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Plant Molecular Biology 17: 183-184, 1991. © 1991 Kluwer Academic Publishers. Printed in Belgium. Update section Sequence Nucleotide sequence of a cDNA clone encoding chloroplast phosphoribulokinase from Arabidopsis thaliana 183 Philip R. Horsnell and Christine A. Raines* Biology Department, University of Essex, Wivenhoe Park, Colchester C04 3SQ, UK (*authorfor correspondence) Received 3 April 1991; accepted 5 April 1991 Key words: Arabidopsis, cDNA, chloroplast, ribulose-5-phosphate kinase Ribulose-5-phosphate kinase (PRKase) catalyses the final step in the regeneration of ribulose-l,5- bisphosphate in the photosynthetic carbon re- duction cycle. This enzyme is unique to this cycle and is only found in photosynthetic organisms. The activity of this enzyme is significantly increased by light which is mediated via the thioredoxin system; two cysteine residues (Cys- 16 and Cys-53) of the mature protein have been shown to be involved in this process [1, 3]. PRKase is one of a number of enzymes believed to play a prominent role in regulating the flow of intermediates through this pathway [6]. We wish to consider the relative importance of individual enzymes of this cycle and as a first step towards this we have isolated a cDNA clone encoding Arabidopsis PRKase. An Arabidopsis thaliana (C24) cDNA library constructed in Lambda ZaplI was screened using a wheat phosphoribulokinase (PRKase) probe [4] and a number of positive clones isolated. One cDNA clone, APRK1, was selected for sequence analysis. Using the plasmid rescue technique the bluescript plasmid containing this clone was isolated from the Lambda Zap vector (Strata- gene). This clone was sequenced in both di- rections using a double-stranded sequencing method [4]. The nucleotide sequence encoding the Arabidopsis chloroplast PRKase along with its deduced amino acid sequence is shown in Fig. 1. The cDNA clone contained an insert of 1495 bp with a 1185 bp open reading frame encoding the 395 amino acid precursor protein which com- prises the mature chloroplast protein (349 amino acids) and its transit peptide (46 amino acids). Comparison of the Arabidopsis PRKase deduced amino acid sequence with those of wheat and spinach [2, 4, 5] shows that the mature proteins are about 90~o identical, whilst the transit se- quences are much less similar. We have prepared PRKase sense and antisense constructs which will be used to produce Arabidopsis mutants with altered levels of this enzyme. This clone is also being used to isolate corresponding genomic se- quences to study the transcriptional regulation of this gene. Acknowledgements This work was supported by an AFRC Plant Molecular Biology Initiative Grant (AFRC PG 84/502). The nucleotide sequence reported will aplJear in the EMB, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X58149.

Nucleotide sequence of a cDNA clone encoding chloroplast phosphoribulokinase fromArabidopsis thaliana

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Page 1: Nucleotide sequence of a cDNA clone encoding chloroplast phosphoribulokinase fromArabidopsis thaliana

Plant Molecular Biology 17: 183-184, 1991. © 1991 Kluwer Academic Publishers. Printed in Belgium.

Update section Sequence

Nucleotide sequence of a cDNA clone encoding chloroplast phosphoribulokinase from Arabidopsis thaliana

183

Philip R. Horsnell and Christine A. Raines* Biology Department, University of Essex, Wivenhoe Park, Colchester C04 3SQ, UK (*author for correspondence)

Received 3 April 1991; accepted 5 April 1991

Key words: Arabidopsis, cDNA, chloroplast, ribulose-5-phosphate kinase

Ribulose-5-phosphate kinase (PRKase) catalyses the final step in the regeneration of ribulose-l,5- bisphosphate in the photosynthetic carbon re- duction cycle. This enzyme is unique to this cycle and is only found in photosynthetic organisms. The activity of this enzyme is significantly increased by light which is mediated via the thioredoxin system; two cysteine residues (Cys- 16 and Cys-53) of the mature protein have been shown to be involved in this process [1, 3]. PRKase is one of a number of enzymes believed to play a prominent role in regulating the flow of intermediates through this pathway [6]. We wish to consider the relative importance of individual enzymes of this cycle and as a first step towards this we have isolated a cDNA clone encoding Arabidopsis PRKase.

