Plant Molecular Biology 20: 565-567, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.
565
Update section Sequence
Nucleotide sequence of a metallothionein gene of the thermophilic cyanobacterium Synechococcus vulcanus Tokurou Shimizu 1, Tetsuo Hiyama 1,,, Masahiko Ikeuchi 2 and Yorinao Inoue 2
1Department of Biochemistry, Saitama University, Urawa 338, Japan (* author for correspondence)," 2Solar Energy Research Group, Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan Received 12 February 1992; accepted in revised form 5 June 1992
Key words:cyanobacterium, gene, metallothionein, nucleotide sequences, Synechococcus vulcanus, thermophile
Metallothioneins (MTs), cysteine-rich heavymetal-binding proteins, are ubiquitously present in eukaryotes [1] and in some prokaryotes, Synechococcus sp. [2] and Pseudomonas putida [3]. The amino acid sequence of the protein of Synechococcus sp. was previously determined; significant differences from eukaryotic counterparts were noted [4]. Genes for eukaryotic MTs have been reported in yeast [5] and sea urchin [6]. Recently, Robinson et al. [7] reported the identification of a gene for metallothionein (smtA) from cyanobacterium Synechococeus PCC6301 and demonstrated that a smtA homologue was also expressed in Synechococcus PCC7942 (Anacystis nidulans R2). We previously reported the nucleotide sequence of the psaC gene for a photosystem- 1 subunit of a thermophilic cyanobacterium, Synechococeus vulcanus [8]. Subsequently, we determined the nucleotide sequence of the upstream region of this gene and found an ORF which was expected to direct a protein 57 amino acids long and rich in cysteine (Fig. 1). This ORF57 is located at the 1757 bp upstream
region of the psaC gene with the same direction of transcription. The ORF57 was found to be single-copy as well as the psaC gene by Southern hybridization analysis of the genomic D N A (data not shown). The nucleotide sequence G G A G G A is located -12 to - 7 from the start codon of ORF57 and similar to the Shine-Dalgarno sequence [9]. The nucleotide sequences T T G A C A and TATACT which are also found at -75 and -51 from the start codon of ORF57 are quite similar to the Escherichia coli consensus -35 sequence (TTGACA) and -10 sequence (TATAAT), respectively. A preliminary northern hybridization analysis revealed that the ORF57 was mainly transcribed as a 320 bp long RNA together with a small amount of other longer transcripts which remained even after a stringent washing. The expected amino acid sequence has homology to those reported for an MT from Synechococcus sp. [2, 4], especially in terms of cysteine residues (Fig. 2). The ORF57 also shows a high homology with that of the gene for MT from Synechococeus PCC6301 [7] in terms of the nucle-
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X53839.
566 CTAGAGCCACGGCAATATCCCTGACACAAAACTCCCCTTGGGCCAGGAGGGCCACAATGC
60
GCACTCGATTCGTGTCAGCCAGTAGGCCAAAAAACTGTGCCATCCGCTGGGCTTTTTCTG
120
TAGAGAGCAGGCGATCGCTCACCTGCGCTAAAGCCTCTGGATGATGAGGATCAAGAGTAT
180
TCATATCTTCATCATAGGCGCTGGTTAGGCAATAACCAATTGACATATGAACAGTTGTTC
240
AAGTATACTGAACTAGAGGCGGAAAACAACCTCACTGATTCGTCATTTGGAGGAAAAGCC
300
ATGACAACCGTGACCCAAATGAAATGCGCCTGTCCCCACTGCCTGTGCATCGTCTCCCTC
360
H
T
T
V
T
Q
H
K
C
A
C
P
H
C
T
C
I
V
S
T
AACGATGCCATTATGGTCGACGGCAAACCCTACTGCTCCGAAGTCTGTGCCAATGGCACC N
D
A
I
H
V
D
G
K
P
Y
C
S
E
V
C
A
N
G
TGCAAAGAGAATAGCGGCTGTGGCCACGCAGGTTGCGGCTGTGGCTCTGCCTAGAAGTTG C
K
E
N S
G C
G H A
G C
G C
G S
420
T
480
A
CAATCTGTAACAGTGGATAATGACGAGGCGATCGCCCCATCGACTCTATACAATTAAGGA
540
GCTAGAGATTGTCCTCGTCATTTCCTATTAGTTGCGATCCAGCGTGGCGGACCTCCCATC
600
GATTTCCTTGATTACAGCCACCTATAACGCTGCCCAGCACTTGCCGGGGCTGATTGAGTC
660
GGTTCGGCAACAGAGCGATCGCGCCTTCGAATGGATTGTGATTGATGGCGCCTCTGAGGA
720
TGGCACCCTTGATCTCATCAGAGCGGCCCAAGATGTGCTCACAGACTATGTGAGTGAGCC
780
AGATTTTGGCATCTTTCATGCATTGAACAAAGGAATCCAACGGGCAACCGGAGACTATTA
840
CCTTGTGGTTGGCGCAGATGATCGCCTCGACCCCCTAGCGATCGCCAACTACAAACAAGC
900
CGTGAGAATGAGTCATGCCGACATTATTGCAGCTGATATCTATTCAGAAAATCAACGACA ATATACAACCCAAAAAAACACCCGTTTGGTTATCAGGTGC
960 1000
Fig. 1. Nucleotide sequence of the rntnA gene. The putative promoter sequences are underlined.
