Electrophoresis 1991, 12, 425-431
Jeri Welsh Higginbotham J. Stephen C. Smith Oscar S. Smith Pioneer Hi-Bred International, Inc., Plant Breeding Division, Departments of Biotechnology and Data Management, Johnston, IA
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Quantitative analysis of two-dimensional protein profiles of inbred lines of maize (Zea mays L.) Two-dimensional electrophoresis and fluorography of ["Slmethionine labeled maize germinated embryo proteins were performed at Cold Spring Harbor Laboratory. Fluorographs of 63 gels representing 37 inbred lines were subsequently scanned and spot-detected at Protein and DNAImageware Systems (Huntington Station,NY).The digitized images were then matched with the aid of PDQUESTI1 computer software. Over 1500 different protein spots were included in the resulting dataset. The optical density data were normalized to parts per million, then transformed to their natural logarithms. Analyses of variance were performed on each spot in order to select for further study those spots with most of their variation partitioned among inbred lines rather than within inbred lines. Using this method of spot selection, over 100 protein spots were included in the set of spots which display significant differences among inbred lines of maize.
1 Introduction Two-dimensional polyacrylamide gel electrophoresis (2DPAGE) of denatured proteins, as developed byO'Farrel1 [l], is a technique which can resolve hundreds of gene products. Because of this, the technique has been used to assay for genetic polymorphisms in diverse groups of organisms. In plants, genetic information obtained from 2D-PAGE has been used to gain a better understanding of gene expression and gene regulation [2-41, to determine relationships among organisms [5,6], to distinguish closely related organisms [7-121, and to construct linkage maps [13].Notwithstanding those references listed above, relatively little work has been done using 2D-PAGE to acquire genetic information about plants. The technique has been applied to maize (Zea mays L.) somewhat more ambitiously than it has been to most other species, due in part to the commercial value of maize. Leonardi et al. [14,15]demonstrated both qualitative and quantitative variation in leaf sheath, leaf blade,and mesocotyl proteins of two maize lines and their reciprocal hybrids. This work was extended by de Vienne etal. [16] to include three additional organs and 9 additional genotypes. DamerVal etal. [17] generated distance measurements using two different sets of protein spots for five inbred lines and compared those distance measurements to distances based on morphological characters. Higginbotham et al. [ 181 examined the variation among protein profiles due to different environmental sources of kernels relative to the variation due to different genotypes. In another study, Higginbotham etal. [19] compared the impact of slightlyvarying the developmental age of the tissue on the 2D-PAGE protein profile relative to the impact of three different genotypes.
Correspondence: Dr. Jeri Welsh Higginbotham, Pioner Hi-Bred International, Inc., Plant Breeding Division, Departments of Biotechnology and Data Management, 7250 NW 62nd Avenue, P.O. Box 1004, Johnston, IA 50131, USA Abbreviations: ANOVA, analysis of variance; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis.
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
Though the studies cited here confirm the value of 2DPAGE data in genetic research, the genotypes actually compared in most of these studies have been few in number. This is an important point because when only a few genotypes are studied, they can be electrophoresed together during every replicate electrophoretic run. Genetic studies require the analysis of many genotypes, and 2D-PAGE protein profiles from different electrophoretic runs must be comparable. Moreover, as more genotypes are included, the perceived relationships among the first few genotypes may change. In this paper, we report the construction of a 2D-PAGE dataset from fluorographs of 63 gels representing 37 inbred lines ofmaize.The methods used to construct the dataset and the methods used to select important spots are detailed.
2 Materials and methods 2.1 Plant material Thirty-nine inbred lines of maize and 10 hybrids were initially selected. The inbred lines represent a broad range of diversity from the central U.S. Corn Belt and include the parents of the 10 hybrids. Thirty-seven of these lines have been used in earlier studies [20, 211.
2.2 Sample preparation For each sample prepared, ten kernels were placed on moist filter paper, embryo side down, and maintained without light in a humid environment at 29 "C to 33 "C. Four kernels were selected which had demonstrably imbibed water. At most, the kernels had just emerged radicles. Embryos were excised from the kernels, washed of debris, dried, and weighed. Embryos were placed scutellum side down in a solution of 1.50 uCi [35S]rnethioninelabel [Amersham (Arlington Heights, IL) SJ.2041 and 150pL distilled water. They were maintained at 29 "C to 33 "C in a dark, humid environment for 17 h. Labeled embryos were washed twice in distilled water, dried, and weighed. Total imbibition time ranged from 45.5 to 71.5 h. Most embryos were imbibed for a total 0173-0835/91/0606-0425 $3.50+.25/0
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of 45.5 to 49 h. The rest of the sample preparation procedure was similar to that reported in Damerval etal. [lo]. Samples were homogenized in 12 mL of ice cold acetone with, at least, two 10 s bursts at the highest setting on a Brinkman (Des Plaines, IL) polytron using a 10 mm probe. Samples were kept chilled in an ice bath and centrifuged under refrigeration at 14 000 g for 10 min. The supernatant was decanted, and the defatted embryo tissue dried under vacuum. The pellet was then resuspended in 6 mL of extraction solution which consisted of 9 M urea, 5 mM K, C03,6% Nonidet P-40, 1.25% sodium dodecyl sulfate (SDS), 0.5% dithiothreitol, and 0.05% protarnine sulfate. Protamine sulfate was added to aid in precipitating DNA [22].A sintered glass pestle was used to aid resuspension. The temperature of this step was maintained at 37°C. Samples were then centrifuged at 11000 gfor 10 min at room temperature to remove cellular debris and precipitated DNA. The supernatant was decanted and stored at -70°C. A total of 112 samples were prepared representing 39 inbred lines and 10 hybrid lines. 2.3 Tests of the protein extraction procedure
Three components of the protein extraction procedure were tested. The trichloroacetic acid (TCA) precipitable radioactivity in the acetone supernatant was assayed to determine how much protein was being lost in the initial extraction [24]. The effectiveness of protamine sulfate in precipitating nucleic acids in a high molar urea solution was examined as follows. Samples with and without protamine sulfate were applied to gels. The gels were subsequently silver stained using the GelcodeTMkit (Gelcode is a trademark of Health Products, Inc., South Haven, MI) to visualize nucleic acids and proteins. Lastly, the effectiveness of the procedure in preventing degradation due to proteinase activity was investigated. The details of all tests listed above have been presented elsewhere [23].
