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A Homolog of the Vertebrate Thyrostimulin Glycoprotein Hormone α Subunit(GPA2) is Expressed in Amphioxus NeuronsAuthor(s): Yukiko Tando and Kaoru KubokawaSource: Zoological Science, 26(6):409-414. 2009.Published By: Zoological Society of JapanDOI: http://dx.doi.org/10.2108/zsj.26.409URL: http://www.bioone.org/doi/full/10.2108/zsj.26.409

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2009 Zoological Society of JapanZOOLOGICAL SCIENCE 26: 409–414 (2009)

A Homolog of the Vertebrate Thyrostimulin Glycoprotein

Hormone α Subunit (GPA2) is Expressed

in Amphioxus Neurons

Yukiko Tando and Kaoru Kubokawa*

Center for Advanced Marine Research, Ocean Research Institute,

The University of Tokyo, Tokyo 164-8639, Japan

The cystine-knot glycoprotein hormone α (GPA) family regulates gonadal and thyroid functions in ver-

tebrates. Little is known concerning GPA family members in primitive chordates. A previous genomic

analysis revealed the presence of two genes homologous to the thyrostimulin α subunit (GPA2) in an

amphioxus (Branchiostoma floridae); however only one GPA2 homolog contained both the cystine-

knot structure and N-glycosylation site characteristic of family members. Gene-specific PCR was used

to obtain the cDNA and genomic sequences of the GPA2 homolog of the amphioxus Branchiostoma belcheri. Whole-mount in situ hybridization revealed GPA2 mRNA expression in the anterior part of the

nerve cord and on the left side of the central canal. Because amphioxus possesses only one true GPA2

homolog, while vertebrates possess two glycoprotein hormone α subunits (thyrostimulin α, or GPA2,

and the common α subunit of gonadal and thyroid glycoprotein hormones, GPA1), our results suggest

that GPA1 was acquired later in the vertebrate lineage through gene duplication.

Key words: amphioxus, hormone, expression, evolution, comparative endocrinology

INTRODUCTION

A family of glycoprotein hormones containing the

cystine-knot motif regulates gonadal and thyroid function in

vertebrates (Hearn and Gomme, 2000; Isaacs, 1995).

Gonadotropins regulate the growth and differentiation of the

ovary and testis, while thyroid-stimulating hormone (TSH) is

essential for energy balance (Kendall et al., 1995). The

active hormones are non-covalent heterodimers of α and βsubunits. Two α subunits and five β subunits are present in

humans, where a common glycoprotein hormone α subunit

(GPA1) heterodimerizes with different β subunits specific for

TSH, follicle-stimulating hormone (FSH), luteinizing

hormone (LH), or chorionic gonadotropin (CG). In the case

of thyrostimulin, which is a recently discovered pituitary gly-

coprotein hormone expressed in diverse human tissues

(Nakabayashi et al., 2002), a single thyrostimulin-specific

glycoprotein hormone α subunit (GPA2) heterodimerizes

with a specific β subunit (GPB5) to form the active hormone.

These glycoprotein hormones are generally synthesized in

the pituitary gland, with the exception of CG, which in

mammals is synthesized in the placenta.

These pituitary glycoprotein hormones were believed to

be unique to vertebrates. However, a homolog of thyrostimulin

was found to be present in invertebrates (Hsu et al., 2002).

Species of amphioxus, also known as lancelets (Subphylum

Cephalochordata), constitute a group of primitive chordates

phylogenetically close to the origin of vertebrates (Bourlat et

al., 2006). A draft genomic sequence of an amphioxus

(Branchiostoma floridae) (Putnam et al., 2008) has revealed

the presence of homologs of several genes encoding

proteins involved in thyroid and hypothalamo-pituitary axis

regulation. These include homologs of thyrotropin-releasing

hormone (TRH) and the TSH receptor (TSHR), although the

TRH receptor and TSH were not found (Mathilde et al.,

2008). Type I GnRH has also been isolated from amphioxus

by means of HPLC and mass spectrometry; this hormone

stimulates LH release from rat pituitary glands in vitro

(Chambery et al., 2009). Previous studies demonstrated

immunoreactivity of human gonadotropins in Hatchek’s pit,

which is the organ considered to be the primitive pituitary

gland (Zhang et al., 1982; Nozaki and Gorbman, 1992).

