Two c- myc genes from a tetraploid fish, the common carp (Cyprinus carpio)

Gene, 153 (1995) 231-236 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50

231

GENE 08602

Two c-myc genes from a tetraploid fish, the c o m m o n carp (Cyprinus carpio) (Recombinant DNA; cloning; nucleotide sequence; genomic organization; tetraploidy)

H u a n Zhang, N o b u a k i O k a m o t o and Yayoi Ikeda Department of Aquatic Biosciences, Tokyo University of Fisheries, Tokyo 108, Japan Received by T. Sekiya: 17 August 1994; Revised/Accepted:4 October 1994; Received at publishers: 11 November 1994

SUMMARY

Two c-myc genes have been isolated from a tetraploid fish, the common carp (Cyprinus carpio). The nucleotide (nt) sequences of the two genes and their flanking regions (CAM1, 8675 bp and CAM2, 5784 bp) have been determined. Both genes contain two coding exons which are homologs of c-myc exons 2 and 3 in higher vertebrates. The nt sequence homologous to c-myc exon 1 of higher vertebrates has not been found. Carp CAM1 and CAM2 presumably encode peptides of 394 and 401 amino acids (aa), respectively. These two peptides share 55-91% aa identity with those of humans, chickens, frog, rainbow trout and zebra fish. The two carp c-Myc have a Glu stretch at the beginning region of exon 3. This Glu stretch is also present in zebra fish, suggesting that it may be characteristic to c-myc genes of cyprinid fishes. The existence of two c-myc in carp is attributed to the tetraploid nature of carp. These two genes share 92.4% nt identity in the coding region and 84.2% in the intron. The two deduced peptides share 94.2% aa identity, suggesting that the tetraploid event probably occurred 58 million years ago. Both of the genes' cDNA clones were obtained from a cDNA library of carp peripheral blood leukocytes, implying that both of the genes are expressed.

INTRODUCTION

Polyploidy has recently been recognized as a potentially important process in the evolution of vertebrates (Ohno, 1970; Lundin, 1993). It is believed that a tetraploid event took place about 500 Myr ago in a common ancestor of all vertebrates (Allendorf et al., 1984). Higher vertebrates have evolved delicate sex determination based on heteromorphic sex chromosomes, which prevent successful polyploidization (Ohno, 1970). Polyploidy seems to be common in teleosts that appear sexually Correspondence to: Dr. N. Okamoto, Department of Aquatic Biosciences,Tokyo University of Fisheries, Konan 4, Minato-ku, Tokyo 108, Japan. Tel. (81-3) 5463-054'7;Fax (81-3) 5463-0552; e-mail: [email protected] Abbreviations: aa, amino acid(s);bp, base pair(s); CAM1, carp c-mycl encoding c-Mycl; CAM2, carp c-myc2 encoding c-Myc2; c-myc, gene(s) encoding c-Myc; c-Myc, cellular Myc; CR, coding region(s); FR, flanking region(s);kb, kilobase(s) or 1000 bp; Myr, million years; nt, nucleotide(s); UTR, untranslated region(s).

SSDI 0378-1119(94)00813-2

undifferentiated (Ojima, 1983). The studies on gene duplication in tetraploid teleosts are important to investigate the evolutionary processes following the tetraploid event (Ohno, 1993). In this study, myc genes in a tetraploid fish, Cyprinus carpio, the common carp, have been selected for investigation. Genes myc are highly conserved in vertebrates, their precise expression (both specifically and quantitatively) being crucial for cell division and differentiation. In mammalian genomes, there are a number of myc genes forming the myc family. Each member is structurally similar to one another, however, the nucleotide (nt) and amino acid (aa) identities shared by them are less than 50% (Kuchino, 1992). Evolutionary origins and relationships of each mye member remain unknown. The study of myc duplicates in tetraploid fish can help us understand the evolution of mammalian myc genes. The aim of the present study was to isolate two c-myc genes from carp and compare with those of other vertebrates.

232 (Zhang et al., 1993) was screened again with C1. Another six positive clones were isolated. Sequence data indicated that two of them corresponded to CAM1, and the other four to C A M 2 (C1 and C2, the two longest cDNA clones of CAM1 and CAM2, are shown in Fig. 1C). These results suggest that both C A M 1 and C A M 2 are transcribed.

EXPERIMENTAL AND DISCUSSION

(a) Cloning of two carp c-myc genes Two c-myc genes have been cloned from carp. Their genomic organizations are determined by four genomic and two cDNA clones (Fig. 1A). Carp genomic DNA was digested completely with EcoRI and hybridized with C1 (1334 bp), a c-myc cDNA clone previously isolated from a cDNA library of carp peripheral blood leukocytes (Zhang et al., 1993, also shown in Fig. 1C). Two bands (8 and 5 kb) were observed (data not shown). DNA fragments of these sizes were used to construct two partial genomic libraries. These two libraries were screened with C1 and two positive clones, E1 (8088 bp) and E2 (5054bp) (Fig. 1B) were obtained. Sequencing results showed that E1 and E2 were independent c-myc genes and that C1 was a cDNA clone of El. To investigate the 5' upstream regions of these two c-myc genes, carp genomic DNA was digested completely with BamHI and hybridized with El-5', a 455-bp EcoRIBamHI fragment in the 5' region of E1 (Fig. 1B). Two bands (1.1 kb and 8 kb) were obtained (data not shown). Two new partial genomic libraries were constructed using DNA fragments of these sizes and screened with El-5'. Two positive clones, B1 (1042 bp) and B2 (approx. 8 kb) were obtained (Fig. 1B). Sequencing results showed that B1 contained the 5' upstream region of E1 and B2 contained the whole length of E2 and also its FR. To investigate the transcription of these two carp c-myc genes, C A M I (for B1 and El) and C A M 2 (for B2), the previous carp peripheral blood leukocytes cDNA library