2.4 Electrophoresis and fluorography The 2D-PAGE and fluorography were performed at the 2D gel lab of Cold Spring Harbor Laboratory according to the method of Garrels [24].In the beginning of the study, about 400 000 dpm ofTCA precipitable radioactivity were applied to each gel. This was increased to 600 000 dpm to shorten the fluorographic exposure time. A pH 4-8 gradient was used in the first dimension, and a 10% SDS-polyacrylamide gel was used in the second dimension. Over 200 gels were generated from the 112 samples. Each gel was exposed to film for three periods of time. This generated a light, medium, and dark exposure for each gel. The length of all three exposures varied with the amount of radioactivity initially applied to the gel. Over 600 fluorographic exposures were produced.
2.5 Computer manipulation and analysis Fluorographs from 64 gels were chosen for scanning and computer digitization. Only one gel of one hybrid was used, and the dark exposure of this gel was used as the standard image against which the other images were matched. Fluorographs representing the inbred lines were chosen so that as many genotypes would be represented as possible with-
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out having to use gels with a low quality protein profile. Table 1 lists the genotype code, the date the gel was run, and gel and exposure numbers of the fluorographs. The entries in Table 1 are sorted according to genotype. Figure 1 shows fluorographs selected to represent the range of germplasm involved. The fluorographs in Figure 1 have been trimmed to show only the area included in the study. Some, but not all, fluorographs had additional spots more basic than those shown. A total of 69 fluorographs were laser scanned and the images processed using the PDQUEST computer analysis programs based on the initial design and appropriate algorithms of the QUEST program of Garrels [25]. Both the medium and dark exposure of five gels were included because, for these gels, the more appropriate exposure was difficult to determine. Following scanning, spot detection, and spot quantification of each fluorograph at Protein and DNA Imageware Systems, the resulting 69 fluorographic images were matched with the aid of PDQUEST-IITMcomputer software (PDQUEST-I1 is a trademark of Protein and DNA Imagewave Systems, Huntington Station, NY). The list of matched spots was then transferred to the VAXTM cluster (VAX is a trademark of Digital Equipment Corporation, Bedford, MA) located at Pioneer Hi-Bred International, Inc. (Johnston, IA). The raw data were in units of optical density. These data were treated in two different ways. In one approach, all densities at or below 10% of the average spot density for each image were set to zero. The dataset was trimmed in this manner to delete those faint spots which were detected preferentially in darker exposures. In the other approach, no trim was employed; all nonzero spot values were retained. In both methods, the optical density data were normalized so that the total density of each image was one million. The normalized data were then transformed to their natural logarithms and a value for the absent spots determined. No fluorograph had all spots. The value of those absent spots was set to the highest whole integer that was less than the lowest log value in the dataset. For the trimmed dataset, the base value was 4. For the untrimmed dataset, the base value was 3. This was done to minimize the difference between an absent spot and a faint, but present, spot and to preserve the quantitative nature of the data. The normalized transformed spot values ranged between these low values and 11. Four analyses of variance (ANOVAs) were performed on each spot in the trimmed and untrimmed datasets in order to select for further study those spots with most of their variation partitioned among inbred lines rather than within inbred lines. Two ANOVAs were performed on every spot after normalization and two more ANOVAs were performed on every spot after log-transformation. In each case, one of the two ANOVAs was performed using a “short”1ist of 21 image pairs, and the other ANOVA was performed using a “long” list of 27 image pairs. An image pair consists of two images of the same genotype. Table 1 lists those image pairs which were used in the ANOVAs. From both the trimmed and untrimmed datasets, a subset of spots was selected such that every spot had at least 80% of its variation partitioned among inbred lines rather than within lines.
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A
C
421
B
D
Figure 1.Fluorographic exposures offour different genotypes of maize. Gel and exposure numbers (see Table 1) are given below. (A) 22166.2. Molecular masses in kilodaltons are indicated on the right. Apparent isoelectric points are indicated on the bottom. (B) 23089.3. (C) 23164.3. (D) 23232.3.
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Table 1. Gel and exposure numbers, genotype codes, and dates the gels were run for the 64 gels (69 fluorographs) in the dataseta) Gel & exposure number
Genotype code
Date of electrophoresis
23408.2b) 23420.2 22832.2 23716.2 22801.2 22802.2 23708.2 23710.2 23088.2 23406.2 23418.2 22833.2 23089.2 22829.2 237 13.2 23714.2 23177.2 23208.2 23166.2 23604.2 23415.2 22837.2 22410.2 23409.2 23414.2 22808.2 23487.2 22835.2 23700.2 2370 1.2 23494.2 23542.2 23416.2 23483.2 23715.2 23539.2 23030.2 23195.2 22803.2 23602.2 23020.2 23085.2 22830.2 23233.2 23233.3 23598.2 23598.3 23401.2 23413.2 23019.2 23196.2 23232.2 23022.2 23220.2 23220.3 23486.2 23540.2 23540.3 23018.2 23411.2 23090.2 22166.2 22279.2 22270.2 22859.2 23184.2 23184.3 23164.2 23209.2
A632** A632** A-3** A-3** A-4** A-4** A-5* A-5* A-6 A-7** A-7** A_8** A-8** A.9 B64* B64* B73** B73** BJ** BJ** B-2 8-3 B-4** B4** B-5 B-6* B-6* B-7 B-9** B-9** C-1** C-1** c3** c-3** c-4 c-5 C-6** C-6** c-7** c-7** C-8** C-8** c-9 D_l** D-l* D-l** D_1* D_3** D3** D-4 D_5** D..S** D_6* D-6* D-6 D_8** D-8** D-8 D_9** D_9** E-1 E-2* E-2* E-3** E-3** H Y ~ Hyb Mo17** Mo17**
9-Mar-89 9-Mar-89 14-Dec-88 26-Apr-89 12-Dec-88 12-Dec-88 26-Apr-89 26-Apr-89 25-Jan-89 9-Mar-89 9-Mar-89 14-Dec-88 25-Jan-89 14-Dec-88 26-Apr-89 26-Apr-89 7-Feb-89 8-Feb-89 2-Feb-89 11-Apr-89 9-Mar-89 14-Dec-88 24-0ct-88 9-Mar-89 9-Mar-89 12-Dec-88 16-Mar-89 14-Dec-88 25-Apr-89 25-Apr-89 21-Mar-89 3-Apr-89 9-Mar-89 16-Mar-89 26-Apr-89 3-Apr-89 18-Jan-89 7-Feb-89 12-Dec-88 11-Apr-89 18-Jan-89 25-Jan-89 14-Dec-88 13-Feb-89 13-Feb-89 11-Apr-89 11-Apr-89 9-Mar-89 9-Mar-89 18-Jan-89 7-Feb-89 13-Feb-89 18-Jan-89 13-Feb-89 13-Feb-89 16-Mar-89 3-Apr-89 3-Apr-89 18-Jan-89 9-Mar-89 25-Jan-89 22-Sep-88 6-Oct-88 6-Oct-88 19-Dec-88 7-Feb-89 7-Feb-89 2-Feb-89 8-Feb-89
A
I
10 20 30 40 50 60 70 80 901001101200130140
SUM MIDPOINT (X 1000)
B
m250300350400450500550600650700750800850 SPOT NUMBER MIDPOINT Figure 2. (A) Distribution of the sums of densities in each of the 67 fluoro-
graphic exposures of inbred lines of maize. The two exposures of the hybrid line were not included. (B) Distribution of the total number ofspots in each of the 67 fluorographic exposures of inbred lines of maize. Films 23598.2,23220.2, and 23540.3 are less like the rest of the images than the alternative exposure for those gels (23598.3,23220.3, and 23540.2).
