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.