(b) Nucleotide sequence analysis We have determined the whole nt sequences of B1 (1042 bp), E1 (8088 bp) and part of B2 (5784 bp, including E2 and its 5' upstream region) (Fig. 2). Because the cDNA clones are incomplete in 5' region, start codon ATG of C A M 1 and C A M 2 were predicted by comparison with those of other vertebrates (Fig. 3). The 'A' of the putative start codon ATG is numbered as + 1. The two carp c-myc genes contain two coding exons interrupted by an intron. These two exons are called exon 2 and 3, since they are homologous to c-myc exon 2 and 3 of higher vertebrates (see section e). The CR of exon 2 is 619bp in both C A M 1 (nt 1-619) and C A M 2 (nt 1-619), while that of exon 3 is 566bp in CAM1 (nt 1043-1608) and 587 bp in C A M 2 (nt 1040-1626). Both of the introns begin with GT and end with AG, following the GT-AG rule (Breathnach et al., 1978). The intron is 423 bp in CAM1 (nt 620-1042), and 420 bp in C A M 2 (nt 620-1039). Two TATA-box like sequences (TATAAA and TATAAT) are found in C A M 1 (nt -1233 and -1200) while three (TATAAA, TATAAA, TATAAA) are found in C A M 2 (nt -1389, -1254 and -904). One potential sequence which may form a hairpin structure (italicized)

5 0 0 bp

A

CAM1 //--exon 2 >619 bp

B

423 k^

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CAM2

~

exon3

II

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exon 2 >619 bp

1039 bp

420 bp

I

//----

exon 3 1066 bp

E2 (5054 bp)

E1 (8068 bp)

/P'-

B1 (1042 bp)

/,1___ //---

B2 (-s kb)

E1-5' (455 bp) C1 (1334 bp)

C

C2 (1142 bp)

"-,r"

-t'"

D

i r

i

J

I

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Fig. 1. Genomic organization of the two carp c-myc genes (CAM1 and CAM2). (A) Genomic structure. Exons are indicated by boxes, putative CR are shaded, UTR unshaded. Intron is indicated by double lines. Sequenced 5' and 3' FR are indicated by single lines. (B) Genomic clones. (C) The longest cDNA clones of CAM1 and CAM2. (D) Restriction enzyme map. Only the enzymes used for subcloning are indicated. Ba, BamHI; Bg, BgllI; E, EcoRI; H, HindlII; K, KpnI; M, MboI; P, PstI; Sa, SacI; Sp, SphI; X, XbaI.

233

CAm

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CA~

GGATCCCGTGCCCCTGGTTG-AGAGACACTGTTCTGGATAAC-TTG---CATTTTGGTGACATG

-1537

......

A.G..T

.......

CA ....

- .....

C .......

-1316

G..T...ACTT.CA,.T

....

T...

CAN CA~

CTGAGGTGGTAGTTGAAGATGGTTACATTTCTTGATTGTTTTGTTGCAACAACATACTCCATAGGCTACATG---T~TGCATTCTTATAAACTTATTGATTTCTATGAACATGGTTTCTATAJtTCAATGCT~TCA~TAGTTTTT

CAN CA~

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .T T T T T A T A T G C - T T T G T C A C A G T C A T A C A A A T T C T A G A A A C A C T T T G T A G T C A G C A C A C A A A A T A T A A G A T

CAN CA~

A-CAAACAGGGGTATAAGAAGATATCTAATTTCACATTAA-GAGAGTGCTTCTAGGCTTTG

~

AACA---GAAT

CA~

TTTGTTT.TT.GTTTGTTT.TTGATTTTTTT.C.G.AT...TT.T.A...TTA.A

CAN CA~

CAATGACGTCAAATCCCCGCCCACCA--G-:TTATCG

CAN CA~

TTTTTAGTTCTTATGTGTGAG~TGGC--GrGACATTTAATA~GCAAACAACGGTGTTTTCGTTTGG~GGGAAAC~TTAGTGCGCAGTCATAAAGTCAGTGGGCGCTGCTTCAGCTCGCGGGAGCTACAGAACTTATTCATCCTGCCACT

CA~

GACACCCAGG•TATCAGTCTGTCCAGCACCrGAGGGGTGCTCGAGTGCGTACGTCGATTACGGTTTC•ATCACTTTCTATTTGGCCAGGACTCTGATTCCACTTATGCTGCAAAAGACCGGAGTAAACTATTGGATCTTACGTTTTGTTC

CA~

.T...A...A

CA~ CA~

CTTATTTTTTGCTCTCTTTTCATTGCATTGCGTCTCGAACGAAATCATTCTGAAACTGCATTTGTTAAA

. . . . . TACACGGTAAGGATCCGACATTTGATGCTGCATGCATCTTTTAGCCTT

..G ........