1
S. sp. HT
10
20
30
40
50
TSTTLVKCACEPCLCNVDPSKAIDRNGLYYCCEACADGHTGGSKGCGHTGCNC
S. vulcanus HT
HTTVTQHKCACPHCLCIVSLNDAIHVDGKPYCSEVCANGTCKENSGCGHAGCGC
S. PCC6301 HT
TSTTLVKCACEPCLCNVDPSKATDRNGLYYCSEACADGHTGGSKGCGHTGCNC
Fig. 2. Comparison of amino acid sequences of cyanobacterial MTs. Asterisks indicate conserved amino acids between the S. vulcanus mtnA protein and other cyanobacterial MTs (see the text for references).
otide sequence (63.2~o) as well as the amino acid sequence (52.1 ~o). It should be noted that nine out of ten cysteine residues are also conserved (Fig. 2). While the present sequence shows little homology with those of eukaryote proteins, a predicted secondary structure by the Chou-Fasman method indicates that the ORF57 protein has some tendency to form a {/-structure near the amino terminal side and a/%turn near the carboxyl terminal which is reminiscent of eukaryote MTs [1]. The ORF57 is tentatively designated as the mtnA gene.
Acknowledgement The authors are grateful to Prof. M. Sugiura of Nagoya University for providing the tobacco chloroplast D N A library.
References 1. Hamer DH: Metallothionein. Annu Rev Biochem 55:
913-951 (1986). 2. Olaf son RW: Prokaryotic metallothionein. Int J Pept Protein Res 24:303-308 (1984). 3. Higham DP, Sadler PJ, Scawen MD: Cadmium-resistant
567 Pseudomonas putida synthesizes novel cadmium proteins. Science 225:1043-1046 (1984). 4. Olafson RW, McCubbin WD, Kay CM: Primary and secondary structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium. Biochem J 251:691-699 (1988). 5. Butt TR, Sternberg EJ, Gorman JA, Clark P, Hamer D, Rosenberg M, Crooke ST: Copper metallothionein of yeast; structure of the gene, and regulation of expression. Proc Natl Acad Sci U S A 81:3332-3336 (1984). 6. Nemer M, Wilkinson DF, Travaglini EC, Sternber EJ, Butt TR: Sea urchin metallothionein sequence: key to an evolutionary diversity. Proc Natl Acad Sci USA 82: 4992-4994 (1985).
7. Robinson NJ, Gupta A, Fordham-Skelton AP, Croy RRD, Whitton BA, Huckle JW: Prokaryotic metallothionein gene characterization and expression: chromosome crawling by ligation-mediated PCR. Proc R Soc London B 242:241-247 (1990). 8. Shimizu T, Hiyama T, Ikeuchi M, Koike H, Inoue Y: Nucleotide sequence of the psaC gene of the cyanobacterium Synechococcus vulcanus. Nucl Acids Res 18:3644 (1990). 9. Shine J, Dalgarno L: The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342-1346 (1974).
