C terminus
a transmembrane domain followed by a stop-transfer sequence composed of
several positively charged amino acids (1, 36). Although this domain is
absent from viral-encoded ecdysteroid UDP-glucose transferases and some
plant UGTs (11), mutations in either the transmembrane domain or the
stop-transfer sequence of vertebrate UGT2B1 eliminate and reduce its
activity, respectively (31).
More surprising, however, is the high degree of primary sequence
identity between the Drosophila members of the UGT35 family and vertebrate sequences in a stretch immediately
NH2-terminal to and partially overlapping with the putative
transmembrane domain (61 and 70% identity to the human UGT1A1 gene
over a 31 amino acid stretch for the a and b
genes, respectively). In addition, although the sequences of members of
the UGT37 family are very different from those of the UGT35 family in
this region, they are also highly conserved within this second family
(Fig. 5). This parallel conservation of primary sequences suggests that the C-terminal domains of UGTs are involved in specific interactions at
the membrane surface that differ between UGT35a, -b and the vertebrate
enzymes on one hand and the members of the UGT37 family on the other.
Despite these differences, the domains of the two classes of proteins
have some shared features. In all cases except for UGT37b1*, the two
amino acids at positions 15 and 16 after the start of sequence
divergence are LD, which are immediately followed by a series of
hydrophobic residues likely to be part of the transmembrane domain
(Ref. 36 and Fig. 5). In the case of UGT37b1*, a three amino acid
insertion moves LD to positions 18 and 19 and the putative
transmembrane domain starts at position 20. These similarities suggest
that despite the divergent sequences the two different types of
C-terminal domains have similar secondary structures and may therefore
interact with related proteins.
What is the function of these alternative C-terminal domains? The
primary sequence conservation within each family suggests it may be
involved in an interaction with another protein that occurs at least in
part within the membrane. The enzymatic reactions catalyzed by UGTs
take place in the lumen of the endoplasmic reticulum and are therefore
dependent on specific transporters that allow entry of nucleotides into
this subcellular compartment (41). One intriguing possibility is that
the C-terminal domain of UGTs participates in interactions with
specific transporters, perhaps corresponding to the specificities of
these enzymes for different glycosyl donors. However, because
permeabilization of membranes with detergent does not restore activity
to proteins with mutations in the C-terminal domain (31), substrate
transport cannot be its only function.
Although scans of the existing data bases have not revealed any UGT
from any organism with a C-terminal domain similar to that of the UGT37
family, the ongoing sequence of the human genome may yet uncover such
genes. Alternatively, if this domain arose after the divergence of the
ancestors of insects and vertebrates, it may constitute an
insect-specific domain and therefore a possible target for rational
pesticide design.
The availability of the genes coding for all these enzymes in
Drosophila should allow the test of their involvement in
olfaction as well as a dissection of the function of the UGT C-terminal domains using both biochemical and reverse genetic
approaches.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Several novel biodegradation enzymes are
preferentially expressed in Drosophila antennae.
Northern blots were performed with RNA extracted from different parts
of the fly (see "Experimental Procedures") as indicated above each
lane. Appendages: legs, third antennal segments, and wings;
bodies: decapitated bodies without legs or wings;
heads: heads without third antennal segments. RNA from third
antennal segments and legs was obtained after manual dissection. To
ensure that each signal corresponds to expression from a single gene,
32P probes were generated from relatively nonconserved
regions of each gene that give rise to a single band on genomic
Southern blots under identical hybridization conditions (data not
shown). Expression of the ubiquitously expressed rp49 gene
(42) was monitored in all fractions as a loading control. 1 µg of
total RNA was loaded in each lane. Probes used for the two UGTs were 5'
cDNA fragments of 560 base pairs (EcoRI-NheI)
and 610 base pairs (EcoRI-NruI) for DmeUgt35a and
DmeUgt35b, respectively. The probe used for AntDH
was the full-length cDNA clone and that for AntP450 was
the partial cDNA clone obtained in the subtracted library.
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Fig. 2.
AntDH is specifically expressed in
the third antennal segment. Horizontal cryosections of heads were
hybridized with digoxygenin-labeled DNA probes that were visualized
using standard experimental procedures with anti-digoxygenin antibodies
conjugated to alkaline phosphatase (21). In the presence of a
chromogenic substrate, a blue/purple precipitate is formed. The section
shown is at the level of the third antennal segment and is typical of
many others. In no case was signal observed in other parts of the head
or in the second antennal segment (data not shown).
-dehydrogenase
(dhi1_mouse in Fig. 3) there is no apparent amino-terminal signal
sequence in the AntDH ORF. Because the short sequence preceding the
apparent translational start in our AntDH clone does not
contain any stop codon, we cannot entirely rule out the possibility
that we are missing some 5' sequences that code for a signal peptide.
However, the presence of an AUG at the almost identical position as it
is found in many cytoplasmic SDRs (Fig. 3) suggests that we have
identified the correct amino terminus and that AntDH is a cytoplasmic
protein.
