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OCR for page 25
Colloquium
Molecular evolution of the insect chemoreceptor
gene superfamily in Drosophila me/anogaster
Hugh M. Robertson*t, Coral G. Warrt§, and John R. Carlson'
*Department of Entomology, University of Illinois, 505 South Goodwin Avenue, Urbana, IL 61801; tSchool of Biological Sciences, Monash University,
Clayton VIC 3800, Australia; and §Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520
The insect chemoreceptor superfamily in Drosophila melanogaster
is predicted to consist of 62 odorant receptor (Or) and 68 gustatory
receptor (Gr) proteins, encoded by families of 60 Or and 60 Gr genes
through alternative splicing. We include two previously unde-
scribed Or genes and two previously undescribed Gr genes; two
previously predicted Or genes are shown to be alternative splice
forms. Three polymorphic pseudogenes and one highly defective
pseudogene are recognized. Phylogenetic analysis reveals deep
branches connecting multiple highly divergent clades within the Gr
family, and the Or family appears to be a single highly expanded
lineage within the superfamily. The genes are spread throughout
the Drosophila genome, with some relatively recently diverged
genes still clustered in the genome. The Gr5a gene on the X
chromosome, which encodes a receptor for the sugar trehalose,
has transposed from one such tandem cluster of six genes
at cytological location 64, as has Greta, and all eight of these
receptors might bind sugars. Analysis of intron evolution suggests
that the common ancestor consisted of a long N-terminal exon
encoding transmembrane domains 1-5 followed by three exons
encoding transmembrane domains 6-7. As many as 57 additional
introns have been acquired idiosyncratically during the evolution
of the superfamily, whereas the ancestral introns and some of the
older idiosyncratic introns have been lost at least 48 times inde-
pendently. Altogether, these patterns of molecular evolution
suggest that this is an ancient superfamily of chemoreceptors,
probably dating back at least to the origin of the arthropods.
odorant receptor I gustatory receptor I olfaction I taste I gustation
Chemoreception in insects has long been a major focus of
insect chemical ecology; however, despite many efforts, it
was only with the sequencing of the genome of Dro~ophila
melanogaster that candidate receptor proteins mediating olfac-
tion and gustation were identified. These discoveries depended
on the use of bioinformatic methods to identify genes encoding
novel candidate G protein-coupled seven-transmembrane recep-
tor proteins (1-5~.
Members of the first family of these genes, the odorant
receptor (Or) genes, were found to be expressed in subsets of
olfactory neurons in the antenna and maxillary palp, the olfac-
tory organs of this fly (1, 3, 6, 7~. Completion of the genome
sequence allowed extension of the Or family to 60 receptors with
a unified naming system based on their chromosomal location
(~. Immunolocalization showed ex~ression in dendrites. as
A ,
expected of odorant receptors (9, 10~. Functional evidence for a
role in odor reception was provided by Wetzel et al. (11) and
Stortkuhl and Kettler (12), who used heterologous expression in
Xenopus oocytes and overexpression in the Drosophila antenna,
respectively, to show that Or43a mediates responses to a subset
of odorants. Recently Dobritsa et al. (10) have shown through
mutant and transgenic rescue analysis that Or22a is required in
vivo for response to ethyl butyrate and certain other odorants.
Moreover, several other Or genes were shown to confer response
www.pnas.org/cgi/doi/10. 1 073/pnas.23358471 00
to particular odorants or were mapped to particular functional
classes of neurons, either by receptor substitution experiments in
a mutant neuron or by analysis of strains in which Or promoters
were used to drive reporter genes (10~. Finally, we note that this
family is conserved in other insects. Anopheles gambiae contains
as many as 79 Or genes (13, 14), with few simple orthologs of
Drosophila Or genes and largely species-specific expansion of
gene subfamily lineages. In a study of four of these genes, all were
detected exclusively in the antenna, and one is female-specific
and down-regulated after a bloodmeal, as expected of a receptor
for host odors (13 ).
