1
Searching for neuronal left/right asymmetry:
Genome wide analysis of nematode receptor-type guanylyl cyclases
Christopher O. Ortiz 1, John F. Etchberger 1, Shoshana L. Posy 1,2, Christian Frøkjær-
Jensen 3, Shawn Lockery 3, Barry Honig 1,2 and Oliver Hobert 1,3
1 Howard Hughes Medical Institute
Department of Biochemistry and Molecular Biophysics
Columbia University Medical Center
701 W.168th Street
New York, NY 10032
2 Howard Hughes Medical Institute
Department of Biochemistry and Molecular Biophysics
Center for Computational Biology and Bioinformatics
Columbia University Medical Center
1130 St. Nicholas Avenue, Room 815
New York, NY 10032
3 Institute of Neuroscience
University of Oregon
Eugene, OR 97403
3 Corresponding author at [email protected]
9 Figures, 4 Tables, 2 Supplementary Figures
Running title: Guanylyl cyclases in nematodes
Key Words: guanylyl cyclase, C. elegans, sensory neurons, left/right asymmetry
Genetics: Published Articles Ahead of Print, published on March 17, 2006 as 10.1534/genetics.106.055749
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ABSTRACT
Functional left/right asymmetry (“laterality”) is a fundamental feature of many
nervous systems, but only very few molecular correlates to functional laterality are
known. At least two classes of chemosensory neurons in the nematode Caenorhabditis
elegans are functionally lateralized. The gustatory neurons ASE Left (ASEL) and ASE
Right (ASER) are two bilaterally symmetric neurons which sense distinct chemosensory
cues and express a distinct set of four known chemoreceptors of the guanylyl cyclase
(gcy) gene family. To examine the extent of lateralization of gcy gene expression
patterns in the ASE neurons, we have undertaken a genome wide analysis of all gcy
genes. We report the existence of a total of 27 receptor-type gcy genes and of seven
soluble gcy genes in C. elegans. We describe the expression pattern of all previously
uncharacterized receptor-type gcy genes and find them to be highly biased but not
exclusively restricted to the nervous system. We find that more than 41% (11/27) of all
gcy genes are expressed in the ASE gustatory neurons and that one third of all gcy
genes (9/27) are expressed in a lateral, left/right asymmetric manner in the ASE
neurons. The expression of all laterally expressed gcy genes is under the control of a
gene regulatory network composed of several transcription factors and miRNAs. The
complement of gcy genes in the related nematode C. briggsae differs from C. elegans
as evidenced by differences in chromosomal localization, number of gcy genes and
expression patterns. Differences in gcy expression patterns in the ASE neurons of C.
briggsae arise from a difference in cis-regulatory elements and trans-acting factors that
control ASE laterality. In sum, our results indicate the existence of a surprising multitude
of putative chemoreceptors in the gustatory ASE neurons and suggest the existence of
a substantial degree of laterality in gustatory signaling mechanisms in nematodes.
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INTRODUCTION
The diversification of neuronal fate and function across the left/right axis of
nervous systems is poorly understood but represents a fundamental problem in the
neurosciences. This problem is well illustrated by a cursory comparison of structure and
function of nervous systems. While the organization of nervous systems is largely
bilaterally symmetric on a morphological level, brain functions are often highly
lateralized (HUGDAHL and DAVIDSON 2003). Functional lateralization is presumably
brought about by the diversification of neuronal function on a subanatomical level, such
as differential gene expression in bilaterally symmetric structures. Indeed, quantitative
comparison of transcript levels have recently revealed left/right asymmetries in gene
expression profiles in the human brain (SUN et al. 2005).
The nematode C. elegans provides a simple model organism to study the
lateralization of nervous system function (HOBERT et al. 2002). Such lateralization can
be observed in the chemosensory system of the nematode. The best studied
chemosensory neurons are a group of 12 classes of neurons called the amphid sensory
neurons (Fig.1A). Each class consists of one pair of two bilaterally symmetric and
morphologically indistinguishable neurons, most of which are chemosensory neurons
(Fig.1B). At least two classes of these chemosensory neurons, the AWC odor sensory
neuron class and the ASE gustatory neuron class are functionally lateralized, allowing
the animal to sense and discriminate different sensory cues with the left and right
neuron (PIERCE-SHIMOMURA et al. 2001; WES and BARGMANN 2001)(Fig.1B). Functional
lateralization of AWCL/R and ASEL/R correlates with the left/right asymmetric
expression of putative chemoreceptors (JOHNSTON et al. 2005; TROEMEL et al. 1999; YU
et al. 1997) but the extent of lateralization of chemoreceptor gene expression in these
neurons is still unclear. In the AWCL/R sensory neurons, only one left/right
asymmetrically expressed chemoreceptor has been reported, a 7-transmembrane
receptor (TROEMEL et al. 1999)(Fig.1B). In the ASEL/R sensory neurons, a total of four
asymmetrically expressed, putative chemoreceptors were known prior to this study,
which all belong to the family of receptor guanylyl cyclases, encoded by the gcy genes
(JOHNSTON et al. 2005; YU et al. 1997)(Fig.1B). gcy-5 and gcy-22 are expressed in the
right ASE neuron (ASER), whereas gcy-6 and gcy-7 are expressed exclusively in the
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left ASE neuron (ASEL).
To further analyze the extent of lateralization of the ASE gustatory neurons, we
identified the complete set of gcy genes in the C. elegans genome and undertook a
genome wide analysis of their expression patterns. Previous counts of C. elegans
receptor-type gcy genes were preliminary given the incomplete nature of the C. elegans
genome sequencing project, but estimated to be in the higher twenties (BIRNBY et al.
2000; YU et al. 1997). Expression patterns had been determined for eight receptor-type
gcy genes (BIRNBY et al. 2000; L'ETOILE and BARGMANN 2000; YU et al. 1997). We now
report the final count of receptor-type guanylyl cyclases in the complete C. elegans
genome to be 27. We present a comparative sequence analysis of all gcy genes and
describe the expression patterns of all previously uncharacterized receptor-type gcy
genes using gfp reporter gene fusions. We analyze the mechanisms of regulation of gcy
gene expression in the context of the ASE gustatory neurons, we investigate the
consequence of removal of one ASE-expressed gcy gene on ASE neuron function and
we examine the evolutionary divergence of gcy gene structure and expression.
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MATERIALS AND METHODS
Strains and transgenes
Wild-type and mutant strains: N2 wild-type Bristol isolate; C. briggsae AF16 wild-
type strain; OH4349 lsy-6(ot71)dpy-11(e224); OH110 lim-6(nr2073) (HOBERT et al.
1999). RB1000 gcy-5(ok921), 4x outcrossed; RB1010 gcy-5(ok930), 4x outcrossed;
OH2957 gcy-5(tm897), not outcrossed.
Transgenes: otIs3: Is[gcy-7prom::gfp; lin-15(+)] (CHANG et al. 2003); this transgene
is expressed in ASEL and the excretory canal cell. otIs151: Is[ceh-36prom::dsRed2; rol-
6(d)] (JOHNSTON and HOBERT 2003); this transgene is expressed in ASEL/R and
AWCL/R. otIs133: Is[ttx-3promB::rfp; pNC4.2(unc-4(+)] (WENICK and HOBERT 2004); this
transgene is expressed in AIYL/R. oyIs17: Is[gcy-8prom::gfp; lin-15(+)], expressed in
AFDL/R, and oyIs51: Is[srh-142prom::rfp; lin-15(+)], expressed in ADFL/R, both gifts from
Piali Sengupta.
Sequence analysis
To identify GCY sequences, the sets of predicted proteins for C. elegans and
C. briggsae were obtained from the Sanger Institute
(http://www.sanger.ac.uk/Projects/C_elegans/WORMBASE/current/wormpep_download
.shtml and ftp://ftp.sanger.ac.uk/pub/wormbase/cbriggsae/cb25.agp8/). Representative
GCYs were used as PSI-BLAST queries to search the two proteomes. The HMMER
2.3.2 package (EDDY 1998) was used to construct a hidden Markov model from an
alignment of GCY catalytic domains and search for additional GCYs.
The intracellular regions of the transmembrane GCYs and the complete soluble
GCY sequences were aligned with T-coffee version 2.03 (NOTREDAME et al. 2000).
Maximum parsimony phylogenetic trees were found via heuristic search with PAUP*
version 4.0 beta 10 (SWOFFORD 2003). The trees were generated in 100 repeated
searches with random addition of taxa to obtain the starting tree. Robustness of the tree
partitions was evaluated by constructing a bootstrap consensus tree with 1000
replicates. The trees were visualized with TreeView version 1.6.6 (PAGE 1996).
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Nomenclature of gcy genes and gcy gene predictions
Most but not all gcy names were previously assigned (http://www.wormbase.org).
We named two previously unnamed gcy genes gcy-28 (T01A4.1) and gcy-29
(C04H5.3). Both code for receptor-type proteins. A few gcy genes have been double-
named in the past. The most current names are (old names in parenthesis): gcy-18 (=
gcy-26), gcy-20 (= gcy-16), gcy-17 (= gcy-24), odr-1 (= gcy-10), gcy-28 (gcy-38 in
WS149), gcy-29 (gcy-39 in WS149).
Since we detected several cases where individual parts of one C. elegans gene
are homologous to separate, adjacent predicted C. briggsae genes (gene prediction in
Wormbase WS149), we suspected that C. briggsae genes may have been incorrectly
predicted. We therefore ran the FGENESH program at www.softberry.com (SALAMOV
and SOLOVYEV 2000) on chromosomal regions that contained the following predicted C.
briggsae genes (from WS149): CBG07423(CBP15915) + CBG07424(CBP15916) +
CBG07425(CBP15917), CBG20867(CBP04902) + CBG20868(CBP04903) and
CBG19454(CBP11205) + CBG19453(CBP11204). In each of these three cases, we
found that FGENESH predicted only one gene, whose product was homologous over its
entire length with putative C. elegans orthologs. However, in two cases, the revised
gene prediction overlooked exons predicted in the original prediction which contained
homology to GCY proteins. We therefore assembled alternate gene prediction by hand,
based on the similarity to known GCY proteins. We named these revised predictions
CBP15915*, CBP04902* and CBP11205* (see Supp.Fig.1 and 2). Based on primary
sequence homology, we also suspect that one of the two C. briggsae orthologs of C.
elegans gcy-35 has been incorrectly predicted as two separate genes CBG20390
(CBP04780) and CBG20392 (CBP04781). Similarly, the CBG10472 (CBP08561) and
CBG10474 (CBP08562) likely constitute one gene. However, neither of these two
suspicions could be corroborated by FGENESH.
Generation of gfp reporter gene fusions, transgenic animals and identification of
reporter gene expressing cells.
Most reporter genes were created by PCR fusion (HOBERT 2002) and some were
generated by subcloning PCR amplicons into pPD95.75 (see Table 1). Primer
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sequences and resulting transgenic arrays are shown in Table 1. DNA was injected at
~10-50 ng/µl using either unc-122::gfp or rol-6 as injection marker. Cell identifications
were done based on overall cell position and morphology and was significantly aided by
the uses of the following four co-labeling procedures. (1) Most gfp reporters were
injected into animals carrying the otIs151 transgene in which ASEL/R and AWCL/R are
labeled with dsRed2. Since the otIs151 transgene already contains the rol-6(d) injection
marker we used unc-122::gfp (LORIA et al. 2004) as injection marker for most injections.
This marker is expressed in coelomocytes but also yields occasional and mosaic gfp
expression within the pharynx, often in the I5 neuron. With the exception of the broadly
pharyngeal expression of one gcy gene, we therefore ignored any cell-type specific
pharyngeal expression of gcy reporter genes. Injections into wild-type C. elegans or C.
briggsae were done using rol-6(d) as injection marker. (2) Some gcy::gfp transgenic
animals were crossed with animals carrying the otIs133 transgene in which the AIY
interneurons are marked with dsRed2, thereby facilitating cell identification either by
red/green overlap (gcy-1 prom::gfp) or by the determination of relative cell position (gcy-
18 prom::gfp). (3) In many cases, a subset of the amphid neuron classes of gcy::gfp
transgenic animals was filled with DiI. DiI fills the ASK, ADL, ASI, AWB, ASH and ASJ in
the head in the PHA and PHB neuron classes in the tail (HEDGECOCK et al. 1985),
thereby facilitating cell identification either by an overlap of red and green fluorescence
or by the determination of relative cell position. DiI was dissolved in DMF, diluted to 10
µg/ml in M9 or in ddH20 plus 50 mM calcium-acetate to additionally fill the IL2 neurons.
Worms were soaked in the DiI solution for at least 1 hour. (4) Expression of gcy genes
in the AFD thermosensory neurons was assessed using the oyIs17 transgene in which
the AFD sensory neurons are marked with GFP. Extrachromosomal arrays carrying
gcy-18prom::gfp and gcy-23 prom::gfp were crossed with oyIs17-containing animals and
the number of GFP expressing amphid sensory neurons was counted. To exclude
expression in the closely adjacent ADF neuron class, gfp transgenes were also crossed
with an ADF-expressed dsRed2 reporter construct, oyIs52, kindly provided by P.
Sengupta.
Chemotaxis assays
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Radial population chemotaxis assays were done as previously described (CHANG
et al. 2004). Assay plates were 10 cm tissue culture dishes containing 20 g/L agar, 5
mM potassium phosphate (pH = 6.0), 1 mM CaCl2, 1 mM MgSO4. To set up the
chemical gradients on the assay plates a 10 µL drop of attractant was placed 15 mm
from the edge of the plate at the “attractive spot”. A 10 µL drop of ddH20 was placed
diametrically opposite and was considered the “negative control spot”. The attractant
was allowed to diffuse for 14-16 hours at room temperature. To increase the steepness
of the chemical gradient, 4 to 4.5 hours prior to chemotaxis assay 4 µL attractant was
added to the “attractive spot” and 4 µL ddH20 added to the “negative control spot”. The
attractants NaCl and NH4Cl (Sigma, MO, USA) were dissolved in ddH2O to a
concentration of 2.5 M and were adjusted to pH6.0 with either NH4OH or acetic acid.
Worms were washed three times in sterile water to remove food and salts. Worms were
then placed at the center of the plate and allowed to chemotax for an hour. Worms
reaching either the attractant peak or the negative control spot (sterile water) were
immobilized with sodium azide. Results were quantified by counting worms that were
located at (A) the attractant, (B) the center of the plate or (C) the negative control. Since
animals carrying two of the three gcy-5 knockout alleles (ok921 and ok930) did not
disperse well from the center of the plate, we calculated a modified chemotaxis index,
defined as C.I. = A/(A+C). This index therefore disregards worms that do not reach
either the attractant or the negative control spot. It is doubtful that the dispersion defects
of ok921 and ok930 alleles are significant since the putative null allele tm897 does not
show these defects.
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RESULTS
Identification of the complete set of GCY proteins in C. elegans
To identify the complete set of guanylyl cyclases in C. elegans we employed the
Position-Specific Iterated Basic Local Alignment Search Tool (PSI-BLAST) to search
the latest release of the complete C. elegans genome databases, using a set of known
GCY proteins as queries. We identified a total of 34 gcy genes, several more than
reported in previous searches of incomplete genome sequence databases (BARGMANN
1998; BIRNBY et al. 2000; YU et al. 1997). The identified gcy genes fall into two distinct
families, receptor type guanylyl cyclases (27 genes) and soluble cyclases (7 genes).
Both families contain a guanylyl cyclase catalytic domain (Pfam domain PF00211; Fig.2;
Supp.Fig.1). In several GCY proteins catalytically important residues are not conserved
in the cyclase domain (Supp.Fig.1) and it has been speculated that in these cases,
heterodimerization with a catalytically active GCY protein ensures activity of the dimer
(MORTON 2004). Apart from the presence of the cyclase domain, receptor-type and
soluble GCY proteins differ significantly (Fig.2). Soluble GCY proteins contain one other
characteristic domain, a HNOB domain (for “heme NO binding “domain Pfam domain
PF07700). In contrast, receptor-type guanylyl cyclases lack this HNOB domain but
always contain an additional protein kinase domain (PF00069), which is likely to be
inactive since it lacks a critical catalytic aspartate residue that is present in the catalytic
core of all active protein kinases (“HRD motif”; Supp.Fig.2). In addition to this protein
kinase-like domain, all but one of the C. elegans receptor-type GCY proteins contain a
single transmembrane domain and a signal sequence. Moreover, the majority of
receptor-type GCY proteins contain a conserved extracellular domain of unknown
function that is also present in many mammalian GCY proteins and conserved in amino
acid receptors in bacteria (“RFLBP” = Receptor family ligand binding region; Pfam
PF01094; Fig.2). The presence of this domain strongly suggests that the C. elegans
GCY proteins are indeed ligand-binding receptor proteins.
The overall domain topology of C. elegans GCY proteins is similar to that of
mammalian GCY proteins. Multiple transmembrane-containing GCY proteins which can
be found in unicellular eukaryotes (WEDEL and GARBERS 2001) are not present in C.
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elegans.
GCY-27 is an unusual receptor-type GCY protein (Fig.2). While containing all the
intracellular signature motifs of receptor-type GCYs, the predicted GCY-27 protein is
several hundred amino acids shorter than all other predicted receptor-type GCY
proteins and does not contain a predicted signal sequence (SS), transmembrane
domain (TM) or other extracellular motifs (Fig.2). In the absence of complete cDNA/EST
sequences, we cannot rule out a gene prediction error, but we consider the failure to
predict a complete extracellular domain to be unlikely for two reasons: (a) The upstream
gene adjacent to gcy-27 is relatively close by (Fig.4), leaving little room for such a
prediction oversight, particularly since in other gcy genes the extracellular domains are
large and composed of many exons; (b) a C. briggsae ortholog (described in more detail
in a later section) also lacks the extracellular domain. While receptor-type GCY proteins
that lack a TM and SS domain have been described before (MORTON 2004), they do not
appear to contain the protein kinase domain that is present in GCY-27 and all other
receptor-type GCY proteins. It is interesting to note that GCY-27 is most closely related
to the intracellular domain of ODR-1 (Fig.3A), a transmembrane GCY protein whose
extracellular domain (which contains no canonical RFLBP domain) has previously been
shown to be dispensable for its function in chemosensory signal transduction (L'ETOILE
and BARGMANN 2000).
We analyzed the relatedness of soluble and receptor-type GCY proteins in more
detail by generating a maximum parsimony bootstrap tree using the intracellular domain
of the receptor-type GCYs and the complete sequences of the soluble GCYs. The
soluble GCYs branch separately from the transmembrane-type proteins and are
clustered into two subgroups. Many receptor-type GCY proteins also fall into small and
well-defined subgroups (Fig.3A).
Notably, in a substantial number of cases, the degree of sequence relation
correlates with proximity in the genome sequence (Fig.3B). For example, all six
members of one subgroup (“gcy-5 subgroup”) are located within a ~7 Mb interval on
chromosome II, five of these (gcy-1 through gcy-5) map to an ~800 Kb interval and
three of them are directly adjacent genes, separated by only a few hundred base pairs
(Fig.3B, Fig.4). This suggests that these genes arose by relatively recent gene
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duplications. Likewise, all seven members of what we term the “gcy-7 subgroup” of gcy
genes (Fig.3A) reside on a single chromosome and five of them in a <8 Mb interval
(Fig.3B, Fig.4). As we will demonstrate below, the degree of sequence relation and
chromosomal location also correlates with similarities in gene expression patterns.
Expression patterns of receptor-type gcy genes.
While the expression of all seven soluble gcy genes has already been described
(CHEUNG et al. 2004; GRAY et al. 2004; YU et al. 1997), the expression patterns of only
eight of the 27 receptor-type gcy genes was previously reported, six in the context of a
preliminary and incomplete genome analysis (gcy-5, gcy-6, gcy-7, gcy-8, gcy-10/odr-1,
gcy-12 and gcy-22) and two in the course of a functional analysis (odr-1 and daf-
11)(BIRNBY et al. 2000; JOHNSTON et al. 2005; L'ETOILE and BARGMANN 2000; YU et al.
1997)(summarized in Table 2). We generated gfp reporter fusions to the putative cis-
regulatory regions of the remaining 19 gcy genes (schematically shown in Fig.4). In all
except one case, sequences to the next upstream genes were included in the reporter
gene fusions (Fig.4). While such upstream sequences are most likely to harbor gene
regulatory elements, it needs to be kept in mind that additional regulatory elements may
be located elsewhere and that therefore the expression patterns of the gfp reporter
genes can only provide a first approximation of endogenous gene expression profiles.
Since gcy genes are likely to primarily function as chemoreceptors in the mature
nervous system, we restricted our gene expression analysis of transgenic animals that
harbor the respective reporter genes to larval and adult stages. To allow us to reliably
detect expression in the ASEL/R gustatory neurons, the neurons in which our laboratory
is most interested in, we used a transgenic reporter array in the background in which
the ASEL/R neurons are labeled with dsRed2 (otIs151; see Material and Methods),
thereby enabling us to score for an overlap between green and red fluorescent signals.
In most cases, multiple lines were analyzed per construct (see Table 1) and we did not
observe any notable differences between individual lines. Reporter gene expression
was observed for each of the gcy genes examined. Expression patterns are shown in
Fig.5 and summarized in Table 2, which also lists previously described expression
patterns. The expression patterns of all receptor-type gcy genes can be summarized as
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follows.
(1) Broad versus cell-type specific: With the exception of the very broadly but not
ubiquitously expressed gcy-28 gene, all receptor-type gcy genes are expressed in a
tissue- and cell-type restricted manner (Table 2; Fig.5).
