Molecular Immunology, Vol. 29, No. 9, pp. 1157-1158, Printed
in Great
1992
0161-5890/92 $5.00 + 0.00 Pergamon Press Ltd
Britain.
BRIEF NUCLEOTIDE
STRUCTURAL
DATA REPORT
SEQUENCE OF A NURSE SHARK IMMUNOGLOBULJN HEAVY CHAIN cDNA CLONE
Miriam Vaxquex, Nobuyuki Mix&i, Martin F. Flajnik, E. Churchill McKinney, and Masanori Kasahara Department of Microbiology and Immunology, University of Miami School of Medicine, Miami. FL 33101. U.S.A. (Received 22 April 1992; accepted 26 May 1992)
The polymerase chain reaction (PCR) with degenerate oligonucleotides enables one to isolate homologous genes from distantly related species. Recently, we used this approach to isolate major histocompatibility complex (MHC) cDNA from nurse sharks, Ginglymosfoma cirratum &asaham et al., 1992). In the course of this study, one of the PCR primers originally designed for the isolation of MHC genes amplified cDNA encoding an immunoglobulin (Ig) heavy chain. Here we describe the complete nucleotide sequence of a nurse shark Ig heavy chain cDNA clone. One-sided PCR was performed as described (Kasahara et al.. 1992) using muse shark spleen cDNA as a template. The sequence of the gene-specific primer that led to the amplification of Ig heavy chain cDNA was: 5’-CAAAGCTTGTGACNGRYYCC-3’ (the 5’ eight bases containing an artificial HindI11 site are underlined). The 3’end 18 bases of this primer were designed based on the fact that most MHC class II Bchains have Val-Thr-(Gly or Asp)-PheTyr-Pro in the second l&strand region of the Ig-like domain. The PCR product encoding an Ig heavy chain, which encompassed a part of the C,J domain, the secretory segment, and the 3’-untranslated region, was used to screen a nurse shark spleen cDNA library to obtain full-length cDNA clones. The longest cDNA clone thus isolated, A4-2, was sequenced in its entirety (Fig. 1). The deduced protein, made up of 557 amino acids excluding the signal peptide. had a structure characteristic of a heavy chain corresponding to the mammalian IgM isotype. The nurse shark V, domain was most similar to horned shark V, domains (Litman et al., 1985; -70% amino acid identity), followed by little skate VH domains (Harding et al., 1990; -52% amino acid sequence identity). The nurse shark C, domains (four domains combined) showed -65% and -50% amino acid sequence identity to the horned shark (Kokubu et al., 1988) and little skate (Harding et al., 1990) C, domains. Among the C, domains, the least conserved region was the junction between Cal and C,2. where the nurse shark Ig had a smaller number of residues than the homed shark Ig. Southern blot hybridization of nurse shark genomic DNA with a C,, probe derived from clone A4-2 showed that the nurse shark (Superfamily Orectoloboidea) has multiple copies of C, genes as in the homed shark (Superfamily Heterodontoideu). Acknowledgments. This work was supported by grants ROI RR06603 from NIH and DCB-8911552 from NSF.
REFERENCES Harding, F.A., Amemiya, C.T., Litman, R.T., Cohen, N. and Litman, G.W. (1990) Two distinct immunoglobulin heavy chain isotypes in a primitive, cartilaginous fish. Raja erinacea. Nucl. Acids Res. 18,63696376. Kasahara. M.. Vaquex, M., Sate. K., McKinney, E.C. and Flajnik, M.F. (1992) Evolution of the major histocompatibility complex: Isolation of class II A cDNA clones from the cartilaginous fsh. hoc. Nutn. Acad. Sci. USA. 89, in the press. Kokubu. F.. Hinds, K., Liunan, R.. Shamblott, MJ. and Litman, G.W. (1988) Complete structure and organization of immunoglobulin heavy chain constant region genes in a phylogenetically primitive vertebrate. EMEO J. 7. 1979-1988. Litman. G.W., Berger, L., Murphy. K.. Litman. R.. Hinds, K. aud Erickson, B.W. (1985) Immunoglobulin V gene structure and diversity in Heterodontus, a phylogenetically primitive shark. Proc. Natn. Ad. SC; USA. 82. 2082-2086.