Clyne et al. (2) subsequently used bioinformatics to identify
another set of 42 seven-transmembrane genes, the gustatory
receptor (Gr) genes. A role in gustatory reception was suggested
by their expression profile. RT-PCR analysis revealed expression
primarily in the proboscis but also in other organs containing
gustatory neurons; moreover, expression was absent in mutants
lacking gustatory neurons. After completion of the genome
sequence, Scott et al. (15) and Dunipace et al. (16) extended the
Gr family to at least 54 and 56 members, respectively, and used
in situ hybridization and reporter gene constructs to reveal the
detailed expression patterns of a subset of the Gr genes. Mem-
bers of this family are expressed in subsets of neurons in
proboscis, pharynx, and leg as well as in larval chemosensory
organs. Some members are expressed in the antenna, suggesting
a role for some members of the Gr family in olfaction. Evidence
that a Gr gene functions ir~ taste perception was provided by
genetic analysis of GrSa, which showed that it is required for
response to the sugar trehalose (17, 18), and heterologous
expression experiments show that Gr5a is a taste receptor timed
to trehalose (19~.
Dunipace et al. (16) noted that the G~ proteins are distantly
related to Or83b, whereas Scott et al. (15) suggested that the Or
and Gr families belong together in a superfamily of insect
chemoreceptors based on conservation of a few amino acid
residues in transmembrane domain 7 (TM7~. Here we extend the
Or family to 62 receptors and the Gr family to 68 receptors, and
we analyze their molecular evolution in an insect chemoreceptor
superfamily.
Materials and Methods
The public DNA database of the Drosophila genome sequences
at National Center for Biotechnology Information (20) was
searched with all available Or and Gr proteins by using TBLASTN
(21) to find additional genes encoding proteins in these families,
This paper results from the Arthur M. Sackier Colioquium of the Nationai Acaclemy of
Sciences, "Chemical Communication in a Post-GenomicWorid," helci January 17-19, 2003,
at the Arnoicl and Mabei Beckman Center of the Nationai Acaclemies of Science anci
Engineering in Irvine, CA.
Abbreviation: TM, transmembrane domain.
tTo whom corresponclence should be adc~ressed. E-maii: hughrobe~uluc.edu.
2003 by The Nationai Acaclemy of Sciences of the USA
PNAS 1 November 25, 2003 1 vol. 100 1 suppl. 2 1 14537-14542
OCR for page 26
lkbp
Or46a locus
Or69a locus
Gr28b locus
` ~ ~ ~ 3
.3'
Fig. 1. Alternativesplicingof Or46a, Or69a, and Gr28b.Thegrayboxesindicatethe N-terminal exonsthatareuniquetothedifferentlyspliced products, labeled
with a letter designating the splice product, whereas the black boxes indicate the shared exons. In the case of Or46a, the encoded protei ns share TM7; for Or69a,
they share TM6 and -7; and for 6r28b, they share TM~7. See Clyne et a/. (2) for alternative splicing of Gr23a and -39a.
which were in turn used in searches to find more genes in an
iterative process. These searches included the updated genomic
sequences available as Release 3.1 of the genome (224. Multiple
PSI-BLAS~ searches were initiated with divergent Ors and Grs to
find any additional already annotated proteins that might belong
to these families, and up to 10 iterations were used. The genes
were reconstructed manually in the PAUP editor (23) by using the
expected exon/intron structures as guides and the spit program
(Softberry, www.softberry.com/berry.phtml) to locate predicted
introns. In addition, protein alignments were used to indicate
instances of unusual gene structure, as were comparisons with
orthologs in the draft Drosophila pseudoobscura genome se-
quence. Proteins were aligned by using CLUST4X (24), with
considerable testing of alternative settings (see ref. 25~. To
facilitate alignment, the unusually long extracellular loop 2
between TM4 and -5 was removed from Or83a, 83b, and 85e, and
Gr33a, 43a, and -66a, as were the long N and C termini of GrSa,
-21a, -32a, -61a, -63a, and Gr64a, -e, and -f. Relaxing the pairwise
and multiple alignment gap and extension penalties by 10% to 9
and 0.09, respectively, yielded the best alignment of the seven
TMs. Amino acid distances calculated between each pair of
proteins were corrected for multiple amino acid changes in the
past by using the maximum likelihood model in TREE-PUZZLE
Ver. 5 (26), with the BLOSUM62 amino acid exchange matrix
and uniform rates based on the actual sequences. A phylogenetic
tree was constructed by using neighbor joining followed by a
heuristic search for better trees by using tree-bisection-
reconnection branch-swapping in PAUP* Ver. 4.0blO (23~. Boot-
strap analysis was performed by using 1,000 neighborjoining
replications with uncorrected distances. RT-PCR was per-
formed as in Clyne et al. (1~.