(2) Cell types: The expression of receptor-type gcy genes is strongly biased
toward the nervous system. With the exception of two non-neuronally expressed gcy
genes (gcy-9 and gcy-11), all gcy genes are expressed in a restricted subset of
neurons. 21/27 genes are exclusively expressed in the nervous system and 4/27 genes
are expressed in restricted sets of both neuronal and non-neuronal cells (Table 2;
Fig.5).
(3) Expression within the nervous system: Within the nervous system, most but
not all C. elegans gcy genes are expressed in sensory neurons. All except two pairs
(ASH and ADF) of the 12 amphid sensory neuron classes shown in Fig.1A express at
least one gcy gene. Unlike what the previous analyses of eight gcy genes seemed to
indicate (BIRNBY et al. 2000; JOHNSTON et al. 2005; L'ETOILE and BARGMANN 2000; YU et
al. 1997), neuronal gcy gene expression is, however, not restricted to sensory neurons.
Non-sensory neurons that express gcy genes include the AIY, AIM, AVK, RIA and PVT
interneuron classes and the RIM neuron, which is both a motorneuron and interneuron.
In addition, as mentioned above, many neurons in the nervous system, including, for
example, ventral cord motor neurons, express the widely-expressed gcy-28 gene. As
summarized in Table 3, 25/27 of the C. elegans receptor-type gcy genes are expressed
in the nervous system, 25/27 are expressed in various types of sensory neurons plus
other neurons, 15/27 are expressed exclusively in sensory neurons and 9/27 are
restricted to single neuron classes. The expression of mammalian gcy genes, of which
there are only 7, show roughly comparable patterns; some are expressed in non-
neuronal cells and those that are expressed in neurons are strongly biased to
expression in sensory structures (WEDEL and GARBERS 2001).
(4) Co-expression: A notable general feature of C. elegans gcy gene expression
profiles, both receptor- and non-receptor-type, is that a small number of neuron classes
co-express a substantial number of gcy genes (Fig.5; summarized in Table 4). The most
striking examples are the ASE gustatory neuron class, which expresses a total of 11
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gcy genes, more than one third of all receptor-type gcy genes. In addition, six gcy genes
are co-expressed in the ASI chemosensory neurons, five gcy genes are co-expressed
in AWC olfactory neurons, four are co-expressed in the AFD thermosensory neurons
and two are co-expressed in the ASG and PHA phasmid sensory neurons, respectively
(Table 4). Besides the overlap in individual neurons, there are also a few examples of
gcy genes that show similar combinations of cellular expression profiles. gcy-7 and gcy-
20 are co-expressed in ASEL and in the excretory canal cell. Moreover, as previously
reported, daf-19 and odr-1 are expressed in precisely the same subset of amphid
sensory neurons and six soluble gcy genes are co-expressed in the AQR/PQR and
URX neurons (BIRNBY et al. 2000; CHEUNG et al. 2004; GRAY et al. 2004; L'ETOILE and
BARGMANN 2000; YU et al. 1997). A single receptor-type gcy gene, gcy-25, complements
the expression of the six soluble gcy genes in the AQR/PQR and URX neurons (Fig.5O;
Table 4).
(5) Left/right asymmetric expression: Two previous studies identified a total of
four left/right asymmetrically expressed gcy genes in the ASE gustatory neuron class
(gcy-5, gcy-6, gcy-7 and gcy-22; Table 2)(JOHNSTON et al. 2005; YU et al. 1997). We
have identified five more examples of gcy genes that are expressed in a left/right
asymmetric manner in the ASE neuron class. gcy-1, gcy-3 and gcy-4 are expressed
predominantly or exclusively in ASER, while gcy-14 and gcy-20 are expressed
predominantly or exclusively in ASEL (Fig.5; data is quantified in Fig.6). Together with
the previously reported expression patterns, a total of four gcy genes are lateralized to
ASEL and five gcy genes are lateralized to ASER (summarized in Fig.1B). One third
(9/27) of all gcy genes are therefore laterally expressed in the ASE neurons. There are
subtle differences in the degree of laterality of ASE-expressed gcy genes. Five out of
the nine L/R asymmetric gcy genes are exclusively expressed in ASER (gcy-1, gcy-5,
gcy-22) or ASEL (gcy-6, gcy-7) (JOHNSTON et al. 2005)(Fig.6A). The other four gcy
genes are only biased to ASER (gcy-3 and gcy-4) or ASEL (gcy-14 and gcy-20)(Fig.6B);
for example, while expression of gcy-4 is almost always stronger in ASER than in ASEL,
there is often faint, but visible expression in ASEL (Fig.6B). Since C. elegans
transgenes harbor multiple copies of reporter gene constructs, the relevance of such
relatively subtle quantitative details is difficult to assess. However, we note that each
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individual array of each reporter constructs behaves in a similar way (see Table 1 for
number of arrays scored), arguing that these observations are not due to transgene
variance.
gcy genes that are asymmetrically expressed in either ASER or ASEL are not
asymmetrically expressed in other sensory neurons. For example, the gcy-1 and gcy-3
genes, two ASER-expressed genes, are expressed bilaterally in other pairs of neurons.
In addition to the asymmetrically expressed gcy genes, we found two bilaterally,
albeit weakly expressed gcy genes in ASEL/R, gcy-19 and gcy-29, increasing the
fraction of ASE-expressed gcy genes to 41% (11/27) of all gcy genes . The possible
lack of regulatory elements in gfp reporter genes may lead to the oversight of perhaps
even more ASE-expressed gcy genes.
We have not observed any other obvious left/right asymmetric gcy gene patterns
in bilaterally symmetric neurons, including the AWCL/R neurons (which we could easily
identify with the dsRed2-expressing otIs151 transgene). The AWCL/R neurons are the
only other known neuron pair displaying functional laterality (WES and BARGMANN 2001)
and although they express five gcy genes (Table 4), none of them is obviously
lateralized.
(6) Similarity of chromosomal position, primary sequence and gene expression
patterns. Strikingly, the co-expression of gcy genes in ASEL or ASER correlates
extensively with the primary sequence similarity and chromosomal location of the gcy
genes. The ASER-expressed gcy-1, gcy-3, gcy-4 and gcy-5 genes fall into one
sequence subgroup (Fig.3A), localize within ~800 Kb and two of them are directly
adjacent to one another (Fig.3B and Fig.4). Notably, their co-expression is not simply
due to a joint cis-regulatory element; as our reporter constructs clearly demonstrate
(Fig.4), each gene contains separable cis-regulatory elements.
Similarly, all four ASEL-expressed gcy genes (gcy-6, gcy-7, gcy-14, gcy-20) fall
into one sequence subgroup (Fig.3A) and localize at the center of chromosome V
(Fig.3B). These observations argue that gene duplication events that lead to the
generation of these paralogues also duplicated their regulatory regions. The only
exception to this pattern is the gcy-22 gene, which is predominantly expressed in ASER,
but in terms of primary sequence identity clusters more closely with the ASEL-
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expressed genes than with the ASER expressed genes (Fig.3).
A similar correlation of sequence relation and gene expression can also be
observed in non-ASE expressed gcy genes. The gcy-8, gcy-18 and gcy-23 genes are
related by sequence (Fig.3A), localize within a 15.6 Mb interval (Fig.3B), and are all co-
expressed in the AFD sensory neurons (Fig.5). Additionally, the AFD-expressed gcy-29
gene is also closely related to gcy-8, gcy-18 and gcy-23 by primary sequence, but it
localizes to a different chromosome. Lastly, the sequence-related gcy-15 and gcy-21
genes (Fig.3A) are in close chromosomal proximity (~530 Kb; Fig.3B) and are co-
expressed in the ASG chemosensory neurons (Fig.5).
Taken together, all these similarities indicate that related gcy genes arose by
local gene duplication events that not only duplicated the protein-coding region but also
their cis-regulatory information.
Regulation of left/right asymmetric expression of gcy genes
How is the laterality of the newly characterized ASEL/R-expressed gcy genes
controlled? We have previously identified a complex network of transcription factors and
miRNAs that control ASE laterality after animals have passed through an initial hybrid
precursor state (CHANG et al. 2004; CHANG et al. 2003; JOHNSTON and HOBERT 2003;
JOHNSTON et al. 2005; JOHNSTON and HOBERT 2005). These regulatory factors fall into
two broad categories; first, factors that control the activity of a bistable feedback loop
that determines whether an ASE neuron adopts the ASEL or ASER state and, second,
factors that act outside the regulatory loop to determine specific subsets of terminal
differentiation features. To test whether the newly identified, asymmetrically expressed
gcy genes are subject to regulation by this network or controlled by a different set of
regulatory factors, we analyzed gfp reporter expression profiles in two null mutant
backgrounds that are representative for each category, lsy-6 and lim-6. Animals that
lack the lsy-6 miRNA display a complete switch from the ASEL fate to the ASER fate
(JOHNSTON and HOBERT 2003) and animals that lack the lim-6 LIM homeobox gene fail
to activate a defined subset of ASEL features and fail to repress a subset of ASER
features (HOBERT et al. 1999; JOHNSTON et al. 2005). We find that all newly identified
gcy genes are components of the ASEL and ASER state that is controlled by the lsy-6-
16
dependent regulatory feedback loop. The ASER-specific gcy genes are ectopically
activated in ASEL upon loss of the ASEL-inducer lsy-6 and the ASEL-specific gcy
genes are lost in the ASEL neuron of lsy-6 null mutant animals. We note that in the case
of the ASEL-biased gcy-14 gene, which is normally strongly expressed in ASEL and
weakly expressed in ASER, the conversion of the ASEL to the ASER fate does not
entail a loss of gcy-14 expression, but rather the bilateralization of weak expression
(“L=R”; Fig.6B).
How does the lim-6 LIM homeobox gene contribute to the expression of the
newly identified gcy genes? Similar to the previously described ASEL-specific gcy-6 and
gcy-7 genes, we find that the expression of the new ASEL-expressed genes gcy-14 and
gcy-20 are unaffected by loss of lim-6 (Fig.6B), demonstrating that ASEL inducers such
as lsy-6 independently regulate the expression of lim-6 and ASEL-specific gcy genes
(Fig.6C). Similar to the previously described ASER-specific gcy-5 and gcy-22 genes, we
find that lim-6 represses the expression of the ASER-specific gcy-1, gcy-3 and gcy-4
genes in ASEL (Fig.6A,B). In summary, all known asymmetrically expressed gcy genes
are controlled by the same categories of gene regulatory factors.
Functional analysis of gcy-5
Three putative loss of function alleles have been generated by the C. elegans
knockout consortia in one of the nine asymmetrically expressed gcy genes, gcy-5
(Fig.7A). At least one of them is a putative molecular null allele (see legend to Fig.7A).
We analyzed all three mutant gcy-5 alleles in a chemotaxis assay that measures the
functionality of the ASE neurons (BARGMANN and HORVITZ 1991). Our previous work has
demonstrated that the ASEL neuron preferentially senses sodium, but not chloride,
whereas the ASER neuron senses chloride and weakly contributes to sodium sensation
(CHANG et al. 2004; PIERCE-SHIMOMURA et al. 2001). Neither gcy-5 mutant allele shows
any significant defects in attraction to sodium or chloride (Fig.7B). We conclude that
gcy-5 is not required for generic aspects of ASER development or function.
Guanylyl cyclase genes in the nematode Caenorhabditis briggsae
The availability of the genome sequence of the nematode C. briggsae, which
17
diverged about 100 million years ago (STEIN et al. 2003), provided us with the
opportunity to examine the evolution of the gcy gene family. To identify the complete set
of guanylyl cyclase genes in C. briggsae we again employed PSI-BLAST to search the
latest release of the complete genome databases of C. briggsae, using a set of known
GCY proteins as queries. We identified a total of 33 gcy genes in C. briggsae, one less
than in C. elegans (Fig.3A). Compared to C. elegans, C. briggsae contains one
additional soluble gcy gene (total = 8) and contains two receptor-type gcy genes less
(total = 25). 23 nematode receptor- and soluble gcy genes show clear one-to-one
ortholog matches between C. elegans and C. briggsae (Fig.3A). All the other gcy genes
show species-specific gene duplication events. For example, the C. elegans gcy-6 gene
has duplicated to produce two paralogs, while the directly adjacent C. elegans gcy-1,
gcy-2 and gcy-3 genes are paralogs with only a single ortholog in C. briggsae (Fig.3A).
We have mentioned above the existence of a receptor-type gcy protein, GCY-27,
that lacks a predicted extracellular domain. C. elegans GCY-27 is closely related to the
intracellular domain of the receptor-type ODR-1; both proteins are more closely related
to each other than to the two closest C. briggsae homologs (Fig.3A). One of these
homologs, CBP15915* (see Material and Methods for refined gene predictions) is
clearly orthologous to ODR-1 due to synteny in the chromosomal region; that is, all
predicted proteins in the direct neighborhood of ODR-1 are homologous to the
neighboring proteins of the putative C. briggsae ODR-1 ortholog. The protein
designated CBP11205* (see Material and Methods for refined gene prediction) is the
likely ortholog of GCY-27. Both proteins share the unusual feature of not containing an
extracellular domain. As in the case of GCY-27, the absence of an extracellular domain
in the C. briggsae ortholog is unlikely caused by a failure to predict more exons because
neighboring genes are in close proximity. We conclude that the extracellular domain of
an ancestral protein was lost before the C. briggsae/C. elegans split, giving rise to GCY-
27 and its ortholog CBP11205*.
We sought to investigate the issue of orthology and paralogy in more detail by (a)
analyzing on a sequence level the degree of synteny between gcy genes and by (b)
analyzing the expression patterns of some orthologous C. briggsae genes. For the
chromosomal analysis, we focused on one subgroup of left/right asymmetrically
18
expressed gcy subfamilies, the “gcy-5 subfamily” (Fig.3A).
We found that the chromosomal arrangement of members of the gcy-5 subfamily
differ significantly between C. elegans and C. briggsae. The gcy-19 orthologs are
located in distinct environments in C. elegans and C. briggsae (Fig.8A), suggesting a
translocation event after the lineage split. The case of the gcy-1, gcy-2, gcy-3, gcy-4
and gcy-5 genes is more complex and provides a fascinating glimpse into evolutionary
divergence. While gcy-4 and gcy-5 are direct neighbors in both C. elegans and C.
briggsae, they are translocated together with their neighbors to a distinct region in C.
briggsae (Fig.8B). Curiously, this distinct region contains the single C. briggsae ortholog
of three C. elegans paralogs from the gcy-5 subfamily, gcy-1, gcy-2 and gcy-3 (Fig.8B).
Since all paralogous members of the gcy-5 subgroup likely arose by gene duplication,
the C. briggsae gene organization is probably more reflective of the ancestral situation
than C. elegans. One conceivable scenario is that a common ancestor of C. elegans
and C. briggsae contained, like C. briggsae, three adjacent gcy genes (which
themselves arose by gene duplication). In the C. elegans lineage, this cluster split up,
with gcy-4 and gcy-5 translocating to a distinct chromosomal region and the gcy-1/2/3
ortholog also translocating to a distinct location and then subsequently duplicating to
generate gcy-1, gcy-2 and gcy-3 in the C. elegans lineage.
Evolutionary divergence of left/right asymmetric gcy gene expression patterns
Using gfp reporter genes generated from genomic C. briggsae DNA, we
analyzed the expression of five gcy genes in C. briggsae. We generated gfp fusions to
the single C. briggsae ortholog of the C. elegans gcy-1/gcy-2/gcy-3 genes and the C.
briggsae orthologs of C. elegans gcy-4, gcy-5, gcy-7 and gcy-19. We observed no
significant adult expression for C. briggsae gcy-1/2/3 prom::gfp, C. briggsae gcy-5prom::gfp
and C. briggsae gcy-7prom::gfp in several transgenic C. briggsae lines which is possibly
due to the limited size of the reporter gene constructs (due to the size of the intergenic
regions, none of the reporter constructs extended to the previous gene; data not
shown). We did, however, observe interesting expression patterns for C. briggsae gcy-
4prom::gfp and C. briggsae gcy-19prom::gfp and focused our analysis on these genes.
We find that C. briggsae gcy-4prom::gfp is expressed bilaterally in both ASEL and
19
ASER (Fig.9A). This is in striking contrast to C. elegans gcy-4prom::gfp, whose
expression is biased to ASER in C. elegans (Fig.5C, Fig.6B). Such a difference could
be caused by two different mechanisms. The gcy-4 loci may contain distinct cis-
regulatory information in C.elegans and C.briggsae, or, alternatively, the cis-regulatory
information may be the same, but different trans-acting factors in C. elegans and C.
briggsae interpret this information differentially. To distinguish between these
possibilities, we injected the C. briggsae gcy-4prom::gfp construct into C. elegans. If the
C. elegans and C. briggsae gcy-4 reporter constructs contained the same cis-regulatory
information, C. briggsae gcy-4prom::gfp should be expressed in the same pattern in C.
briggsae and C. elegans (bilateral expression in ASEL and ASER). In contrast, if there
are differences in trans-acting factors, then the C. briggsae gcy-4prom::gfp reporter
should be expressed in C. elegans in a manner similar to that of C.elegans gcy-
4prom::gfp (biased to ASER). We found that C. briggsae gcy-4prom::gfp expression
becomes ASER-biased when injected into C. elegans, which mimics the expression of
C.elegans gcy-4prom::gfp (Fig.9A). The difference between gcy-4 expression in C.
elegans and C. briggsae does therefore not appear to be a difference in their cis-
regulatory architecture but a reflection of different trans-acting factors.
The gcy-19 locus represents another example of evolutionary divergence of
left/right asymmetric gene expression. C. elegans gcy-19prom::gfp is strongly expressed
in the IL2 sensory neurons and weakly in both ASEL and ASER and several other head
sensory neurons in C. elegans (Fig.5N). In striking contrast, C. briggsae gcy-19prom::gfp
shows strong and exclusive expression in the C. briggsae ASER neuron, but not any
other head neurons (Fig.9B). In contrast to the case of gcy-4, however, the difference in
expression does not appear to be caused by differences in trans-acting factors. When
injected into C. elegans, the C. briggsae gcy-19prom::gfp reporter is still expressed
exclusively in ASER (Fig.9B). The cis-regulatory architecture of the C. briggsae gcy-19
locus can therefore be “read out” in the same way by the trans-acting factors in both
C.elegans and C.briggsae. Differences in the expression of C. elegans and C. briggsae
gcy-19 are therefore more likely caused by a difference in the cis-regulatory architecture
of these loci. Since C.elegans and C.briggsae gcy-19 reside in non-syntenic
chromosomal regions (Fig.8A), it appears that the alterations in chromosomal context of
20
these two genes not only affected the neighboring genes of the gcy-19 loci but also their
cis-regulatory architecture. As a word of caution, we note the intrinsic limitations of
reporter gene constructs, which may not harbor the complete set of cis-regulatory
elements thereby potentially yielding a misleading impression of gene expression
patterns.
DISCUSSION
Biochemical properties and functions of receptor-type guanylyl cyclases have
been summarized and discussed in several reviews over the past few years (LUCAS et
al. 2000; MORTON 2004; WEDEL and GARBERS 2001). Recent findings on the function of
soluble gcy gene in oxygen sensation (CHEUNG et al. 2004; CHEUNG et al. 2005; GRAY et
al. 2004) have also been reviewed (RANKIN 2005). We focus here on several specific
outcomes of our studies.
The function of nematode receptor-type gcy genes. C. elegans and C.
briggsae contain an unusual number of receptor-type gcy genes. Insects such as
Drosophila melanogaster or Anopheles gambiae contain six receptor-type guanylyl
cyclases (MORTON 2004), mammals contain seven (four orphan and three peptide-
binding receptors) (LUCAS et al. 2000; WEDEL and GARBERS 2001), but C. elegans
contains 27 and C. briggsae 25 (this study). The physiological function of insect gcy
genes is entirely unknown, though the expression of the only two analyzed receptors in
sensory neurons (among other neurons) has been noted (MORTON 2004). Vertebrate
gcy genes are expressed in several different tissue types including chemosensory
neurons (WEDEL and GARBERS 2001).
We propose that the significant expansion of receptor-type gcy genes in the
nematode lineage is a reflection of their employment as chemoreceptors used to sense
and navigate through a complex soil habitat. This hypothesis, which was also put
forward by YU et al. (1997), is mainly based on the observation that almost 90% (24/27)
of gcy genes are expressed in sensory neurons (lack of sensory neuron expression of
three gcy genes may merely be caused by a lack of the complete set of cis-regulatory
21
element in the reporter genes used). Moreover, 41% (11/27) of gcy genes are
expressed in the main gustatory neuron class of C. elegans, ASE. This neuron class
has previously been shown to be functionally lateralized in that it can sense different
chemosensory cues (PIERCE-SHIMOMURA et al. 2001). Consistent with a role of gcy
genes as chemoreceptors we find that 9/11 ASE-expressed gcy genes are expressed in
a left/right asymmetric manner, thereby providing molecular correlates to functional
lateralization.