1157
1158
Brief Structural
Data
Report
Signal peptide 110
I
MNTSTIFLSLLLALL VH
-1 +1
10
30 CDRl
20
220
CDRl
50 CDR2 60 CTACTATAGTTCTTCGATGAATMCTATGCGCCAGCGATTAAAGA YYSSSMNNYAPAIKD
70
SO
90
330
DH
----bloo
CAAGACGCATGAGTGGGTATGAATACTTG
---+llo JH 120 f-b cH1 130 GGCGGCCACTCCGGTTACTG~GACMG~C~TGGT~CCGT~CTACAGCMCACCGTCTTCTCCGA~CTTTATGGCTTAGTCTCCTCCTGTCAG~GGCM~T GGHSGYWGQGTMVTVTTATP SSPTLYGLVSSCQQGNI 150
160
140 550
160 170 TGGCGATGGACTATTCCCCCGACGTCGCCAGTGTGACCTG~G~CATGGGCAGCTGATCACGACTG~GTTCAGACGTACC
190
200
210 GGTTCGACACAGCGGGTCC
220 ---b cH2 230 GACMGAGCACTGGMTGCCATGTCCTGATGGCTTTCCW\TCATCGT
260 CAGTGRCTTCCACTCAAhGT~TCTCCGTTGCTTCTCAG SD F W SK S I S V TW L
440
240
660
770
250 880
270
280 990
K
N
G
R
S
V
D
S
G
I
F
T
S
P
VC
EA
N
G-1 w
300 290 TGACCAGTCGCCTGAGAGTCCCTTATGCTGGTTCGATAC VT S R LR V P Y A EW F D
RAVY
r,
340
(23 330 GTTTCCGAGTGCCATGGTTACACAGCTAAAATACTACCAC VSECHGYTAKILPPPVEQVLLEATVTLTCVVSNLHSG
G TPT C
310 320 AGGTCAAGTATAAAGAGGTCATTCAAAGTTGGAACATCACCGGACCTCAG QV K Y K EV IQ SWIrdG P Q cm 350 360 G TTGTGTCCAATCTTCATTCTGG
1210
n
370 380 390 AGTCMCTTCACCTGGTTACM~CGAGM~CTCT~TCAGAGATTGCTCACGACTCTG~~GCATTCC~TGGTGC~TCAGC~TT~ATATTTCTACTGMG VpijW L QD E KT L K S E I AH D S G E H SD GA I S K L D I ST E au 400 410 420 I-+ 430 cH4 CCTGGCTGAGTGAGGTTGTTTTCG G TGGTGAATCATCAGTATCTACCW\CTCCTCCTCTGAGAGACTCCATCCACMGGAGAGMTT~TCCACTG~GCCATCG AWLSEVVFECVVNHQYLPTPLRDSIHKERIENPLEPS v 440 450 GTGTCTGTCCTCCTGC~C~CAGMGMCTCTCCGCGCAGAGGTTTCTTTCCCTTAC
1100
460
1320
1430
470 1540
480 490 500 CMCGACMGCCGGTTMTCCCAGTMCTA~GMCACCGMGTGACGG~~~GCGACMCACCTCCTTCTTCTTGTA~GCCTGTTATCCATTGCAGCG~GGAGT 1650 ND K P VN P S NY KN T E V T A E S DbT[F F LY S L L S I AA E E cm secretory e.egment 520 530 + 540 510 GGGCCAGCGGTGCTTCTTACTC G TGGTGGGACATGMGCGATTCCATTGAAGATCATCATCMCAGMCGGTTGAT~TCCAGCGGT~CCGAGTTTTGT~TATT 1760 WASGASYSCVVGHEAIPLKII~~VDKSSGKPSFV~ "T'F c-n am 557 550 TCACTTGCACTGATGGACACCATTMTTCATGAAC(A)n 1850 -~LALLDTINSCQTER
Fig. 1. Nucleotide and predicted amino acid sequences of a nurse shark Ig heavy chain cDNA clone A4-2. The deduced amino acid sequence is shown below the nucleotide sequence and numbered from -20 to -1 for the signal peptide and from 1 to 557 for the mature protein. The assignment of domains and segments is based on comparisons with published homed shark (Kolcubu et al., 1988) and little skate (Harding et al.. 1990) Ig heavy chain sequences. Ambiguities in domain or segment boundaries are indicated by dashed lines. Cysteinyl residues assumed to form intradomain disulphide bridges are boxed. Also boxed are complementarity determining regions (CDR) and potential A%nkd glycosylation sites (CHO). Putative polyadenylation signals are underlined. (A)n, a poly A tail. DNA sequencing was performed using custom-made gene-specific primers in addition to standard T3/17 primers. Sequence ambiguities were eliminated by replacing dG’lB with dlTP. This sequence has been submitted to the EMBL/GenBank/DDBJ nucleotide sequence databases under the accession number M92851.
in Great
1992
0161-5890/92 $5.00 + 0.00 Pergamon Press Ltd
Britain.
