Monoclonal antibodies used to characterize cDNA clones expressing specific wheat endosperm proteins

Journal of Cereal Science 9 (1989) 97-111

Monoclonal Antibodies used to Characterize cDNA Clones Expressing Specific Wheat Endosperm Proteins G. R. DONOVAN*t, J. H. SKERRITT* and S. L. CASTLEt

* CSIRO Wheat Research Unit, Division of Plant Industry, P.O. Box 7, North Ryde, New South Wales, Australia and t Canberra City, Australian Capital Territory, 2601, Australia Received 30 March 1988 A wheat cDNA library, prepared from grain endosperm poly A+-mRNA and cloned into the Escherichia coli expression vector lambda gtll, has been screened with nine monoclonal antibodies having specificities for different wheat endosperm proteins. At least one positive cDNA clone was isolated, and purified, from those selected with each antibody. Each purified cDNA clone was induced to express fusion proteins, and the nitrocellulose membranes to which the proteins were transferred were incubated with each of the other antibodies at two or more concentrations to investigate the extent of homologies between expressed fusion proteins. The specificities of the antibodies were determined using immunoQ)otting under the same conditions used for binding to the fusion proteins from the expressed cDNA clones. Denatured DNA from each antibody-selected cDNA clone was also characterized by hybridization to ct-/~-genomic gliadin and genomic high molecular weight glutenin subunit DNA probes. Northern hybridizations using the isolated cDNAs as probes for endosperm mRNA were also used to assist clone identification. Some monoclonal antibodies with overlapping specificities (identified from blotting and ELISA experiments) crossreacted with several expressed cDNA clones. However, in other instances, positive identifications were made of the proteins coded for by single families of the cDNA clones. Monoclonal antibody screening of a wheat cDNA library is useful in identifying families of cDNA clones corresponding to different wheat polypeptides at the primary screening stage, obviating the need in many instances for the application of more tedious methods of clone identification.

Introduction The gliadin and glutenin fractions of wheat endosperm comprise several dozen polypeptides, many of which hayehigh degrees of homology with each other 1 . 2. Their, unusualsolubilityproperties&n
t

Present address: Kolling Institute, Royal North Shore Hospital, St Leonards, New South Wales,

Australia.

Abbreviations used: SOS = sodium dodecyl sulphate; SOS-PAGE = sodium dodecyl sulphate polyacrylamide electrophoresis; cONA = complementary deoxyribonucleic acid; poly A"-mRNA = polyadenylated messenger ribonucleic acid; EDTA = ethylene-diamine tetra acetic acid, di-sodium salt; oligo(dT) = oligo-deoxythymidylic acid; HMW gtn = high molecular weight glutenin subunit; LMW gtn = low molecular weight glutenin subunit; ELISA = enzyme-linked immunosorbant assay. 0733-5210/89/020111

+ 15 $03.00/0

© 1989 Academic Press Limited CER 9

98

G. R. DONOVAN ET AL.

potential means of separating and characterizing both cDNA and genomic clones encoding these proteins, and in many cases both the primary sequences of the proteins and the putative controlling regions of the genes have been deduced 3 • The extensive homologies of the proteins, however, has also been evidenced in high frequency of crosshybridization of cDNAs with the result that the primary screening and characterization of the clones can be laborious and tedious. Polyclonal antibodies have been used previously to detect expressed cDNA clones in Escherichia coli for maize prolamins4.5, maize glutelin-2 6 and a wheat HMW-glutenin subunit'. The usefulness of polyclonal antibodies for the identification and characterization of wheat gluten proteins has been hampered by the structural homologies that exist between these proteins and further complicated by high endogenous titres of gluten antibodies present in the serum of unimmunized animals B• The generation of a library of monoclonal antibodies to wheat endosperm proteins 9 , 10, provides an alternative means for primary screening of a wheat endosperm cDNA library in a suitable expression vector. Lambda gtll has its site of insertion of foreign DNA within the structural gene for ~-galactosidase, and, thus, these foreign DNAs can be expressed as fusion proteins of ~-galactosidasell. In this study the screening of a wheat eDNA library in lambda gtll using monoclonal antibodies was performed and monoclonal antibodies used in conjunction with RNA and DNA hybridization techniques to characterize selected eDNA clones. Experimental

Determination of monoclonal antibody specificities by electrophoresis and immunoblotting SOS-PAGE immunoblotting was performed as described earlier iO , except that gels 1'5 mm thick were used, and total wheat flour proteins were extracted (4 h, 55°C) with 2 % (w Iv) SDS-2 % (v Iv) 2-mercaptoethanol in 62 mM Tris-HCI, pH 7'5 (8 ml/g). The gliadin antigenic specificity of the antibodies was examined in greater detail by electrophoresis under acidic-buffer (4 mM Na lactate, pH 3'1) conditions and immunoblotting 1o • Under these electrophoresis conditions, gliadin fractionation on the basis of both molecular size and charge occurs, producing zones termed cr-, ~-, 'Y-, fast-w- and slow-ro-gliadin 12 . 13 . Double one-dimensional gel electrophoresis was carried out using the procedure described by Gupta and Shepherd 14 .

