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Isolation, partial purification and differential DNA-binding properties of putative high-mobility-group proteins from rice
Plant Science 139 (1998) 117 – 129
Isolation, partial purification and differential DNA-binding properties of putative high-mobility-group proteins from rice Keng-Hock Pwee a,*, Sharon Joanne Ooi a, R. Manjunatha Kini b a
Department of Biological Sciences, National Uni6ersity of Singapore, Lower Kent Ridge Road, Singapore 119260, Singapore b Bioscience Centre, National Uni6ersity of Singapore, Lower Kent Ridge Road, Singapore 119260, Singapore Received 16 June 1998; received in revised form 5 September 1998; accepted 7 September 1998
Abstract Putative high-mobility-group (HMG) proteins were isolated from 10 – 11-day-old rice (Oryza sati6a L. cv. IR36) shoots and roots, and from ungerminated rice grains. Nine proteins below 30 kDa which fulfilled the criteria for HMG proteins were identified from rice shoots and subjected to reversed-phase perfusion chromatography as an initial purification step. Southwestern hybridisation analysis established novel selective and differential binding of individual pea, wheat and rice HMG proteins to A/T- and G/C-rich DNA probes. Gel retardation assays using sequences derived from the pea plastocyanin and oat phytochrome A3 gene promoters have suggested that rice shoot HMG protein extracts contain a DNA-binding protein factor resembling PCF1 from pea in its binding characteristics, and that this factor is closely related to the HMG-I/Y-like PF1 protein from rice. An analysis of fractionated rice shoot HMG proteins suggests that specific promoter-binding activity resides in the 26 kDa HMGa protein. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: A/T-rich sequences; DNA-binding proteins; G/C-rich sequences; High-mobility-group proteins; Oryza (chromosomal proteins); Southwestern hybridization
1. Introduction Despite the relative abundance of high-mobility-group (HMG) proteins in chromatin, their role within the nucleus has yet to be determined conclusively. Lacking a functional framework for * Corresponding author. Tel.: +65-874-2787; Fax: +65779-5671; e-mail: [email protected].
classification, HMG proteins continue to be defined operationally as small (B 30 kDa) proteins rich in basic and acidic amino acid residues, which are extracted from chromatin with 0.35 M NaCl and are soluble in 2% trichloroacetic acid or 2–5% perchloric acid [1]. Based on operational criteria, plant HMG proteins have been isolated from a variety of sources, including wheat germ [2], barley leaves
0168-9452/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 8 ) 0 0 1 7 6 - 9
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[3,4], Arabidopsis leaves [5], maize endosperm and leaves [6–8], soybean leaves and nodules [9], pea embryos [10] and shoots [11], Vicia faba cotyledons [12], and broccoli heads [13]. These direct extracts of plant tissue have yielded HMG proteins from 12–28 kDa on SDS-PAGE, often with the most abundant HMG proteins \ 14 kDa. The four archetypal HMG proteins from wheat germ have been subjected to analyses of amino acid composition, peptide mapping [2] and partial N-terminal protein sequencing [14], revealing no persuasive similarities to animal HMG proteins. Vertebrate HMG proteins have been subdivided into three groups based on conserved primary motifs, viz the HMG 1/2, the HMG 14/17 and the HMG I/Y families [15]. Sequencing of plant cDNA clones for HMG proteins obtained by Western, Southwestern or homology screening has revealed two sets with sequence identity to characteristic HMG protein motifs (reviewed in [16]). Plant HMG 1/2 homologues contain a highly acidic carboxyl-terminal domain and a single DNA-binding ‘HMG box’ (compared to two in mammals), while the proline-rich plant HMG I/Y homologues possess sequence similarity at the amino-terminus to plant H1 histones and contain three to four repeats of the ‘A/T-hook’, a DNAbinding motif which interacts with the minor groove of A/T-rich DNA. Several reports of the DNA-binding ability of plant HMG proteins exist. Wheat HMG proteins are able to interact with nucleosomes, although only HMGa does so with apparent specificity [17]. Plant HMG proteins also bind to the promoter regions of several genes, including seed-specific genes [7,18–20], photosynthesis-associated genes [11,18,21], soybean nodulin [9] and heat shock [22] genes, and intrinsically-bent regions from the intergenic spacer of plant rRNA genes [23,24]. Many of the proteins responsible are thought to be HMG I/Y homologues, which interact with runs of \5 A/T bp in mammalian genes [25]. HMG I/Y is known to function as a transcription factor in humans, stimulating the binding of NFkB and ATF-2 to the promoters of the interferonb [26,27] and E-selectin [28] genes for the activation of transcription. Plant HMG 1/2 ho-
mologues are also known to bind to plant promoters [18,21,23,29,37], although the binding specificity is uncertain. Like mammalian HMG 1/2, the maize homologue HMGa has been shown to display a similar affinity for distorted DNA structures such as four-way junctions or DNA minicircles, and also mediates DNA bending [29]. The small genome of rice and its relatively larger size compared to Arabidopsis make it appropriate for both molecular and biochemical studies of HMG proteins. Partial and complete cDNA clones for HMG 1/2 (GenBank Accession No. D41834, Sasaki, T., Miyao, A., and Yamamoto, K., unpublished) and HMG I/Y [30] homologues exist for rice, but little is known about the proteins from chromatin extracts. It is possible that a rice system may allow the future identification and cloning of novel HMG proteins, as well as facilitate molecular genetic and functional studies of existing clones. Three protein bands B 25 kDa from rice embryo nuclear extracts have been attributed to HMG proteins [31], but as the operational criteria were not applied to the extraction of these proteins, their identity is uncertain. In this paper, we describe the isolation of putative HMG proteins from rice shoots, roots and grains which meet the operational criteria and characterise their differential DNA-binding properties, as well as demonstrate the relationship of the HMG-I/Y-like PF1 protein from rice [30] to PCF1 from pea [11].
2. Materials and methods
2.1. Plant materials and growth conditions Grains of Oryza sati6a L. cv. IR36 (International Rice Research Institute, Phillipines) were immersed in water for 20 min before being sown on a double layer of wet muslin. Germinating grains were maintained at 26°C and a 16 h light/8 h dark photoperiod at an average light intensity of 30 mmol photons m − 2 s − 1 in a growth cabinet (model MLR-350T, 3500HT, Sanyo, Japan). Seedlings were fertilised every 3rd day with Trimogreen-63 (N:P:K ratio 21:21:21, Tri Products, Singapore) and harvested for shoots (averaging 8
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cm in height) and roots after 10 – 11 days. Raw Australian wheat germ (embryos) was obtained from a commercial source (Origins Healthcare, Singapore). Peas (Pisum sati6um L. cv. Dundale) were farm grown (Golden Mushroom Enterprise, Singapore) and sown on a mixture of mushroom compost and sawdust after an overnight immersion of seeds in water. Pea shoots were harvested after 8 days of growth at a maximum of 32°C and a minimum of 23°C, and a 12 h light/12 h dark photoperiod at an approximate daylight intensity of 80–100 mmol photons m − 2 s − 1. All harvested tissues were frozen and stored at − 80°C.
2.2. Preparation of HMG proteins Putative HMG proteins were prepared according to [11] with minor adaptations to allow for differences in tissue. An additional step of hand grinding in liquid nitrogen was included for rice (a particularly fibrous material) and pea tissues before homogenisation using a Polytron PT-DA 3020/2 TS aggregate and PT 3000 machine (Kinematica, Switzerland) at a setting of 7 for 10 min and a ratio of 100 g tissue to 300 ml buffer. Ungerminated rice grains were first soaked in water for 1 h before grinding in liquid nitrogen and homogenisation. Wheat germ was homogenised directly in buffer. A low salt wash of purified chromatin was omitted for rice tissues to maximise yield, but included for pea and wheat chromatin. The 60 min precipitation period using 2% trichloroacetic acid was retained for pea and wheat extracts, but increased to 90 min for extracts from rice tissues to reduce contamination by high molecular weight proteins. The yield for rice shoots was about 200 mg of protein for every 100 g of rice tissue.
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2.4. Gel retardation assays Gel retardation assays were performed in 10– 20 ml of binding buffer (25 mM Hepes-KOH pH 7.6, 40 mM KCl, 1 mM EDTA, 1 mM DTT, 10% (v/v) glycerol) in 1% agarose gels using 3–9 ng of [32P]-labelled probe and 10 mg of poly(dIdC).poly(dIdC) as non-specific competitor in each reaction according to [11].
2.5. Southwestern hybridisation analysis Proteins were separated on an 18% denaturing gel by SDS-PAGE [32] and transferred to nitrocellulose by electroblotting [33]. The electrophoresed gel and nitrocellulose membrane were equilibrated in electroblotting buffer (10 mM 3-[Cyclohexylamino]-1-propane-sulfonic acid (CAPS)-NaOH pH 11, 10% methanol) for 5 min before transfer in a Bio-Rad Trans-Blot Cell (Bio-Rad) at 150 mA for 140 min at 4°C. Washing of the blotted membrane and hybridisation to [32P]-end-labelled probe was performed in binding buffer + 0.02% (w/v) bovine serum albumin + 0.02% (w/v) Ficoll 400 +0.02% (w/v) polyvinylpyrrolidone PVP-360 according to [11], except that 30 mg ml − 1 of degraded herring sperm DNA (Sigma, USA) was included as nonspecific competitor during the relevant hybridisation steps, and that 0.06 ml buffer per cm2 of membrane was used during the stationary hybridisation. After autoradiography, hybridised probe was removed by washing membranes for 3× 20 min in a total of 100 ml of 1 M NaCl, so that reprobing of the membrane could take place.