a) Those images which were included on both the long list and the short list for analysis of variance are designated by two asteriks.Those images which were included only o n the long list for analysis of variance are designated by one asterik. b) The first five digits identify the gel. The last digit identifies the exposure. Medium exposures are designated with a 2, while dark exposures are designated with a 3.
Electrophoresis 1991, 12, 425-43 I
3 Results and discussion 3.1 Sample preparation Germination after a 45.5 to 49 h imbibition period was satisfactory for most lines. Some lines failed to germinate within this time interval, and those lines were allowed to imbibe longer so that the developmental stage of all lines would be the same. Even allowing for the slower germination, there was still a significant negative correlation between the total number of hours imbibed and relative gain of the embryos during labeling (combined mass that embryos gained during labeling divided by final combined mass) ( r = -0.43, P=O.OOOl) and between the total number ofhours imbibed and combined mass gained during labeling ( r = - 0.32, P=O.OOl). For each sample of four embryos, between 21 O/o and 52% of their final combined mass was gained during labeling. There was a significant negative correlation between the combined mass of the embryos before labeling
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and relative gain of the embryos during labeling ( r = - 0.38, P = 0.0001) indicating that larger embryos tended not to gain any more than smaller embryos. There was no correlation between the combined mass of the embryos before labeling and combined mass gained during labeling. 3.2 Tests of the protein extraction procedure Repeated assays of the TCA-precipitable radioactivity in the acetone supernatant revealed 1000-fold less labeled protein in the acetone than in the sample. Samples often contained 100 000 cpm/pL of TCA-precipitable radioactivity. Proportional amounts of acetone supernatants contained an average of 100 cpm. Silver stained gels of samples prepared with or without protamine sulfate revealed clear differences. In those without protamine sulfate, numerous horizontal gray streaks extended across the gels. The basic, high molecular weight quadrant of the gels appeared to be
Figure 3. Red crosshairs mark the location of over 1500 spots on the synthetic standard image used to match the 67 h a ges of inbred lines of maize.
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most consistently amicted, although the acidic low molecular weight quadrant had some shorter streaks. Samples prepared with protamine sulfate had a much clearer background due to the almost complete absence of gray streaks. The extraction procedure appeared to be effective in preventing degradation due to proteinase activity. This was determined in three different ways. In the first test, silver stained gels of part of two samples which had been incubated at 37 “C for 3 h prior to electrophoresis were compared with silver stained gels of part of two samples which had been warmed to 37 “Cimmediately prior to electrophoresis. The pairs of protein profiles were essentially identical with no preferential loss of high molecular weight proteins or visible degradation of the protein profiles. No proteinase activity in the samples was detected using Azocoll dyebound collagen substrate (Azocoll is a trademark of Calbiochem, San Diego, CA) even when the reaction was allowed to proceed 30 min at 37°C. When protein extraction solution was used instead of phosphate buffer, no proteinase activity was detected in any of the positive controls either. Proteinase K and trypsin were used as positive controls.
the pairs were generally darker than the others. If only the “short” list had been used which did not include the darker image pairs, then a few spots would have been included which varied within the inbred lines represented by the darker pairs. Similarly, some spots would have been included that were inconsistent in some image pairs if only the logtransformed data had been used for the ANOVAs. If only the “long” list had been used, then a few spots would have been included, based on image density rather than genotypic differences. Figure 2 shows the distribution of the sums of densities and the distribution of total spot number for the 67 fluorographs of the inbred lines. As can be seen in Fig. 2, the densest image is fourteen times denser than the faintest image. With the exception of one image, however, this translates only into a threefold difference among the images for total number of spots.
Associations among inbred lines were studied using fewer than four ANOVA’s per spot. The best results were obtained using the four ANOVAs listed in Section 2.5 in combination. Two lists of image pairs were used because some of
The total density and total spot number of three different images are listed in Fig. 2. For those three gels, the dark exposure of two of them (23598.3 and 23220.3) and the medium exposure of one of them (23540.2) appear to be more comparable to the rest of the images. It was found that the subsets of spots, selected such that every spot had at least 80% of its variation partitioned among inbred lines rather than within lines, produced results as concordant or more concordant with pedigree data than other selection criteria. A schematic illustration of, and a discussion of, the pedigree data for most of the inbred lines included in this study may be found in Smith etal. [20]. Values less than 80%,
A
B
3.3 Computer manipulation and analysis
Figure 4. (A) Triangles mark the location of 108 spots selected using an 80% criterion through 4 ANOVAs on the untrimmed data. Film 23184.2. (B) Squares mark the location of 114 spots selected using an 80% criterion through 4ANOVAs on the trimmed data. Film 23184.3.The letter Aidentifies
those spotswhich are present in one spot set but not the other.Mo1ecularmasses in kilodaltons are indicated on the right.Apparent isoelectric points are indicated on the bottom. The density difference between the medium and dark exposure can be seen in this figure as well.
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where more spots were included in the subsets but more variation was included within lines, yielded less satisfactory results. Values greater than 80% produced results which were as concordant with pedigree data as the results using 80%, but utilized fewer spots.