TAACT

CAN CA~

AATATCTTTGT-TGTTAAATGGGAGTTTGCAT-GTT-GCACGTT•AAAGAATACACT-AGAAGCAGGGCTGTTTTTTAATG-TTTTGATAGCAACCCCGCTTTGAGGTTTAATCGCTCCGTTTTTACACATACTGTCATATAACTTAGTT

CAN

CCTTTATTTAGCTATTTTATCCACCC-CAYGCA~TTATGCA~GTCTAAATATGTCCATTTTAGGTAAAGC . .A ........... G...TC ...... T . A Y G C A ~ C G A T G C A T . . . T ............ --, .... C .....

....... A . . . T . . . T T

.......................

A ..............

TATATATATATATATATATATATATATATATATATA,A,A

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.......... ........

.....

A..-G

.......

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T .... C ............

CAR

....

......

.......

TAT/%AA C ....

C..TATAAA

......

.......

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...........

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..............

C.----.

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......

AAAAAAT--AAAC

G...T..CAAATTTTAGCACGC

.......

.................

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.....

.....

C ....

TT ......

A.T

.......

-1325

AAACAAA-CAA

-1084

.....

GA...ATGAAG...T...GG..

-1179

T .......

A .... G .... C.CC

A ....

-1029

-950

.........

T.A

.....

T..TG...T

.............................

A..GT...AAC.C.----,

G .............

AA ........................

......

.....

C ..............

A.A

.....

A .....

G .....

G ............

AT ..............

GA .............................

A...A..G...G

1175

CG.G..TATATA

---A

......

T ......

T ....

851

CTGCTCGACTATJ~%ACGATCCTTCACTC

-899

TTCCGCGAGAAAATAGTTCCACATTTATCGAATTCAAAAATAGATGTAACATGTCCTCTAAGTCACTAAAGACTAATTATAGTTTCAAAGACGT•GTTGTG

A...A.ATAG.A...AG...T

C .........

C...C

........

ACAATG--TTATTTCTATTTAAAACAACAAAGGGTGATTTCTTTTAACCTATTTTGCTTATCTTTGTGGTA-TTTA

GTTTCCT.AGTA

...........

.......

T .....

C .....

...G.A...A.A.T..T..AA

C,.G.T.TT..G.G..TT

............

T .....

T ..........

A .... T ......... .......

.GCCAACATATGCTC

A..GA.:.GG

............

.......

C.A.T...TAG

AGACAGAAATAAACAGGAACGGTCTGATTTTGTTGTATCGTTGTTTACTAAAATGAAAATTCATCCTTTTTTCAAAAAA-GTGAGCCGTCA

. . . . . . . . . . . . . . . . . . . . . . . T..TA.A...C

......

A.T

TA .......

-1475

.......

C .....

-.C

.....

....

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- ......

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A .... T .........

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A .... G.T

AG.G

.....

...................

T .....

CG..G

T .......

G ...............

..........

-736 -568

C ......

T..

-594

T .....

C.,

-444

AATATGAATGCA

-289

.....

G ....

-294

....

-149

G ...........

................

T ................

T ...........

-716

...........

-418

A .......................

...............

C ....

A..TA..A

A...AAAGTGCAAATTGCGTGT

-144

TG ..........

T ........

C ......

G .....

- ....

...G

..... GTTGTGATGTTTAGTTAGACGTTGCATATGCACGATAATAATTCCGATGTGTA-TTTTTTCACAAGTGTCACA GTTTT

........

A.G

......................

TG..G

.......

A..T

-i

....

T .................

A.

I M P V S A S L A Y K N Y D Y D Z D S I Q P Y F Y F D N D D E D F Y H H Q Q G Q T Q P P A P S E D I W JtTGCCGGTGAGTGCGAGTTTGGCATATAAAAACTACGATTACGA~TACGACTCCATCCAGCCCTACTTCTA~TTCGACAACGACGATGAAGATTTCTATCACCATCAGCAAGGACAGA~T~AACCTCCAGCTCC~AGCGAGGACATTTGG

CAN CA~

ATG

.........

T ..........

IM 5

G .......................

T .................

T .......................

+I 50

G ............................................................

+150

S

1 K K F E L L P T P P L S P S R R Q S L S T A E Q L E M V S E F L G D D V V N Q S F I C D A D Y S Q S AAAAAATTCGAGCTGCTGCCCACACCGCCCCTCTCTCCGAGCCGGAGAcAGTCGCTCTCCACTGCCGAGCAGCTGGAGATGGTCAGCGAGTTTCTGGGAGACGACGTGGTCAATCAGAGCTT•ATTTGCGACGCGGACTATTC••AGTCC

~ CA~

-i

..G ...................................

C .......................

C .....

A .......................

C ....................

C ...........

C .....

T ........

+300

C .........