2.6. Molecular biology techniques 2.3. Re6ersed-phase perfusion chromatography Reversed-phase chromatography was carried out on a BioCAD perfusion chromatography workstation using a POROS R2/H 4.6 mm D × 100 mm L reversed-phase column (PerSeptive Biosystems, USA).
Standard DNA manipulations were performed according to [34]. DNA fragments were isolated from agarose gels using the QIAquick gel extraction kit (QIAGEN, Germany) and radioactively-labelled probes were separated from unincorporated nucleotides by passage through NucTrap columns (Stratagene, USA).
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3. Results
3.1. Extraction of rice HMG proteins Putative HMG proteins were extracted from 10 – 11-day-old rice shoots and roots, and from ungerminated rice grains by adapting a protocol for pea shoots [11]. The putative rice HMG proteins were subjected to 18% SDS-PAGE, and the polypeptide profiles compared to that of previously described HMG proteins from wheat germ [2] and pea shoots [11], as well as that of pea histones (Fig. 1, lanes 1 – 4). All samples contained a few proteins above 30 kDa, but these were, by definition, not belonging to a ‘high-mobility group’. Nine major proteins from 13 – 26 kDa were identified in extracts from rice shoots and labelled HMGa to HMGf (Fig. 1, lanes 4 and 5). Although appearing as a wide band in Fig. 1, rice HMGb could be resolved into two bands (b1 and b2) on long (15×17.5 cm) gels (data not shown). The rice proteins were named partly with reference to the electrophoretic mobility of the four archetypal HMGa, b, c and d proteins from wheat germ (Fig. 1, lane 3); thus
Fig. 1. Coomassie-stained 18% SDS-PAGE of plant HMG proteins. Each lane contained 8 mg of protein. Lane 1, pea shoot histones; subsequently HMG proteins from, lane 2, pea shoots; lane 3, wheat germ; lanes 4 and 5, rice shoots; lane 6, rice roots, and lane 7, rice grains. Specific bands of rice shoot HMG proteins are indicated in Greek letters. Major wheat germ HMG proteins are labelled directly, a–d. Molecular mass markers are given in kDa to the right and left of the figure which is derived from two independent gels. Some bands were too faint for photographic reproduction.
rice HMGa was closest in electrophoretic mobility to wheat HMGa, HMGb1 and b2, to wheat HMGb and so on. The polypeptide profile from rice shoots was substantially different from that of rice grains and roots (Fig. 1, lanes 6 and 7), reflecting either organ-specific expression of rice HMG proteins or differences in protein degradation in different tissues. In general, putative rice grain HMG proteins were of low molecular weight, with two bands of the same mobility as shoot HMGd2 and HMGo. At least eight protein bands were discernible in root extracts, four with similarities in mobility to shoot HMGb, d2, o and f. Further purification of putative rice HMG proteins was focused on shoot extracts because of the relative abundance of the proteins, similar mobility to wheat HMG proteins and the tendency of root polypeptides to degrade.
3.2. Re6ersed-phase perfusion chromatography Putatitive rice shoot HMG proteins were subjected to reversed-phase perfusion chromatography as an initial purification step in the analysis of individual proteins. Fig. 2(a) shows the elution profile of an optimised protocol, with the bulk of proteins eluting between about 28 and 44% of acetonitrile. Fig. 2(b) displays the SDS-PAGE analysis of samples encompassing protein-containing fractions. Concentration of proteins in individual fractions occasionally allowed some bands not seen in the whole shoot HMG extract to be visualised. The bulk of high molecular weight contaminants elute in fractions 3 and 4 (too faint for visualisation in Fig. 2(b), but commonly observed in this and replicate runs), along with a subset of HMG proteins. Most of the HMG proteins elute in fractions 12–19, apparently free of contaminants \ 30 kDa. The g2 band elutes almost purified as a single silverstained polypeptide from fractions 21–26. Many HMG proteins of the same apparent electrophoretic mobility elute both early in fractions 3–4 and again in fractions 12–19 or 21–26. However HMGf only exists in fraction 3, and HMGa is found only in fractions 3 and 4.
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3.3. Gel retardation assays Gel retardation assays were used to investigate the DNA-binding characteristics of putative rice HMG proteins in comparison with pea and wheat HMG proteins. The probe used was P268, an A/T-rich positive regulatory fragment derived from the pea plastocyanin gene promoter [35] which has been shown to bind a pea HMG-I/Ylike factor [11]. Rice shoot, wheat germ and pea shoot HMG proteins formed two complexes with P268: a distinct complex of higher mobility, and a weaker, less distinct complex of lower mobility (Fig. 3a, lanes 2–4). Rice root proteins completely retarded P268 to a broad band with a mobility similar to that of the shoot complexes (Fig. 3a, lane 5), presumably because of the higher concentration of DNA-binding proteins present and even though the reaction was carried out in the presence of 10 mg of the non-specific competitor poly(dIdC).poly(dIdC). Rice grain proteins did not exhibit any P268-binding activity (Fig. 3a, lane 6). The binding characteristics of rice shoot HMG extracts were examined (Fig. 3b). The complex
Fig. 2.
Fig. 2. Fractionation of rice shoot HMG proteins (570 mg) using reversed-phase perfusion chromatography. Proteins dissolved in NEBM buffer (25 mM Hepes-KOH pH 7.6, 40 mM KCl, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, 10% (v/v) glycerol) were made up to 1 ml with Milli-Q water, applied to the column equilibrated with 65% buffer A (0.1% trifluoroacetic acid), 35% buffer B (80% acetonitrile), and eluted at 5 ml min − 1. The acetonitrile gradient used was as follows: from 0 to 8 min after sample injection, a linear gradient from 65% A, 35% B to 45% A, 55% B; from 8 – 12 min, a linear gradient from 45% A, 55% B to 100% B; from 12 to 12.5 min, 100% B was maintained, and from 12.5 to 13 min, a linear gradient from 100% B to 65% A, 35% B. Two ml fractions were collected and lyophilized. (a) Elution profile as detected at 215 nm (solid line). The acetonitrile gradient is represented by % buffer B (dotted line). (b) Silver-stained [39] 18% SDSPAGE of fractions. Lyophilized protein fractions were each resuspended in 60 ml of NEBM and 5 ml was loaded for each fraction. Fraction numbers are indicated below each track; R, unfractionated rice shoot HMG proteins (5 mg). Note that b2, g2 and o bands are negatively stained with silver when protein is in excess. Molecular mass markers are given in kDa on the left. Low-mobility-group proteins ( \ 30 kDa) were too low in abundance to be visible in protein fractions after chromatography, and these portions of the gels were omitted in the diagram. Some bands were too faint for photographic reproduction.
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corresponded to the activity observed in unfractionated rice shoot HMG extracts. An HMG-I/Y homologue from rice, PF1, which binds to the oat phytochrome A (PhyA) promoter has been cloned [30]. The region of the promoter to which PF1 binds is an A/T-rich positive cis-element designated PE1 (position −367 to − 346). Specificity of recombinant PF1 for PE1 was demonstrated in gel retardation assays where a single bound complex was detected using an oat PhyA promoter fragment containing PE1, but not in similar promoter fragments carrying linker substitution mutations [30]. In order to investigate the relationship of the rice PCF1 homologue to rice PF1, we conducted gel retarda-
Fig. 3. (Continued)
could not be competed out by a 143-fold molar excess of unlabelled P268 (Fig. 3b, lane 3), similar to previous studies using pea HMG proteins [11] and indicating an abundant protein, but the binding factor was selective for A/T-rich regions as it could be competed out by 10 mg ( : 1430-fold mass excess) of the DNA alternating copolymer poly(dAdT).poly(dAdT), but not an equivalent amount of poly(dGdC).poly(dGdC) or poly(dIdC).poly(dIdC) (Fig. 3b, lanes 4 – 6). Heat treatment to 90°C did not alter the binding ability of the factor, which was protein in nature as proteinase K treatment of HMG extracts destroyed complex formation (Fig. 3b, lanes 7 and 8). The binding characteristics mimic exactly the behaviour of PCF1, the HMG-I-like protein in pea shoot HMG extracts which binds to P268 [11]. Thus rice shoot HMG extracts may contain the rice homologue of PCF1. After fractionation using reversed-phase chromatography, P268-binding activity was found to be contained only in fractions 3 and 4 (Fig. 3c). A high-concentration of binding activity occurred in fraction 3, causing near-complete retardation of the probe in gel shift assays; the mobility shift observed using fraction 4
Fig. 3. Gel retardation assays of plant HMG proteins. (a) Retardation of pea plastocyanin gene promoter fragment P268 [11] using plant HMG proteins. Lane 1, [32P]-labelled P268 (2000 dpm; 7 ng) in absence of protein; subsequently HMG proteins included from: lane 2, pea shoots (1.3 mg); lane 3, wheat germ (0.5 mg); lane 4, rice shoots; lane 5, rice roots, and lane 6, rice grains (1.8 mg each). Protein loadings were chosen to give similar patterns of retardation. Bound complexes are indicated by arrows. (b) Binding characteristics of rice shoot HMG proteins with P268. Except for lane 1, each track contained 1.8 mg rice shoot HMG proteins. The various molecular species of competitor DNA included was in addition to the 10 mg of non-specific poly(dIdC).poly(dIdC) already present in all gel retardation reactions. Lane 1, [32P]-labelled P268 (2700 dpm; 7 ng) in absence of protein; lane 2, + rice shoot HMG proteins alone; lane 3, + 1 mg of unlabelled P268 as specific competitor ( :143-fold molar excess); lane 4, + 10 mg of poly(dAdT).poly(dAdT); lane 5, +10 mg of poly-(dGdC). poly(dGdC); lane 6, +10 mg of poly(dIdC).poly(dIdC), lane 7, rice shoot HMG proteins were heat-treated (90°C, 10 min) before [32P]P268 was added, and lane 8,+1 mg proteinase K (incubated for 20 min at room temperature with HMG proteins before [32P]P268 was added). (c) Detection of P268-retarding activity in reversed-phase purified fractions of rice shoot HMG proteins. [32P]-labelled P268 fragments (1900 dpm; 7 ng each lane) were incubated with 1 ml aliquots of each resuspended fraction from Fig. 2(b). Only fractions containing visible proteins on SDS-PAGE were assayed and only the gel containing retardation activity is shown. Previous fractionations indicate no retardation using fractions without visible protein (data not shown). Fraction numbers are indicated below each track, no protein added, R, unfractionated rice shoot HMG proteins added (1.8 mg). (d) Binding specificity of rice shoot HMG proteins to normal (PE1) and mutant (OT-614, OT-615, OT616) oligotrimers derived from the oat phytochrome promoter [30]. Oligotrimer fragments ( :1700 dpm; : 7 ng per track) were incubated in the presence ( +) or absence ( −) of 0.9 mg of rice shoot HMG proteins.