3.4 Spot set composition Over 1500 different protein spots were documented in the 37 inbred lines and one hybrid included in the dataset. Figure 3 shows the location of the spots on the synthetic image used to match the gels. Using the method of spot selection described above, 108protein spots were included in the untrimmed set of spots and 114 protein spots were included in the trimmed set of spots which display significant differences among inbred lines of maize. Figure 4 shows the location of spots in both sets. The two sets share 104 spots. Both spot sets are distributed evenly, relative to local spot density, over the protein profile.
4 Concluding remark., The results of the protein extraction procedure tests presented here indicate that the protocol does not contribute significantly to the variability seen on 2-D gels of corn seedling proteins. The variability generated due to quantitative loss of protein into the acetone supernatant during protein extraction appears to be minimal. The removal of nucleic acids by protamine sulfate precludes distortion of the profile due to 1heirpresence.There appears to be little ifanyvariability generated by the action of proteinases in situ. Not only does the extraction solution prevent proteinase activity, but the acetone extraction appears to be an effective inhibitor of activity as well. Furthermore, samples appear to be stable at 37 "C for at least 3 h. Samples can be stored frozen and warmed forreuse many times without risk of degradation. It appears that 2D protein profiles will be useful in determining genetic associations among many inbred genotypes of maize even when gels are run months apart. Based on our stringent spot selection criteria, over 100 spots were included in 2 different subsets. Preliminary analyses show that these subsets do indeed separate and associate inbred lines in a manner concordant with their pedigrees. For example, inbred lines, A632, B64, A-3, and C-4, were shown to be more closely related to each other than to any other inbred line, a result which agrees with their pedigrees. Likewise, inbred lines, A-4, B-4, C-3, D-8, and D-9, were shown to be more closely related to each other than to any other inbred line which reflects their pedigrees. In general, correlations between pedigree data and genetic similarity based on different sets of 2D-PAGE gels and different 2DPAGE spot sets were consistently above 0.70 (P= 0.0001). There are compelling reasons for using this technique in genetic and numerical taxonomic studies. Given good quality gels and fluorographs, these complex molecular profiles appear to be stable enough and reproducible enough to match gels run months apart. The effects of different genotypes of maize on the profile are obvious and measurable even when closely related (95 %-97% by pedigree) genotypes are compared. This technique samples a large portion of the
43 I
total genome. The only other technique which samples as large a portion of the genome is the restriction fragment length polymorphism (RFLP) technique. The two techniques compliment each other in that one samples DNA polymorphisms while the other samples polymorphisms of gene products. Not only are polymorphisms at structural loci revealed through 2D-PAGE of proteins, polymorphisms at regulatory loci are also revealed (through quantitative differences). Such insights are not possible with RFLP data. The 2D-PAGE data appear amenable to traditional methods of numerical taxonomic analysis, such as cluster analysis. Additional studies, utilizing this dataset, are in progress. The authors would like to thank Heidi Sacco of Cold Spring Harbor Laboratory, Cold Spring Harbor, N Y for her electrophoretic and fluorographic expertise, Liza Chan of Protein and DNA Imageware Systems, Huntington Station, NYfor her computer skills, and Steve Wall of Pioneer Hi-Bred International, Inc., Johnston, IA for help with the analysis. Editorial assistance was provided by Steven Briggs, Alan Orr, Gura Rao, and two anonymous reviewers. Received October 18, 1990
5 References [l] O'Farrell, P. H., J. B i d . Chem. 1975, 250, 4007-4021. [2] Colas des Francs,C.andThiellement,H., Theor.Appl. Genet. 1985,7/, 31-38. [3] Bahrman N. and Thiellement, H., Theor. Appl. Genet. 1987, 74,218223. [4] Gottlieb, L. D. and de Vienne, D., Genetics 1988, 119, 705-710. [5] Bahrman, N., Zivy, M. and Thiellement, H., Heredity 1988,61,473480. [6] Beckstrom-Sternberg, S. M., Biochem. Syst. Eco. 1989, /7,573-582. 171 Basha, S. M. M., Plant Physiol. 1979, 63, 301-306. [8] Bahrman, N., de Vienne, D., Thiellement, H. and Hofmann, J.-P., Biochem. Getter. 1985, 23, 247-255. [9] Dunbar, B. D., Bundman, D. S. and Dunbar, B. S., Electrophoresis 1985, 6,39-43. [lo] Damerval, C., de Vienne, D., Zivy, M. and Thiellement, H., Electrophoresis 1986, 7, 52-54. [ l l ] Holloway, P. J. and Arundel, P. H., Anal. Biochem. 1988, 172, 8-15. [I21 Ramagopal, S., Theor. Appl. Genet. 1990, 79, 297-304. [13] Bahrman, N. and Damerval, C., Herediy 1989, 63,267-274. (141 Leonardi, A,, Damerval, C. and de Vienne, D., Genet. Res., Camb. 1987, 50, 1-5. [15] Leonardi, A , , Damerval, C. and de Vienne, D., Genet. Res., Camb. 1988, j2,97-103. [16] de Vienne, D., Leonardi, A. and Damerval, C., Electrophoresis 1988, 9,742-750. [17] Damerval, C., Hebert,Y. and de Vienne, D., Theor. Appl. Genet. 1987, 74, 194-202. [18] Higginbotham, J. W., Smith, J. S. C. and Smith, 0. S., Maize Genet. Coop. Newsl. 1989, 63,84-85. 1191 Higginbotham, J. W., Smith, J. S. C. and Smith, 0. S . , Maize Genet. Coop. Newsl. 1989, 63, 85. [20] Smith,O. S., Smith, J . S. C., Bowen, S. L.,Tenborg, R . 4 . and Wall, S. J., Theor. Appl. Genet. 1990, 80, 833-840. [2l] Smith, J. S. C. and Smith, 0. S., Maydica 1989, 34, 151-161. [22] Mayer, J.E.,Hahne, G.,Palme,K. and Schell, J., Plant CellRep., 1987, 6, 77-8 1. [23] Higginbotham, J. W. and Smith, J. S. C., Maize Genet. Coop. Newsl. 1989, 63, 83-84. [24] Garrels, J. I., Methods Enzymol. 1983, IOO, 411-423. [25] Garrels, J. I., Farrar, J.T. and Burwell, C. B., in: Celis, J. E. and Bravo, R., (Eds.), Two-dimensional Gel Electrophoresis of Proteins,Academic Press, New York 1984, pp. 37-91.
