Nucleic Acids Research, Vol. 18, No. 16 4939
\.) 1990 Oxford University Press
Nucleotide sequence of a cDNA encoding the vacuolar protein strictosidine synthase from Catharanthus roseus T.D.McKnight*, C.A.Roessner1, R.Devagupta1, A.I.Scottl and C.L.Nessler Departments of Biology and 'Chemistry, Texas A & M University, College Station, TX 77843, USA Submitted July 6, 1990
EMBL accession
Strictosidine synthase is a plant vacuolar glycoprotein that catalyses the condensation of tryptamine and secologanin to form strictosidine, a key intermediate in indole-type alkaloid production. Catharanthus roseus (Madagascar periwinkle) produces two clinically useful antitumor alkaloids, vinblastine and vincristine, derived from strictosidine. Characterization of genes encoding enzymes in alkaloid biosynthesis may reveal how this pathway is regulated. We used a labeled oligonucleotide derived from the sequence of the Rauwolfia serpentina strictosidine synthase gene (1) to probe a C. roseus cDNA library (provided by Dr. V.DeLuca). Of 45,000 clones screened, only one hybridized to the probe. The translated region begins with two Met codons, the second of which is found within the Kozak consensus motif (2). The predicted amino acid sequences of both strictosidine synthases are 344 residues long and 274 (80%) of these positions are identical. The C. roseus strictosidine synthase
no.
X53602
signal peptide is unusual in that it does not contain a basic amino acid, as is usually found in signal peptides. This feature does not seem to be absolutely required, since other signal peptides have been reported without a basic residue, e.g. the mouse kappa light chain variable region gene (3). The signal peptide has an unbroken run of 13 hydrophobic amino acids immediately followed by 10 serines. The arrow indicates the presumed signal peptidase cleavage site. The region used as the initial oligonucleotide probe is underlined.
REFERENCES 1. Kutchan,T.M., Hampp,N., Lottspeich,F., Beyreuther,K. and Zenk,M.H. (1988) FEBS Lett. 237, 40-44. 2. Kozak,M. (1989) J. Cell. Biol. 108, 229-241. 3. Xu,D. and Tsang,K.Y. (1990) Nucl. Acids Res. 18, 1912.
1
GAATTCCTAAATCCATGATGGCAGTTTTCTTCATGTTTTTCCTTCTTCTTCTTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCACCAATT
91
181
TTGMAAAGATTTTTATTGAAAGCCCTTCCTATGCTCCGAATGCCTTCACCTTCGATTCAACTGATAAAGGGTTCTACACTTCCGTCCAA L K K I F I E S P S Y A P N A F T F D S T D K G F Y T S V Q GATGGCCGAGTTATCAAATATGAAGGGCCAAATTCAGGCTTCACTGACTTCGCCTACGCATCTCCCTTCTGGAACAAAGCTTTTTGTGAG
271
AACAGCACCGATCCAGAGAAAAGACCATTGTGTGGGAGGACATATGATATTTCCTATGACTATAAGAACAGCCAAATGTACATTGTTGAT
M M A V
D
G
R V
I
K Y
E
G
F
P
F M
N
S
F
G
F
L L L L S
F T
D
S
S
F A Y A S
S
P
S
S
S
F W N
S
S
K A
S
F
P
C
I
E
S T D P E K R P L C G R T Y D I S Y D Y K N S Q M Y I V D GGCCATTACCATCTTTGTGTGGTTGGAAAAGAAGGTGGGTATGCCACACAACTAGCCACAAGTGTGCAAGGAGTGCCATTCAAATGGCTC G H Y H L C V V G K E G G Y A T Q L A T S V Q G V P F K W L TATGCAGTAACTGTTGATCAGAGAACAGGGATTGTTTATTTCACTGATGTTAGCTCCATACATGATGACAGTCCCGAAGGTTGTGAAGAA
N
361 451
Y A V T