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tion assays using rice shoot HMG extracts together with oligotrimers of PE1 (OT-PE1) and the same linker substitution mutations (OT-614, OT615 and OT-616) in the PF1 study as probes. Fig. 3(d) demonstrates that a single bound complex is formed with OT-PE1, but not its mutations when rice shoot HMG extracts were added, indicating that PF1 was present with the same binding specificity. When the binding characteristics of the OT-PE1/PF1 complex were examined, the same binding selectivity, heat stability and proteinase K sensitivity was observed as for rice PCF1 in Fig. 3(b) (data not shown), suggesting that the identities of PF1 and the rice PCF1 homologue may be closely related.
3.4. Southwestern hybridisation studies Southwestern analysis had previously identified a 21 kDa protein in size-fractionated pea HMG protein extracts which bound to P268 [11]. The same approach was used here to define P268binding rice HMG proteins. As the protein factor PF1/PCF1 was selective for A/T-rich DNA (Fig. 3b), we also examined the interaction of plant HMG protein extracts with poly(dAdT).poly(dAdT), and compared the pattern obtained with that using poly(dGdC).poly(dGdC) and poly(dIdC).poly(dIdC). Fig. 4 shows the hybridisation patterns of the different plant HMG protein extracts. When probed with P268, there was virtually no hybridisation in the pea histone track (Fig. 4a, lane 1), demonstrating that binding activity in HMG protein extracts was unlikely to be due to contaminating histones. A faint band of :27 kDa may instead have reflected HMG protein contamination of pea histones, as a 27 kDa hybridising species is a major band in pea shoot HMG proteins, along with bands of 23, 21 and 19 kDa (Fig. 4a, lane 2). All four wheat embryo HMG proteins hybridised to P268, although preferential labelling of HMGa and b is observed (Fig. 4a, lane 3). P268 labelled a strong 26 kDa band and a reasonably intense band of 34 kDa in rice extracts (Fig. 4a, lane 4). No proteins are labelled in rice root or grain HMG protein extracts (Fig. 4a, lanes 5 and 6).
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The pattern obtained by hybridising the same membrane (after washing with high salt) to poly(dAdT).poly(dAdT) was substantially similar to that obtained with P268 (Fig. 4b). However wheat HMGa and b show a strong selectivity for A/Trich DNA, whereas HMGc and d are bound relatively weakly (Fig. 4b, lane 3). Taken together, the inference is that A/T-rich regions of P268 are likely to be responsible for much but not all of the binding observed with P268. Hybridization to poly(dGdC).poly(dGdC) produced a pattern distinct from that obtained by binding to A/T-rich regions. Fig. 4(c) shows that in the pea HMG protein track (Fig. 4c, lane 2), few, if any, of several distinct bands corresponded to that obtained with hybridisation to A/T-rich probes. In strong contrast to the hybridisation with poly(dAdT).poly(dAdT), wheat HMGc and d are strongly labelled while HMGa and b are not (Fig. 4c, lane 3). As in Fig. 4(a and b), a 34 kDa albeit weaker band was labelled for rice shoots, but the strong band observed at 26 kDa under A/T-rich hybridisation was replaced by weak bands at 27, 28 and 29 kDa (Fig. 4c, lane 4). Rice root HMG proteins appear to contain several G/C-binding proteins (Fig. 4c, lane 5), with bands at 27 and 29 kDa coinciding with rice shoot HMG proteins. Interestingly, the major labelled proteins from hybridisation with A/T-rich probes are only weakly labelled, if at all, with poly(dGdC).poly(dGdC), which demonstrates a strong selectivity of DNA-binding of proteins in HMG protein extracts. The pattern after hybridisation with poly(dIdC).poly(dIdC) reveals bands which were labelled also in prior hybridisation’s with A/T- or G/C-rich probes (Fig. 4d). All four wheat HMG proteins are labelled relatively evenly (Fig. 4d, lane 3). The pea HMG lane is more complex, containing bands of similar mobility to bands labelled with either A/T- or G/C-rich probes (Fig. 4d, lane 2). This implies that poly(dIdC).poly(dIdC) has characteristics resembling both poly(dGdC).poly(dGdC) and poly(dAdT).poly(dAdT), thus enabling binding to proteins with either selectivity. However the presence of distinct 19 and 22 kDa bands in the rice shoot HMG protein track (Fig. 4d, lane 4) not
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Fig. 4. Southwestern analysis of plant HMG proteins. Each gel lane contained 8 mg of protein before transfer to a nitrocellulose membrane. The same membrane was hybridised separately with 4.8 × 105 dpm of [32P]-end-labelled (a) P268 (b) poly(dAdT).poly(dAdT) (c) poly(dGdC).poly(dGdC) (d) poly(dIdC).poly(dIdC). Lane 1, pea shoot histones, subsequently HMG proteins from: lane 2, pea shoots; lane 3, wheat germ; lane 4, rice shoots, lane 5 rice roots and lane 6, rice grains. Molecular mass markers are given in kDa to the right of each diagram. The position of where HMGa might run under SDS-PAGE is marked with an ‘a’ in each panel.
seen in Fig. 4(b or c, both lanes 4) suggests that poly(dIdC).poly(dIdC) also has its own unique structural characteristics which are recognised by rice nuclear proteins. In order to identify the protein species involved, Southwestern hybridisation studies were performed on reversed-phase purified rice shoot HMG fractions which contained visible protein bands after silver-staining (Fig. 2b). Binding activity was limited to fractions 3 and 4 (Fig. 5, data not shown for other non-binding fractions), the same fractions which demonstrated P268-binding activity in gel retardation assays (Fig. 3c). Several
DNA-binding species were identified binding to the various probes, but fractions 3 and 4 appeared between them to contain all the DNA-binding species found in the control lane (R) of unfractionated rice shoot HMG proteins (Fig. 5). Moreover fraction 3 contained additional DNA-binding bands not observed in the control lane in all hybridisation’s, probably as a result of the concentration of low abundance DNA-binding protein species during the reversed-phase fractionation. Fraction 4 contained considerably fewer DNA-binding species than fraction 3 in all hybridisation’s. Better transfer of rice shoot
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Fig. 5. Southwestern analysis of reversed-phase fractionated rice shoot HMG proteins. Resuspended proteins (7 ml per fraction) from fractions containing visible protein bands from Fig. 2(b) were subjected to electrophoresis and transfer. Only the membrane displaying hybridising bands is shown. The same blotted nitrocellulose membrane was hybridised separately with 4.8 ×105 dpm of [32P]-end-labelled (a) P268 (b) poly(dAdT).poly(dAdT); (c) poly(dGdC).poly(dGdC) (d) poly(dIdC).poly(dIdC). Fraction numbers are indicated below each track, R, unfractionated rice shoot HMG proteins (8 mg). Molecular mass markers are given in kDa to the left of each diagram. The position of where HMGa might run under SDS-PAGE is marked with an ‘a’ in each panel.
proteins onto this membrane may also have occurred (Fig. 5a–d, all lanes R), as the labelled polypeptide bands were slightly greater in number and more distinct than for the previous membrane (Fig. 4a–d, all lanes 4). As gel retardation assays demonstrated the putative presence of HMG-I in fraction 4 (Fig. 3c), the less complex Southwestern hybridisation profile (compared to fraction 3) could aid in determining the size of the DNA-binding species involved. In all hybridisation’s, a 26 kDa protein was labelled in fraction 4 (Fig. 5). However labelling was strongest with P268 and poly(dAdT).poly(dAdT), followed by poly(dIdC).poly(dIdC), and was weakest with poly(dGdC).poly(dGdC). Thus the 26 kDa rice protein shows a strong selectivity for A/T-rich sequences, and in this respect behaves not only
like an HMG-I protein, but is also a candidate for the protein identified as PF1/PCF1 [11,30].
