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 or38@columbia.edu

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-

15

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.......