+300

51 1 0 1 F I K S I Z I Q D C M W S G F S A A A K L E K V V S E R L A S L H A A R K ~ L M S D S S S N R L N A CA~ TTCATCAAGTCCATCATTATCCAGGACTGCATGTGGAGCGGCTTTTCCGCTGCTGCCAAGTTGGAGAAAGTGGTT•CTGAGAGATTAGCGTCCCTGCACG•TGCTAGGAAGGAACTGATGT•TGACAGCAGcT•AAATCGACT•AATG•A CA~ ................. A..T ............................................................. AGC .................................. C .............................. 101 K I 1 5 1 S Y L Q D V S T S A S E C I D P S V V F P Y P L P E S G K S S K V A P S E P C A N AGTTATTTGCAGGATGTGAGCACCTCTGCATCAGAATGCATCGACCCTTCTGTGGTCTTCCCGTATCCCCTCCCGGAGTCTGGCAAATCTAGCAAGGTTGCTCCATCCGAACCCATGCCTGTCCTGGACACTCCACCTAACAGCTCCAGC CA~ ............... C ......................... T.GA..C..C ........ T..A ......... A.A ..... CA ....... C ................. I5] i C T S 201 S S G S D S CAN AGCAGTGGCAGTGATTCTGgtatgttatgcattatcttgttatttatgacatgcataatcttgatttatctagt CA~

...................................

t...gc

....

c .........

g .........

g..g..g

tactacatgtgcaagtt ......

cttgctttat

g .........

M

P

V

L

D

T

P

P

N

S

S

+450 +450

S +680

A .....

A ....

T ...............................

+600

L

ttaagtgacctttacttgtcaccggtgagggggtgtgccagtcacggga

tagctt..a..t

.........

t ......

a ......

t .......

+750

t ........

g .........

+750

201 CA~ CA~

ctttatcctcatacacttgatgcaatgattggtgcaagtggtagttgtataagatc•ct•cacctcccctctgctagacccaagtcagatctgtaggaaaaactgcagtgtt•aaatatCagccgataaggaaagtaggcggggactgga ..............

c ......

at.g

....

c ....

t ............

c ......................

t .....

t ....

g .............

- ....

g ......

a ...............

t...g

..t .....

g...t...g

207 E CAN CA~

........

t ..........

g ............

c ...........

c ........

t ........

aa ..........

ct...t.at

.......

+1046

t.gtt.g.t

....

a ......

tt..g

.....

t ..........

GAT..

207 209E C A N AGAA ......... CA~ .... G A A G A G G A G 210 E E E

E

E

E

E

E

E

G .....

E

E

E

A..A..A

E

E

E

........

E

E

E

I

D

V

V

T

V

A ..........................

E

K

R

Q

K

C ...........

K

N

E

T .......

T

A

V

G .......

S

D

G.A.AG

S

R

Y

P

S

P

L

V

L

K

R

R

A

C +1187

.....

C .......................

G ..............

+1196

E

2 5 6 H V S T H Q H N Y ~ A H P S T R H D Q P A V K R L R L E A SS N S N S R H V K Q R K C T S P R CAN T~ACGTCTCCACC~ACCAGCACAACTACG~TGCCCACCCCTCCACACGGCATGACCAACCGGCTGTCAAGAGGCTACGACTGGAGGCA---AGCAGC---AA~AGCAA~AGC~--AGGCATGTGAAACAACGCAAGTGCACGAGTC~CC~ CA~ ...T .......................... C .............. G ..... C ..... G ....................... C...AGCAGC ..... TAAC ....... G .... AAC ..... A.G,..G ....................... 260 S S N S N Q G 3 0 3 T S D S E D N D K $ : R T H N V L E R Q R R N E L K L S F F A L R D E I P D V A N N E K A A K V V I L CAN C~CATCGGATTCTGAGGACAATGACAAAC6;CAGGACT~ACAATGTGCTGGAGCGCCAACGCAGAAATGAACTCAAACTCAGCTTTTTCG~ATTAcGGGATGAGATCCcTGATGTTGCAAATAATGAGAAAGC~GCTAAAGTGGTTATCCT CA~ ............................................................ T .............................. C ................... G ........... C ........ T ........ T ..... A., 310 E 3

5

CA~

3

K

K

A

T

E

C

I

H

S

M

Q

L

D

E

Q

R

L

L

S

I

K

E

Q

L

R

R

K

S

E

Q

L

K

H

R

L

Q

L

L

R

S

S

H

...G

.............................................

+1478 +1496

+1628

T ............................................................

A ................

360

TAA

.... C .....

T .........

+1646

Q

CAN

CAAAGTGAAAA-ACTTTTTAAGAAACCCCCTCCCCCTGCTTTTTTATGCAAGTTAAAGACTGATGCTGTATATAGTTGATGTTGCATGTCCAATGCATTTTTTTTCCTTTTGAATA•TGCCAGTTTTTTTCATGTTTGAAACTGCCACAG

CA~

...........

CA~ CAR

TTTAAAATTTAAGGTGGTTAAAATAATTTGTATTTAAGTCATTTATTTTTATTAATAAAATTAGAAAAAATAGGGCTGCACAACCCTGAAGTAAAATTTTTTTTGTATGTTTTTGTATATAATGAATTAATATTTCCTAAGAAAGGTATT .....................................................