An Arabidopsis thaliana (C24) cDNA library constructed in Lambda ZaplI was screened using a wheat phosphoribulokinase (PRKase) probe [4] and a number of positive clones isolated. One cDNA clone, APRK1, was selected for sequence analysis. Using the plasmid rescue technique the bluescript plasmid containing this clone was isolated from the Lambda Zap vector (Strata- gene). This clone was sequenced in both di-

rections using a double-stranded sequencing method [4]. The nucleotide sequence encoding the Arabidopsis chloroplast PRKase along with its deduced amino acid sequence is shown in Fig. 1. The cDNA clone contained an insert of 1495 bp with a 1185 bp open reading frame encoding the 395 amino acid precursor protein which com- prises the mature chloroplast protein (349 amino acids) and its transit peptide (46 amino acids). Comparison of the Arabidopsis PRKase deduced amino acid sequence with those of wheat and spinach [2, 4, 5] shows that the mature proteins are about 90~o identical, whilst the transit se- quences are much less similar. We have prepared PRKase sense and antisense constructs which will be used to produce Arabidopsis mutants with altered levels of this enzyme. This clone is also being used to isolate corresponding genomic se- quences to study the transcriptional regulation of this gene.

Acknowledgements

This work was supported by an AFRC Plant Molecular Biology Initiative Grant (AFRC PG 84/502).

The nucleotide sequence reported will aplJear in the EMB, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X58149.

Page 2: Nucleotide sequence of a cDNA clone encoding chloroplast phosphoribulokinase fromArabidopsis thaliana

184

i0 30 50 70 90 CTGAG~T~A~AAAGCTAAA~AAAAAAAAAAGAGCTTAGG~AG~G~GAGGAGA~CAAAA~ACCAAACAAA~C~TGG~TGTCTC~TATCTA~T

M A V S T I Y S ii0 130 150 170 190

C~CAc~GCTCTC~TTC~CTCATTTCTT~CCTCTTCTTCTTCTTCCAAAC~GTCTTC~TCTAC~T~GT~CCACAAACC~CCGGAGATTC~

T Q A L N S T H F L T S S S S S K Q V F L Y R R Q P Q T N R R F N

210 V 230 250 270 290

cACACTCATCACTTGCGCAc~GAAACCATCGTGATCC4SAcTAGCTGCTGACTCTGGCTGCGGCAAAAGTAc~TTTATGCGGAGGcTCACcAGCGTcTTT T L I T C A Q E T I V I G L A A D S G C G K S T F M R R L T S V F

310 330 350 370 390

GGTGGCGCTGCT~GCCAC~AAAAGGCGGG~CCCTGATTCC~CACACTCATCAGCGACACGACCACTGTGATCTGTCTTGATGATTACCATTCTTT~ G G A A K P P K G G N P D S N T L I S D T T T V I C L D D Y H S L D

410 430 450 470 490

ATAGGTACGGTAGGAAAGAGCAGAAAGTCACCGCTTTGGACCCAcGCGCC~TGACTTTGATCTcATGTATGAGC~GTCAAAGCTCTT~G~TGGTAT

R Y G R K E Q K V T A L D P R A N D F D L M Y E Q V K A L K N G I

510" 530 550 570 590 CGCcGTCGAGAAACCGATTTAT~CCATGTCACTGGACTT~TTGACCCTCCGGAGCTTATTCAGCcTCCT~GATTCTTGTCATcG~GGTCTTCACCCA

A V E K P I Y N H V T G L L D P P E L I Q P P K I L V I E G L H P

610 630 650 670 690

ATGTTTGATGAGCGAGT~GAGACTTACTAGACTTCAGTATCTAcTTGGACATTAGC~CG~GTCAAATTCGCTTGGAAAATTcAGAGGGACATGGC~ M F D E R V R D L L D F S I Y L D I S N E V K F A W K I Q R D M A E