Preparation oj wheat endosperm eDNA libraries in lambda gilD and gtll (a) Preparation of poly A+-mRNA. Polyribosomal RNA was prepared from membrane-bound polyribosomes prepared from immature kernels (20 days after flowering) of bread wheat (Triticum aestivum; cv. Timgalen)15. The RNA was dissolved in oligo-(dT)-cellulose loading buffer (20 mM Tris-HCI pH 7'6,0'5 MNaCl, I mM EDTA, 0·1 % (w/v) SDS) and chromatographed through a 2 ml bed volume column of Sigma-cell Type 50 cellulose to remove carbohydrate which would otherwise obstruct the flow through oligo-(dT)-cellulose 16 • Oligo-(dT)-cellulose chromatography was used to recover poly A+-mRNN7. The poly A+-mRNA was translated in a wheat-germ cellfree translation system 18 and the reaction mixtures extracted with 55 % (v Iv) propan-2-ol and precipitated with TeA in the presence of (unlabelled) carrier wheat gliadin u . The resuspended radioactively labelled pellet was subjected to SDS-PAGE and the radioactive bands identified by fluorography. With the exception of the HMW-glutenin polypeptides, which were only poorly translated, the poly A+-mRNA translated the full array of wheat endosperm gliadins and glutenin subunits identifiable by comparison with in vivo radioactively'labelled wheat prolamins

99

CHARACTERIZATION OF WHEAT PROTEIN eDNA CLONES

using one-dimensional SDS-PAGE and f1uorography. The poor translation of the HMW-glutenin polypeptides was expected, as the wheat-germ system inefficiently translates polypetides greater than M r 70000 19 • (b) Preparation oj double-stranded eDNA and cloning in lambda gtlO and gill. Procedures for the preparation of double-stranded cDNA from poly N -mRNA and their ligation into lambda gtlO were as described by Huynh and co-workers ll • Approximately 10 7 clones in the primary library were derived from 2 Jlg of poly A + -mRNA. Excising the cloned cDNA inserts by digestion with the restriction endonuclease EcoRI, end-labelling them with 32P_dATP followed by agarose gel electrophoresis and autoradiography indicated that the sizes of the inserts ranged from approximately 0·4 kilo bases (kb) to over 3 kb, with the mean clone size being around 0·7 kb. cDNAs from the lambda gtlO library were separated from the parent phage after EeoRl digestion of DNA prepared from the amplified cDNA library. The digest was applied to a 10-40 % (v/v) glycerol gradient in 10 mM Tris-Hel, pH 7'4, I mM EDTA, 0·3 MNa acetate and centrifuged in a Beckman SW41 rotor at 35000 for 16 h 20 . The cDNAs wcre recovered after fractionation by ethanol precipitation followed by electrophoresis in 0'7 % (wIv) low-melting-temperature agarose (Bio-Rad, Richmond, CA, USA) in TBE (0,89 M Tris base, 0'89 M boric acid, 0·002 M EDTA, pH 7'8) buffer. cDNAs having sizes greater than approximately 1 kb were cut from the gel and recovered by chromatography through . NACS' columns (Bethesda Research Laboratories, Gaithersburg, MD, U.S.A.). The recovered cDNAs werc ligated into lambda gtll using a 'Protoclone' lambda gtll system (Promega Biotcc, Madison, WI, U.S.A.). The cDNAs ligated into lambda gtll had a mean size of 1 kb. There were many clones with inserts below this size, however, presumably as a result of annealing or the £CoR I 'sticky' cnds of small rragments during the preparative agarose electrophoresis step or by trailing of smaller fragments in the gel. Nonrecombinant phage representcd about 10% or the library.

Screening of the cDNA library in lambda gt 11 using monoclonal antibodies Murine monoclonal antibodies to wheat endosperm proteins were prepared and characterized using methods described earlieI'll). Nine monoclonal antibodics werc used: six IgG I isotypes (218/17,221/23,222/5,227/22,228/20 and 304/13) and three IgM antibodies (236/9, 237/24 and 246/21). In most experiments the supernatants from in vitro growth of thc hybridoma celliinc were used. In some instances, where thc supernatants had low antibody titres, purilied immunoglobulins derived from ascites fluid were used. In the latter case, the antibody solutions were initially preabsorbed by incubation with nitrocellulose membrane disks that had been soaked in E. coli extract (Promega) followed by blocking with 20 % (vIv) foetal calf serum in Tris-buffered saline, 0,05 % (vIv) Tween 20, to remove anti-E. eoli antibodies found in mouse peritoneal cavity fluids. Antibody concentrations were expressed as micrograms per ml or as dilutions of the supernatant. Dilutions were made with 20 % foetal calf serum in Tris-buffercd saline (l0 mM Tris-HC1, pH 8'0, 0·15 MNaCl). Immunoscreening was carried out using a 'ProtoBlot Immunoscreening System' (Promega). Positive clones were purified by re-plating on 55 mm diameter plates at approximately 100 plaque forming units (pfu) per plate until all plaques produced a positive signal. These clones were amplified by single-plate confluent lysis using 150 mm diameter plates and phage prepared by polyethylene-glycol precipitation and CsCI block gradient ultracentrifugation 11. DNA was prepared from these CsCI phage' stocks' using the formamide method l l• cDNA inserts were excised by EeoRl digestion and sized by electrophoresis in 1 % agarose gels. The eDNA clones are identified using the prefix' L " followed by the code name of the antibody used to select them, and a numeral suffix to distinguish each of the several clones selected with that antibody. For example L246/21-6 is a cDNA clone selected with 246/21 and carrying the number '6' to distinguish it from the other L246/21 clones under study (e.g. L246/21-12 and L246j21-26). To measure the level of expression of B-galactosidase fusion proteins, one filter from each expressed clone was reactcd with mouse anti-~-galactosidase (Promega) at 1: 5000 dilution, 4-2

