Plant Molecular Biology 11:255-269 (1988)

© Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands 255

Molecular cloning and analysis of four potato tuber mRNAs

Willem J. Stiekema, Freek Heidekamp,1 Wim G. Dirkse, Joke van Beckum, Peter de Haan, Carolien ten
Bosch I and Jeanine D. Louwerse 1
Research Institute Ital, P.O. Box 48, 6700 A A Wageningen, Netherlands; 1present address: TNO-CIVO,
P.O. Box 360, 3700 A J Zeist, Netherlands

Received 11 March 1988; accepted in revised form 19 May 1988

Key words: potato, patatin, proteinase inhibitor, cloning, gene expression

Abstract

Tuberization in potato is a complex developmental process involving the expression of a specific set of genes
leading to the synthesis of tuber proteins. We here report the cloning and analysis of mRNAs encoding tuber
proteins. From a potato tuber cDNA library four different recombinants were isolated which hybridized
predominantly with tuber mRNAs. Northern blot hybridization experiments showed that three of them,
pPATB2, p303 and p340, can be regarded as tuber-specific while the fourth, p322, hybridizes to tuber and stem
mRNA. Hybrid-selected in vitro translation and nucleotide sequence analysis indicate that pPATB2 and p303
represent patatin and the proteinase inhibitor II mRNA respectively. Recombinant p322 represents an mRNA
encoding a polypeptide having homology with the soybean Bowman-Birk proteinase inhibitor while p340
represents an mRNA encoding a polypeptide showing homology with the winged bean Kunitz trypsin inhibitor.
In total, these four polypeptides constitute approximately 50°7o of the soluble tuber protein. Using Southern
blot analysis of potato DNA we estimate that these mRNAs are encoded by small multigene families.

Introduction the other hand, coumarin has been described as an

enhancer of tuber formation [49]. Therefore, the-
Tuberization in potato is a complex process leading ories based on both physiological and hormonal fac-
to the differentiation of an underground stem, also tors controlling tuber formation have been put for-
called a stolon, into a specialized storage organ, the ward. However, despite the large amount of
tuber [1]. During this developmental process mor- descriptive work favouring one of these theories, the
phological and genetical changes such as radial ex- molecular mechanisms underlying tuberization re-
pansion of the stolon take place and the expression main totally unknown.
of specific genes is dramatically influenced. This is As shown by Paiva et al. [33], a set of specific pro-
reflected by the synthesis of starch and specific pro- teins are present in mature tubers; the mRNAs of
teins [33, 35]. This differentiation process can be in- only two of them are isolated. Mignery et al. [29] and
fluenced by environmental conditions [3, 5, 7]. Fac- Rosahl et al. [39] reported the molecular cloning of
tors such as short daylength, low temperature or low the patatin mRNA; Sanchez-Serrano et al. [43]
nitrogen supply favour tuber formation. Plant hor- reported the cloning of the proteinase inhibitor II
mones also play a role [15, 22]. Cytokinin [8, 22, 34] mRNA.
enhances tuber formation while gibberellic acid [10, Recently the isolation and analysis of patatin
17, 23, 27] inhibits this developmental process. On genes has been accomplished [2, 36, 40]. Pikaard et
256

al. [37] showed that two classes of patatin genes ex- tein having homology to the Bowman-Birk pro-
ist. Class I is exclusively expressed in the tuber while teinase inhibitor described in soybean [16]. The
Class II patatin genes are expressed in both tubers fourth cloned tuber mRNA encodes a protein show-
and roots. In tubers equal amounts of Class I and ing homology to the Kunitz trypsine inhibitor of
Class II mRNAs are present. In roots the expression winged bean [55].
of Class II genes show a 50-100- fold lower level. Re-
cently, Twell and Ooms [52] presented data showing
that the 5' flanking region of one of the patatin Materials and methods
genes is able to direct tuber-specific gene expression.
A 3 800 basepairs (bp) DNA fragment located up- Growth conditions f o r plants
stream of the patatin protein-coding sequences con-
tains the regulatory signals conferring organ- Potato plants (Solanum tuberosum cv. Bintje and
specific expression upon a reporter gene. monoploid Solanum tuberosum clone AM 79.7322)
Patatin is the most abundant tuber protein [35], were grown from seed potatoes in 4-1itre containers
suggesting that it serves as a storage protein. Recent- in a growth chamber at 18 °C with a light intensity
ly, it has been shown that patatin also displays a lipid o f 10-15 klux for 14 hours followed by 10 hours of
acyl hydrolase activity [38, 41]. This enzymatic ac- darkness at 15 °C.
tivity of patatin might have a function in the transi-
tion of the tuber from dormancy to vegetative
growth [41] or in protection against microbial R N A isolation
growth [38].
The mRNA encoding a second tuber protein, the Tissue was harvested from young potato plants and
proteinase inhibitor II, has been cloned by Sanchez- immediately frozen at - 8 0 °C until use. Frozen tis-
Serrano et al. [43], Keil et al. [19] and Thornburg sue was ground in a mortar to a fine powder under
et al. [51] isolated the corresponding genes. Under liquid nitrogen. To 1 g of tissue a mixture of 2 ml
normal conditions the potato proteinase inhibitor II 0.2 M sodium acetate, pH 5.0, l°70 SDS, 0.01 M
gene is only expressed in the tuber. Upon wounding EDTA and 2 ml distilled phenol containing 0.1°70
the expression o f this gene is systemically induced in 8-hydroxyquinoline was added. After rigorous shak-
potato [13, 43], suggesting that the proteinase inhibi- ing and centrifugation the aqueous phase was re-
tor II protein is involved in the defence response of moved and the phenol phase re-extracted with 0.2 M
the plant against pathogens. As shown by Sanchez- sodium acetate, pH 5.0, 1°70SDS, 0.01 M EDTA. The
Serrano et al. [44] and Thornburg et al. [51] 1000 bp aqueous phases were collected and re-extracted twice
of 5' and 3' flanking regions of the proteinase inhib- with an equal volume of phenol:chloroform (1:1) and
itor II gene are sufficient to confer wound-inducible chloroform, respectively. Subsequently LiC1 was ad-
expression upon a reporter gene in transgenic tobac- ded to a final concentration of 2.5 M. The RNA was
co plants. precipitated overnight at 4 °C, collected by centrifu-
To enlarge our knowledge of tuber proteins and gation, washed once with 2.5 M LiC1 and twice with
the regulatory mechanisms involved in the develop- 70°7o ethanol. The dried pellet was dissolved in water
mental expression of their structural genes we decid- and stored in portions at - 8 0 °C. By this procedure
ed to clone and characterize a number of genes en- total RNA was isolated from different plant tissues.
coding these proteins. As a first step towards this Poly(A) ÷ RNA was isolated by oligo-dT cellulose
goal we report here the cloning and analysis of four chromatography [25].
different tuber mRNAs. Northern blot analysis
shows that these mRNAs are predominantly present
in tubers. Two of them encode the patatin and pro- Construction o f c D N A library
teinase inhibitor II proteins respectively of potato cv.
Bintje. A third cloned mRNA codes for a tuber pro- A quick one-tube method was used for the construc-
 257