AATAAAA.G

CA~ CA~

TTTGGATCTGTATCAGCTACATGTTCTCAUAAACCATATTCTGTTTCA-TATG

........

CA~ CA~

CACATATAGATTAGACACTTTAGAAAGAAUTTTGGAGAATATTCAGAGAAAT~TGATATTTACTTGTTAGTCCAGCAAGCATGATACTGTTACTTTTTCAGTGACAAACTGGTACTTATTTCAGGGATTTTCCCAGT---TAATTTCAGT

CA~

~TAAGTTTTCACCT-GTTT

G ..........

....................

T..TA

........

G..-.G...T

- ..................................

G ...................

A .......

--...T

C ....

TTTTTTTTT

......

G .......

.........

G..T

C ...................

............

A ........

+I 777

- ..................

A...C

......

....

.....

C ...........

A .......

+1942

GTTTTCTTCATGTTTTCACTTAATCATCAATGAAATTCTTTCTGACCTTGACTCTGGTCTCTTTTGGTTTCT•TGCA•TGAGCTCCTCA ....

T ..........

T..A

.......

A ......

T ..........

........

T ......

AG

.... C ...................

G .....

C.A

.....

A ................

+2068 C .....

C ............

A .......

-.A.A

.................

+2092 +2215

. ....

T .....

+I 792 +i 927

T ........

C ......

CAGA..GG

GTUTTTTCACTTTCTCTCATGATATCAGTATAGAAAAGCTTACAAAAGACAAAAATGCTCAACTGCATC~CGCCCATCTAGATGAAAGTTGAAC-TCCTTCATATTTGGCATTGAAAT

AAAATGGT..A..G

G ....

C ..........................................

- ....................................................................................................... ........

T..T

CA~

.....

CA~

---AATAACC-AACAGTG .... G C T T A T T i L ~ A G T T A C T G T G A G G A C ~ G C T G C T G G A A A A C C C A A ~ A A A G G G G A T T T T G A ~ A G C A A G A G A A T C - A C T T T A A G G T G A T T C A C C A A A G A T T T T A C A A G C G A T G A C T ~ A C A G T +2481 ~C, GC. C..A..A.A..AAAG..A.TGC .... C..T ......... T ..... CA,G ......... G .......................... A ..... C ........ T .... T ..T .GAC... TTAAGAT~TTCGAGGTGATGACTCACA

CA~

+I 328 +1346

*

AAAAAAGGCGACAGAGTGCATCCA~AGCATGCAGTTGGATGAGCAAAGGCTG~TGTCCATCAAAGAACAGCTGAGGCGAAAGAGCGAACAGTTAAAACACAGGCTGCAGCTGCTGCGAAGTTCACATTAATGGATCTGTGACACAAGAGT

CA~

+1046

D

GAGGAGGAGGAAGAAGAGGAGGAGGAGGAAGAGGAAGAGGAAGAAATTGATGTGGTGACTGTGGAAAAGCGA~AGAAAAAGAACGAGACGGCAGTGTCAGATTCAAGGTACCCCAGT~CCCTCGTGTTGAAGCGCT~ ...........

+899

E

atgcagagacccccccccca~cccaccctttgttgaaagccaattgtcaaattccattaaattcccactgcgctgttg~atgaggttttaaatgacctgatgtt~taagt-taattatttttctggttttaattcaattgcagAA---GA

........ - - - t i t

+900

.....

A.GG.A

..................

C ....

.....

+2139

....

+2351

AAGC

+2287

+2437

CA~ CA~

TTCCACAGTCGTCAATAGATATAGAGGAACTTTAAATTTTTTTT

+2481

Fig. 2. The nt sequences of CAM1 and CAM2 and deduced aa sequences. The nt sequences in the intron are indicated by lowercase letters, others by capital letters. The nt residues are numbered at fight. Dots (.) in CAM2 indicate the same nt residues as that of CAM1. Gaps ( - ) are introduced to optimize identity. For CAM~! the deduced aa is shown as an italic one letter code above the middle nt residue of each codon and numbered in italics at the left. For CAM2, only aa residues different ~ o m those of CAMI are indicated below the middle nt residue of each codon. The TATAbox like sequences, putative start codon ATG, stop codon TAA and the poly(A)-addition consensus sequence, 5'-AATAAA, are indicated by bold letters. The sequences which may form hairpin structures are italicized. The nt sequences of the two carp c-myc genes have D D B J accession Nos. D37887 (for CAM1, 8675 bp) and D37888 (for CAM2, 5784 bp).

234

MPVSASLAYKNYDYDYD~IQPYFyFDNDDEDFYKH-QQ--GQTQPPA~EDIWKKFELLPTPPLSPSRR~L .... S .............................. RTM

NS...S

XLM

. .LN.NFPS

............... ........

cam

..H ....

HUM

. .LNV.FTNR...L

-L..C

V..E

KWS

V .....

L ~ E L M S D S S S N

................

......

.F.LEE-.N.

Ps . . . . . . . . . v ...... ....

-..--.

...............................

--..-P..L

....

• .--..--SRL..

