Studies on nebulin in amphioxus striated muscle

July 2009

Akira Hanashima

Graduate School of Science

CHIBA UNIVERSITY

(千葉大学学位申請論文)

Studies on nebulin in amphioxus striated muscle

2009 年 7 月

千葉大学大学院理学研究科

地球生命圏科学専攻生物学コース

花島 章

1

Contents

Chapter 1. Primary structure, localization and binding proteins of

the C-terminus of amphioxus nebulin

1. Abstract---------------------------------------------------------------3

2. Introduction----------------------------------------------------------4

3. Materials and Methods-------------------------------------------5

4. Results-----------------------------------------------------------------11

5. Discussion------------------------------------------------------------15

6. Conclusion------------------------------------------------------------17

7. References------------------------------------------------------------18

8. Figures-----------------------------------------------------------------22

Chapter 2. Structure of the amphioxus nebulin gene and

evolution of the nebulin family genes

1. Abstract----------------------------------------------------------------31

2. Introduction-----------------------------------------------------------32

3. Materials and Methods--------------------------------------------34

4. Results------------------------------------------------------------------35

5. Discussion--------------------------------------------------------------39

6. Conclusion-------------------------------------------------------------43

7. References-------------------------------------------------------------44

8. Figures------------------------------------------------------------------47

2

Chapter 1

Primary structure, localization and binding proteins of

the C-terminus of amphioxus nebulin

3

1. Abstruct

Nebulin is a large protein located along the thin filaments of vertebrate

skeletal muscle. I isolated nebulin cDNA from the striated muscle of

cepharochordate (invertebrate) amphioxus. Although the sequence homology

is 26% with the human, the C-terminal of amphioxus nebulin has similar

structural motifs of 35 amino acid nebulin repeats and an SH3 domain. With

indirect immunofluorescence analysis by using specific antibody raised to

the bacteria produced recombinant peptide, I confirm that this nebulin

fragment is located in the Z-line of the sarcomere similar to human nebulin.

Pull-down and co-sedimentation assays in vitro showed that the C-terminal

region binds to actin, α-actinin and connectin (titin). These results suggest

that the C-terminal region of amphioxus nebulin plays a role in maintaining

striate muscle structure similar to that of human. This is the first report that

nebulin is not vertebrate-only protein but chordate protein.

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2. Introduction

Nebulin is a large, 773-kDa protein found in vertebrate skeletal muscles

(Wang, 1982; McElhinny et al., 2003). The primary structure of human

nebulin consists of a N-terminal glutamic acid-rich domain, followed by 185

(M1–185) contiguous nebulin repeats of approximately 35 amino acids with a

central SDXXYK consensus sequence, a serine-rich region and a C-terminal

Src homology 3 (SH3) domain (Labeit and Kolmerer, 1995). Nebulin repeats

(M9–162) in the I-band region of the sarcomere consist of sets of

seven-repeats. These seven-repeats comprise approximately 245 amino-acid

residues (i.e., 35 amino-acid residues per repeat x 7 repeats) to form a

super-repeat, there are 22 super-repeats (SR1–22).

Within the sarcomere, the N-terminal region is localized in the pointed end

of the thin filaments, the central region along the thin filaments and the

C-terminal region in the Z-line (Wang and Wright, 1988). Binding assays

have revealed that each nebulin repeat (SDXXYK) binds to actin (Chen et al.,

1993, Lukoyanova et al., 2002). Furthermore, the nebulin repeat 1–3, repeat

163–170, repeat 185–SH3 domain and the SH3 domain bind to tropomodulin,

desmin, connectin (also called titin), and myopalladin and b-actinin (also

called CapZ), respectively (Bang et al., 2001; Bang et al., 2002; Jin and Wang,

1991; McElhinny et al., 2001; Witt et al., 2006).

Existence of nebulin in non-vertebrate has not been reported; however,

sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

revealed that bands corresponding to the size of nebulin are present in

amphioxus striated muscle (Hu et al., 1986; Locker and Wild, 1986). In 1997,

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Kimura et al. reported that one of several nebulin antibodies reacted with a

750-kDa protein found in amphioxus striated muscle, and in 1999, Fock and

Hinssen prepared an antibody against this protein and showed that it was

located in the I-Z-I region of the sarcomere. However, because these findings

could have resulted from a nebulin-mimicking epitope or simply from

cross-immunoreactivity with an SH3 domain protein, this 750-kDa protein

was not proven to be nebulin.

In this study, for the first time, I cloned the C-terminal region of the

750-kDa protein identified in amphioxus striated muscle, examined its

functional properties and compared it to vertebrate nebulin.

3. Materials and Methods

3.1. cDNA cloning, sequencing and analysis

Approximately 500 specimens of adult amphioxus (Branchiostoma

belcheri) were collected in the Enshu-Nada Sea, Japan (Kubokawa et al.,

1998). Nerve cord and muscle tissues were used to produce a cDNA library

that was constructed in lambda ZAP II (Stratagene, La Jolla, CA, USA)

using oligo (dT) primers. The fragments encoding the homologue of nebulin

were obtained from the cDNA library using an EST-analysis system

established by the sequencing laboratory in RIKEN, Kobe, Japan (Mineta et

al., 2003). Three clones containing the nebulin homologues were isolated

from approximately 7000 clones of the cDNA library. The domains were

aligned with reference to Labeit and Kolmerer (1995). The sequence analysis

and homology search were performed using GENETYX-WIN Ver. 4.0.4

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(Genetyx, Tokyo, Japan).

3.2. SDS-PAGE and immunoblotting

A piece of freshly excised amphioxus striated muscle was dissolved in 5

volumes of a solution containing 10% SDS, 40 mM dithiothreitol, 10 mM

EDTA and 100 mM Tris-HCl (pH 8.0). The solution with the dissolved muscle

was boiled for 3 min and clarified by centrifugation at 13,500rpm for 10 min.

The supernatant proteins were separated by SDS-PAGE using 2–6%

gradient polyacrylamide gels (acrylamide:methylenebisacrylamide, 30:1.5;

w/w) or 2–15% gradient polyacrylamide gels

(acrylamide:methylenebisacrylamide, 30:0.8; w/w) (Laemmli, 1970). The

protein bands were electrically transferred onto a nitrocellulose membrane

(Towbin et al., 1979) and treated with primary antibody and horseradish

peroxidase-conjugate anti-rabbit IgG secondary antibody (Code number:

P-0399, Dako, Carpinteria, CA, USA).

3.3. Antibody production

The cDNA fragment of amphioxus nebulin (1007–1848 bp of clone 5361)

was generated by restriction enzyme digestion (SmaI/SalI) and inserted into

the PvuII/SalI site of the pGEX4T-3 vector (GE Healthcare, Piscataway, NJ,

USA). Recombinant GST-tagged protein was expressed in Escherichia coli (E.

coli) XL1 Blue-MRF’ with 3 l of LB + ampicillin culture medium under the

conditions of OD600 = 0.5, IPTG 0.1mM, 37°C and 3 h. The bacteria were

harvested by centrifugation (4000rpm, 4°C, 10min), dialysed with 120ml

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PBS, treated with 80ml sample buffer, boiled for 10min and subjected to 227

sheets of 10% SDS-PAGE. The bands of recombinant protein were excised

and the recombinant protein was electrically extracted from the gels into a

running buffer solution (0.1% SDS, 25mM Tris, 192mM glycine) at 400mA for

16h. The protein was dialysed with PBS, and GST was deleted using

thrombin (Sigma-Aldrich Japan, Tokyo, Japan) (1/1000 volume of

recombinant protein solution) at 22°C for 16h. The solution was added to the

sample buffer and subjected to 88 sheets of 12.5% SDS-PAGE. The nebulin

fragment without GST was electrically extracted from the gel, dialyzed with

PBS, conjugated with an equal volume of Freund’s incomplete adjuvant

(Difco Laboratories, Detroit, MI, USA) and injected into three times (0.25,

0.9 and 0.1mg protein) into a rabbit. The antiserum was separated from

blood by centrifugation.

3.4. Immunofluorescence microscopy

After skinning the amphioxus specimens and collecting the muscle tissue

under a stereoscopic microscope, the myofibers were stretched with relaxing

buffer (50 mM KCl, 10 mM EGTA, 10 mM NaPO4, 3 mM ATP and 0.5%

Triton X-100; pH 7.5) and fixed with buffer (3.5% formaldehyde, 45 mM KCl,

9 mM EGTA, 9 mM NaPO4, 2.7 mM ATP and 0.45% Triton X-100; pH 7.5) for

1 min. The tissue was homogenized five times for 2 s each in phosphate

buffer saline (PBS) containing 0.5 mM leupeptin using Ultra-turrax T-25

(IKA-Labortechnik, Staufen, Germany) and then fixed on a glass slide. The

samples were fixed with 3.7% formaldehyde in PBS for 15min, washed two

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times with PBS for 5min and blocked with 1% bovine serum albumin(BSA)

in PBS for 15min. The fixed samples were stained with amphioxus nebulin

(1:50) and a-actinin A7811 (1:800; Sigma-Aldrich Japan, Tokyo, Japan)

antibodies for 12h, washed three times with PBS for 10 min and reacted with

Alexa 488 (1:2500) and Alexa 546 (1:4000) conjugated secondary antibodies

(Invitrogen, Carlsbad, CA, USA). The samples were then washed three times

with PBS for 15min, fixed with 3.7% formaldehyde in PBS for 15 min and

washed with PBS for 15 min. Anti-fador was added to the samples and they

were covered with a cover glass. Fluorescence was observed with

fluorescence microscope (Zeiss Axioskop 2 plus; Carl Zeiss, Oberkochen,

Germany).

3.5. Protein preparation

The cDNA fragments of amphioxus nebulin repeats 3–9 (AN3–9) and the

unique region of the SH3 domain (U–SH3) generated by restriction enzyme

digestion were cloned into the pGEX6P series (GE Healthcare, Piscataway,

NJ, USA). The recombinant proteins tagged with GST were expressed in E.

coli BL21 (DE3) pLysS in 250ml LB containing ampicillin. The conditions

were OD600 = 0.5, IPTG 0.5 mM, 37°C and 3 h. The bacteria were harvested

by centrifugation (3000rpm, 4°C, 10min). The bacteria with the GST-fusion

proteins were dissolved in PBS (140 mM NaCl, 10 mM Na2HPO4, 2.7 mM

KCl and 1.8 mM KH2PO4; pH7.3), frozen at –80°C for 20 min, sonicated at

output 2 for 1min (TOMY UD-200; Tomy Seiko, Tokyo, Japan) and

centrifuged at 13,000rpm for 30min at 4°C. The supernatant was loaded onto

9

a 2ml glutathione Sepharose 4B column (GE Healthcare, Piscataway, NJ,

USA), washed with PBS and eluted with 10mM glutathione (reduced) in 50

mM Tris-HCl (pH 8.0). For the co-sedimentation assay, GST was deleted

from GST–AN3–9 on a column using PreScission Protease (GE Healthcare,

Piscataway, NJ, USA) with buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA

and 1 mM DTT; pH7.0) at 4°C for 12 h. Actin was prepared from acetone

powder of rabbit skeletal muscle using the protocol described by Spudich and

Watt (1971). G-actin was polymerized in 0.1 M KCl. a-Actinin was prepared

from rabbit skeletal muscle by the method of Goll et al. (1972). Connectin

was prepared according to Kimura et al. (1992).

3.6. Far-western blot assay

GST-fusion proteins mixed with GST, and the bacteria with the His-tag

proteins were dissolved in SDS sample buffer, subjected to SDS-PAGE and

transferred onto a nitrocellulose membrane. The membranes were blocked

with 20 mg/ml BSA in buffer (80 mM KCl, 2mM MgCl2, 10 mM Hepes, 1 mM

DTT and 2% Triton X-100, pH 7.3, for α-actinin or connectin) or 0.5% casein

in buffer (2 mM Tris, 0.01% NaN3, 0.1 mM CaCl2 and 100 mM KCl, pH8.0,

for F-actin) at 25°C for 1 h. The membranes were incubated with F-actin (0.3

mg/ml), α-actinin (10 µg/ml) or connectin (80 µg/ml) in each buffer at 25°C for

1.5 h. After washing two times with TBS for 10 min, binding was detected

with anti-actin (Sigma A4700; Sigma-Aldrich Japan), anti α-actinin (Sigma

A7811; Sigma-Aldrich Japan) or anti-connectin (Pc1200) (Kimura et al.,

1992) antibody.