Jeri Welsh Higginbotham J. Stephen C. Smith Oscar S. Smith Pioneer Hi-Bred International, Inc., Plant Breeding Division, Departments of Biotechnology and Data Management, Johnston, IA
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Quantitative analysis of two-dimensional protein profiles of inbred lines of maize (Zea mays L.) Two-dimensional electrophoresis and fluorography of ["Slmethionine labeled maize germinated embryo proteins were performed at Cold Spring Harbor Laboratory. Fluorographs of 63 gels representing 37 inbred lines were subsequently scanned and spot-detected at Protein and DNAImageware Systems (Huntington Station,NY).The digitized images were then matched with the aid of PDQUESTI1 computer software. Over 1500 different protein spots were included in the resulting dataset. The optical density data were normalized to parts per million, then transformed to their natural logarithms. Analyses of variance were performed on each spot in order to select for further study those spots with most of their variation partitioned among inbred lines rather than within inbred lines. Using this method of spot selection, over 100 protein spots were included in the set of spots which display significant differences among inbred lines of maize.
1 Introduction Two-dimensional polyacrylamide gel electrophoresis (2DPAGE) of denatured proteins, as developed byO'Farrel1 [l], is a technique which can resolve hundreds of gene products. Because of this, the technique has been used to assay for genetic polymorphisms in diverse groups of organisms. In plants, genetic information obtained from 2D-PAGE has been used to gain a better understanding of gene expression and gene regulation [2-41, to determine relationships among organisms [5,6], to distinguish closely related organisms [7-121, and to construct linkage maps [13].Notwithstanding those references listed above, relatively little work has been done using 2D-PAGE to acquire genetic information about plants. The technique has been applied to maize (Zea mays L.) somewhat more ambitiously than it has been to most other species, due in part to the commercial value of maize. Leonardi et al. [14,15]demonstrated both qualitative and quantitative variation in leaf sheath, leaf blade,and mesocotyl proteins of two maize lines and their reciprocal hybrids. This work was extended by de Vienne etal. [16] to include three additional organs and 9 additional genotypes. DamerVal etal. [17] generated distance measurements using two different sets of protein spots for five inbred lines and compared those distance measurements to distances based on morphological characters. Higginbotham et al. [ 181 examined the variation among protein profiles due to different environmental sources of kernels relative to the variation due to different genotypes. In another study, Higginbotham etal. [19] compared the impact of slightlyvarying the developmental age of the tissue on the 2D-PAGE protein profile relative to the impact of three different genotypes.
Correspondence: Dr. Jeri Welsh Higginbotham, Pioner Hi-Bred International, Inc., Plant Breeding Division, Departments of Biotechnology and Data Management, 7250 NW 62nd Avenue, P.O. Box 1004, Johnston, IA 50131, USA Abbreviations: ANOVA, analysis of variance; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis.
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
Though the studies cited here confirm the value of 2DPAGE data in genetic research, the genotypes actually compared in most of these studies have been few in number. This is an important point because when only a few genotypes are studied, they can be electrophoresed together during every replicate electrophoretic run. Genetic studies require the analysis of many genotypes, and 2D-PAGE protein profiles from different electrophoretic runs must be comparable. Moreover, as more genotypes are included, the perceived relationships among the first few genotypes may change. In this paper, we report the construction of a 2D-PAGE dataset from fluorographs of 63 gels representing 37 inbred lines ofmaize.The methods used to construct the dataset and the methods used to select important spots are detailed.
2 Materials and methods 2.1 Plant material Thirty-nine inbred lines of maize and 10 hybrids were initially selected. The inbred lines represent a broad range of diversity from the central U.S. Corn Belt and include the parents of the 10 hybrids. Thirty-seven of these lines have been used in earlier studies [20, 211.
2.2 Sample preparation For each sample prepared, ten kernels were placed on moist filter paper, embryo side down, and maintained without light in a humid environment at 29 "C to 33 "C. Four kernels were selected which had demonstrably imbibed water. At most, the kernels had just emerged radicles. Embryos were excised from the kernels, washed of debris, dried, and weighed. Embryos were placed scutellum side down in a solution of 1.50 uCi [35S]rnethioninelabel [Amersham (Arlington Heights, IL) SJ.2041 and 150pL distilled water. They were maintained at 29 "C to 33 "C in a dark, humid environment for 17 h. Labeled embryos were washed twice in distilled water, dried, and weighed. Total imbibition time ranged from 45.5 to 71.5 h. Most embryos were imbibed for a total 0173-0835/91/0606-0425 $3.50+.25/0
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of 45.5 to 49 h. The rest of the sample preparation procedure was similar to that reported in Damerval etal. [lo]. Samples were homogenized in 12 mL of ice cold acetone with, at least, two 10 s bursts at the highest setting on a Brinkman (Des Plaines, IL) polytron using a 10 mm probe. Samples were kept chilled in an ice bath and centrifuged under refrigeration at 14 000 g for 10 min. The supernatant was decanted, and the defatted embryo tissue dried under vacuum. The pellet was then resuspended in 6 mL of extraction solution which consisted of 9 M urea, 5 mM K, C03,6% Nonidet P-40, 1.25% sodium dodecyl sulfate (SDS), 0.5% dithiothreitol, and 0.05% protarnine sulfate. Protamine sulfate was added to aid in precipitating DNA [22].A sintered glass pestle was used to aid resuspension. The temperature of this step was maintained at 37°C. Samples were then centrifuged at 11000 gfor 10 min at room temperature to remove cellular debris and precipitated DNA. The supernatant was decanted and stored at -70°C. A total of 112 samples were prepared representing 39 inbred lines and 10 hybrid lines. 2.3 Tests of the protein extraction procedure
Three components of the protein extraction procedure were tested. The trichloroacetic acid (TCA) precipitable radioactivity in the acetone supernatant was assayed to determine how much protein was being lost in the initial extraction [24]. The effectiveness of protamine sulfate in precipitating nucleic acids in a high molar urea solution was examined as follows. Samples with and without protamine sulfate were applied to gels. The gels were subsequently silver stained using the GelcodeTMkit (Gelcode is a trademark of Health Products, Inc., South Haven, MI) to visualize nucleic acids and proteins. Lastly, the effectiveness of the procedure in preventing degradation due to proteinase activity was investigated. The details of all tests listed above have been presented elsewhere [23].