541
631
721
V D Q R T G I V Y F T D V S S I H D D S P E G V E E ATCATGAATACAAGTGATAGAACAGGGAGATTAATGAAGTATGATCCTTCAACAAAAGAAACCACCTTATTATTGAAAGAGCTACATGTT I M N T S D R T G R L M K Y D P S T K E T T L L L K E L H V CCCGGCGGTGCAGAAATCAGCGCAGATGGTTCCTTTGTTGTAGTAGCAGAATTTTTAAGCAATCGGATAGTGAAGTATTGGCTAGAAGGG P G G A E I S A D G S F V V V A E F L. S N R I V K Y W L E G CCAAAGAAAGGCAGTGCAGAGTTCTTAGTTACAATCCCAAATCCAGGAAATATAAAGAGGAATTCTGATGGCCATTTTTGGGTGTCTTCA P K K G S A E F L V T I P N P G N I K R N S D G H F W V S S
AGTGAAGATTAGATGGAGGTCAACATGGAAGTGTTGTTTCAAGAGGAATTMGTTTGATGGATTTGGGAATATTCTTCMGTTATACCA S E E L D G G Q H G S V V S R G I K F D G F G N I L Q V I P 901 CTTCCACCACCATATGAAGGTGAACATTTGAACAGATTCAAGAGCACGATGGTTTGTTATACATTGGAAGTCTCTCCCATAGCTCTGTG L P P P Y E G E H F E Q I Q E H D G L L Y I G S L S H S S V 991 GGTATATTAGTGTATGATGATCATGATAACAAGGGAAATTCTTATGTTTCTCAGCTAGTCATTAATTGAATATTTGTTTTGATTTGTAAC G I L V Y D D H D N K G N S Y V S Q L V I N * 1081 TTTTTAATGATTTCTGCAAATTCTATGGCTTTTTGAAGGTTACAAAGTCTTAGGAGGAGATTTTGAGTGTCTGCTGCTACATATATGTTC 811
1171 *
AATAAGAAGCCAATTCCATGAAAAAAAGGAATTC
To whom correspondence should be addressed
\.) 1990 Oxford University Press
Nucleotide sequence of a cDNA encoding the vacuolar protein strictosidine synthase from Catharanthus roseus T.D.McKnight*, C.A.Roessner1, R.Devagupta1, A.I.Scottl and C.L.Nessler Departments of Biology and 'Chemistry, Texas A & M University, College Station, TX 77843, USA Submitted July 6, 1990
EMBL accession
Strictosidine synthase is a plant vacuolar glycoprotein that catalyses the condensation of tryptamine and secologanin to form strictosidine, a key intermediate in indole-type alkaloid production. Catharanthus roseus (Madagascar periwinkle) produces two clinically useful antitumor alkaloids, vinblastine and vincristine, derived from strictosidine. Characterization of genes encoding enzymes in alkaloid biosynthesis may reveal how this pathway is regulated. We used a labeled oligonucleotide derived from the sequence of the Rauwolfia serpentina strictosidine synthase gene (1) to probe a C. roseus cDNA library (provided by Dr. V.DeLuca). Of 45,000 clones screened, only one hybridized to the probe. The translated region begins with two Met codons, the second of which is found within the Kozak consensus motif (2). The predicted amino acid sequences of both strictosidine synthases are 344 residues long and 274 (80%) of these positions are identical. The C. roseus strictosidine synthase
no.
X53602
signal peptide is unusual in that it does not contain a basic amino acid, as is usually found in signal peptides. This feature does not seem to be absolutely required, since other signal peptides have been reported without a basic residue, e.g. the mouse kappa light chain variable region gene (3). The signal peptide has an unbroken run of 13 hydrophobic amino acids immediately followed by 10 serines. The arrow indicates the presumed signal peptidase cleavage site. The region used as the initial oligonucleotide probe is underlined.