tion of the cDNA library using tuber poly(A) ÷ microtitre plates were grown on GeneScreen Plus
RNA as a template. Tuber poly(A) ÷ RNA (5 #g) (New England Nuclear) and hybridized to radioac-
and oligo-dT (1.5 #g) were incubated at 80 °C for tively labelled probes essentially as described by
30 s in a volume o f 20/~1 double-distilled water. Sub- Franssen et al. [9]. For selection 10 #g/ml tetracy-
sequently mix I (20 units reverse transcriptase cline was used in Luria Broth (LB) agar. Single-
(Boehringer) in 30/~I 0.1 M Tris-HCl pH 8.3, 0.01 M stranded cDNA probes for differential screening of
MgC12, 0.15 M KCI, 0.02 M DTT, 1 mM dCTP, the cDNA library were prepared from poly(A) ÷
1 mM dGTP, 1 mM dTTP, 1 mM dATP) was added RNA according to the first-strand synthesis proce-
and the mixture incubated at 43 °C for 45 min. After dure used in the construction of the cDNA library
the first-strand synthesis 150/~l ice-cold mix II except that 50 #Ci o f ot-32p-dATP and o~-32p-dCTP
(40 mM Tris-HCl pH 7.5, 10 mM MgCI2, 75 mM (specific activity 3 200 Ci/mmol; New England Nu-
KC1, 25 #g/ml BSA) was added and the mixture put clear) and 0.2 mM unlabelled dATP and dCTP were
on ice for 5 min. Second-strand synthesis was per- added.
formed by adding 2 #l o~-32p-dATP (3 000 Ci/mmol
-- 110000 GBq/mmol; 10 mCi/ml; New England
Nuclear), 8 tzl DNA polymerase I (5 U//~l; Hybrid-selected translation
Boehringer) and 1 #l E. coli RNase H (2 U/#l;
Boehringer), and the mixture incubated at 11 °C for PIasmid DNA was isolated by the alkaline lysis
60 min. Subsequently 2/xl 20 m M ATP and 1 #l T4 method [25]. For hybrid-selected translation inserts
DNA ligase (1 U//~l; Boehringer) were added and the o f selected recombinants ( 5 - 1 0 #g of DNA) were
incubation prolonged for 2 h at 18 °C. To stop the denatured and applied to 0.5 cm 2 discs of di-
reaction 200 #l 0.4 M EDTA and 1 #120% SDS was azophenylthioether paper (BioRad) essentially as
added and the ds-cDNA separated from unincorpo- described [25]. Total potato tuber RNA (750 #g) was
rated a-32p-dATP by Sephadex G-50 column chro- hybridized to the filter-bound DNA in 300/zl 50%
matography in 0.01 M Tris, 1 mM EDTA, 0.3 NaCl (v/v) deionized formamide, 0.1% SDS, 0.6 M NaC1,
pH 7.5. The ds-cDNA was precipitated with ethanol, 4 mM EDTA, 80 mM Tris-HC1 pH 7.8. Hybridiza-
dried and dissolved in double-distilled water. Ds- tion was initiated at 56 °C and the temperature was
cDNA was size-fractionated on a 5 - 30% sucrose slowly decreased to 37 °C over a period of 6 hours.
gradient in 0.1 M NaC1, 0.01 M Tris-HC1 pH 7.5, After thorough washing, the bound RNA was eluted
1 mM EDTA for 16 h at 4°C and 30000 rpm. Ds- at 90°C in H20, ethanol-precipitated, dried and
cDNA having a length of 500 bp or more was used dissolved in 5 #1 H20; 1.5/~1 was translated in a
for the tailing reaction in 120 mM potassium wheat germ extract (Bethesda Research Laborato-
cacodylate pH 6.9, 1 mM dCTP, 1 mM CoCI 2, 50 ries) in a 7.5/~1 mixture to which 20 #Ci [35S]-
units terminal deoxynucleotidyltrans ferase methionine, 2.5 #1 wheat germ extract in 20 mM
(Boehringer). Tailed ds-cDNA (1 ng) was precipitat- magnesium acetate, 30 mM potassium acetate,
ed with ethanol, dried and annealed to 10, 20, 50 or 0.5 mM each of 19 amino acids was added. Transla-
100 ng Pst I-cut oligo-dG-tailed pBR322 (Bethesda tion products were separated by electrophoresis on
Research Laboratories) in 50/~1 0.1 M NaC1, 1 mM a 12.5% SDS-polyacrylamide slab gel according to
Tris-HC1 pH 7.5, 0.1 mM EDTA at 58 °C f o r 2 h. Dorssers [12]. The translation products were visual-
The annealed mixture was used to transform E. coli ized by fluorography using a Kodak XAR-5 film.
M H 1 [11]. For selection o f transformants 10 #g/ml
tetracycline was used. On the average 3 000 transfor-
mants were obtained per/~g o f poly(A) + RNA. Nothern blot analysis

Total RNA was denatured in dimethylsulphoxide

Differential screening of the cDNA library and glyoxyl, subject to electrophoresis in 1%0agarose
[25] transferred to GeneScreen and treated further
Individual transformants stored in 96-well according to the manufacturer's manual. The blots
258

