Development-dependent changes in the tight DNA-protein complexes of barley on chromosome and gene level
© Sjakste et al; licensee BioMed Central Ltd. 2009
Received: 23 December 2008
Accepted: 12 May 2009
Published: 12 May 2009
The tightly bound to DNA proteins (TBPs) is a protein group that remains attached to DNA with covalent or non-covalent bonds after its deproteinisation. The functional role of this group is as yet not completely understood. The main goal of this study was to evaluate tissue specific changes in the TBP distribution in barley genes and chromosomes in different phases of shoot and seed development. We have: 1. investigated the TBP distribution along Amy32b and Bmy1 genes encoding low pI α-amylase A and endosperm specific β-amylase correspondingly using oligonucleotide DNA arrays; 2. characterized the polypeptide spectrum of TBP and proteins with affinity to TBP-associated DNA; 3. localized the distribution of DNA complexes with TBP (TBP-DNA) on barley 1H and 7H chromosomes using mapped markers; 4. compared the chromosomal distribution of TBP-DNA complexes to the distribution of the nuclear matrix attachment sites.
In the Amy32b gene transition from watery ripe to the milky ripeness stage of seed development was followed by the decrease of TBP binding along the whole gene, especially in the promoter region and intron II. Expression of the Bmy1 gene coupled to ripening was followed by release of the exon III and intron III sequences from complexes with TBPs. Marker analysis revealed changes in the association of chromosome 1H and 7H sites with TBPs between first leaf and coleoptile and at Zadoks 07 and Zadoks 10 stages of barley shoot development. Tight DNA-protein complexes of the nuclear matrix and those detected by NPC-chromatography were revealed as also involved in tissue- and development-dependent transitions, however, in sites different from TBP-DNA interactions. The spectrum of TBPs appeared to be organ and developmental-stage specific. Development of the first leaf and root system (from Zadoks 07 to Zadoks 10 stage) was shown as followed by a drastic increase in the TBP number in contrast to coleoptile, where the TBPs spectrum became poor during senescence. It was demonstrated that a nuclear protein of low molecular weight similar to the described TBPs possessed a high affinity to the DNA involved in TBP-DNA complexes.
Plant development is followed by redistribution of TBP along individual genes and chromosomes.
Reorganization of the chromatin structure is one of the main mechanisms for regulation of gene expression in plants, chromatin rearrangements take place in response to light and tissue-specific signalling molecules . Despite rapid progress in the field, the functional significance of some groups of nuclear proteins, including the tightly bound to DNA proteins (TBP), remains obscure. TBP is a specific nuclear protein group that remains attached to DNA with covalent or non-covalent bonds after its deproteinisation independently of the deproteinisation method applied: protease digestion, phenol extraction, chloroform extraction or salting-out. The TBP have been found in the DNA of numerous evolutionary distant species . Both the function of the TBPs and the nature of DNA sequences involved in the tight complexes remain to be detailed. Enrichment of the TBPs in specific DNA sequences is of special interest in connection with speculation on the potential function of such sequences in higher order structures of the genome of different organisms including humans, mouse, and chicken [3–6]. In our recent work  we have applied the DNA microarray technique to study the distribution of TBPs along the chicken alpha-globin domain in cell lines that expressed the gene, did not express it or conducted abortive expression. In this study we have shown profound transcription-dependent changes in the TBP-distribution pattern in the alpha-globin domain. Other preliminary results from of our team  also indicated the existence of tissue- and development specificity in the patterns of TBP distribution in barley (Hordeum vulgare) shoots.
The life cycle of the barley plant, especially the shoot and seed provide an excellent model for plant development studies. Barley shoot and seed developmental stages are well characterized and classified . Etiolated barley seedlings provide cell populations with different proliferation, differentiation and senescence status including synchronously dividing cell populations of primary leaf [10–12], senescent coleoptiles and a mixed cell population from roots . In the present work we used dry grains (Zadoks 0) and grains after 20 h of imbibition (Zadoks 1); coleoptiles, first leaves and roots were dissected from shoots of Zadoks 07 (coleoptile emerged stage, classification according to ) and Zadoks 10 (first leaf through coleoptile) development stages as well as seeds of watery ripe (Zadoks 71) and medium milk development (Zadoks 75).
Moreover, expression of several specific enzymes of carbohydrate metabolism in barley is development- or/and tissue specific and is restricted to well-defined stages of the plant development . It was reported that transcription of α-amylase genes is low and decreases during seed development, but β-amylase expression in endosperm is up-regulated during its development [13–15]. Two representatives of each group of amylase genes, the Amy32b and Bmy1 genes provide prospective model systems. Highly conserved in barley cultivars, Amy32b gene  is located on chromosome 7H in the centromere region . Gene encodes a low-pI α-amylase and is expressed in barley aleurone cells under the control of gibberellic acid and abscisic acid . Highly polymorphic in structure, barley Bmy1 gene [18, 19] is located in the long arm of chromosome 4H , encodes endosperm specific β-amylase and is expressed only during seed development . Several allelic forms of Bmy1 structural gene were sequenced and analyzed in relation to their functionality [21–24].