Results
The Or Family. Fifty-four members of the Or family had been
annotated by Celera Genomics and the Drosophila Annotation
Jamboree (27) when this work was begun in mid-2000, whereas
six more were recognized subsequently (6, 8~. In the original
Celera Genomics scaffold sequences, there were two identical
copies of Orl9a in inverted orientation ~50 kb apart; however,
resequencing of the genome by the Berkeley Drosophila Ge-
nome Project showed that these copies differ at seven nucleotide
positions, yielding three changes in amino acid sequence; the
proximal gene retains the Orl9a name, and the distal gene we
have named Orl 9b. The duplication extends ~850 bp beyond the
predicted N termini and ~700 bp beyond the predicted C
termini. This gene pair represents an unusually recent segmental
duplication of the kind that might have been responsible for
some of the expansion of the family; however, the separated and
14538 1 www.pnas.org/cgi/doi/10.1073/pnas.2335847100
inverted nature of the duplication is not typical of the tandem
pairs and triplets of related genes seen for the rest of the family.
One additional divergent Or gene, Or67d, was discovered in
Ts~AsTN searches, located near the three known Or genes in
chromosomal division 67. Twenty-eight of the annotations pro-
vided by Celera Genomics for the 54 annotated proteins seemed
unlikely to be entirely correct, as judged from alignment of the
protein family and common features of their structures, partic-
ularly a short final exon that encodes part of TM7 and that
follows a final intron at a conserved location. These revisions
were communicated to Swiss-Prot and FLYBASE, and most are
incorporated in Release 3.1 of the genome annotations (22~.
Despite intensive searching, satisfactory C-terminal exons
could not be identified for two of the genes, Or46a and Or69b,
leading us to hypothesize that they undergo alternative splicing
similar to that noted by Clyne et al. (2) for two Drosophila Gr
genes and Hill et al. (~14) for several Anopheles Gr genes. An
appropriate final exon encoding the end of TM7 was not found
for Or46a, suggesting that the exons encoding most of Or46a are
spliced to the final exon of Or46b. Or69a might similarly be
spliced to the final two exons of Or69b (Fig. 1~. We have
confirmed both of these models by RT-PCR analysis, by using
maxillary palp cDNA for Or46a and antenna! cDNA for Or69a.
These annotation changes require name changes from Or46a and
Or46b to Or46a~1 and Or46aB, and from Or69b and Or69a to
Or69aA and Or69aB.
Pseudogenes are rare in the Drosophila genome; however,
several are found in the superfamily. In the sequenced Canton-
S-derivedy; cn bw sp strain, Or85e has suffered a deletion of the
3' end of the gene relative to an intact cDNA obtained by
Vosshall et al. (3) from the Oregon-it strain. In addition, a
fragmentary pseudogene was found just upstream of, and in
tandem with, Or98a and was named 0r98P. Its sequence is
comparable to that of Or98a, but it has suffered a ~1-kbp
internal deletion that leaves 138 bp encoding the 46 N-terminal
amino acids, the final intron, and the final exon of 69 bp that
encodes the 23 C-terminal amino acids. Large deletions like this
are thought to be responsible for the paucity of pseudogenes in
the Drosophila genome and its small size (e.g., ref. 28~.
The locations and orientations of these Or genes are shown in
Fig. 2. In addition to the recent Orl9a/b duplication, a number
of- them are in short tandem arrays of two or three genes,
indicating relatively recent gene duplication, and indeed in some
cases the encoded proteins are closely related (Fig. 3; Or22a/b,
Or33a-c, OrS9b/c, 0r65a-c, Or85b-d, and Or94a/b). However,
the majority of Or genes are widely spread through the genome,
indicating that, in agreement with their high sequence diver-
gence, they are old members of this gene family that have been
Robertson et a/.