However, genes homologous to LH, FSH, and TSH were not

found in the amphioxus genome (Holland et al., 2008),

whereas genes homologous to both the α (GPA2) and β(GPB5) subunits of thyrostimulin were present (Holland et

al., 2008). In amphioxus, two genes homologous to GPA2

and a single GPB5 homolog were found, while vertebrates

possess single copies of both the GPA2 and GPB5 genes.

To address whether the two genes corresponding to the

thyrostimulin α subunit GPA2 are functional genes or

pseudogenes, we sought to obtain homologous cDNA and

genomic clones from the amphioxus Branchiostoma

belcheri and to use these to investigate the pattern of

expression in amphioxus.

MATERIALS AND METHODS

Animals

Specimens of Branchiostoma belcheri were collected from the

Enshu Nada Sea, Japan, and maintained in a tank at the Ocean

Research Institute, University of Tokyo as previously described

* Corresponding author. Phone: +81-3-5351-6529;

Fax : +81-3-5351-6820;

E-mail : kubokawa@ori.u-tokyo.ac.jp

doi:10.2108/zsj.26.409

Y. Tando and K. Kubokawa410

(Kubokawa et al., 2003; Mizuta and Kubokawa 2004).

Cloning of cDNA

Total RNA was isolated as described by Mizuta and Kubokawa

(2007), eluted in sterile (DEPC) water, and stored at –80°C. First-

strand cDNA was prepared by using a PrimeScriptTM RNA PCR Kit

(TaKaRa, Japan) and an oligo-dT primer at 45°C for 30 min, 55°C

for 10 min, 65°C for 10 min, and 70°C for 10 min. The enzyme was

denatured at 95°C for 5 min, and the cDNA was stored at –30°C

until use. For amplification of cDNA, we used GPA2-specific primers

5’-TAGAGGCTACTGTGAGTCCATA-3’ and 5’-GGTTATCGTCGCA-

GATGCTACA-3’, and GPA2-LP- (GPA2-like protein) specific primers

5’-GCGCTATATGCCAATGTTAGCCGTAC-3’ and 5’-CAGCA-

GACTCGATAGTGTAGGTC-3’, designed from genomic sequences

(Holland et al., 2008); PCR conditions were 95°C for 1 min; 40

cycles of 95°C for 10 sec, 60°C for 10 sec, and 72°C for 30 sec; and

72°C for 1 min. PCR products of the expected size (GPA2, 209 bp;

GPA2-LP, 425 bp) were cloned into the plasmid vector pCR4 TOPO

(Invitrogen, Carlsbad, CA) and sequenced.

To obtain the full-length clone of GPA2, a GeneRacer Kit (Invit-

rogen) with an oligo-dT Primer was used. For reverse transcription,

we employed Superscript III transcriptase (Invitrogen) at 50°C for

30 min, 55°C for 10 min, 60°C for 10 min, and 65°C for 10 min, fol-

lowed by inactivation at 70°C for 15 min. The 5’ and 3’ ends of

GPA2 cDNA were amplified by RACE using the GeneRacer Kit

(Invitrogen). For 5’-RACE, we used the GeneRacer 5’ primer and a

GSP-R primer (5’-CGCAGATGCTACAGGCGCAGCTGGAT-3’ for

GPA2 or 5’-GCAGGCGTTGATGAGAACCGTAG-3’ for GPA2-LP);

for 3’-RACE, we used the GeneRacer 3’ primer and a GSP-F primer

(5’-GCAGGGGTCGAGCGGGAACCACGTCAT-3’ for GPA2 or 5’-

GATGAACGGGAGCAGAACGAAATAGG-3’ for GPA2-LP). PCR

products were used as templates for nested PCR. For 5’ nested

RACE, we used the GeneRacer 5’ nested primer and a GSP-

R_Nest primer (5’-GCGCAGCTGGATGCCGAGTAGAGGGTTT-3’ for

GPA2 or 5’-GCCAGATCCCACTCAGACGTCAG-3’ for GPA2-LP);