were prehybridized for 4 hours in 50% formamide, Results

1 M NaC1, 0.05 M Tris-HCl pH 7.5, 10× Den-
hardt's solution, 1% SDS, 100/~g denatured salmon Molecular cloning o f tuber-specific m R N A s
sperm D N A per ml and hybridized with nick-
translated [25] probes. Hybridization was per- Poly(A) + R N A isolated from potato tubers was
formed for 16 h at 42 °C. Blots were washed in 0.3 M transcribed into ds-cDNA and cloned after GC-
NaC1, 0.03 M sodium citrate, 0.1% SDS at 42 °C and tailing into the Pst I site of pBR322. A quick one-
subsequently several times in 75 mM NaCI, 7.5 mM tube method was used which abolished both hairpin
sodium citrate, 0.1°70 SDS. formation normally used for priming the second-
strand synthesis as well as Sl nuclease digestion
necessary for removal of this hairpin. It is most likely
Southern blot analysis that less cloning artefacts are introduced using this
strategy than in the standard approach [50]. Part of
D N A was isolated from young potato leaves as the 6000 c D N A recombinants obtained were
described by Dellaporta [6]. Southern blot analysis screened by differential colony filter hybridization
was performed on nitrocellulose according to Mani- using 32p-labelled cDNA probes against leaf and
atis et al. [25]. tuber mRNAs. About 20O7o of the recombinants
hybridized predominantly to the tuber m R N A
probe. Cross-hybridization analysis showed that
D N A sequence analysis several of these c D N A clones could be divided into
one of four groups. Subsequently one clone of each
Standard procedures were used for cloning into M13 group was analysed further by Northern blot
vectors [28], for the chain termination [45, 46] and hybridizations as shown in Fig. 1. Total R N A isolat-
the chemical sequencing methods [26]. The D N A se- ed from tubers, stolons (subsoil and aerial), stems,
quence data were stored and analysed using Staden leaves and roots was separated on a denaturing
programs [48] on a VAX computer. glyoxal-agarose gel and transferred onto nylon
membranes. Recombinant p303 hybridized exclu-

Fig. 1. Occurrenceof tuber mRNAsin different tissues of potato. Autoradiographsare shownof Northern blots containing RNA isolated
from underground stolon (1), aerial stolon (2), root (3), tuber (4), stem (5) and leaf (6) tissue and separated by electrophoresis on 1°70
agarose gels, blotted onto GeneScreen filters and hybridized against 32p-labelled p303, p322, p340 and p207. The position of the
ribosomal RNAs, visualized after ethidium bromide staining of the agarose gel, is indicated.
 259

sively to a 800 nucleotides long tuber m R N A and

thus this recombinant contained a copy o f a tuber-
specific mRNA. The three other c D N A recom-
binants, p322, 340 and p207, hybridized mainly to
tuber m R N A having a length of 600, 900 and 1 500
nucleotides respectively. The m R N A s cloned in
recombinants p207 and p340 could also be detected
in underground stolons and stems. We estimated the
amount of these two m R N A s in stolons and stems
as approximately 1% of the amount present in
tubers. Based on these data we also classified recom-
binants p340 and p207 as containing copies of tuber-
specific mRNAs. Recombinant p322 contained a
copy of an m R N A which is about 10 times more
abundant in tubers than in stems. Therefore, we
regarded p322 as a stem-tuber-specific m R N A con-
taining cDNA recombinant.

Protein coding capacity of the cloned tuber

mRNAs

The translation products of total tuber m R N A have Fig. 2. Characterization of the coding capacity of tuber cDNA
clones by hybrid selected translation. Tuber RNA eluted from
been analysed by electrophoresis on a denaturing
filter-bound p207, p303, p322and p340 as well as total RNA iso-
SDS-polyacrylamide gel. A number o f abundant lated from tuber, leaf and stem tissue was translated in a wheat
polypeptide bands could be identified having a germ extract in the presence of 35S-methionine. The products ob-
molecular weight of 10000, 12500, 16000, 21000, tained were separated on a 12.5% polyacrylamide gel and
23 000 and 43 000 (Fig. 2). Only three potato tuber fluorographed. The molecular weight of marker proteins is indi-
proteins have been characterized thoroughly until cated as Mr × 10-3.
now: the patatin protein of Mr 43 000 [29], the pro-
teinase inhibitor I of M r 12600 [4] and the pro-
teinase inhibitor II o f M r 16000 [51, 55]. Patatin can copy of the cv. Bintje proteinase inhibitor II mRNA.
account for up to 40% of the tuber protein [35] while Recombinant p322 selected a m R N A encoding a
the proteinase inhibitors can represent up to 10% M r 10000 polypeptide while p340 hybrid-selected
[4]. So, most likely the dominant M r 43000, 16000 m R N A s appeared to code for polypeptides of M r
and 12 500 polypeptide bands detected after in vitro 23 000, 21000 and 19000. This might be explained by
translation of tuber poly(A) ÷ R N A represented the assuming that p340 contains a copy of a m R N A en-
patatin and the proteinase inhibitor I and II poly- coded by a diverged multigene family. Tuber proteins
peptides, respectively. showing these molecular weights have not been
The identity o f the cloned tuber m R N A s was es- described in the literature.
tablished by hybrid selection and in vitro translation.
Polyacrylamide gel electrophoresis (PAGE) demon-
strated that polypeptides of M r 16000 and 43000 Nucleotide sequence analysis of the tuber
were encoded by m R N A s selected by c D N A clone m R N A s containing cDNA recombinants
p303 and p207 (Fig. 2) respectively. This strongly
suggested that p207 contained a copy of cv. Bintje pPA TB1 and pPA TB2
patatin m R N A while c D N A clone p303 contained a Recombinant p207 contained an insert of ca. 750
260

I i0 20 30 40 50 60
B2 GAAAACACTTTGAACATTTGCAAA ATG GCA ACT ACT AAA TCT TTT TTA ATT TTA TTT TTT
BI
B2 Met Ala Thr Thr Lys Ser Phe Leu Ile Leu Phe Phe
BI

70 80 90 i00 ii0 120

B2 ATG ATA TTA GCA ACT ACT AGT TCA ACA TGT GCT AAG TTG GAA GAA ATG GTT ACT GTT CTA
BI C
B2 Met lle Leu Ala Thr Thr Ser Ser Thr Cys Ala Lys Leu Glu Glu Met Val Thr Val Leu
BI

130 140 150 160 170 180

B2 AGT ATT GAT GGA GGT GGA ATT AAG GGA ATC ATT CCA GCT ATC ATT CTC GAA TTT CTT GAA
BI
B2 Ser lle Asp Gly Gly Gly lle Lys Gly lle lle Pro Ala lle lle Leu Glu Phe Leu Glu
BI

190 200 210 220 230 240

B2 GGA CAA CTT CAG GAA GTG GAC AAT AAT AAA GAT GCA AGA CTT GCA GAT TAC TTT GAT GTA
B1
B2 Gly Gln Leu Gln Glu Val Asp Asn Asn Lys Asp Ala Arg Leu Ala Asp Tyr Phe Asp Val
BI

250 260 270 280 290 300

B2 ATT GGA GGA ACA AGT ACA GGA GGT TTA TTG ACT GCT ATG ATA ACT ACT CCA AAT GAA AAC
BI
B2 lle Gly Gly Thr Ser Thr Gly Gly Leu Leu Thr Ala Met lle Thr Thr Pro Asn Glu Asn
BI