OCR for page 27
Or la 2a 7a 9a lea 13a l9b/a
X 1 ~
Gr 2a 5a 8a 9a 10a/b
Or 22a/b 22c 23a 24a 30a 33a 35a
^. ~ / 1 1 1 1 1 _ _ _
~~ ~ ~ 1 — ~ ~ 1 1 1\ —
Gr 21a 22a-f 23aA/B 28a 28bA-E 32a 33a 36a 39aA-D39b
Or 42a 42b 43a43b 45a45b46aA/B47a47b 49a 49b
2R~V 1 1 \ 1 1 \/ 1 /
l
Gr 43a
56a
59a 59b/c
\,
- T /\
57a 5& 5&1b 59a/b 59c/d 59e/f
Or 63a
3L - I
Gr
1 1
47a 47b
65a~c 67a 67b 67c 67d 69aA/B 71a 74a
1 N1 1/ 1
1 1 _
I _
68a 77a
, _ T
66a
61a
63a 64a-f
its','
~ ~ ~ ~ ~ 44' ~ ~ ~ t '' i' ~
Or 82a 83a 83b 83c 85a 85b d 85c 85f 88a 92a 94a/b 98P/a 98b
3R · ~ l '' I /` I I ~ ~
Gr 85a 89a 92a 93a 93~d 94a 97a 98a
~ ~ ~ ~ ·~4 ~ ~ i,',
Fig. 2. Genomic locations of the Or and Gr genes. The Or genes are shown above, and the Gr genes below, central lines representing each of the five major
chromosome arms drawn to scale following Adams et a/. (27). Genes with inferred independent origins are designated by thin lines, whereas clusters of related
adjacent genes, or alternatively spliced genes, are shown by thick lines. Orientation of transcription is shown with an arrow; the arrows for the alternatively
spliced products are contiguous. The fragmentary Or pseudogene is indicated as 98P. All gene locations and orientations are based on data from Release 3.1 of
the genome.
distributed around the genome by the processes of genome flux
(e.g., ref. 29~. This genomic distribution of the family is in
contrast with the patterns observed with the mammalian olfac-
tory receptors (e.g., refs. 30, 31) and the nematode chemore-
ceptors in the str, srh, and ski families (32, 33), which commonly
are highly clustered on particular chromosomes, in part reflect-
ing the relatively recent expansions of these chemoreceptor
families.
The ancestral and idiosyncratic intron locations within the
coding regions of the Or genes are shown schematically in Fig.
4, along with those of the Gr genes. Three intron locations
appear to be ancestral within the superfamily, as determined by
their common location and insertion phase among multiple
highly divergent Or and/or Gr lineages. We have named these
intron positions 1, 2, and 3 (intron 2 is not present in the Or
family, as if it were lost at the base of the Or lineage). There are
27 idiosyncratic Or introns that are not shared in the same
location and phase with any Gr lineage but are generally present
in one or only a few closely related Or genes. The exceptions are
introns x and y, in phases 2 and 0, respectively, which are present
in divergent Or lineages and were likely acquired near the base
of the Or family tree. It seems likely that at least 25 introns were
independently acquired within these single genes or small lin-
eages relatively recently. They are unlikely to be ancient, because
then the original Or gene must have been extraordinarily frag-
mented by introns, and multiple independent losses must have
occurred in multiple different Or lineages. Twenty-seven inde-
pendent losses of introns are inferred on the tree in Fig. 3.
Twelve Or genes have lost all but one of their older introns
without acquiring any new ones and hence have only one intron
within their coding regions; at the other extreme, Or63a and
Or67b acquired five new introns in addition to four older ones for
a total of nine introns each.
Vosshall et al. (6) detected expression of 40 of the 57 Or genes
they examined in sensory neurons of the antenna and maxillary
Robertson et a/.
. . .
palp by in situ hybridization, and we have been able to detect
mRNAs representing 48 of 61 Or transcripts (the analysis did not
distinguish between Orl9a and -b) in olfactory organs by a
combination of in situ hybridization and RT-PCR (ref. 1;
C.G.W., unpublished data), leaving 13 transcripts for which
there is no evidence of expression in adult olfactory organs.
There is no phylogenetic pattern to these 13 Or transcripts in the
tree in Fig. 3 (highlighted in bold italics), suggesting that they do
not represent a single lineage of genes. It is possible that they
have been recruited to expression in other cell types or life
stages.