Amino acids are among the several classes of chemicals that can be sensed by
the ASE neurons (BARGMANN and HORVITZ 1991). The presence of a domain in the
extracellular parts of many GCY proteins that is homologous to bacterial amino acid
binding proteins makes receptor-type GCY proteins good candidates to be amino acid
receptors. More sensitive assays (FAUMONT and LOCKERY 2005; FAUMONT et al. 2005;
MILLER et al. 2005; WICKS et al. 2000), than those previously used (WARD 1973) will be
required to establish the full spectrum of amino acids and other possible sensory cues
that signal through ASE. Such a systematic cataloguing of sensory cues processed by
C. elegans needs to be followed by a systematic analysis of strains harboring deletions
in gcy genes as well as misexpressing gcy genes to establish their roles as amino acid
receptors. A role for GCY proteins as salt receptors is also conceivable but highly
speculative at present. There is as yet no consensus about the molecular identity of salt
receptors in the vertebrate gustatory system. Notably, the crystal structure of the ANP
receptor, a mammalian GCY protein that like most C. elegans GCY proteins contains an
extracellular RFLPB domain, revealed a high affinity chloride binding site (VAN DEN
AKKER et al. 2000).
Our attempt to establish a mutant phenotype for a gcy gene, the ASER-
expressed gcy-5 gene, has failed so far, but we note that we have only tested one of the
two known cues sensed in an ASER-specific manner, namely chloride ions. The failure
to detect a mutant phenotype does, however, allow the conclusion that gcy genes such
as gcy-5 are unlikely to control a fundamental, non-redundant aspect of the
development or overall function of the neuron. It appears more likely that gcy-5 and, by
inference, other ASE-expressed gcy genes fulfill a sensory modality specific function in
ASE such as being a receptor for a specific class of gustatory cues.
22
Whereas two thirds of C. elegans gcy genes (16/24) are expressed exclusively in
sensory neurons, one third are also expressed in non-sensory neurons, including
inter/motorneurons and non-neuronal cells, suggesting that GCY proteins also respond
to endogenously produced ligands. Since all known ligands for mammalian GCY
proteins are peptidergic signaling molecules (LUCAS et al. 2000) and since C. elegans
contains scores of neuropeptide encoding genes (LI et al. 1999), we propose that non-
sensory C. elegans GCY proteins may be receptors for peptidergic ligands.
The function of receptor-type GCY proteins may not be restricted to a receptor
function. The C. elegans ODR-1 protein does not require its extracellular domain to fulfill
its function in transducing odorsensory signals (L'ETOILE and BARGMANN 2000).
Moreover, one GCY protein that we describe here, the ODR-1-related GCY-27 protein,
entirely lacks an extracellular domain. These proteins may heterodimerize with ligand-
binding GCY receptors to constitute a receptor complex and/or they may serve as
second-messenger producing signaling proteins that are embedded in signal
transduction cascades triggered by other receptor systems.
Co-expression of gcy genes. Another notable feature of gcy gene expression
patterns is their degree of co-expression. Six gcy genes are co-expressed in the ASI
sensory neuron class, five are co-expressed in ASER, four in ASEL, five in the AWC
olfactory neuron class, four in the AFD thermosensory neuron class and two gcy genes
are each co-expressed in the ASG and PHA phasmid sensory neuron classes. In
addition, several gcy genes that are expressed in multiple cell types show similar and
non-intuitive combinations of expression patterns. gcy-7 and gcy-20 are co-expressed in
ASEL and the excretory cell (two cells of no obvious relation) and daf-11 and odr-1
show a precisely overlapping expression pattern in five amphid sensory neurons
(BIRNBY et al. 2000; L'ETOILE and BARGMANN 2000). Co-expression of receptor-type
GCY proteins raises at least two different possibilities: (1) The proteins are independent
receptors for distinct sensory inputs. While an attractive possibility for the ASE-
expressed gcy genes, this is unlikely to be the case for daf-11 and odr-1, which are both
independently required for AWC-mediated olfaction to several distinct odorants (BIRNBY
et al. 2000; L'ETOILE and BARGMANN 2000). (2) Since GCY proteins dimerize, it is
possible that the defined set of GCY proteins of one cell-type can homo- and
23
heterodimerize to form an even larger repertoire of dimerized receptor complexes. As
previously suggested, such heterodimerization may be obligatory in those cases where
a given GCY protein lacks residues that are necessary for cyclase activity (MORTON
2004). The cyclase domains of several GCY proteins, including at least GCY-11, DAF-
11 and GCY-29, are predicted to be inactive based on the substitution of conserved
residues required for activity (Supp.Fig.1), but all three genes are co-expressed with
other GCY proteins that are predicted to be active (Table 4).
Laterality in the nematode nervous system. With the identification of a total of
nine asymmetrically expressed gcy genes, the ASE neurons present so far the most
striking example of a lateralized chemosensory neuron. As mentioned above, our
previous work has provided a conceptual framework for the functional relevance of
laterality in the ASE neurons (PIERCE-SHIMOMURA et al. 2001). We expect that other
neurons may similarly employ the principle of lateralizing chemosensory function.
However, our expression analysis has not revealed further examples of laterality in gcy
gene expression profiles in other bilaterally symmetric amphid neurons, therefore
leaving only the AWC odorsensory and ASE gustatory neurons as neuron pairs with
lateralized functions (Fig.1B). Considering the potential usefulness of lateralizing
chemoreceptor function, it is our expectation that the analysis of expression of the
hundreds of chemoreceptors of the 7-transmembrane receptor family (BARGMANN 1998)
will reveal more examples of laterality in the nervous system.
Our analysis of the laterality of gcy gene expression in C. briggsae revealed
several striking examples of evolutionary plasticity in the laterality of the gustatory
system of nematodes. The variation that we observe appears to be caused by a
variation in both cis-acting elements and trans-acting factors, a conclusion that we can
draw from our comparison of C. briggsae gcy promoter activity in C. briggsae and C.
elegans. Variations in cis-regulatory control have been recognized to be a major feature
of evolutionary processes (CARROLL et al. 2001) but our cross-species analysis also
provides strong support for the more conventional view of evolution of trans-acting
factors. The clearly distinct nature of cis-regulatory architecture of at least some gcy
genes in C. elegans and C. briggsae is a strong reminder that the use of bioinformatic
tools which use phylogenetic conservation to identify cis-regulatory elements in genomic
24
sequences (e.g. (BIGELOW et al. 2004); for review see (BULYK 2003)) may easily lead to
false-negative predictions. It is difficult at this point to speculate about differences in the
trans-acting factors that control laterality in C. elegans and C. briggsae. We have
demonstrated that so far all known left/right asymmetrically expressed terminal
differentiation markers in the ASEL/R neurons, including all gcy genes, are under
control of a previously described bistable feedback loop that is composed of several
transcription factors and miRNAs (JOHNSTON et al. 2005). The activity of transcription
factors that are controlled by the output of the loop, for example the lim-6 LIM
homeobox gene (Fig.6B), may have functionally diverged in C. briggsae. This can
perhaps be best illustrated with the gcy-4 gene, which in C. elegans is repressed by lim-
6 in ASEL. In C. briggsae, gcy-4 is not repressed in ASEL, yet the C. briggsae gcy-4
promoter can be repressed in C. elegans. These observations are consistent with the
presence of a lim-6 responsive repressor element in both C. elegans and C. briggsae
gcy-4 promoters but with an inability of C. briggsae lim-6 to control this element in C.
briggsae.
Mutant screening approaches that identify the complete set of trans-acting
factors controlling L/R asymmetric gcy gene expression in C. elegans and experimental
promoter dissection approaches that identify cis-regulatory elements through which
these trans-acting factors act are currently ongoing in our laboratory and are likely to
reveal the molecular control of laterality in the gustatory system of C. elegans and its
divergence in C. briggsae.
25
ACKNOWLEDGEMENTS
We thank Qi Chen for expert technical assistance, the C. elegans gene knockout
consortia at Tokyo Women’s Medical University School of Medicine (led by Shohei
Mitani) and at the Oklahoma Medical Research Foundation (led by Bob Barstead) for
providing mutant strains, the CGC and members of the worm community for providing
strains, Piali Sengupta for help with cell identifications and members of the Hobert lab
and Cori Bargmann for comments on the manuscript. This work was supported by the
NIH Medical Scientist Training Program (to C.O.O.), NIH R01 NS050266-01 (O.H.) and
R01 NS39996-05 (O.H.). B.H. and O.H. are Investigators of the HHMI.
26
FIGURE LEGENDS
Fig.1: An introduction to C. elegans sensory anatomy.
A: A prominent and well-characterized subset of C. elegans sensory neurons, the
amphid sensory neurons, are schematically shown. Like most other neuron classes,
amphid sensory neuron classes consist of one pair of two bilaterally symmetric cells
(see also panel B). Each of the 12 pairs of amphid sensory neurons extends a dendrite
to the tip of the nose and an axon into the nerve ring, a nerve bundle where synaptic
connections are made (WHITE et al. 1986). Delineated functions of amphid sensory
neurons are indicated (BARGMANN and MORI 1997).
B: Amphid sensory neuron classes consist of two bilaterally symmetric pairs of neurons,
two of which, AWCL/R and ASEL/R, are functionally lateralized. While some other
amphid sensory neurons appear to contribute to gustation, ASE is the main gustatory
neuron class in C. elegans, mediating responses to salts, amino acids and small
metabolites (BARGMANN and HORVITZ 1991). ASE not only mediates attractive, but also
repulsive responses to specific chemicals (SAMBONGI et al. 1999). The salts sodium,
chloride and potassium are sensed in a left/right asymmetric manner, with ASEL
sensing sodium, but not chloride and potassium and ASER sensing chloride and
potassium (PIERCE-SHIMOMURA et al. 2001). It is not yet known whether other ASE-
sensed chemicals may also activate ASEL and ASER differentially. ASE-expressed gcy
genes are shown; those newly described in this paper are shaded; those that are
asymmetric are colored. The AWCL/R neurons can discriminate benzaldehyde and
butanone based on the left/right asymmetric expression of str-2 (colored), which is
stochastically expressed in either AWCL or AWCR. Newly identified gcy genes in AWC
are shown and shaded.
Fig.2: Domain structure of GCY proteins.
SS = signal sequence, TM = transmembrane domain, RFLPB = “Receptor family ligand
binding region” (Pfam PF01094), Protein Kinase-like = ‘Protein kinase domain”
(PF00069), Cyclase = “Adenylate and Guanylate cyclase catalytic domain ” (PF00211),
HNOB = “heme NO binding domain” (PF07700). See Supp.Fig.1 and Supp.Fig.2 for the
27
primary sequence alignment of individual domains. We note that several of the receptor-
type GCY proteins, such as GCY-11, lack a clear SS at the N-terminus but the presence
of a clear TM and/or RFLPB domains make us suspect that the absence is due to an
incorrectly predicted N-terminus of the respective genes and we therefore grouped
these genes together with other clear-cut SS/TM-containing proteins. GCY-22 has an
unusual and phylogenetically conserved insertion between the transmembrane and
protein kinase domain (Supp.Fig.2).
Fig.3: Sequence similarity and chromosomal localization of gcy genes.
A: Phylogenetic tree based on the intracellular domain of the receptor-type guanylyl
cyclases and the complete sequences of the soluble guanylyl cyclases. Numbers at the
tree nodes are bootstrap values, which indicate the frequency (in %) of occurrence of a
given partition in the 1000 replicate trees. C. elegans proteins are shaded; C. briggsae
proteins all carry the prefix “CBP”. A select number of cells that co-express multiple gcy
genes are indicated by color-coded shades, as indicated in the figure. See Material and
Methods for comments on the gcy gene names. Note that the CBP15915*, CBP11205*
and CBP04902* proteins used here differ from those in Wormbase WS149 based on an
alternative gene prediction that we performed (see Material and Methods).
B: Chromosomal localization of gcy genes. See Table 2 for detailed map position.
Chromosome III does not contain any gcy genes.
Fig.4: Reporter gene constructs.
Representation of genomic loci are adapted from www.wormbase.org. Reporter gene
constructs are indicated by the 5’ upstream region used (red box), usually the intergenic
region up to the next gene, and the gfp coding region (green box; not drawn to scale).
See Table 1 for primer sequences and a list of transgenic arrays containing the
individual reporter gene constructs.
Fig.5: Expression patterns of gcy reporter gene fusions.
Transgenic animals expressing gfp reporter gene fusions are shown. Images shown are
representative animals from several independent lines (see Table 1 for a list of
28
transgenic reporter arrays used). Most transgenic animals are scored in the late larval
and adult stage and contain otIs151 in the background to facilitate the identification of
the ASE neurons (see Material and Methods); blue circles indicate ASER, red circles
indicate ASEL. The quantification of the L/R asymmetric expression in ASE is shown in
Fig.6.
A: gcy-1prom::gfp. Dorsal view (left panel) and ventral view (right panel) of two different
focal plains of the head region. The inset in the right panel shows the overlap of the gfp
signal with otIs133, an AIY-expressed rfp marker (lateral view).
B: gcy-3prom::gfp. Dorsal view of the head region (left panel) of an animal whose amphid
sensory neurons have been filled with DiI. The right panel shows a full length worm with
expression in the PVT interneuron.
C: gcy-4prom::gfp. Dorsal view of the head region.
D: gcy-14prom::gfp. Dorsal view of the head region.
E: gcy-20prom::gfp. Dorsal view of the head region (left panel). The right panel shows a
full length worm with expression in the excretory system. EXG = excretory gland cell;
EXC = excretory canal cell.
F: gcy-7prom::gfp. Lateral view. Expression in ASEL, but not expression in the excretory
canal cell (EXC), had been previously reported (YU et al. 1997).
G: gcy-2prom::gfp. Lateral view of the head region. A defined subset of the amphid
neurons are filled with DiI to allow for easier assessment of cell position. The inset
shows a dorsal view to illustrate bilateral symmetry of gfp expressing cells.
H: gcy-11prom::gfp. Lateral view of the head region. The strong neuronal expression (N)
in the pharynx is likely due to the injection marker, but pharyngeal muscle expression is
due to the reporter gene.
I: gcy-13prom::gfp. Dorsal view of the head region. The inset shows a lateral view of a
DiI-filled animal.
J: gcy-15prom::gfp. Lateral view of the head region. A defined subset of the amphid
neurons are filled with DiI to allow for easier assessment of cell position. The inset
shows a ventral view to illustrate bilateral symmetry.
K: gcy-17prom::gfp. Dorsal view of the tail region. The inset shows a DiI-filled animal.
L: gcy-18prom::gfp. Lateral view of the head region. The inset shows a lateral view of gfp
29
expression in relation to the rfp expression from otIs133, an AIY-specific cell marker.
M: gcy-21prom::gfp. Lateral view of the head region of a DiI-filled animal. The inset shows
a ventral view to illustrate bilateral symmetry.
N: gcy-19prom::gfp. Lateral view (left panel) and dorsal view (right panel) of the head
region. The inset in left panel shows the overlap of the gfp signal with DiI-filled IL2
neurons
O: gcy-25prom::gfp. Left panel shows an oblique view of the head region, the right panel
shows a lateral view of the tail region.
P: gcy-23prom::gfp. Ventral view of the head region.
Q: gcy-27prom::gfp. Dorsal view of the head region. The inset shows a lateral view of a
DiI-filled animal.
R: gcy-28prom::gfp. VNC = ventral nerve cord; HG = head ganglia; N = non-neuronal
cells. The animal is mosaic and does not show muscle expression.
S: gcy-29prom::gfp. Ventral view of the head region. The reporter is also expressed in
AWCL/R and AVKL/R (not shown in this animal).
Fig.6: Regulation of the expression of asymmetric gcy genes.
A, B: Quantification of the asymmetry of ASE-expressed gcy genes in wild-type and
mutant backgrounds. In panel A, the quantification and a representative example of gcy-
1prom::gfp reporter gene-carrying animals are shown, in panel B only the quantification of
the results obtained with the other asymmetrically expressed and previously
uncharacterized gcy reporter gene constructs are shown. Black, white and grey circles
indicate relative expression levels of gfp in the ASEL and ASER neurons. Data is shown
for one representative array each (otEx2419 for gcy-1prom::gfp, otEx2423 for gcy-
3prom::gfp, otEx2409 for gcy-4prom::gfp, otEx2322 for gcy-14prom::gfp, otEx2327 for gcy-
20prom::gfp). These arrays were each crossed into the indicated mutant backgrounds.
Comparable results were obtained with several independent arrays (not shown). Like in
other figures, color coding is red to indicate ASEL expression and blue for ASER
expression.
C: Summary of the gene regulatory interactions. Grey shading indicates genes
identified in this paper. For more details on the ASEL and ASER inducers, see
30
JOHNSTON et al., 2005.
Fig.7: Functional analysis of gcy-5.
A: Mutant alleles of gcy-5. Color coding for the domains encoded by the individual
exons is shown in Fig.2. Reading frames are indicated to illustrate the effects of the
respective deletion alleles. tm897 contains a 691 bp deletion from position 462 - 1045 of
the coding sequence and replacement with 5’-GGGGTAGAAGAGGC. Within the
genomic locus, the deletion starts in exon 4 and ends in exon 7. With the deletion and
insertion, a frame-shift is created leading to an early stop codon. This allele is therefore
a putative null allele. The effect of the other alleles is more difficult to predict since the
respective deletions start in exons and end in introns. If one assumes splicing around
the half-deleted exons, then the two ok alleles produce large, but in-frame deletions.
B: Chemotaxis to soluble ions of wild-type and gcy-5 mutant animals. NaCl measures
the functionality of both ASEL and ASER. NH4Cl mainly measures ASER function since
NH4+ is sensed in a ASE-independent manner (NH4
+ sensation is unaffected in che-1
mutants in which ASEL/R fail to develop (CHANG et al. 2004)). All strains were grown
and assayed at room temperature (21-23°C). Population chemotaxis assays were
performed in a radial gradient of the indicated salt (see Material and Methods). Each
experiment was done with at least two plates in parallel and for each assay plate at
least 20 worms reached either attractant or negative control spot. 3-9 independent
experiments were done for each condition. Error bars indicate standard error of the
mean. For statistical analysis, an one-way ANOVA was performed for each attractant,
with Dunnet’s post-test comparing all three alleles to wild-type control data. None of the
means were significantly different.
Fig.8: Analysis of synteny of gcy genes in C. elegans and C. briggsae.
Gene predictions were taken from WormBase release WS149 (www.wormbase.org).
The red line indicates the genomic regions included in gfp reporter gene constructs. The
C. elegans reporter constructs are also shown in Fig.4. The size of the C. briggsae gcy-
19 construct is 2 kb, the size of the C. briggsae gcy-4 construct is 433 bp (up to
preceding gene).
31
Fig.9: Phylogenetic conservation of gcy gene expression profiles.
C. briggsae reporter gene constructs are shown in Fig.8, C. elegans reporter gene
constructs are shown in Fig.4. Species names in pictures indicate into which species
the respective reporter gene was injected.
A: gcy-4 expression. C. briggsae gcy-4prom::gfp is expressed in both ASEL and ASER in
C. briggsae (3 lines; data for one representative array, otEx2508, is shown), but is
expressed predominantly in ASER in C. elegans (3 lines; n>40 each; data for one
representative array, otEx2510, is shown). Expression of C. elegans gcy-4prom::gfp
(construct in Fig.4) in C. elegans is also biased to ASER (Fig.5C, Fig.6B).
B: gcy-19 expression. C. briggsae gcy-19prom::gfp is expressed exclusively in ASER in
C. briggsae (3 lines; data for one representative array, otEx2139, is shown) and in C.
elegans (3 lines; data for one representative array, otEx2141, is shown).
Supp.Fig.1: Alignment of the guanylyl cyclase domain of C. elegans GCY
proteins. An asparagine and arginine residue essential for catalytic activity (MORTON
2004; YAN et al. 1997) are boxed. Note that one or both of these two residues is
mutated in several of the receptor and soluble-type kinases suggesting that they may be
catalytically inactive.
Supp.Fig.2: Alignment of the protein kinase-like domain of C. elegans GCY
proteins. A set of canonical protein kinase domains is included in this alignment of the
kinase domains of the GCY proteins. Note the labeled HRD motif (boxed in figure); the
D is conserved in all active protein kinases and is required for catalytic activity (TAYLOR
et al. 1992). It is not conserved in GCY proteins, therefore making it unlikely that these
protein kinases have catalytic activity.