for 3’ nested RACE, we used the GeneRacer 3’ nested primer and

a GSP-F_Nest primer (5’-AGCCTGCTGTGACATCGCCTCCACA-

CAT-3’ for GPA2 or 5’-CAGACCTCCCTGTCCTCTTACGC-3’ for

GPA2-LP). Both 5’- and 3’-RACE were performed by touchdown

PCR; after an initial denaturation at 95°C for 2 min, multistep PCR

was performed. Five PCR cycles of 95°C for 30 sec and 72°C for

2 min were followed by a further five cycles of 95°C for 30 sec, 70°C

for 30 sec, and 72°C for 2 min; 25 cycles of 95°C for 30 sec, 68°C

for 30 sec, and 72°C for 2 min; and a final extension at 72°C for

10 min. Amplified fragments were cloned into pCR4 vector and

sequenced. Partial sequences obtained from 5’ and 3’ RACE were

assembled with ATGC software (Genetyx, Tokyo, Japan). The full-

length cDNA encoding GPA2 was confirmed to be a single gene.

Comparison of the structure of glycoprotein hormone α genes

Whole-body genomic DNA was isolated from B. belcheri with

QuickGene-800 (Fujifilm, Kyoto, Japan) according to manufac-

turer’s instructions. Genomic DNA encoding GPA2 was amplified by

PCR using GPA2-specific primers (GPA2-F, 5’-CGACCACCTTAAG-

CAATCAC-3’; GPA2-R, 5’-TCCTGCATGAGGTTGTTGGA-3’). PCR

conditions were 95°C for 2 min; 30 cycles of 95°C for 30 sec, 60°C

for 30 sec, and 72°C for 5 min; and 72°C for 10 min. Amplified

fragments were sequenced. Sequences of human glycoprotein

hormone α subunit genes were obtained from the NCBI database

(Entrez; http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene).

Phylogenetic analysis

Sequences were aligned by using the program Clustal W

(Thompson, 1994). Phylogenetic trees were constructed by the

neighbor-joining method, and evolutionary distances (Poisson

correction) were calculated, with MEGA version 3.1 software

(Kumer, 2004).

In situ hybridization

For whole-mount in situ hybridization (WISH), we obtained a B.

belcheri GPA2 probe by PCR amplification with primers GPA2-F

(5’-CGACCACCTTAAGCAATCAC-3’) and GPA2-R (5’-TCCTGCAT-

GAGGTTGTTGGA-3’), which flanked nucleotides 8–1162 of the

GPA2 gene and generated a product 1155 bp long. We generated

digoxigenin (DIG)-labeled sense and antisense RNA probes spe-

cific for GPA2 by using a DIG RNA Labeling Kit (Roche, Penzberg,

Germany) according to manufacturer’s instructions. Labeled RNA

probes were fragmented in fragmentation buffer (42 mM NaHCO3,

63 mM Na2CO3, 62.5 mM DTT) at 60°C for 10 min, purified with

Ultrafree-MC Centrifugal Filter Units (Millipore), and eluted in 40 μl.

Animals were fixed in 4% paraformaldehyde (PFA) in 0.5 M

NaCl, 0.1 M MOPS buffer (pH 7.5) at 4°C for 12 h and dehydrated

in increasing concentrations of methanol (25%, 50%, 75%, and

100% v/v) in phosphate buffered saline (PBS) over 30 min; the

dehydrated specimens were stored at –30°C until use. Nerve cords

were dissected from fixed heads in 80% v/v methanol/PBS. WISH

was carried out as described by Ogasawara et al. (2006), with mod-

ifications. Specimens in 80% methanol were rehydrated, washed

with PBS/Tween 20 (PBT), treated with 20 μg/ml proteinase K for

20 min, and post-fixed with 4% PFA/PBT for 20 min. Tissues were

immersed in InSitu Chip (ALOKA, Tokyo, Japan) and incubated in

prehybridization buffer (5X standard saline citrate [SSC], 50% v/v

formamide, 1% w/v SDS, 50 μg/ml yeast tRNA, 50 μg/ml heparin,

0.1% CHAPS, 5 mM EDTA, pH 8.0) at 50°C for 1 h, and then

hybridized in hybridization buffer (prehybridization buffer containing

the DIG-labeled RNA probe) at 50°C for 16 h. After hybridization,

tissues were washed sequentially at 50°C in wash buffer 1 (WB1:

4X SSC, 50% formamide, 0.1% Tween 20), WB2 (2X SSC, 50%

formamide, 0.1% Tween 20), WB3 (1X SSC, 50% formamide, 0.1%

Tween 20), and WB4 (0.1X SSC, 50% formamide, 0.1% Tween 20),

each for 20 min. Hybridized tissues were treated with 20 μg/ml

RNase A in PBST for 20 min and rinsed in WB5 (1X SSC, 0.1%

Tween 20). After color development, nerve cords were embedded

in 5% agar and prepared as 50-μm sections by using a linear slicer

(Douhan EM, Kyoto, Japan).