The Gr Family. The combined efforts of Clyne et al. (2), Scott et
al. (15), and Dunipace et al. (16) led to the recognition of ~64
proteins in this family. In addition to the examples described by
Clyne et al. (2) of alternatively spliced transcripts from two genes
(`Gr23a and -39a), together encoding six substantially different
proteins, Gr28b encodes five predicted proteins (Fig. 1), which
were only partially recognized by Scott et al. (15) and Dunipace
et al. (16~. We also add two previously undescribed members to
the family, Gina and -89a, bringing the total to 68 proteins
encoded by 60 genes. Most of these were poorly annotated or
missed by the automated Celera Genomics annotation. The
principal authors of these three papers (2, 15, 16) have coordi-
nated a naming convention for these proteins analogous to that
agreed to for the Or proteins; improved annotations for them
have been agreed to by all groups and submitted to Swiss-Prot,
and most are available in Release 3.1 of the Drosophila anno-
tation (three genes originally designated as members of this
family, Gr36d, 43b, and -65a, are no longer considered to be
members of the superfamily). Comparison with Gr orthologs in
the draft D. pseudoobscura genome indicated that eight of these
annotations require further revision; the updated versions have
been communicated to FLYBASE and are utilized here (see Or/Gr
Proteins, which is published as supporting information on the
PNAS I November 25, 2003 1 vol. 100 | suppl. 2 | 14539
OCR for page 28
50% conected distance
- Oreo
ur4-za
Ot43b
x ~ Or22b
Or7a
Or33a
, 1 ~ Or33b
~ -- - , on3a
y ~ Or59a
I ~ ~ Or19b
_
Or
Fig. 3. Tree of the insect chemoreceptor superfamily. The tree is rooted at the midpoint. The Or and Gr families are indicated on the right, and the scale bar indicates
50% divergence in corrected distancesffarlargerthantheuncorrected distanceswhen comparing distantly related proteins). Brancheswith 7~100% bootstrapsupport
are indicated with a square and can be considered to be confident, whereas branches with 60-75% bootstrap support are indicated with a diamond and can be
considered somewhat confident. Inferred intron gains within the superfamily are indicated above branches in bold uppercase letters, whereas inferred intron losses
are shown in lowercase letters (intron losses that are not confidently independent according to the bootstrap support for branches are shown in italics). The Or and
Grfamilies have separate sets of intron letter designations (see Fig. 4), and the putatively ancient ancestral phase-0 C-terminal introns 1-3 are shown as numbers. The
Or genes for which no evidence of expression in antenna or maxillary palp has been detected by Vosshall et a/. (6) or by us are highlighted in bold italics, as are the
four Gr genes that Scott et a/. (15) and Dunipace et a/. (16) showed to be expressed in the antenna and/or maxillary palp.
14540 1 www.pnas.org/cgi/doi/10. 1 073/pnas.2335847 100
Robertson et al.
OCR for page 29
a he
a- d e f g h i jk 1
Or
0 0 1 2 1 2021
m nap q rs tavw x
1 201 0 01 0211 2
a' 3
O O
2 22
a be d e
Gr -
2 11 10
f g hi j klm n-
0 1 21 1 100 0
o
p q rs t u v w x y Z
a' b'c' d' 1 2 3
2 1 010 2 0 1 2 0 2 2 1 0 1 0 0 0
I I I ~ , ~ ~ , , , I , , ~ ~ I I I ~ ~ I , , r, ~ , , , , I , , , , ~ , , I , I
1 100 200 300 400
Fig. 4. Locations and phases of introns in the Or and Gr genes. The intron locations (above the lines) and phases (below the lines) are shown separately for
the two families, relative to a scale of the average receptor size in amino acids (determined by excluding the large insertions in some of the receptors). The
ancestral phase-0 C-terminal introns 1 and 3 are shared between the two families.
PNAS web site; see also ClustalX Multiple Sequence Alignment
and Table 1 (CG numbers, locations, lengths, and intron num-
bers), which are published as supporting information on the
PNAS web site.