E~E,.N..U~.

. .......................

QLF24VSEFLGDDVVNQSF

- .............................

T ..............

- ......

T .... G.M

.................

• . .D .....

QSS--LFP

.................

- - .D .....

/

C.EE-.N..QQQ..--SEL.

• "''1"

.........................

. . . . . . . . . . . . . . . . . . . 7. .

RLNASYLQDVSTSASEC

CSPSYVAVTPFSLRGDNDGGGGSF...D

I DP ..........

SVVFP

y pLHE

SGKS

.....

SKVA9

- ........................

. ........

L ........

G.- .........

. .......

T..S

........................

. ........

L ........

G.- .........

. .......

T.C..AG...SPQ..-

. . . . . 1. . . . . Q T . . . D S A V G D N A E C P T ...................... ..... ~... YQ.S... SALS... PCQSQPPPSPLKSPSCHGSLSLGGTH.

....N .... PN ....... G. NTSASECZGP SSHGF... P.--.D.V...........

,T T YQ.S.R.GGPAAA.RPGPPPSGPPPPPAGHAASAG .........

L.. H.LG~.AD

.... - .........

IC- DADYSQSFIKS

...........

T.L

DEA-LL

I ~ I QDCMWSGFSAAAKLE

. . . .~. . . . . . . . . .

T ....

.......

-E..

r.L..Q.M

. . . . . . . . . P.DES-..V..] . . . . . . . . . . . . . . . .

T.L..G.M

. . . . . . .

- . P . D E T - . . . N

SE pM ......................

I ...... E .... I...N..-

RTM XLM

.......

STAE

- .......................

p~[.. S S - - I F P

"''I" ....................

. . . . . . .

H~

~ ....................

. . . . . . . . . . . . . . . . . . . . . . . .

. . . ..[. . . . . . . . . . . . . . . . . . . . . . . . l.~-,scrP . . . . . . . . . . . . . . . . . . . . D.....

H~SEH

ZEM

CI~4

.,.

PVL--

- .....................

L..--.

.....................

.--H

...................

. ~. . . . . . . . . . . . . . .

DTPPN--

SSSS~G

SDSREEE

.... --.

.........

D..

.... --.

.........

D..

....... IT.TP.P ..... PTI>- ...................... . ....... NDTISNASSPCQD.......................

A~A. --. .... SG .......... DDD ~LI.--E...I--..N..S.E .... P

. .......

G~G. V - - . . . . -

S .RAgRAAPPGAN.ALL

....................

.....

TTS

......

.... [~[._,Q.... DS~.P,W~G~SVCS~SS. . . . . . . . . . . . . . . . . . . . ~.... r ~ . . . . . . . . . . . . . . . . . . . . . . . . . rm.SSPKSC.SQt~S~S;SSDSH~SS~SS~SPE .~E . . . . . . . . . ~S ...... EEEEEEEEEEEEE

....

E I E~VTVE

: . ZEM

............. . .D ..........

= = EEEE

............ ............

KIIQKKNE

RTM

• • .DD. D...-

XLM

.D.D.

TAVSDSHY

SPLVLKRCHVSTHQHNYAAHPSTRH~-SN-SNS

P ........

-

..... R I I V K Q R K C ~ S

P R TS DS E DNDKRR

T

N

RQ RRNELIQhS

FTALRDE

Q

9

I P DVA

/

DCD...-

...... .....

- = . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RH • .DA. E .... - ....... .. . . . . . . . . . . . . . . . . . . . . . . . . . .

. ..........

AVI~RCDP.

T. ETR ......

. ..~........

SASKRVE.

S. HSQ--PSRPHY

HH .........................

i

~" ° ° . .. ... ... ... ... .. ... ... ... ... .. .. . . . . HUM .D

- ~~ =" . c~ .H~ H ~ S~ ,-i:. .; _~_ .' . .~~ G

ZHM

................................................

XLM C~M

S .... P .......... ..... P ..........

HUM

.....

P .........

yAI. YVL. AY.L.

..........

PI .......

E. S...KV

"H~.~I: .. ... ... ... ... .. .. . . . . . ~. .. ... ... ... . . ~ .... ~

NNHSI..

..... SSSSN

.S.H-VLK

.....

QISSN

.........

NIR-VLK

.....

QISNN

.....

.....

o ~~. . . . : p .. . . . .. . . . . .~..~.~.~ .. ~ . . . . .. ... ... ... ... .. ... ... ... ... .. ... ... . . . . Q

~: :~:~:l:-°H-~ -w-~ .....

oo ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................... E •. T..Y

................................

S .... E .... K ......................

QV.E..

..... H....... ~o .....

* "'"

E..R.. IRET...KIRK .... Q...Q..NFV* ~.S..H..IAE ...... RR ..... K.EQ..N.RA* %1. A N . . K . I . E E D L . . K R R

.....

