Proc. Natl. Acad. Sci. USAVol. 83, pp. 6820-6824, September 1986Botany

Ethylene-regulated gene expression: Molecular cloning of the genesencoding an endochitinase from Phaseolus vulgaris

(phytohormone/cDNA clones)

KAREN E. BROGLIE*, JOHN J. GAYNORt, AND RICHARD M. BROGLIE*Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399

Communicated by Charles J. Arntzen, May 27, 1986

ABSTRACT A full-length copy of bean leaf chitinasemRNA has been cloned. The 1146-base-pair insert of pCH18encodes the 27-residue amino-terminal signal peptide of theprecursor and 301 residues of the mature protein. UtilizingpCH18 as a hybridization probe, we have shown that theincrease in translatable chitinase mRNA seen upon ethylenetreatment of bean seedlings is due to a 75- to 100-fold increasein steady-state mRNA levels. Southern blot analysis of beangenomic DNA revealed that chitinase is encoded by a small,multigene family consisting of approximately four members.From our nucleotide sequence analysis of five additionalchitinase cDNA clones, it appears that at least two of thesegenes are expressed. Three of the bean chitinase genes havebeen isolated from a Sau3A genomic library and partiallycharacterized.

The development of disease resistance in higher plants ismanifested by the accumulation of a number of host-synthe-sized polypeptides that are produced in response to pathogenattack. Among these are the following: (i) enzymes involvedin the synthesis of phytoalexins (secondary metabolites thatare toxic to bacteria and fungi) (1), (ii) enzymes leading to theformation of physical barriers to fungal invasion throughmodifications of the plant cell wall (2), (iii) inhibitors of serineendoproteases (2, 3), and (iv) lytic enzymes (e.g., chitinaseand ,-1,3-glucanase) that are capable of degrading fungal cellwalls (4, 5). While inhibitor studies indicate that host RNAand protein synthesis are required for the induction of theseproteins (6), there is a paucity of information concerning theregulation and expression of the genes involved.The activities of several of these enzymes (2, 4, 7) can be

increased by exposure to exogenous ethylene. Increasedsynthesis of this phytohormone has been associated withvarious kinds of plant stresses including mechanical wound-ing (8) and infection by bacteria and fungi (9, 10). Theseobservations have led to the suggestion that ethylene maymediate the host response to pathogen attack (2, 4, 5, 11).Because of its potential role in plant defense, we haveundertaken an investigation of ethylene regulation of geneexpression in higher plants. As a model system for thesestudies, we have selected the enzyme chitinase (EC 3.2.1.14).

Chitinase is a basic protein that catalyzes the hydrolysis ofthe P-1,4 linkages of N-acetyl-D-glucosamine polymers ofchitin, a major component of fungal cell walls. Since higherplants do not contain a substrate for this enzyme, it has beenproposed that chitinase functions as a defense against chitin-containing pathogens (4, 5). Chitinase activity has beendetected in both woody and herbaceous plants including anumber of important crop species (4). Boller et al. (4) haveshown that the major (95%) ethylene-induced chitinolyticactivity in bean leaves is attributable to an endochitinase that

they subsequently purified to homogeneity. The purified Mr-30,000 protein was found to exhibit both chitinase andlysozyme activities.As a prelude to investigating the molecular mechanisms

responsible for regulating chitinase gene expression, we haveconstructed a cDNA library complementary to mRNA iso-lated from ethylene-treated bean seedlings.. Here, we reportthe identification and complete nucleotide sequence of afull-length chitinase cDNA clone. This information has al-lowed us to deduce the pritnary structure of the chitinasepolypeptide and its amino-terminal signal peptide. Using thisclone as a hybridization probe, we present evidence thatethylene treatment results in an increase in steady-statechitinase mRNA levels, indicating that ethylene control ofchitinase gene expression occurs at the level of gene tran-scription.