Dunipace et al. (16) noted that Gr22b and -d are pseudogenes
because of an in-frame stop codon and a single base-pair
deletion in their first exons, respectively. Scott et al. (15) avoided
these mutations by starting their annotations downstream, but
the resulting proteins do not have TM1. We amplified and
sequenced the relevant regions of these two genes from the
Oregon-it strain, and both are intact genes in this strain. The
apparent TGA stop codon in Gr22b corresponds to a CGA
encoding arginine, whereas the single base deletion in Gr22d,
located in a string of four Ts, corresponds to a string of five Ts
in Oregon-it. Like Or85e, these differences therefore reflect
strain polymorphisms also seen in some Anopheles receptors
(14~; the intact versions are used herein.
The genomic locations of the Gr genes are shown in Fig. 2.
Like the Or genes, they are distributed widely around the
genome; however, there are some larger clusters (Gr22a-f and
Gr64a-f contain six genes each). Some Gr genes appear to have
transposed relatively recently: within the Gr22a-f cluster, Gr22e
is in the opposite orientation, and Gr22f is ~28 kb downstream
(see also ref. 16~. Perhaps the most interesting cases are Gr61a
and -5a, which phylogenetically cluster confidently with the
Gr64a-f set but are now located elsewhere. GrSa is the only Gr
gene for which a function has been shown; it is required for
response to trehalose (17-19), and it is possible that Gr61a and
Gr64a-f encode additional sugar receptors. These genes share
five idiosyncratic introns and are highly divergent from other
receptors within the family. In contrast, the GrS8a-c genes are
adjacent to each other, yet only GrS8a/b weakly cluster in the
tree, and all three share as little amino acid similarity with each
other as many other pairs of Gr proteins (11-16%~. We note that
Orl Oa is immediately upstream of and in the same orientation as
Grl Oa/b, with only ~350 bp separating the stop codon of Orl Oa
and the start codon of Grl Oa. This proximity might be related to
the expression of GrlOa in the antenna (15~.
Most Gr proteins are extraordinarily divergent, sharing as
little as 8% amino acid identity. Multiple alignment and phylo-
genetic analysis of such highly divergent proteins is difficult. We
have used the entire lengths of the proteins, as aligned by
CLUST4X by using slightly relied gap penalties, which are
particularly capable of achieving alignment of the hydrophobic
TMs. In this manner, relationships of the more closely related
proteins are well resolved, possibly at the expense of the more
distant relationships of the backbone of the tree. As with the Or
family, more distant relationships have no bootstrap support
(Fig. 3~.
The Gr genes contain even more idiosyncratic introns than the
Or family. The most recently shared ancestral Gr gene is inferred
to have had a long exon encoding TM1-5 followed by three phase
0 introns separating three exons encoding the C-terminal region
beyond TM6 (Figs. 3 and 4; see also figure 1 in ref. 2~. Mapping
of intron losses on the tree is complicated by uncertainty in the
backbone of their relationships. However, it seems most likely
Robertson et a/.
that there have been 30 idiosyncratic intron gains and at least 21
intron losses, bringing the superfamily totals to 57 gains beyond
the ancestral three phase 0 introns and at least 48 losses. Within
the Gr family, two genes, Gr68a and -94a, have lost all introns
within the coding region. Both of these genes are within the
introns of other genes, and Gr68a may have lost its three introns
simultaneously through reverse transcription and insertion of a
retrocopy. It is possible that these two Gr genes and others have
introns in their 5' UTRs that cannot easily be recognized
bioinformatically. In contrast, Gr64e has eight introns.
Discussion
We describe here what may be the full complement of 60 Or and
60 Gr family genes and 130 predicted chemoreceptor proteins
that they encode in D. melanogaster. We cannot exclude the
possibility of additional highly divergent, or evolutionarily inde-
pendent, insect chemoreceptors. Scott et al. (15) introduced the
notion that these two families of odorant and gustatory receptors
are evolutionarily related in an insect chemoreceptor superfam-
ily, and we endorse this view. This superfamily provides a
remarkable diversity of receptors that could underlie the entire
range of chemoreceptive capabilities of this fly. The Or genes
appear to be one lineage, albeit highly expanded in gene number,
w~thin the larger Gr family (Fig. 3~. To preserve nomenclatural
clarity, however, we prefer to retain the Or and Gr designations.