K.EQ..N.CA*

Fig. 3. Comparison of deduced aa sequences of c-Myc of carp (CAM1 and CAM2), zebra fish (ZEM), rainbow trout (RTM), Xenopus laevis (XLM), chicken (CHM) and human (HUM). Dots (.) represent aa identical with those of CAM1. Gaps (-) are introduced to optimize identity. The functionally important regions are boxed (for details, see Taya, 1991; Scbreiber-Agus, et al., 1993). Asterisks (*) represent the stop codons. Arrow indicates the boundary of exon 2 and 3. GENETYX-MAC, a computer algorithm developed by Software Development, was used for sequence comparison.

is

found

in

the

(ATGCATGTTATGCAT, (ATGCATACGATGCAT,

5'

region nt - 1 1 6 nt - 1 2 0

of

both

to - 1 0 2 )

CAM1

and CAM2

to - 1 0 6 ) .

(c)

Comparison

of the two

carp

c-myc

genes

C a r p C A M 1 a n d C A M 2 a r e v e r y s i m i l a r in t h e i r g e n o -

T h e r e is

m i c o r g a n i z a t i o n a n d nt a n d a a s e q u e n c e s as s h o w n in

(nt 1831) a n d

Fig. 1 a n d T a b l e I. T h e nt identities of C A M 1 a n d C A M 2

C A M 2 (nt 1846); h o w e v e r , p o l y ( A ) a r e a d d e d at different sites in c D N A c l o n e s of C A M 1 (nt 1864 a n d 2021) a n d C A M 2 (nt 2023, 2042, 2055 a n d 2105). T h e u s a g e of

in the i n t r o n . T h e 5' F R c a n be d i v i d e d i n t o t w o d e p e n d -

o n l y o n e A A T A A A s e q u e n c e in C A M 1

different p o l y ( A ) - a d d i t i o n sites m a y c o r r e s p o n d to t h e result of N o r t h e r n b l o t a n a l y s i s in w h i c h several b a n d s w e r e o b s e r v e d ( Z h a n g et al., 1993).

are 9 2 . 4 % in the C R , 9 0 . 5 % in the 3' U T R a n d 8 4 . 2 % ing o n the nt identity. T h e nt i d e n t i t y of the 5' FR-1 (nt - 1 3 7 4 to - 8 1 7 of C A M I a n d - 1 5 7 3 to - 8 3 7 of C A M 2 ) is m u c h l o w e r ( 5 7 . 5 % ) t h a n t h a t of the 5' FR-2 (nt - 8 1 6 to - 1 o f C A M 1 a n d - 8 3 6 to - 1 of C A M 2 )

TABLE I Comparison of two carp c-myc genes, CAM1 and CAM2 and their products Region

CR

Intron

Size

(aa)

1 2 1+ 2

L (nt)

I I 2 l +2

I I

3' UTR b FR

5'-1 5'-2 3'-1 3'-2

Identity (%)~

CAM1 or CAM1

CAM2 or CAM2

206 aa 188 aa 394 aa

206 aa 195 aa 401 aa

619 bp (1-619) 566 bp (1043-1608) 1182 bp 423 bp (620-1042) 473 bp (1609-2081) 558 bp (-1374 to --817) 816 bp ( - 816 to - 1) 378 bp (2082-2459) (2460 and over)

619 bp (I-619) 587 bp (1040-1626) 1203 bp 420 bp (620-1039) 479 bp (1627-2105) 705 bp (-1537 to -837) 836 bp ( - 836 to -- 1) 299 bp (2106-2404) (2405 and over)

96.1 92.3 94.2 94.2 88.3 92.4 84.2 90.5 57.5 81.7 57.2 < 50.0

" Identity is calculated using GENETYX-MAC, a computer algorithm developed by Software Development (Tokyo, Japan). b From the nt residues immediate 3' side of the stop codon to the most 3' poly(A)-addition site in CAM2 or the corresponding region in CAM1.

235 (81.7%), suggesting that 5'FR-1 m a y be already outside of the gene. There is only one c - m y c gene in diploid organisms (Bernard et al., 1983; W a t s o n et al., 1983; King et al., 1986; Schreiber-Agus et al., 1993). The existence of two c - m y c genes in carp is t h o u g h t to be caused by tetraploidy. It is k n o w n that mouse and h u m a n separated about 80 M y r ago, and mouse and chicken about 300 M y r ago (Kimura, 1983). The aa identity shared by mouse and h u m a n and by mouse and chicken c-Myc are 92% and 70%, respectively (Bernard et al., 1983). The evolutionary drift of c - M y c could be roughly estimated to be a b o u t 1% in 10 M y r for related pairs. The aa identity shared by the two carp c-Myc (94.2%) suggests that the doubling of chromosorae numbers p r o b a b l y occurred 58 M y r ago, in agreement with the estimation by isoenzyme p o l y m o r p h i s m (Ferris et al., 1977).

h o m o l o g o u s sequence to h u m a n or mouse exon 1. Using exon 1 of h u m a n c - m y c gene as a probe, carp genomic D N A was analyzed by Southern hybridization, and no signal was detected ( Z h a n g et al, 1993). E x o n 1 was also not detected in rainbow trout (Van Beneden et al., 1986). It has been reported that exon 1 evolved quicker than exon 2 and exon 3 (Bernard et al., 1983; Hayashi, 1990). Exon 1 m a y be exist in carp c - m y c genes, but their nt sequences are too different to be detected by the h u m a n exon 1 probe.