MATERIALS AND METHODSReagents and Enzymes. L-(2-Aminoethoxyvinyl)glycine

was a generous gift from Hoffmann-La Roche Co. Deoxy-and dideoxyribonucleotides were purchased from P-L Bio-chemicals. Reverse transcriptase from avian myeloblastosisvirus was obtained from Life Sciences (St. Petersburg, FL).M13 pentadecamer sequencing primer was purchased fromNew England Biolabs. DNase I was from Cooper Biochem-icals (Malvern, PA). Restriction endonucleases and DNAmodifying enzymes were obtained from Bethesda ResearchLaboratories, New England Biolabs, or Boehringer Mann-heim.

Plant Growth Conditions. Seeds of Phaseolus vulgaris L.cv. Saxa were purchased from Samen Mauser, Zurich,Switzerland. Plants were grown in controlled environmentalchambers with a day/night photoperiod of 16 (22°C):8 (18°C)and used 7-10 days after imbibition of dry seeds. Ethylenetreatment was achieved by spraying with a solution ofethephon (1 mg/ml) (2-chlorethylphosphonic acid; Sigma)and enclosing the plants in plastic bags. Similar results havebeen obtained by exposing plants to 10 ppm gaseous ethylene(unpublished results).

Preparation of Chitinase Antibodies, Chitinase was purifiedfrom ethylene-treated leaves by affinity chromatography (14)and applied to preparative NaDodSO4 slab gels. The 30-kDaband was excised, and the protein was eluted by electrodial-ysis. Antibodies to purified chitinase were raised in rabbits(12).

Protein Analysis. Soluble protein was extracted (12) andanalyzed by two-dimensional polyacrylamide gel electropho-resis using nonequilibrium pH gradient electrophoresis (13)in

Abbreviations: kb, kilobase(s); bp, base pair(s).*Present address: The E. I. du Pont de Nemours & Co., Inc., CentralResearch and Development Department, Experimental Station,Wilmington, DE 19898.

tPresent address: Department of Botany, Rutgers University, New-ark, NJ 07102.

6820

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 83 (1986) 6821

the first dimension and 10% NaDodSO4/polyacrylamide inthe second dimension (12). Immunoblots were by the methodof Blake et al. (14) at an antibody concentration of 85 ,pg/ml.RNA Isolation. Poly(A)+ RNA was isolated as described

(15) and translated using a wheat germ cell-free system. Invitro synthesized polypeptides were analyzed by polyacryl-amide gel electrophoresis and fluorography (16). Immuno-precipitation of translation products was performed withanti-chitinase IgG.

Construction and Screening of a Bean cDNA Library.Double-stranded cDNA was synthesized from poly(A)+RNA (17) and cloned into the EcoRI site of Xgtll (18). Therecombinant DNA molecules were packaged in vitro andused to infect Escherichia coli RY1088.cDNA clones corresponding to ethylene-induced mRNAs

were identified using a combination of differential plaquehybridization (19) and hybridization to size-fractionatedmRNA (20). Chitinase cDNA clones were identified byhybridization-selection and translation (21).DNA Sequence Analysis. Chitinase cDNA inserts were

excised by EcoRI digestion and inserted into pEMBL8' (22).Two different approaches were employed to determine thenucleotide sequence of bean chitinase cDNA. Progressivedeletions were constructed as described by Barnes et al. (23)and sequenced using the dideoxy method (24). For chitinasecDNA clones (pCH6, pCH18, pCH20, and pCH28) restric-tion enzyme fragments, generated by Taq I and HindIIdigestions, were inserted into the M13 vectors mplO andmpll and sequenced by the dideoxy method. In all cases, thesequence of complementary strands was determined.Amino Acid Sequence Analysis. A partial amino-terminal

amino acid sequence was determined by subjecting 3 nmol ofgel-purified chitinase to automated Edman degradation in aBeckman Model 890B Sequence Analyzer using the DiluteQuadrol program. The phenylthiohydantoin derivatives fromeach cycle were identified by HPLC (Hewlett-PackardModel 1084A).