32
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35
Table 1: PCR primers used to generate reporter constructs
PCR fusion primers 1Promoter“A” primer “A*” primer “B” primer
Resulting transgenicarrays 2
gcy-1 gaagctggagtcaagtgtg gtgtactacaacaagggactttg agtcgacctgcaggcatgcaagctgaatatttgcatcgaaaag otEx2419, otEx2420, otEx2421gcy-2 ccattacagggacatcag ggatacattgaaagtagcg agtcgacctgcaggcatgcaagctgaccattttgaattaatgattcc otEx2312, otEx2313, otEx2314gcy-3 gagctctcatggatcacg cacattgaaagtagcgaattag agtcgacctgcaggcatgcaagctgttttttcaaacaaagatcag otEx2422, otEx2423
gcy-4 gaagtttcagtggtacttgc gattgattcagaattcgagatc agtcgacctgcaggcatgcaagctgagtatcataattcatggaagtag otEx2407, otEx2408, otEx2409gcy-9 gttatctccgatagccaag ggttgttgaaagccatatc agtcgacctgcaggcatgcaagctcggaagtggatagatgg otEx2307, otEx2308, otEx25414,
otEx25424
gcy-11 ctgatttgatgccatcac gatttgtctcttcactggtcac agtcgacctgcaggcatgcaagctgtccagcatgcttctgc otEx2304, otEx2305, otEx2306
gcy-13 gccacaatgacaattatctc ctcaaaatccattgtgtaagttc agtcgacctgcaggcatgcaagctccatcctacagaggaggc otEx2410, otEx2411, otEx2412gcy-14 gtgcattaatcagaatgagc cagaacactgaaagctacc agtcgacctgcaggcatgcaagctgcacatgttagatgatgaatg otEx2322, otEx2323, otEx2324
gcy-15 gttatcctaatagcaactacatcacc cttcaataatagtttctctcacttg agtcgacctgcaggcatgcaagctcgcaactgttgcattg otEx2497gcy-17 cagttgaacatctccctgg caacaacgttcaagctcc agtcgacctgcaggcatgcaagctcatcatgttcttagagtaggc otEx2413, otEx2414, otEx2415gcy-18 ccgaaagaagtcgagttg ggtcgagagcagcaaatc agtcgacctgcaggcatgcaagctcattttctgatgctccgac otEx2498 3
gcy-19 gataccgtgagcaagatgg gatagattgcattgttgtgg agtcgacctgcaggcatgcaagctggacattggccatcctatac otEx2309, otEx2310, otEx2311,otEx25356, otEx25366
gcy-20 gatgatttatacccacatacattg ggagagttttcaatgatttg agtcgacctgcaggcatgcaagctccgcattgttgaatgaac otEx2325, otEx2326, otEx2327
gcy-21 gtaaactgggagtgaaagc ctcgacgagcattatgtg agtcgacctgcaggcatgcaagctctgaaagcatagcagaataatatg otEx2416, otEx2417, otEx2418gcy-23 caaacatcttacgcttctcac ctcgagtgtcgacacg agtcgacctgcaggcatgcaagctccaacttttacctttaaagttaatg otEx2499, otEx2500, otEx2501
gcy-25 caagattcgatttcaaaactg ccaacctgattttgtgg agtcgacctgcaggcatgcaagctgagaagcatctccgaatg otEx2424, otEx2425, otEx2426gcy-27 gatactttggaaaagaacaatg cgaatagacaaccaaacttc agtcgacctgcaggcatgcaagctgttgtatctcgagaaagactctg otEx25024, otEx2503,4 otEx25404
gcy-28 caagacctccaaagttttg cggtcttgaagcgattc agtcgacctgcaggcatgcaagctgagcatagcctcataggtac otEx2504, otEx2505, otEx2537gcy-29 cacagacaagcagagcac gcaaacgagtcccagc agtcgacctgcaggcatgcaagctgagcattttttactgtttttc otEx2490, otEx2491, otEx2492
Subcloning 5
5’ primer 3’ primerC.b.gcy-4 ttaagcttAAAACGTATCCCTCTTCCT ttggatcCTAGATGGCTGAGATCTACG otEx25066, otEx25076, otEx25086
otEx2509, otEx2510, otEx2511C.b.gcy-19 ttaagcttGCCGCGGTTGAAAACAAAAATTG ttggatccAATACTTGGCGAGTAGCCCC otEx21386, otEx21396, otEx21406
otEx2141, otEx2142
1 Primer nomenclature according to Hobert, 2002. All sequences go 5’ to 3’. 2 If not indicated otherwise, C.elegans animals containing the otIs151transgene were injected, using unc-122::gfp as injection marker. 3 Injection was done into otIs133 animals, using rol-6(d) as injection marker.4 Injectionwas done into N2 animals, using rol-6(d) as injection marker. 5 Primers used for subcloning contained restriction sites at their end and were subcloned intopPD95.75. 6 Injected into C.briggsae, using rol-6(d) as injection marker.
36
Table 2: Receptor guanylyl cyclases in C.elegans
Expression pattern in adult animalsGene Other names 1 Mapposition 2 Previous reports 3-7 This report
daf-11 B0240.3 V +3.27 ASIL/R, ASJL/R, ASKL/R,AWBL/R, AWCL/R 3
-
gcy-1 AH6.1 II +1.49 no signal 4 ASER, ASIL/R, PVT, URXL/R, AIYL/R, intestinegcy-2 R134.2 II +1.48 - AWAL/R, ASIL/R, RIAL/R, PVT,gcy-3 R134.1 II +1.48 - ASER, ASIL/R, PVTgcy-4 ZK970.5 II +2.31 no signal 4 ASER-biased 8
gcy-5 ZK970.6 II +2.32 ASER 4 -gcy-6 B0024.6 V +2.46 ASEL 4,7 -gcy-7 F52E1.4 V +1.37 ASEL 4,7 also expressed in excretory canal cell (only in adults)gcy-8 C49H3.1 IV +3.50 AFDL/R 4 -gcy-9 ZK455.2 X +3.0 no signal 4 weak, occasional and variable expression in non-neuronal tissuesgcy-11 C30G4.3 X +24.07 - pharyngeal musclegcy-12 F08B1.2 II –1.80 PHAL/R 4 -gcy-13 F23H12.6 V +4.02 no signal 4 RIML/Rgcy-14 ZC412.2 V +6.93 no signal 4 ASEL-biased 8, AWCL/R (faint), PVTgcy-15 ZC239.7 II –8.08 - ASGL/R (faint)gcy-17 W03F11.2, gcy-24 V –10.32 - PHAL/Rgcy-18 ZK896.8, gcy-26 IV +6.48 - AFDL/R, AIML/Rgcy-19 C17F4.6 II –7.80 - IL2 (strong), ASEL/R (faint)9, additional faint sensory neurons (3 pairs)gcy-20 F21H7.9, gcy-16 V +9.87 - ASEL, AWCL/R (faint) excretory gland and canal cellsgcy-21 F22E5.3 II -12.28 - ASGL/R, ADLL/R (faint)gcy-22 T03D8.5 V +25.25 ASER 6 -gcy-23 T26C12.4 IV -5.01 - AFDL/Rgcy-25 Y105C5B.2 IV +14.17 - AQR, PQR, URXL/Rgcy-27 C06A12.4 IV +16.74 - ASKL/R, ASIL/R, ASJL/Rgcy-28 T01A4.1 I –1.17 - Many head neurons, ventral cord and tail neurons, body wall muscle,
hypodermis, somatic gonad, intestine 10
gcy-29 C04H5.3 II +23.04 - ASEL/R, AWCL/R, AVKL/R, AFDL/R, few variable other neurons(weak)
odr-1 R01E6.1, gcy-10 X +12.7 ASIL/R, ASJL/R, ASKL/R,AWBL/R, AWCL/R 4,5
-
This Table summarizes previously characterized and newly determined receptor-gcy expression patterns. “-“ indicates that expression was not analyzed.1 See Material and Methods for comments on gene nomenclature. 2 From www.wormbase.org. 3 Birnby et al., 2000. 4 Yu et al., 1997. We suppose thatwe detected clear expression in several cases where no signal was observed by Yu et al. since we (a) used a gfp variant that is much brighter than theold gfp version used by Yu et al. and (b) since our reporter constructs may encompass more cis-regulatory information that those of Yu et al. 5 L’Etoileand Bargmann, 2000. 6 Johnston et al., 2005. Additional weak expression that fades in adults is observed in two additional, unidentified head neurons. 7
37
As described in Johnston et al., 2005, gcy-6 and gcy-7 are embryonically expressed in both ASEL and ASER and only become restricted to ASELpostembryonically. A similar scenario may apply for other ASEL-expressed gcy genes, but has not been explicitly examined. 8 “ASER-biased”incorporates two categories of expression patterns in a given transgenic line: expression only in ASER in some animals and stronger expression inASER than in ASEL in other animals. The opposite holds for “ASEL-biased” expression. Such weak and occasional expression in the other cell could becaused by array-overexpression artifacts and we therefore do not wish to emphasize that biased expression is fundamentally different from exclusiveexpression. 9 Expression in ASEL/R is very dim and not completely penetrant and potential biases to ASEL or ASER are therefore difficult to determine.10 Expression in all tissue types is often mosaic. Expression in the ASE and AWC neuron classes could not be observed, but as with any other reporterconstruct described in this Table it is possible that additional regulatory elements not contained within the respective constructs may yield expression inthese neurons.
38
Table 3: Summary of cell-type specificity of gcy reporter gene expression.
gcy gene expression pattern #Non-neuronal only 2/27
Nervous system 25/27
Nervous system only 21/27
Sensory neurons + other neurons 25/27
Sensory neurons only 15/27
Single neuron class-specific 9/27
Table 4: Summary of co-expressed gcy reporter genes.
Co-expressed gcy genesSensory neuronclass # nameASE 11 gcy-1, gcy-3, gcy-4, gcy-5, gcy-6, gcy-7,
gcy-14, gcy-19, gcy-20, gcy-22, gcy-29
AQR, PQR, URX 7 gcy-25, six soluble gcy genes
ASI 6 daf-11, gcy-1, gcy-2, gcy-3, gcy-27, odr-1
AWC 5 daf-11, odr-1, gcy-14, gcy-20, gcy-29
AFD 4 gcy-8, gcy-18, gcy-23, gcy-29
ASG 2 gcy-15, gcy-21
PHA 2 gcy-12, gcy-17
Anterior Posterior
LeftASEL
AWBL
AWCL
AFDL
Figure 1
LEFT
Lateral view
RIGHT
Posterior
Dorsoventral view
ASEL
AWCR
A B
ASKL ASIL
ASJL
ASGL
ADLL
ADFLASHL
Salts Amino acids
Small metabolites
gcy-14gcy-20
gcy-6gcy-7
gcy-1gcy-3gcy-4
gcy-5gcy-22
AWA, AWB, AWC: odorsensoryAFD: thermosensoryASE, ASG, ADF,ASI, ASJ, ADL, ASK: chemosensoryASH: polymodal
AWAL
gcy-19gcy-29
gcy-19gcy-29
str-2odr-1daf-11
AWCL
odr-1daf-11
gcy-14gcy-20gcy-29
gcy-14gcy-20gcy-29
ASER
Odorants
ASEL/R AWCL/R
HNOB
Receptor-type GCYs
Soluble GCYs
GCY-31, GCY-32, GCY-33,GCY-34, GCY-35, GCY-36,GCY-37
GCY-1, GCY-12, GCY-13, GCY-14,GCY-17, GCY-18, GCY-19, GCY-2,GCY-20, GCY-22, GCY-23, GCY-3,GCY-28, GCY-29, GCY-4, GCY-5,GCY-6, GCY-7, GCY-8, GCY-9
GCY-11, GCY-15, GCY-21,GCY-25, DAF-11, ODR-1
GCY-27
SS TMRFLBPProteinKinase-like Cyclase
Figure 2
Extracellular domain Intracellular domain
CBP 00094GCY-25
CBP15915*CBP11205*
66
ODR-1GCY-27
96
100
CBP 01192GCY-32
90
CBP 01705GCY-34
99
100
CBP 01899GCY-36
100
100
GCY-35
CBP 04780CBP 04781
95100
50
CBP 17959GCY-37 S
olub
le G
CY
s
100
99
CBP 05524GCY-33
100
CBP 21055GCY-31
100
75
100
GCY-3CBP 00282
GCY-1
"gcy-5 subgroup"
"gcy-7 subgroup"
GCY-2
ASEL neuron
Expression:
Figure 3
ASER neuron
AFDL/R neurons
ASGL/R neurons
AQR, PQR, URX neurons
10058
99
CBP 13905GCY-5
96
57
CBP 13906GCY-4
99
81
CBP 04086GCY-19
100
100
CBP 15625CBP 01237GCY-20GCY-14
56100
CBP14936GCY-17
99
98
CBP02750GCY-7
100
65
CBP08562CBP 16612GCY-6
85100
94
CBP01427GCY-22
100
55
CBP05559GCY-13
100
63
100
CBP00662GCY-12
100
CBP03512GCY-28
100
73
CBP 04548GCY-15GCY-21
100100
CBP18607GCY-11
100
69
68
CBP20608DAF-11
100
CBP04125GCY-8
100
CBP13439GCY-18
100
CBP14192GCY-23
100
67
GCY-29CBP04902*
100
100
72
CBP 04194GCY-9
100
100
0
5
10
15
20
25
-5
-10
-15
-20
-25
gcy-28
gcy-4gcy-5
gcy-29
gcy-12
gcy-21
gcy-19gcy-15
gcy-23
gcy-8
gcy-36
gcy-31
gcy-18
gcy-25gcy-27 gcy-32
gcy-34gcy-33
gcy-13daf-11 gcy-9
odr-1
gcy-11
I II IV V X
gcy-1 gcy-2gcy-3
gcy-35
gcy-22
gcy-6gcy-7
gcy-17
gcy-14
gcy-20
gcy-37
ASEL+ASER neurons
A B
gcy-17
PHA
gcy-13
RIM
gcy-18
AFD
AIM
PQR
gcy-25
gcy-21
ASG
gcy-11
gcy-27
ASKASI
ASJ
ASK ASI ASJ
gcy-4
ASER
ASEL
ASER
ASEL
ASI PVT
ASER
ASEL
gcy-20
ASEL
EXG
EXC
gcy-14
ASER
ASEL
ASEL
EXC
muscle
gcy-7
ASER
ASEL
URX ASI
gcy-1
AIYR
AIYL
gcy-15
gcy-23
AFDR
AFDL
ASG
gcy-2
RIA NASI
AWA
gcy-28
gcy-19
gcy-29
ASEL
ASER
AFDR
AFDL
HG
VNC
N
gcy-3
URX
AQR
ASEL
IL2
ASER
ASEL
A B
EDC
F G H I
MLKJ
N O
P Q R S
Figure 5
C
A
B
gcy-5gcy-22
gcy-6
lim-6
gcy-7
gcy-3
gcy-1
red - ASEL
wild type
lim-6(nr2073)
lsy-6(ot71)
gcy-4gcy-3gcy-1
gcy-4
blue - ASER
wild type
lim-6(nr2073)
lsy-6(ot71)
gcy-20
gcy-14
gcy-14gcy-20
ASEL-inducing genes
(die-1, lsy-6)
ASER-inducing genes(mir-273, cog-1)
Figure 6
77
100
% a
nim
als
47
100
% a
nim
als
34
100
% a
nim
als
wild type
lim-6(nr2073)
lsy-6(ot71)
wild
type
lsy-
6(ot
71)
ASEL ASER
ASERASEL
n100
% a
nim
als
58
100
% a
nim
als
47
100
% a
nim
als
65
96
100
% a
nim
als
80
100%
ani
mal
s
35
100
% a
nim
als
53
100
% a
nim
als
66
100
% a
nim
als
47
100
% a
nim
als
55
100
% a
nim
als
60
100
% a
nim
als
39
100
% a
nim
als
n n
n
nleft-biased right-biasedequal
left-biased right-biasedequalleft-biased right-biasedequal
left-biased right-biasedequal left-biased right-biasedequal
tm897 ok921reading frame
ok930
gcy-5
NaCl NH4ClGradient
mo
dified
C.I.
B
A
Figure 7
1 kb
wild-type
gcy-5(tm897)
gcy-5(ok921)
gcy-5(ok930)
0.5
0.6
0.7
0.8
0.9
1.0
Orthologs at~2220K
CBG17049≈CBP04086
Orthologueon contig cb25.fpc4248
cb25.fpc4055
C.elegans
C.briggsae
LG II
CBG00847≈CBP00282CBG00851≈CBP13905CBG00850≈CBP13906
LG II C.elegans
C.briggsae
Cb25.fpc0022
B
No obvious ortholog
Ortholog as shown
A
Orthologous gcy genes as shown
Genomic region includedin gfp reporter construct
Figure 8
C.briggsae gcy-4 (CBP13906)
C.briggsae gcy-19 (CBP04086)
A
B
C. briggsae
C. briggsae
C. elegans
C. elegans
Figure 9
ASELASER
50
100
% a
nim
als
nleft-biased right-biasedequal
61
100
% a
nim
als
nleft-biased right-biasedequal
32
100
% a
nim
als
nleft-biased right-biasedequal
22
100
% a
nim
als
nleft-biased right-biasedequal
.