2.4 Electrophoresis and fluorography The 2D-PAGE and fluorography were performed at the 2D gel lab of Cold Spring Harbor Laboratory according to the method of Garrels [24].In the beginning of the study, about 400 000 dpm ofTCA precipitable radioactivity were applied to each gel. This was increased to 600 000 dpm to shorten the fluorographic exposure time. A pH 4-8 gradient was used in the first dimension, and a 10% SDS-polyacrylamide gel was used in the second dimension. Over 200 gels were generated from the 112 samples. Each gel was exposed to film for three periods of time. This generated a light, medium, and dark exposure for each gel. The length of all three exposures varied with the amount of radioactivity initially applied to the gel. Over 600 fluorographic exposures were produced.
2.5 Computer manipulation and analysis Fluorographs from 64 gels were chosen for scanning and computer digitization. Only one gel of one hybrid was used, and the dark exposure of this gel was used as the standard image against which the other images were matched. Fluorographs representing the inbred lines were chosen so that as many genotypes would be represented as possible with-
Electrophoresis 1991, 12, 425-431
out having to use gels with a low quality protein profile. Table 1 lists the genotype code, the date the gel was run, and gel and exposure numbers of the fluorographs. The entries in Table 1 are sorted according to genotype. Figure 1 shows fluorographs selected to represent the range of germplasm involved. The fluorographs in Figure 1 have been trimmed to show only the area included in the study. Some, but not all, fluorographs had additional spots more basic than those shown. A total of 69 fluorographs were laser scanned and the images processed using the PDQUEST computer analysis programs based on the initial design and appropriate algorithms of the QUEST program of Garrels [25]. Both the medium and dark exposure of five gels were included because, for these gels, the more appropriate exposure was difficult to determine. Following scanning, spot detection, and spot quantification of each fluorograph at Protein and DNA Imageware Systems, the resulting 69 fluorographic images were matched with the aid of PDQUEST-IITMcomputer software (PDQUEST-I1 is a trademark of Protein and DNA Imagewave Systems, Huntington Station, NY). The list of matched spots was then transferred to the VAXTM cluster (VAX is a trademark of Digital Equipment Corporation, Bedford, MA) located at Pioneer Hi-Bred International, Inc. (Johnston, IA). The raw data were in units of optical density. These data were treated in two different ways. In one approach, all densities at or below 10% of the average spot density for each image were set to zero. The dataset was trimmed in this manner to delete those faint spots which were detected preferentially in darker exposures. In the other approach, no trim was employed; all nonzero spot values were retained. In both methods, the optical density data were normalized so that the total density of each image was one million. The normalized data were then transformed to their natural logarithms and a value for the absent spots determined. No fluorograph had all spots. The value of those absent spots was set to the highest whole integer that was less than the lowest log value in the dataset. For the trimmed dataset, the base value was 4. For the untrimmed dataset, the base value was 3. This was done to minimize the difference between an absent spot and a faint, but present, spot and to preserve the quantitative nature of the data. The normalized transformed spot values ranged between these low values and 11. Four analyses of variance (ANOVAs) were performed on each spot in the trimmed and untrimmed datasets in order to select for further study those spots with most of their variation partitioned among inbred lines rather than within inbred lines. Two ANOVAs were performed on every spot after normalization and two more ANOVAs were performed on every spot after log-transformation. In each case, one of the two ANOVAs was performed using a “short”1ist of 21 image pairs, and the other ANOVA was performed using a “long” list of 27 image pairs. An image pair consists of two images of the same genotype. Table 1 lists those image pairs which were used in the ANOVAs. From both the trimmed and untrimmed datasets, a subset of spots was selected such that every spot had at least 80% of its variation partitioned among inbred lines rather than within lines.
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Elertrophoresis 1991, 12, 425-431
A
C
421
B
D
Figure 1.Fluorographic exposures offour different genotypes of maize. Gel and exposure numbers (see Table 1) are given below. (A) 22166.2. Molecular masses in kilodaltons are indicated on the right. Apparent isoelectric points are indicated on the bottom. (B) 23089.3. (C) 23164.3. (D) 23232.3.
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Table 1. Gel and exposure numbers, genotype codes, and dates the gels were run for the 64 gels (69 fluorographs) in the dataseta) Gel & exposure number
Genotype code
Date of electrophoresis
23408.2b) 23420.2 22832.2 23716.2 22801.2 22802.2 23708.2 23710.2 23088.2 23406.2 23418.2 22833.2 23089.2 22829.2 237 13.2 23714.2 23177.2 23208.2 23166.2 23604.2 23415.2 22837.2 22410.2 23409.2 23414.2 22808.2 23487.2 22835.2 23700.2 2370 1.2 23494.2 23542.2 23416.2 23483.2 23715.2 23539.2 23030.2 23195.2 22803.2 23602.2 23020.2 23085.2 22830.2 23233.2 23233.3 23598.2 23598.3 23401.2 23413.2 23019.2 23196.2 23232.2 23022.2 23220.2 23220.3 23486.2 23540.2 23540.3 23018.2 23411.2 23090.2 22166.2 22279.2 22270.2 22859.2 23184.2 23184.3 23164.2 23209.2
A632** A632** A-3** A-3** A-4** A-4** A-5* A-5* A-6 A-7** A-7** A_8** A-8** A.9 B64* B64* B73** B73** BJ** BJ** B-2 8-3 B-4** B4** B-5 B-6* B-6* B-7 B-9** B-9** C-1** C-1** c3** c-3** c-4 c-5 C-6** C-6** c-7** c-7** C-8** C-8** c-9 D_l** D-l* D-l** D_1* D_3** D3** D-4 D_5** D..S** D_6* D-6* D-6 D_8** D-8** D-8 D_9** D_9** E-1 E-2* E-2* E-3** E-3** H Y ~ Hyb Mo17** Mo17**
9-Mar-89 9-Mar-89 14-Dec-88 26-Apr-89 12-Dec-88 12-Dec-88 26-Apr-89 26-Apr-89 25-Jan-89 9-Mar-89 9-Mar-89 14-Dec-88 25-Jan-89 14-Dec-88 26-Apr-89 26-Apr-89 7-Feb-89 8-Feb-89 2-Feb-89 11-Apr-89 9-Mar-89 14-Dec-88 24-0ct-88 9-Mar-89 9-Mar-89 12-Dec-88 16-Mar-89 14-Dec-88 25-Apr-89 25-Apr-89 21-Mar-89 3-Apr-89 9-Mar-89 16-Mar-89 26-Apr-89 3-Apr-89 18-Jan-89 7-Feb-89 12-Dec-88 11-Apr-89 18-Jan-89 25-Jan-89 14-Dec-88 13-Feb-89 13-Feb-89 11-Apr-89 11-Apr-89 9-Mar-89 9-Mar-89 18-Jan-89 7-Feb-89 13-Feb-89 18-Jan-89 13-Feb-89 13-Feb-89 16-Mar-89 3-Apr-89 3-Apr-89 18-Jan-89 9-Mar-89 25-Jan-89 22-Sep-88 6-Oct-88 6-Oct-88 19-Dec-88 7-Feb-89 7-Feb-89 2-Feb-89 8-Feb-89
A
I
10 20 30 40 50 60 70 80 901001101200130140
SUM MIDPOINT (X 1000)
B
m250300350400450500550600650700750800850 SPOT NUMBER MIDPOINT Figure 2. (A) Distribution of the sums of densities in each of the 67 fluoro-
graphic exposures of inbred lines of maize. The two exposures of the hybrid line were not included. (B) Distribution of the total number ofspots in each of the 67 fluorographic exposures of inbred lines of maize. Films 23598.2,23220.2, and 23540.3 are less like the rest of the images than the alternative exposure for those gels (23598.3,23220.3, and 23540.2).