310 320 330 340 350 360

B2 AAT CGA CCC TTT GCT GCT GCC AAA GAT ATT GTA CCC TTT TAC TTC GAA CAT GGC CCT CAT
B1
B2 Asn Arg Pro Phe Ala Ala Ala Lys Asp lie Val Pro Phe Tyr Phe Glu His Gly Pro His
B1

370 380 390 400 410 420

B2 ATT TTT AAT TAT AGT GGT TCA ATT TTA GGC CCA ATG TAT GAT GGA AAA TAT CTT CTG CAA
BI T G
B2 lle PhelAsn Tyr SerlGly Ser lie Leu Gly Pro Met Tyr Asp Gly Lys Tyr Leu Leu Gln
BI Phe Arg

430 440 450 460 470 480

B2 GTT CTT CAA GAA AAA CTT GGA GAA ACT CGT GTG CAT CAA GCT TTG ACA GAA GTT GCC ATC
BI
B2 Val Leu Gin Glu Lys Leu Gly Glu Thr Arg Val His Gin Ala Leu Thr Glu Val Ala lie
BI

490 500 510 520 530 540

B2 TCA AGC TTT GAC ATC AAA ACA AAT AAG CCA GTA ATA TTC ACT AAG TCA AAT TTA GCA AAG
BI A
B2 Ser Ser Phe Asp lle Lys Thr Asn Lys Pro Val lle Phe Thr Lys Ser Asn Leu Ala Lys

550 560 570 580 590 600

B2 TCT CCA GAA TTG GAT GCT AAG ATG TAT GAC ATA TGC TAT TCC ACA GCA GCA GCT CCA ATA
BI T
B2 Ser Pro Glu Leu Asp Ala Lys Met Tyr Asp lie Cys Tyr Ser Thr Ala Ala Ala Pro lie
BI lie

610 620 630 640 650 660

B2 TAT TTT CCT CCA CAT CAC TTT GTT ACT CAT ACT AGT AAT GGT GCT AGA TAT GAG TTC AAT
BI C
B2 Tyr Phe Pro Pro His His Phe Val Thr His Thr Set Asn Gly Ala Arg Tyr Glu Phe Asn
BI Thr
 261

670 680 690 700 710 720

B2 CTT GTT GAT GGT GCT GTT GCT ACT GTT GGT GAT CCG GCG TTA TTA TCC CTT AGC GTT GCA
BI G
B2 Leu Val Asp Gly Ala Val Ala Thr Val Gly Asp Pro Ala Leu Leu Ser Leu Ser Val Ala
BI Gly
730 740 750 760 770 780
B2 ACG AGA CTT GCA CAA GAG GAT CCA GCA TTT TCT TCA ATT AAG TCA TTG GAT TAC AAG CAA
B1
B2 Thr Ar~ Leu Ala Gln Glu Asp Pro Ala Phe Ser Ser lie Lys Ser Leu Asp Tyr Lys Gin
B1
790 800 810 820 830 840
B2 ATG TTG TTG CTC TCA TTA GGC ACT GGC ACT AAT TCA GAG TTT GAT AAA ACA TAT ACA GCA
B1
B2 Met Leu Leu Leu Ser Leu Gly Thr Gly Thr Asn Ser Glu Phe Asp Lys Thr Tyr Thr Ala
B1
850 860 870 880 890 900
B2 GAA GAG GCA GCT AAA TGG GGT CCT CTA CGA TGG ATG TTA GCT ATA CAG CAA ATG ACT AAT
BI T
B2 Glu Glu Ala Ala Lys Trp Gly Pro Leu Arg Trp Met Leu Ala lle Glu Glu Met Thr Asn
BI Leu
910 920 930 940 950 960
B2 GCA GCA AGT TCT TAC ATG ACT GAT TAT TAC ATT TCT ACT GTT TTT CAA GCT CGT CAT TCA
B1
B2 Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr lie Ser Thr Val Phe Gin Ala Arg His Ser
BI
970 980 990 i000 i010 1020
B2 CAA AAC AAT TAC CTC AGG GTT CAA GAA AAT GCA TTA AAT GGC ACA ACT ACT GAA ATG GAT
BI CA
B2 Gin Asn Asn Tyr Leu Arg Val Gln Glu Asn Ala Leu Asn Gly Thr Thr Thr Glu Met Asp
BI Thr
1030 1040 1050 1060 1070 1080
B2 GAT GCG TCT GAG GCT AAT ATG GAA TTA TTA GTA CAA GTT GGT GAA ACA TTA TTG AAG AAA
B1
B2 Asp Ala Ser Glu Ala Asn Met Glu Leu Leu Val Gln Val Gly Glu Thr Leu Leu Lys Lys
B1
1090 II00 iii0 1120 1130 1140
B2 CCA GTT TCC AAA GAC AGT CCT GAA ACC TAT GAG GAA GCT CTA AAG AGA TTT GCA AAA TTG
BI G
B2 Pro Val Ser Lys Asp Ser Pro Glu Thr Tyr Glu GLu Ala Leu Lys Arg Phe Ala Lys Leu
BI
1150 1160 1170 1180 1190 1200
B2 CTC TCT GAT AGG AAG AAA CTC CGA GCA AAC AAA GCT TCT CAT TAATTCAAGGTCCCGGGTTGTAG
BI A G T T
B2 Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala Ser His
BI Asn Tyr
1210 1220 1230 1240 1250 1260 1270 1280
B2 TAGT- TAAATAATAAGCGCTTGCAATATTTATGATCTGCACGCATTTAAATATTTCAACCCTCAAAC
BI G AACCTTACTATGC -T G T A