GCY-8 LFSDIVGFTEMCQNASPLEV---------VAVLNGIFDGFDQFIARKDAYKV------ETIGDAYMVVSGVPEENG-HRHINEIASIALDVHKFLSEFIVP-HKRDTKVQCRLGFHTGPVAAAVVGLNAPRYCLFGDTVNMASRMESNS-E-----------------------------------------PGKTQISETAKNLLLKEY-PDYICEQRGEIPIKGKGLCMTYWLMG 177
CBP00094 LVVDVCQFTKFCEAFIPVHI---------LETLQELYSSFDYIVQKNKAFKV------ENVGDAYLICSGIPEMTA-CRHLREVSKISLKLQEFMKTFTVR-HRPSHILQIKMGITSGAVAAGILGSIAPRI-ITGICKNLAYKILNNI-Q-----------------------------------------PGTIQLSELTANTMMEKF-PSFIIEERGMIDVKGKGTCLTFWLTG 176
GCY-25 LVVDVCQFTKFCEAFIPVHI---------LETLQELYSSFDNIVQKNKAFKV------ENVGDAYLICSGIPEMSG-FRHLREICKISLKLQAFMKTFKVR-HRPSHTLQIKMGITSGAVAAGILGSTAPRFCIFGDTVNMACRMASTG-N-----------------------------------------PGSIQLSELTANTLMEKF-PSFMLEERGMIDVKGKGACLTFWLTG 177
CBP15915* MFVQICEFNALMKKSTPNQT---------IMFLNDVFDQFDNVIKNHDAYKV------ETTGETYMVASGVPEENE-GRHVYEIAEMSLEIREVSYRYKLS-YNPSYVLRVRIGFHMGPIAAGVIGIRSPRYCLFGDTVNFASRMQSNS-A-----------------------------------------PNQIQTSEITANLLMAS--KMYTFKKRGYVHVKGKGDVKCFWLNQ 176
GCY-10 MFVQICDFNALMKRSSPEQV---------IAFLNDIYDQFDTVIKRHDAYKV------ETTGETYMVASGVPHENE-GRHIFEVAEISLEIREISYIYVLQ-HDKNYKLRIRIGFHAGPIAAGVIGIRSPRYCLFGDTVNFASRMQSNC-P-----------------------------------------PNQIQTSEITARLLFDS--HEYKFVKRGIVHVKGKGNAARLKICC 176
GCY-27 MFVQICDFIVILKRRPPKEV---------IGFLNDIFDQFDTVIKRHD---V------ETTGETYMVASGVPNENE-GRHVFEVAEMSLEIRAISLSYTLE-NDKNYKLRVRIGFHAGPIAAGVIGIKNPRYCLFGDTVNFASRMQSNC-P-----------------------------------------PLQIQTSEITARMLLAT--HEYKLVKRGIVHVKGKGEVNCYWLNE 173
CBP11205* MFVQICEFNVLMKKSTPDQV---------IAFLNDVFDQFDSVIKHHVAYKV------ETTGETYMVASGVPEEND-GQHIFEIAEIALEIREVAAVYVLP-HDPKYILRIRTGFHMGPIAAGVIGIKSPRYCLFGDTVNYASRMQSTC-A-----------------------------------------PNQIQTSEITAKVLENS--KRYILDDKKLVYVKGKGDIKCYLLTG 176
DAF-11 LYSDIVGFTSLCSQSQPMEV---------VTLLSGMYQRFDLIISQQGGYKVILNRIMETIGDAYCVAAGLPVVME-KDHVKSICMIALLQRDCLHHFEIP-HRPGTFLNCRWGFNSGPVFAGVIGQKAPRYACFGEAVILASKMESSG-V-----------------------------------------EDRIQMTLASQQLLEENF-PQFVCSNRGGRTIEGIGRILTYWLEG 183
CBP04125 LFSDIVGFTEMCQHATPLEV---------VSVLNGIFDGFDQFIARKDAYKV------ETIGDAYMVVSGVPEENG-HRHINEIASIALDVHKFLSEFIVP-HKRDTKVQCRLGFHTGPVAAAVVGLNAPRYCLFGDTVNMASRMESNG-E-----------------------------------------PGKTQISEAAKNLLLKEY-PDYICEQRGEIPIKGKGMCMTYWLMG 177
CBP13439 LFSDIVGFTTICSGSTPLEV---------VTMLNGLYTGFDECITRNKSYKV------ETIGDAYMVVSGIPEENG-NDHSKNIANAALDMRQYLTGYQIP-HRPSTRVRCRWGFHTGSVAAGVVGLTSPRFCLFGDTVNVASRMESTG-T-----------------------------------------PGMIQMSEDAHNHIRSYH-PVFSTTKRGEVQVKGKGSCCTFWLED 177
GCY-18/26 LFSDIVGFTTICSGSTPLEV---------VNMLNGLYTGFDECITRNKSYKV------ETIGDAYMVVSGIPEENE-YNHSRNIANTALDMRQYLTGYQIP-HRPTHRVRCRWGFHTGSVAAGVVGLTCPRYCLFGDTVNVSSRMESTG-T-----------------------------------------PGMIQMSEEAHMHIRAHH-PVFTTTERGEVQVKGKGTCRTFWLED 177
CBP14192 MFSDIVGFTTICSSSSPLEV---------VSMLNSIYSKFDDAINKHSAYKV------ETIGDAYMIVSGIPEENG-NEHIRNICNTALELMLLLKTYEIP-HRRNVKLRIRLGIHTGTVAAGVVGLTAPRYCLFGDTVNVASRMESTS-E-----------------------------------------PEKIQMSQEARDFCLRYY-QEFQIVLRGVVEAKGKGPVTTYWLLG 177
GCY-23 MFSDIVGFTTICSSSTPLEV---------VSMLNSIYSKFDDAINKHGSYKV------ETIGDAYMIVSGIPEENG-NEHIRNICNTALELMLLLKTYEIP-HRRNVKLRIRLGIHTGTVAAGVVGLTAPRYCLFGDTVNVASRMESTS-E-----------------------------------------PEKIQMSQEARDFCVRYY-SEFQITLRGTVEAKGKGPVTSYWLLG 177
GCY-29 MFSDIVGFTKLCSASTPIEV---------VNLLNKLYSEFDTVISKHDCYKV------ETIGDAYMVVSGIPIENG-QRHVANISAVTLGIMDLLKVFEVP-HRRDYRLTIRLGFASGQVSAAVVGLSSPRYCLFGETVNIAAVMESSG-E-----------------------------------------GGRVQITETSKILLENEY-PEFIIEIRGINKDVKQDDFVTYWLTG 177
CBP04902* MFSDIVGFTKLCSASTPIEV---------VNLLNKLYSEFDSILNKQDCYKV------ETIGDAYMVVSGIPMENG-RRHVANIASVTLEIMELLKTFVVP-HRQNHRLSIRLGFASGPVAAAVVGLNSPRFCLFGETVNIASIMESSG-E-----------------------------------------GERIQITDSSKILLANEY-PEYIIEIRGINKEVNQSEFTTHWLVG 177
CBP04194 LFSDIRGFTRISSTSTPLQVTKKLLVFQVVTFLNDMFSGFDAIIAKHDAYKV------ETIGDAYMIVSGVPTENG-NNHAQNIADVALKMRAFICNFKLA-HRPEELMMVRIGFHSGPVAAGVVGLAAPRYCLFGDTVNTASRMESTG-V-----------------------------------------ANKIQISEGAYNLLHCFF-PQFQMVERGKIEVKVS---------- 176
GCY-9 LFSDIRGFTRISSTSTPLQVCKLRQKIIVVTFLNDMFSGFDAIIAKHDAYKV------ETIGDAYMIVSGVPTENG-NSHAQNIADVALKMRAFICNFKLA-HRPEELMMVRIGFHSGPVAAGVVGLAAPRYCLFGDTVNTASRMESTG-V-----------------------------------------ANKIQISEGAYNLLHCFF-PQFQMVERGKIEVKVS---------E 177
CBP00282 FFSDVVKFTQLSQKCSPFQV---------VNLLNDLYSNFDTIIEEHGVYKV------ESIGDGYLCVSGLPTRNG-YAHIKQIVDMSLQFMEYCRKFKIP-HLPREQVELRIGVNSG---------------------------------------------------------------------------------------------------------------------- 101
CBP04086 FFSDVVKFTQLSQKCSPFQV---------VNLLNDLYSNFDAIIEEHGVYKV------ESIGDGYLCVSGLPQRNG-NAHIKCIVELSLDFMAYCKAFKIP-HLPREKVELRVGVNSGPCVAGVVGLSMPRYCLFGDTVNTASRMESNG-KRLR---------------------------------IDGPAPSHIHLSAAAYTLLMKHYPNQYNTASRGDVIIKGKGVMETFWVFE 186
GCY-19 FFSDVVKFTQLAAKCSPFQV---------VNLLNDLYSNFDAIIEEHGCYKV------ESIGDGYLCVSGLPSKNG-NAHIKQIVELSLDFMSYCKSFKIP-HLPREKVELRIGVNSGPCVAGVVGLSMPRYCLFGDTVNTASRMESNG-KGRQ---------------------------------MALKSSSHIHLSAASYTLLMKHYPNQYNTASRGDVIIKGKGVMETFWVFE 186
CBP13905 FFSDVVKFTQLSQKCSPFQV---------VNLLNDLYSNFDTIIEEHGVYKV------ESIGDGYLCVSGLPTRNG-YAHIKQIVDMSLQFMEYCRKFKIP-HLPREQVELRIGVNSGPCVAGVVGLSMPRYCLFGDTVNTASRMESNG-K-----------------------------------------PSQIHMSEAAHSLLTSHYPHQYETSSRGEVIIKGKGVMETFWVHG 178
GCY-5 LFSDVVKFTQLAAKCSPFQV---------VNLLNDLYSNFDTIIEEHGVYKV------ESIGDGYLCVSGLPTKNG-YAHIKQIVDMSLKFMDYCKSFKVP-HLPREKVELRIGINSGPCVAGVVGLSMPRYCLFGDTVNTASRMESNG-K-----------------------------------------PSMIHMSEAAHSLLTDHYPHQYETSSRGEVIIKGKGVMETFWVLG 178
GCY-1 FFSDVVKFTILASKCSPFQT---------VNLLNDLYSNFDTIIEQHGVYKV------ESIGDGYLCVSGLPTRNG-YAHIKQIVDMSLKFMEYCKSFNIP-HLPRENVELRIGVNSGPCVAGVVGLSMPRYCLFGDTVNTASRMESNG-K-----------------------------------------PSLIHLTNDAHSLLTTHYPNQYETSSRGEVIIKGKGVMETFWVHG 178
GCY-2 FFSDVVKFTILASKCSPFQT---------VNLLNDLYSNFDTIIEQHGVYKV------ESIGDGYLCVSGLPTRNG-YAHIKQIVDMSLKFMEYCRSFKIP-HLPRENVELRIGVNSGPCVAGVVGLSMPRYCLFGDTVNTASRMESNG-K-----------------------------------------PSLIHLTSDAHLLLLTHYPNHYDTSSRGEVIIKGKGVMETFWVHG 178
GCY-3 FFSDVVKFTILASKCTPFQV---------VNLLNDLYSNFDTIIEEHGVYKV------ESIGDGYLCVSGLPTRNG-FNHIKQIVDMSLKFMDYCKNFKIP-HLPRERVELRIGVNSGPCVAGVVGLSMPRYCLFGDTVNTASRMESNG-K-----------------------------------------PSLIHLTSDAHLLLMKHFPHQYETNSRGEVIIKGKGVMETFWVLG 178
CBP13906 FFSDLVKFTELANKCSPFQV---------VNLLNEVFSNFDAIIEKHDVYKV------ESIGDGFLCVSGLPNRNG-VEHIRQIVEMALGFLEFCDKFRIP-HLPRERVELRVGVNSGSCVAGVVGLSMPRYCLFGDTVNTASRMESNG-K-----------------------------------------ASLIHMSEIAHAFLVDHFPYQYETNSRGEADNWSFRVEKP----- 173
GCY-4 FFSDLVKFTDLASKCSPFQV---------VNLLNDVFSNFDSIIEKHDVYKV------ESIGDGFLCVSGLPNRNG-MEHIRQIVGMSLCFMEFCRNFRIP-HLPRERVELRVGINSGPCVAGVVGLSMPRYCLFGDTVNTASRMESNG-K-----------------------------------------PSMIHMSEAAHSLLVNNYPYQFETNSRGEVIIKGKGVMETYWLLG 178
CBP01237 FFSDVVQFTTLAGKCTPLQV---------VTLLNDLYTIFDGIIEQNDVYKV------ETIGDGYLCVSGLPHRNG-NEHIRHIARMALGFLSSLQYFRVQ-HLPAERINLRIGINCGSVVAGVVGLTMPRYCLFGDAVNTASRMESNG-K-----------------------------------------PGQIHVTAEANRMLTQVV-GGFKTESRGEVIIKGKGVMETFWLLG 177
GCY-16/20 FFSDVVQFTTLAGKCTPLQV---------VTLLNDLYTIFDGIIEQNDVYKV------ETIGDGYLCVSGLPHRNG-NDHIRHIARMSLGFLSSLEFFRVQ-HLPAERINLRIGINCGSVVAGVVGLTMPRYCLFGDAVNTASRMESNG-K-----------------------------------------PGQIHVTAEANRMLTQVV-GGFRTESRGEVIIKGKGVMETFWLLG 177
GCY-14 FFSDVVQFTTLAGKCTPLQV---------VTLLNDLYTIFDGIIEQNDVYKV------ETIGDGYLCVSGLPHRNG-NEHIRHIARMSLGFLSSLEFFRVQ-HLPSERINLRIGINCGSVVAGVVGLTMPRYCLFGDAVNTASRMESNG-K-----------------------------------------PGKIHVTAEANQMLTQVV-GGFRTESRGEVIIKGKGVMETYWLLG 177
CBP15625 FFSDVVQFTNLAAKCTPLQV---------V----------------------------ETIGDGYLCVSGLPHRNG-NEHIRHIAKMSLGFLTSLKFFRVQ-HLPNERINLRIGINCGSVVAGVVGLTMPRYCLFGDAVNTASRMESNG-K-----------------------------------------PGQIHVTADANRMLTEVV-GGFRTESRGEVLIKGKGVMETYWLLG 155
CBP14936 FFSDVVQFTVLAGKCTPLQV---------VQLLNDLYTIFDSIIEQHDVYKV------ETIGDGYLCVSGLPHRNG-NEHIRHIARMSLAFLSSLAKFRIS-HMPNERINLRIGINCGSVVAGVVGLTMPRYCLFGDAVNTASRMESNG-K-----------------------------------------PGRIHVSSEANRLLTKVV-GGFRTEARGEVIIKGKGVMETFWLLE 177
GCY-17/24 FFSDVVQFTTLASKCTPLQV---------VQLLNDLYTIFDSIIEQNDVYKV------ETIGDGYLCVSGLPHRNG-HDHIKHIARMSLAFLSSLAEFRVA-HMPSERINLRIGINCGSVVAGVVGLTMPRYCLFGDAVNTASRMESNG-K-----------------------------------------PGRIHVSSEANHLLTHVV-GGFRTEERGEVIIKGKGVMNTYWLLG 177
CBP02750 FFSDVVQFTTLASKCTPLQV---------VNLLNDLYTIFDGIIEKHDVYKV------ETIGDGYLCVSGLPHRNG-NEHVRQIALMSLAFLSSLEFFRIP-HLPSERINLRIGMNCGSVVAGVVGLTMPRFCLFGDAVNTASRMESNG-K-----------------------------------------PGKIHLSAEANRLLTETV-GGFATESRGEVIIKGKGVMETFWLLG 177
GCY-7 FFSDVVQFTTLASKCTPLQV---------VNLLNDLYTIFDGIIEKHDVYKV------ETIGDGYLCVSGLPHRNG-NEHVRQIALMSLAFLSSLQFFRVP-HLPSERINLRIGMNCGSVVAGVVGLTMPRFCLFGDAVNTASRMESNG-K-----------------------------------------RMDFGFPPFSDKLNI------------------------------ 148
CBP16612 FFSDVVSFTTLAGKCTPLQV---------VNLLNGLYTIFDGIIEQHDVYKV------ETIGDGYFVASGVPRRNG-NEHTRNIASMSLCFVKSLADFNIP-HLPGEKINIRVGFHCGSVVAGVVGLTMPRYCLFGDAVNTASRMESNS-K-----------------------------------------PGHVHISDEANRMLMTL--GGFTTETRGEVIIKGKGVMTTYWLLK 176
GCY-6 FFSDVVSFTTLAGKCTPLQV---------VNLLNGLYTIFDGIIEQHDVYKV------ETIGDGYFVASGVPRRNG-NEHTRNIASMSINFVKSLADFSIP-HLPGEKIKIRVGFHCGSVVAGVVGLTMPRYCLFGDAVNTASRMESNS-KRKIFQKSTN-----------------------ISSSFHLFSAGQIHLSEEANQMLMRL--GGFTTEPRGEVIIKGKGVMATYWLLK 194
CBP08562 FFSDVVSFTTLAGKCTPLQV---------INLLNGLYTIFDGIIEQHDVYKV------ETIGDGYFVASGVPRWNG-NEHTRNIASMSLCFVKSIAGYKVP-HLPGEKINIRVGFHCGSVVAGVVGLTMPRYCLFGDAVNTASRMESNS-K-----------------------------------------PGQIHISQEANEMLMRL--GGFTTEPRGEVIVKGKGVMNTYWLKE 176
CBP01427 FFSDVVSFTTLASRCTPLQV---------VNLLNDLYTTFDAIIEQHDVYKV------ETIGDGYLCVSGLPHRNG-NEHAKEISSMSFALLKAIKTFRVP-HLPKERINIRVGLHTGPVVTGVVGMTMPRYCLFGDSVNTASRMESNG-K-----------------------------------------PGRVHISTECKNFLTDTI-GGYQTEPRGEVIVKGKGAVQTYWLLT 177
GCY-22 FFSDVVSFTTLASRCTPLQV---------VNLLNDLYTTFDAIIEQHDVYKV------ETIGDGYLCVSGLPHRNG-NEHAKEISSMSFSLLKAIKTFRVP-HLPKERINIRVGLHTGPVVTGVVGMTMPRYCLFGDSVNTASRMESNG-K-----------------------------------------PGRVHISTECMKFLTEVI-GGYQTEPRGEVIVKGKGAVQTHWLLT 177
CBP05559 FFSDVVGFT----------V---------VNLLNDLYTTFDAIIERNDSYKV------ETIGDAYLVVSGLPRRNG-TEHVNNIANMSLELMDSLQSYKIP-HLPQEKVQIRIGMHSGSCVAGVVGLTMPRYCLFGDTVNTASRMESNG-K-----------------------------------------PGFIHLSSDAHELLTSLY-KEYNTENRGEVIIKGKGVMQTFWLLG 167
GCY-13 FFSDVVGFT----------V---------VNLLNDLYTTFDAIIEKNDSYKV------ETIGDAYLVVSGLPRRNG-TEHVANIANMSLELMDSLQAFKIP-HLPQEKVQIRIGMHSGSCVAGVVGLTMPRYCLFGDTVNTASRMESNG-K-----------------------------------------PGFIHLSSDCYDLLTSLY-KEYNTESRGEVIIKGKGVMQTYWLLG 167
CBP00662 YFSDIVGFTTISGESTPMEV---------VTFLNKLYTLFDGIIRRYDVYKV------ETIGDAYMVVSGVPQYKTMEYHAEQIAMMAIHILSAVRTFTIP-HRIGEQLMIRIGMHTGPCVAGVVGKTMPRYTLFGDTVNTASRMESNG-EA-----------------------------------------LRIHCSSSTQKVLASID-QGFLLEERGTMAIKGKGQMTTYWLNG 178
GCY-12 YFSDIVGFTTISGESTPMEV---------VTFLNKLYTLFDSIIRRYDVYKV------ETIGDAYMVVSGVPQYKTMEYHAEQIAMMAIHILSAVRSFSIP-HRSCEPLMIRIGMHTGPCVAGVVGKTMPRYTLFGDTVNTASRMESNG-EA-----------------------------------------LRIHCSSSTQKVLTSID-QGFLLEERGSLAIKGKGQMTTYWLNG 178
CBP03512 YFSDIVGFTSLSSQSTPMQV---------VTLLNDLYLAFDGVVDNFKVYKV------ETIGDAYMVVSGLPERR--DDHANQIAQMSLSLLHKVKNFVIR-HRPHEQLKLRIGMHSGSVVAGVVGSKMPRYCLFGDTVNTSSRMESNG-LRKFLLEIEASENRKFRKPEKSEIFKFRNSEIFKFQNIQKYRTLKIHVSQQTYDILMQE--AGFKLELRGSVEMKGKGMQTTYWLRG 216
GCY-28 YFSDIVGFTSLSSQSTPMQV---------VTLLNDLYLAFDGVVDNFKVYKV------ETIGDAYMVVSGLPERR--DDHANQIAQMSLSLLHKVKNFVIR-HRPHEQLKLRIGMHSGSVVAGVVGSKMPRYCLFGDTVNTSSRMESNG-L-----------------------------------------PLKIHVSQQTYDILMQE--AGFKLELRGSVEMKGKGMQTTYWLRG 175
CBP04548 FFSDCPGFVEMSATSKPIDI---------VNFLNDLYTVFDRIIDQFDVYKV------ETIADAYMVASGLPVPNG-NHHAGEIASLGLALLSAIDAFKIR-HLPNEKVRLRIGMNSGPCVAGVVGLKVFRKLKKIKPFSIQKLLFIND-LKIHFQL---------------------------------FPALRINCSESAKQVLDQL--GGYVMEERGIVEMKGKGKQMTYFVRG 184
GCY-15 FFSDCPGFVEMSATSKPIDI---------VQFLNDLYTVFDRIIDQFDVYKV------ETIADAYMVASGLPVPNG-NHHAGEIASLGLALLKAVESFKIR-HLPNEKVRLRIGMNSGPCVAGVVGLKMPRYCLFGDTVNTASRMESNG-IRKFFWVAEP------------------------------VSSLRINCSGTAKEILDQL--GGYEIEERGIVEMKGKGKQMTYFVRG 187
GCY-21 FFSDCPGFVEMSATSKPIDI---------VQFLNDLYTVFDRIIDQFDVYKV------ETIADAYMVASGLPVPNG-NHHAGEIASLGLALLKAVESFKIR-HLPNEKVRLRIGMNSGPCVAGVVGLKMPRYCLFGDTVNTASRMESNG-IP-----------------------------------------LRINCSGTAKEILDQL--GGYEIEERGIVEMKGKGKQMTYFVRG 176
CBP18607 YFSDIVGFTSLSSKSTPLQI---------VNMLNNLYTNFDTIIDKFDCYKV------ETIGDAYMFVSGLPELNS-YLHAGEVAAASLELLDSIKTFIVP-HCPDEKLRLRIGNHAGPVVTGVVGIRMPRYCLFGDTVIVANNMESSG-EP-----------------------------------------MRIQVSSDSYELLLKC--GGFVTEQREKIVLKNKMEVMTYWMND 176
GCY-11 YFSDIVGFTALSSKSTPLQV---------VNMLNNLYTNFDTIIDKFDCYKV------ETIGDAYMFVSGLPEVNS-YLHAGEVASASLELLDSIKTFTVS-HCPDEKLRLRIGNHTGPVVTGVVGIRMPRYCLFGDTVIIANMMESSG-EP-----------------------------------------MRIQISSDAYELILKC--GGYVTEQREKIVLKNKLEVMTYWMND 176
CBP20608 LYSDIVGFTSLCSESKPLEV---------VNLLSGMYQRFDQIISTNGGYKM------ETIGDAYCVAAGLPEERA-MEHVKIICMIALYQRDSLHHFEIP-HRTGQYLNCRWGFNSGEVFAGIIGQRAPRYACFGEAVMLASKMESSG-V-----------------------------------------EDRIQMTLVSKQLLEDHY-PFFKCSFRGGVTVEPFGHMLTYWLEG 177
CBP01192 MFCDLPTFQQTIPQSSPKEI---------VRILNEIFKKLDRIIVIRGVYKV------ETVSDSYMAVSGITEYTP--EHAENMCHVALGMMWEARSVVDP--ISKNPFLLRIGLHSGKIVAGVVGTKSPKYCLFGETVTLASQMECQG-MA-----------------------------------------GKIQCSKWTYLQASAT--GRFEFSPRGRIAVKDRGLTETYFLTR 174
GCY-32 MFCDLPAFQQAIPQCSPKDI---------VNMLNEIFRKLDRIVVIRGVYKV------ETVSDSYMAVSGIPDYTP--EHAENMCHVALGMMWEARSVIDP--VSKTPFLLRIGIHSGTITAGVVGTVHPKYCLFGETVTLASQMESLG-MA-----------------------------------------GKIQCSKWAYQKAMET--GRFEFSPRGRIDVKQRGLTETYFLTR 174
CBP01705 MFCDLPAFQQIIPVCQPKNI---------VKLLNEVFHKLDRIVVLRGVYKV------ETVSDSYMTVSGIPDYTS--EHSETMCHVALGMMWEARSVIDP--VNNTPFLLRIGLHSGTIIAGVVGTKMPRYCLFGETVTLASQMESLG-LP-----------------------------------------GKIQCSKWTYSKAMET--GRFEFAPRGRINVKGRGELETYFLTR 174
GCY-34 MFCDLPAFQQIIPVCQPKNI---------VKLLNEVFFKLDRIVVLRGVYKV------ETVSDSYMTVSGIPDYTS--EHAENMCHVALGMMWEARSVMDP--VNKTPFLLRIGLHSGTIIAGVVGTKMPRYCLFGETVTLASQMESLG-VA-----------------------------------------GKIQCSSWTYSKAMET--GRFEFSPRGRINVKGRGDVETYFLMR 174
CBP01899 MFTDVPTFQQIVPLCQPKDI---------VHLLNELFTKFDRLIGIQKAYKV------ETVGDSYMSVGGIPDTVD--DHCEVICHLALGMVMEARTVCDP--ITSTPLHIRAGIHSGPVVAGVVGAKMPRYCLFGDTVNTASRMESHS-PI-----------------------------------------GRIHCSENAKKCAEST--GRFEFEPRGKVQIKGKGEMNTYFLLR 174
GCY-36 MFTDVPTFQQIVPLCTPKDI---------VHLLNELFTKFDRLIGIQKAYKV------ETVGDSYMSVGGIPDLVD--DHCEVICHLALGMVMEARTVCDP--ITNTPLHIRAGIHSGPVVAGVVGAKMPRYCLFGDTVNTSSRMESHS-PI-----------------------------------------GRIHCSENAKKCAEST--GRFEFEPRGRVQIKGKGEMNTYFLLR 174
G20390+CBG20392 FTDIVTFTNICALCTPYDV---------VTLLNDLYLRFDRLVGLHDAYKV------ETIGDAYMIV-GVPEHCE--NHAERVLNISIGMLMESKLVLSP--ITHKPIKIRLGVHCGPVVAGVVGIKMPRYCLFGDTVNVANKMESNG-IQ-----------------------------------------CKIHVSESAKLNGLKAN-PSYVFIDRGNTEIRGKGMMYTFFLER 173
GCY-35 LFTDIVTFTNICAMCTPYDV---------VTLLNDLYLRFDRLVGLHDAYKV------ETIGDAYMIVGGVPERCE--NHAERVLNISIGMLMESKLVLSP--ITHKPIKIRLGVHCGPVVAGVVGIKMPRYCLFGDTVNVANKMESNG-IQ-----------------------------------------CKIHVSETGKLNGLKAN-PSYVFIDRGNTEIRGKGMMYTYFLER 175
CBP17959 IFTDIPDFFTISVNCSPKEI---------ITLVTDLFHRFDRIIEKHKGYKV------LSLMDSYLIVGGVPNANQ--YHCEDSLNLALGLLFEARQVEVP--KLGQYVRLRIGVHCGPVVAGIVSQQKPRFCVLGNTVNITKNICAHS-QP-----------------------------------------GKALVSNAVRTMVTKHLKSIFVFNANGYLELAT-GKVLTHFLEK 175
GCY-37 IFTDIPDFFTISVNCSPTEI---------ITVVTDLFHRFDRIIEKHKGYKV------LSLMDSYLIVGGVPNANQ--YHCEDSLNLALGLLFEAKQVVVP--KLERSVRLRIGVHCGPVVAGIVSQQKPRFCVLGNTVNVTKSICSHS-SP-----------------------------------------GKVLVSNAVRTMVTKHLKSIFVFNANGYLELQS-GKVLTHFLEK 175
CBP05524 GFIRVCDFAKISLFIEAFEV---------VNLLNTIYSHLDEIVDTHGVYKV------ETIGESYMISAGCPYRDD-H-DAEMVADCCLEMVAHIRSFEYQSHDAVKKVLIKCGIFTGPVVGGVVGVRTPRYCLFGDTVNTASRMESSNQTA-----------------------------------------MTIQVGQRTKDRVEKQASGSFRIKPKGNIFVKGKGDMRVYEIEK 179
GCY-33 GFIRVCDFSKISLFIEAFEV---------VNLLNTIYSHLDSIVDTHGVYKV------ETIGESYMISAGCPYRDD-Y-DAEMVSDCCLEMVSHIKSFEYQSHDAVKKVLIKCGIFTGPVVGGVVGVRTPRYCLFGDTVNTASRMESSNQTP-----------------------------------------MTIQIGQRTKDRVEKQASGAFRIKPKGNVFVKGKGDMRVYEIEK 179
CBP21055 LFTDIVEFTKMCSSLTPLEV---------IEFLKVIYTNFDKIIDDHGVYKV------ETIGDAYMVVSGAPTKTE-H-DAEFILDCASQFLIEAAKMVNM-NAKVPRIDIRAGVHSGAVVAGVVGLSMPRYCLFGETVYVANKMEQSS-SP-----------------------------------------MKILVSETTHNKIEESDPGLYQFEMRDEIEIKDDQTIQTFFVVS 177
GCY-31 LFTDIVEFTKMCSSLTPLEV---------IEFLKVIYTNFDKIIDTHGVYKV------ETIGDAYMVVSGAPTKTE-H-DAEFILDCASQFLVEAGKMVNM-NNKIHKIDIRAGVHSGSVVAGVVGLSMPRYCLFGETVYVANKMEQNS-SP-----------------------------------------MKILVSETTHNKIEESDPGLYQFERREEIEIKDDQTIQTFFVVS 177
ruler 1.......10........20........30........40........50........60........70........80........90.......100.......110.......120.......130.......140.......150.......160.......170.......180.......190.......200.......210.......220.......230.......