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3.7. Yeast two hybrid assay

The cDNA fragments coding the repeat sequence 1–9 of amphioxus nebulin

or each of the regions of the chicken a-actinin 2 were generated by restriction

enzyme digestion and inserted into pGBT9 or pGAD424 vector (Clontech

Labratories, Palo Alto, CA, USA). The nebulin clone was co-transformed with

the a-actinin clones into Saccharomyces cerevisiae Y170. The transformants

grown on –Leu, –Trp plates were transferred on to a nitrocellulose

membrane and their b-galactosidase activity was examined.

3.8. Co-sedimentation assay

F-actin (2.5 mM) or BSA(5 mM) was mixed with AN3–9 (12.5 mM) in 500 ml

buffer (2 mM Tris, 0.1 mM CaCl2, 0.1M KCl and 0.01% NaN3; pH 8.0) at 25°C

for 1 h. The mixture was centrifuged (13,000 rpm, 4°C, 30 min), and the

supernatant and pellet were diluted with SDS sample buffer and subjected

to SDS-PAGE.

3.9. GST pull down assay

GST–AN3–9 (2mg for a-actinin pull down) or GST–U–SH3 (2mg for

connectin pull-down) were bound to 20 ml glutathione Sepharose 4B beads

according to the manufacture’s method. The beads were washed two times

with 150ml buffer (80 mM KCl, 2 mM MgCl2, 10 mM HEPES, 1 mM DTT and

2% TritonX-100; pH7.3), and bound with a-actinin (4 mg, 20 ml) or connectin

(5 mg, 150 ml) in buffer at 4°C for 1h. They were washed four times with 150

ml buffer, dissolved in 20 ml SDS sample buffer, and subjected to SDS-PAGE.

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The gels were stained with Coomassie Brilliant Blue.

4. Results

4.1. Primary structure of amphioxus nebulin C-terminus

Three clones were obtained from a mixed cDNA library of amphioxus

muscle tissues and nerve cord. Sequencing revealed a 2527-bp sequence

containing a poly-A tail and a stop codon, suggesting that it contains the

C-terminal region of the protein (DDBJ accession no. AB244086). The

amphioxus sequence contains 13 nebulin repeats and an SH3 domain with

unique regions between these modules (Fig. 1A).

Amphioxus nebulin repeats were assigned the numbers Neb-1–13 for

descriptive purposes. The consensus sequence observed in each nebulin

repeat was a PEXXRXKXVXKIQ motif in the N-terminal region and a

GKXYTXVXDT/D motif in the C-terminal region, as well as a SE and a Y in

the SEXXYX-motif (Fig. 1B,C). P1, E2, R5 and K7 in the N-terminal and S14

and Y18 in the SEXXYX motif of the amphioxus nebulin repeats correspond

to the human C-terminal nebulin repeats (M172–185) (Fig. 1C). The

SSVLYKEN-box, which is present in human M174–181, was not found in the

amphioxus nebulin repeats.

4.2. Comparison between amphioxus and vertebrate nebulins

The amphioxus nebulin repeats shared an approximately 26% homology

with human C-terminal nebulin repeats. I predicted the secondary structure

of the amphioxus nebulin repeat region using the Multivariate Linear

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Regression Combiner secondary structure prediction program

(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_mlrc.ht

ml). The secondary structure was composed of 48% a-helix, 0% b-structure

and 47% random coil. The composition was similar to that of the secondary

structure of the C-terminal region of the human nebulin repeat (41% a-helix,

0% b-structure and 52% random coil) predicted using the same program.

Calculation of the isoelectric points (pIs) based on the amphioxus C-terminal

amino acid sequence revealed pIs of 5.6 and 7.1–10.8 (total pI=9.9) for Neb-1

and Neb-2–13, respectively indicating that the pIs of the amphioxus nebulin

repeats are in accordance with those of the human nebulin repeats (pI = 5.6

for M172 and pI = 10.0 for M173–185) (Labeit and Kolmerer, 1995) (Fig. 2).

The PGSIFDYEP of the last nebulin repeat (Neb-13) in amphioxus (Fig.

1B) is conserved across species, and corresponds to the last human nebulin

repeat M185 (X83957) (Labeit and Kolmerer, 1995), chicken nebulin repeat

M65 (AB024330) (Suzuki et al., 2000), the zebrafish nebulin-like protein

(AL974314) and the nebulin-like protein (nebulette) of vertebrate cardiac

muscle (Y16350) (Millevoi et al., 1998; Moncman and Wang, 1995).

Furthermore, when I compared the C-terminal SH3 domain of amphioxus

nebulin to human (X83957), chicken (AB024330) and zebrafish (AL974314), I

observed a high homology (approximately 65%) with human nebulin (Fig. 2),

revealing that the amino acid sequence of the SH3 domain was well

conserved across species. However, two unique regions, which are between

Neb-12 and Neb-13 and between Neb-13 and the SH3 domain, showed no

homology with any region of the human nebulin (Fig. 2).

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The domain structure, secondary structure and the pIs of the C-terminal

region of amphioxus nebulin are similar to those of vertebrate nebulin.

4.3. Localization of the amphioxus nebulin C-terminal region in the

sarcomere

To confirm that the cDNA was from amphioxus nebulin and to localize the

C-terminal region within the sarcomere, I prepared an antibody (PcAmpN)

using the recombinant protein from Neb 10 to the former half of the unique

region as an antigen (Figs. 1A, 3A). Immunoblot analysis showed that the

antibody reacted only with the 750-kDa band and not with other proteins

such as nebulette (107 kDa) (Fig. 3B,C). This confirmed the sequence to be

that of amphioxus nebulin cDNA.

Double immunostaining of amphioxus myofibrils with PcAmpN and an

a-actinin monoclonal antibody revealed that the C-terminal region of

amphioxus nebulin is localized to the Z-line of the sarcomere and co-localizes

with a-actinin (Fig. 4).

4.4. Binding of amphioxus nebulin to F-actin, a-actinin and connectin

It is known that vertebrate nebulin is localized along thin filaments and

the nebulin repeats binds to actin. To clarify whether amphioxus nebulin

repeats bind to actin, I examined the binding of nebulin repeats 3–9 to

F-actin by far-western blot analysis and a co-sedimentation assay. The

far-western blot analysis showed that F-actin binds to the amphioxus

nebulin repeats, similar to human nebulin repeats (Fig. 5A). Furthermore,

14

the results of the co-sedimentation assay showed that F-actin alone did not

precipitate after centrifugation at 13,000 rpm for 30 min, however, F-actin

co-precipitated with the nebulin repeats, indicating that amphioxus nebulin

repeats bind to F-actin (Fig. 5B).

Next, I examined binding of the amphioxus nebulin repeats with purified

a-actinin by far-western blot analysis, a yeast two-hybrid assay and a GST

pull-down assay. The results of the far-western blot analysis showed that

a-actinin bind to nebulin repeats 3-9 (Fig. 6A). I also created cDNA

fragments encoding each region of a-actinin 2 in the striated muscles, and

examined their binding ability to repeats 1–9 of amphioxus nebulin using the

yeast two-hybrid system. The 343–804 amino-acid fragments of a-actinin

showed b-galactosidase activity, while the fragments without this region did

not (Fig. 6B), demonstrating the direct binding of 343–804 amino-acid

fragments of a-actinin to amphioxus nebulin. Furthermore, the results of the

GST pull-down assay showed that a-actinin precipitated when added to the

GST-fusion protein with nebulin repeats 3–9 (lane 5 in Fig. 6C), but not

when it was added only to GST (lane 6 in Fig. 6C). The results of a control

experiment showed that amphioxus nebulin repeats 3–9 do not bind to BSA

(Fig. 6D).

I examined binding of the SH3 domain from the unique region (U–SH3) in

amphioxus nebulin with purified rabbit connectin by far-western blot

analysis and a pull-down assay. The results of the far-western blot analysis

showed that connectin binds to the U–SH3 domain (Fig. 7A). Furthermore,

the result of the GST pull-down assay revealed that connectin binds to the

15

U–SH3 domain of amphioxus nebulin, but not to GST alone (Fig. 7B).

5. Discussion

The present study elucidated the primary structure, localization and

binding proteins of the C-terminal region of amphioxus nebulin. These

results confirm earlier findings that nebulin is expressed in amphioxus and

suggest that it plays a role similar to that of vertebrate nebulin.

In human nebulin, repeats M167–172 are negatively charged and repeats

M173–185 are positively charged under physiological conditions. Similarly,

in amphioxus nebulin, Neb-1 is negatively charged and Neb-2–13 is

positively charged. Thus, the charge transition is conserved between human

and amphioxus nebulins. Although the function of the charge transition

region is unknown, its localization within or around the Z-line suggests that

it is involved in the formation and structural maintenance of the Z-line.

a-Actinin is the main component of the Z-line of the sarcomere, and is also

the location of the N-terminus of connectin (titin) (Tskhovrebova and Trinick,

2003). I showed that amphioxus nebulin bind to a-actinin and connectin

(titin) and that the C-terminal region of amphioxus nebulin is localized in

the Z-line of the sarcomere (Fig. 4). These results suggest that amphioxus

nebulin maintains the structure of the Z line by binding to a-actinin and

connectin, similar to the C-terminal region of human nebulin.

I predicted that the secondary structure of the amphioxus nebulin repeat

region was 48% a-helix and 0% b-structure. The secondary structure was

similar to that of the C-terminal region of the human nebulin repeat. This is

16

because both amphioxus and human nebulins have the consensus sequences

PEXXRXK at the N terminal, SXXXYX in the middle and GKXXTXXXXT at

the C-terminal of each repeat. As a result, amphioxus nebulin functions

similar to human nebulin, even though there is only a 26% homology at the

sequence level. Because the helical part of the protein can present the

conserved surface and interact to other proteins (McLachlan and Karn,

1982).

The thin filaments in the striated muscle of arthropods and molluscs do

not have a uniform length of 1 mm as in vertebrate skeletal muscle, whereas

the thin filaments of amphioxus striated muscle have a uniform length of 1

mm (Hagiwara et al., 1971). Although amphioxus is not a vertebrate and is

the phylogenetically lowest chordate, its nebulin has a similar function to

vertebrate nebulin, which might explain the uniform 1 μm size of the thin

filaments in amphioxus striated muscle. Taken together with my result of

the interaction of amphioxus nebulin with actin, a-actinin and connectin, the

thin filaments in amphioxus striated muscle might be maintained in a

manner identical or similar to that in the thin filaments of vertebrate

skeletal muscle.

The existence of nebulin protein have not be seen in invertebrate without

amohioxus. In fact, nebulin gene cannot be seen in Drosophila melanogaster,

Caenorhabditis elegans and Strongylocentrotus purpuratus genome.

However urochordate ciona, which is evolved from cepharochordate, have

nebulin gene in its genome. These insights indicate chordate (vertebrate,

urochordate and cepharochordate) have nebulin but the other creatures don’t

17

have nebulin.

6. Conclusion

Invertebrate amphioxus have nebulin and its structure and functions are

similar to human nebulin. These insights show that nebulin is not

vertebrate-only protein but chordate protein. Detailed elucidation of the

differences of thin filaments between chordate and non-chordate will lead to

a better understanding of the nebulin function such as length regulator of

the thin filaments.

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tropomyosin-troponin complex with actin and the proteolytic fragments of

myosin. J. Biol. Chem. 246, 4866-4871.

Suzuki, T., Yajima, H., Maruyama, K. and Kimura, S. (2000) A 7.5-kb 3'

terminal cDNA sequence of chicken skeletal muscle nebulin reveals its actin

binding regions. Zool. Sci. 17, 1095-1099.

21

Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of

proteins from polyacrylamide gels to nitrocellulose sheets: procedure and

some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354.

Tskhovrebova, L. and Trinick, J. (2003) Titin: properties and family

relationship. Nat. Rev. Mol. Cell Biol. 4, 679-689.

Wang, K. (1982) Purification of titin and nebulin. Methods Enzymol. 85,

264-274.

Wang, K. and Wright, J. (1988) Architecture of the sarcomere matrix of

skeletal muscle: immunoelectron microscopic evidence that suggests a set of

parallel inextensible nebulin filaments anchored at the Z line. J. Cell Biol.

107, 2199-2212.

Witt, C. C., Burkart, C., Labeit, D., McNabb, M., Wu, Y., Granzier, H. and

Labeit, S. (2006) Nebulin regulates thin filament length, contractility, and

Z-disk structure in vivo. EMBO J. 25, 3843-3855, 2006.