1290 1300 1310 1320 1330 1340 1350 1360

B2 TAAAAGGAGTTTGAGGGATAAATTTCAATAGAAATGTCTCTCTATGTAATGTGTGCTTGGATTATGTAACCTTTTGGTT
BI

1370 1380 1390 1400

B2 G T G T T A A A T A T T T A A A T A A - T T A T C C T T T A ~
B1 A G ATTTATGTTCAAGT

Fig. 3. Nucleotide sequence of the insert of clones pPATBI and pPATB2. The copy of the patatin m R N A was cloned by GC tailing in
the Pst I site of pBR322. These tails are not shown here. The 5' -terminal nucleotide of the insert of pPATB1 is indicated by the triangle.
Putative polyadenylation sites are underlined, and the glycosylation site is boxed.
262

nucleotides (data not shown) while it hybridized to genes which are expressed in tubers and to a very low
an mRNA having a length of ca. 1 500 nucleotides extent in roots [37]. Class II mRNAs contain a 22 bp
(Fig. 1). Obviously p207 did not contain a full- insert 9 nucleotides upstream of the ATG transla-
length copy o f the cloned mRNA and therefore the tion initiation codon, as opposed to Class I mRNAs.
cDNA library was rescreened using the insert of p207 In accordance with the in vivo glycosylated nature
as a probe. A number of cross-hybridizing cDNA of the patatin polypeptide the patatin precursor pro-
clones were detected, two of which, pPATB1 and tein contains a signal peptide of 23 amino acids. A
pPATB2, were analysed further. putative glycosylation site in the mature protein
The complete nucleotide sequence of the inserts could be assumed on the basis of the N-
of pPATB1 and pPATB2 was determined (Fig. 3). glycosylation site rule as formulated by Sharon and
The insert of pPATB2 showed a length of 1 377 bp Lis [47] and formed by Asn-Tyr-Ser in both pPATB1
(the GC tails and the poly(A) ÷ tail not included). and pPATB2.
The insert of pPATB1, lacking 50 nucleotides at the The 3' untranslated regions contained four puta-
5' end compared to pPATB2, showed 13 nucleotide tive polyadenylation signals (AATAA) two of which
changes and 9 amino acid substitutions resulting in were located near the polyadenylation site. It is
an overall homology of more than 97% with tempting to speculate that the pPATB2 mRNA tran-
pPATB2. In both recombinants one open reading script has used the more distal of these two poly-
frame could be detected. In pPATB2 this sequence adenylation sites (Fig. 3) for termination in compar-
could accommodate a potential polypeptide of 386 ison to the pPATB1 transcript.
amino acids having a molecular weight of 42610.
The deduced amino acid sequence of this polypep- p303
tide shows more than 95 % homology with the pata- The insert of cDNA p303 has been partly (200
tin amino acid sequence as determined by Mignery nucleotides) analysed by DNA sequence analysis
et al. [29]. The homology of pPATB2 with the pata- (data not shown). Comparison of the determined
tin amino acid sequence as determined by Rosahl et nucleotide sequence with the potato inhibitor II
al. [39] in another potato cultivar extends even to 385 mRNA nucleotide sequence as published recently by
out o f 386 amino acids. Only the carboxy terminal Sanchez-Serrano et aL [43] shows more than 90°70
(C-terminal) tyrosine residue as determined by homology. So, this cDNA clone contained a copy of
Rosahl et al. has changed to histidine in pPATB2 the cv. Bintje proteinase inhibitor II mRNA. We did
(TAT--CAT). These data infer that pPATB1 and not examine this clone further.
pPATB2 contain copies of two different cv. Bintje
patatin mRNAs. In total, we have isolated cDNA p322
clones containing copies of five different patatin As shown in Fig. 4, the insert of recombinant p322
mRNAs (data not shown) which indicates that at has a length of 470 bp (except GC tails and
least five different patatin genes are actively ex- poly(A) ÷ tails of 30 residues). The determined
pressed in the tetraploid potato cv. Bintje. nucleotide sequence allowed for one open reading
The patatin mRNA copy cloned in pPATB2 con- frame coding for a polypeptide o f 76 amino acids
tained an untranslated leader sequence of 23 nucleo- having a molecular weight of 8 614. The coding ca-
tides. Recently Pickaard et al. [37] reported a Class pacity of p322 as determined by hybrid-release
I patatin mRNA leader of 37 nucleotides in cv. Su- translation and SDS-polyacrylamide gel electropho-
perior as determined by S 1 nuclease protection ex- resis has been estimated at ca. M r 10000 (Fig. 2).
periments. The 23 nucleotide long cv. Bintje patatin This discrepancy might be explained by anomalous
leader showed complete homology to the cor- migration of the polypeptide on the gel system used,
responding part o f the cv. Superior patatin mRNA possibly due to differences in the extent of SDS bind-
leader. By analogy we concluded that the pPATB2 ing [18] or by assuming that the first methionine
mRNA is encoded by a Class I patatin gene which codon (nucleotides 7 - 9) does not function as a start
is only expressed in tubers in contrast to Class II codon. The sequences surrounding this triplet do
 263

i i0 20 30 40 50 60
CTA TCC ATG CGT TTC TTT GCT ACT TTC TTT CTT CTA GCT ATG CTT GTC GTG GCT ACT AAG
Leu Ser Met Arg Phe Phe Ala Thr Phe Phe Leu Leu Ala Met Leu Val Val Ala Thr Lys

70 80 90 I00 ii0 120

ATG GGA CCA ATG AGA ATT GCA GAG GCA AGA CAT TGC GAG TCG TTG AGC CAT CGT TTC AAG
Met Gly Pro Met Arg lle Ala Glu Ala~Arg His Cys Glu Ser Leu Ser His Arg Phe Lys
i
130 140 150 160 170 180
GGA CCA TGT ACG AGA GAT AGC AAT TGT GCT TCG GTC TGT GAG ACC GAA AGA TTT TCC GGT
Gly Pro Cys Thr Arg Asp Ser Asu Cys Ala Ser Val Cys Glu Thr Glu Arg Phe Ser Gly

190 200 210 220 230 240

GGC AAT TGC CAT GGA TTC CGT CGC CGT TGC TTT TGC ACT AAG CCA TGC TAAATGAGTATTAAAAAT
Gly Asu Cys His Gly Phe Arg Arg Arg Cys Phe Cys Thr Lys Pro Cys

250 260 270 280 290 300 310 320

TATGTGTAATAGAAGAAGTTTGAGAAAAAAATTATGTACTCTTGAATAAAGTACACTATGATTGTTCAAAGATATATGTGGT

330 340 350 360 370 380 390 400

GCTAGTTTTGTTTGTAAAACTAGTCGTGATCTTTGAATTTATATGCAATTATGGTGCACTAGACTTGTTAATTTCTTCATG

410 420 430 440 450 460 470 480

TGATGTATTTTTTGCTCTTTTGTTATGAAATATTATGGATAAAATTTGTCTTTTAGTCTTTA~

490 500
A ~
Fig. 4. Nucleotidesequenceof the insert of clone p322 (exceptthe GC tails). The putativepolyadenylationsignals are underlinedas well
as the putative methionineinitiation codon (nucleotide7-9). The direct invertedrepeat in the 3' untranslated region is overlined.The
putative proteolytic cleavagesite is indicated by an arrow.