GCY-8 ---KSM-L---------------------------------------------SLGST---R-TK------------------------------------------------------------------------------------------------------MSNMNYG------SRN-H-AIVGTNTH-----AIYHKY----VQRRPIVFNRADKTLI-----QLAKNFS----------------------------------------------------------------------------QNP--KEAGLPNYFKHLFYDRNHIQMKA-AVHD-NINPFLGMVWNE--R-----------EEMLLVW 110
CBP00094 ---RRVS----------------------------------------------TVSTA---R-AS------------------------------------------------------------------------------------------------------YSSIF--SA---NVSE-H--AHVNKQK-----VSIKRY----VQRRPITFTRQEMEML---------------------------------------------------------------------------------------------------------------NQLKY-MSHT-NINPFTGICFNQ--G-----------SELIVMW 82
GCY-25 ---RRVS----------------------------------------------TISTA---R-AS------------------------------------------------------------------------------------------------------YSSIF--SG---NVAE-H--AIVNKQK-----VSVKRH----VQRRAITFSRQEMEML---------------------------------------------------------------------------------------------------------------NQLKY-MSHT-NINPFTGICFNQ--G-----------SELIVMW 82
CBP15915* ---RELE----------------------------------------------NRTNK---D-EN------------------------------------------------------------------------------------------------------T--LISRRR---VFGS-Y--ALVGTQR-----AEYLQF----KQDKKLSLSEATLDYL---------------------------------------------------------------------------------------------------------------YSLKQ-LQND-NLAKFFGIQCND--DN---------MPTLTVLHTLV 87
GCY-10 ---RELE----------------------------------------------NRASN---T-DN------------------------------------------------------------------------------------------------------AAALTSRRR---VFGS-Y--ALVGTQR-----AEYVQF----KQIRKINFPETTLDYL---------------------------------------------------------------------------------------------------------------YSLKQ-LQHD-NLAKFYGIQVND--D----------IMTMTILH 85
GCY-27 ---RELE----------------------------------------------NRTAK---D-EA------------------------------------------------------------------------------------------------------N-ALTSRRR---VFGS-Y--ALIGTQR-----AEFIQF----RQIKHIDITNASLDFL---------------------------------------------------------------------------------------------------------------YNLKQ-LKHD-NLANFYGIQLND--D----------LNTMTILH 84
CBP11205* ---RELE----------------------------------------------NKNSK---E-EN--------------------------------------------------------------------------------------------------------PIISKRR---VFGS-Y--ALVGTQR-----AEFLQF----KQFTMIHLSPDVIDYL---------------------------------------------------------------------------------------------------------------YTLKQ-LQND-NLAKFLGIQCND--DT---------MPTLTVLH 84
CBP20608 ---MSVQ----------------------------------------------SLKEK---M-AE------------------------------------------------------------------------------------------------------RAHLKSI------ART-KLIATVDQTY-----VLVDKF----VVRDKIRYEKSDINLL---------------------------------------------------------------------------------------------------------------YQMKSQVQHD-NLNQFVGLSLDK--S-----------SHIYVIW 84
DAF-11 ---MSIQ----------------------------------------------SLQRN---M-EE------------------------------------------------------------------------------------------------------KQRLQSL------ART-KHIATVDQVY-----VLADKY----VMRDKLRYDKIDINLL---------------------------------------------------------------------------------------------------------------YQMKSHLQHD-NLNSFVGITIDK--A-----------SHMYIIW 84
CBP04125 ---KSM-L---------------------------------------------SLGST---R-TK------------------------------------------------------------------------------------------------------MSNMNYG------SRN-H-AIVGTNTH-----AIYHKY----VQRRPIVFNRADKTLI-----QLKDSFK----------------------------------------------------------------------------FAL--NLLKFSKQLLKSRYCYDCSKMNA-AVHD-NINPFLGMVWNE--R-----------EEMLLVW 110
CBP13439 ---RSM-M---------------------------------------------SVGSV---T-TK------------------------------------------------------------------------------------------------------LSNMQTG------QKQ-H-AIIGVNTH-----STYHKY----PQRRPIKFVKEDMQLL---------------------------------------------------------------------------------------------------------------TQMKQ-AVHD-NLNPFLGVAFNE--K-----------EEMLVLW 82
GCY-18/26 ---KSM-M---------------------------------------------SVGSV---T-TK------------------------------------------------------------------------------------------------------LSNIQTG------QKQ-H-AIIGVNTH-----TTYHRY----KQRRPIKFIKEDMQLL---------------------------------------------------------------------------------------------------------------TQMKQ-AVHD-NLNPFLGAAFNE--K-----------EEMLVLW 82
CBP14192 ---KSM-L---------------------------------------------SIGSS---K-TK------------------------------------------------------------------------------------------------------MSNMSMF------VKH-H-AVVGTNTH-----ASFHLY----PQRRPIVFNRQDLQLL---------------------------------------------------------------------------------------------------------------NQMKQ-AVHD-NLNPFLGMSFNE--K-----------EEMVVLW 82
GCY-23 ---KSM-L---------------------------------------------SIGSS---K-TK------------------------------------------------------------------------------------------------------MSNMSMF------VKH-H-AVVGTNTH-----ASFHLY----PQRRPIVFNRQDLQLL---------------------------------------------------------------------------------------------------------------NQMKQ-AVHD-NLNPFLGMSFNE--K-----------EEMVVLW 82
GCY-29 ---KSV-V---------------------------------------------SLNSA---S-TK------------------------------------------------------------------------------------------------------MSQVETK-----LIKN-H-AIVGVNTH-----AVYDLY----EQRQNIRFTREDLILL---------------------------------------------------------------------------------------------------------------TKMKQ-AVHD-NINPFIGVSFNE--K-----------SELLLLW 83
CBP04902* ---KSV-I---------------------------------------------SLGSA---T-TK------------------------------------------------------------------------------------------------------MSQVETK-----LVKN-H-AIVGVNTH-----AIYDIF----EQRQNIKFKREDLILL---------------------------------------------------------------------------------------------------------------TKMKQ-AVHD-NINPFIGVSFNE--K-----------SELLLLW 83
CBP04194 ---ASKSQ---------------------------------------------SSGSF---S-GS------------------------------------------------------------------------------------------------------MNSKQNG-----LIAA-K-QAVSNGVK-----LAIKRY----QQVRNITFPKSELKLL---------------------------------------------------------------------------------------------------------------KELKI-LEND-NLNKFYGISFNQ--Q-----------NEFIVMW 84
GCY-9 ---ASKSQ---------------------------------------------SSGSF---S-GS------------------------------------------------------------------------------------------------------MNSKQNG-----LIAA-K-QAVSNGVK-----LAIKRY----QQVRNITFPKSELRLL---------------------------------------------------------------------------------------------------------------KELKI-CEND-NLNKFYGISFNQ--Q-----------NEFIVMW 84
CBP00282 ---RSLQ----------------------------------------------SAQT-------G------------------------------------------------------------------------------------------------------ESKTTAVSD---FCEN-Y-TIMIYEKDM----VLTMK------YQYM-NLTKSDMEK---------------------------------------------------------------------------------------------------------------FVKLRK-LEHE-NLNKFIGLSID---G-----------SQFISVT 79
CBP04086 ---RSIQ----------------------------------------------SGPSN---I-TD------------------------------------------------------------------------------------------------------TSKMTFDNTF----SN-Y-SIFYLDKEP----VLSTA------HPAS-NLDRTDYDT---------------------------------------------------------------------------------------------------------------FVKLRK-LDHD-NINKFVGLSID---G-----------AEYLAVW 81
GCY-19 ---RSLQ----------------------------------------------SGPSN---I-TD------------------------------------------------------------------------------------------------------TSKMTFDNTF----SN-Y-SIFYLDKEP----VLSTA------HPAS-NLIRTDYDT---------------------------------------------------------------------------------------------------------------FVRLRK-LDHE-NVNKFVGMSID---G-----------PEYLAVW 81
CBP13905 ---RSIN----------------------------------------------SGPST---V-TG------------------------------------------------------------------------------------------------------DSKMTGEGLY-GFHEN-Y-TVLIYEKDL----VLTTR------HHGI-SLNSMEKEK---------------------------------------------------------------------------------------------------------------YAKLRK-LDHD-NLNKFIGLSID---G-----------PLWISVW 84
GCY-5 ---RSLQ----------------------------------------------SGPST---I-TG------------------------------------------------------------------------------------------------------ESKMTIDGG---FHEN-Y-TVQMFEKDL----VLTTK------HHSM-QMNKEEKEK---------------------------------------------------------------------------------------------------------------FVKLRK-LEHD-NLNKFIGLSID---G-----------PQFVAVW 82
GCY-1 ---RSLQ----------------------------------------------SAPSI---S-TG------------------------------------------------------------------------------------------------------HSGVTTVSD---FCEN-Y-TMMMYEKEM----VLTAK------YQYT-HLTKADKER---------------------------------------------------------------------------------------------------------------FVKMRK-LDHE-NINRFIGLSID---S-----------AHFISVT 82
GCY-2 ---RSLQ----------------------------------------------SAPSI---S-TG------------------------------------------------------------------------------------------------------HSGVTTVSD---FCEN-Y-TMMMYEKEM----VLTAK------YQYT-HLTKADKER---------------------------------------------------------------------------------------------------------------FVKMRK-LDHE-NINRFIGLSID---S-----------AHFIAVT 82
GCY-3 ---RSLQ----------------------------------------------SAPST---I-TG------------------------------------------------------------------------------------------------------ESKVSTGSE---FCEN-Y-EVKMFEKDM----VLTMK------FQYM-NLNKADMDK---------------------------------------------------------------------------------------------------------------FVKLRK-LDHE-NLNKFIGLSID---S-----------SQFISVT 82
CBP13906 ---DSEN----------------------------------------------STVTKSSK--GS------------------------------------------------------------------------------------------------------SSKNFETSE---FNEN-Y-EIQFLENDL----VLTTA------HQVQ-ELSNLDKMR---------------------------------------------------------------------------------------------------------------LVKLRK-LDHE-NLNKFIGLSID---G-----------SRYLAVW 84
GCY-4 ---ESET----------------------------------------------ESASETENF-TS------------------------------------------------------------------------------------------------------KSGDTMTSE---FKET-Y-TIQYLENDL----VLTTA------HQVQ-ELSQAEMMK---------------------------------------------------------------------------------------------------------------FVKLRK-LDHE-NLNKFIGLSID---G-----------SRFVSVW 85
CBP01237 ---HSFA----------------------------------------------SGPST---S-TK------------------------------------------------------------------------------------------------------IT-IESRS----ETTN-F-IFYYHKQEI----VAAMK------HDLILLFDTNEKSE---------------------------------------------------------------------------------------------------------------FRQMRN-FDND-NLNKFIGLCLD---G-----------PQLLSLW 81
GCY-16/20 ---HSFA----------------------------------------------SGPST---S-TK------------------------------------------------------------------------------------------------------MT-VESRT----ETTN-F-IFYHYHQEV----VAAKK------HDLLVLFDANQKSE---------------------------------------------------------------------------------------------------------------FRQMRN-FDND-NLNKFIGLCLD---G-----------PQLLSLW 81
GCY-14 ---HSFA----------------------------------------------SGPST---S-TK------------------------------------------------------------------------------------------------------IT-VESRS----ETIN-F-IFYYYQQDI----LAAMK------HDLILQFDAEQKAE---------------------------------------------------------------------------------------------------------------FRQMRN-FDND-NLNKFIGLCLD---G-----------PQLFSLW 81
CBP15625 ---HSFA----------------------------------------------SGPST---A-TK------------------------------------------------------------------------------------------------------IT-VESRS----ETTN-F-IFYFYKQEI----LAGMK------HELMQIFDQDQKSE---------------------------------------------------------------------------------------------------------------FRQIRN-FDND-NLNKFIGLCLD---G-----------PQLLSLW 81
CBP14936 ---RSFA----------------------------------------------SGPST---S-TK------------------------------------------------------------------------------------------------------LT-LESRT----ETNR-F-SFYLYKNEM----VAANK------HNARPSLTEQERYQ---------------------------------------------------------------------------------------------------------------LRQMRS-LDHD-NINKFIGLCLN---G-----------PQLMSVY 81
GCY-17/24 ---RSFA----------------------------------------------SGPST---S-TK------------------------------------------------------------------------------------------------------LT-VESRT----ETTR-F-IFYIYQNEV----VAANK------HDFRPQLTDVERSEL-----RQVWPRDEMVTSHLFHYARRKVTSHVRHFVIFWHFHSKKLTTRGRRDFRACLKPARLTPAFRLFSTFLCFVEAGGGLLKVRQIGVL--TPTSMPYLKPKLSYFSSMHHMRS-LDHD-NLNKFIGLCLN---S-----------QQLLSIW 185
CBP02750 ---RSLQ----------------------------------------------SGTSTM--S-SR------------------------------------------------------------------------------------------------------TT-VSFKT----ETRN-F-LFFSLQREADYEPVVAKK------HAYRPRLDDERCAF---------------------------------------------------------------------------------------------------------------MRTLRT-LDHD-NLNRFIGLCLD---G-----------PQMLSVW 86
GCY-7 ---RSLQ----------------------------------------------SGTSTL--S-SR------------------------------------------------------------------------------------------------------TT-VSFKT----ESRN-F-LFFSLQRESDYEPVVAKK------HAYRPRLDDDKCTF---------------------------------------------------------------------------------------------------------------MRSLRN-LDQD-NLNRFIGLCLD---G-----------PQMLSVW 86
CBP16612 ---RSIS----------------------------------------------SVPSTI--S-STRS----------------------------------------------------------------------------------------------------ST-LSEVG----ETRN-Y-LFFQIQNEVEIEKVAARK------YTIRTLFDNKICAN---------------------------------------------------------------------------------------------------------------MRQMRL-IDHS-NLNKFIGMSLD---A-----------PQLLSVW 88
GCY-6 ---RSIS----------------------------------------------SVPSTI--S-STRS----------------------------------------------------------------------------------------------------ST-LSEVG----ETRN-Y-LFFQIQNDVEMERVAAKK------HSIRMVFDNKTCATM-----RQ-------------------------------------------------------------------------------------------------------VVEMRL-IDHA-NLNKFIGMSLD---A-----------PQLYSVW 91
CBP08562 ----SWK----------------------------------------------NAPCDC--V-PANF----------------------------------------------------------------------------------------------------AG-LIYYD----DPAY-YPASFVAQNEINRQKCI---------------------------------------------------------------------------------------------------------------------------------------------------------------A-----------SVYANPSI 51
CBP01427 ---RSLQ----------------------------------------------STVTT---S-TK------------------------------------------------------------------------------------------------------VT-INSKK----DTER-H-SFYFLNNDS----VVARK------HNFRAVFTKNDRAM---------------------------------------------------------------------------------------------------------------FRKMRN-VDHD-NLCKFIGLSLD---S-----------PTLISVW 81
GCY-22 ---RSLQ----------------------------------------------STVTT---S-TK------------------------------------------------------------------------------------------------------VT-INSKK----DTER-H-SFYFLNNDS----VVARK------HNFRATFTKNDRAM---------------------------------------------------------------------------------------------------------------FRKMRN-VDND-NLCKFIGLSLD---S-----------PTLISIW 81
CBP05559 ---RSLQ----------------------------------------------SNSTT---T-TG------------------------------------------------------------------------------------------------------TTGISSRSVFFPETES-Q-GFFIYMNEP----VLARK------YQLRVPILKADRAE---------------------------------------------------------------------------------------------------------------LRMLRS-IEHD-NVNRFIGLSID---G-----------PVYMSFW 86