565
Update section Sequence
Nucleotide sequence of a metallothionein gene of the thermophilic cyanobacterium Synechococcus vulcanus Tokurou Shimizu 1, Tetsuo Hiyama 1,,, Masahiko Ikeuchi 2 and Yorinao Inoue 2
1Department of Biochemistry, Saitama University, Urawa 338, Japan (* author for correspondence)," 2Solar Energy Research Group, Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan Received 12 February 1992; accepted in revised form 5 June 1992
Key words:cyanobacterium, gene, metallothionein, nucleotide sequences, Synechococcus vulcanus, thermophile
Metallothioneins (MTs), cysteine-rich heavymetal-binding proteins, are ubiquitously present in eukaryotes [1] and in some prokaryotes, Synechococcus sp. [2] and Pseudomonas putida [3]. The amino acid sequence of the protein of Synechococcus sp. was previously determined; significant differences from eukaryotic counterparts were noted [4]. Genes for eukaryotic MTs have been reported in yeast [5] and sea urchin [6]. Recently, Robinson et al. [7] reported the identification of a gene for metallothionein (smtA) from cyanobacterium Synechococeus PCC6301 and demonstrated that a smtA homologue was also expressed in Synechococcus PCC7942 (Anacystis nidulans R2). We previously reported the nucleotide sequence of the psaC gene for a photosystem- 1 subunit of a thermophilic cyanobacterium, Synechococeus vulcanus [8]. Subsequently, we determined the nucleotide sequence of the upstream region of this gene and found an ORF which was expected to direct a protein 57 amino acids long and rich in cysteine (Fig. 1). This ORF57 is located at the 1757 bp upstream
region of the psaC gene with the same direction of transcription. The ORF57 was found to be single-copy as well as the psaC gene by Southern hybridization analysis of the genomic D N A (data not shown). The nucleotide sequence G G A G G A is located -12 to - 7 from the start codon of ORF57 and similar to the Shine-Dalgarno sequence [9]. The nucleotide sequences T T G A C A and TATACT which are also found at -75 and -51 from the start codon of ORF57 are quite similar to the Escherichia coli consensus -35 sequence (TTGACA) and -10 sequence (TATAAT), respectively. A preliminary northern hybridization analysis revealed that the ORF57 was mainly transcribed as a 320 bp long RNA together with a small amount of other longer transcripts which remained even after a stringent washing. The expected amino acid sequence has homology to those reported for an MT from Synechococcus sp. [2, 4], especially in terms of cysteine residues (Fig. 2). The ORF57 also shows a high homology with that of the gene for MT from Synechococeus PCC6301 [7] in terms of the nucle-
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X53839.
566 CTAGAGCCACGGCAATATCCCTGACACAAAACTCCCCTTGGGCCAGGAGGGCCACAATGC
60
GCACTCGATTCGTGTCAGCCAGTAGGCCAAAAAACTGTGCCATCCGCTGGGCTTTTTCTG
120
TAGAGAGCAGGCGATCGCTCACCTGCGCTAAAGCCTCTGGATGATGAGGATCAAGAGTAT
180
TCATATCTTCATCATAGGCGCTGGTTAGGCAATAACCAATTGACATATGAACAGTTGTTC
240
AAGTATACTGAACTAGAGGCGGAAAACAACCTCACTGATTCGTCATTTGGAGGAAAAGCC
300
ATGACAACCGTGACCCAAATGAAATGCGCCTGTCCCCACTGCCTGTGCATCGTCTCCCTC
360
H
T
T
V
T
Q
H
K
C
A
C
P
H
C
T
C
I
V
S
T
AACGATGCCATTATGGTCGACGGCAAACCCTACTGCTCCGAAGTCTGTGCCAATGGCACC N
D
A
I
H
V
D
G
K
P
Y
C
S
E
V
C
A
N
G
TGCAAAGAGAATAGCGGCTGTGGCCACGCAGGTTGCGGCTGTGGCTCTGCCTAGAAGTTG C
K
E
N S
G C
G H A
G C
G C
G S
420
T
480
A
CAATCTGTAACAGTGGATAATGACGAGGCGATCGCCCCATCGACTCTATACAATTAAGGA
540
GCTAGAGATTGTCCTCGTCATTTCCTATTAGTTGCGATCCAGCGTGGCGGACCTCCCATC
600
GATTTCCTTGATTACAGCCACCTATAACGCTGCCCAGCACTTGCCGGGGCTGATTGAGTC
660
GGTTCGGCAACAGAGCGATCGCGCCTTCGAATGGATTGTGATTGATGGCGCCTCTGAGGA
720
TGGCACCCTTGATCTCATCAGAGCGGCCCAAGATGTGCTCACAGACTATGTGAGTGAGCC
780
AGATTTTGGCATCTTTCATGCATTGAACAAAGGAATCCAACGGGCAACCGGAGACTATTA
840
CCTTGTGGTTGGCGCAGATGATCGCCTCGACCCCCTAGCGATCGCCAACTACAAACAAGC
900
CGTGAGAATGAGTCATGCCGACATTATTGCAGCTGATATCTATTCAGAAAATCAACGACA ATATACAACCCAAAAAAACACCCGTTTGGTTATCAGGTGC
960 1000
Fig. 1. Nucleotide sequence of the rntnA gene. The putative promoter sequences are underlined.