not obey the Kozak rule [20]. Instead o f purines a ala-29 and arg-30. This site obeys the ( - 3 , - 1 ) rule
thymidine residue is found at the position - 3 while proposed by Von Heye [53] which suggests a small
a cytidine residue is located at position + 4. There- residue at - 1 (ala) whereas at - 3 the amino acid
fore, recombinant p322 might not contain a full- may not be aromatic, charged or large and polar
length copy of the corresponding mRNA. On the (ala). Moreover, the putative signal peptide contains
other hand comparison o f the amino terminal (N- a hydrophobic core of 14 amino acids which is
terminal) amino acid sequence to published protein flanked at its N-terminal site by an arginine at posi-
sequences showed homology to some of these. Start- tion 4 and at its C-terminal site by 10 amino acids,
ing at the methionine residue mentioned the p322 o f which two are basic (lys-20, arg-25) and one is
polypeptide showed 54% identity to the N-terminal acidic (glu-28). Cloning of the p322 gene and $1
part of the signal peptide of human lactalbumin nuclease analysis or primer extension experiments
precursor protein [14] (7 out of 13 N-terminal amino will ultimately show whether or not the p322 cDNA
acids), suggesting the presence o f a signal peptide in recombinant contains a full-length copy of the
the N-terminal domain o f p322. This was further mRNA.
supported by the presence of the amino acid se- At the C-terminus of the p322 polypeptide anoth-
quence phe-phe-leu-leu-ala (nucleotides 25 to 40) er remarkable homology is found (Fig. 5). Five of
also found in the signal peptide of the proteinase in- the six amino acids o f the C-terminal sequence phe-
hibitor I of potato ([4], W. J. Stiekema, unpublished cys-tyr-pro-cys appeared to be identical to the C-
results). Inspection of the hydrophobicity profile terminal part of the Bowman-Birk trypsin inhibitor
(data not shown) indeed showed a strongly o f soybean [31, 32]. Comparison of the mature part
hydrophobic region at the N-terminus of the p322 o f this proteinase inhibitor to the putative mature
polypeptide. The cleavage site is predicted between part of the p322 polypeptide showed an overall ho-
264

4, tained an insert of 786 nucleotides (besides A and

1 I0 20 ~30 ~ 40 GC tails). This insert completely overlapped p340,
I LSMRFFATFFLLAMLVVATKMGPMRIAEARHCESLSHRFK
0 0 0 ~ 0 • 0 O0
but additionally it contained extra nucleotides at the
II SDQSSSYDDDEYSKPCCDLCMCTRSMPPQCSCEDIRLN 5' end compared to p340. The nucleotide sequence
i i0 20 30 • of only strategic regions of the p34021 insert were de-
termined (Fig. 6) including the 5' and 3' terminal
50 60 70 regions: bp 1 - 303 (50 bp overlap with the 5' end of
I Gp CTRNSNCASVCETERFS GGNCHGFRRRCFCTKP C p340) and bp 6 0 9 - 819 (complete overlap with the 3'
• • • • • O • O OOQOOO 010000 end of p340). The overlapping regions of p340 and
II - S C - - H S D C K S - C M C T R S Q P G Q C R C L D T N D F C Y K P C K S R D D
40 50 60 70
p34021 showed complete sequence identity. A com-
Fig. 5. Comparison of the amino acid sequence of the p322 poly- bination of both sequences would lead to an open
peptide (I) and the mature amino acid sequence of the proteinase reading frame of 220 amino acids having M r of
inhibitor CII (Bowman-Birk) of soybean (II) as determined by 23 915 in agreement with the hybrid-selected transla-
protein sequencing. The arrow shows the putative proteolytic tion data. Unfortunately, p34021 also did not con-
cleavage site in I. Identical amino acids are depicted by e , whereas
tain a putative N-terminal methionine. Nevertheless
conservative amino acid substitutions by o. The putative active
site of the proteinase inhibitor II is Arg-38 as indicated by *. the hydrophobicity pattern (data not shown) predict-
ed a partial signal peptide of at least 20 amino acids.
The proteolytic processing site is located most likely
mology of 3007o. If conservative amino acid changes between ala-20 and arg-21. The ( - 3 , - 1 ) rule [53]
are disregarded this homology increases to more is obeyed in this region. This suggests that p34021
than 5007o. It is also striking that 8 o f the 14 cysteine contained a nearly full-length copy of the cloned
residues of the soybean proteinase inhibitor are mRNA. Comparison of the p34021 amino acid se-
found in similar positions in the p322 polypeptide, quence to published protein sequences showed 30°70
while the other six cysteine residues are part of an homology to the C-terminal 90 amino acids of the
N-terminal domain of the soybean mature inhibitor Kunitz trypsin inhibitor of winged bean (Fig. 7) [55].
not present in the p322 polypeptide. This suggests This homology would increase to more than 5007o if
similar types of disulphide bridges in both polypep- conservative amino acid changes were to be dis-
tides. regarded. It is remarkable that in a small region of
The 3' terminal untranslated region of the p322 the C-terminal part an even larger homology could
mRNA contains a number o f palindromes. The be detected: 7007o homology was shown in the region
most remarkable one consists of 25 nucleotides pres- comprising amino acids 199 to 207. Moreover three
ent immediately after the stop codon. Putative poly- of the four cysteine residues were located in identical
adenylation sites (GATAA and AATAT) are found positions while the fourth was found in correspond-
18 bp and 30 bp upstream of the poly(A)+ tail ing regions of both peptides.
respectively. Two overlapping AATAAA sequences are present
26 nucleotides upstream of the polyadenylation site
p340 in agreement with the consensus sequence AATAAA
The nucleotide sequence of the insert of cDNA clone found 2 5 - 3 0 nucleotides in front of the poly(A) ÷
p340 and part of the sequence of the insert of a cross- addition site in most eukaryotic genes analysed. It
hybridizing clone p34021 were determined (Fig. 6). is therefore likely that this sequence serves as a
The insert of p340 has a length of 560 nucleotides poly(A) ÷ additional signal. A second AATAAA se-
(excluding the A and GC tails). One open reading quence is present 83 nucleotides upstream of the
frame could be detected potentially encoding a poly- poly(A) ÷ tail. The significance of this sequence is
peptide of 145 amino acids having a M r of 16445. unknown.
No N-terminal methionine was found indicating that
p340 did not contain a full-length copy of the cloned
mRNA. The cross-hybridizing clone p34021 con-
 265

i I0 20 30 40 50 60
p34021TCG ATT AAT ATT TTG AGT TTC CTC TTG CTT TCA AGT ACC CTC TCT TTG GTT GCC TTT GCT
P34021Ser lie Asn lle Leu Ser Phe Leu Leu Leu Ser Ser Thr Leu Ser Leu Val Ala Phe Ala~