4. Discussion
4.1. Isolation of rice HMG proteins and organ-specific differences Nine polypeptide bands of varying abundance have been isolated from rice shoot HMG extracts which meet the operational definition of HMG proteins. However not all may be distinct protein species, as HMG proteins are susceptible to proteolytic degradation and are known to be posttranslationally modified [1]. Several putative rice HMG proteins (including possible b,g,d and o bands) of the same electrophoretic mobility elute
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early in fractions 3 and/or 4, then again in fractions 12–26. These bands may represent posttranslationally modified species (e.g. phosphorylated forms which differ in protein net charge), or be derived from distinct genetic alleles or isoforms, or be completely different proteins. The different polypeptide profiles from shoot, root and grain extracts of rice (Fig. 1) may suggest organ-specific accumulation of different HMG proteins, as has been observed in extracts of maize tissues where the HMGa and d proteins were found in the endosperm, but not in leaves [8]. However Northern and Western studies have subsequently detected maize HMGa in several different tissues including leaves and endosperm [36], suggesting that the different profiles observed in different HMG protein organ extracts may in part be accounted for by organ-specific protein degradation or the differential ease of protein extraction associated with different tissues. The presence of a retarding factor with characteristics of HMG I/Y proteins in rice shoots but not in roots or grains, implies either an organ-specificity of the PCF1/PF1 factor, or a greater lability in root or grain extracts. While the root extract had retarding ability, the extent of the bandshift became greater as more protein was added (data not shown), implying non-specific complex formation. Considerable differences also exist in the DNA-binding ability of rice shoot, root and grain HMG extracts as observed in Southwestern assays (Fig. 4). Retardation of a zein gene promoter by an HMG-like protein has also exhibited organ-specificity, but in this case, activity was present in the endosperm, embryo, stem and root, but not leaf [20]. The 5% upstream region of the winged bean Kunitz chymotrypsin inhibitor gene has however been described to bind to ubiquitous protein factors which had characteristics of HMG proteins [19]. The complex profile of rice shoot HMG proteins is reminiscent of maize leaf HMG extracts [8], rather than the four distinct bands observed for wheat germ or maize endosperm proteins. Several low molecular weight polypeptides are in evidence in rice and maize shoot HMG extracts. This contrasts with the more compact pattern obtained in legume shoot extracts
(see [9,11,12] and Fig. 1, lane 3). This may reflect greater proteolytic degradation in cereal shoots despite the presence of the protease inhibitor phenylmethylsulphonyl fluoride (PMSF) in the extraction buffers. A detailed structural analysis of or sequence data from these peptides would clarify if they were distinct proteins or degradation products, and help to ascertain if they truly belonged to the HMG family of proteins as defined by amino acid sequence.
4.2. Rice HMG protein extracts may contain a PCF1 /PF1 -like factor The binding characteristics of the protein factor responsible for the retardation of P268 suggests the presence of a rice homologue to the pea PCF1 protein in rice shoot extracts (Fig. 3b), as well as the presence of rice PF1 protein (Fig. 3d). Reversed-phase perfusion chromatography has facilitated separation of most rice HMG proteins away from high molecular weight contaminants (Fig. 2b). Specific P268 retardation activity (in the presence of excess poly(dIdC).poly(dIdC) included in each assay) was concentrated only in fractions 3 and 4, which enabled the elimination of several polypeptide bands in fractions 12–19 and 21–26 as the active candidates for PCF1/PF1. Several polypeptide species were nevertheless present in fractions 3 and 4, and the results of the Southwestern hybridisations have indicated potential candidates for the DNA-binding proteins. Fraction 3 contained the bulk of DNA-binding activity, but fraction 4, which also contained PCF1-type retardation activity and considerably fewer DNA-binding species in Southwesterns was likely to facilitate analysis (Fig. 5). The poly(dAdT).poly(dAdT) and P268 (A/T-rich) hybridisations of fraction 4 revealed a strong 26 kDa band, which was present but at lesser intensity in poly(dGdC).poly(dGdC) and poly-(dIdC).poly(dIdC) hybridisations, demonstrating the A/T selectivity of the protein. For the A/T-rich probes, the 26 kDa band was virtually the only protein labelled in fraction 4, thus indicating a strong candidate for rice PCF1/PF1. The protein is of the same electrophoretic mobility as HMGa and could possibly represent that protein.
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However caution must be advised in the interpretation of Southwestern assay results, as the membrane-bound DNA-binding species from any fraction may not reflect the full set of DNA-binding proteins available during a gel retardation assay due to non-equal transfer of proteins during blotting and the denaturation of some species during SDS-PAGE. The next most intense band in whole shoot extracts labelled with A/T-rich probes in Southwestern assays was a 34 kDa polypeptide, and this was largely contained in fractions 3 and 4. We believe that the 34 kDa band could represent the full-length PCF1/PF1 protein, and the 26 kDa band its proteolytic or processed product. This is because polyclonal antibodies raised against invitro synthesised recombinant PF1 recognise two proteins in rice nuclear extracts of 34 and 26 kDa [30]. In that study, it was speculated that the 26 kDa band was either a cleavage product derived during extract preparation, a protein which shared the same epitopes as PF1, or a mature polypeptide processed in vivo from the 34 kDa form. We believe that the 26 kDa protein/HMGa represents an authentic form of PF1 as it is the major species labelled with P268 and virtually the only A/T-selective DNA-binding protein in fraction 4. However it cannot be excluded that the proteolysis represents an artefact of sample preparation. Extensive biochemical evidence also exists for the proteolytic cleavage of HMG 1/2 and I/Y proteins from pea shoot extracts at the aminoand carboxyl-terminus respectively [37]. Data obtained in that study is consistent with the two most heavily-labelled bands of 27 and 21 kDa in A/T-probed pea shoot HMG extracts (Fig. 4a and b) representing the full-length, and proteolytically-processed, pea HMG I/Y homologue respectively.
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extracts (Fig. 1), and could be regarded as nuclear protein contaminants or minor HMG protein species, although the strong affinity and selectivity of these low abundance proteins is intriguing. Of the bands which could be associated with major HMG proteins, the strong selectivity of wheat HMGa and b for A/T-rich DNA and of wheat HMGc and d for G/C-rich DNA is of particular interest. Wheat HMGa and b have both been reported to interact specifically with A/T-rich sequences upstream of the pea ferredoxin 1 gene, whereas HMGc and d could not [18]. As wheat HMGa represents the wheat HMG I/Y homologue [16,17], it could thus be expected to bind to A/T runs of more than 5 bp [25]. Wheat HMGb represents the HMG 1/2 homologue [14] and the recombinant maize HMG 1/2 homologue is known to interact with the P2 promoter region of an A/T-rich zein gene [29]. No prior reports of G/C-selectivity have ever been ascribed to HMG proteins as observed here with wheat HMGc and d, and in rice root extracts in particular. This property might in future be used as a relatively simple biochemical test for HMG protein classification. Wheat HMGc and d have also been distinguished from HMGa and b in being preferentially released from wheat embryo nuclei by ethidium bromide treatment, a property shared by vertebrate HMG 14/17 proteins [38]. In contrast to the four distinct wheat germ HMG proteins, rice and pea shoot HMG protein extracts exhibit a greater heterogeneity of selectively-binding proteins (Fig. 4), and (except for the A/T-selective PCF1/PF1 protein) are of low abundance and difficult to characterise. A/T- and G/C-rich regions of the genome may play significant roles in nuclear organisation and activities, and the A/T- and G/Cselective chromosomal proteins described here can certainly be subjected to further investigations in this respect.
4.3. A/T-and G/C-selecti6e DNA-binding proteins Southwestern assays of rice, pea and wheat HMG proteins have detected several DNA-binding bands which are selective for either A/T- or G/C-rich probes (Fig. 4). Many of the bands do not represent major polypeptides visible by Coomassie blue or silver staining in HMG protein
Acknowledgements We wish to thank Bun Tuck Weng for technical assistance, Dr Gurdev S. Khush for his frequent gifts of rice IR36 grains, and Professor Peter H. Quail and Jim Tepperman for the gift of plasmids
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with cloned PE1 and mutant oligotrimers. This research was supported by grant RP920357 from the National University of Singapore.
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[16] K.D. Grasser, Plant chromosomal high mobility group proteins, Plant J. 7 (1995) 185 – 192. [17] L.J. Arwood, S. Spiker, Binding of wheat and chicken high mobility group chromosomal proteins to DNA and to wheat and chicken mononucleosomes, J. Biol. Chem. 265 (1990) 9771 – 9777. [18] T.J. Pedersen, L.A. Arwood, S. Spiker, M.J. Guiltinan, W.F. Thompson, High mobility group chromosomal proteins bind to AT-rich tracts flanking plant genes, Plant Mol. Biol. 16 (1991) 95 – 104. [19] Y. Habu, Y. Sakata, K. Fukusawa, T. Ohno, Ubiquitous nuclear proteins bind to 5% upstream region of major Kunitz chymotrypsin inhibitor gene in winged bean, Plant Mol. Biol. 23 (1993) 1139 – 1150. [20] I. Ponte, P. Guille´n, R.M. Debo´n, et al., Narrow A/T-rich zones present at the distal 5%-flanking sequences of the zein genes Zc1 and Zc2 bind a unique 30 kDa HMG-like protein, Plant Mol. Biol. 26 (1994) 1893 – 1906. [21] S. Yanagisawa, K. Izui, Molecular cloning of two DNAbinding proteins of maize that are structurally different but interact with the same sequence motif, J. Biol. Chem. 268 (1993) 16028 – 16036. [22] E. Czarnecka, J.C. Ingersoll, W.B. Gurley, AT-rich promoter elements of soybean heat shock gene Gmhsp17.5E bind two distinct sets of nuclear proteins in vitro, Plant Mol. Biol. 19 (1992) 985 – 1000. [23] E.A. Greiss, K.D. Grasser, G. Feix, Repeat units from a maize rDNA external spacer region exhibit DNA curvature and interact with high-mobility-group proteins, Planta 191 (1993) 524 – 531. [24] C. Kneidl, E. Dinkl, F. Grummt, An intrinsically bent region upstream of the transcription start site of the rRNA genes of Arabidopsis thaliana interacts with an HMG-related protein, Plant Mol. Biol. 27 (1995) 705 – 713. [25] M.J. Solomon, F. Strauss, A. Varshavsky, A mammalian high mobility group protein recognises any stretch of six AT base pairs in duplex DNA, Proc. Natl. Acad. Sci. USA 83 (1986) 1276 – 1280. [26] D. Thanos, T. Maniatis, The high mobility group protein HMG I(Y) is required for NF-kB-dependent virus induction of the human IFN-b gene, Cell 71 (1992) 777 – 789. [27] W. Du, D. Thanos, T. Maniatis, Mechanisms of transcriptional synergism between distinct virus-inducible enhancer elements, Cell 74 (1993) 887 – 898. [28] H. Lewis, W. Kaszubska, J.F. DeLamarter, J. Whelan, Cooperativity between two NF-kB complexes, mediated by high-mobility-group protein I(Y), is essential for cytokine-induced expression of the E-selectin promoter, Mol. Cell. Biol. 14 (1994) 5701 – 5709. [29] K.D. Grasser, A.B. Krech, G. Feix, The maize chromosomal HMGa protein recognises structural features of DNA and increases DNA flexibility, Plant J. 6 (1994) 351 – 358. [30] J. Nieto-Sotelo, A. Ichida, P.H. Quail, PF1: an A-T hook-containing DNA binding protein from rice that interacts with a functionally defined d(AT)-rich element in the oat phytochrome A3 gene promoter, Plant Cell 6 (1994) 287 – 301.