100

G. R. DONOVAN ET AL.

followed by the anti-mouse IgG/alkaline phosphatase conjugate, using wash and colour development methods described by Promega (Proto Blot Immunoscreening System, Technical Manual).

Specificity of selection of cDNA clones using monoclonal antibodies cDNA clones in lambda gtll were plated on to 55 mm diameter plates at approximately 100 pfulplate, and the nitrocellulose disks were reacted with antibodies using separate disks for all nine monoclonal antibodies (including the selecting antibody). A range of antibody dilutions was used. In this part of the study supernatant fractions of the monoclonal antibodies were used exclusively to prevent the possibility of misleading results resulting from the possible presence of antibodies to E. coli proteins in purified preparations derived from ascites fluid. Plates were scored over a range of 0-5 according to colour intensities rather than the numbers of plaques identified.

Analysis of poly A+-mRNA by northern hybridization Poly A+-mRNA from 20-day-old wheat kernels was electrophoresed on formaldehyde gels and transferred to nitrocellulose 21 . cDNA inserts from each clone were radioactively labelled by nicktranslation 17 and hybridized to the northern transfers in 50 % (v Iv) formamide at 42 °C17. Washes were with 0·1 x SSC buffer 17 at 58°C or where specified at 68 0c.

Hybridization of eDNA clones to gliadin and glutenin DNA probes Amplified lambda gtll bacteriophage eDNA stocks (> 10 10 pfu/ml) were titrated. To aliquots (0'1 ml) of the stocks was added 10 M NaOH (2 Ill) and the mixture allowed to stand at room temperature for 15 min before transfer to an ice bath. To each tube was added a mixture (7 f.Ll) comprising 10 M HCl (2111) and I M Tris-HCl buffer pH 8·0 (5 Ill). The mixture was made to 6 x SSCjust prior to application to the nylon membrane by the addition of20 x SSC (47 111)17. The nylon membrane (Hybond N, Amersham) was placed upon a 3MM Whatman filter dampened with 6 x SSC above two dry sheets of Whatman 3MM paper and the denatured DNA solution (15 f.L1) prepared as above was applied in 3 x 5 !-II applications with a micropipettor. The DNA was covalently fixed to the nylon membrane using a 2 min exposure to the ultraviolet emission from a TM 360 Transilluminator (302 nm, UV Products, Ca, USA). The filters were pre-hybridized using the same conditions for northern transfers. The genomic C(-/~-gliadin clone pW8233 22 and the 1-8 kb HindIII fragment of the HMW-glutenin genomic clone of the IBy sub-unit of cv. Cheyenne 23 were nick-translated 17 and hybridized to the filters. Washes were in 2 x SSC at 65°C.

Results and Discussion

Specificities of monoclonal antibodies for mature flour proteins

lmmunoblots produced after both acidic buffer electrophoresis and SOS-PAGE, and showing the specificities of the monoclonal antibodies used in this study, are presented in Fig. 1(A) and (B) and summarized in Table 1. Two main specificity groups of antibodies were used in this studylo. Firstly, several antibodies were reactive with clusters of high-mobility gliadins (221/23, 222/5, 227/22 and 228/20) and a second group of antibodies bound rJ.- and w-gliadins and HMW-glutenin subunits (218/17, 23619, 237/24, 246/21 and 304/13). However, the fine specificities of each antibody differed. For example, while 304/13 bound strongly to a small group of high-mobility co-gliadins

CHARACTERIZATION OF WHEAT PROTEIN eDNA CLONES

101

and to HMW-glutenin subunits, 246/21 bound well to most ro-gliadins, some y-gliadins and HMW glutenin subunits. While the acidic-buffer gel blots indicate that antibodies 304/13 and 246/21 bound ro-; and y- and ro-gliadins respectively, SDS-PAGE blots showed little binding of these antibodies to gliadins but strong binding to HMWglutenin subunits. This result is attributed to SDS 'denaturation' of gliadin. These antibodies bound more strongly to glutenin subunits; gliadin interaction on SDS-PAGE blots could be demonstrated at higher antibody concentrations. It should be noted that while binding to particular gliadins and HMW-glutenin subunits could be clearly established using the electrophoretic techniques described in this paper, additional specificities may exist. For example, on immunoblots, some of these antibodies (e.g. 236/9 and 246/21) bound to certain starch granule-associated proteins 24 at similar dilutions to which they bind to gluten proteins. However, the other antibodies investigated in this study bound to starch-granule proteins only at considerably higher antibody concentrations (Skerritt, Greenwell and Robson, unpublished). By the use of double-one-dimensional electrophoresis 14 and immunoblotting methods, the LMW-glutenin subunits specificities of the antibodies were determined (Skerritt and Robson, unpublished). Several of the antibodies (218/17, 222/5, 228/20 and 246/21) bound well to LMW-glutenin subunits, while 227/22 and 236/9 bound less strongly at the antibody concentrations used in this study. In contrast, the antibodies 221/23 and 304/13 exhibited binding to LMW-glutenin subunits only at somewhat higher concentrations, and 228/20 has yet to be tested. Thus, there is a possibility that a DNA sequence identified as coding for epitopes binding to these antibodies may be a cDNA of a structural gene for one of these less-well-characterized but, in the case of LMW-glutenin subunits, rather abundant endosperm proteins. Monoclonal antibody binding to expressed cDNA clone fusion proteins - comparison with DNA hybridization analysis