Isolation of Chitinase Genomic Clones. Total DNA wasisolated from etiolated leaves (25) and purified by two cyclesof CsCl/ethidium bromide density gradient centrifugation.Purified DNA was partially digested with Sau3A and frag-ments 10-20 kilobases (kb) long were cloned in XEMBL4(26). The bean genomic library (5 x 105 recombinant phage)was screened for chitinase genomic sequences using nick-translated (26) cDNA clone pCH18.RNA and DNA Filter Hybridizations. RNA was denatured

with glyoxal, separated by electrophoresis on a 1% agarose

H OH- H+

(A)

gel, and blotted onto nitrocellulose filters (27). DNA sampleswere digested with the appropriate restriction enzyme, frac-tionated by agarose gel electrophoresis, and transferred tonitrocellulose filters (28). Filters were hybridized with nick-translated probes and washed as described (25).

RESULTS

Chitinase activity in bean seedlings is increased 30-foldfollowing exposure to exogenous ethylene (4, 5). Whensoluble leaf proteins are analyzed by two-dimensional poly-acrylamide gel electrophoresis, approximately 20-25 addi-tional polypeptides are found to accumulate to stainablelevels with ethephon treatment. By immunological methods,at least two of these have been identified as isoelectric formsof chitinase (Fig. iB). In vitro translation ofmRNA extractedfrom treated and untreated plants reveals a translationproduct, precipitated by anti-chitinase IgG and observed onlyafter ethephon treatment (Fig. 2, lane 4). No chitinasepolypeptide can be detected in the translation products fromcontrol mRNA (Fig. 2, lane 2). Thus, the elevation ofchitinase activity, observed in earlier studies, is reflected atthe mRNA level, where an increased amount of translatablechitinase mRNA directs the synthesis and accumulation ofthe polypeptide following ethylene treatment.To examine more closely the ethylene effect on chitinase

gene expression, we constructed a cDNA library usingpoly(A)+ RNA from ethephon-treated leaves as template.cDNA clones corresponding to mRNAs expressed afterethephon treatment were selected by a combination ofdifferential plaque hybridization and hybridization to anenriched mRNA fraction (19). Using these procedures, twocDNA clones (pCH4 and pCH6) were isolated that, uponhybrid-selection, yielded a 35-kDa translation product thatwas precipitated by chitinase antiserum (Fig. 2). The 850-base-pair (bp) EcoRI insert of one of these recombinants,pCH6, was nick-translated and used as a probe to rescreenthe cDNA library. This second screen produced four addi-tional clones harboring chitinase cDNA inserts (pCH18,pCH20, pCH22, and pCH28).The six recombinant clones were found to contain chitinase

cDNA inserts ranging in size from 750 to 1200 bp in length.The nucleotide sequence of a full-length cDNA clone(pCH18) shows that the 1146-bp insert contains a short,A+T-rich segment (33 bases) of the 5'-untranslated regionand a 984-nucleotide open reading frame that terminates 115nucleotides from the poly(A) tail (Fig. 3). The TGA termi-

* OH-

(B)

97-

6 8- __

43- - 0 X

2-*

26- _ t

6

z

FIG. 1. Two-dimensional profiles oftotal soluble leaf protein extracted fromcontrol (A) and ethephon-treated (B)bean seedlings. The positions of molec-ular size standards in kDa are indicated inthe vertical axis at the left. The two majorisoelectric forms of chitinase (indicatedby the arrows in B) were identified byimmunoblot analysis as described (14)using an IgG concentration of 85 /.g/ml.

18-

14- w

.W

-P

400- a 0

Oft. 4.0

- 4wwlw

Botany: Broglie et al.