BRIEF NUCLEOTIDE
STRUCTURAL
DATA REPORT
SEQUENCE OF A NURSE SHARK IMMUNOGLOBULJN HEAVY CHAIN cDNA CLONE
Miriam Vaxquex, Nobuyuki Mix&i, Martin F. Flajnik, E. Churchill McKinney, and Masanori Kasahara Department of Microbiology and Immunology, University of Miami School of Medicine, Miami. FL 33101. U.S.A. (Received 22 April 1992; accepted 26 May 1992)
The polymerase chain reaction (PCR) with degenerate oligonucleotides enables one to isolate homologous genes from distantly related species. Recently, we used this approach to isolate major histocompatibility complex (MHC) cDNA from nurse sharks, Ginglymosfoma cirratum &asaham et al., 1992). In the course of this study, one of the PCR primers originally designed for the isolation of MHC genes amplified cDNA encoding an immunoglobulin (Ig) heavy chain. Here we describe the complete nucleotide sequence of a nurse shark Ig heavy chain cDNA clone. One-sided PCR was performed as described (Kasahara et al.. 1992) using muse shark spleen cDNA as a template. The sequence of the gene-specific primer that led to the amplification of Ig heavy chain cDNA was: 5’-CAAAGCTTGTGACNGRYYCC-3’ (the 5’ eight bases containing an artificial HindI11 site are underlined). The 3’end 18 bases of this primer were designed based on the fact that most MHC class II Bchains have Val-Thr-(Gly or Asp)-PheTyr-Pro in the second l&strand region of the Ig-like domain. The PCR product encoding an Ig heavy chain, which encompassed a part of the C,J domain, the secretory segment, and the 3’-untranslated region, was used to screen a nurse shark spleen cDNA library to obtain full-length cDNA clones. The longest cDNA clone thus isolated, A4-2, was sequenced in its entirety (Fig. 1). The deduced protein, made up of 557 amino acids excluding the signal peptide. had a structure characteristic of a heavy chain corresponding to the mammalian IgM isotype. The nurse shark V, domain was most similar to horned shark V, domains (Litman et al., 1985; -70% amino acid identity), followed by little skate VH domains (Harding et al., 1990; -52% amino acid sequence identity). The nurse shark C, domains (four domains combined) showed -65% and -50% amino acid sequence identity to the horned shark (Kokubu et al., 1988) and little skate (Harding et al., 1990) C, domains. Among the C, domains, the least conserved region was the junction between Cal and C,2. where the nurse shark Ig had a smaller number of residues than the homed shark Ig. Southern blot hybridization of nurse shark genomic DNA with a C,, probe derived from clone A4-2 showed that the nurse shark (Superfamily Orectoloboidea) has multiple copies of C, genes as in the homed shark (Superfamily Heterodontoideu). Acknowledgments. This work was supported by grants ROI RR06603 from NIH and DCB-8911552 from NSF.
REFERENCES Harding, F.A., Amemiya, C.T., Litman, R.T., Cohen, N. and Litman, G.W. (1990) Two distinct immunoglobulin heavy chain isotypes in a primitive, cartilaginous fish. Raja erinacea. Nucl. Acids Res. 18,63696376. Kasahara. M.. Vaquex, M., Sate. K., McKinney, E.C. and Flajnik, M.F. (1992) Evolution of the major histocompatibility complex: Isolation of class II A cDNA clones from the cartilaginous fsh. hoc. Nutn. Acad. Sci. USA. 89, in the press. Kokubu. F.. Hinds, K., Liunan, R.. Shamblott, MJ. and Litman, G.W. (1988) Complete structure and organization of immunoglobulin heavy chain constant region genes in a phylogenetically primitive vertebrate. EMEO J. 7. 1979-1988. Litman. G.W., Berger, L., Murphy. K.. Litman. R.. Hinds, K. aud Erickson, B.W. (1985) Immunoglobulin V gene structure and diversity in Heterodontus, a phylogenetically primitive shark. Proc. Natn. Ad. SC; USA. 82. 2082-2086.
