Plant Molecular Biology 13: 249-250, 1989. © 1989 Kluwer Academic Publishers. Printed in Belgium. 249

Isolation and nucleotide sequence of cDNA clones encoding potato chitinase genes

Daniel Laflamme and Robert Roxby Biochemistry Department, University of Maine, Orono, M E 04469, USA

Received 23 January 1989; accepted 18 April 1989

Chitinases are among a group of proteins expressed by plants in response to infection by pathogens [6]. Broglie et al. [ 1] and Shinshi et al. [9] have published sequences of basic chitinases from bean and tobacco, respectively, and immunologically related acidic chitinases are among the pathogenesis-related proteins that have been described in tobacco [7]. Multiple chitinase genes exist in bean [ 1 ] and several pro- teins having chitinase activity have been isolated from potato [5]. We have isolated chitinase cDNA and genomic clones from potato which restriction mapping and sequencing data indicate represent at least four different genes. This com- munication reports the sequence of one cDNA clone.

A cDNA encoding bean chitinase [ 1 ] was used to isolate a chitinase containing genomic fragment from a lambda library of potato D N A [3]. This fragment was subcloned and used to identify cDNA clones in a library constructed from mRNA of ethephon-treated potato plants [4]. Northern blots of this RNA show that all mes- sages are about the same size, 1.2 kb. Northern and slot-blots comparing RNA from ethephon- treated and untreated plants show that these mes- sages are only moderately, 3- to 5-fold, inducible by 24-hour treatment of whole, cultured plants with ethephon. The sequence of one of the cDNA clones, CCH4, is given in Figure 1. This sequence can be translated in a single 315 amino acid read- ing frame to give a deduced protein sequence rich in cysteine (5~o mol/mol) and proline (6.6~o mol/mol). The 5' end encodes a sequence having

the properties of a signal peptide, which, accord- ing to consensus patterns [2, 8], is probably cleaved after glycine 16 or asparagine 18. The calculated isoionic point is at pH 7.0, with or without the signal peptide. The nucleotide se- quence is 86~o homologous to that of the tobacco gene [9]. The size of the deduced protein and the high homology to the tobacco gene suggest that CCH4 is similar to the basic chitinases previously described.

Note and acknowledgements

The CCH4 sequence is in the EMBL/GenBank/ DDBJ nucleotide sequence databases under the accession number X14133. This work was sup- ported by U S D A Competitive Grant 85-CRCR- 1-1558 and Grant ME8406 from the Maine Agri- culture Experiment Station, of which this is publi- cation number 1352.

References

1. Broglie K, Gaynor J, Broglie R: Ethylene-regulated gene expression: molecular cloning of the genes encoding a chitinase from Phaseolis vulgaris. Proc Natl Acad Sci USA 83:6820-6824 (1986).

2. von Heijne G: Patterns of amino acids near signal- sequence cleavage sites. EurJ Biochem 133:17-21 (1983).

3. Horovitz D: Construction of a potato genomic library and screening for sequences homologous to a bean chitinase gene. M.S. thesis, University of Maine (1986).

4. Jendrisak J, Young R, Engel J: Cloning into 2gtl0 and 2gtll. In: Berger SL, Kimmel AR (eds) Methods in Enzymology, vol. 152:358-371 (1987).

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ACT ATT TTT TCT TTA CTA TTC TCT CTC CTT TTG CTG AAC GCC TCG GGG AGC AM GTG GTT T I F S L L F S L L L L N A S G S N V V