RESULTS

Cloning of B. belcheri GPA2 cDNAs

Full-length cDNAs encoding two B. belcheri GPA2

homologs (GPA2 and GPA2-LP; GenBank accession Nos.

AB468062 and AB479090, respectively) were isolated from

amphioxus heads by using RT-PCR and RACE (Fig. 1A–C).

The cDNA encoding GPA2 was 1205 bp in length and con-

tained an open reading frame of 381 bp (126 amino acids)

(Fig. 1A). Amino acid and nucleotide sequence similarities

between B. belcheri GPA2 and the corresponding coding

region of B. floridae (protein ID, 117901) were 93% and

91%, respectively. A putative N-linked glycosylation site was

found at Asn97. The second cDNA, encoding a GPA2-like

protein (GPA2-LP), was 1349 bp in length and contained an

open reading frame of 468 bp (155 amino acids) (Fig. 1B).

Amino acid and nucleotide sequence similarities of this gene

with the B. floridae equivalent (protein ID, 63816) were 82%

and 77%, respectively; however, the predicted amino acid

sequence failed to contain any consensus site for N-linked

glycosylation, and this homolog was therefore excluded as

a potential member of the glycoprotein hormone family. Our

results indicate that GPA2 is the only glycoprotein hormone

of this group in B. belcheri.

Conservation of the cystine-knot domain structure

At the amino acid sequence level, the B. belcheri GPA2

Amphioxus Thyrostimulin α 411

Fig. 1. (A) Nucleotide and amino acid sequences of amphioxus (B. belcheri) GPA2. The

putative signal peptide is underlined and the potential N-linked glycosylation site is circled.

(B) Nucleotide and amino acid sequences of amphioxus GPA2-like protein (GPA2-LP).

The putative signal peptide is underlined. (C) Sequence alignment between the deduced

B. belcheri GPA2 and GPA2-LP polypeptides; identical amino acid residues are marked

with asterisks. Potential N-linked glycosylation site is boxed. (D) Comparison of the amino

acid sequences of GPA2 polypeptides from various species. Identical amino acid residues

are marked with asterisks and conserved cysteine residues are shaded. Numbers above

cysteine residues indicate pairs of residues thought to form disulfide bonds; those num-

bered from 1 to 3 and connected with a line are necessary for constructing the cystine-

knot structure. Putative N-linked glycosylation sites are boxed. Fingers 1 and 2 indicate

the outer parts of the GPA2 molecule consisting of β-strands; the heel indicates the outer

part of α-helical structure (described in E). (E) Schematic drawing of the cystine-knot

structure. Dotted lines connecting cysteine residues indicate disulfide bonds; three disul-

fide bonds contribute to the cysteine-knot structure. C, cysteine; G, glycine.

Y. Tando and K. Kubokawa412

sequence shared 21.6% and 39.9% identity with human

GPA1 and GPA2, respectively. An alignment of sequences

of human, puffer, sea urchin, fly, and amphioxus GPA2

(Fig. 1D) indicated that B. belcheri GPA2 contains con-

served cysteine residues consistent with five S-S bonds in

the mature polypeptide. Three S-S bonds contribute to the

cystine-knot structure (Vitt et al., 2001) (Fig. 1E). A poten-

tial N-linked glycosylation site at Asn97 was located within

the finger2 region of the cystine-knot structure; vertebrate

GPA2s possess two glycosylation sites, one in the finger1

region and the other in the heel region of the cystine-knog

structure (Vitt et al., 2001).

Genomic structure of B. belcheri GPA2

The genomic structure of the B. belcheri GPA2

homolog was elucidated by PCR amplification of genomic

DNA and sequencing. The B. belcheri GPA2 sequence

extended over 4.2 kb and comprised four exons (Fig. 2),

with three introns comprising 1583 bp, 200 bp, and 1180

bp. In human, the coding sequences for GPA1 and GPA2

extend over 9.6 and 1.4 kb, respectively. Notably, the sec-

ond exon of B. belcheri GPA2 contained not only the open

reading frame but also a non-coding region absent from

human glycoprotein hormone α genes.