DNA hybridization analysis was carried out under only moderately stringent hybridization/washing conditions. The genomic Ci-/~-gliadin probe pW8233 hybridized to the clones L22l/23, L222/5 and L228/20, indicating that these clones had sequences related to Ci-/~-gliadins. The genomic HMW-glutenin subunit probe corresponding to the HindUI fragment of the lBy gene of cv. Cheyenne hybridized to the clones L246/2l-6, L246/2l-l2, L246/2l-26, L236/9-l6, L236/9-l7, L304/13-7, L304/l3-9, L237/24-l5 and L237/24-22. This HMW-glutenin subunit genomic clone restriction fragment is reported to hybridize to all the HMW-glutenin subunits (Shewry, personal communication) and therefore these cDNA clones must all represent HMW-glutenin subunits or sequences closely related to these polypeptides. The colour intensities ofthe expressed cDNA clone plaques bound to the nitrocellulose disks (after reaction with mouse anti-~-galactosidase followed by anti-mouse IgG/alkaline phosphatase conjugate) indicated that the levels of expression of ~­ galactosidase fusion proteins was sufficiently similar on different disks to ensure that there were only small differences in antigen concentration in different expressed cDNA clones. Table II shows the results of cross-affinity analysis of the monoclonal antibodies used

G. R. DONOVAN ET AL.

02

... D

£IIPO£

0

D

lUg

~

0

... D

t>UL£~

0

D

6/9£i?:

0

OU9i?:i?:

... .0

ZZILZZ

0

... .0

~nai?:

a

... .0

£UIZZ

0

... .0

0

ct

is :>

§~

'0

~'O

X

3

I

I

C)