Proc. Natl. Acad. Sci. USA 83 (1986)

kDa FIG. 2. Immunoprecip-itation of chitinase precursor

68- polypeptide (pCh) and identifi-cation of cDNA clone pCH6.Lanes 1 and 3, translation pro-

43-- file of poly(A)+ RNA isolated

il* _pCh from control and ethephon-treated plants, respectively.Lanes 2 and 4, immunoselect-26- ed translation products. Lane

5, translation profile ofmRNA18- £ hybrid-selected by 10 gg of

pCH6 bound to nitrocellulose14- U filters (21). Lane 6, immuno-U ^ ^ precipitation of hybrid-select-*UV ed translation products. The

1 2 3 4 5 6 positions of molecular sizestandards are indicated.

nation codon, at position 1018 is repeated again 54 residuesdownstream. Three hexanucleotide consensus sequences(29) for polyadenylylation (AAUAAA) are present in the3'-untranslated region. Two of these sequences at positions1038 and 1042 overlap, while the third is found at nucleotide1113. Both of these potential poly(A) addition sites are usedin bean chitinase mRNA. Five of the six analyzed clones(pCH4, pCH6, pCH18, pCH20, and pCH28) employ the mostdownstream site, while the remaining clone (pCH22) uses themore upstream site.DNA sequence analysis of the chitinase cDNA clones

indicates that they are derived from two different chitinasemRNAs. Five clones (pCH4, pCH6, pCH20, pCH22, andpCH28) have identical DNA sequences and differ from theremaining clone, pCH18, by 16 nucleotides. Of these differ-ences, 12 occur within the open reading frame. While pCH18is a full-length clone, the largest insert representing the firstmRNA species is 1060 bp long (pCH4). This cDNA insert

contains an open reading frame of 935 nucleotides andpossesses 115 nucleotides of 3'-untranslated sequence.Deduced Amino Acid Sequence of Chitinase. The 984-bp

open reading frame of pCH18 encodes a protein of Mr 35,400,a size consistent with that observed upon NaDodSO4/poly-acrylamide gel electrophoresis of the in vitro translationproduct (Fig. 2). This value is larger than that determined forthe isolated protein (4) and suggests that chitinase is initiallysynthesized as a larger precursor.A partial amino acid sequence of 30 residues has been

obtained from the amino terminus of bean chitinase (30). Bycomparing the deduced amino acid sequence of pCH18 withdata obtained from the purified protein, we have localized theamino terminus of mature chitinase to the 28th residue of theprecursor polypeptide. pCH18, therefore, encodes the entire301-amino acid residues of the mature protein and a 27-residue amino-terminal sequence (Fig. 3). This leader se-quence displays the primary structural properties character-istic of other eukaryotic signal sequences (31, 32). Althoughthe biosynthetic pathway of chitinase has yet to be elucidat-ed, the presence of a signal sequence suggests its synthesis onmembrane-bound ribosomes. Similar results have been ob-tained for two protease inhibitors that accumulate in vacuolesof tomato leaves upon wounding (3).The deduced amino acid sequence of pCH18 was found to

differ from pCH4 by six nucleotide changes in the openreading frame. At residue 141 an alanine is exchanged forthreonine while at residues 61 and 293 there are conservativereplacements of methionine with valine and of leucine withphenylalanine, respectively. The changes at residues 33 and34 involve substitution of a neutral amino acid with a basicand an acidic residue, respectively. Despite the amino aciddifferences, the net charge of the two polypeptides is notaltered. The remaining nucleotide changes in the openreading frame reside in the third base of the codon and aresilent.

-27 -20 -10met lys lys asn arg met met met met ile trp ser val gly val val trp mt lu leu leu

CACCTrATCATTTAGAGGAAAAGAGAGAGAGAA ATG AAG AAG MAT AGG ATG AT ATG ATG ATA TOG AGC GTA GGA GTG GTG TGG ATG CTO TIM TTG

I1 10 20val gly gly mer tyr glyVglu gin cys gly arg gin ala gly gly ala leu cys pro gly gly asn cys cys ser gin phe gly trp cysGTT GGA GGA AGC TAC GGA GAG CAG TGT GGA AGG CAA GCA GGA GGT GCA CTC TGC CCA GGG GGC AAC TGT TGC AGC CAM TTC GGG TOG TGC

30 40 50gly ser thr thr asp tyr cys gly pro gly cym gin ser gin cys gly gly pro ser pro ala pro thr asp leu ser ala leu iie merGGC TCC ACC ACC GAC TAC TGC GGC W GGT TGC CAG AGC CAG TGC GGG GGA CCG TCT CCT GCT CCT ACT GAT CTC AGC GCC CTC ATA TCC