1
S. sp. HT
10
20
30
40
50
TSTTLVKCACEPCLCNVDPSKAIDRNGLYYCCEACADGHTGGSKGCGHTGCNC
S. vulcanus HT
HTTVTQHKCACPHCLCIVSLNDAIHVDGKPYCSEVCANGTCKENSGCGHAGCGC
S. PCC6301 HT
TSTTLVKCACEPCLCNVDPSKATDRNGLYYCSEACADGHTGGSKGCGHTGCNC
Fig. 2. Comparison of amino acid sequences of cyanobacterial MTs. Asterisks indicate conserved amino acids between the S. vulcanus mtnA protein and other cyanobacterial MTs (see the text for references).
otide sequence (63.2~o) as well as the amino acid sequence (52.1 ~o). It should be noted that nine out of ten cysteine residues are also conserved (Fig. 2). While the present sequence shows little homology with those of eukaryote proteins, a predicted secondary structure by the Chou-Fasman method indicates that the ORF57 protein has some tendency to form a {/-structure near the amino terminal side and a/%turn near the carboxyl terminal which is reminiscent of eukaryote MTs [1]. The ORF57 is tentatively designated as the mtnA gene.
Acknowledgement The authors are grateful to Prof. M. Sugiura of Nagoya University for providing the tobacco chloroplast D N A library.
References 1. Hamer DH: Metallothionein. Annu Rev Biochem 55:
913-951 (1986). 2. Olaf son RW: Prokaryotic metallothionein. Int J Pept Protein Res 24:303-308 (1984). 3. Higham DP, Sadler PJ, Scawen MD: Cadmium-resistant
567 Pseudomonas putida synthesizes novel cadmium proteins. Science 225:1043-1046 (1984). 4. Olafson RW, McCubbin WD, Kay CM: Primary and secondary structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium. Biochem J 251:691-699 (1988). 5. Butt TR, Sternberg EJ, Gorman JA, Clark P, Hamer D, Rosenberg M, Crooke ST: Copper metallothionein of yeast; structure of the gene, and regulation of expression. Proc Natl Acad Sci U S A 81:3332-3336 (1984). 6. Nemer M, Wilkinson DF, Travaglini EC, Sternber EJ, Butt TR: Sea urchin metallothionein sequence: key to an evolutionary diversity. Proc Natl Acad Sci USA 82: 4992-4994 (1985).
7. Robinson NJ, Gupta A, Fordham-Skelton AP, Croy RRD, Whitton BA, Huckle JW: Prokaryotic metallothionein gene characterization and expression: chromosome crawling by ligation-mediated PCR. Proc R Soc London B 242:241-247 (1990). 8. Shimizu T, Hiyama T, Ikeuchi M, Koike H, Inoue Y: Nucleotide sequence of the psaC gene of the cyanobacterium Synechococcus vulcanus. Nucl Acids Res 18:3644 (1990). 9. Shine J, Dalgarno L: The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342-1346 (1974).