Phylogenetic analysis

For the phylogenetic analysis, we used a multiple align-

ment of known GPA2 sequences from diverse species. As

shown in Fig. 3, the coding sequence obtained here clearly

falls into the GPA2 branch of glycoprotein hormone αsequences.

Expression of B. belcheri GPA2

In situ hybridization of whole-mount specimens of B.

belcheri was used to investigate GPA2 expression patterns.

GPA2 mRNA was detected around the sixth dorsal nerve

root, included in the intercalated region (Wicht and Lacalli,

2005) of the nerve cord (Fig. 4A). No expression was

detected in other parts of the nerve cord. In trans-

verse view, GPA2 mRNA was detected in cells on

the right side of the central canal of the nerve cord

(Fig. 4B–D). Here the cell bodies of the neurons

are present on both sides of the canal; these cells

gave positive signals for GPA2. Notably, positive

cells formed clusters on the dorsal side of the

canal (Fig. 4C). Expression of B. belcheri GPA2

was not detected in Hatschek’s pit (data not

shown), which is a groove-like structure in the

mouth cavity located between myomeres 3 and 4,

and is considered to be homologous with the ver-

tebrate pituitary gland.

DISCUSSION

We report the cloning of a thyrostimulin αGPA2 homolog from the amphioxus Branchiostoma

belcheri. The genomic sequence of the related

amphioxus B. floridae contains two homologs of

vertebrate GPA2, but the pituitary common α sub-

unit of vertebrates (GPA1) was not found (Holland

et al., 2008). However, a search of the B. floridae

genome sequence using a motif search program

Fig. 2. Comparison of the amphioxus (B. belcheri) GPA2 gene with

human glycoprotein hormone α genes. Vertical bars indicate exons:

open bars indicate untranslated regions; black bars indicate coding

regions. h, human; Ampbb, amphioxus Branchiostoma belcheri.

Fig. 3. Molecular phylogenetic tree of glycoprotein hormone α sub-

units, constructed with the neighbor-joining method. Numbers on the

branches indicate bootstrap values from analysis of 1000 pseudorepli-

cates. The scale bar represents the calculated evolutionary distance

(Poisson correction) in substitutions per site. GenBank accession

numbers for the taxa included are: human GPA1, J00152; quail GPA1,

S70833; xenopus GPA1, L07619; sturgeon GPA1, AJ310342;

zebrafish GPA1, AY522553; human GPA2, AF260739; pufferfish

GPA2, Q4S0U2; amphioxus (Branchiostoma belcheri) GPA2,

AB468062; sea urchin GPA2 (GLEAN3), 15344; fly GPA2, AY940435.

Fig. 4. Whole-mount in situ hybridization of amphioxus GPA2. (A) Dorsal view

showing localization of GPA2 mRNA in the anterior part of the nerve cord. Arrows

indicate cells expressing GPA2. (B–D) Transverse sections of the nerve cord,

including the GPA2-expressing cells indicated in A. Arrowheads indicate pigment

cells. Scale bars: 100 μm (A), 25 μm (B–D).

Amphioxus Thyrostimulin α 413

(Niimrura, pers. comm.) for the characteristic cystine-knot

structure and N-glycosylation site (N-X-T/S) identified only a

single GPA2 homolog. We isolated cDNA clones corre-

sponding to the two GPA2 homologs. The first, GPA2,

presented all the features of a glycoprotein hormone while

the second, GPA2-like protein (GPA2-LP), lacked any

consensus site for N-linked glycosylation. We conclude that

GPA2 is the only cystine-knot glycoprotein hormone α in

amphioxus.

Phylogenetic analysis indicated that B. belcheri GPA2 is

more similar to vertebrate GPA2 subunits than to GPA1,

confirming that the amphioxus sequence studied is indeed a

member of the GPA2 family. It is noteworthy that genes

homologous to GPA2 are present in several invertebrates,

while the GPA1 family appears to be specific to vertebrates

(Hsu et al., 2002). In a recent review, Sower et al. (2009)

noted that lamprey has only GPA2 and not GPA1, and

concluded that GPA2 is the ancestral α subunit of the

glycoprotein family. Accordingly, we suggest that only one αsubunit, GPA2, was present from invertebrates to

agnathans, and that GPA1 appeared in the gnathostome

lineage through gene duplication.