22

A

B

C Fig 1. Domain structures and primary structure of the nebulin C-terminal region in amphioxus striated muscle cDNA. Domain structures (A) and amino acid sequence (B) of amphioxus nebulin, and consensus sequences of amphioxus and human nebulin (C). Fine bar, size of each clone. Bold bar, Pc AmpN antigen.

Neb1-12 Neb13 SH3

Unique region SH3 domain

Antigen

Clone:53616024

907polyApolyApolyA

Nebulin repeat

Neb1-12 Neb13 SH3

Unique region SH3 domain

Antigen

Clone:53616024

907polyApolyApolyA

Nebulin repeat

K----ERAELEKHK-YTQLPDT

Neb1 PDISFAKEVHDITSETKYK----EEYNKERGK-YSMVAQT

Neb2 PLYETAQKANKLTSETLYK----KAYKALQGKGY-ALIDT

Neb3 PEFERCKQVNKLASRTKYH----EDYEKSKIKGYTPVVDT

Neb4 PENIRLRQQSKIQSQNEYQ----KDYKALSGK-FHYTADN

Neb5 PEHNRIRSVSKIQSQIHYG----PSRLEQHGQKYHAAIDD

Neb6 PELKRLQEVGKFQSQVLYGEITDKDDASKGRGKYTQIQQD

Neb7 PEMKRLKEANAYASEAQVA----VRRFDEK-NRYTTVLDD

Neb8 PELNRMKNVGKFQSELQYK----DKWTRKTGARYTNVLDD

Neb9 PEMNRIKGVTKMLSEVQYR----DAEL-KTG-RLTAVTDD

Neb10 LDTKRVKRNTKIQSENEYR-------KGRKINTYNAVLDT

Neb11 PDMLRIRNATKISSEVKYH----EDFEKQKGKAYHTVIDD

Neb12 PELTRVKKNTVIQSNLAYQGVKEKLQTMESRRQMI

Unique PGEPSMPPPAQQLGTQMEARMAADASYAQFKAKGKGKV

Neb13 GGRYMPIRLGDPGSIFDYEPYQGKEEAGRAIYVTTATWK

Unique PAKPIRTIEDFVVEEESAEEALEARDKITEMDMEYERRRGEGLSYEYDPQKYSHHYWS

KTTSELGGVVTTSGMKYRKIEDFLNLNADLIYSTENESTRESGSETSGMWDYDPDKYW

RARGYRISAEQRYQQVRLKPGKIEDYDPMTHPDAQPKFEAPAAVSEPEPVIQSGNVMR

SH3 AVFDYAAAEDDEISFMDGDVIVNCVKIDDGWMTGTVQRTGQSGMLPANYVEPMS

Amphioxus PE--R-K-V-KIQSEXXYX-----------GK-YT-V-DT/D

** * * * * ** * *

Human PEM-RVK-NQENISSVLYKEN---------GK--TP-P-T

23

Fig 2. Comparison of the C-terminal region between amphioxus and human nebulins. The domain structure and isoelectric point (pI) are presented according to Labeit and Kolmerer (1995), and the I-Z border is presented according to Millevoi et al. (1998). Amphioxus striated muscle nebulin, Amphioxus; Human skeletal muscle nebulin, Human.

N2-12N1N13

M169-172

SH3

SH3

pI=10.0pI=5.6

pI=9.9pI=5.6

26% 11% 65%

Z-line

Amphioxus

HumanM173-185

Unique region SH3 domainNebulin repeat

N2-12N1N13

M169-172

SH3

SH3

pI=10.0pI=5.6

pI=9.9pI=5.6

26% 11% 65%

Z-line

Amphioxus

HumanM173-185

Unique region SH3 domainNebulin repeat

24

Fig 3. SDS-PAGE and immunoblot detection of amphioxus striated muscle. Antigen for amphioxus nebulin (A). Molecular size marker, lane M; Pc AmpN antigen (bold bar of Fig. 1A), lane 1. Immunoblot detection of amphioxus striated muscle using Pc AmpN (B, C). Electrophoresis was performed on 2–6% (B) or 2–15% (C) polyacrylamide gradient gels. Molecular size marker, lane M; amido black stains, lane 1; immunoblot detection by Pc AmpN, lane 2. Connectin, C; Nebulin, N; Myosin heavy chain, MHC.

2209766

45

24

18

(kDa) M 1A B

N

MHC

C

21C

220

9766

45

2418

(kDa) M 1 2

N220

9766

45

24

18

(kDa) M 1A

2209766

45

24

18

2209766

45

24

18

(kDa) M 1A B

N

MHC

C

21B

N

MHC

C

21

N

MHC

C

21C

220

9766

45

2418

(kDa) M 1 2

N

C

220

9766

45

2418

(kDa) M 1 2

N

25

Fig. 4. Immunofluorescence microscopic observations of amphioxus striated muscle sarcomeres using Pc AmpN (B) and Mc a-actinin (C). Phase-contrast image (A) and merged image of Pc AmpN and Mc a-actinin (D). Arrow head, Z line. Bar, 10 mm.

A

C

D

B

A

C

D

B

26

A B Fig. 5. Binding of amphioxus nebulin and F-actin. (A) Binding of amphioxus nebulin and F-actin by far-western blotting. 1, Molecular weight marker; 2, Amido black of (AN3-9 + GST); 3, + F-actin, treated with actin antibody; 4, – F-actin, treated with actin antibody (negative control of 3). (B) Binding of amphioxus nebulin and F-actin by co-sedimentation. Actin only, Actin; amphioxus nebulin repeats 3–9 dissolved in GST, AN3–9; amphioxus nebulin repeats 3–9 + actin, AN3–9 + Actin; amphioxus nebulin repeats 3–9 + BSA, AN3–9 + BSA. Before centrifugation, W; supernatant after centrifugation, S; pellet after centrifugation, P. Electrophoresis was performed on a 12.5% polyacrylamide gel.

W S P W S P W S P W S PActin AN3-9 AN3-9+ActinAN3-9+BSA

BSAActin

AN3-9

220

9766

45

24

(kDa)

GST3-9

1 2 3M

27

A B

C D

1 2 3 4 5 6220

97

66

45

24

(kDa)

a-Actinin

GST-AN3-9

GST

1 2 3 4 5 6220

97

66

45

24

(kDa)

a-Actinin

GST-AN3-9

GST

18

9766

45

24

(kDa)

His1-21 2 1 2 1 2 1 2

His3-9

His10-U

HisU-SH3

M

220

97

66

45

24

(kDa)1 2 3 4

CH SPEC EFEFSPECSPECSPECCHa-act

343-897343-804426-804343-729

Amphioxus Nebulin binding-++-

343-897 343-804 426-804343-729

CH SPEC EFEFSPECSPECSPECCHa-act

343-897343-804426-804343-729

Amphioxus Nebulin binding-++-

343-897 343-804 426-804343-729

28

Fig. 6. Binding of amphioxus nebulin and a-actinin. (A) Binding of amphioxus nebulin C-terminal region and a-actinin by far-western blotting. 1, treated with polyhistidine antibody; 2, treated with a-actinin antibody. U, unique region; SH3, SH3 domain. (B) Binding of amphioxus nebulin and a-actinin by the yeast two-hybrid system. a-act, Domain structure of a-actinin; CH, calponin homology domain; SPEC, spectrin repeat; EF, EF hand. +, b-Gal (b-Galactosidase) positive; –, b-Gal negative. (C) Binding of amphioxus nebulin and a-actinin by GST pull-down assay. 1, Molecular size marker; 2, a-actinin used for the pull-down assay; lane3-6, after the pull-down assay.; 3, GST fusion protein for amphioxus nebulin repeats 3–9–a-actinin; 4, GST only–a-actinin; 5, GST fusion protein for amphioxus nebulin repeats 3–9+a-actinin; 6, GST only+a-actinin; GST–AN3–9, GST fusion protein for amphioxus nebulin repeats 3–9. Electrophoresis was performed on a 10% polyacrylamide gel. (D) Binding of amphioxus nebulin repeats 3−9 and BSA as a control for binding of nebulin repeats 3−9 and α-actinin, by a GST pull-down assay. 1, Molecular size marker; 2, BSA used for pull-down assay; lane3 and 4, after pull-down assay; 3, GST fusion protein for amphioxus nebulin repeats 3−9 + BSA; 4, GST fusion protein for amphioxus nebulin repeats 3−9 − BSA; GST−AN3−9, the GST fusion protein for amphioxus nebulin repeats 3−9. Electrophoresis was performed on a 10% polyacrylamide gel.

29

A B Fig. 7. Binding of amphioxus nebulin and connectin. (A) Binding of amphioxus nebulin and connectin by far-western blotting. 1, Molecular weight marker; 2, Amido black of (U–SH3 + GST); 3, + connectin, treated with connectin antibody; 4, – connectin, treated with connectin antibody (negative control of 3). U, unique region; SH3, SH3 domain. (B) Binding of amphioxus nebulin and connectin by GST pull-down assay. 1, Molecular size marker; 2, rabbit skeletal muscle; 3, connectin used for the pull-down assay; lane4-7, after the pull-down assay; 4, GST–U–SH3 – connectin; 5, GST only–connectin; 6, GST–U–SH3+connectin; 7, GST only+connectin; GST-U–SH3, GST fusion protein for amphioxus nebulin U–SH3 region. Electrophoresis was performed on a 2–15% polyacrylamide gel.

1 2 3 4 5 6 7

220

9766

45

24

(kDa)

18

Connectin

GST-U-SH3GST

1 2 3 4 5 6 7

220

9766

45

24

(kDa)

18

Connectin

GST-U-SH3GST

18

9766

45

24

(kDa)2 3

GSTU-SH3

M 1

30

Chapter 2

Structure of the amphioxus nebulin gene and evolution of

the nebulin family genes

31

1. Abstruct

Nebulin family genes are believed to have diverged from a single gene

during the evolution of vertebrates. I determined the structure of the

amphioxus nebulin gene and showed that in addition to the features of the

human nebulin gene, this gene had a LIM domain, secondary super repeats

and a giant exon with 98 nebulin repeats containing unique sequences. A

transcript of this gene amplified by reverse transcriptase-polymerase chain

reaction had an LIM domain, three nebulin repeats and an SH3 domain.

This transcript was similar to an isoform of human nebulette (Lasp-2).

Phylogenetic analysis using the LIM and SH3 domains of the nebulin family

proteins showed that amphioxus nebulin is located outside the vertebrate

nebulin family group in the phylogenetic tree. These results indicated that

the amphioxus nebulin gene had a unified structure among nebulin,

nebulette, lasp-1 and N-RAP of vertebrates, and that these nebulin family

genes diverged from the amphioxus nebulin gene during the course of

vertebrate evolution.

32

2. Introduction

The nebulin family of proteins includes Nebulin, Nebulette, N-RAP and

Lasp-1 actin binding proteins, which are characterized by nebulin repeats.

Nebulin is a large protein (molecular mass: 773 kDa) found on the thin

filaments of vertebrate skeletal muscles (Wang, 1982; McElhinny et al.,

2003). The primary structure of human nebulin is an N-terminal glutamic

acid-rich domain, followed by 185 (M1–185) contiguous nebulin repeats of

approximately 35 amino acids with a central SDXXYK consensus sequence, a

C-terminal serine-rich region and an Src homology 3 (SH3) domain. Nebulin

repeats (M9–162) in the I-band region of the sarcomere comprise 22 sets of

seven-repeats, called super repeats (SR1-22) (Labeit and Kolmerer, 1995).

The gene for the production of human nebulin is located in chromosome

2q21-22 and is composed of 183 exons spanning 249 kb of the genome

sequence, an area that contains 29 equivalent super repeats (Donner et al.

2004).

Nebulette is a 107 kDa protein located in the Z-line of cardiac muscle

(Moncman and Wang. 1995). The primary structure of human nebulette is a

N-terminus glutamate acid-rich domain, followed by 23 nebulin repeats, a

C-terminal ser-rich region and an SH3 domain (Millevoi et al. 1998). A

34-kDa splicing isoform of nebulette is Lasp-2 (LIM-nebulette), observed in

various tissues and has an N-terminus LIM domain, followed by 3 nebulin

repeats, a C-terminus ser-rich region and an SH3 domain (Katoh and Katoh.

2003; Terasaki et al. 2004; Li et al. 2004). The gene producing human

nebulette is located in chromosome 10p12 and spans 393 kb—an area

33

containing 28 exons (exon 1–28) for nebulette protein (Arimura et al. 2000)

and four additional 5’ exons (1a-4a) only expressed in Lasp-2 protein

(Katoh and Katoh. 2003). Lasp-2 is coded by exons 1a–4a, 24, 27 and 28.