K.-H. Pwee et al. / Plant Science 139 (1998) 117–129 [31] J. Yamaguchi, P.-Y. Lim, K. Aratani, T. Akazawa, Isolation and characterisation of nuclei from rice embryos, Cell Struc. Func. 17 (1992) 87–92. [32] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of the bacteriophage T4, Nature 227 (1970) 680 – 685. [33] P. Matsudaira, Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes, J. Biol. Chem. 262 (1987) 10035–10038. [34] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edn, Laboratory Press, Cold Spring Harbour, 1989. [35] K.H. Pwee, J.C. Gray, The pea plastocyanin promoter directs cell-specific but not full light-regulated expression in transgenic tobacco plants, Plant J. 3 (1993) 437–449.
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[36] K.D. Grasser, M.M. Haaß, E.A. Greiss, G. Feix, Detection of the chromosomal HMGa protein in crude maize extracts, J. Plant Physiol. 140 (1992) 634 – 636. [37] C.I. Webster, L.C. Packman, K.H. Pwee, J.C. Gray, High mobility group proteins HMG-1 and HMG-I/Y bind to a positive regulatory region of the pea plastocyanin gene promoter, Plant J. 11 (1997) 703 – 715. [38] L.J. Arwood, E. Hill, S. Spiker, Ethidium bromide preferentially releases high mobility group proteins HMGc and HMGd from wheat embryo nuclei, Physiol. Plant. 82 (1991) 419 – 422. [39] H. Blum, H. Beier, H.J. Gross, Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels, Electrophoresis 8 (1987) 93 – 99.
Isolation, partial purification and differential DNA-binding properties of putative high-mobility-group proteins from rice Keng-Hock Pwee a,*, Sharon Joanne Ooi a, R. Manjunatha Kini b a
Department of Biological Sciences, National Uni6ersity of Singapore, Lower Kent Ridge Road, Singapore 119260, Singapore b Bioscience Centre, National Uni6ersity of Singapore, Lower Kent Ridge Road, Singapore 119260, Singapore Received 16 June 1998; received in revised form 5 September 1998; accepted 7 September 1998
Abstract Putative high-mobility-group (HMG) proteins were isolated from 10 – 11-day-old rice (Oryza sati6a L. cv. IR36) shoots and roots, and from ungerminated rice grains. Nine proteins below 30 kDa which fulfilled the criteria for HMG proteins were identified from rice shoots and subjected to reversed-phase perfusion chromatography as an initial purification step. Southwestern hybridisation analysis established novel selective and differential binding of individual pea, wheat and rice HMG proteins to A/T- and G/C-rich DNA probes. Gel retardation assays using sequences derived from the pea plastocyanin and oat phytochrome A3 gene promoters have suggested that rice shoot HMG protein extracts contain a DNA-binding protein factor resembling PCF1 from pea in its binding characteristics, and that this factor is closely related to the HMG-I/Y-like PF1 protein from rice. An analysis of fractionated rice shoot HMG proteins suggests that specific promoter-binding activity resides in the 26 kDa HMGa protein. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: A/T-rich sequences; DNA-binding proteins; G/C-rich sequences; High-mobility-group proteins; Oryza (chromosomal proteins); Southwestern hybridization
1. Introduction Despite the relative abundance of high-mobility-group (HMG) proteins in chromatin, their role within the nucleus has yet to be determined conclusively. Lacking a functional framework for * Corresponding author. Tel.: +65-874-2787; Fax: +65779-5671; e-mail: [email protected].
classification, HMG proteins continue to be defined operationally as small (B 30 kDa) proteins rich in basic and acidic amino acid residues, which are extracted from chromatin with 0.35 M NaCl and are soluble in 2% trichloroacetic acid or 2–5% perchloric acid [1]. Based on operational criteria, plant HMG proteins have been isolated from a variety of sources, including wheat germ [2], barley leaves
0168-9452/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 8 ) 0 0 1 7 6 - 9
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[3,4], Arabidopsis leaves [5], maize endosperm and leaves [6–8], soybean leaves and nodules [9], pea embryos [10] and shoots [11], Vicia faba cotyledons [12], and broccoli heads [13]. These direct extracts of plant tissue have yielded HMG proteins from 12–28 kDa on SDS-PAGE, often with the most abundant HMG proteins \ 14 kDa. The four archetypal HMG proteins from wheat germ have been subjected to analyses of amino acid composition, peptide mapping [2] and partial N-terminal protein sequencing [14], revealing no persuasive similarities to animal HMG proteins. Vertebrate HMG proteins have been subdivided into three groups based on conserved primary motifs, viz the HMG 1/2, the HMG 14/17 and the HMG I/Y families [15]. Sequencing of plant cDNA clones for HMG proteins obtained by Western, Southwestern or homology screening has revealed two sets with sequence identity to characteristic HMG protein motifs (reviewed in [16]). Plant HMG 1/2 homologues contain a highly acidic carboxyl-terminal domain and a single DNA-binding ‘HMG box’ (compared to two in mammals), while the proline-rich plant HMG I/Y homologues possess sequence similarity at the amino-terminus to plant H1 histones and contain three to four repeats of the ‘A/T-hook’, a DNAbinding motif which interacts with the minor groove of A/T-rich DNA. Several reports of the DNA-binding ability of plant HMG proteins exist. Wheat HMG proteins are able to interact with nucleosomes, although only HMGa does so with apparent specificity [17]. Plant HMG proteins also bind to the promoter regions of several genes, including seed-specific genes [7,18–20], photosynthesis-associated genes [11,18,21], soybean nodulin [9] and heat shock [22] genes, and intrinsically-bent regions from the intergenic spacer of plant rRNA genes [23,24]. Many of the proteins responsible are thought to be HMG I/Y homologues, which interact with runs of \5 A/T bp in mammalian genes [25]. HMG I/Y is known to function as a transcription factor in humans, stimulating the binding of NFkB and ATF-2 to the promoters of the interferonb [26,27] and E-selectin [28] genes for the activation of transcription. Plant HMG 1/2 ho-
mologues are also known to bind to plant promoters [18,21,23,29,37], although the binding specificity is uncertain. Like mammalian HMG 1/2, the maize homologue HMGa has been shown to display a similar affinity for distorted DNA structures such as four-way junctions or DNA minicircles, and also mediates DNA bending [29]. The small genome of rice and its relatively larger size compared to Arabidopsis make it appropriate for both molecular and biochemical studies of HMG proteins. Partial and complete cDNA clones for HMG 1/2 (GenBank Accession No. D41834, Sasaki, T., Miyao, A., and Yamamoto, K., unpublished) and HMG I/Y [30] homologues exist for rice, but little is known about the proteins from chromatin extracts. It is possible that a rice system may allow the future identification and cloning of novel HMG proteins, as well as facilitate molecular genetic and functional studies of existing clones. Three protein bands B 25 kDa from rice embryo nuclear extracts have been attributed to HMG proteins [31], but as the operational criteria were not applied to the extraction of these proteins, their identity is uncertain. In this paper, we describe the isolation of putative HMG proteins from rice shoots, roots and grains which meet the operational criteria and characterise their differential DNA-binding properties, as well as demonstrate the relationship of the HMG-I/Y-like PF1 protein from rice [30] to PCF1 from pea [11].
2. Materials and methods
2.1. Plant materials and growth conditions Grains of Oryza sati6a L. cv. IR36 (International Rice Research Institute, Phillipines) were immersed in water for 20 min before being sown on a double layer of wet muslin. Germinating grains were maintained at 26°C and a 16 h light/8 h dark photoperiod at an average light intensity of 30 mmol photons m − 2 s − 1 in a growth cabinet (model MLR-350T, 3500HT, Sanyo, Japan). Seedlings were fertilised every 3rd day with Trimogreen-63 (N:P:K ratio 21:21:21, Tri Products, Singapore) and harvested for shoots (averaging 8
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cm in height) and roots after 10 – 11 days. Raw Australian wheat germ (embryos) was obtained from a commercial source (Origins Healthcare, Singapore). Peas (Pisum sati6um L. cv. Dundale) were farm grown (Golden Mushroom Enterprise, Singapore) and sown on a mixture of mushroom compost and sawdust after an overnight immersion of seeds in water. Pea shoots were harvested after 8 days of growth at a maximum of 32°C and a minimum of 23°C, and a 12 h light/12 h dark photoperiod at an approximate daylight intensity of 80–100 mmol photons m − 2 s − 1. All harvested tissues were frozen and stored at − 80°C.