60 70 80arg ser thr phe asp gln met leu lys his arg asn asp gly ala cys pro ala lys gly phe tyr thr tyr asp ala ph. ile ala alaAGG TCC ACC TTC GAC CMG ATG CTC AAM CAT CGC AAC GAC GGA GCC TGC CCA GCC AM GGC TTC TAC ACC TAC GAT GCC TTC ATC GCC GCC

90 100 110ala lys ala tyr pro mer phe gly asn thr gly asp thr ala thr arg lys arg glu ile ala ala phe leu gly gln thr ser his gluGCC AAG GCT TAC CCC AGC TTC GGA AAC ACC GGA GAC ACG GCC ACT CGC AAG AGG GAG ATT GCG GCC TTC TTG GGG CAM ACG TCT CAC GAA

120 130 140thr thr gly gly trp ala thr ala pro asp gly pro tyr ala trp gly tyr cys phe val arg glu arg asn pro ser thr tyr cys serACA ACC GGG GGA TOO GCC ACT GCG CCC GAC GOA CCA TAC GCA TGG GGA TAC TGC TTC GTG AGO GAG CGG MC CCC AGI ACG TAC TGC TCC

150 160 170ala thr pro gin ph. pro cys ala pro gly gin gin tyr tyr gly arg gly pro ile gin He ser trp asn tyr asn tyr gly gin cysGCC ACT CCC CAG TTC CCC TGC CCC CCT 0G0 CAG CAG TAC TAC GGC AGG GOT CCC ATC CAG ATA TCC TGG MC TAC MC TAI GOT CMG TGC

180 190 200gly arg ala ile gly val asp leu leu asn lys pro asp leu val ala thr asp ser val ile ser phe lys ser ala leu trp ph. trpGGA AGM GCC ATT GGG OTT GAC TTt CTC MC MA CCT CAT CTA GTC CCC ACT GAC TCT GTC ATC TCC TTC AAG TCC CCC CTC TOG TTC TGO

210 220 230met thr ala gln ser pro lym pro ser ser his asp val ile thr ser arg trp thr pro ser ser ala asp val ala ala arg arg leuATS ACC CCA CAG TCC CCC AAG CCT TCC TCC CAC GAC GTC ATC ACC TCT CGA TGG ACC CCC TCC TCT GCC GAC GTC 0CC GCC CGC CGO CTT

A240 250 260

pro gly tyr gly thr val thr amn ile il- asn gly gly leu glu cys gly arg gly gin asp ser arg val gln asp *rg il gly ph.CCC GCC TAC GGC ACT MTG ACG MC ATC ATC MC GGA GGC CTG GAG TGC GGG CCA GGA CAG GAC AGC AGG GTT CAP GAC CCC ATC GGA TIC

270 280 290ph. lys arg tyr cys asp l-u leu gly val gly tyr gly asn asn leu asp cys tyr ser gin thr pro phe gly asn ser leu leu leuTTC AM AGA TAC TGT OAT CTC CTT GGA GTC GGT TAT GGC MC AAC CTT GAC TGC TAC TCT CAG ACT CCA TTT GGA AAT TCA CTC TTA CTC

A A300 301

smr asp lou val thr mer gin OPTCT GMC CTT GT ACC TCT CMG TGA CACTG

TCAATMAATCACTACTCTATMAAAAAAAAAAMATCATCCCATCAGMTAAATAAACTCATMAGTCTOTOTrCACTITCGATCACAACTTTCTATA.CTTTCCcTCA.A - A A A

ccAc

FIG. 3. Nucleotide sequence ofcDNA clone pCH18. The deduced amino acid residues are shown above the nucleotide triplets. Nucleotidesin pCH18 that differ from the other five clones analyzed are indicated by (A) below the nucleotide; those changes that affect the amino acid codonare discussed in the text. Putative polyadenylylation signals are underlined. The alternative polyadenylylation site used by pCH22 is indicatedby the arrow A.