GCY-13 ---RSLQ----------------------------------------------SNSTT---T-TG------------------------------------------------------------------------------------------------------TTGISSRSVFFPETET-Q-GYFVYMNEP----VLARK------YQLRVPIFKQDRSE---------------------------------------------------------------------------------------------------------------LRMLRS-IEHD-NVNRFIGLSID---G-----------PVYMSFW 86
CBP00662 ---ESTA----------------------------------------------SLDHVKLKK--------------DQRKPPPPWWSKDAIYGSG-----------------------------------------------------------------------------------------MRGLASYKGTL----VGLKDFLYN---RKQKEITREAKKE---------------------------------------------------------------------------------------------------------------LRAMRQ-LAHP-NVNNFLGIIPN---S-----------YKITIVR 94
GCY-12 ---ESTA----------------------------------------------SLMQSDVMQ---------------FAKTKTPWWSKAPVQGTG-----------------------------------------------------------------------------------------MRGLASYKGTL----VGLKDLMYG---RKPKDLTREAKKE---------------------------------------------------------------------------------------------------------------LRAMRQ-LAHP-NVNNFLGIIVC---Q-----------YSVTVVR 93
CBP03512 ---QKK-EKKKAKKRKNHDGYLPESDPLLRSTSRSSVNSDK-------------RSSSSGTT-RKISAMIDRKLSIFTRKKSTPPSDKEQKNGGLTPNSLQKAENGDCSPINEVQFRLPLNDRRVSSPSSDA-RKKNSNEEDPDNGAKKSLSLKNRKLSFGMVSFKSGSGGSVETIAQNNTQI-YTKTAIFKGVV----VAIKKLNIDPKKYPRLDLSRAQLME--------------------------------------------------------------------------------------------------------------LKKMKD-LQHD-HITRFTGACID---F-----------PHYCVVTE 230
GCY-28 ---QKKNEKKKAKKRKNHNDYLPESDPLLRSTSRSSVNSDKFDEDSLIPIRFRLRSSSSGTT-RKISAMIDRKLSIFTRKKSTPPSES-QKNGGLTPNSLQKAENGDCSPINEVQFRLPLNDRRVSSPSSEATRKKNSNEEDPENGAKKSLSLKNRKLSFGMVSFKSGSGGSVETIAQNNTQI-YTKTAIFKGVV----VAIKKLNIDPKKYPRLDLSRAQLME--------------------------------------------------------------------------------------------------------------LKKMKD-LQHD-HITRFTGACID---F-----------PHYCVVTE 244
CBP04548 ---ASLR----------------------------------------------SMASIHGSF-AAASQTLRDSHFFIEDYGNHPP-------------------------------QDSSQMSSIFHTGSTA-----------------------RANPFGQFPGFGVSPGNSEDEKWHQLKE-FG-VGLFEGRM----IGLKRIY-----RTDVELTRNVRLE---------------------------------------------------------------------------------------------------------------IAQLLE-ATNS-NTLEFVGMVIH---S-----------PDVFLVH 138
GCY-15 ---QSLR----------------------------------------------SMASIHGSV-VAASQTLRDSHFFIEDYNN----------------------------------ASSINASSIFNTGSTA-----------------------RAGPFGPIPGFGGVTGASEDEKWHQIPD-FG-VGLYEGRT----VALKRIY-----RSDVEFTRSNRLE---------------------------------------------------------------------------------------------------------------IAKLQE-SVNS-NVIEFVGMVVQ---S-----------PDVFVVY 135
GCY-21 ---QSLR----------------------------------------------SMASIHGSV-VAASQTLRDSHFFIEDYNN----------------------------------ASSINASSIFNTGSTA-----------------------RAGPFGPIPGFGGVTGASEDEKWHQIPD-FG-VGLYEGRT----VALKRIY-----RSDVEFTRSIRLE---------------------------------------------------------------------------------------------------------------IAKLQE-SVNS-NVIEFVGMVVQ---S-----------PDVFVVY 135
CBP18607 ---TKFG----------------------------------------------S--SRAGSI-MQSAISMRSQY----DDDP-AP-------------------------------AGGATMN-FYTNGIRK-----------------------AA-RRNQMARDNRALGNNKDD-WLEIMD-WH-LAKYDNTL----VTVRKIN-----KSQLKLTREMKQE---------------------------------------------------------------------------------------------------------------IDLLMN-ETHE-NLNRFFGLINE---S-----------DLIFTIH 128
GCY-11 ---TKFG----------------------------------------------S--SRGGSM-IQSAMSMRSQY----EDS--GP-------------------------------VGGATMN-FYTNGIRK-----------------------AAIRRNQMQRSDKGPAANRDE-WLEIVD-WH-LAKYENTL----VTVRKIN-----KTQLKLTREMKQE---------------------------------------------------------------------------------------------------------------IDLLMN-ETHE-NLNRFFGLINE---S-----------DLIFTIH 128
CBP04780 ---VHMA-------------------------------------------------KFELGTESEIAHYYND------------------------------------------------------------------------------------DETLRLVNSMANVIGIPIEE-IWEAYG-GFLIQFTMETGW--------------DELLRAMAPDLEGFL--------------------------------------------------------------------------------------------------------------------DSLD-SLHYFIDHVVYKTKLRGPSFRCDVQADGTLLLHY 111
FUSED_DROME/4-254 YAVSSL-V---------------------------------------------GQGSF---G-CV------------------------------------------------------------------------------------------------------YKATRKD------DSKVV-AI---------------KV----ISKRGRATKELKNLRR-----ECDIQAR----------------------------------------------------------------------------L-K---HPHVIEMIESFESKTDLFVVTE-FALM-DLHR-----YLS--Y-----------NGA---- 93
AKT_MLVAT/171-429 FEYLKL-L---------------------------------------------GKGTF---G-KV------------------------------------------------------------------------------------------------------ILVKEKA------TGRYY-AM---------------KI----LKKEVIVAKDEVAHTLT----ENRVLQN----------------------------------------------------------------------------S-R---HPFLTALKYSFQTHDRLCFVME-YANGGELFF-----HLS--R-----------ERV---- 95
KIN82_YEAST/324-602 FEKIRL-L---------------------------------------------GQGDV---G-KV------------------------------------------------------------------------------------------------------YLMRERD------TNQIF-AL---------------KV----LNKHEMIKRKKIKRVLT----EQEILAT----------------------------------------------------------------------------S-D---HPFIVTLYHSFQTKDYLYLCME-YCMGGEFFR-----ALQTRK-----------SKC---- 97
PKD1_DICDI/36-291 FNFYGS-L---------------------------------------------GSGSF---G-TA------------------------------------------------------------------------------------------------------KLCRHRG------SGLFF-CS---------------KT----LRRETIVHEKHKEHVNN----EINIMLN----------------------------------------------------------------------------I-S---HPYIVKTYSTFNTPTKIHFIME-YAGKKDLFH-----HLR--A-----------NKC---- 95
KGP1_DROME/457-717 LEVVST-L---------------------------------------------GIGGF---G-RVEL----------------------------------------------------------------------------------------------------VKAHHQD------RVDIF-AL---------------KC----LKKRHIVDTKQEEHIFS----ERHIMLS----------------------------------------------------------------------------S-R---SPFICRLYRTFRDEKYVYMLLE-ACMGGEIWT-----MLR--D-----------RGS---- 97
ARBK1_BOVIN/191-453 FSVHRI-I---------------------------------------------GRGGF---G-EV------------------------------------------------------------------------------------------------------YGCRKAD------TGKMY-AM---------------KC----LDKKRIKMKQGETLAL-----NERIMLS----------------------------------------------------------------------------LVSTGDCPFIVCMSYAFHTPDKLSFILD-LMNGGDLHY-----HLS--Q-----------HGV---- 98
CHK1_SCHPO/10-272 YHIGRE-I---------------------------------------------GTGAF---A-SV------------------------------------------------------------------------------------------------------RLCYDDN------AKI-Y-AV---------------KF----VNKKHATSCMNAGVWARRMASEIQLHKL----------------------------------------------------------------------------CNG---HKNIIHFYNTAENPQWRWVVLE-FAQGGDLFD-----KIE--P-----------DVG---- 99
CDC5_YEAST/82-337 YHRGHF-L---------------------------------------------GEGGF---A-RC------------------------------------------------------------------------------------------------------FQIKDDS------GEI-F-AA---------------KT----VAKASIKSEKTRKKLLS----EIQIHKS----------------------------------------------------------------------------M-S---HPNIVQFIDCFEDDSNVYILLE-ICPNGSLME-----LLK--R-----------RKV---- 94
TTK_HUMAN/509-775 YSILKQ-I---------------------------------------------GSGGS---S-KV------------------------------------------------------------------------------------------------------FQVLNEK------KQI-Y-AI---------------KY----VNLEEADNQTLDSYRN-----EIAYLNK----------------------------------------------------------------------------LQQ--HSDKIIRLYDYEITDQYIYMVME-CGNI-DLNS-----WLK--K-----------KKS---- 94
ruler 1.......10........20........30........40........50........60........70........80........90.......100.......110.......120.......130.......140.......150.......160.......170.......180.......190.......200.......210.......220.......230.......240.......250.......260.......270.......280.......290.......300.......310.......320.......330.......340.......350.......360.......370.......380
GCY-8 KFCSRGTLQDIIYNESIQLDT--KFHGAFIRDIL-------------------------------------AGL----EYLHA-SQIGYHGS-----------------------------------LTPWSCLIDR-NWMIKLTDYGIADPLERW-------------EK------SQSI-SRD----GLTSDD--DKSQAT------QATSILYESPEMLKNREKNRVRRVDQDWMRQTQTRRQLGDVYAF------GLVMYEIIFRALPFPE----------------G-------T-NQ-SELVEWL----RDGSKVVKP---TIPQNK------VLNMDLSALIQDCWNTTPEMRPS-----LRRIKLNVE- 325
CBP00094 QFTARYSLEDLIFGKEQKFGR--NFQSTFIKHIM-------------------------------------HGI----NFIHN-SSIKVHGT-----------------------------------LYLSNCVVDS-YWVVKLTDFGIKNILKER-------------MH--HKELAPNS-SF---------------DIDA------IHYKYLQLAPEHLSSLLEKHE--EP--------MGTVEGDIYQL------AMVIYQILFYMKPFAE-RQEDIKAVKK----LF-------FL----ELATLLSN---QSTAPLHP---KVPEGN------SFTMRLLSIIQQCWLYKPAARPT-----LIKITDAVN- 292
GCY-25 QFTTRYSLEDLIFVKEQKFGR--NFQSTFIKHIV-------------------------------------HGI----NYIHN-SSIKVHGA-----------------------------------LYLSNCVVDS-YWVVKLTDFGIKGILKER-------------TN--HKELAPSS-AF---------------DVDA------IHYKYLQLAPEHISAILEKLE--EP--------RGTVEGDIYQL------AMCIYQILFYMRPFAE-RQESIKVYSG----EI-------FLKL-PELAHLLSS---QSTAPLHP---KVPEGN------SFTMRLLSIIQQCWLYKPAARPA-----LIKITDAVN- 295
CBP15915* ERGTLEEFCLDRDFEMNE--TFKSAFMRDML-------------------------------------KGL----NYLHK-GPIAHHGF-----------------------------------LQASTCLIDI-NWVLKLTLYGVSNFICDA-------------FD--STNVKLLD-HA---------------APMI------SYPQYVCFPPEHIKEYDPTGK--LPPRIV----RGSQKGDMYCV------GMILYMMIEREDPFML-IHSLERPNPN-----------------------LVKD---IIDNGKMP---RVADEH------NPEAKLLQKCQECWNRNPEERPD-----VRKILETIA- 290
GCY-10 TLVERGTLEEFCLDRDFGMDD--TFKSAFMRDIL-------------------------------------KGL----QYLHK-SSIGYHGH-----------------------------------LQASTCLIDI-NWVLKLTLYGVSNFMSDQ-------------LD--AENIKVPE-QA---------------AHMI------TYPQYVCFPPEHIREYDDSGK--QPPRVV----RGSPKGDIYCV------GMIFYMMVEREDPYHL-IHSVERPNAT-----------------------LIKQ---ILNENHMP---RITDDY------RQENMLLEMCKECWDRNPDKRPT-----IKKLIESIS- 291
GCY-27 ALVERGTLEEFCLDRDFGMDE--TFKSAFMRDIL-------------------------------------KGL----QYLHL-SPVAYHGH-----------------------------------LHAATCLIDI-NWVLKIALYGVTNFVCDN-------------FD--AENITMPD-RS---------------DYTI------SYAQYVCFPPEHIREYDATGK--LPTRFV----RGSKQGDIYCV------GMIFYMMIEREDPYRL-IHSVERPGSG-----------------------LMME---ILDHNLMP---FISNNE------TQEDTLLDKCKECWNRDPEKRPT-----IENLRNAIA- 290
CBP11205* TLVERGTLEEFCLNQDFSMNE--TFKSAFMRDML-------------------------------------RGL----NYLHK-GPIGYHGF-----------------------------------LQAATCLIDI-NWVLKLSLYGVTNFICDA-------------LD--NKNIQAPD---------------------------------------FL---DRTGE--LPSIMV----RGSQKGDMYSV------GMILYMMIERDDPYMF-SHELGRPDQR-----------------------LVKD---IIVNNKLP---RIAEEH------NSEALLLEKCKECWSRKPEERPD-----VRDIMDTIS- 270
CBP20608 NQCFRGSLYDHIFTKERSRDTATNFEGAFLRDIL-------------------------------------KGL----DYLHN-SSLEYHGN-----------------------------------LTLHNCLLDS-HWIVKISGFGVNRLLVKW-------------KA------SGQI-FTEDHTPVLKSEG--IEIIEL------KVEEIHYFDPLMKAVWKSMNGTKQDKGMISGDMG--RRGDMYSF------GVILYEICFKKKMVPPLFD-------------Y-------PKGE-DESVLIDDE--NDAIASKYPLPLDLPPNH------DMHNDLIKLLENCFGT---NRPD-----VKLARKIID- 309
DAF-11 NQCFRGSLHDHIFTKERQRGTATRFEGLFLRDIL-------------------------------------KGL----EYIHA-SAIDFHGN-----------------------------------LTLHNCMLDS-HWIVKLSGFGVNRLLVKW-------------KT------SGQI-FTEDHTPVIK------------------SEELHYFDPAMKKIWKNY-ADRNERALITPQFG--KKCDMYSF------GVILHEIILKKKFVEQLFD-------------S-------PREE-DDSVLIDDE--NDAIASRFPLPIIIPEGI------EMHNDLIKMLENCFGS---VRPD-----IALARKIID- 298
CBP04125 KFCSRGTLQDIIYNDNITLDT--KFHGAFIRDIL-------------------------------------AGC----EYLHA-SQIGYHGS-----------------------------------LTPWSCLIDR-NWMIKLTDYGIADPLERW-------------EK------QQSI-SRD----ALTSDD--DKSQAT------QSTSILYEAPEMLKNREKNRTRRVDQEWIRQTQTRRQLGDIYAF------GLVMYEIIFRSLPFPE----------------G-------T-NQ-MELVEWL----RDGTKVVKP---TIAQNK------VLNMDLTALIQDCWNTTPEMRPS-----LRRIKLNVE- 325
CBP13439 KFCSRGTIQDIIYNMNVVLDE--KFHGAFVRDITLGRKPLTREMSMKLGDITGTYICIKWEMEEKPKNGGSQGL----EYLHA-SPIGYHGS-----------------------------------LTPWCCLIDR-NWMVKLSDYGIANPLERW-------------EK------QGAI-SIN----VAKDSD--DKSEAA------QTTSILYMAPELLKNRETNKRRGVDQTWVKQSMARRQAGDIYSF------GMVMYEILFRSLPFRD----------------N-------V-NV-TELIAYL----ADGSKTVTP---EIQNQM------GLHPDLNALLRDCWSENPEIRPS-----IRRVRLNTE- 334
GCY-18/26 KFCSRGTIQDIIYNANVVLDE--KFHGAFVRDIT-------------------------------------LGL----EYLHA-SPIGYHGS-----------------------------------LTPWCCLIDR-NWMVKLSDYGIANPLERW-------------EK------QGAI-EIA----AAKDSD--DKSQAS------QATSIIYMAPELLKNRETNKRRGMDQSWVKQSMLRRQAGDIYSF------GMVMYEILFRSLPFRD----------------N-------T-NI-SELVDYL----ADGSKTVSP---EIQNQM------GLHPDLNALLRDCWSENPEIRPS-----IRRVRLNTE- 297
CBP14192 KFCSRGTIQDMIYNQEVVLDA--KFHGAFIRDIT-------------------------------------LGL----EYLHS-SIIGYHGS-----------------------------------LTPWSCLIDR-NWMIKLTDFGIANPLERW-------------EK------MGLI-STE----TLKEGD--DKSGSA------QKTSILYQPPEMLKNRESNRLRRMDQSWVKQSQARRQMGDIYAF------GMVMHEILFRALPFPN----------------G-------T-NI-SEVLDYI----RDGTRNFRP---TIHDRT------QIHPDLVALLLDCWNENPEVRPS-----IRRVRLNTE- 297
GCY-23 KFCSRGTIQDMIYNQEVSLDS--KFHGAFIRDIT-------------------------------------LGL----EYLHS-SIIGYHGS-----------------------------------LTPWSCLIDR-NWMIKLTDYGIANPLERW-------------EK------LGLI-STE----TLKEGD--DKSGSA------QKTSLLYQPPEMLKNKESNRTRRMDQSWVKQSQARRQMGDIYAF------GMVMHEILFRALPFPN----------------G-------T-NV-SEVMDYI----RDGTKTFRP---TVHDRT------QIHPDLVALLLDCWNENPEVRPS-----IRRVRLNTE- 297
GCY-29 KFCSRGTLQDVIYCEKFAMDE--KFQGAFVRDIT-------------------------------------MGL----EYLHS-SPIGYHGG-----------------------------------LACWSVLIDK-NWMLKLTDYAVCDPLKRW-------------EK------HGRI-NCK----V---DN--EAEKQW------QKMASLYVPPEIRTANEKNRLKRMDQKWQGQTILKRQQSDIYAF------GVIIYEILFRSLPYDE----------------K-------V-DL-TELAQKA----AEGDKIQKP---SIQRNK------KLNPDLIALLQDCWSDQPDMRPT-----IRRVRLATE- 295
CBP04902* KFCSRGTLQDVIYCDKFNMDE--KFHGAFVRDIT-------------------------------------LGL----EYLHS-SSIGFHGG-----------------------------------LASWTALIDK-NWMLKLTDYAIGDPLKRW-------------EK------HGRI-NCK----V---DN--ESEQEW------QKMASLYVPPEIRTANEKNRMKRMDQKWQAQSILRRQQSDIYAF------GVIIYEILFRSLPYDE----------------K-------V-DL-TELAQKA----SEGEKIQRP---SIQKNK------KLNPDMIALLQDCWSGQPDMRPT-----IRRVRLATE- 295
CBP04194 VLCSRGSLEDILFNDELKLGR--NFQVSFAKDVV-------------------------------------KGL----NFLHT-SPLLHHGM-----------------------------------LCLQNCLVDS-NWTVKLTNFATEQIIFEK-------------LD--HNELRPFV-NAD----SESAEDVPDPTKDF------ARKKYLQQAPEIIREIVTTKV----------IPDGSQAADIYAL------GMVLYQILFRVEPFHE----------------R-------NKSI-NKLMEMLAMA-NDDDQLIRP---TFPSSNTG---EGYNLQLLSCIEACWLEIPEMRPP-----IKKVRTMVN- 302
GCY-9 VLCSRGSLEDILFNDELKLGR--NFQVSFAKDVV-------------------------------------KGL----NFLHT-SPLLHHGM-----------------------------------LCLQNCLVDS-NWTVKLTNFATEAVIFEK-------------LD--HNELRPFI-NTD----SESADDVSDPTKDF------ARKKYLQQAPEIIREIVTTKT----------IPEGSQSADIYAL------GMVLYQILFRVEPFHE----------------R-------NKSI-NKLMETLAMA-NDDDQLIRP---TFPSSNTG---EGYNLQLLSCIEACWLEIPEMRPP-----IKKVRTMVN- 302
CBP00282 KLCSRGSLQDILSRGNFSMDY--FFMFCIIKDVAE-------------------------------------GL----NYLHK-SFLRLHGN-----------------------------------LRSATCLVND-SWQVKLAEFGIENLVENE--------------IPPK-------------------------------------KRLLWVAPEVLRGSLTVS-------------QMDPSADVYSF------AIVASEILTKKEAWDF-LD-------------R-------KEDS-EEIVYMVKKG---GVFPIRP---ELITD-----IPDVNPDLTERLKAGQTVEPEGFDS-----VTVFFSDVV- 267
CBP04086 KMCMRGSLQDIIGQGNFSIDP--FFMFCVIRDMAE-------------------------------------GL----KYLHN-SFLHVHAN-----------------------------------LRSGTVLINE-SWQAKLTDFGLGNLAEEK--------------KPMK-------------------------------------RRQLWMAPEVIRGTLLPH-------------QVEKPADIYSL------AIIASEVLTRKEAWNM-SE-------------R-------KDTV-DEIVYRIKKG---GPNSIRP---DLDMD-----GVEINHSLLVLIRDCWSEDPTDRPG-----ADIICNLLK- 269
GCY-19 KLCMRGSLQDIIGQGNFSIDP--FFMFCVIRDMAE-------------------------------------GL----KYLHN-SFLHVHAN-----------------------------------LRSGTVLVNE-SWQAKLTDFGLGTLAEEK--------------KPMK-------------------------------------RRQLWMAPEVIRGTLLPH-------------QIEKSADIYSL------AVIASEVLTRKEAWNM-AE-------------R-------KDTV-DEIVYRIKKG---GPNAPRP---ELDMD-----GVEINHNLLILIRDCWSEEPADRPS-----ADVICNLLK- 269