N-RAP is a 196 kDa protein that has been reported in striated muscles

(Luo et al. 1997). The N-terminus of human N-RAP is a LIM domain, and is

followed by 11 nebulin repeats and 5 super repeats (35 nebulin repeats). The

gene responsible for the production of human N-RAP is located in

chromosome 10q25.3 and spans 75 kb, an area that contains 42 exons

(Mohiddin et al. 2003).

Lasp-1 is a 38 kDa protein (Tomasetto et al. 1995) that is highly expressed

within epithelial and brain tissues. It consists of an N-terminus LIM domain,

followed by two nebulin repeats, a C-terminal unique region and an SH3

domain. The gene encoding human Lasp-1 production is located in

chromosome 17q12-21 and spans 52 kb.

The genes of the above four proteins are believed to have diverged from a

single gene by gene duplication during vertebrate evolution. In order to

explore this hypothesis thoroughly, it was deemed necessary to find a gene

that was common to the nebulin family in amphioxus, a cephalochordate (i.e.,

an invertebrate chordate), and the nearest ancestor to the vertebrates. In

chapter 1, I demonstrated the existence of amphioxus nebulin by revealing

its C-terminal primary structure, localization and binding protein. In

chapter 2, I reveal the structure of the amphioxus nebulin gene and analyze

the hypothesis that the nebulin family arose from a single gene.

34

3. Materials and Methods

3.1. Genome analysis

The scaffold coding amphioxus nebulin gene was obtained from the

Branchiostoma floridae genome assembly release v2.0 (Putnam et al. 2008)

on the JGI portal site (http://genome.jgi-psf.org/Brafl1/Brafl1.home.html) by

BLAST search using the sequence of the SH3 domain of the amphioxus

(Branchiostoma belcheri) nebulin (Chapter 1). The complementary sequence

of No. 205 in 398 connected sets of allelic scaffolds was identified (amino acid

homology 98%), and translated to 3 frame amino acids. The exons were

visually determined referred to the C-terminus sequence of amphioxus

nebulin (Chapter 1), an EST sequence of B. floridae (BW805004) and the

sequence of human nebulin (Labeit and Kolmerer. 1995). The connecting

error of the No. 205 set was proofed by comparison with scaffold 169 of the B.

floridae v1.0 assembly.

3.2. Reverse transcriptase-polymerase chain reaction (RT-PCR) and

sequence

Collection of amphioxus, isolation of RNA and construction of the first

strand of cDNA by reverse transcription (RT) were performed by Mizuta and

Kubokawa (2007). PCR reactions were performed using a forward primer

(cgcggatccATGAATCCGAAGTGTGCACG; BamHI site plus N-terminus of

LIM domain) and a reverse primer

(cgcgaattcCGACATGGGCTCCACGTAGT; EcoRI site plus C-terminus of SH3

domain) with a condition schedule of 1 min at 95°C, followed by 30 s at 95°C,

35

30 s at 50°C and 1 min at 72°C for 25 cycles, followed by 3 min at 72°C; the

product obtained was stored at 4°C. Sequences were analyzed using an ABI

PRISM 310 sequencer (Applied Biosystems, Foster City, CA, USA).

3.3. Phylogenetic analysis

The amino acid sequences of the LIM and SH3 domains were aligned in

various species and proteins and 1000 bootstrap values were calculated

using the CLUSTALW algorithm (Thompson et al. 1994). Phylogenetic trees

were visualized using TreeView (Page, 1996).

4. Results

4.1. The structure of the amphioxus nebulin gene

Using the amino acid sequence of the SH3 domain of amphioxus nebulin

(Chapter 1), I searched for an amphioxus nebulin gene in the B. floridae v2.0

genome assembly (Putnam et al. 2008) and identified a complementary

sequence of No. 205 in 398 connected sets of allelic scaffolds encoding

nebulin genes. The exons encoding nebulin repeats were visually determined

with reference to human nebulin sequences (Labeit and Kolmerer, 1995),

whereas the exons encoding the unique sequence of the amphioxus nebulin

C-terminus and the SH3 domain were searched using homology with the

C-terminal sequence of amphioxus nebulin (Chapter 1). Furthermore, exons

encoding the N-terminal LIM domain and unique sequences were searched

based on homology with the EST sequence(BW805004).

The predicted amphioxus nebulin gene comprised 208 kb with 283 exons

36

(Fig. 1), and was smaller and had more exons than the human nebulin gene

(249 kb with 183 exons). The size range of the exons were very various from

18 b (exon 277) to 15 kb (exon 260).

All exons were connected and aligned (Fig.2), and exons 1 and 2, encoding

the LIM domain, were not observed in the human nebulin gene. These exons

were separated by 24.7 kb from the other exons.

Exon 3–269 encoded nebulin repeats, while in exon 9–245, nebulin super

repeats were observed, including those super repeats observed in human

nebulin. However, the amphioxus nebulin gene has 2 sets of similar super

repeats, one containing 21 super repeats (SR01–21) and the other containing

20 super repeats (SR22–41). These sets were different from those in the

human nebulin gene, which has one set of 22 super repeats. Each set of super

repeats had amino acids that were highly conserved. In particular, repeat 2

among the super repeats was more similar than other repeats within the

super repeats. Furthermore, the WLKGIGW motif observed in the super

repeat sequences of human nebulin was not observed in that of amphioxus

nebulin.

Exon 260 is a large exon with 98 nebulin repeats containing unique

sequences at the N- and C-terminus, which show no similarities to other

proteins in the database (Fig. 3). This exon is located between the exons that

encode Neb04/05 and Neb05/06, and is not observed at the C-terminus

sequence of either amphioxus (Chapter 1) or human nebulin. Expression of

the C-terminus of this exon was observed in the neurula stage EST clones of

B. floridae (DDBJ accession BW855343; underlined in Fig. 3).

37

Exons 266–267 were two additional nebulin repeats that could not be

observed in the sequence of the amphioxus nebulin C-terminus

(Neb00/01–Neb11/12) (Chapter 1). The C-terminus of human nebulin has a

splicing isoform region called a ‘hot spot’, but the C-terminus of amphioxus

nebulin has few alternatively spliced exons compared to that of human

nebulin. Exon 270–282 encoded unique sequences and exon 283 encoded the

SH3 domain. While exons 271–283 are expressed in amphioxus nebulin

(Chapter 1), exon 270 is not.

These results indicate that the amphioxus nebulin gene has exons

encoding a LIM domain, a set of secondary super repeats and 98 nebulin

repeats containing unique sequences, in addition to having exons that are

present in the human nebulin gene.

4.2. A small transcript from the amphioxus nebulin gene

In order to search for a transcript that had an LIM domain in the

amphioxus nebulin gene that was not observed in human nebulin, I

performed RT-PCR using amphioxus (B. belcheri) total RNA and primers

that encoded the N-terminus of the LIM domain as well as the C-terminus of

the SH3 domain. A transcript was identified (Fig. 4A) that had an LIM

domain and a unique sequence at the N-terminus, three nebulin repeats and

a unique sequence and an SH3 domain at the C-terminus (Fig. 4B). This

sequence was encoded by exons 1, 2, 3, 258, 269, 270, 282 and 283 (Fig. 1). In

particular, exons 258, 269, 282 and 283 were visible on the C-terminus of

amphioxus nebulin (Chapter 1), but exon 270 was not. Domain structures

38

and splicing pathways observed in this transcript were not observed in

human nebulin. Instead, they were similar to human Lasp-2, which is a

splicing isoform of nebulette (Li et al. 2004; Terasaki et al. 2004) (Fig. 4C).

These results indicate that the nebulette gene is merged with the nebulin

gene in amphioxus.

4.3. The evolution of vertebrate nebulin family genes from the amphioxus

nebulin gene

The results show that the amphioxus nebulin gene has many sequences in

addition to those observed in human nebulin, and that a small transcript

from the amphioxus nebulin gene is similar to the Lasp-2 isoform of human

nebulette. Therefore, I investigated the possibility that vertebrate nebulin

family genes diverged from an amphioxus nebulin gene.

Molecular phylogenetic analysis using amino acid sequences of the SH3

domains in nebulin, nebulette and Lasp-1 showed that each vertebrate

protein, from fish to human, formed its own specific group (i.e., a vertebrate

nebulin group, a vertebrate nebulette group and a vertebrate Lasp-1 group),

and that each group formed a vertebrate super-group (Fig. 5A). Furthermore,

the analysis showed that Ciona nebulin (urochordate) and amphioxus

nebulin (cephalochordate) were located outside of the vertebrate

super-group.

Similar analysis using amino acid sequences of LIM domains in nebulette,

Lasp-1 and N-RAP showed that each protein of vertebrate formed one group,

and that amphioxus nebulin was located outside the vertebrate group (Fig.

39

5B).

These results indicate that the genes of vertebrate nebulin, nebulette,

Lasp-1 and N-RAP diverged from an amphioxus nebulin gene.

5. Discussion

In this study, I have determined the structure of the amphioxus nebulin

gene and have shown that this gene has exons encoding the LIM domain,

secondary super repeats and 98 nebulin repeats containing unique

sequences, in addition to having exons similar to those of the human nebulin

gene. I have also identified a small isoform from this amphioxus nebulin

gene and have shown that the isoform is similar to the Lasp-2 isoform of

human nebulette. Furthermore, I have revealed the evolution of nebulin

family proteins from the amphioxus nebulin gene.

5.1. LIM domain

The presence of the exons encoding an LIM domain in the amphioxus

nebulin gene has been demonstrated and I have also demonstrated the

expression of these exons in a small isoform. These results indicate that in

amphioxus, the nebulette gene is the same as the nebulin gene. However,

this insight did not lead to the conclusion that the N-terminus of amphioxus

nebulin is an LIM domain. In fact, the exon encoding the LIM domain is 24.7

kb away from other exons. Among exons, this is a relatively long distance

and a similar great amount of separation is only observed in a small isoform

of human nebulette, (Lasp-2; 152.2 kb away from other exons). Instead of an

40

LIM domain, nebulette has a unique sequence at the N-terminus. This

indicates that nebulette does not require an LIM domain, even if the exons at

the N-terminus of the gene encode an LIM domain. I performed an analysis

of the N-terminal amino acids within the amphioxus nebulin protein, using a

750-kDa band transferred onto a polyvinylidene fluoride membrane; however,

I could not determine the sequence of amphioxus nebulin due to the high

molecular weight of nebulin (data not shown). Thus, the determination of the

N-terminal sequence of amphioxus nebulin is a future task.

5.2. Super repeats

My data show that the amphioxus nebulin gene has two super repeat

groups: one contains 21 super repeats and the other contains 20 super

repeats. In human nebulin, there are 29 super repeats (Donner et al. 2004).

Twenty-two of those are expressed in skeletal muscles (Labeit and Kolmerer.

1995) and the other seven are expressed only in foetal muscles (Donner et al.

2004). Since the molecular weight of nebulin in amphioxus striated muscles

(750 kDa, Chapter 1) is slightly lower than that in human adult skeletal

muscles (773 kDa, Labeit and Kolmerer 1995), the expression of one group of

21 or 20 super repeats is possible in amphioxus nebulin. On the other hand,

the expression of both groups of 21 and 20 super repeats is unlikely in

amphioxus nebulin, because the length of thin filament (about 1 mm) in

amphioxus striated muscle has similar length to that in human skeletal

muscle (Hagiwara et al. 1971). My phylogenetic analysis showed that the

N-RAP gene diverged from the amphioxus nebulin gene. This may mean that

41

one group of the super repeats expresses nebulin, whereas the other

expresses N-RAP. Two unresolved issues for future studies are: the reasons

for the presence of two groups of super repeats in the amphioxus nebulin

gene, and identification of the group from these two groups that is expressed

in amphioxus nebulin.

All amino acid sequences in the super repeats from the amphioxus nebulin

gene lack a WLKGIGW-motif, which is assumed to be a

tropomyosin/troponin binding motif. However, because the second repeat in

every repeat is more conservative than those between the super repeats of

each group, this second repeat may be important for binding to the

tropomyosin/troponin complex.

5.3. Giant exon

Here, I report the detection of a giant exon in amphioxus that encodes 98

nebulin repeats and has unique sequences at the N- and C- terminals. The

expression of this exon was observed in the EST sequence (BW855343). This

giant exon does not exist in nebulin, nebulette or N-RAP genes of vertebrates.