c

~

'0

!i c

:; '0 I

~~~ o oJ

t.Ii <

L1/91i?:

8

n

:r

»

~

;p n

w

~

tTl ~

y

N

IJ

o z

~

o'T1

Q

~

Gel

a

b

,..

::::: III N

c

a

b

,.., N .... N N

a

b

on

....

~

N

c

0

b N N

....

~

N

c

b

0

....2III

N N

..,....

01

~

N

b

0

,......,..,~

N

a

b

N ....

~ N

a

b

.., .,.::::: ..,

c

0

FIGURE 1. The binding of monoclonal antibodies to blots of mature wheat flour proteins after (A) SDS-PAGE and (B) acidicbuffer polyacrylamide gradient gel electrophoresis. Antibody supernatant concentrations used were: (a) 1/50, (b) 1/500 and (c) 1/5000 for antibodies 218/17, 222/5 and 221/23 (SDS blots only); (a) 1/50 and (b) 1/500 for antibodies 236/9, 221/23 and 246/21; (a) 1/20, (b) 1/200 and (c) 1/2000 for antibody 304/13 and 237/24 (SDS blots only); (a) 1/20 and (b) 1/200 for antibody 237/24; (a) 1/5, (b) 1/50 and (c), 1/500 for antibody 227/22; 1/25 for antibody 228/20.. Gel' refers to a lane of the gel which was stained with Coomassie Blue.

::r: tTl >~ "t! ~

o

~

ttl

Z

n

o

Z

;p

n

r-'

oZ

tIl

tn

o w

o

.j:>.

TABLE 1. Specificities of monoclonal antibodies with blots from acidic-buffer polyacrylamide gradient-gel electrophoresis (GG blots), onedimensional SDS-PAGE blots (Fig. 1A and B), double one-dimensional SDS-PAGE blots and with starch granule proteins (surface and intrinsic) GG blots

Clone

Subclone

Isotype

At highest concentration used

218/17

7E8

IgGI

/3,

221/23 222/5

SOl1CS 9F9

IgGl IgGI

ct/3

227/22 228/20 236/9

12Hl2 13C6

IgGI IgGl IgM

ct,

y, 0)

Y,O)

237/24

4Hll

IgM

Y, 0)

y,O)

246/21

18F2

IgM

y,O)

y, 0)

304/13

B2

IgGl

Fast o}, slight 0)

Fast

y, 0)

cx, (3, y, co

/3

/3

Antibody concentrations were as shown in Fig. 1.

At higher dilution

SDS blots (one-dimensional)

y/Fast 0)

y/Fast 0)+ HMW glutenin a-gliadin Gliadin, mainly ct, /3, Y /3-Gliadin ct- and /3-gliadin Y,O)+HMW glutenin y,O)+HMW glutenin y,O}+HMW glutenin Fast 0), weak HMW glutenin

ct ct,

/3 ct,

/3,

Y

/3

0}

SDS blots (LMW glutenins double one-dimensional)

Starch granule proteins

++

+, Surface

!'l

+

+, Surface +, Surface and intrinsic +, Extrinsic

o z o ~

++

:..

++

++

+

Intrinsic

+, Intrinsic

+

+ +, Intrinsic +, Intrinsic

P

o

Z

...,t>J ~

CHARACTERIZATION OF WHEAT PROTEIN eDNA CLONES

105

to select the cDNA clones together with the antibody concentrations used in the primary screening and plaque purification steps. Only the ~-gliadin specific antibody, 227/22 and the cr-/~-gliadin specific antibodies, 221/23 and 228/20, showed no cross-reaction with expressed cDNA clones other than those identified with their respective selecting antibody. This observation is in only partial agreement with the DNA-hybridization analysis, which indicates that they are all structurally similar. The clone L227/22 selected with a ~-gliadin-specific monoclonal did not hybridize to pW8233, the cr-/Pgliadin genomic clone. This apparent conflict can be explained since the proteins to which antibody 227/22 bind seem not to be typical p-gliadins; this antibody also recognizes an epitope in maize cr-zeins (known to have little sequence to gliadins). Furthermore, DNA sequence analysis of this clone has indicated a lack of homology with the gliadins (unpublished observation). The clone L228/20, however, is clearly identified as a ~-gliadin clone as it both hybridizes to pW8233 and is identified by the ~-gliadin-specific antibody (228/20) binding to the expressed cDNA clone. The clone L221/23 is almost certainly an cr-gliadin on the basis of its hybridization to pW8233, its binding to the cx-/p-specific antibody 221/23 and its failure to bind the two ~-gliadin-specific antibodies 227/22 and 228/20. The cx-/~-Ir-gliadin plus LMWglutenin subunit specific antibody 222/5 and the HMW-glutenin subunit plus ro-IYgliadin specific antibody 218/17 showed restricted cross-reaction with other expressed cDNA clones. In the case of 225/5, it cross-reacted moderately at both the higher and lower concentrations of antibody with the L221/23 cDNA clone. This result is in keeping with antibody reactions with immunoblots, as both antibodies bind ex-gliadin. The cDNA clone L222/5 also hybridized to pW8233, indicating that its sequence was related to ex-/~-gliadin. Antibody 218/17, which had specificity for HMW-glutenin subunits and y-/ro-gliadins, bound both the expressed cDNA clones L218/17-4 and L222/5. The clone L218/17-4 is certainly not an C(-/~-gliadin and probably not a ygliadin, since at 1700 bases in length it is too large. These characteristics suggest that it encodes an ro-gliadin or an HMW-glutenin subunit. Its failure to hybridize to the HMWglutenin subunit genomic restriction fragment argues against its being an HMWglutenin. It is therefore possibly an ro-gliadin. Within the group of antibodies that bound various y-/ro-gliadins and HMW-glutenin subunits (236/9, 237/24,218/17, 246/21 and 304/13), the observed cross-reactions with the expressed cDNA clones were in most instances in keeping with the specificities noted from the immunoblots. All the clones in this grouping hybridized to the genomic HMWglutenin subunit probe. As the sequence of this probe is thought to be related to neither y-gliadins nor ro-gliadins, it would appear that the antibodies selected HMW.g1utenin subunits in all cases despite the apparent breadth of antibody specificity. The antibodies 236/9, 237/24 and 246/21 had specificities for y-/ro-gliadins and HMW-g1utenin subunits. The antibodies 236/9 and 246/21 also bound certain LMW-glutenin subunits, while 304/13 has specificity for fast-ro-gliadins with a weaker cross-reaction with HMWglutenin subunits. At the higher antibody concentration, all these antibodies gave crossreactions with the expressed cDNA clones; these cross-reactions were more or less equivalent with some exceptions. For example, the expressed cDNA clone L246/21-26 appeared to bind the antibody 304/13 more strongly than it did the other antibodies. At lower concentrations of the antibodies some clear distinctions in their binding to