ACKNOWLEDGEMENTS This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, J a p a n (Nos. 05404014, 05044136) and the Japanese Fisheries Agency.

(d) Comparison of c-myc genes of carp and other

organisms As shown in Fig. 3 and Table II, the C R of c - m y c genes in vertebrates are highly conserved, especially in the functionally i m p o r t a n t regions boxed in Fig. 3 (for details, see Taya, 1991; Schreiber-Agus et al., 1993) where there are few aa differences a m o n g them. The two carp c - M y c share 5 5 - 9 1 % aa identity with those of h u m a n (Bernard et al., 1983), chicken (Watson et al., 1983), frog (King et al., 1986), rainbow trout (Van Beneden et al., 1986) and zebra fish (Schreiber-Agus et al., 1993) (Table II), being consistent with their evolutionary distances. The major differences which should be pointed out is the existence of a Glu stretch (18 in C A M 1 and 21 in C A M 2 ) in the beginning of exon 3 of the two carp c - m y c genes. A similar Glu stretch (20) is also present in zebra fish (SchreiberAgus et al., 1993), which belongs to the same family (Cyprinidae) as carp. This Glu stretch, which appears to be inherent in cyprinid fishes c - m y c genes, is partially deleted or substituted in rainbow trout and other higher vertebrates (Fig. 3). In m a m m a l i a n c - m y c genes, there is a n o n c o d i n g exon 1, which plays a regulatory role in the transcription of c - m y c (Hayashi, 1990). In the sequenced 5' regions of carp C A M 1 (1376 bp) and C A M 2 (1542 bp), there is no TABLE II Amino-acid identities (%) of c-Myc of carp and other vertebrates Carp ~

CAM 1 CAM2

Other vertebratesa ZEM

RTM

XLM

CHM

HUM

90.4 90.9

72.9 72.8

59.2 57.4

57.1 57.6

55.3 56.7

a See legend to Fig. 3 for abbreviations.

REFERENCES Allendorf, F.W. and Thorgaard, G.H.: Tetraploidy and the evolution of salmonid fishes. In: Turner, B.J. (Ed.), Evolutionary Genetics of Fishes. Plenum Press, New York, NY, 1984, pp. 1-53. Bernard, O., Cory, S., Gerondakis, S., Webb, E. and Adams, J.M.: Sequence of the murine and human cellular myc oncogenes and two modes of myc transcription resulting from chromosome translocation in B lymphoid tumors. EMBO J. 2 (1983) 2375-2383. Breathnach, R., Benosit, C., O'Hare, K., Gannon, F. and Chambon, P.: Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries. Proc. Natl. Acad. Sci. USA 75 (1978) 4853-4857. Ferris, S.D. and Whitt, G.S.: The evolution of duplicate gene expression in the carp (Cyprinus carpio). Experientia 33 (1977) 1299-1301. Hayashi, K.: The expression controlling of c-myc gene. Exp. Med. 8 (1990) 152-155 (in Japanese). Kimura, M: The Neutral Theory of Molecular Evolution. Cambridge University Press, Cambridge, 1983. King, M.W., Roberts, J.M. and Eisenman, RIN.: Expression of the c-myc proto-oncogene during development of Xenopus laevis. Mol. Cell. Biol. 6 (1986) 4499-4508. Kuchino, Y.: The s-myc gene. Protein Nucleic Acid Enzyme 37 (1992) 865-867 (in Japanese). Lundin, L.G.: Evolution of the vertebrate genome as reflected in paralogous chromosomal regions in man and the house mouse. Genomics 16 (1993) 1-19. Ohno, S.: Evolution by Gene Duplication. Springer, Heidelberg, 1970. Ohno, S.: Pattern in genome evolution. Curr. Opin. Genet. Dev. 3 (1993) 911-914. Ojima, Y: Fish Cytogenetics. Suikosha Press, Tokyo, 1983(in Japanese). Schreiber-Agus, N., Horner, J., Tortes, R., Chiu, F.C. and Depinho, R.A.: Zebra fish myc family and max genes: differential expression and oncogenic activity throughout vertebrate evolution. Mol. Cell. Biol. 13 (1993) 2765-2775. Taya, Y.: The myc genes and signal transduction. Exp. Med. 9 (1991) 169-172 (in Japanese). Van Beneden, RJ., Watson, D.K., Chen, T.T., Lautenberger, J.A. and Papas, T.S.: Cellular myc (c-myc) in fish (rainbow trout): its relationship to other vertebrate myc genes and to the transforming genes

236 of the MC29 of viruses. Proc. Natl. Acad. Sci. USA 83 (1986) 3698 3702. Watson, D.K., Reddy, E.P., Duesberg, P.H. and Papas, T.S.: Nucleotide sequence analysis of the chicken c-myc gene reveals homologous and unique coding regions by comparison with the transforming

gene of avian myelocytimatosis virus MC29, Agag-myc. Proc. Natl. Acad. Sci. USA 80 (1983) 2146 2150. Zhang, H., Okamoto, N., Yamamoto, N. and Ikeda, Y.: Molecular cloning of carp cellular myc (c-myc) cDNA. Gyobyo Kenkyu (Fish Pathol.) 28 (1993) 111-117.