CAC AGG CCG GAC GCG CTT TGT GCC CCA GGA CTC TGT TGT AGC AAA TTC GGC TGG TGT GGT H R P D A L C A P G L C C S K F G W C G

AAT ACA AAT GAC TAT TGT GGT CCA GGT AAT TGC CAG AGC CAG TGT CCT GGC GGC CCC GGT N T N D Y C G P G N C Q S G C P G G P G

CCT TCA GGG GAC TTA GGC GGT GTT ATT TCA AAT TCC ATG TTT GAT CAG ATG CTT AAT CAT P S G D L G G V I S N S H F D Q M L N H

CGC AAC GAT AAT GCT TGT CAA GGA AAG AAT AAT TTC TAT AGT TAC AAT GCC TTC ATC AGT R N D N A C q G K N N F Y S Y N A F I S

GCT GCT GGG TCT TTT CCT GGC TTT GGC ACT ACT GGT GAT ATA ACT GCC CGT AAA AGG GAA A A G S F P G F G T T G D I T A R K R E

ATT GCT GCT TTC CTT GCC CAA ACT TCC CAT GAA ACT ACT GGA GGA TGG CCT TCA GCA CCT I A A F L A q T S H E T T G G W P S A P

GAT GGA CCA TAC GCA TGG GGT TAC TGC TTC CTT AGA GAA CAA GGT AGC CCG GGC GAT TAC D G P Y A W G Y C F L R E Q G S P G D Y

TGT ACA CCA AGT AGT CAA TGG CCT TGT GCT CCT GGA AGG AAA TAT TTC GGA CGA GGC CCA C T P S S Q W P C A P G R K Y F G R G P

ATC CAA ATT TCA CAC AAC TAC AAC TAT GGG CCA TGT GGA AGA GCC MC GGA GTG GAC CTT I Q I S H N Y N Y G P C G R A I G V D L

TTA AAC AAT CCT GAT TTA GTA GCC ACA GAC TCA GTC ATC TCA TTT AAA TCA GCT ATC TGG L N N P D L V A T D S V I S F K S A I W

TTC TGG MG ACA CCT CAA TCA CCA AAG CCT TCT YGC CAC GAT GTC ATC ACC GGA AGA TGG F W M T P Q S P K P S C H D V I T G R W

CAA CCA TCT GGC GCT GAC CAA GCA GCT AAT CGT GTC CCT GGA TTC GGT GTC ATC ACA AAC G P S G A D G A A N R V P G F G V I T A

ATC ATC AAT GGT GGC TTG GAA TGT GGT CAT GGA AGT GAC AGC AGG GTC CAG GAC CGA ATT I I N G G L G C G H G S D S R V Q D R I

GGA TTT TAC AGG AGG TAT TGC GGA ATT CTT GGA GTT AGT CCT GGT GAC AAT CTT GAT TGT G F Y R R Y C G I L G V S P G D N L D C

GGC AAC CAG AGG TCT TTT GGA AAC GGA CTC TTA GTC GAT ACT GTG TAA GCATCGACTTCATCA G N G R S F G N G L L V D T V

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TATAAGGCCCCAACTATAAATAAAGTTATGTATBTACGAAATTGTATGAATTGTGATTGTAATATGATGGAACCACGAC 1042

TTATAATTGTTTGTTTTTTATAATAAACAAGACTATTGGTTCTTCTTCT 1091

Fig. l. Nucleotide and deduced amino acid sequences ofpotato chitinase cDNA clone CCH4. Polyadenylation signals are underlined.

5. Kombrink E, Schr6der M, Hahlbrock K: Several 'patho- genesis related' proteins of potato are 1,3-~-glucanases and chitinases. Proc Natl Acad Sci USA 85:782-786 (1988).

6. Lamb C, Lawton M, Dron M, Dixon R: Signals and transduction mechanisms for activation of plant defenses against microbial attack. Cell 56:215-224 (1989).

7. van Loon L, Gerritson Y, Ritter C: Identification, purifi- cation and characterization of pathogenesis-related pro-

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teins from virus-infected Samsun NN tobacco leaves. Plant Mol Biol 9:593-609 (1987). Perlman D, Halvorson H: A putative signal-peptidase recognition site and sequence in eukaryotic and pro- karyotic signal peptides. J Mol Biol 167:391-409 (1983). Shinshi H, Mohnen D, Meins, Jr., F: Regulation of a plant pathogenesis related enzyme: inhibition of chitinase and chitinase mRNA accumulation in tobacco tissues by auxin and cytokinin. Proc Natl Acad Sci USA 84:89-93 (1987),