2.2. Preparation of HMG proteins Putative HMG proteins were prepared according to [11] with minor adaptations to allow for differences in tissue. An additional step of hand grinding in liquid nitrogen was included for rice (a particularly fibrous material) and pea tissues before homogenisation using a Polytron PT-DA 3020/2 TS aggregate and PT 3000 machine (Kinematica, Switzerland) at a setting of 7 for 10 min and a ratio of 100 g tissue to 300 ml buffer. Ungerminated rice grains were first soaked in water for 1 h before grinding in liquid nitrogen and homogenisation. Wheat germ was homogenised directly in buffer. A low salt wash of purified chromatin was omitted for rice tissues to maximise yield, but included for pea and wheat chromatin. The 60 min precipitation period using 2% trichloroacetic acid was retained for pea and wheat extracts, but increased to 90 min for extracts from rice tissues to reduce contamination by high molecular weight proteins. The yield for rice shoots was about 200 mg of protein for every 100 g of rice tissue.
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2.4. Gel retardation assays Gel retardation assays were performed in 10– 20 ml of binding buffer (25 mM Hepes-KOH pH 7.6, 40 mM KCl, 1 mM EDTA, 1 mM DTT, 10% (v/v) glycerol) in 1% agarose gels using 3–9 ng of [32P]-labelled probe and 10 mg of poly(dIdC).poly(dIdC) as non-specific competitor in each reaction according to [11].
2.5. Southwestern hybridisation analysis Proteins were separated on an 18% denaturing gel by SDS-PAGE [32] and transferred to nitrocellulose by electroblotting [33]. The electrophoresed gel and nitrocellulose membrane were equilibrated in electroblotting buffer (10 mM 3-[Cyclohexylamino]-1-propane-sulfonic acid (CAPS)-NaOH pH 11, 10% methanol) for 5 min before transfer in a Bio-Rad Trans-Blot Cell (Bio-Rad) at 150 mA for 140 min at 4°C. Washing of the blotted membrane and hybridisation to [32P]-end-labelled probe was performed in binding buffer + 0.02% (w/v) bovine serum albumin + 0.02% (w/v) Ficoll 400 +0.02% (w/v) polyvinylpyrrolidone PVP-360 according to [11], except that 30 mg ml − 1 of degraded herring sperm DNA (Sigma, USA) was included as nonspecific competitor during the relevant hybridisation steps, and that 0.06 ml buffer per cm2 of membrane was used during the stationary hybridisation. After autoradiography, hybridised probe was removed by washing membranes for 3× 20 min in a total of 100 ml of 1 M NaCl, so that reprobing of the membrane could take place.
2.6. Molecular biology techniques 2.3. Re6ersed-phase perfusion chromatography Reversed-phase chromatography was carried out on a BioCAD perfusion chromatography workstation using a POROS R2/H 4.6 mm D × 100 mm L reversed-phase column (PerSeptive Biosystems, USA).
Standard DNA manipulations were performed according to [34]. DNA fragments were isolated from agarose gels using the QIAquick gel extraction kit (QIAGEN, Germany) and radioactively-labelled probes were separated from unincorporated nucleotides by passage through NucTrap columns (Stratagene, USA).
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3. Results
3.1. Extraction of rice HMG proteins Putative HMG proteins were extracted from 10 – 11-day-old rice shoots and roots, and from ungerminated rice grains by adapting a protocol for pea shoots [11]. The putative rice HMG proteins were subjected to 18% SDS-PAGE, and the polypeptide profiles compared to that of previously described HMG proteins from wheat germ [2] and pea shoots [11], as well as that of pea histones (Fig. 1, lanes 1 – 4). All samples contained a few proteins above 30 kDa, but these were, by definition, not belonging to a ‘high-mobility group’. Nine major proteins from 13 – 26 kDa were identified in extracts from rice shoots and labelled HMGa to HMGf (Fig. 1, lanes 4 and 5). Although appearing as a wide band in Fig. 1, rice HMGb could be resolved into two bands (b1 and b2) on long (15×17.5 cm) gels (data not shown). The rice proteins were named partly with reference to the electrophoretic mobility of the four archetypal HMGa, b, c and d proteins from wheat germ (Fig. 1, lane 3); thus
Fig. 1. Coomassie-stained 18% SDS-PAGE of plant HMG proteins. Each lane contained 8 mg of protein. Lane 1, pea shoot histones; subsequently HMG proteins from, lane 2, pea shoots; lane 3, wheat germ; lanes 4 and 5, rice shoots; lane 6, rice roots, and lane 7, rice grains. Specific bands of rice shoot HMG proteins are indicated in Greek letters. Major wheat germ HMG proteins are labelled directly, a–d. Molecular mass markers are given in kDa to the right and left of the figure which is derived from two independent gels. Some bands were too faint for photographic reproduction.
rice HMGa was closest in electrophoretic mobility to wheat HMGa, HMGb1 and b2, to wheat HMGb and so on. The polypeptide profile from rice shoots was substantially different from that of rice grains and roots (Fig. 1, lanes 6 and 7), reflecting either organ-specific expression of rice HMG proteins or differences in protein degradation in different tissues. In general, putative rice grain HMG proteins were of low molecular weight, with two bands of the same mobility as shoot HMGd2 and HMGo. At least eight protein bands were discernible in root extracts, four with similarities in mobility to shoot HMGb, d2, o and f. Further purification of putative rice HMG proteins was focused on shoot extracts because of the relative abundance of the proteins, similar mobility to wheat HMG proteins and the tendency of root polypeptides to degrade.
3.2. Re6ersed-phase perfusion chromatography Putatitive rice shoot HMG proteins were subjected to reversed-phase perfusion chromatography as an initial purification step in the analysis of individual proteins. Fig. 2(a) shows the elution profile of an optimised protocol, with the bulk of proteins eluting between about 28 and 44% of acetonitrile. Fig. 2(b) displays the SDS-PAGE analysis of samples encompassing protein-containing fractions. Concentration of proteins in individual fractions occasionally allowed some bands not seen in the whole shoot HMG extract to be visualised. The bulk of high molecular weight contaminants elute in fractions 3 and 4 (too faint for visualisation in Fig. 2(b), but commonly observed in this and replicate runs), along with a subset of HMG proteins. Most of the HMG proteins elute in fractions 12–19, apparently free of contaminants \ 30 kDa. The g2 band elutes almost purified as a single silverstained polypeptide from fractions 21–26. Many HMG proteins of the same apparent electrophoretic mobility elute both early in fractions 3–4 and again in fractions 12–19 or 21–26. However HMGf only exists in fraction 3, and HMGa is found only in fractions 3 and 4.
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3.3. Gel retardation assays Gel retardation assays were used to investigate the DNA-binding characteristics of putative rice HMG proteins in comparison with pea and wheat HMG proteins. The probe used was P268, an A/T-rich positive regulatory fragment derived from the pea plastocyanin gene promoter [35] which has been shown to bind a pea HMG-I/Ylike factor [11]. Rice shoot, wheat germ and pea shoot HMG proteins formed two complexes with P268: a distinct complex of higher mobility, and a weaker, less distinct complex of lower mobility (Fig. 3a, lanes 2–4). Rice root proteins completely retarded P268 to a broad band with a mobility similar to that of the shoot complexes (Fig. 3a, lane 5), presumably because of the higher concentration of DNA-binding proteins present and even though the reaction was carried out in the presence of 10 mg of the non-specific competitor poly(dIdC).poly(dIdC). Rice grain proteins did not exhibit any P268-binding activity (Fig. 3a, lane 6). The binding characteristics of rice shoot HMG extracts were examined (Fig. 3b). The complex
Fig. 2.
Fig. 2. Fractionation of rice shoot HMG proteins (570 mg) using reversed-phase perfusion chromatography. Proteins dissolved in NEBM buffer (25 mM Hepes-KOH pH 7.6, 40 mM KCl, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, 10% (v/v) glycerol) were made up to 1 ml with Milli-Q water, applied to the column equilibrated with 65% buffer A (0.1% trifluoroacetic acid), 35% buffer B (80% acetonitrile), and eluted at 5 ml min − 1. The acetonitrile gradient used was as follows: from 0 to 8 min after sample injection, a linear gradient from 65% A, 35% B to 45% A, 55% B; from 8 – 12 min, a linear gradient from 45% A, 55% B to 100% B; from 12 to 12.5 min, 100% B was maintained, and from 12.5 to 13 min, a linear gradient from 100% B to 65% A, 35% B. Two ml fractions were collected and lyophilized. (a) Elution profile as detected at 215 nm (solid line). The acetonitrile gradient is represented by % buffer B (dotted line). (b) Silver-stained [39] 18% SDSPAGE of fractions. Lyophilized protein fractions were each resuspended in 60 ml of NEBM and 5 ml was loaded for each fraction. Fraction numbers are indicated below each track; R, unfractionated rice shoot HMG proteins (5 mg). Note that b2, g2 and o bands are negatively stained with silver when protein is in excess. Molecular mass markers are given in kDa on the left. Low-mobility-group proteins ( \ 30 kDa) were too low in abundance to be visible in protein fractions after chromatography, and these portions of the gels were omitted in the diagram. Some bands were too faint for photographic reproduction.
