Plant Molecular Biology 11:871-872 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands

871

Cloning and nucleotide sequence of a petunia gene encoding a chloroplastlocalized superoxide dismutase J. Tepperman, C. K a t a y a m a a n d P. D u n s m u i r Advanced Genetic Sciences, Inc., 6701 San Pablo Avenue, Oakland, CA 94611, USA

Received and accepted 19 September 1988

S u p e r o x i d e d i s m u t a s e s (EC 1.15.1.1) are a class o f m e t a l l o p r o t e i n s f o u n d in all a e r o b i c o r g a n i s m s which catalyse the d i s p r o p o r t i o n a t i o n o f the sup e r o x i d e a n i o n (O 3) to H 2 0 2 a n d 02 p r o v i d i n g a n i m p o r t a n t defense a g a i n s t oxygen toxicity [1]. In the leaves o f higher p l a n t s at least two f o r m s o f the c o p p e r - z i n c e n z y m e are f o u n d , a s s o c i a t e d with the cytosol a n d the c h l o r o p l a s t respectively [2]. U s i n g a n a n t i b o d y raised a g a i n s t p u r i f i e d c h l o r o p l a s t S O D f r o m Petunia hybrida cv. Mitchell, we screened a k g t l l l i b r a r y o f - 5 0 0 0 0 0 c D N A clones. O n e o f the positive phage, c o n t a i n i n g a p o r t i o n o f the sequence for the S O D c o d i n g region, was used to isolate a fulll e n g t h c D N A f r o m Xgtl0 l i b r a r y o f - 100000. Figure 1 is the n u c l e o t i d e a n d d e d u c e d a m i n o acid seq u e n c e o f this c D N A . T h e first 65 a m i n o acids constitute a transit p e p t i d e sequence t h a t is cleaved after i m p o r t into the c h l o r o p l a s t . T h e m a t u r e c o d i n g reg i o n o f 154 a m i n o acids possesses 94°70 i d e n t i t y with the sequence o f s p i n a c h c h l o r o p l a s t S O D [3].

Acknowledgements We are grateful to Drs K. S t e i n b a c k a n d P. W o l b e r for h e l p f u l suggestions o n p r o t e i n p u r i f i c a t i o n .

References 1. Fridovich I: The biology of oxygen radicals. Science 201: 875-880 (1978). 2. Foster JG, Edwards GE: Localization of superoxide dismutase in leaves of C3 and C4 plants. Plant and Cell Physiol 21: 895-906 (1980). 3. Kitigawa Y, Tsunasawa S, Tanaka N, Matsube Y, Sakiyama F, Asada K: Amino acid sequence of copper, zinc-superoxide dismutase from spinach leaves. J Biochem 99: 1289-1298 (1986). Accession no. M20792.