To address whether the B. belcheri GPA2 polypeptide

forms a typical cystine-knot structure, we employed the

SWISS-MODEL protein folding program (http://swissmodel.

expasy.org/) using the experimentally determined structure

of human GPA1 (Protein Data Bank accession code,

1HRPA) as a template. Although the program correctly

predicted the formation of a cystine knot in the human

polypeptide, it predicted no such structure from the B.

belcheri GPA2 polypeptide sequence. Because cystine-knot

formation is thought to contribute to heterodimerization in

this family, it is possible that the amphioxus protein

dimerizes by a different conformational mechanism. Further

studies with GPA2 and GPB5 polypeptides will be required

to address this possibility.

The B. belcheri GPA2 polypeptide sequence contained

only one N-glycosylation site, unlike human GPA2, which

contains two sites. In human thyrostimulin, site-specific

disruption of either of the two GPA2 glycosylation sites did

not affect heterodimerization with GPB5 but decreased the

efficiency of TSHR activation (Sudo et al., 2005). The

Drosophila melanogaster GPA2 has only one glycosylation

site, but the GPA2/GPB5 heterodimer efficiently activates

the G-protein coupled receptor (DLGR1) thought to be the

invertebrate counterpart of the vertebrate TSH, LH, and FSH

receptors (Okajima et al., 2008). The dependence of GPA2

receptor targeting on ligand glycosylation therefore appears

to vary according to species. The amphioxus genome con-

tains a gene equivalent to vertebrate TSHR; further studies

will be required to address the potential interaction between

amphioxus GPA2 and this receptor.

Branchiostoma belcheri GPA2 mRNA expression was

detected in the anterior part of the nerve cord and at the

right side of the central canal. This location corresponds to

RB cells (Retzius bipolar cells), which are thought to be

somatic sensory neurons (Bone, 1960). RB cells are bipolar

neurons that form two continuous columns along the dorsal

part of nerve cord, on either side of the central canal imme-

diately below the cord roof, and have both ascending and

descending fibers. However, the peripheral connections of

GPA2-expressing cells have not yet been examined.

Antisera directed against vertebrate neurotransmitters and

neuropeptides have been used for immunohistochemical

investigations of the amphioxus nerve cord (Nieuwenhuys,

1998), but no cross-reacting material was detected in RB

cells. Further studies using antisera directed against differ-

ent neuronal markers will be required to confirm the identity

of the cells expressing GPA2. In vertebrates, GPA2 genes

are predominantly expressed in the pituitary (Hsu et al.,

2002; Nagasaki et al., 2006; Okada et al., 2006). The

present study indicates that in amphioxus, GPA2 is not

expressed in Hatschek’s pit, a potential homolog of the pitu-

itary gland (Tjoa and Welsch, 1974). Morphological studies

have indicated the presence of secretory granules in

Hatschek’s pit (Tjoa and Welsch, 1974), while Candiani and

Pestarino (1998) reported that a pituitary-specific transcrip-

tion factor (Pit-1) is present in the pit. The absence of GPA2

expression in Hatschek’s pit suggests it is controversial to

regard this organ as homologous to the pituitary, although

immunoreactivity of gonadtropins in the pit has previously

been reported (Zhang et al., 1982; Nozaki and Gorbman,

1992). However, it is possible that a heterodimer of

thyrostimulin may occur in Hatchek’s pit after processing of

subunits, because we examined the localization only of

mRNA, rather than protein. Immunohistochemical studies

using an antibody against amphioxus GPA2 should be done.

A gene homologous to GPB5 is present in the

amphioxus genome (Holland et al., 2008), but it is unknown

whether the GPB5 and GPA2 polypeptides co-localize in

this species or otherwise interact. Further studies will be

required to address this possibility.

ACKNOWLEDGMENTS

We thank Dr. Niimura (Tokyo Medical and Dental University)

for assistance with motif searching of the B. floridae genome. This

study was supported by Kakenhi (Grants-in-Aid for Scientific

Research) in the priority area ‘Comparative Genomics’ from the

Ministry of Education, Culture, Sports, Science and Technology of

Japan (KK), by a Sasakawa Scientific Research Grant from The

Japan Science Society (YT), and by a Research Fellowship of the

Japan Society for the Promotion of Science for Young Scientists

(YT).

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(Received February 12, 2009 / Accepted March 24, 2009)