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Fig. 3.
AntDH is a member of the short chain
dehydrogenase family of proteins. The sequence of AntDH was
aligned with those of representative members of the large family of
SDRs using Pileup. Residues identical between at least three of the
five sequences are boxed in black and similar
residues in gray. The NAD/NADP-binding domain and residues
required for activity in other SDRs are indicated by
asterisks under the sequences. PksB_Dd, PksB gene product
from Dictyostelium discoideum (accession number AF019986);
Cla9_Sc, Cla9 clavulanate-9-aldehyde reductase from Streptomyces
clavuligerus (accession number AJ000671); dhi1_mouse,
corticosteroid 11- -dehydrogenase from mouse (accession number
P50172); adh_drome, alcohol dehydrogenase from Drosophila
melanogaster (accession number P00334).
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Fig. 4.
Nine putative Drosophila
UGTs. A, nine Drosophila UGTs have
putative NH2-terminal dimerization sequences. The
amino-terminal portion of the nine Drosophila putative UGTs
were included in a multiple sequence alignment with representative UGTs
from vertebrates and baculovirus. The organism or group of organisms
from which each sequence was obtained is abbreviated on the left.
Dm, Drosophila melanogaster; Bac, baculoviruses;
Vert, vertebrates. An asterisk indicates the
location of a leucine residue required for dimerization of the UGT2B1
gene (34). Although the nomenclature for full-length members of the
UGT35 and UGT37 protein families is discussed under "Experimental
Procedures," four additional Drosophila putative
Ugts are presently only known by an EST and are indicated
according to the name of the cDNA clone within the Berkeley
Drosophila Genome Project EST database. UGTs from
vertebrates and baculoviruses are named as suggested by the UGT
Nomenclature Committee (11). UGT31A2 (accession number Q88168), UGT31D3
(accession number P18569), and UGT32 (accession number Q98166) are
encoded by different baculoviruses. UGT1A1(accession number M84125) and
UGT2B1 (accession number P09875) are two UDP-glucuronosyltransferases
from different vertebrate families; UGT8, UDP-galactosyltransferase
from human brain (accession number Q09426). B, five
Drosophila UGTs contain the "UGT signature sequence."
The UGT signature sequence (11) is indicated below a multiple sequence
alignment of UGTs from a variety of organisms. The five full-length
Drosophila UGTs are indicated by arrowheads. UGTs
18E1, 16B1, and 17A1 are from Caenorhabditis elegans and
71A1, 77A1, and 78C1 are from plants (see Ref. 11 for accession
numbers).
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Fig. 5.
Drosophila UGT35a and -b have C-terminal
domains similar to those of vertebrate UGTs but different from those of
Drosophila UGT37a1, -b1 and -c1. Vertebrate UGTs
(UGT1A1, UGT2A1, and UGT8) can be aligned with all five
Drosophila UGTs (indicated by arrowheads) up to a
point where the sequences of the two families diverge dramatically. The
three alignments were generated independently using Pileup, but the
sequences shown are contiguous and their order within the alignments
has been preserved and does not correspond to similarity scores.
Boxed residues indicate identities in five of eight, three
of five, and two of three for the left, top, and bottom alignments,
respectively. The transmembrane domain indicated for members of the
UGT35 family and its vertebrate relatives is the one proposed for
vertebrate UGT2B1 (36). Hydropathy calculations for members of both
Drosophila families are consistent with the prediction that
the transmembrane domains start immediately after the LD sequence (data
not shown). Hydropathy was calculated by the Kyte and Doolittle method
using the peptides structure program of Genetics Computer Group. See
the legend to Fig. 4 for UGT nomenclature and accession numbers.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Yashi Ahmed and Ren-Ping Zhou for helpful comments on the manuscript and express gratitude also to Michael Rosbash in whose laboratory this work was initiated.
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FOOTNOTES |
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* This research was supported in part by a Shannon award from the NIDCD, National Institutes of Health and a small grant from the Foundation for the University of Medicine and Dentistry of New Jersey (to C. P.) and a Biotechnology Career Fellowship from the Rockefeller Foundation (to G. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF116553, AF116555, AF116554.
¶ To whom correspondence should be addressed: Dept. of Neuroscience and Cell Biology, Robert Wood Johnson Medical School/UMDNJ, 675 Hoes Lane, Piscataway, NJ 08854. Tel.: 732-235-5658; Fax: 732-235-4029; E-mail: pikielcl{at}umdnj.edu.
2 S.-K. Park, S. Shanbhag, A. Dubin, G. Hasan, Q. Wang, P. Yu, G. Harris, A. Steinbrecht, and C. W. Pikielny, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: SDR, short chain dehydrogenase/reductase; UGT, UDP-glycosyltransferase; ORF, open reading frame; EST, expressed sequence tag.
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REFERENCES |
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