6822 Botany: Broglie et A

Botany: Broglie et al.

o

O a

1D ><: LU

B

Abp

- 1350

-1070

- 870

kb

_ -23

-9.6w

-6.6

- -44qw-4.4

- 600

-2.3-2.0

2 1 2 3

FIG. 4. (A) RNA gel blot analysis ofbean chitinase mRNA levels.Control plants were cut at the root-shoot interface, and the excisedstems were placed in a 100 ,uM solution of the ethylene biosynthesisinhibitor aminoethoxyvinylglycine (AVG) for 6 hr prior to RNAisolation (lane 1). Ethephon-treated plants were sprayed and placedin an enclosed chamber for 30 hr. Total RNA was fractionated on a1% agarose gel and transferred to nitrocellulose. The probe was anick-translated cDNA insert from clone pCH18. Glyoxylated Hae IIIrestriction fragments of OX174 were used as molecular size stan-dards. (B) Southern blot analysis ofbean genomic DNA digested withEcoRI, BamHI, or HindIII. DNA fragments were fractionated on a0.65% agarose gel and transferred to nitrocellulose. The filter washybridized to nick-translated cDNA insert of pCH18. X DNAdigested with HindIII and 4X174 digested with Hae III were used asthe molecular size standards.

Proc. Nati. Acad. Sci. USA 83 (1986) 6823

this RNA in ethephon-treated plants is 75- to 100-fold higherthan in control plants treated with the ethylene biosynthesisinhibitor L-(2-aminoethoxyvinyl)-glycine.

Isolation of Chitinase Genomic Clones. Our nucleotidesequencing data strongly suggests that multiple copies of thechitinase gene are present in the bean genome. To determinethe number of chitinase genes, high molecular weight chro-mosomal DNA was digested to completion with the restric-tion enzymes EcoRI, BamHI, and HindIII and then subjectedto Southern blot analysis. Three or four bands, ranging in sizefrom 4 to 21 kb, were found to hybridize with pCH18 insertDNA (Fig. 4B). Since each ofthese fragments are of sufficientsize to contain the entire chitinase gene and since no EcoRI,BamHI, or HindIII sites are found in pCH4 and pCH18cDNA, these results suggest that bean chitinase is encodedby a multigene family consisting of 3 or 4 members.To isolate and characterize these genes, we constructed a

genomic library of 15- to 20-kb Sau3A partial digestionproducts in the vector, XEMBL4. Utilizing pCH18 insertDNA as a probe, six independent clones were isolated. Therestriction maps of these clones indicate that they comprisethree different chitinase genes (Fig. 5). XCH3A and XCH3Bcontain overlapping segments of DNA as also do XCH2A,XCH5B, and XCH1OA. XCH5A is a unique isolate. The threechitinase genes have been localized on the 5.7-kb EcoRIfragment of XCH3B, the 4.5-kb HindIll fragment of XCH5Band the 5.2-kb HindIII fragment of XCH5A. Correspondingrestriction enzyme fragments are evident in the Southern blotof genomic DNA (Fig. 4B). Preliminary sequence dataindicate that XCH5A and XCH5B encode the cDNAs ofpCH18 and pCH4, respectively. XCH3B represents a thirdchitinase gene that was not detected in our cDNA library.

RNA Gel Blot Analysis. To quantitate the effect of ethyleneon steady-state chitinase mRNA levels, an RNA gel blotanalysis was performed. Equal amounts of total RNA fromcontrol and ethephon-treated plants were fractionated byagarose gel electrophoresis and transferred to nitrocellulosefilters. In both samples, a 1200-nucleotide mRNA was de-tected after hybridization with nick-translated pCH18 insertDNA (Fig. 4A). Quantitation by silver grain counting (33) andslot-blot analysis (data not shown) revealed that the level of