There are three possible reasons for the expression of this giant exon. The

first possibility is that partial exons within the giant exon are expressed and

play a role similar to that of the hot spot of alternatively spliced exons in the

human nebulin C-terminus. The reason for considering this possibility is

that this giant exon is inserted between the exons encoding Neb3/4 and

Neb4/5 of the C-terminus of nebulin amphioxus (Chapter 1) and human

nebulin, and that this region is a hot spot of alternatively spliced exons. The

42

second possibility is that this giant exon is expressed with other exons,

especially with the SH3 domain, and it performs the functions of amphioxus

nebulette, because like human nebulette, this giant exon has no super repeat.

The third possibility is that only this giant exon is expressed, it performs

functions specific to the striated muscles of amphioxus and these functions

differ from those of nebulin, nebulette, N-RAP and Lasp-1. Future

investigations of the expression and functional analysis of this exon should

lead to new insights in amphioxus striated muscle.

5.4. Gene separation

It is believed that vertebrates have more genes than their closest

invertebrate relatives (amphioxus) because of gene duplication (Holland et al.

1994, 2003). This belief leads to the hypothesis that nebulette, N-RAP and

lasp-1 genes emerged through nebulin gene duplication at an early stage of

vertebrate evolution. This hypothesis is partially supported by the

observation that the cardiac muscle of primitive vertebrates, such as jawless

lamprey and hagfish, have nebulin, but that this nebulin has changed to

nebulette in further evolved vertebrates, such as sharks (Fock and Hinssen

2002). However, there is no direct evidence for this hypothesis at the gene

level, and my results show that there are nebulette exons and possibly

N-RAP exons in the amphioxus nebulin gene. Furthermore, other than the

nebulin gene, I could not find genes corresponding to the production of

nebulette, N-RAP or lasp-1 in the amphioxus genome. My results are the

first direct data to support the fact that nebulin, nebulette, N-RAP and

43

lasp-1 are present in a single gene, and that these results will assist in the

analysis of nebulin family evolution in vertebrates.

6. Conclusion

The amphioxus nebulin gene has many exons that are observed not only in

vertebrate nebulin, but also in other nebulin family genes in vertebrates.

Elucidation of this gene and its transcripts will lead to a better

understanding of the evolution of nebulin family proteins and their

functions.

44

7. References

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Donner, K., Sandbacka, M., Lehtokari, V.L., Wallgren-Pettersson, C., Pelin,

K. (2004) Complete genomic structure of the human nebulin gene and

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Fock, U., Hinssen, H. (2002) Nebulin is a thin filament protein of the cardiac

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75-84.

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the cytoskeletal proteins LIM-nebulette and Lasp-1. J. Biol. Chem. 279,

45

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cardiac muscle. Cell Motil. Cytoskeleton 32, 205-225.

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46

Dubchak I, Garcia-Fernàndez J, Gibson-Brown JJ, Grigoriev IV, Horton AC,

de Jong PJ, Jurka J, Kapitonov VV, Kohara Y, Kuroki Y, Lindquist E, Lucas

S, Osoegawa K, Pennacchio LA, Salamov AA, Satou Y, Sauka-Spengler T,

Schmutz J, Shin-I T, Toyoda A, Bronner-Fraser M, Fujiyama A, Holland LZ,

Holland PW, Satoh N, Rokhsar DS (2008) The amphioxus genome and the

evolution of the chordate karyotype. Nature 453, 1064-1071.

Terasaki, A.G., Suzuki, H., Nishioka, T., Matsuzawa, E., Katsuki, M.,

Nakagawa, H., Miyamoto, S., Ohashi, K. (2004) A novel LIM and SH3

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M.C. (1995) Lasp-1 (MLN 50) defines a new LIM protein subfamily

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47

Fig. 1 The structure of the amphioxus nebulin gene.The exons are shown as boxes and the introns as lines. Yellow boxes, untranslated regions; red, nebulin repeats; purple, LIM domains; dark orange, nebulin repeats (i.e. super repeat set 1); light orange, nebulin repeats (i.e. super repeat set 2); pink, a large exon with 98 nebulin repeats containing unique sequences at the N- and C-terminus; grey, unique regions; green, SH3 domain. Double solidus marks, connecting error sites of allelic scaffolds. Arrowheads, exons coding an isoform of nebulin (shown in Fig. 4).

5‘UTR

10 20 30

40 50 60

70 80 90

100 110 120

130 140 150

160 170 180

190 200210

SR1 SR2 SR3 SR4 SR5 SR6 SR7 SR8 SR9 SR10

SR11 SR12 SR13 SR14 SR15 SR16 SR17 SR18 SR19

SR20 SR21 SR22 SR23 SR24 SR25 SR26 SR27 SR28 SR29 SR30

SR31 SR32 SR33 SR34 SR35 SR36 SR37 SR38 SR39

kb

SR40 SR41

LIM

SH33‘UTR

5‘UTR

10 20 30

40 50 60

70 80 90

100 110 120

130 140 150

160 170 180

190 200210

SR1 SR2 SR3 SR4 SR5 SR6 SR7 SR8 SR9 SR10

SR11 SR12 SR13 SR14 SR15 SR16 SR17 SR18 SR19

SR20 SR21 SR22 SR23 SR24 SR25 SR26 SR27 SR28 SR29 SR30

SR31 SR32 SR33 SR34 SR35 SR36 SR37 SR38 SR39

kb

SR40 SR41

LIM

SH33‘UTR

48

Fig. 2 The connected sequence of amphioxus nebulin exons. The tyrosines conserved among the nebulin repeats are marked with black. The common amino acids conserved among super repeats of each group are marked with grey. LIM, LIM domain; Neb, nebulin repeat; SR01–42, super repeat 01–42; Uni, unique region; SH3, SH3 domain. The exons coded by Chapter 1 are shown as <Neb00/01> – <SH3>. Exon 270, which codes a unique sequence, but was not expressed in Chapter 1 was not added.

(LIM) MNPKCARCEKTVYPMEKLNCLDKYWHKGCFNCEECRMTLNMKNYKGFNKKPYCNA

(Neb) HYPQSKPTVVSETPEIARVMQQSQQQSL

(Neb) *TNKQTNMLTVVLMFQADQPWDTPETKRVREMGTVQSE

(Neb) *AEMMAEMTAGSGKRG DQSWETPEMVRCREMKDLQSE

(Neb) RVYKEKAKERMKAYTHHPDAPEMARLKEIGKVCSE

(Neb) VKYHEAYEKSKAKGYTSVVDTPENIRVKESGKIASQ

(Neb) *GTRQAKCIEDFEKLPKPGMLPADTSEIVRMKKVQQIQSE

(SR01) IPYKKEVKESTGKFFDVQDTPGYQHNLQLTKYQSD-----NVYMDEAKYIMEKGHSQYPFTPEMTRVQGAEKVKNE--RLYKESYEKYKSTFTLPPDTPGIVHAKNMGALHSEMKYHELY-QKELGQANKLPPDAILLRGAKQAVKIASEIEYSKKALQETRKMPYTAV-DSLAYMTAKAVSGLISQVNYTKQLGQALSHYNITADTPAIKQAKKAQQIQSQNTYSKDRPTKYTTVYDSPAMKQASNLSKIQSD

(SR02) IPYKQEVKESTGKFFPVQDTPTYQHHLQLTKYESD-----NVYKDEAKHIMQQGHSQYPFTPEMFLVGDAERIKSE--RLYKANYEKWKSTYTMHADEPSITRAKDMGTLSSENTYRKMY-EDSKAQGNTLPADAINIKRAKEAMEISSEINYVKKARK--DNKPHIAV-DSLSYMTAKAVNGLVSQVKYKENLDKALASYSLPSDTPEILLARKAQQLQSQHRYTKDKPSKYTTVYETPLMMQASNVSKMQSD

(SR03) IPYKREVKENTGSFFQVQDTPTYQHHLQLTKYESD-----HRYKDEAKFIMEKGHSQYPFTPEMSRVEGAEKIKSE--RLYKENYEKWKSRYNMGVDDPQIVHAKEVASLQSENKYKEQY-EKDKEQGKSLTVDSVNIQRAKDAMEMASEIKYMREAQKVAQKTPFNAV-ESLTYMTAKAVSNLNSQLNYTAKLREALSQYQLTADIPAIKEARRAQQIQSQHNYTKDRPTKYTTVPDTPLMLQASNVAKLQSD

(SR04) IPYKKEVKESTGKFFPVQDTPTYQHMLELTKYESD-----HKYKDEAKFIMQKGHSQYPFTPEMTLVEDAEKIKSQ--RLYKENYEKWKSRYTMQVDDPQIVHAKEVATLQSENKYKEQY-EKDKEQGKSLTVDSVNIQRAKDAMEMASEINYVKDAKKVAQKNTHGAV-DSLSYLTAKAVSNLTSQVKYTEKLHEALTQYQVTPDMPAILEARRVQQIQSQHNYTKDKPTKYTTVYDTPLMLQASNVAKLQSD

(SR05) IPYKKEVKESTGKFFPVQVTPTYQHQLELTKYESD-----HKYKDEAKFIMEKGHSQYPFTPEMTLVEDAEKIKSQ--RLYKENYEKWKSRYTMQVDDPQIVHAKEVATLQSENKYKEQY-EKDKEQGKSLTVDSVNVQRAKNAMELASEINYMKDAKKVAQQNTHGAV-DSLSYLTAKAVSNLTSQVKYTEKLREALTQYQVTPDMPAILEARRVQQIQSQHNYTKDRPTNYTTVLDTPLLKQAANAAKWQSD

(SR06) VPYKKDVKESTGKFFPVQVTPTYQHMLELTKYESD-----HKYKDEAKFIMEKGHSQYPFTPEMFRVDDAEKIKSE--RLYKANYEKWKTGYTMLVDDPNIIHAREVAALQSENKYKEQY-EKDKGQGKSLTVDAVIIRGAKDAMELASEIKYVEESRKEAQKMPFNAV-DSLAYLTVKAVSNLNSQLKYTEKLPEALSQYHISADIPAIKEARRAQEIQSQYNYTKDRPTKYTTVYETPLMKQAELAAKHQSD

(SR07) SLYKKEVKESTGKFFPVQVTPTYQHMLELTKYESD-----RNYKEEAKYIMEQGHSQYPFTPEFTRVEDAEKVKSE--RLYKENYEKWKSRYNMQVDDPQIVHAKEMASLQSENKYKEQY-EKDKEQGKSLTVDSVNIQRAKDAMEMASEINYVKEAKKGAQKNTHGAV-DSLSYLTAKAVSNLTSQVKYTEKLREALTHYHITADMPAIHDARRAQEIQSQHNYTRDRPTSYTTVYETPLMKQAELVAKLQSD

(SR08) IPYKKDVREAEVKQFPVHDTPTYEHMLQLTKYQSD-----RVYKDDAKYIMEKGHSQYPFTPEISRVEDAEKVKSE--RLYKENYEKWKSRYTMLVDDPQIVHARDVALLQSENKYKEQY-EKDKEHGKSLTVDSMNVQRAKDAMEMASEINYVKEAKKVAQKNVHGAV-DSLSYLTAKAVSSLNSQVKYTEKLREALTHYQITHDIPAIKDARRAQEIQSQHYYTKDRPTSYTTVYDTPLMKQAELVARLQSD

(SR09) IPYKKEMREAEVKQFPVHDTPTYEHMLQLTKYQSD-----HVYKDEAKEIMEKGHSQYPFTPEMFRVDDAEKIKSE--RLYKENYEKWKSRYTMLVDDPQIVHARDVALLQSENKYKEQY-EKDKEQGKSLTVDSVNIQRAKDAMEMASEINYVKEARKEAQRKPFSAV-DSLTYLTAKAVSELNSQVKYKEKLREALTQYHITADMPAIHDARKAQEIQSQHNYTKDRPTSYTTVYDTPLMKQAELVARLQSD

(SR10) IPYKKEMREAEVKQFPVHDTPTYEHMLQLTKYQSD-----RVYKDEARYIMEKGHSQYPFTPEMFLVEDAEKIKSQ--RLYKENYEKWKSRYTMLVDDPNIIHAKEVALLQSENKYKEQY-EKDKEQGKSLTVDSVNIQRAKDAMEMASEINYAKDARSEAHSKAYNAV-DSLPYMTAKAAAEISSEVKYTEKLREALSQYLITHDVPAIQEARRAQQIQSQHNYTKDRPTSYTTVYDTPLMKQAELVARLQSD