CBP13905 KMCSRGSLQDIISRGNFSMDG--FFMFCIIRDIAE-------------------------------------GL----NFLHK-SFLRLHGN-----------------------------------LRSATCLVND-SWQVKLAEFGMDDLLEEH--------------TPTK-------------------------------------RRLLWAAPEVLRGSLTVS-------------QMDPSADVYSF------AIVASEILTKREAWDF-AN-------------R-------KEGA-DEILYMIKKG---GARAVRP---ELIPD------AEVSPSLCTLVKDCWSEVPEDRPK-----SEQICKLLF- 271
GCY-5 KMCSRGSLQDIIARGNFSMDG--FFMFCIITDIAE-------------------------------------GM----NFLHK-SFLHLHGN-----------------------------------LRSATCLVND-SWQVKLTDFGLGALLEEH--------------TPSK-------------------------------------KRLLWAAPEVLRGSLTIH-------------QMDPSADVYSF------AIIASEILTKREAWDI-SN-------------R-------KEGA-DEILYMVKKG---GNRTIRP---ELILD------AEVSPRLTTLVKDCWSEQPEDRPK-----AEQICKLLS- 269
GCY-1 KLCSRGSLQDILSRGNFSMDY--FFMFCIIRDVAK-------------------------------------GL----EYLHK-TFLRLHGN-----------------------------------LRSATCLVND-SWQVKLAEYGMDNLVEEQ--------------TPPK-------------------------------------KRLLWVAPEVLRGSLSVS-------------QMEPSADIYSF------AIIASEILTKKEAWDI-LD-------------R-------KEDC-EEIVYNVKKG---GLFPIRP---EIITD-----IHDVNPALIALVKDCWAEVPEDRPT-----AENICSQMK- 270
GCY-2 KLCSRGSLQDILSRGNFSMDY--FFMFCIIRDVAK-------------------------------------GL----EYLHK-TFLRLHGN-----------------------------------LRSATCLVND-SWQVKLAEYGMDNLVEEQ--------------TPPK-------------------------------------KRLLWVAPEVLRGSLSVS-------------QMEPSADIYSF------AIIASEILTKKEAWDI-LD-------------R-------KEDC-E--------------------------------------ALIALVKDCWAEVPEDRPT-----AENICSQMK- 243
GCY-3 KLCSRGSLLDILYKGNFSMDF--FFMYCIIKDVAE-------------------------------------GM----SYLHK-SFLRLHGN-----------------------------------LRSATCLVND-SWQVKLAEFGFDQLLEEI--------------TPTK-------------------------------------RRLLWAAPEVLRGSLTVS-------------QMDPSADVFSF------AIIASEILTRKEAWDL-KE-------------R-------KEGY-DEIIYRVKKG---GSFPIRP---DIITD-----VPDVNPTLIALVKDCWAEAPEDRPT-----AENICEQLR- 270
CBP13906 KMCTRGSIQDIMSRGNFSMDY--FFMFCMIRDIAE-------------------------------------GL----NFLHK-SFLRLHGN-----------------------------------LRSATCLVND-SWQVKLAEFGLEFLQDDE-------------ERPTQ-------------------------------------KRLLWAAPEVLRGSLTVS-------------QMDPSADVYSF------AIVASEILTKKEAWDL-HK-------------R-------KEGY-EEIIYNVKKG---GPHPFRP---TLLTD------SDVNKSLLALVKDCWSENPEARPN-----TENICKIIL- 272
GCY-4 KMCSRGSLQDIISKGSFSMDY--FFMFCMIRDIAE-------------------------------------GL----HYIHK-SFLRHHGN-----------------------------------LRSATCLVND-SWQVKLADFGLQFLQDEE-------------EKPFK-------------------------------------KNMLWAAPEVIRGSLSIE-------------QMDSSADIYSF------AIVASEILTKREAWDL-SR-------------R-------KE----EIKYAVKKG---GQFVLRP---DLHID------IEVNQTLLALVKDCWCENPEERPS-----AENVCKVLF- 270
CBP01237 RFCSRGSLSDVISKSSMQMDS--FFMFSLIRDISN-------------------------------------GL----SFIHN-SFLKYHGH-----------------------------------LTSRCCLIDD-RWQVKISGYGLKSVRTFE--------------NPKK-------------------------------------EDLLWTAPEHLRNESG---------------EKTSEGDIYSF------GIICSEILTRSSAFDL-EN-------------R-------KEKP-DVIIYQVKKG---GHNPMRP---SLETS----ETVEVNPALLHLIRDCWTERPSERPS-----IEQVRSHLN- 268
GCY-16/20 RFCSRGSLSDVISKSSMQMDS--FFMFSLIRDISN-------------------------------------GL----YFIHS-SFLKCHGQ-----------------------------------LTSRCCLIDD-RWQIKISGFGLKSVRTFE--------------NPKK-------------------------------------EDLLWASPEYLRNEDQ---------------ERLPEGDIYSF------GIICAEILTRSSAFDL-EN-------------R-------KEKP-DVIIYQVKKG---GHNPTRP---SLDTG----ETVVINPALLHLVRDCWTERPSERPS-----IEQVRSHLN- 268
GCY-14 RFCSRGSLSDVISKSSMQMDS--FFMFSLIRDISN-------------------------------------GL----LFIHN-SFLKCHGH-----------------------------------LTSRCCLIDD-RWQIKISGYGLKSVRTFE--------------NPKK-------------------------------------EDLLWTPPENLRNENE---------------ERLPEGDIYSF------GIICSEILTRSSAFDL-EN-------------R-------KEKP-DVIIYQVKKG---GHNPMRP---SLDTG----ETVEINPALLHLIRDCWTERPSERPS-----IEQVRGHLN- 268
CBP15625 RFCSRGSLSDVISKSSMQMDS--FFMFSLIRDISN-------------------------------------GL----SFIHN-SFLGFHGH-----------------------------------LTSRCCLIDD-RWQIKISGYGLKSIRSFE--------------KPNP-------------------------------------KDLLWTAPEHLRNENN---------------ERTSEGDIYSF------GIICSEILTRSSAFDM-EN-------------R-------KEKP-DVIIYQVKKG---GHNPMRP---SLETM----ESVEVNPALLHLIRDCWTERPSERPN-----IDQVRGHLN- 268
CBP14936 RYCSRGSLADVIERSSMQMDA--FFMFSLIHDIAN-------------------------------------GL----AFIHS-SILHLHGY-----------------------------------LSSKNCLIDD-RWQVKISSFGIPMIRQSD--------------EVSK-------------------------------------KGMLWSAPEVLRNETE---------------ERTQEGDIYSF------GIICSEIITRSSAFDI-EN-------------R-------KEKP-EEIIYQLKKG---GFNAIRP---SLLTD----ESLEINPALTHLIRDCWTEKPSERPP-----IDQVKSLLK- 268
GCY-17/24 RYCSRGSLADVISRSSMQMDS--FFMLSLIRDIAN-------------------------------------GL----GFIHT-SMLHFHGY-----------------------------------LSSRSCLIDD-RWQVKISDFGLNEVRGMD--------------KLST-------------------------------------ENMLWWAPEVLRGLE----------------QRSKEADIYSF------GIICSEVITRSSAFDL-EN-------------R-------KEKP-EEIIYQLKKG---GFNAIRP---SLLTD----EALEINPALVHLIRDCWTEKPSERPP-----IDQVRSLLR- 371
CBP02750 RFCSRGSMADVILKATIQMDN--FFIYSLIKDIVQ-------------------------------------GL----VFLHG-SIVGCHGM-----------------------------------LTSKCCLIDD-RWQVKISNYGLKDIRSPE--------------MYEK-------------------------------------KDLLWSAPELLRAEDI---------------KGTKEGDVYSL------GIICAELITRKSVFNM-ED-------------R-------KEDP-EEIIYLLKKG---GMKSPRP---DLDYD----HTIEINPALLHLIRDCFTERPSERPS-----IDTVRSQLR- 273
GCY-7 RFCSRGSIADVILKATIQMDN--FFIYSLIKDMVH-------------------------------------GL----VFLHG-SMVGYHGM-----------------------------------LTSKCCLIDD-RWQVKISNYGLQDLRSPE--------------MYEK-------------------------------------KDLLWSAPELLRAEDI---------------KGSKEGDVYSL------GIICAELITRKGVFNM-ED-------------R-------KEDP-EEIIYLLKKG---GLKSPRP---DLEYD----HTIEINPALLHLVRDCFTERPSERPS-----IETVRSQLR- 273
CBP16612 RFCSRGSLADVIRKASLQMDG--FFIYSLMKDIVN-------------------------------------GL----TWIHD-SSHEYHGM-----------------------------------LTSKNCLLND-RWQLKITDFGLRNFRTHD--------------QYTK-------------------------------------MDRLWTAPELLRNDDI---------------MGSREGDVYSL------GIISAELITRSSVFDL-EN-------------R-------KEDA-EEIVYMLKKG---GLQSPRP---HLDHD----ESIEINPGLLHLVRDCWTERPSERPD-----IKQVASQLR- 275
GCY-6 RFCSRGSLADVIRKASMQMDG--FFIYSLMKDIIN-------------------------------------GL----TWIHE-SSHEFHGM-----------------------------------LTSKNCLLND-RWQLKITDFGLRIFRTHD--------------QYNK-------------------------------------SDRLWTSPELLRTDDI---------------LGSREGDIYSF------GIISAELITRSSVFDL-EN-------------R-------KEDA-EEIIYMLKKG---GLQSPRP---SLEHD----ESIEINPALLHLVRDCWTERPSERPD-----IKQVASQLR- 278
CBP08562 FCARGSLADVIAKASLQMDG--FFIYSLMKDIVN-------------------------------------GL----TWIHE-SSHGCHGM-----------------------------------LTSKCCLLND-RWQLKITDFGLSWFRTHD--------------QLTK-------------------------------------TDRLWTAPELLRNDDL---------------LGSREGDIYSL------GIISAELITRSSVFGL-EN-------------R-------KEDP-EEIVYKLKKG---GLQSPRP---NLEHD----ESLEINPALLHLVRDCWTERPSERPD-----IKQVGSQLR- 237
CBP01427 RYCSRGSLQDVIAKGSLQMDW--FFKYSLMRDVAE-------------------------------------AI----YYLHH-SPIGPHGW-----------------------------------LSSSTCLVDE-RWQVKVTFFGLSAIKQFE--------------VKEQ-------------------------------------KDFLHTAPEHIRDVHL---------------PITKEMDIYSF------AIICSELITKKSAWDL-EN-------------E-------TFDI-EELVYKIKKG---GRTPVRP---SLETE------DEHNGSMSLLVRDCWNENPDQRPT-----SEQIKTLMK- 266
GCY-22 RYCSRGSLQDVIAKGSLQMDW--FFKYSLMRDVAD-------------------------------------AI----YYLHH-SPIGPHGW-----------------------------------LSSSTCLVDE-RWQVKVSFFGLSAIKQYE--------------VKEQ-------------------------------------RDFLHTAPEHIRDTNL---------------PITKEMDIYSF------AIICSELITKKSAWDL-EN-------------E-------TFDI-EELVYKIKKG---GRSPPRP---SLETE------DEHNGSMSLLVRDCWNENPDQRPT-----SEQIKTLMK- 266
CBP05559 RYCSRGSIKDVIAKSSINMDG--FFIYCLMKDIAAPTI------------------------------QQLQGL----QYIHH-SPIRQHGS-----------------------------------LTSECCYIND-RWQVKIGAYGLSFMHGVE--------------KRSE-------------------------------------DSMLHTAPEVLREGLA---------------SGSQLGDIYSF------AIVCSELVGHCSAWNL-EN-------------R-------KEEA-DEIIFMVKRG---GRIPFRP---SLDEA-----DEDINPAMLHLIRDCWDEDPKQRPN-----IDMVNKLMK- 279
GCY-13 RYCSRGSIKDVIAKSSINMDG--FFIYCLIKDIASVSR------------------------------HENPGL----QYIHH-SPIKQHGS-----------------------------------LTSECCYIND-RWQVKIGSYGLSFMQGVE--------------KRTE-------------------------------------DGLLHTAPEVLREGLT---------------SGTQAGDVYSF------SIVCSELVGHSSAWNL-EN-------------R-------KEEA-DEIIFMVKRG---GRTPFRP---SLDDV-----DDDINPAMLHLIRDCWDEDPKQRPN-----IDMVNKLMK- 279
CBP00662 EYCSKGSLHDILRNENLKLDH--MYVASFVDDLVK-------------------------------------GM----VYIHA-SELKYHGN-----------------------------------LKSTNCLITS-RWTLQVADFGMRQLREDI--------------LYGSNFN--------------------------------IWENFLWTAPEGMTINGNVPT---------LN-PPTTKSDIYSF------GIIFNEIFTREGPYKI-FVQRGDVNGDGA-KPD-------STEC-RELVEKTVRRVY-SDPYFRP---DTSD-------IEVQNYVKEVMAACWHNDPSQRPE-----FKSIKKKLN- 302
GCY-12 EYCSKGSLHDILRNENLKLDH--MYVASFVDDLVK-------------------------------------GM----VYIHD-SELKMHGN-----------------------------------LKSTNCLITS-RWTLQIADFGLRELREGI--------------MYDSSYN--------------------------------IWENFLWTAPEAMTINGSLAI---------SN-PPTPKADAYSF------GIIFHEIFTREGPYKI-YVQRGDVNGEAAPKKD-------SVEC-RALVEKTVRRVY-SDPYFRP---DTSD-------LEVQNYVKEVMAACWHHDPYQRPE-----FKTIKNKLK- 302
CBP03512 YCPKGSLEDILENEKIELDK--LMKYSLLHDLVK-------------------------------------GL----FFLHN-SEIRSHGR-----------------------------------LKSSNCVVDS-RFVLKVTDFGLHKLHCLE--------------EINLEEIGEHA----------------------------YYKKMLWTAPELLRDSNAPP-------------MGTQKGDIYSF------AIILHEMMFRKGVFAL-EN-------------E-------DLSP-NEIVQRVRKPVSEDQEPLRP---WVSETADG--DDALNDTLLSLMVACWSEDPHERPE-----VSSVRKAVR- 432
GCY-28 YCPKGSLEDILENEKIELDK--LMKYSLLHDLVK-------------------------------------GL----FFLHN-SEIRSHGR-----------------------------------LKSSNCVVDS-RFVLKVTDFGLHRLHCLE--------------EINLEEIGEHA----------------------------YYKKMLWTAPELLRDSNAPP-------------MGTQKGDIYSF------AIILHEMMFRKGVFAL-EN-------------E-------DLSP-NEIVQRVRKPVSEDQEPLRP---WVSETGEGEGDDALNDTLLSLMVACWSEDPHERPE-----VSSVRKAVR- 448
CBP04548 ELAQRGSLKDILDNEDMALDD--VFRSQMTKDIIA-------------------------------------GL----EYLHS-SPIGCHGR-----------------------------------LKSTNCLIDG-RWMVRLSSFGLREMRSEE--------------EWQREEGVQE------------------------------GKDDLWTAPELLRWSTGLGQ---------CGHLLVQKADVYSL------SIVLYELFGRLGPWG---D-------------E-------PMEP-REIVSCVKRLPSSGKKVFRP---DLAVIK------EAPQIVRDTVAAAWTEEPLDRPS-----LFQIKRKLK- 337
GCY-15 ELAQRGSLKDILDNDDMPLDD--VFRSQMTKDIIA-------------------------------------GL----EYLHS-SPVGCHGR-----------------------------------LKSTNCLIDA-RWMVRLSSFGLRELRGEE--------------TWQQEDDVQE------------------------------GKDQLWTSPELLRWSTGLSQ---------CGVLLVQKSDVYSL------AIVLYELFGRLGPWG---D-------------E-------PMEP-REIVSLVKREALAGKKPFRP---DMAVLK------ESPRIVQETVVAAWTEDPLNRPS-----LHQIKRKLK- 334
GCY-21 ELAQRGSLKDILDNDDMPLDD--VFRSQMTKDIIA-------------------------------------GL----EYLHS-SPIGCHGR-----------------------------------LKSTNCLIDA-RWMVRLSSFGLRELRGEE--------------TWQQEDDVQE------------------------------GKDQLWTSPELLRWSTGLSQ---------CGVLLVQKSDVYSL------AIVLYELFGRLGPWG---D-------------E-------PMEP-REIVSLVKREALAGKKPFRP---DMAVLK------ESPRIVQETVVAAWTEDPLNRPS-----LHQIKRKLK- 334
CBP18607 HYGPRKSLMDLLRNDDLRLDR--MFRVSFVEDVVK-------------------------------------GL----QFLHENSKIGYHGN-----------------------------------LKSSNCVVDA-YWRIKLSNYGMEQIRVDE--------------PEAKP------------------------------------DDLLWFAPEIIRRYAVKHD---------LSKIELAKADIYSF------SIILYEIYGRQGPFG---D-------------D-------LLDS-DEIISQLKF--PDGQALTRP---DIHLIT------KAPYPVSSVVEQCWAEDPDARPN-----IKKVKELLK- 320
GCY-11 SYGPRKSLMDLLRNDDLRLDR--MFKVSFVEDVVK-------------------------------------GL----QFLHEGSKIGYHGN-----------------------------------LKSSNCIVDA-YWRIKLSNYGMEQIRVEE--------------PESKPDVFYDL-VIF----------SNKYNSKTKNPIKKFSTDLLWFAPEIIRRYAVKHD---------LSKLELAKADIYSL------AIVLYEIYGRQGPFG---D-------------D-------LLDS-DEIIEQLKF--PDGGALTRP---DIHLIT------KAPYPLASVVEKCWVEDPASRPS-----IQKVRELLE- 345
CBP04780 YSKRSGLYPIVKGVVREVARR-IYDTEVVMKVQE----------------------------------RKQEHLDAFVTEH--VVFVITQIENANSVQQKSISSKADSQIDLSTGIYEISSTDFGLAFPYHICFDP-DLFLEHFGNFIKKTYPNA-------------T-----------------------------------RQETRVTDLLELVHPEVPFSYESIK--------------YYKNSLFVFRLKGLG-----------DIVHN---------------PS-------DEAK-----TVLLKG--------------------------SMVFIDEGKYILYMCSVNVTTV-----RELIERNLH- 317
FUSED_DROME/4-254 ------------------MGE--EPARRVTGHLV-------------------------------------SAL----YYLHS-NRI-LHRD-----------------------------------LKPQNVLL-DKNMHAKLCDFGLARNM-----------------T------LGTH-VLT-----------------S------IKGTPLYMAPELLA----------------EQPYDHH-ADMWSL------GCIAYESMAGQPPFCA----------------S---------SILHLVKMIK----HEDVKWPST---LT--S-----------ECRSFLQGLLEKDPGLRIS-----WTQLLCHPFV 251
AKT_MLVAT/171-429 ------------------FSE--DRARFYGAEIV-------------------------------------SAL----DYLHSEKNV-VYRD-----------------------------------LKLENLML-DKDGHIKITDFGLCKEG-----------------I------KDGA-TMK-----------------T------FCGTPEYLAPEVLE----------------DNDYGRA-VDWWGL------GVVMYEMMCGRLPFYN----------------Q---------DHEKLFELIL----MEEIRFPRT---LG--P-----------EAKSLLSGLLKKDPTQRLGGGSEDAKEIMQHRFF 259
KIN82_YEAST/324-602 ------------------IAE--EDAKFYASEVV-------------------------------------AAL----EYLHL-LGF-IYRD-----------------------------------LKPENILL-HQSGHVMLSDFDLSIQATGSKKPTMKDSTYLDTKI------CSDGFRTN-----------------S------FVGTEEYLAPEVIR----------------GNGHTAA-VDWWTL------GILIYEMLFGCTPFKG----------------D---------NSNETFSNIL----TKDVKFPHD---KEVSK-----------NCKDLIKKLLNKNEAKRLGSK-SGAADIKRHPFF 279
PKD1_DICDI/36-291 ------------------FTE--QTTKLIVAEIV-------------------------------------LAI----EYLHA-ENI-IYRD-----------------------------------LKPENILI-DEKGHIKLTDFGFSKK------------------T------VGGK-NTS-----------------S------VCGTFDYMAPEILN---------------SSNGHGKP-VDWWAL------GVVVYELVTGKLPFSN----------------SKESLLNRK-ADFQLIFQNS----YLS-------------D-----------EIKDFIFQLLSVDPSKRLGT--FDSCSIRNHKWF 256
KGP1_DROME/457-717 ------------------FED--NAAQFIIGCVL-------------------------------------QAF----EYLHA-RGI-IYRD-----------------------------------LKPENLML-DERGYVKIVDFGFAKQ------------------I------GTSS-KTW-----------------T------FCGTPEYVAPEIIL----------------NKGHDRA-VDYWAL------GILIHELLNGTPPFSA----------------P---------DPMQTYNLIL----KGIDMIAFP---KHISR-----------WAVQLIKRLCRDVPSERLGYQTGGIQDIKKHKWF 261
ARBK1_BOVIN/191-453 ------------------FSE--ADMRFYAAEII-------------------------------------LGL----EHMHN-RFV-VYRD-----------------------------------LKPANILL-DEHGHVRISDLGLAC-------------------D------FSKK-KPH-----------------A------SVGTHGYMAPEVLQ---------------KGVAYDSS-ADWFSL------GCMLFKLLRGHSPFRQ----------------H-----KTK-DKHEIDRMTL----TMAVELPDS---FS--P-----------ELRSLLEGLLQRDVNRRLGCLGRGAQEVKESPFF 263
CHK1_SCHPO/10-272 ------------------IDE--DVAQFYFAQLM-------------------------------------EGI----SFMHS-KGV-AHRD-----------------------------------LKPENILL-DYNGNLKISDFGFASLFS---------------YK------GKSR-LLN-----------------S------PVGSPPYAAPEI-----------------TQQYDGSK-VDVWSC------GIILFALLLGNTPWDE----------------A-----ISN-TGDYLLYKKQ----CERPSYHPW---NLLSP-----------GAYSIITGMLRSDPFKRYS-----VKHVVQHPWL 263
CDC5_YEAST/82-337 ------------------LTE--PEVRFFTTQIC-------------------------------------GAI----KYMHS-RRV-IHRD-----------------------------------LKLGNIFF-DSNYNLKIGDFGLAAVL-----------------A------NESE-RKY-----------------T------ICGTPNYIAPEVLM--------------GKHSGHSFE-VDIWSL------GVMLYALLIGKPPFQA----------------R---------DVNTIYERIK----CRDFSFPRD---KPISD-----------EGKILIRDILSLDPIERPS-----LTEIMDYVWF 256
TTK_HUMAN/509-775 ------------------IDP--WERKSYWKNML-------------------------------------EAV----HTIHQ-HGI-VHSD-----------------------------------LKPANFLI-V-DGMLKLIDFGIANQMQ---------------PD------TTSV-VKD-----------------S------QVGTVNYMPPEAIKDMSSSR-----ENGKSKSKISPK-SDVWSL------GCILYYMTYGKTPFQQ----------------I-------I-NQISKLHAII----DPNHEIEFP---DIPEK-----------DLQDVLKCCLKRDPKQRIS-----IPELLAHPYV 267
ruler .......390.......400.......410.......420.......430.......440.......450.......460.......470.......480.......490.......500.......510.......520.......530.......540.......550.......560.......570.......580.......590.......600.......610.......620.......630.......640.......650.......660.......670.......680.......690.......700.......710.......720.......730.......740.......750.......