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corresponded to the activity observed in unfractionated rice shoot HMG extracts. An HMG-I/Y homologue from rice, PF1, which binds to the oat phytochrome A (PhyA) promoter has been cloned [30]. The region of the promoter to which PF1 binds is an A/T-rich positive cis-element designated PE1 (position −367 to − 346). Specificity of recombinant PF1 for PE1 was demonstrated in gel retardation assays where a single bound complex was detected using an oat PhyA promoter fragment containing PE1, but not in similar promoter fragments carrying linker substitution mutations [30]. In order to investigate the relationship of the rice PCF1 homologue to rice PF1, we conducted gel retarda-
Fig. 3. (Continued)
could not be competed out by a 143-fold molar excess of unlabelled P268 (Fig. 3b, lane 3), similar to previous studies using pea HMG proteins [11] and indicating an abundant protein, but the binding factor was selective for A/T-rich regions as it could be competed out by 10 mg ( : 1430-fold mass excess) of the DNA alternating copolymer poly(dAdT).poly(dAdT), but not an equivalent amount of poly(dGdC).poly(dGdC) or poly(dIdC).poly(dIdC) (Fig. 3b, lanes 4 – 6). Heat treatment to 90°C did not alter the binding ability of the factor, which was protein in nature as proteinase K treatment of HMG extracts destroyed complex formation (Fig. 3b, lanes 7 and 8). The binding characteristics mimic exactly the behaviour of PCF1, the HMG-I-like protein in pea shoot HMG extracts which binds to P268 [11]. Thus rice shoot HMG extracts may contain the rice homologue of PCF1. After fractionation using reversed-phase chromatography, P268-binding activity was found to be contained only in fractions 3 and 4 (Fig. 3c). A high-concentration of binding activity occurred in fraction 3, causing near-complete retardation of the probe in gel shift assays; the mobility shift observed using fraction 4
Fig. 3. Gel retardation assays of plant HMG proteins. (a) Retardation of pea plastocyanin gene promoter fragment P268 [11] using plant HMG proteins. Lane 1, [32P]-labelled P268 (2000 dpm; 7 ng) in absence of protein; subsequently HMG proteins included from: lane 2, pea shoots (1.3 mg); lane 3, wheat germ (0.5 mg); lane 4, rice shoots; lane 5, rice roots, and lane 6, rice grains (1.8 mg each). Protein loadings were chosen to give similar patterns of retardation. Bound complexes are indicated by arrows. (b) Binding characteristics of rice shoot HMG proteins with P268. Except for lane 1, each track contained 1.8 mg rice shoot HMG proteins. The various molecular species of competitor DNA included was in addition to the 10 mg of non-specific poly(dIdC).poly(dIdC) already present in all gel retardation reactions. Lane 1, [32P]-labelled P268 (2700 dpm; 7 ng) in absence of protein; lane 2, + rice shoot HMG proteins alone; lane 3, + 1 mg of unlabelled P268 as specific competitor ( :143-fold molar excess); lane 4, + 10 mg of poly(dAdT).poly(dAdT); lane 5, +10 mg of poly-(dGdC). poly(dGdC); lane 6, +10 mg of poly(dIdC).poly(dIdC), lane 7, rice shoot HMG proteins were heat-treated (90°C, 10 min) before [32P]P268 was added, and lane 8,+1 mg proteinase K (incubated for 20 min at room temperature with HMG proteins before [32P]P268 was added). (c) Detection of P268-retarding activity in reversed-phase purified fractions of rice shoot HMG proteins. [32P]-labelled P268 fragments (1900 dpm; 7 ng each lane) were incubated with 1 ml aliquots of each resuspended fraction from Fig. 2(b). Only fractions containing visible proteins on SDS-PAGE were assayed and only the gel containing retardation activity is shown. Previous fractionations indicate no retardation using fractions without visible protein (data not shown). Fraction numbers are indicated below each track, no protein added, R, unfractionated rice shoot HMG proteins added (1.8 mg). (d) Binding specificity of rice shoot HMG proteins to normal (PE1) and mutant (OT-614, OT-615, OT616) oligotrimers derived from the oat phytochrome promoter [30]. Oligotrimer fragments ( :1700 dpm; : 7 ng per track) were incubated in the presence ( +) or absence ( −) of 0.9 mg of rice shoot HMG proteins.
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tion assays using rice shoot HMG extracts together with oligotrimers of PE1 (OT-PE1) and the same linker substitution mutations (OT-614, OT615 and OT-616) in the PF1 study as probes. Fig. 3(d) demonstrates that a single bound complex is formed with OT-PE1, but not its mutations when rice shoot HMG extracts were added, indicating that PF1 was present with the same binding specificity. When the binding characteristics of the OT-PE1/PF1 complex were examined, the same binding selectivity, heat stability and proteinase K sensitivity was observed as for rice PCF1 in Fig. 3(b) (data not shown), suggesting that the identities of PF1 and the rice PCF1 homologue may be closely related.
3.4. Southwestern hybridisation studies Southwestern analysis had previously identified a 21 kDa protein in size-fractionated pea HMG protein extracts which bound to P268 [11]. The same approach was used here to define P268binding rice HMG proteins. As the protein factor PF1/PCF1 was selective for A/T-rich DNA (Fig. 3b), we also examined the interaction of plant HMG protein extracts with poly(dAdT).poly(dAdT), and compared the pattern obtained with that using poly(dGdC).poly(dGdC) and poly(dIdC).poly(dIdC). Fig. 4 shows the hybridisation patterns of the different plant HMG protein extracts. When probed with P268, there was virtually no hybridisation in the pea histone track (Fig. 4a, lane 1), demonstrating that binding activity in HMG protein extracts was unlikely to be due to contaminating histones. A faint band of :27 kDa may instead have reflected HMG protein contamination of pea histones, as a 27 kDa hybridising species is a major band in pea shoot HMG proteins, along with bands of 23, 21 and 19 kDa (Fig. 4a, lane 2). All four wheat embryo HMG proteins hybridised to P268, although preferential labelling of HMGa and b is observed (Fig. 4a, lane 3). P268 labelled a strong 26 kDa band and a reasonably intense band of 34 kDa in rice extracts (Fig. 4a, lane 4). No proteins are labelled in rice root or grain HMG protein extracts (Fig. 4a, lanes 5 and 6).
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The pattern obtained by hybridising the same membrane (after washing with high salt) to poly(dAdT).poly(dAdT) was substantially similar to that obtained with P268 (Fig. 4b). However wheat HMGa and b show a strong selectivity for A/Trich DNA, whereas HMGc and d are bound relatively weakly (Fig. 4b, lane 3). Taken together, the inference is that A/T-rich regions of P268 are likely to be responsible for much but not all of the binding observed with P268. Hybridization to poly(dGdC).poly(dGdC) produced a pattern distinct from that obtained by binding to A/T-rich regions. Fig. 4(c) shows that in the pea HMG protein track (Fig. 4c, lane 2), few, if any, of several distinct bands corresponded to that obtained with hybridisation to A/T-rich probes. In strong contrast to the hybridisation with poly(dAdT).poly(dAdT), wheat HMGc and d are strongly labelled while HMGa and b are not (Fig. 4c, lane 3). As in Fig. 4(a and b), a 34 kDa albeit weaker band was labelled for rice shoots, but the strong band observed at 26 kDa under A/T-rich hybridisation was replaced by weak bands at 27, 28 and 29 kDa (Fig. 4c, lane 4). Rice root HMG proteins appear to contain several G/C-binding proteins (Fig. 4c, lane 5), with bands at 27 and 29 kDa coinciding with rice shoot HMG proteins. Interestingly, the major labelled proteins from hybridisation with A/T-rich probes are only weakly labelled, if at all, with poly(dGdC).poly(dGdC), which demonstrates a strong selectivity of DNA-binding of proteins in HMG protein extracts. The pattern after hybridisation with poly(dIdC).poly(dIdC) reveals bands which were labelled also in prior hybridisation’s with A/T- or G/C-rich probes (Fig. 4d). All four wheat HMG proteins are labelled relatively evenly (Fig. 4d, lane 3). The pea HMG lane is more complex, containing bands of similar mobility to bands labelled with either A/T- or G/C-rich probes (Fig. 4d, lane 2). This implies that poly(dIdC).poly(dIdC) has characteristics resembling both poly(dGdC).poly(dGdC) and poly(dAdT).poly(dAdT), thus enabling binding to proteins with either selectivity. However the presence of distinct 19 and 22 kDa bands in the rice shoot HMG protein track (Fig. 4d, lane 4) not
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Fig. 4. Southwestern analysis of plant HMG proteins. Each gel lane contained 8 mg of protein before transfer to a nitrocellulose membrane. The same membrane was hybridised separately with 4.8 × 105 dpm of [32P]-end-labelled (a) P268 (b) poly(dAdT).poly(dAdT) (c) poly(dGdC).poly(dGdC) (d) poly(dIdC).poly(dIdC). Lane 1, pea shoot histones, subsequently HMG proteins from: lane 2, pea shoots; lane 3, wheat germ; lane 4, rice shoots, lane 5 rice roots and lane 6, rice grains. Molecular mass markers are given in kDa to the right of each diagram. The position of where HMGa might run under SDS-PAGE is marked with an ‘a’ in each panel.
seen in Fig. 4(b or c, both lanes 4) suggests that poly(dIdC).poly(dIdC) also has its own unique structural characteristics which are recognised by rice nuclear proteins. In order to identify the protein species involved, Southwestern hybridisation studies were performed on reversed-phase purified rice shoot HMG fractions which contained visible protein bands after silver-staining (Fig. 2b). Binding activity was limited to fractions 3 and 4 (Fig. 5, data not shown for other non-binding fractions), the same fractions which demonstrated P268-binding activity in gel retardation assays (Fig. 3c). Several
DNA-binding species were identified binding to the various probes, but fractions 3 and 4 appeared between them to contain all the DNA-binding species found in the control lane (R) of unfractionated rice shoot HMG proteins (Fig. 5). Moreover fraction 3 contained additional DNA-binding bands not observed in the control lane in all hybridisation’s, probably as a result of the concentration of low abundance DNA-binding protein species during the reversed-phase fractionation. Fraction 4 contained considerably fewer DNA-binding species than fraction 3 in all hybridisation’s. Better transfer of rice shoot
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Fig. 5. Southwestern analysis of reversed-phase fractionated rice shoot HMG proteins. Resuspended proteins (7 ml per fraction) from fractions containing visible protein bands from Fig. 2(b) were subjected to electrophoresis and transfer. Only the membrane displaying hybridising bands is shown. The same blotted nitrocellulose membrane was hybridised separately with 4.8 ×105 dpm of [32P]-end-labelled (a) P268 (b) poly(dAdT).poly(dAdT); (c) poly(dGdC).poly(dGdC) (d) poly(dIdC).poly(dIdC). Fraction numbers are indicated below each track, R, unfractionated rice shoot HMG proteins (8 mg). Molecular mass markers are given in kDa to the left of each diagram. The position of where HMGa might run under SDS-PAGE is marked with an ‘a’ in each panel.