70 80 90 i00 ii0 120

p34021 CGA TCT TTC ACT TCT GAG AAT CCA ATT GTC CTC CCC ACA ACT TGT CAT GAT GAT GAT AAT
p 3 4 0 2 1 A r g Ser Phe The Ser Glu Asn Pro lle Val Leu Pro Thr Thr Cys His Asp Asp Asp Asn

130 140 150 160 170 180

p34021CTT GTA CTC CCT GAA GTT TAT GAC CAA GAT GGC AAT CCG CTG AGG ATT GTG AGA GGT ACA
p34021Leu Val Leu Pro Glu Val Tyr Asp Gin Asp Gly Asn Pro Leu Arg lie Val Arg Gly Thr

190 200 210 220 230 240

p340 ATA TTG GAA ACC TTC
p 3 4 0 2 1 T T A TTA ACA ATC CTC TCC TCG GGG CCG GAG CCG TAT ACT TGT ACA
p340 lle Leu Glu Thr Phe
p 3 4 0 2 1 L e u Leu Thr lle Leu Ser Ser Gly Pro Glu Pro Tyr Thr Cys Thr . . . . .

250 260 270 280 290 300

p340 AAT GCC CAA ATG CAC GTG TTG CAG CAC ATG TCG ATT CCC CAA TTT TTG GGA GAA GGC ACG
p34021
p340 Asu Ala Gln Met His Val Leu Glu Gis Met Ser lle Pro Glu Phe Leu Gly Glu Gly Thr
p34021 . . . . . . . . . . . .

310 320 330 340 35O 360

p340 CCC GTC GTG TTC GTT CGT AAG TCG GAG TCG GAT TAT GGT GAT GTG GTG CGT GTA ATG ACT
p340 Pro Val Val Phe Val Arg Lys Ser Glu Ser Asp Tyr Gly Asp Val Val Arg Van Met Thr

370 380 390 400 410 420

p340 GTT GTT TAT ATC AAG TTC TTT GTT AAA ACA ACA AAG TTG TGT GTT GAC CAA ACT GTT TGG
p340 Val Val Tyr lie Lys Phe Phe Val Lys Thr Thr Lys Leu Cys Val Asp Gln Thr Val Trp

~30 440 450 460 470 480

p340 AAA GTT AAT GAT GAA CAG TTG GTG GTA ACT GGT GGT AAG GTA GGA AAT GAA AAC GAC ATC
p340 Lys Val Asn Asp Glu Gin Leu Val Val Thr Gly Gly Lys Val Gly Asn Glu Asn Asp lle

490 500 510 520 530 540

p340 TTC AAG ATT ATG AAA ACT GAC TTG GTG ACA CCA AGA GGT TCC AAA TAT GTA TAC AAG TTA
p340 Phe Lys lle Met Lys Thr Asp Leu Val Thr Pro Arg Gly Ser Lys Tyr Val Tyr Lys Leu

550 560 570 580 590 600

p340 CTG CAT TGT CCC TCT CAT CTT GGG TGC AAA AAT ATC GGC GGC AAC TTT AAA AAT GGA TAT
p340 Leu His Cys Pro Ser His Leu Gly Cys Lys Asn lle Gly Gly Asn Phe Lys Asn Gly Tyr

610 620 630 640 650 660

p340 CCT CGT CTG GTG ACT GTC GAT GAC GAT AAG GAC TTT ATT CCA TTT GTG TTC ATC AAG GCG
p34021
p340 Pro Arg Leu Val Thr Val Asp Asp Asp Lys Asp Phe lle Pro Phe Val Phe lle Lys Ala
p34021

670 680 690 700 710 720 730 740

p340 TAGAATGCTAATTAGCTGGCTAGCTTGTAGCTTTCTAAATAAAGTGCATATATCCTTCTATCGCTCCATGTAAATTAATG
p34021

750 760 770 780 790 800 810 820

p340 TATGCTTATCAATAAATAAACAAGCTAGCAATTAGCCTATTACCTT~
P34021

830
p340 AAAAAAAAAAAAAAAA
Fig. 6. Nucleotide sequence of the insert of clone p340 (except GC tails) and part of the nucleotide sequence of p34021 (nucleotides 1- 303
and 609-786). Nucleotides and amino acids identical in p34021 and p340 are indicated by -. The putative proteolytic cleavage site is
indicated by the arrow, and putative polyadenylation signals are underlined.
266

130 140 150 160 Estimation o f the number o f genes encoding the
I FVKTTKLCVDQTVWKVNDEQLVVTGGKVGN-ENDI cloned tuber m R N A s
@0 • I0 0 O0 • 00@0 0 @<DO 0
II FANPPK-CAPSPWWIVVEDQPQQPSVKLSELKSTK
30 • 90 I00 ii0 Figure 8 shows the result of a Southern blot hybridi-
zation experiment performed with the four different
cDNA clones as probes. Potato cultivar Bintje or
170 180 190
I FKIM-KTDLVTPRGSKYVYKLLHCPSHLGCKNIGG-N monoploid potato AM 79.7322 DNA was cut by re-
• 0 • 0 O0 0 • 000 000 00~ O@ striction enzymes Eco RI, Hind III and Barn HI
II FDYLFKFEKVTSKFSS--YKLKYCAKRDTCKDIGIYR and hybridized against the different clones. With all
120 130 A AI40
enzymes and all probes a number of hybridizing
DNA fragments appeared. This might be partly ex-
200 210 220 plained by restriction enzyme polymorphism since
I FKNGYPRLVTVDDDKDF IPFVF IKA part of the experiment was performed with DNA of
OO OOOO O
II DQKGYARLVVTDENPLVV I FKKVE S S the tetraploid cv. Bintje. Still it can be concluded that
150 160 170 all cloned tuber mRNAs are encoded by small mul-
Fig. 7. C o m p a r i s o n of the C-terminal region of the p34021 (I) tigene families. The patatin mRNAs were shown to
a n d the Kunitz trypsine inhibitor of winged bean (II) polypeptide be encoded by the largest family of ca. 10-15 mem-
as determined by protein sequencing. Identical a m i n o acids are bers per haploid genome, in agreement with data
depicted by e , a n d conservative substitutions by o. Conserved presented by Bevan et al. [22]. The p303 proteinase
cysteine residues are indicated by A.