(SR11) IPYKKEMKEAEVKQFPVHDTPTYEHMLQLTKYQSD-----HVYKDEAKYIMEKGHSQYPFTPEMSRVEDAEKIKSE--RLYKANYEKWRADYSKSVDDPDIIHAREVGEQQSENKYRELY-KKERGKTKALPPDTMDNVRAKAASEISSDVRYTQDAQKEARSKPFNAM-MSLATLTAKVVAGLTSQKKYTDRAARIRSRFNMPADTPTIQEAKRVQELQSEYLYTKDRPTTYTTVYDTPYIKQAEQVAKLQSD

(SR12) IPYKKEMKEAEVKQFPVHETPVYLNQLQVSGFQSD-----HVYKDEAREIMEKGHSQYPFTPEMSRVEEAEKIKSE--KLYKEAFEKQRGHFTVRVDDPQITHAKETTDLQSENKYKEQY-RQDKLKGHMLPPDAMDNMRAKEAMDIASEVKYRKKTRDEMQNQMFTSV-DTLGNLTAKACMDMISMVKYKEEAEKMRPQLKMPPDLPEVKLAKEVQQLQSYQNYTKDRPTSYTTVYDTPLMKQAELVARLQSD

(SR13) IPYKKEMKEAEVKQFPVHETPVYLNQLQVSGYQSD-----HVYRDEAKDIMEKGHSQYPFTPEMTRVEGAEKLKSE--KLYREAFEKQRGHFTVRVDDPQITHAKETTDLQSENKYKEQY-RQDKLKGHMLPLDAMENMRAKEAMDIASEVKYVKKTRDEMQNQMFTSV-DTLGNLTAKACMDMISMVKYKEEAEKMRPQLKMPPDLPEVKLAKEVQQLQSQQNYTKDRPTSYTTVYDTPFIKQAELVAQLQSD

(SR14) IPYRREVKEAEVKQFPVHETPVYLNQLQVSGYQSD-----RNYRDEAKYIMEKGHSQYPFTPEMSRVEETEKLKSE--KLYKEGFEKQRGHFTVRVDDPQITHAKETTDLQSENKYKEQY-LQDRGKGQMLPLDAMDNMRAKEAMDIASEVKYVKKTRDEMQNQMFTSV-DSLSNLTAKACMDMVSQVKYKEEAEKMRPQLKMPPDLPEVKLAKEVQQLQSYKNYTKDRPTSYTTVYDTPFIKQAELVAQLQSD

(SR15) IPYKKEMKEAEVKQFPVHETEQYKYMLQVSGYQSE-----NNYKDEAKYIMHQGHSQYPFTPEMSRVEETEKLKSE--KLYKENFEKQRGQYSVRVDDPQILHAKEADSLQSENKYKEQY-LQDKLKSQFLPLDAMDNMRAKEAMDIASEVKYTRPTRDEMQNQMFTSV-DTLGNLTAKACMDMISQVKYKEAAEKMRPQLKMPADLPEVKLAKEVQQLQSHQNYTKDRPTSYTTVYDTPLIKQAELVAKLQSD

(SR16) IPYKKEMKEAEVKQFPVHETPVYLNQLQVSGYQSD-----HVYRDEAKTIMEEGHSQYPFTPEMTRVEEAEKLKSD--KLYKENYEKMKGQFSVRLDHPALIHAKEVADLQSERKYKEQY-LQDKLKSQFYPVDAMENIRAKEATDIASEVKYTDKARKEMQNQMYLAV-DSLTNMSAKLVHDLVSQVKYKEAAEKMRPQLKMPPDLPEVKWAKEVQELQSYKNYTKDRPTNYMTVYDTPAIKQAEQATMIQSD

(SR17) IPYKKDVKEAEVKQFPVHETSQYEFMLQVSGYQSD-----HNYRDEAKYIMEKGHSQYPFTPEMTRVEEAEKLKSE--KLYKENSEKQRGHYSVRLDDPQIQHAKEADSLQSENKYREQY-LKEKGQGLMLPLDSMDNQRAKEVSELVSEVKYAQDARREAQEKMYNTV-DSLTSMTAQVVSQLTSQVKYKEKADKERSQHKVPADTPEVAWAKEVQQLQSTKSYTKDRPTNYIPVYDSPHIKQAEEATKLQSS

(SR18) LQYSDRDAGTLHSEFRGQRYTPLETLEKKHTDDIRNIVSDVVYKDEAKEIMQKGHSQYPFTPEMTMVEEAEKLKSN--HLYQENYKKYLTNFTIRADYPEFVFAKETSDLQSENKYREQY-NKEKGQGLILPLDTMDNLHAKEAVALASEVKYTQDARKEAQEKMYSPV-NSLTLMTAKMVTDLTSQAKYREQVQSTLAQHKMPPDTPEILWAKEVGKLQSQNEYIKGKSTQYTPVFDTPAIRQADEASRLQSD

(SR19) IPYKYEVKNLVGKQFPVHETEQYKYMLQVSGYQSD-----HVYRDEAKTIMEEGHSQYPFTPEMTRVEEAEKLKSD--HLYKENYEKWKQKYTSHPNQLGFVTAKEIAEVQSESNYREKY-LKERGTGYFVPADNLEAVRVKEATELASEVKYSQDALNEAHNKKFDAVGDSVVVKTAQEVSELTSEVKYKEQAEKIKSQHKVPADAPDFVWAKEVGDLQNQRLYAKDKSDKYVPVLDTPVNLHAKEAGALQSH

(SR20) LPYSDRNAGTLFSEYRGQRYTPLVTLEKKHTDEVQSMISDVIYKDEAKEIMQKGHSQYPFTPEMSRVEEAEKLKSE--NLYKANYEKHKQVYTQTPNTAHITACKEAAELQSEMKYREQY-EKGKGHGYFLPADSMENVRAKEASQLASQILYTHEAQKEAQTKPYNPV-SSLTLLTAQAISELTSEVKYSEEAEKTKSQHKMPPDAPEIQRAKEVGQLQSQVEYAKNKTNQYTAVLDTPSNRHAVEATRLTSE

(SR21) LPYKEEAEDLIRNKAFAAQDTPVMDRVKKVSDLQSD----VVYKADAKHIMSLGHSQYPWTPDMARVQEAETIKSE--RLYTRDSNDQKKKFTILPETPEVALAKEVAKLQSEKQYKEQY-QKERGKGLHLPPDAVENLRAKEANQLASQIKYTEEARKSAEGKKFDAVAETPVFKTAVEAAENASETNYMKDYEKGKTKYNLSSDTPLIKTARQSANLASEFKYQQPVKGAKFEQICDGMREVHCATVSKLQSD

(SR22) IEYKQPLE-NYTVITDTPEFRKLASLQEIVSD--------IFYKEEAKEIIERCCSQAPIYLEMDRMARLQDLLNM—-TQYREKWEMDKKKYTVLTDSPEIIRVVEAGKQQSDLEYKKGM—QKQQKKGYALVPDSTSFVAAKEAAKIASDVEYKTKDLIDMRKASFKNVHESPWYLQAKLSAENASGIKYIKDYEKEKVHYLTPVDRVDFVAAKAVSGMTD-RKYRQKLGKPISIPDTPEIVRSKKLEKLKD

(SR23) QTYKQEPG-PFISTPDSMEMDRVRTLQQITSE--------QMYKEDAEWLVQRCLSQYPFSVEIDRVTKVKFQNDP--RVYQWEADRDKQKYTVLADSPEITRVVNAAKMQSDRVYHQGY-EKQKQGGYRLAADAPTFTTAKEAAKNASDYNYQSGLREA-RGKGFPNFQESPLFLQNKLSAQLASGAEYTKDYEKQKVKYTLPADRVDMVTAKNILELSD-KKYRQEPGKFISIPDTMEQVRCTKAAKLASD

(SR24) KHYKQEPG-AFISTPDSMEMERVKTLQEITSD--------QYYKEDAQWLVQRCLSQYPFSVELDRVNKVKFQNDP--RIYQWEADKEKQKYTVLADSPEITRVVNAAKMQSDRLYHQDY-EKQKEGGYRLAADAPVFLTAKEAAINASDHVYQSGLREA-RGKGFPNFQESPLFLQNKLSAQLASGAEYTKDYEKQKVKYTLPADRVDMVTAKNILEQSD-RKYRQEPGKFISIPDTMEQVRCTKAAKLASD

(SR25) KHYKQEPG-AFISTPDSMEMERVRTLQDITSD--------QYYKEDAQWLVQRCLSQYPFSVELDRVNKVKFQNDP--RLYQEEADREKQKYTVLADSPEITRVVNAAKMQSDRVYHEGY-EKQKQGGYRLAADAPVFLTAKEAAINASDYNYQSGLREA-RGKGFPNFQESPLFLQNKLSAQLASGAVYTKDYEQQKLKYTLPADRVDMVAAKAVLEQSD-KKYRQKPGKFISIPDNMEQVRCNAVAKLASD

(SR26) KHYKQEPG-AFISIPDSMEMERVKTLQEITSD--------QYYKEDAQWLVQRCLSQYPYSVELDRVNKVKFQNDP--QLYREEWDRDRQKYTVLADSPEVTRVCEVAKLQSDLVYHQGY-EKQKQAGYKLAADAPAFLTAKESAKNASDYNYQSGLREA-RGKGFPNFQESPLFLQNKLSAQLASGALYTKDYEQQKLKYTLPADRVDMVAAKNILELSD-RKYRKEPGKFISIPDNVEQVRCNAVAKLASD

(SR27) KNYKQEPG-KFISIPDSMEMERVKSLQEITSD--------QYYKEDAQWLVQRCLSQYPYSVELDRVNKVKFQNDP--QLYREEWDRDRQKYTVLADSPEVTRVCEVAKLQSDLVYHQGY-EKQKQAGYKLAADAPAFLTAKESAKNASDYNYQSGLREA-RGRGFPNFQESPLFLQNKLSAQLASGALYTKDYEQQKLKYTLPADRVDMVAAKNILDLSE-RKYRQRPGKFISIPDNVEQIRCNAVAKLASD

(SR28) KHYKQEPG-KFISIPDSMEMERVKTLQEITSD--------LYYKEDAKELVERCLSQYPFSVELDRVNKVSMMQDP--RLYREEWDRDRQNYTVLADSPEVTRVCEVAKLQSDLVYHQGY-EKQKQAGYKLAADAPAFLTAKESAKNASDYNYQTGLREA-RGRGFPNFQESPLFLQNKLSAQLASGALYTKDYQQQKLKYTLPADRVDMVAAKAVLDLSD-KKYRQEPGKFISIPDNVEQVRCNAVAKLASD

(SR29) KYYKQEPG-KFISIPDSMEIERVKTLQDITSD--------YYYKEEAKELVERCLSQYPFSVEIDRVNKVSLMQDP--RLYREEWDRDRQKYTVLADSPEVTRVCEVAKLQSDLVYHQGY-EKQKQAGYKLAADAPLFLTAKESAKNASDYNYQTGLREA-RGRGFPNFQESPLFLQNKLSAQLASGALYVKDYQQQKLKYTLPADRVDMKAAKAVLDLSD-RKYKQQPGKPISIPDTVEQVRCNEVAKLASD

(SR30) KHYKQEPG-RFISTNDTPEILRLKSLQEIVSD--------HYYKEEAKEIVERCLSQYPFSVEIDRVHKVSEMQDP--RLYREEWDRDRQKYTVIPDSPEVARVQEVAKMQSDLVYRQGY-EKQKQGGYKLAADAPLFLTAKESAKNASDYNYQTGLRAA-RGSGFPNFQESPLFLQNKLSAQLASGALYIKDYQQQKLKYTLPADRVDMVAAKAVMDLSD-RKYKQKPAVPKLPPDSVEQVHFKSLAPLKD

(SR31) KVYKHEPG-GFTSEIDTPETRRLKSLQEIVSD--------LYYKEEAKEIVNKCDSQYPFSVEIDRVHKVSEMQDP--RLYREEWDRDRQKYTVIPDSPEVARVCEVAKLQSDRIYQLGY-EKQKQEGYKMIPDTPLFLQCRESARNASDYVYQTALKEA-RGRGFPNFQESPLFLQNKLSAQLASGALYTKDYEQQKLKYTLPADRVDMVAAKNILDLSD-RKYKQKPAVPKVFPDSVEQVHFKSLAPLKD

(SR32) KVYKHEPG-GFTSEIDTPETRRMRTLLETVSD--------YYYKEEAKEIVEKCCSQFPFTVEIERVEKVTEMQDD--RRYRADYERARHNYTVIPDSPEVARVIEAARMQSDRFYHEGY-EKQKQEGYKMVSDTPLFLQCRESAWNASDYNYKTALEEA-RKSQYKNVHESPLFLQNKLSAQLASGPLYVKDYEQQKLKYTLPADRVDMVAAKNILDLSD-RKYKQKPGVPKVFPDSVEQLHVKSLAPLKD