o

C1'

TABLE II. The cross-reaction of monoclonal antibodies with the fusion proteins of the expressed cDNA clones cDNA clone Size (b.p.) alb select concentration mRNA size (bases) 221/23 (l/50) 221/23 (1/500) 222/5 (1/50) 222/5 (1/500) 21&/17 (l/50) 218/17 (1/500) 246/21 (1/50) 246/21 (1/500) 236/9 (1/50) 236/9 (1/500) 304/13 (1/20) 304/ I3 (1/200) 237/24 (1/20) 237/24 (1/200) 227/22 (1/5) 227/22 (1/50) 22&/20 (1/25)

L221/23

L222/5

L218/17-4

L218/17-5

L246/21-6

L246/21-12

L246/21-26

L236/9-16

740 2-5 Jlg/ml 1250

1200 2·5 Ilg/ ml 1270

1700 20 Ilg/ ml

&00 20 Jlg/ml 1400

++++ ++ ++ + ++

a

0 0 0 0

°

660 20 Ilg/ m1 *3000, 2400, 680-1400

1160 20 Ilg/ m1 *3000, 2400, 680-1400 0 0

2360 20 Jlg/1l1 *3000, 2400, 680-1400 0 0 0 0 0 0

1440 1/10 SIN 2250,2000, 740-1700 0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0

++++ ++++ ++++ +++ 0 0 0

0

0 0 0 0 0 0 0

-

+++ + 0 0 0 0 0 0 0 0 0

0 0

0 0 0 0 0 0 0 0 0 0

0

0 0 0 0 0

a

0 0 0 0 0

+++ +++ ++++ +++ ++++ +++ +++ + 0

0

0

0

0 0 0

+++++ ++++ +++ ++ +++ +++ ++ 0 0 0 0

++ + + + +++ + 0 0 0 0 0

++++ ++ +++++ +++ +++++ +++ ++++ +++ 0 0 0

P

?O 0

0

z 0 < > Z

t>J '"-l A

r--

TABLE cDNA clone Size (b.p.) alb select concentration mRNA size (bases)

221/23 (1/50) 222/23 (1/500) 222/5 (l/50)\ 222/5 (1/500) 218/17 (1150) 218/17 (1/500) 246/21 (1/50) 246/21 (1/500) 236/9 (/50) 236/9 (1/500) 304/13 (1/20) 304/13 (11200) 237/24(1/20) 237/24 (l/200) 227/22(1/5) 227/22 (1/50) 228/20 (1/25)

n

(contd)

L236/9-17

L304/13-7

L304/13-9

L237/24-15

L237/24-22

L227/22

L228/20

740 1/10 SIN 1250

1200 1/20 SIN *2900, 2400, 2000, 560--1600 0 0 0 0 0 0 0 0

650 1/20 SIN

1200 1/20 SIN *3350, 2600, 680--1950

650 1/20 SIN *3700, 3200, 2900,2400, 680--2150 0 0 0 0 0 0

700 25 J.lg/ml

350 1/25 SIN 1440

0 0 0 0 0 0

+++ +++ ++++ ++++ +++++ +++ +++ 0 0 0

0

-

0 0 0 0 0 0

+++++ ++++ +++++ +++ ++++ ++

++++ +++ ++++ ++++ ++++ +++ +++ +

0

0

0

0

0

0

0 0 0 0 0 0

+++++ ++++ +++ +++ +++ + +++ 0 0 0 0

++++ +++ ++++ +++ ++++ +++ +++ 0 0

0 0

-

()

=r:

:> ;:0 :> ()

-l l'!'1

;:0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

++++ +++ 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0

+ 0

++

N :> ::J 0

Z

0 ..,.,

~

=r:

tTl

:>

-l

"tl ;:0

SltTl

Z

()

0

Z :> ()

r

0 Z

tTl

(n

cDNA clones were selected using the antibody used to name the clone. All nine antibodies were reacted with the expressed fusion proteins of each eDNA clone and scored from 0 to 5. Northern hybridizations to detennine the mRNA sizes complementary to the cDNAs were carried out using washes in 0·1 x sse, O' I % SDS at 58°C (*68 0c). Score: + + + + + Very strong; + + + + strong; + + + moderate; + + weak; + weak; 0 zero.

;; .....

108

G. R. DONOVAN ET AL.

the expressed cDNA clones could be seen. The antibody 237/24 at 1/200 dilution bound most strongly with the eDNA clone L236/9-16, yet failed to bind to the other expressed cDNA clones selected in the primary screen with that antibody, where one concentration of that antibody had been used. The cDNA clones L304/13-7 and L304/13-9, on the basis of antibody specificity of their selecting antibodies, are both most likely to encode co-gliadins or HMW-glutenin subunits since the et-/~-/'Y-g1iadin specific monoclonal antibody 222/5 failed to bind to the expressed fusion polypeptides of either clone. A notable observation, however, was that the expressed clone L304/13-7 did not bind monoclonal antibody 246/21 even at the higher antibody concentration. This would clearly indicate that the other clone, L304/13-9, encodes quite a different protein, since U04/ 13-7 is a larger clone, and would be expected to encompass the smaller L304/13-9 if they are cDNAs representing equivalent proteins. In addition, the binding of the other antibodies to these two expressed L304/ 13 clones differed, supporting the view that they encode different proteins. Since this whole cluster of clones hybridized to the HMWglutenin subunit probe, they are almost certainly all structurally related to HMWglutenin subunits. However, the differences in antibody binding of the expressed clones indicates that this affords a potential means of subclassification by the presence of specific epitopes. Northern hybridization analysis of the monoclonal antibody-selected clones