872 1

TCC

TTC

ACA

TTA

TCA

TTC

ATC

ACT

CAC

ACT

ATC

ATC

TTA

GCC

ACC

TGT

GTC

TGT

GTG

GCA

1 61

TCC

MET ATG

ALA GCA

ALA GCC

HIS CAT

THR ACA

ILE ATC

PHE TTC

THR ACC

THR ACC

THR ACA

SER AGC

THR ACT

THR ACC

ASN AAT

SER TCT

PHE TTC

LEU TTA

PHE TTC

PRO CCA

20 121

ILE ATT

ALA GCT

SER TCC

SER TCT

ASN AAC

THR ACC

ASN AAC

SER TCT

ALA GCC

PRO CCT

SER TCA

LEU CTT

SER TCC

SER TCT

SER TCT

PHE TTC

HIS CAT

GLY GGT

VAL GTT

SER TCC

40 181

LEU CTT

LYS AAA

VAL GTC

LYS AAG

SER TCA

LYS AAA

THR ACT

PRO CCT

GLN CAA

SER TCA

LEU TTG

THR ACC

LEU CTT

SER TCA

SER TCT

VAL GTC

THR ACT

SER TCT

PRO CCT

LYS AAA

60 241

PRO CCT

PHE TTC

ILE ATT

VAL GTT

PHE TTT

ALAIALA GCT GCT

THR ACT

LYS AAG

LYS AAA

ALA GCT

VAL GTT

ALA GCT

VAL GTA

LEU CTT

LYS AAA

GLY THR GC-C A C T

SER TCC

ASN AAT

80 301

VAL GTT

GLU GAG

GLY GGT

VAL GTT

VAL GTC

THR ACT

LEU CTT

THR ACT

GLN CAA

ASP GAT

ASP GAT

ASP GAT

GLY GGT

PRO CCA

THR ACC

THR ACT

VAL GTG

LYS AAA

VAL GTT

ARG CGT

i00 361

ILE ATA

THR ACT

GLY GGA

LEU CTT

ALA GCT

PRO CCT

GLY GGA

LEU CTT

HIS CAT

GLY GGG

PHE TTT

HIS CAT

LEU TTG

HIS CAC

GLU GAG

PHE TTT

GLY GGT

ASP GAC

THR ACT

THR ACC

120 421

ASN AAT

GLY GGG

CYS TGT

MET ATG

SER TCC

THR ACA

GLY GGA

PRO CCC

HIS CAT

PHE TTC

ASN AAT

PRO CCT

ASN AAC

GLY GGC

LEU TTG

THR ACA

HIS CAT

GLY GGA

ALA GCT

PRO CCT

140 481

GLY GGA

ASP GAT

GLU GAA

VAL GTC

ARG CGT

HIS CAT

~ GCG

GLY GGT

ASP GAC

LEU CTG

GLY GGA

ASN AAC

ILE ATA

GLU GAG

ALA GCC

ASN AAT

ALA GCC

SER AGT

GLY GGT

VAL GTG

160 541

ALA GCT

GLU GAA

ALA GCA

THR ACA

LEU CTT

VAL GTA

ASP GAT

ASN AAT

GLN CAG

ILE ATA

PRO CCA

LEU TTG

SER TCT

GLY GGT

PRO CCA

ASN AAC

SER TCA

VAL GTC

VAL GTT

GLY GGA

180 601

ARG AGA

ALA GCG

LEU CTT

VAL GTA

VAL GTT

HIS CAC

GLU GAG

LEU CTT

GLU GAG

ASP GAT

ASP GAT

LEU CTT

GLY GGA

LYS AAG

GLY GGT

GLY GGC

HIS CAC

GLU GAA

LEU CTC

SER AGC

200 661

LEU CTT

THR ACC

THR ACT

GLY GGA

ASN AAT

ALA GCT

GLY GGT

GLY GGA

ARG CGA

LEU TTG

ALA GCC

CYS TGT

GLY GGT

VAL GTG

VAL GTT

GLY GGT

LEU TTG

THR ACT

PRO CCA

ILE ATA

721

TGA

AGT

CAA

GAT

AGC

AGT

AAT

TCT

TTG

GTA

TGA

ATT

ATA

ATG

AGT

GGT

TAT

CTC

CTC

CCC

781

ACG

TTG

CCA

ATA

AAG

CTC

TAC

GTG

AAT

GTA

TTT

TGA

GAG

ATT

CTT

GAT

GGA

CAA

ATT

TGA

841

GCT

TGC

ATG

TAC

ACT

TGC

CAA

GTG

TAG

TGC

ACC

ACT

TTT

TAA

CTC

AAC

ACT

GTA

ATT

GGA

901

GTC

GGC

TAT

TTT

ACC

ATC

CTT

AAG

AGT

TTT

TTC

TAT

CAA

AAA

AAA

!

Fig. 1. Nucleotide and deduced amino acid sequence of a lambda gtl0 c D N A encoding chloroplast SOD. The arrow indicates the transit peptide cleavage site.

Cloning and nucleotide sequence of a petunia gene encoding a chloroplast-localized superoxide dismutase.

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