710 730 750 770 790 AAAGAGGTCACAGTTTGGAGAGcAT~AAAGCGAGTATCG~GCCCGAAAGCCcGACTTCGATGCATTCATCGACCCGcAAAAGCAGTACGCGGATGC~T

R G H S L E S I K A S I E A R K P D F D A F I D P Q K Q Y A D A V

810 830 850 870 890

CATAG~GTGCTTccTACGACTCTGAT~c~AGATGAC~cG~GGGAAAGTGTTGAGAGTGAGATTGAT~TG~GG~GGTGTT~GTACTTcAGCCCG I E V L P T T L I P D D N E G K V L R V R L I M K E G V K Y F S P

910 930 950 970 990 GTTTACCTATTCGATG~GGTTc~CCAT~TCGTGGATTCCTTGCGGcCGCAAACTCACTTGcTcGTACCCTGGCATC~GTTC~CTACG~CCTGACT

V Y L F D E G S T I S W I P C G R K L T C S Y P G I K F N Y E P D S i010 1030 1050 1070 1090

ccTACTTCGACCATGAGGTATCAGTTTTGGAGATGGATGGAC~TTTGATAGAcTGGAcGAGCTGATTTACGTGGAAAGTcA~TTGAGC~CCTcTCGAC Y F D H E V S V L E M D G Q F D R L D E L I Y V E S H L S N L S T

iii0 1130 1150 1170 1190

cAAATTcTACGGAG~GTcAcTC~cAAATGCT~AAACATGCTGATTTCcCGGGTAGC~C~CGGTA~TGGTCTTTTCCAAACCATTGTTGGATTG~G

K F Y G E V T Q Q M L K H A D F P G S N N G T G L F Q T I V G L K

1210 1230 1250 1270 1290

ATcAGAGATCTCTATGAGCAGCTCATTGcC~cAAAGCCAcTGCTCGTGCAG~GCTAAAGccTAAAACAAACG~GCG~GCcAAATAG~CGCGTCGA I R D L Y E Q L I A N K A T A R A E A K A *

1310 1330 1350 1370 1390

TAAATCcATCCGcAGTTT•TTCTTTTTCTTATTTcTTTcTTTCTTTATGCGTTGTTTACcGcGGATTTTcATGGCGAAAAGGAcGGTcTTGCTTGTTTGT 1410 1430 1450 1470

~TTTGTGTGGAGATAAAAAGAAAAAGCcTGT~TGTAGAG~GTG~TAGCAGAGC~T~cAGTTcAGTTTATAG~cGCcAAAAAAAAAAAA

Fig. 1. Nucleotide sequence and derived amino acid sequence of an Arabidops~ PRKase cDNA clone. The arrow indic~es the likely cleav~e site ~r removal of the transit sequence. Nucleotides are numbered above the line.

References

1. Krieger TJ, Mende-Muller L, Miziorko HM: Phosphori- bulokinase: isolation sequence determination of the cysteine-containing active site peptide by 5-p-fluoro- sulphonyl-benzoyladneonsine. Biochim Biophys Acta 915:112-119 (1987).

2. Milanez S, Mural RJ: Cloning and sequencing of cDNA encoding the mature form of phosphoribulokinase from spinach. Gene 66:55-63 (1988).

3. Porter MA, Stringer CD, Hartman FC: Characterisation of the regulatory thioredoxin site ofphosphoribulokinase. J Biol Chem 263:123-129 (1988)

4. Raines CA, Longstaff M, Lloyd JC, Dyer TA: Complete coding sequence of wheat phosphoribulokinase: Develop- mental and light-dependent expression of the mRNA. Mol Gen Genet 220:43-48 (1989).

5. Roesler KR, Ogen WL: Nucleotide sequence of spinach cDNA encoding phosphoribulokinase. Nucl Acids Res 16:7192 (1988).

6. Woodrow IE, Furbank RT, Brocks A, Murphy DJ: The requirements for a steady state in the C3 reductive pentose phosphate pathway of photosynthesis. Biochim Biophys Acta 807:263-271 (1985).