Northern hybridization analysis, using the cDNA inserts as probes for poly A+-mRNA isolated from developing endosperm, indicated that some clones (e.g. L221/23, L222/5, L218/17 and L228/20) exhibited quite specific hybridization patterns, as shown by the hybridizing of the probes to the Northern transfers to give single bands. Other probes hybridized to several RNA bands even when the wash stringency was increased by raising the temperature from 58 to 68 °e. The overall effect of increased wash temperatures cannot be precisely estimated, however, without knowing the T mS of the DNA/RNA hybrids, and it was determined empirically!? The results from the 'crosshybridization' of antibodies and expressed cDNA fusion proteins only reflects the extent of conservation of antigenic determinants composed of only relatively short amino acid sequences. The results from northern hybridization (in which hybridization over much longer nucleic acid sequences may occur) indicated that this conservation of sequence is not restricted in general only to the antigenic determinants, but occurs throughout much of the prolamin amino acid sequence. They do, however, allow some distinctions to be made within some of the clones selected with the same antibody. Although L236/9-16 and L236/9-17 show similar patterns of antibody cross-reaction (Table I), and both hybridize to the HMW-g1utenin subunit DNA probe, suggesting that both are HMWglutenin subunit clones, they have clearly different DNA sequences, since the latter hybridized to only a single band representing a mRNA size of 1250 bases, whereas the former has several bands from 2250 to 740 bases. In some instances, no hybridization was obtained (e.g. 227/22 and 218/17-4). In the case of 227/22, hybridization of the clone to the original cDNA library revealed that it represented less than 0·03 % of the total eDNA sequences, and presumably would represent a similar proportion of the mRNA. Therefore, under the conditions used, there may have been insufficient specific mRNA to detect by hybridization to the eDNA probe.

CHARACTERIZATION OF WHEAT PROTEIN eDNA CLONES

109

Use of monoclonal antibodies in selection and identification of cDNA clones The use of monoclonal antibodies as tools to identify the p-galactosidase fusion proteins expressed by cDNA clones, on the basis of the specificity data from immunoblotting of flour proteins transferred to nitrocellulose after polyacrylamide gel electrophoresis, involves a number of variables that are not identical in each system. The colour intensity after antibody binding and colour development of the expressed cDNA clones on the nitrocellulose disks depends upon several factors: (1) The concentration of the antigen, which will be determined, in part, by the level of expression of the cloned cDNAs. (2) The frequency of the antibody-binding epitope within an individual clone, which may be dependent upon the size of the eDNA, if the protein that the cDNA encodes contains more than a single copy of that epitope. (3) The affinity of the primary antibody for the antigen and the antibody concentration. (4) The extent of masking of epitopes by formation of secondary structure or loss of epitope affinity resulting from disulphide bond cleavage and the use of detergents such as SDS. In all cases the level of expression of the cDNA clones, as determined by the level of expressed p-galactosidase, was similar, and therefore the antigen concentrations were approximately equivalent. However, the amount of antigen in an expressed cDNA clone transferred to nitrocellulose membrane may differ from that obtained on an immunoblot from gel electrophoresis. For example, a minor band on a blot may react only weakly with an antibody because the antigen concentration is limiting. Therefore, it is possible for the antigen concentration to be different from that observed on immunoblots of endosperm proteins, and protein species expressed to a quantitatively minor extent (e.g. that encoded by L227/22) may well give plaque colour intensities on the nitrocellulose disks equal to major protein types. Epitopes in a single polypeptide may exist singly or multiply. In the expressed fusion polypeptides, the size of the eDNA may be relevant where an epitope is present in several regions of the sequence of the corresponding full-length protein. This may be especially important in detection of the clones in a library, where epitopes are concentrated toward the N-terminus of the protein. Thus, certain antibodies that may bind determinants near the N-terminus of the mature protein sequence may not bind any fusion polypeptides because the cDNAs may not be full length. Indeed, other monoclonal antibodies not reported in this study (e.g. 122/24,405/7), which bound well to nanogram quantities of gluten proteins following their direct immobilization on nitrocellulose, did not detect any fusion polypeptides even at very high antibody concentrations. cDNA synthesis in our library was primed using oligo-dT, and therefore the cDNAs will represent a family originating from the C-terminus with few full-length cDNAs. This will result in only a relatively small number of fusion polypeptides having full-length wheat protein polypeptide inserts with an N-terminus. The use of monoclonal antibodies to screen a cDNA library in an expression vector, such as lambda gt 11, has some advantages over alternative methods. For example, selection of cDNAs based upon northern hybridization of groups of clones, and from the cross-hybridization of those clones to establish related families of cloned DNA

110

G. R. DONOVAN ET AL.