a) Those images which were included on both the long list and the short list for analysis of variance are designated by two asteriks.Those images which were included only o n the long list for analysis of variance are designated by one asterik. b) The first five digits identify the gel. The last digit identifies the exposure. Medium exposures are designated with a 2, while dark exposures are designated with a 3.
Electrophoresis 1991, 12, 425-43 I
3 Results and discussion 3.1 Sample preparation Germination after a 45.5 to 49 h imbibition period was satisfactory for most lines. Some lines failed to germinate within this time interval, and those lines were allowed to imbibe longer so that the developmental stage of all lines would be the same. Even allowing for the slower germination, there was still a significant negative correlation between the total number of hours imbibed and relative gain of the embryos during labeling (combined mass that embryos gained during labeling divided by final combined mass) ( r = -0.43, P=O.OOOl) and between the total number ofhours imbibed and combined mass gained during labeling ( r = - 0.32, P=O.OOl). For each sample of four embryos, between 21 O/o and 52% of their final combined mass was gained during labeling. There was a significant negative correlation between the combined mass of the embryos before labeling
Two-dimensional protein profiles of maize
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and relative gain of the embryos during labeling ( r = - 0.38, P = 0.0001) indicating that larger embryos tended not to gain any more than smaller embryos. There was no correlation between the combined mass of the embryos before labeling and combined mass gained during labeling. 3.2 Tests of the protein extraction procedure Repeated assays of the TCA-precipitable radioactivity in the acetone supernatant revealed 1000-fold less labeled protein in the acetone than in the sample. Samples often contained 100 000 cpm/pL of TCA-precipitable radioactivity. Proportional amounts of acetone supernatants contained an average of 100 cpm. Silver stained gels of samples prepared with or without protamine sulfate revealed clear differences. In those without protamine sulfate, numerous horizontal gray streaks extended across the gels. The basic, high molecular weight quadrant of the gels appeared to be
Figure 3. Red crosshairs mark the location of over 1500 spots on the synthetic standard image used to match the 67 h a ges of inbred lines of maize.
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most consistently amicted, although the acidic low molecular weight quadrant had some shorter streaks. Samples prepared with protamine sulfate had a much clearer background due to the almost complete absence of gray streaks. The extraction procedure appeared to be effective in preventing degradation due to proteinase activity. This was determined in three different ways. In the first test, silver stained gels of part of two samples which had been incubated at 37 “C for 3 h prior to electrophoresis were compared with silver stained gels of part of two samples which had been warmed to 37 “Cimmediately prior to electrophoresis. The pairs of protein profiles were essentially identical with no preferential loss of high molecular weight proteins or visible degradation of the protein profiles. No proteinase activity in the samples was detected using Azocoll dyebound collagen substrate (Azocoll is a trademark of Calbiochem, San Diego, CA) even when the reaction was allowed to proceed 30 min at 37°C. When protein extraction solution was used instead of phosphate buffer, no proteinase activity was detected in any of the positive controls either. Proteinase K and trypsin were used as positive controls.
the pairs were generally darker than the others. If only the “short” list had been used which did not include the darker image pairs, then a few spots would have been included which varied within the inbred lines represented by the darker pairs. Similarly, some spots would have been included that were inconsistent in some image pairs if only the logtransformed data had been used for the ANOVAs. If only the “long” list had been used, then a few spots would have been included, based on image density rather than genotypic differences. Figure 2 shows the distribution of the sums of densities and the distribution of total spot number for the 67 fluorographs of the inbred lines. As can be seen in Fig. 2, the densest image is fourteen times denser than the faintest image. With the exception of one image, however, this translates only into a threefold difference among the images for total number of spots.
Associations among inbred lines were studied using fewer than four ANOVA’s per spot. The best results were obtained using the four ANOVAs listed in Section 2.5 in combination. Two lists of image pairs were used because some of
The total density and total spot number of three different images are listed in Fig. 2. For those three gels, the dark exposure of two of them (23598.3 and 23220.3) and the medium exposure of one of them (23540.2) appear to be more comparable to the rest of the images. It was found that the subsets of spots, selected such that every spot had at least 80% of its variation partitioned among inbred lines rather than within lines, produced results as concordant or more concordant with pedigree data than other selection criteria. A schematic illustration of, and a discussion of, the pedigree data for most of the inbred lines included in this study may be found in Smith etal. [20]. Values less than 80%,
A
B
3.3 Computer manipulation and analysis
Figure 4. (A) Triangles mark the location of 108 spots selected using an 80% criterion through 4 ANOVAs on the untrimmed data. Film 23184.2. (B) Squares mark the location of 114 spots selected using an 80% criterion through 4ANOVAs on the trimmed data. Film 23184.3.The letter Aidentifies
those spotswhich are present in one spot set but not the other.Mo1ecularmasses in kilodaltons are indicated on the right.Apparent isoelectric points are indicated on the bottom. The density difference between the medium and dark exposure can be seen in this figure as well.
Two-dimensionalprotein profiles of maize
Electrophoresis 1991, 12, 425-431
where more spots were included in the subsets but more variation was included within lines, yielded less satisfactory results. Values greater than 80% produced results which were as concordant with pedigree data as the results using 80%, but utilized fewer spots.