In addition to this phylogenetic interrelationship, Scott et al. (15)
and Dunipace et al. (16) found that four Gr genes are expressed
in subsets of neurons in the antenna and/or maxillary palp
(indicated in bold italics in Fig. 3~. Neurons expressing Gr21a
project axons to glomeruli in the antenna! lobe of the brain (15~.
If these antenna! Grs in fact function as odorant receptors, then
it would appear that olfactory receptor function has evolved
separately several times within the superfamily, perhaps in
conjunction with the evolution of terrestrial insects from aquatic
arthropod ancestors ~400 million years ago.
Or83b is extremely divergent from the other Or proteins and
is expressed in most olfactory receptor neurons (3~. Or83b is also
unusual in having an ortholog in A. gambiae that shares 78~o
amino acid identity, much higher than any other orthologous pair
in these two distantly related dipterans (14), as well as 68%
identity to CR2 from the moth Heliothis virescens (344. This
conservation is sustained throughout the Endopterygota (meta-
morphosing insects) (ref. 35; G. New, H. Patch, K. Walden, and
H.M.R., unpublished results). Dunipace et al. (16) noted that
among the Ors, Or83b appears most similar to the Gr family
proteins. Our phylogenetic analysis supports this observation
(Fig. 3), and although this placement is not supported by
bootstrapping, it is obtained regularly when the alignment and
tree methodologies are modified considerably. This extraordi-
nary conservation suggests that Or83b serves a function unlike
that of other chemoreceptors (14, 35~.
The antiquity of this insect chemoreceptor superfamily is
supported by several lines of evidence. First, the genes encoding
these proteins are roughly evenly spread throughout the genome
(Fig. 2 ). Although there are a few clusters of related proteins that
represent recent in situ expansions of gene lineages, the pro-
PNAS I November25, 2003 1 vol. ~oo 1 suppl. 2 1 1454'
OCR for page 30
cesses of genome flux that led to the current distribution of the
genes are clearly evident (for example, the translocation of GrSa
to the X chromosome from the Gr64 cluster on chromosome 3L).
This flux is reminiscent of several other ancient gene families in
the Drosophila genome, e.g., the tetraspanin superfamily (36~.
Second, the amino acid divergences between the Gr and Or
proteins, and particularly among the Gr proteins, are extremely
high; indeed, Gr proteins commonly share only 8-12% amino
acid identity. Some of this divergence could be attributed to an
evolving need to adapt to new ecological niches. Nevertheless,
the extreme divergence within the family is consistent with an
ancient origin. Identification of Or and Gr family members in the
moths H. virescens (34) and Manduca sexta (H. Patch, K. Walden,
and H.M.R., unpublished results) confirms the antiquity of the
families.
Third, the vast majority of introns appear to have been
idiosyncratically acquired by limited lineages of genes and com-
monly single genes (Fig. 3~. This pattern of intron evolution is
found in other old gene superfamilies (e.g., ref. 36J. The
ancestral insect chemoreceptor genes appear to have had only
three phase 0 introns near their C termini.
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Fourth, extended PSI-BLASTP searches initiated with various
Grs detected similarities with proteins encoded by five gustatory
related (gur) genes, putative seven-TM receptors in the nematode
Caenorhabdit~s elegans (`C. Stoetzner and H.M.R., unpublished
results). The gur genes are quite distinct from the ~1,000
candidate chemoreceptors already identified in this nematode
(32, 33, 37, 38~. The similarity between Gr and GUR proteins
suggests that the superfamily predates the arthropod/nematode
split.
Note Added in Proof. Bray and Amrein (39) demonstate that Gr68a is
expressed in neurons of male-specific contact-chemosensory sensilla on
male forelegs and implicate Gr68a in recognition of females in an early
step of courtship, when males tap the abdomen of a female with their
forelegs, presumably sampling their sex- and species-specific cuticular
hydrocarbons. This study provides further support for a gustatory role for
most Gr proteins.
We thank P. Clyne, J. Daniels, J. Kim, E. Moriyama, and A. Ray for
assistance with annotation of these genes; and H. Amrein and K. Scott
for cooperation in annotation/naming of the Grs. This work was funded
by National Science Foundation Grant 9604095 (to H.M.R.) and Na-
tional Institutes of Health Grants DC04729 and GM63364 (to J.R.C.~.
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Robertson et al.
Representative terms from entire chapter:
insect chemoreceptor