Fig. 8. Autoradiographs of Southern blots are shown containing nuclear D N A of potato cv. Bintje (A, B) or monohaploid A M 79.7322
(A) digested by Barn HI, Eco RI or H i n d III, separated on a 0.7°/0 agarose gel and blotted onto nitrocellulose filters. These filters were
hybridized against 32p-labelled pPATB2 (A) or p303, p322 and p340 (B). Molecular weight markers of k D N A digested with H i n d III
are indicated in kilobases.
 267

inhibitor II gene family most likely consists of only hydrophobic N-terminal region strongly resembling
a few members. a signal peptide as expected for a putative proteinase
inhibitor polypeptide. This domain possesses all fea-
tures expected to be present in such a region.
Discussion A similar degree o f homology can be detected be-
tween p340, p34021 and the Kunitz trypsin inhibitor
Tuberization in potato is a complex developmental of winged bean [55]. Again three of the four cysteine
process involving morphological changes and also residues are found in the same position while the
the expression o f a specific set of genes leading to fourth residue is located in a similar region in both
the synthesis of tuber proteins. A precise under- polypeptides, suggesting a similar secondary struc-
standing of this process is a prerequisite to any at- ture. Also in the C-terminal domain a region of ex-
tempt to manipulate genetically the properties of the tensive homology can be identified. Moreover, in
tuber of this important crop. analogy to the Kunitz trypsin inhibitor the p34021
To increase our knowledge of this process at the polypeptide contains a very hydrophobic N-terminal
molecular level we decided to analyse the mechanism region indicating the presence of a signal peptide.
involved in the developmental regulation of genes Overall, the mature domain shows 50°70 amino acid
specifically expressed in the tuber tissue. homology (conservative amino acid changes dis-
As a first step we report the molecular cloning of regarded) to the C-terminal part of winged bean pro-
four mRNAs encoding tuber proteins. All four teinase inhibitor.
mRNAs are mainly present in tuber tissue as shown These homologies suggest that p34021 and p322
by Northern blot hybridization. However, one of contain copies of mRNAs encoding proteinase
them can also be detected at a ten-fold lower level inhibitor-like polypeptides. The amino terminal
in stem tissue. Two o f the cloned tuber mRNAs, hydrophobic signal sequences present in both poly-
pPATB2 and p303, encode patatin and proteinase in- peptides are in agreement with the expected cellular
hibitor II proteins respectively. These two proteins location of proteinase inhibitors in vacuoles [54].
have been described and analysed in detail [29, 39, The signal sequence-mediated translocation across
40, 37, 36, 2, 41, 38, 43, 19, 51] which will not be the membrane of the endoplasmic reticulum is the
repeated here. Nevertheless it is remarkable that the first step in sorting o f this kind of proteins to cell or-
nucleotide sequence o f pPATB2 is almost complete- ganelles. The homology found in the positively
ly homologous to the patatin mRNA analysed by charged carboxy terminal domain can also have a
Rosahl et al. [39]. This m R N A was isolated from a function in the sorting event. Recently it has been
diploid potato. This clearly shows that some o f the shown for the M13 procoat protein [30] and for
expressed patatin genes are extremely conserved. honeybee prepromellitin [21] that properties of the
Two other tuber mRNAs were cloned and ana- whole mature domain determines this process. The
lysed. They encode polypeptides which have not presence and distribution of charged amino acids
been described for potato before. One of them, p322, within the mature domain of these precursors can
shows homology to a Bowman-Birk type proteinase play especially distinct roles.
inhibitor present in soybean [31, 32]. Eight cysteine As yet nothing is known for certain about the
residues in both polypeptides are located in the same physiological function of the proteinase inhibitor II,
position, suggesting a similar secondary structure in the patatin and the 2 proteinase inhibitor-like poly-
both polypeptides. Another remarkable homology peptides described here which constitute about 50070
has been found at the C-terminal region of these of the soluble tuber protein [4, 35]. All these four
polypeptides. Overall, the mature domain shows polypeptides are present at very high concentrations
46°70 amino acid homology (conservative amino acid in developing (data not shown) as well as mature
changes disregarded) to the Bowman-Birk pro- tubers. Certainly they will have some function as
teinase inhibitor including the putative reactive site, storage proteins. But it is tempting to speculate that
arg-49. Also the p322 polypeptide contains a the proteinase inhibitors are also involved in the
268

defence mechanism against invading predators such Characterization of cDNA for nodulin-75 of soybean: a gene
as larvae or fungi. The systemic inducibility of the product involved in early stages of root nodule development.
Proc Natl Acad Sci USA 84:4459-4499 (1987).
proteinase inhibitor II genes [43] upon wounding
10. Garcia-Torres L, Gomez-Campo: In vitro tuberization of
potato plants supports this idea. On the other hand, potato sprouts as effected by Ethel and gibberellic acid. Pota-
the patatin mRNA levels decrease dramatically after to Res 16:73-79 (1973).
wounding [24]. Further analysis of the expression of 11. Goddard JM, Caput D, Williams SR, Martin DW" Cloning
the genes encoding the p322 and p34021 polypep- of human purinenucleotide phosphorylase cDNA sequences
by complementation in Escherichia coli. Proc Natl Acad Sci
tides upon wounding have to be performed to fur-
USA 80:4281-4285 (1983).
ther support this hypothesis. 12. Govers F, Nap J-P, Moerman M, Franssen H J, Van Kammen
Cloning of the genes encoding these four tuber A, Bisseling q2. cDNA cloning and developmental expression
proteins and comparative analysis of their regulato- of pea nodulin genes. Plant Mol Biol 8:425-435 (1987).
ry domains may reveal homologous sequences. Also 13. Green TR, Ryan CA: Wound-inducible proteinase inhibitor
in plant leaves: a possible defense mechanism against insects.
putative regulatory proteins and the regions they
Science 175:776-777 (1972).
bind can be identified and compared. This kind of 14. Hall L, Craig RK, Edbrooke MR, Campbell PN: Compari-
study will possibly shed light on the complex de- son of the nucleotide sequence of cloned human and guinea-
velopmental process of tuber formation. pig pre-a-lactalbumin cDNA with that of chick pre-lysozyme
cDNA suggests evolution from a common ancestral gene.
Nucl Acids Res 10:3503-3515 (1982).
15. Hammes PS, Nel PC: Control mechanisms in the tuberiza-
Acknowledgements tion process. Potato Res 18" 262-272 (1975).
16. Hammond RW, Foard DE, Larkins BA: Molecular cloning
We thank Dr L. Visser and Dr L. van Vloten-Doting and analysis of a gene coding for the Bowman-Birk protease
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17. Hannapel JD, Creighton-Miller J, Park WD: Regulation of
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