(SR33) KVYKHEPG-GFTSEIDTPETRRMKSLWEMVSD--------YYYKEEAKEIVEKCCSQFPFTVEIERVEKVTEMQDD--RRYRADYERDRHNYKVIPDSPEVARVIEANRMQSDLYYKEGF-EQQKKEGYKMVSDTPLFNQCRDSARNASDYNYKTAIKQA-QEAQYKNVHESPLYLQNKLSAQLASGPLYVKDYEQQKLKYTLPADRVDMVAAKNILDLSE-RKYKQKPGVPKVFPDSVEQLHVKSLDPLKD

(SR34) KVYKSEPG-RFTSEIDTPETRRMKSLWEMVSD--------HYYKEEAREIVEKCCSQFPFTVEIERVEKVSEMQDA--KKYREKYDEEKQKYTVIPDSPEVLRVIEAAKMQSDLHYKDGF-EQQKKEGYKMVSDTPLYNQCRDSARNASDYIYKTAIKQA-QEAQYKNVHESPLYLQNKLSAQLASGPLYVKDYEQQKLKYTLPADRVDMVAAKNILDLAD-RKYKQKPGVPKVFPDSVEQLHVKSLDPLKD

(SR35) KVYKSEPG-RFTSEIDTPETRRMKSLWEMVSD--------HYYKEEAREIVEKCCSQFPFTVEIERVEKVSEMQDA--KKYREKYDEEKQKYTVIPDSPEVLRVIEAARMQSDLHYKEGL-EKQKKEGYKMVSDTPLFLQARESAWNASDYNYKTALKEA-QEKQYKNVHESPLYLQNKFSAQLASGALYIKDYETGKVKYALPPDRVDIVAAKNYQNFSD-RKYKQKPGVPKVFPDTIEGVRVKSLEPLKD

(SR36) KIYKQEPG-RFTSEIDTPETRRMKSLLEIVSD--------HYYKEEAKEIVEKCCSQFPFTVEIERVEKVSEMQDA--KKYREAWDEAKQNYTVIPDSPEVLRVLEANRMQSDRIYHEGL-LKQRDEGYKMVSDTPEYHRARFAAALASDYLYKSALKKA-QETQYKNVHESPLYLQNKLSAELSSGARYIKDYGENKAKYQLPADVWDMVAAKEAIARSD-RHYKQKLGSPKVFPDSIEGVRVKSLEPMKD

(SR37) KVYKQEPG-RFTSEIDTPETRRLKSLLEIVSD--------HYYKEEAKEIVEKCCSQYPFTVEIERLEKVAEMQDA--KKYREKWDEDKQLYKVIPDSPEVLRVLEAAKMQSDRLYQKGL-DKQRDEGYKLVADTPEYRRILQTTAQASDYLYKAALREA-REKQYKNVHESPLYLQNKLSAEMASGARYVKDYETMKVKYSCPADRVDLKTAKLVAGFSD-LKYKHIEARYTPVADTVQNEHAKTVAGFSD

(SR38) LKYVQKVSAKSLSETDTPVMRRIKSLQEITSD--------NLYKEEAKEIVEKCCSQYPFTVEMERFEKLLELNDKASKLYKKDYEEERQKYHVYPDTPEVKRVIDAAILQSDAKYREKYEKEMKGKGNFPQHITPVY-ASQKEAAGLTDAVYSRDAREKMQEKSYKNFHESPLYRTLKDDTDMASMSNYVSDWEKRKVDYKVVSDTPDIKRVKKAGTLASPRTYVQKQEKYETVADTPANILANESGKLVGD

(SR39) RHYKQVVKGKGIMDTDTPEMRKIKSLREITSD--------NIYKEEAKEIVEKCCTQYPFTVDMERFEKLMELMDQ--KRYKKNFEEERNNYHVYPDAPDIKRTLAMAQLYSDLKYKEIYESQIKGKGNFPQHITPVY-KSQIEAAKITDAVYKKDAHDEMQKKSYKNFHESPLYRTLKDDTDMASMTRYRDQYEKEKTNYKITTDTPDIKRVKKGGILASERLYKQKCDSYETVADTPANIHAKETGKLHSD

(SR40) LPYSNRDAGTLRSVYRGQRFTPFDTPESNRLKSLRELASDNVYKEEAKELYEKCCTQFPYTVEMERLDKLKELYDK--GLYMKLVDEARQ-YHLFPDCPEVKRVIDADKIQSDLVYKKAY-EEARGKGYFKVTETPLYKTLVKAALDASPDHYSRQALTEMREKNYTAVHESPLYKMQKENTDNQSPHKYNKFPETRDNF-KTPVDTPDLKRVKKAGDIASEIKYQKDKQTEKYIPIADTVAVRHAQHMGEIQGY

(SR41) LPYEKHDAGTVRARYRGQRFTPVDTPESNRLKSLKEIVSDTIYKEEAKEINEKCCSQFPLTPEMERFWRLQDLYSD--ADYRQKALDTRGDFHLYPDIPEFERLNKMNDVQSDLKYQERYMKELRGKGYKLTPDMPLYQRMKRVTAQNSDNVYKGEAKKLLGKSIDVTQTPEYARQQEAQKISSQ--SEYVAKDKERTHQYETTPDTPEYRRQRKVAKFTPDKAYQTAKPDLDKYHPVKDTPSNERADQAQKIQSD

(Neb) LWYQEQGRDLKGKAYTEVDTPVNRRVKSLQEITSD

(Neb) TFYKEEAQHLWHVACTQYPYTPDMERHEDNLRKFSD

(Neb) VLYKNKYEMNKHEYHPVPDSPELNLAKENAVRHSG

(Neb) FKYREKYEELKKKPKFNIAETQLYRRMRQVAADTCD

(Neb) DNYMGDYNKNIKGKGVANYAETPLYQRNKEAGDASSE

(Neb) LKYGKDLKKIREGKGYTPTADTPQHRHVKKMEEIQSS

(Neb) VKYPKALKDIAKYATVLDTPDNQRAISTYKNQST

(Neb) LYYGADAKNEMIGKAFKEVNETPASAHHKKVLDMSD

(Neb) VVYKDEAKRLHTVACTQYPYTPENLRMAELSRTQSD

(Neb) QMYKERGELEKHKYTTLANTPDLLHAKEVHDITSE (Neb00/01)

(Neb) NKYKEEYNKERGKYSMVAQTPLYETAQKANKLTSE (Neb01/02)

(Neb) TLYKKAYKALQGKGYALIDTPEFERCKQVNKLASR (Neb02/03)

(Neb) TKYHEDYEKSKIKGYTPVVDTPENIRLRQQSKIQSQ (Neb03/04)

(Neb) NEYQKGYKALAGKFSYTADNPEHNRIRSVSKIQSQ (Neb04/05)

(98 nebulin repeats with Unique sequence)

(Neb) IHYGHTRLEQHGQKYHAAIDDPELKRLQEVGKFQSQ (Neb05/06)

(Neb) VLYGEIKDKDDASKGRGKYTTIQQDPEMKRLKEANAYASE (Neb06/07)

(Neb) AQVAVRKFDEKNRYTTVLDDPELNRMKNVGKFQSE (Neb07/08)

(Neb) LQYRDKKTRKTGARYTNVLDDPEMNRIKGVTKMLSE (Neb08/09)

(Neb) VQYRDRELKAGRLTAVTDDLDTARVKRNTKIQSE (Neb09/10)

(Neb) LQYRDEELKVGKLHVVTDDIDAERIRRNTKIQSE

(Neb) TLYRKDFSKGRGTFTGTNGIMDSLDYERIKRASKLCSE (Neb) NEYRKGRKINMYNAVLDTPDMARIRNATKISSE (Neb10/11)

(Neb) VKYHEDFEKQKGKAYHTVIDDPELTRVKKNTVIQSN (Neb11/12)

(Uni) LAYQGVKEKLQTMESRRQMIPGEHPMPQPAGQQLGQMEARMAADASYAQFKAKGKGKVGG (Neb12/Uni)

(Neb) RYMPIRLGDPGSIFDYEPYQGKEEAGRAIY (Neb13)

(Uni) VTTATWKPAKPIRTIEDFVVEESTDVSAAKVEEAMEARDKLTEMDMEYERRQGEGLSYEYDPEKYSHHYWSKTTGELGGVVTTSGMKYRKIEDFLNLNADLIYTTENESSTRESASESSGMWDYDPDKYWKARGYRISAEQRYQQVRLKPGKIEDYDPMTHPDAQQPKFEAPAAVSEPEPTIQSG (Uni)

(SH3) QNVMRAVFDYTAAEDDEISFMDGDVIVNCVKIDDGWMTGTVQRTGQSGMLPANYVEPMS (SH3)

49

Fig. 3 The giant exon of 98 nebulin repeats with a unique sequence. The tyrosines conserved among the nebulin repeats are marked with black. Underlined area, coding site in a B. floridae neurula stage EST clone (DDBJ accession BW855343).