1157
1158
Brief Structural
Data
Report
Signal peptide 110
I
MNTSTIFLSLLLALL VH
-1 +1
10
30 CDRl
20
220
CDRl
50 CDR2 60 CTACTATAGTTCTTCGATGAATMCTATGCGCCAGCGATTAAAGA YYSSSMNNYAPAIKD
70
SO
90
330
DH
----bloo
CAAGACGCATGAGTGGGTATGAATACTTG
---+llo JH 120 f-b cH1 130 GGCGGCCACTCCGGTTACTG~GACMG~C~TGGT~CCGT~CTACAGCMCACCGTCTTCTCCGA~CTTTATGGCTTAGTCTCCTCCTGTCAG~GGCM~T GGHSGYWGQGTMVTVTTATP SSPTLYGLVSSCQQGNI 150
160
140 550
160 170 TGGCGATGGACTATTCCCCCGACGTCGCCAGTGTGACCTG~G~CATGGGCAGCTGATCACGACTG~GTTCAGACGTACC
190
200
210 GGTTCGACACAGCGGGTCC
220 ---b cH2 230 GACMGAGCACTGGMTGCCATGTCCTGATGGCTTTCCW\TCATCGT
260 CAGTGRCTTCCACTCAAhGT~TCTCCGTTGCTTCTCAG SD F W SK S I S V TW L
440
240
660
770
250 880
270
280 990
K
N
G
R
S
V
D
S
G
I
F
T
S
P
VC
EA
N
G-1 w
300 290 TGACCAGTCGCCTGAGAGTCCCTTATGCTGGTTCGATAC VT S R LR V P Y A EW F D
RAVY
r,
340
(23 330 GTTTCCGAGTGCCATGGTTACACAGCTAAAATACTACCAC VSECHGYTAKILPPPVEQVLLEATVTLTCVVSNLHSG
G TPT C
310 320 AGGTCAAGTATAAAGAGGTCATTCAAAGTTGGAACATCACCGGACCTCAG QV K Y K EV IQ SWIrdG P Q cm 350 360 G TTGTGTCCAATCTTCATTCTGG
1210
n
370 380 390 AGTCMCTTCACCTGGTTACM~CGAGM~CTCT~TCAGAGATTGCTCACGACTCTG~~GCATTCC~TGGTGC~TCAGC~TT~ATATTTCTACTGMG VpijW L QD E KT L K S E I AH D S G E H SD GA I S K L D I ST E au 400 410 420 I-+ 430 cH4 CCTGGCTGAGTGAGGTTGTTTTCG G TGGTGAATCATCAGTATCTACCW\CTCCTCCTCTGAGAGACTCCATCCACMGGAGAGMTT~TCCACTG~GCCATCG AWLSEVVFECVVNHQYLPTPLRDSIHKERIENPLEPS v 440 450 GTGTCTGTCCTCCTGC~C~CAGMGMCTCTCCGCGCAGAGGTTTCTTTCCCTTAC
1100
460
1320
1430
470 1540
480 490 500 CMCGACMGCCGGTTMTCCCAGTMCTA~GMCACCGMGTGACGG~~~GCGACMCACCTCCTTCTTCTTGTA~GCCTGTTATCCATTGCAGCG~GGAGT 1650 ND K P VN P S NY KN T E V T A E S DbT[F F LY S L L S I AA E E cm secretory e.egment 520 530 + 540 510 GGGCCAGCGGTGCTTCTTACTC G TGGTGGGACATGMGCGATTCCATTGAAGATCATCATCMCAGMCGGTTGAT~TCCAGCGGT~CCGAGTTTTGT~TATT 1760 WASGASYSCVVGHEAIPLKII~~VDKSSGKPSFV~ "T'F c-n am 557 550 TCACTTGCACTGATGGACACCATTMTTCATGAAC(A)n 1850 -~LALLDTINSCQTER
Fig. 1. Nucleotide and predicted amino acid sequences of a nurse shark Ig heavy chain cDNA clone A4-2. The deduced amino acid sequence is shown below the nucleotide sequence and numbered from -20 to -1 for the signal peptide and from 1 to 557 for the mature protein. The assignment of domains and segments is based on comparisons with published homed shark (Kolcubu et al., 1988) and little skate (Harding et al.. 1990) Ig heavy chain sequences. Ambiguities in domain or segment boundaries are indicated by dashed lines. Cysteinyl residues assumed to form intradomain disulphide bridges are boxed. Also boxed are complementarity determining regions (CDR) and potential A%nkd glycosylation sites (CHO). Putative polyadenylation signals are underlined. (A)n, a poly A tail. DNA sequencing was performed using custom-made gene-specific primers in addition to standard T3/17 primers. Sequence ambiguities were eliminated by replacing dG’lB with dlTP. This sequence has been submitted to the EMBL/GenBank/DDBJ nucleotide sequence databases under the accession number M92851.