E H E

DISCUSSIONIn this paper we report the isolation and characterization ofseveral bean chitinase cDNA clones. The identity of theseclones has been verified by the following two independentmeans: (i) hybrid-selection of a 35-kDa translation productthat was precipitated by chitinase antiserum and (ii) compar-ison of the deduced amino acid sequence with a partialamino-terminal sequence obtained by direct amino acid

H H E K B

H H E K B

H,,,,,,

XCH3A

H EII I i

H E B E H", AV

E H B B,)(

K E

E BH HEEE B,, AI(( ,

H E H(~~~~~~ B E

XCH3B

xCH5A

XCH2A

H

H HEB E H

E BHE H B BI (

E BHE H B B,, t

H E HNI

H

B

B-.,,,,,",

xCH5B

xCH1OA

FIG. 5. Restriction enzyme profiles of chitinase genomic clones. The restriction sites for EcoRI (E), BamHI (B), HindIII (H), and Kpn I(K) were mapped on the insert DNA by a combination of single, double, and partial digestions. DNA fragments containing the chitinase gene(indicated by the solid bars) were identified by hybridization to nick-translated pCH18 insert DNA.

Proc. Natl. Acad. Sci. USA 83 (1986)

sequencing of the purified protein. Nucleotide sequenceanalysis of a full-length cDNA clone (pCH18) has permittedthe elucidation of the primary structure of the maturechitinase polypeptide as well as the 27-residue amino-termi-nal leader peptide. The synthesis of chitinase as a largerprecursor is consistent with its vacuolar localization (34).The analysis of six independent chitinase cDNA clones

reveals that they represent copies of two different mRNAs.Of the 16 nucleotide differences between pCH4 and pCH18,12 are found to occur in the open reading frame. Althoughnine of these changes are silent or conservative in nature, theremaining three differences involve the substitution of aneutral residue with an amino acid bearing a charged sidechain. The mature protein encoded by pCH18 contains24-basic and 22-acidic residues while in pCH4, these numbersare increased to 25-basic and 23-acidic residues. Theisoelectric properties of the two polypeptides thus remainunchanged. The different mRNA species isolated from ourcDNA library cannot explain the two isoelectric formsdetected by two-dimensional polyacrylamide gel electropho-resis. Southern blot analysis of bean genomic DNA indicatesthat chitinase is encoded by a small multigene family con-sisting of approximately four members. It is possible that apolypeptide encoded by other chitinase genes may accountfor the additional isoelectric form. It is also possible thatposttranslational modifications (acetylation, glycosylation,methylation, phosphorylation, etc.) are responsible for gen-erating multiple isoelectric forms of chitinase.

Ethylene treatment of carrot roots and soybean hypocotylshas been shown to affect the expression of specific mRNAs,as assayed by changes in the levels of in vitro translationproducts (35). Nichols and Laties (36) have demonstratedincreased transcription of three ethylene-induced mRNAs inisolated carrot nuclei. Using our chitinase cDNA clone as ahybridization probe, we have found that ethylene treatmentof bean seedlings promotes a 75- to 100-fold increase in thesteady-state level of chitinase mRNA. While we cannot ruleout the possibility that this effect is due to changes in the ratesof mRNA turnover or RNA processing, the increase inchitinase mRNA most likely represents an enhanced level ofgene transcription.

It is interesting to note that five of six analyzed cDNAclones are complementary to the same mRNA. Since this ispresumably indicative of the relative abundance of thismRNA in ethylene-treated leaves, it is possible that theindividual chitinase genes are differentially regulated. Theavailability of chitinase genomic clones will allow the furtherinvestigation and identification of the DNA sequence ele-ments responsible for ethylene regulation of chitinase geneexpression.

We thank N. Ahmed for excellent technical assistance; Dr. T.Boller for providing seeds and for helpful discussions; and Drs. G.Coruzzi, S. Tingey, N.-H. Chua for many helpful discussions. Wealso thank W. Roine for typing and assembling the manuscript. Thiswork was supported by Grant GM-31500 (R.M.B.) from the NationalInstitutes of Health and a National Research Service AwardGM-09514 (K.E.B.).

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