3.4 Spot set composition Over 1500 different protein spots were documented in the 37 inbred lines and one hybrid included in the dataset. Figure 3 shows the location of the spots on the synthetic image used to match the gels. Using the method of spot selection described above, 108protein spots were included in the untrimmed set of spots and 114 protein spots were included in the trimmed set of spots which display significant differences among inbred lines of maize. Figure 4 shows the location of spots in both sets. The two sets share 104 spots. Both spot sets are distributed evenly, relative to local spot density, over the protein profile.
4 Concluding remark., The results of the protein extraction procedure tests presented here indicate that the protocol does not contribute significantly to the variability seen on 2-D gels of corn seedling proteins. The variability generated due to quantitative loss of protein into the acetone supernatant during protein extraction appears to be minimal. The removal of nucleic acids by protamine sulfate precludes distortion of the profile due to 1heirpresence.There appears to be little ifanyvariability generated by the action of proteinases in situ. Not only does the extraction solution prevent proteinase activity, but the acetone extraction appears to be an effective inhibitor of activity as well. Furthermore, samples appear to be stable at 37 "C for at least 3 h. Samples can be stored frozen and warmed forreuse many times without risk of degradation. It appears that 2D protein profiles will be useful in determining genetic associations among many inbred genotypes of maize even when gels are run months apart. Based on our stringent spot selection criteria, over 100 spots were included in 2 different subsets. Preliminary analyses show that these subsets do indeed separate and associate inbred lines in a manner concordant with their pedigrees. For example, inbred lines, A632, B64, A-3, and C-4, were shown to be more closely related to each other than to any other inbred line, a result which agrees with their pedigrees. Likewise, inbred lines, A-4, B-4, C-3, D-8, and D-9, were shown to be more closely related to each other than to any other inbred line which reflects their pedigrees. In general, correlations between pedigree data and genetic similarity based on different sets of 2D-PAGE gels and different 2DPAGE spot sets were consistently above 0.70 (P= 0.0001). There are compelling reasons for using this technique in genetic and numerical taxonomic studies. Given good quality gels and fluorographs, these complex molecular profiles appear to be stable enough and reproducible enough to match gels run months apart. The effects of different genotypes of maize on the profile are obvious and measurable even when closely related (95 %-97% by pedigree) genotypes are compared. This technique samples a large portion of the
43 I
total genome. The only other technique which samples as large a portion of the genome is the restriction fragment length polymorphism (RFLP) technique. The two techniques compliment each other in that one samples DNA polymorphisms while the other samples polymorphisms of gene products. Not only are polymorphisms at structural loci revealed through 2D-PAGE of proteins, polymorphisms at regulatory loci are also revealed (through quantitative differences). Such insights are not possible with RFLP data. The 2D-PAGE data appear amenable to traditional methods of numerical taxonomic analysis, such as cluster analysis. Additional studies, utilizing this dataset, are in progress. The authors would like to thank Heidi Sacco of Cold Spring Harbor Laboratory, Cold Spring Harbor, N Y for her electrophoretic and fluorographic expertise, Liza Chan of Protein and DNA Imageware Systems, Huntington Station, NYfor her computer skills, and Steve Wall of Pioneer Hi-Bred International, Inc., Johnston, IA for help with the analysis. Editorial assistance was provided by Steven Briggs, Alan Orr, Gura Rao, and two anonymous reviewers. Received October 18, 1990
5 References [l] O'Farrell, P. H., J. B i d . Chem. 1975, 250, 4007-4021. [2] Colas des Francs,C.andThiellement,H., Theor.Appl. Genet. 1985,7/, 31-38. [3] Bahrman N. and Thiellement, H., Theor. Appl. Genet. 1987, 74,218223. [4] Gottlieb, L. D. and de Vienne, D., Genetics 1988, 119, 705-710. [5] Bahrman, N., Zivy, M. and Thiellement, H., Heredity 1988,61,473480. [6] Beckstrom-Sternberg, S. M., Biochem. Syst. Eco. 1989, /7,573-582. 171 Basha, S. M. M., Plant Physiol. 1979, 63, 301-306. [8] Bahrman, N., de Vienne, D., Thiellement, H. and Hofmann, J.-P., Biochem. Getter. 1985, 23, 247-255. [9] Dunbar, B. D., Bundman, D. S. and Dunbar, B. S., Electrophoresis 1985, 6,39-43. [lo] Damerval, C., de Vienne, D., Zivy, M. and Thiellement, H., Electrophoresis 1986, 7, 52-54. [ l l ] Holloway, P. J. and Arundel, P. H., Anal. Biochem. 1988, 172, 8-15. [I21 Ramagopal, S., Theor. Appl. Genet. 1990, 79, 297-304. [13] Bahrman, N. and Damerval, C., Herediy 1989, 63,267-274. (141 Leonardi, A,, Damerval, C. and de Vienne, D., Genet. Res., Camb. 1987, 50, 1-5. [15] Leonardi, A , , Damerval, C. and de Vienne, D., Genet. Res., Camb. 1988, j2,97-103. [16] de Vienne, D., Leonardi, A. and Damerval, C., Electrophoresis 1988, 9,742-750. [17] Damerval, C., Hebert,Y. and de Vienne, D., Theor. Appl. Genet. 1987, 74, 194-202. [18] Higginbotham, J. W., Smith, J. S. C. and Smith, 0. S., Maize Genet. Coop. Newsl. 1989, 63,84-85. 1191 Higginbotham, J. W., Smith, J. S. C. and Smith, 0. S . , Maize Genet. Coop. Newsl. 1989, 63, 85. [20] Smith,O. S., Smith, J . S. C., Bowen, S. L.,Tenborg, R . 4 . and Wall, S. J., Theor. Appl. Genet. 1990, 80, 833-840. [2l] Smith, J. S. C. and Smith, 0. S., Maydica 1989, 34, 151-161. [22] Mayer, J.E.,Hahne, G.,Palme,K. and Schell, J., Plant CellRep., 1987, 6, 77-8 1. [23] Higginbotham, J. W. and Smith, J. S. C., Maize Genet. Coop. Newsl. 1989, 63, 83-84. [24] Garrels, J. I., Methods Enzymol. 1983, IOO, 411-423. [25] Garrels, J. I., Farrar, J.T. and Burwell, C. B., in: Celis, J. E. and Bravo, R., (Eds.), Two-dimensional Gel Electrophoresis of Proteins,Academic Press, New York 1984, pp. 37-91.