MSHPKFDSQESAPYQQGAMWVLTGCSRLALVNCNNNNNNG

SSDDENDGNGNAQPGEHETPAAYNSEQNPGQEDTESDNAD

QAEGNGAFGTEGVQDQGQGDDSPPPGEAQEEEPPQESPSI

EVTPPEDSPPQQDVFEEETQPKEPEGPIIEMAPSPTVEEA

GIEQAMEAEEGPSEQFTELPLSTDAPLEPIRTPVVEEPEE EQPAEYEEAEDALIDFDLPSPPPEKVSPDEYRAVSPREQP

TPEERPVSPRATSPAEEAVPPERPPSPMEEDVFVEPAFPS

PAEATGRPDVPTPPYMEQAPSPVVPDALMEAVEPEVPETP

MEPDFPSPPSYNTLTEDVVRDVPVHVEKPSQRTASVFTNP

DELDEEEVGNPHPLAGNLGHKTGNPHPHNPGNPHPHNPGT PPPIEEKSSSPQPVQASGGRLVKTGSYSSYQYEESVREVR

SSTKQTIREVVSSDGKVISKQESEETSHAEASQARAKSET

SRSDGKQTVTTKREAEESMQALEMQKKKKTTSGGVTTVEP

TQGMRAMEMERKAKASTTVQQAVGAAMEVEPPPPIPPLPV EIGQVTIKQEPGTLRGISLKGVSKSRGGVKHTIKTTNALS

EERSSQVTTSAMSEQRSSHVTTSSQSEQRASHVSSSSMSQ

ERASHVATSAMTQERASHVSTSAMSQERASHVTSVRAAKA

VHTGVTKKVESQPKIVVARRTETVKIQLPRGSRERVLRIK

IRFGKKDKPNRLTAYAWSQLRWPAITEVKTQEDVSEDTRY VYILLKEISPDRVAKESCLRVQVPPVIEQSIMKISAETSR

MLVQQQKVIYKAHERKYGSGQKYKFTVQQAVPPELDESPT

LSARDAKSRFEMARAEELRQRRHVTRHEVGQMKEMFERGA

SDAEDYEREPAQITRQEVGEKKEAFERGVSEVRMAEREEI QVARQDIGQKKQLFEKVAEGHWASRYMDSDEGGEAPERPP

PPRFDVGQKKEIFENVGQSWRRTDSETEEALQMITQQDVV

RKRELFERGRSGSEEETPERPPPPRQDVGQKAELFEKVAV

GQFVRQEGEERERVIPIRPPTPSHDMIQKREMFQQFSQGR

WSSSETEEREEIQIAAGVSTKKELFEKAAEAKLLPPPSPR RMRRRIRSDSFRRSMSSDDEGTPERPPPPRPPPPAPAPPT

PSPPASPVTTAPPSRPPPPPEGEEGEDWMASVSKCVEMYE

REVQIRGERMKLKTIRRDPTPVRRQPRSWPPTPPSSPAEP

ISPVRRTRRSPSPGPPVADRPRGREPPPVARRFRDRSPAV HREHSSERRLTNLAQNILSQEGFEEYRAVPQDTYQVVVDP

FLQKAQEVTKLLSR

AEYEKEAKDRMWRYQGGLPDEAEARRRAELARMQSD

VRYRDYPRYLEAKPGMVAVVDRWMIGQKKQTHEQST

VEYPRDHPKDMTWRLKYQSTPDDTVTKRAQEVTHISSE LEYQKRAKQTKDRYTAITDELQLETQKDAARMQSS

VSYVKDHPLAMEKRHAYTSVEEDPSSKHAKDAYKMSSQ

LEYEKRYKQEVLGHVGYIELDTPEQERIRRMKTLQSQ

LEYQKDYPMDFERRKDYQSVHDDPNTIRAKEATHLASK VEYESRYHKEKDIYKTVADTPEGVRVKDVGKMQSE

IEYQKPDKERRQYYQTITDAKDSQRLKEVGHLQSQ

IEYQKDHPMGFEGRRDYQAVSDDPETLRVKDVTKLSSG

VEYVRKDKPRREVYQTVTDDPEGKRMKEVGKLHSQ

LEYQKDHPMTFEGRLDYTSIADDPETRRVKDVTMMASG IEYERKDKPRREVYQTVTDDMESKRMKEVGHLQSQ

IEYQRDHPMTLEGRLDYTAVADDPETRRVKDMTIMASG

IEYERKDKPRREIYQTVTDDMESNRMREVGKLHSQ

IEYQKDHPMTLEGRLDYTAVADDPETLRVKDMTLMASG

VEYQRKDKPRREIYQTVTDDMESNRMKEVGKLHSQ

IEYQKDHPMTLEGRLDYTAIADDPETRRVKDVTMMASG

IEYERKDKPRREVYQTVTDDMESKRMKEVGHMQSQ

IEYQRDHPMTLEGRLDYTAVADDPETRRVKDMTIMASG

VEYERKDKPRREMYQTVTDDMESNRMREVGKLHSQ

VEYQKDHPMTLEGRLDYTAIADDPETRRVKDVTMMASE

IEYERKDKPRREIYQTVTDDMESKRMKEVGKLHSQ

MEYQKDHPMTLEGRLDYTAVADDPETLRVKDVTKLSSG IEYQKKDKPRREIYQSVTDDVATVQAKDSQRLQSQ

LEYERLYREKKGTGIFTMIADDKNLQHVKDVSKLGSQ

QGYTKDFPMELEKRGGYTSVEADPTTERAKEVYQFQSD

VEYQRNFRQRRGSFTAITDDAATKHAQESGKVSNE LAYTKNFRTRRGSFTAVVDTPEAQRVKEVNRYQSE

LEYKKRALQRRGSFTAVVDDPVTERVKDSTRLASD

TIYKAAAHERKQAYTIALDDPNTERVKNVSKMVSD

FEYQRMYREKRGTGIFTSIADDPFIKHAKETAEYLSS

NVYKKDFPENMERRSGYTTIVDASGLKHAKEASEIQSL IKYYEKGKEGKSKYTLVTDDPESMRMKDVNKMQSR

IEYEKDYEASKDKYTMVGDDPQTRRVQEMTKLQSQ

YEYQRTFLEERGLGWYTAWLDYDKEMERQLGMKHMYSQ

VKYKKDFPEQMTGREGYQMVADDVQTVHASDMSKMQSR VEYEKDYHEKKGLYTTTTDEMRATHAKEVGKLASS

VEYPVDHPMEWERRRKYTAVADDPNTQRVKETTKYQSP

YEYEREHRRKKGQYTMVADDREQVRVVEMNKLHSL

VQYHETFEKEKGQFTTVLFDPETKRVVELSKLQSM

NYYQQEFEKNKGNYITVLFDPESQRYKEVGKLASD

VEYQKEFKDLRGKYTATADTVATKHATEAGKLQSQ

LEYQKDFPQQMTERAGYTPVTDKETIRVVEMSQLHNL

IKYREQFEKDKDRFTTVLFDPETKRVVELSSIQSV

IQYEKKYQANKDKYTTVMFDPETSRTMEMSKLQSL

NEYHLQFERDKGQYQLVTDDPVSERIREVSRHQSQ

YEYVRKWLEERGIYHSAPDDKEHMRIVEVNKLQSL

LHYTDEYNKEKGKYTTILFDPETKRVVELSKLQSA

VQYHEEFEKNKGNYITVLFDPESKRYKEVGKWQSD

VEYQKEFRGLRGKYTSVEEDVQTKRAKDVGKYQSQ LEYQKDFPQEFEGRTGYTPVADKETVRVVEMSQLHSL

LKYQEEFEKDKGKYTTILFDPETKRVVELSSIQSA

IAYQKEYQKDKDQFTTILYDPETQRFTELSKIQSL

VSYHDEYEKEKGKYHSVADDIDSKRLKDVGKFASE TEYQKEYQKLKAFYTMVPDKEMLRVIEMSQLHSV

VKYHEEYEKDKGDFTTVLYDPETRRVVELSSIQSI

NKYQEEFEKNKGKYTTILFDPETERVQEVGKIQSN

VEYQKAYHEMKGSYRTVTDDPESERLKSVSKWQSM

SEYQKDFPSKFEGRPEYQSLAADKETLRIVDVTKLQSL IKYQEQHKDDIGKFTTVLFDPETKRVLELSKVQSL

LKYKEEFESEKGGYTTVLYDPETQRFIDLSSKQSD

VHYHEQYEKEKGQYTSVVDDVETRRQTGITKVQSS

VVYPKDYPLRYEGRSGYTTIPDDKELLRVVNLQKIHSL

IRYNKEYEDEKGAYTTVMFDPETKRLIELSKIQSL

IKYQEQFQKEKGSFTTVLFDPETKRAIEVGKIQSQ

VEYQKDFKEGRAAQSGAEADKEILRLVDMSQLHSI LKYQEEFEQGKGKYTTILYDPETQRVVELSSIQSA

VKYEDEYKKNKGQYTTVMFDPETKRLLELSEIQSA

FKYQEDFERGKSIYSQIPDDVQTKRAEEVSHFQST

VEYGKEARAQLPQYTPVADEKEVVRMVNMTKLHSL

INYHERYEKERGGYTTILFDPETRRTMELSNVQSL VKYQEQFQQQKGNYTTVLYDPETKRVVELSKTQSQ

LEYQKDFPVAMEGRDAGVIDDQDKEVLRLVDISKLQSL

SKYQEEFQRGKGNYTTVLYDPETKRVVEVSSIQSL

IKYEEDYRRSKGQYTTIMFDPETQRLMEMSEIQSA

FKYHEDFEKGKGNIMQITDDVETRRATDVTHLQST

FEYRRDYPLEYQQRPGYTSSTTDKEMVRLVEMSKLHSL

AEYRSEYEKGRGGYTTILFDPETKRLLEISRVQSL

INYQEQFYREKGNYTTVLWDPRTKTAVESTKLQSQ

IQYQKDFPVGMEGRGGKGADEADKEILRLVDVSKLQSL

AQYHEEFKKDKGKYTTVLFDPETQRVIELSAVQSL

VKYEDEYKRNVGQYQTVLYDPETQRVIEMQKMQSV

FKYHEQFEQEKGQYTLATDSIDSRRAQEASRVQST

LLYRRDFPQEFEGRAAYIPIPDDKETLRIIELTKLQSL IQYQKEYDLSKGDYTTCMYDPETQRVLELTRIQSY

VNYKKNFEKEKGHYATIIYDPETRRCIELSKTESQ

IGYKKDFPITFEGRPHISTYGDKELLRLIDISNVQSV

IKYQEEFLRDKDKYTTVMYDPETKRVVELSRILSL

SKYQEEHQKGRGKYSTILFDPETQRALDLTQVQSS

VRYRKDYPQTMEGRAAYSASIDMSILRVIEMSQAIGV

TRYHEDYDIAKANYNAILYDPETRHVMEISKIQHL

DKYKEAFTHQVGPHVAVLYDPATKKAMNLSSISSQ

KDFRNEYVETFETIVKRAKNKVVLRLIDLTRVDNL

VAFEEELIIETSNYRTLLFNSENSRVIEISRLDSF

VNYEKRFKETKGSYTTVLYDPETQRALTLSKVTAQ

AEYQRQVFESSFGDRVSESTQFEEPQWSLDIESMA

KFYEDVASGKHREPEIPSYTEGTSSSKGAPKYRGKKKVGFSL

SGEELRGVDSAGKAELEVIYKGGDVVETKEQAEEVKQTIEQV GLKAAGAVKDEKPGVSVTSRRSDTPPEALGDGGQEEDDDTPM

TQDMVRRLIELHEQSMRGIVRTLDEDLAALNGNNNNNKVVEA

TRYRQPTWTVCTMADPEALTYCQVHGPRNYPLTVLNS

50

Fig. 4 A small transcript from the amphioxus nebulin gene. (A) Reverse transcriptase-polymerase chain reaction (RT-PCR) product using LIM and SH3 domain primers. (B) Amino acid sequence of the RT-PCR product. (C) Domain structure of the RT-PCR product and the product’s homology with human Lasp-2.

LIM METKCARCQKTVYPMEKLNCLDKYWHKGCFNCEVCHMTLNMKNYKGYNKIPYCNAUni HYPQSKPTVVSETNeb PEIARVMQQSQQQSLTKYHEDYEKSKIKGYTPVVDTNeb PENIRLRQQAKIQSQVKYHEDFEKQKGKAYHTVIDDNeb PELTRVKKNTVIQSNLAYQGVKEKLQTMESRRQMIUni PGEPSMPPPAQQLGTLMDPTHGMESMSIQDGHERMMQQQQQEEDRHLASHRRQE

NPPISIQSYQQVRLKPGKIEDYDPMTHPDAQPKFEAPAAVSEPEPVIQSGNVMRSH3 AVFDYAAAEDDEISFMDGDVIVNCVKIDDGWMTGTVQRTGQSGMLPANYVEPMS

337 aa, 1011 bp, Molecular weight 38,677

(B)

(C)

(A)

1000

500

(bp)M AM

1000

500

(bp)M AM

80%

46%

50%

53%

40%

65%

7%

AmphioxusLasp-like protein

Human Lasp-2

80%

46%

50%

53%

40%

65%

7%

AmphioxusLasp-like protein

Human Lasp-2

51

Fig. 5 Phylogenetic analysis of nebulin family proteins. (A) Phylogenetic tree based on SH3 domains. (B) Phylogenetic tree based on LIM domains. Percentage derived from 1000 bootstrap probabilities. The scale bar is 0.1 estimated amino acid substitutions per site.

Human src8Amphioxus nebulin

Ciona nebulinZebrafish nebulin

Chicken nebulinHuman nebulin

Zebrafish nebuletteChicken nebulette

Human nebulette

Human lasp-1Chicken lasp-1

Zebrafish lasp-1

34.8%42.9%

20.9%

50.0%56.7% 99.3%

66.3%87.2%

66.9%

(A)

Vertebrate

CepharochordateUrochordate

0.1

Human src8Amphioxus nebulin

Ciona nebulinZebrafish nebulin

Chicken nebulinHuman nebulin

Zebrafish nebuletteChicken nebulette

Human nebulette

Human lasp-1Chicken lasp-1

Zebrafish lasp-1

34.8%42.9%

20.9%

50.0%56.7% 99.3%

66.3%87.2%

66.9%

Human src8Amphioxus nebulin

Ciona nebulinZebrafish nebulin

Chicken nebulinHuman nebulin

Zebrafish nebuletteChicken nebulette

Human nebulette

Human lasp-1Chicken lasp-1

Zebrafish lasp-1

Human src8Amphioxus nebulin

Ciona nebulinZebrafish nebulin

Chicken nebulinHuman nebulin

Zebrafish nebuletteChicken nebulette

Human nebulette

Human lasp-1Chicken lasp-1

Zebrafish lasp-1

34.8%42.9%

20.9%

50.0%56.7% 99.3%

66.3%87.2%

66.9%

(A)

Vertebrate

CepharochordateUrochordate

0.1

C.elegans laspAmphioxus nebulin

Zebrafish lasp-1Human lasp-1

Human nebuletteChicken nebulette

Human N-RAP

(B)

97.5%

47.4%92.2%

38.3%

Vertebrate

Cepharochordate

0.1

C.elegans laspAmphioxus nebulin

Zebrafish lasp-1Human lasp-1

Human nebuletteChicken nebulette

Human N-RAP

(B)

97.5%

47.4%92.2%

38.3%

Vertebrate

Cepharochordate

0.1

52

Acknowledgements

I am indebted to Professor S. Kimura of Chiba University for her

continuing guidance and stimulating advice during my course of study. I

wish to express my gratitude to Professor K. Ohashi of Chiba University for

his thoughtful and helpful discussions. I am grateful to Professor K.

Kubokawa of University of Tokyo for collaborations on this study. Finally, let

me thank the members of Professor Kimura’s laboratory for their technical

assistances and useful discussions.