REFERENCES 1. Kutchan,T.M., Hampp,N., Lottspeich,F., Beyreuther,K. and Zenk,M.H. (1988) FEBS Lett. 237, 40-44. 2. Kozak,M. (1989) J. Cell. Biol. 108, 229-241. 3. Xu,D. and Tsang,K.Y. (1990) Nucl. Acids Res. 18, 1912.
1
GAATTCCTAAATCCATGATGGCAGTTTTCTTCATGTTTTTCCTTCTTCTTCTTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCACCAATT
91
181
TTGMAAAGATTTTTATTGAAAGCCCTTCCTATGCTCCGAATGCCTTCACCTTCGATTCAACTGATAAAGGGTTCTACACTTCCGTCCAA L K K I F I E S P S Y A P N A F T F D S T D K G F Y T S V Q GATGGCCGAGTTATCAAATATGAAGGGCCAAATTCAGGCTTCACTGACTTCGCCTACGCATCTCCCTTCTGGAACAAAGCTTTTTGTGAG
271
AACAGCACCGATCCAGAGAAAAGACCATTGTGTGGGAGGACATATGATATTTCCTATGACTATAAGAACAGCCAAATGTACATTGTTGAT
M M A V
D
G
R V
I
K Y
E
G
F
P
F M
N
S
F
G
F
L L L L S
F T
D
S
S
F A Y A S
S
P
S
S
S
F W N
S
S
K A
S
F
P
C
I
E
S T D P E K R P L C G R T Y D I S Y D Y K N S Q M Y I V D GGCCATTACCATCTTTGTGTGGTTGGAAAAGAAGGTGGGTATGCCACACAACTAGCCACAAGTGTGCAAGGAGTGCCATTCAAATGGCTC G H Y H L C V V G K E G G Y A T Q L A T S V Q G V P F K W L TATGCAGTAACTGTTGATCAGAGAACAGGGATTGTTTATTTCACTGATGTTAGCTCCATACATGATGACAGTCCCGAAGGTTGTGAAGAA
N
361 451
Y A V T
541
631
721
V D Q R T G I V Y F T D V S S I H D D S P E G V E E ATCATGAATACAAGTGATAGAACAGGGAGATTAATGAAGTATGATCCTTCAACAAAAGAAACCACCTTATTATTGAAAGAGCTACATGTT I M N T S D R T G R L M K Y D P S T K E T T L L L K E L H V CCCGGCGGTGCAGAAATCAGCGCAGATGGTTCCTTTGTTGTAGTAGCAGAATTTTTAAGCAATCGGATAGTGAAGTATTGGCTAGAAGGG P G G A E I S A D G S F V V V A E F L. S N R I V K Y W L E G CCAAAGAAAGGCAGTGCAGAGTTCTTAGTTACAATCCCAAATCCAGGAAATATAAAGAGGAATTCTGATGGCCATTTTTGGGTGTCTTCA P K K G S A E F L V T I P N P G N I K R N S D G H F W V S S
AGTGAAGATTAGATGGAGGTCAACATGGAAGTGTTGTTTCAAGAGGAATTMGTTTGATGGATTTGGGAATATTCTTCMGTTATACCA S E E L D G G Q H G S V V S R G I K F D G F G N I L Q V I P 901 CTTCCACCACCATATGAAGGTGAACATTTGAACAGATTCAAGAGCACGATGGTTTGTTATACATTGGAAGTCTCTCCCATAGCTCTGTG L P P P Y E G E H F E Q I Q E H D G L L Y I G S L S H S S V 991 GGTATATTAGTGTATGATGATCATGATAACAAGGGAAATTCTTATGTTTCTCAGCTAGTCATTAATTGAATATTTGTTTTGATTTGTAAC G I L V Y D D H D N K G N S Y V S Q L V I N * 1081 TTTTTAATGATTTCTGCAAATTCTATGGCTTTTTGAAGGTTACAAAGTCTTAGGAGGAGATTTTGAGTGTCTGCTGCTACATATATGTTC 811
1171 *
AATAAGAAGCCAATTCCATGAAAAAAAGGAATTC
To whom correspondence should be addressed