proteins onto this membrane may also have occurred (Fig. 5a–d, all lanes R), as the labelled polypeptide bands were slightly greater in number and more distinct than for the previous membrane (Fig. 4a–d, all lanes 4). As gel retardation assays demonstrated the putative presence of HMG-I in fraction 4 (Fig. 3c), the less complex Southwestern hybridisation profile (compared to fraction 3) could aid in determining the size of the DNA-binding species involved. In all hybridisation’s, a 26 kDa protein was labelled in fraction 4 (Fig. 5). However labelling was strongest with P268 and poly(dAdT).poly(dAdT), followed by poly(dIdC).poly(dIdC), and was weakest with poly(dGdC).poly(dGdC). Thus the 26 kDa rice protein shows a strong selectivity for A/T-rich sequences, and in this respect behaves not only
like an HMG-I protein, but is also a candidate for the protein identified as PF1/PCF1 [11,30].
4. Discussion
4.1. Isolation of rice HMG proteins and organ-specific differences Nine polypeptide bands of varying abundance have been isolated from rice shoot HMG extracts which meet the operational definition of HMG proteins. However not all may be distinct protein species, as HMG proteins are susceptible to proteolytic degradation and are known to be posttranslationally modified [1]. Several putative rice HMG proteins (including possible b,g,d and o bands) of the same electrophoretic mobility elute
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early in fractions 3 and/or 4, then again in fractions 12–26. These bands may represent posttranslationally modified species (e.g. phosphorylated forms which differ in protein net charge), or be derived from distinct genetic alleles or isoforms, or be completely different proteins. The different polypeptide profiles from shoot, root and grain extracts of rice (Fig. 1) may suggest organ-specific accumulation of different HMG proteins, as has been observed in extracts of maize tissues where the HMGa and d proteins were found in the endosperm, but not in leaves [8]. However Northern and Western studies have subsequently detected maize HMGa in several different tissues including leaves and endosperm [36], suggesting that the different profiles observed in different HMG protein organ extracts may in part be accounted for by organ-specific protein degradation or the differential ease of protein extraction associated with different tissues. The presence of a retarding factor with characteristics of HMG I/Y proteins in rice shoots but not in roots or grains, implies either an organ-specificity of the PCF1/PF1 factor, or a greater lability in root or grain extracts. While the root extract had retarding ability, the extent of the bandshift became greater as more protein was added (data not shown), implying non-specific complex formation. Considerable differences also exist in the DNA-binding ability of rice shoot, root and grain HMG extracts as observed in Southwestern assays (Fig. 4). Retardation of a zein gene promoter by an HMG-like protein has also exhibited organ-specificity, but in this case, activity was present in the endosperm, embryo, stem and root, but not leaf [20]. The 5% upstream region of the winged bean Kunitz chymotrypsin inhibitor gene has however been described to bind to ubiquitous protein factors which had characteristics of HMG proteins [19]. The complex profile of rice shoot HMG proteins is reminiscent of maize leaf HMG extracts [8], rather than the four distinct bands observed for wheat germ or maize endosperm proteins. Several low molecular weight polypeptides are in evidence in rice and maize shoot HMG extracts. This contrasts with the more compact pattern obtained in legume shoot extracts
(see [9,11,12] and Fig. 1, lane 3). This may reflect greater proteolytic degradation in cereal shoots despite the presence of the protease inhibitor phenylmethylsulphonyl fluoride (PMSF) in the extraction buffers. A detailed structural analysis of or sequence data from these peptides would clarify if they were distinct proteins or degradation products, and help to ascertain if they truly belonged to the HMG family of proteins as defined by amino acid sequence.
4.2. Rice HMG protein extracts may contain a PCF1 /PF1 -like factor The binding characteristics of the protein factor responsible for the retardation of P268 suggests the presence of a rice homologue to the pea PCF1 protein in rice shoot extracts (Fig. 3b), as well as the presence of rice PF1 protein (Fig. 3d). Reversed-phase perfusion chromatography has facilitated separation of most rice HMG proteins away from high molecular weight contaminants (Fig. 2b). Specific P268 retardation activity (in the presence of excess poly(dIdC).poly(dIdC) included in each assay) was concentrated only in fractions 3 and 4, which enabled the elimination of several polypeptide bands in fractions 12–19 and 21–26 as the active candidates for PCF1/PF1. Several polypeptide species were nevertheless present in fractions 3 and 4, and the results of the Southwestern hybridisations have indicated potential candidates for the DNA-binding proteins. Fraction 3 contained the bulk of DNA-binding activity, but fraction 4, which also contained PCF1-type retardation activity and considerably fewer DNA-binding species in Southwesterns was likely to facilitate analysis (Fig. 5). The poly(dAdT).poly(dAdT) and P268 (A/T-rich) hybridisations of fraction 4 revealed a strong 26 kDa band, which was present but at lesser intensity in poly(dGdC).poly(dGdC) and poly-(dIdC).poly(dIdC) hybridisations, demonstrating the A/T selectivity of the protein. For the A/T-rich probes, the 26 kDa band was virtually the only protein labelled in fraction 4, thus indicating a strong candidate for rice PCF1/PF1. The protein is of the same electrophoretic mobility as HMGa and could possibly represent that protein.
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However caution must be advised in the interpretation of Southwestern assay results, as the membrane-bound DNA-binding species from any fraction may not reflect the full set of DNA-binding proteins available during a gel retardation assay due to non-equal transfer of proteins during blotting and the denaturation of some species during SDS-PAGE. The next most intense band in whole shoot extracts labelled with A/T-rich probes in Southwestern assays was a 34 kDa polypeptide, and this was largely contained in fractions 3 and 4. We believe that the 34 kDa band could represent the full-length PCF1/PF1 protein, and the 26 kDa band its proteolytic or processed product. This is because polyclonal antibodies raised against invitro synthesised recombinant PF1 recognise two proteins in rice nuclear extracts of 34 and 26 kDa [30]. In that study, it was speculated that the 26 kDa band was either a cleavage product derived during extract preparation, a protein which shared the same epitopes as PF1, or a mature polypeptide processed in vivo from the 34 kDa form. We believe that the 26 kDa protein/HMGa represents an authentic form of PF1 as it is the major species labelled with P268 and virtually the only A/T-selective DNA-binding protein in fraction 4. However it cannot be excluded that the proteolysis represents an artefact of sample preparation. Extensive biochemical evidence also exists for the proteolytic cleavage of HMG 1/2 and I/Y proteins from pea shoot extracts at the aminoand carboxyl-terminus respectively [37]. Data obtained in that study is consistent with the two most heavily-labelled bands of 27 and 21 kDa in A/T-probed pea shoot HMG extracts (Fig. 4a and b) representing the full-length, and proteolytically-processed, pea HMG I/Y homologue respectively.
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extracts (Fig. 1), and could be regarded as nuclear protein contaminants or minor HMG protein species, although the strong affinity and selectivity of these low abundance proteins is intriguing. Of the bands which could be associated with major HMG proteins, the strong selectivity of wheat HMGa and b for A/T-rich DNA and of wheat HMGc and d for G/C-rich DNA is of particular interest. Wheat HMGa and b have both been reported to interact specifically with A/T-rich sequences upstream of the pea ferredoxin 1 gene, whereas HMGc and d could not [18]. As wheat HMGa represents the wheat HMG I/Y homologue [16,17], it could thus be expected to bind to A/T runs of more than 5 bp [25]. Wheat HMGb represents the HMG 1/2 homologue [14] and the recombinant maize HMG 1/2 homologue is known to interact with the P2 promoter region of an A/T-rich zein gene [29]. No prior reports of G/C-selectivity have ever been ascribed to HMG proteins as observed here with wheat HMGc and d, and in rice root extracts in particular. This property might in future be used as a relatively simple biochemical test for HMG protein classification. Wheat HMGc and d have also been distinguished from HMGa and b in being preferentially released from wheat embryo nuclei by ethidium bromide treatment, a property shared by vertebrate HMG 14/17 proteins [38]. In contrast to the four distinct wheat germ HMG proteins, rice and pea shoot HMG protein extracts exhibit a greater heterogeneity of selectively-binding proteins (Fig. 4), and (except for the A/T-selective PCF1/PF1 protein) are of low abundance and difficult to characterise. A/T- and G/C-rich regions of the genome may play significant roles in nuclear organisation and activities, and the A/T- and G/Cselective chromosomal proteins described here can certainly be subjected to further investigations in this respect.
4.3. A/T-and G/C-selecti6e DNA-binding proteins Southwestern assays of rice, pea and wheat HMG proteins have detected several DNA-binding bands which are selective for either A/T- or G/C-rich probes (Fig. 4). Many of the bands do not represent major polypeptides visible by Coomassie blue or silver staining in HMG protein
Acknowledgements We wish to thank Bun Tuck Weng for technical assistance, Dr Gurdev S. Khush for his frequent gifts of rice IR36 grains, and Professor Peter H. Quail and Jim Tepperman for the gift of plasmids
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with cloned PE1 and mutant oligotrimers. This research was supported by grant RP920357 from the National University of Singapore.
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