sequences, perhaps followed by hybrid-selected translation 19 to identify the polypeptides encoded by the mRNAs homologous to those cDNAs, are tedious. The use of monoclonal antibodies is, in fact, quite comparable to the use of DNA/DNA crosshybridization to establish cDNA families, but in the former case the particular protein epitopes encoded by specific DNA sequences are used to classify the eDNA clones into groups. This obviates at least one tedious step, and allows, in some instances, direct and definitive clone identification. The results obtained from antibody clone identification, and that from DNA hybridization using bona fide clones (pW8233 and the 1·8 kb lBy9 HMW-glutenin subunit restriction fragment from cv. Cheyenne), are, with some exceptions, in reasonably good agreement. However, the antibody cross-affinity analysis was able to demonstrate clear differences within clones identified by DNA hybridization. The cross-affinity study of the binding of the monoclonal antibodies used in the selection of the expressed cDNA clones gave patterns that were in general agreement with those predicted from immunoblots of electrophoretically separated mature endosperm proteins. From this it is concluded that the monoclonal antibodies are equally efficacious in the identification of eDNA clones as they are in identifying and characterizing wheat flour proteins. The results from northern hybridization indicated that the epitopes identified with monoclonal antibodies are expressed in several mRNA species, and these results generally concur with the observations made from analysis of the immunoblots of electrophoretically separated proteins. There are, however, a number of instances where there were clear and definitive identification of cDNA clones, and a few of these are currently being DNA-sequenced. An obvious extension to the present work will be the use of the cDNA clones characterized in this study for the isolation and identification of genomic DNA clones and in studies of the structure of the storage protein gene loci. Another possible use of cDNA clones can also be envisaged for the mapping of antibody epitopes. Such a recombinant DNA expression strategy has been used previously to characterize some of the antigenic determinants of a single protein of Myobacterium leprae, the aetiologic agent of leprosy25. Antigenic sequences encoded within a wheat endosperm protein cDNA could be characterized by the use of a range of techniques such as deletions and in vitro mutagenesis, combined with the expression of fusion proteins and antibody screening. The authors wish to thank Mrs Helen Carpenter and Mrs Lisa Robson for their skilled technical assistance, Dr D. G. Soll for his kind donation of the genomic o:-/p-gliadin clone pW8233, and Dr P. R. Shewry for his kind donation of the 1·8 kb genomic fragment of the lBy HMW-glutenin subunit gene from cv. Cheyenne. Dr G. R. Donovan wishes to specially thank Dr Trevor Lockett, CSIRO Division of Biotechnology, for use of his laboratory and for his guidance in the preparation of the wheat endosperm cDNA library.

References I. Lasztity, R. in 'The Chemistry of Cereal Proteins'. CRC Press, Forida (1984) pp 13-88. 2. Shewry, P. R. and Millin, B. J. in 'Advances in General Science and Technology' Vol. 7. (Y. Pomeramz, ed.), American Association of Cereal Chemists, Minnesota (1985) pp 1-83. 3. Shewry, P. R., Tatham, A. S., Forde, 1., Kreis, M. and Miflin, B. J. J. Cereal Sci. 4 (1986) 97-106. 4. Wang, S.-Z. and Esen, A. Gene 37 (1985) 267-269. 5. Wang, S.-Z. and Esen, A. Plant Sci. 42 (1985) 49-54.

CHARACTERIZATION OF WHEAT PROTEIN eDNA CLONES

III

6. Prat, S., Cortadas, J., Puigdomenech, P. and Palau, J. Nucleic Acids Res. 13 (1985) 1493-1504. 7. Bartels, D., Thompson, R. D. and Rothstein, S. Gene 35 (1985) 159-167. 8. Johnson, R. B., Le Brooy, J. T., Shearman, D. J. and Davidson, G. P. Aust. J. Exp. Bioi. Med. Sci. 63 (1985) 299-304. 9. Skerritt, J. H., Smith, R. A., Wrigley, C. W. and Underwood, P. A. J. Cereal Sci. 2 (1984) 215-224. 10. Skerritt, J. H. and Underwood, P. A. Biochim. Biophys. Acta 874 (1986) 245-254. I!. Huynh, T. V., Young, R. A. and Davis, R. W. in 'DNA Cloning: A Practical Approach' Vol. I (D. M. Glover, ed.), IRL Press, Oxford (1985) pp 49-78. 12. Jones, R. W., Taylor, N. W. and Senti, F. R. Arch. Biochem. Biophys. 84 (1959) 363-367. 13. Woychik, J. H., Boundy, J. A. and Dimler, R. J. Arch. Biochem. Biophys. 94 (l961) 477-482. 14. Gupta, R. B. and Shepherd, K. W. in 'Gluten Proteins: Proceedings of the 3rd International Workshop' (R. Lasztity and F. Bekes, eds) (1987) pp 13-19. 15. Donovan, G. R.. Lee, J. W. and Longhurst, T. J. Aust. J. Plant Physiol. 9 (198!) 59-68. 16. Mozer, 1. J. Plant Physiol. 65 (1980) 834-837. 17. Maniatis, T., Fritsch, E. F. and Sambrook, J. in 'Molecular Cloning: A Laboratory Manual', Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). 18. Marcu, K. and Dudock, B. Nucleic Acids Res. 1 (1974) 1385-1397. 19. Forde, 1. and Miflin, B. J. Planta 157 (1983) 567-576. 20. Kemp, D. J., Coppel, R. L., Cowman, A. F .. Saint, R. F., Brown, G. F. and Anders, R. F. Proc. Natl. Acad. Sci. USA 80 (1985) 3787-3791. 21. Sherness, D. and Gardiner, M. Mol. and Cell. Bioi. 4 (1984) 1206-1212. 22. Rafalski • .I. A., Scheets, K., Metzler, M., Peterson, D., Hedgecoth, C. and Soli, D. G. EMBO J.3 (1984) 1409-1415. 23. Halford, N. G .. Forde, .I., Anderson, O. D., Greene, F. C. and Shewry, P. R. TheOl·. Appl. Genet. 75 (1987) 117-126. 24. Greenwell, P. and Schofield, J. D. Cereal Chem. 63 (1986) 379-380. 25. Mehra, V., Sweetser, D. and Young, R. A. Proc. Natl. Acad. Sci. USA 83 (1986) 7013-7017.