Despite the fact that barley genome is not completely sequenced, work with this plant provides the opportunity to use the technique of mapped barley genomic markers developed during recent decades [25, 26]. The tool allows chromosome profiling to be performed in any application.
In order to study transcription, tissue and development dependent changes in TBPs distribution in barley genes and chromosomes at different phases of shoot and seed development, we have formulated the following as the goals of the present study: 1. to investigate the distribution of TBPs along Amy32b and Bmy1 genes using oligonucleotide DNA arrays; 2. to characterize the polypeptide spectrum of TBP in different shoot organs and during different development stages. 3. to apply mapped barley MS as a tool to study tissue and developmental specificity in the distribution of DNA complexes with TBP (TBP-DNA) in the barley chromosomes 1H and 7H; 4. to compare the chromosomal distribution of TBP-DNA complexes and distribution of tight DNA-protein complexes separated using other than TBP isolation approaches including nuclear matrix isolation and nucleoprotein chromatography on celite (NPC-chromatography).
The R and F fractions obtained from watery ripe seeds hybridized also with different intensity with Bmy 1 gene microarray. An R vs F ratio close to or less than 1 was observed for positions 3 and 11, and 4 and 12 correspondingly. In positions 1, 2, 5, and 8 hybridization with R probes was somewhat stronger than with F probes, however the R vs F ratio did not exceed 2. The R vs F ratio was rather high in oligonucleotide positions 6, 9 (more than 2) and 13 (near to 3). Finally, in positions 7 and 10, the intensity of hybridization with R probe was 4 times stronger than with F probe. The highest ratio in the milky ripeness stage (Figure 3, panel B) was close to 2 in position 10, and slightly exceeded 2 in position 13. In all other oligonucleotide positions the R vs F ratio was around 1. Thus, similar to the results obtained with Amy32b gene, the overall decrease of the R vs F ratio is revealed along the Bmy1 gene during seed development. The process is more pronounced in Exon 3 and upstream the microsatellite locus in Intron III.
Bioinformatic analysis of Amy32b and Bmy 1 gene sequences
Identification of specific areas of TBP binding in both genes raised the question of peculiarities in the gene sequences in these areas. A search for nuclear matrix attachment regions (MARs) performed by the MatInspector program (Rel. 7.4), revealed a possible MAR in Intron 3 of the Bmy1 gene (Figure 3). No MARs were detected in the Amy32b gene. DiAlign TF, Release 3.1 revealed 13 homology regions between the two genes (Figures 2 and 3), however no common features in the distribution of transcription factor binding sites along the genes and transcription factor binding modules were revealed. Comparison of the predicted MAR with the TBP binding sites in the Bmy1 gene Intron 3 indicates that this site is enriched in TBPs. Similar sequences of the two genes differed in their affinity to TBPs. The only exception was observed in the area of similarity S9; these sites were TBP-enriched in both genes.
Profiling of DNA-protein complexes along chromosomes
Positive data on intragenic changes in TBP distribution in the course of development encouraged us to upscale the investigation and to study the long-range distribution of tightly bound proteins along barley chromosomes 1H and 7H. Lack of barley genome sequence information makes it impossible to apply microarray technology to study long-range distribution of TBPs along chromosomes. To at least partly reach the goal we have used the tool of mapped markers well developed in barley studies [25–27]. Profiling experiments were designed as PCR-based identification of the DNA matrices in DNA-protein complexes obtained by different fractionation approaches. Involvement of the DNA stretch in interactions with a given protein group (TBP, nuclear matrix) was scored in terms of the presence/absence of amplification with mapped markers of barley chromosomes 1H and 7H. Data are organized as graphical representations of chromosome 1H and 7H with an indication of the PCR result at each MS locus in all DNA-protein fractions analyzed. The study was performed on DNA from barley shoot organs; this model enables us to obtain a sufficient quantity of material for fractionation of nuclear structures.
Panel B in Figure 4 summarizes the trends of transitions during development of different organs compared with the situation in dry seed. Panel C gives a summary of the involvement of the Chromosome 1 H and 7H sites of the R ↔ F transitions. It emerges that all the sites on chromosome 7H are involved, and only four sites of 1H are not involved in the dynamics. Thus, the development of the barley seedling is coupled to rearrangements in the interactions of DNA with TBPs both on chromosome 1H and 7H. The process is organ specific and can be termed "tightening" of the DNA-TBP interactions in the leaves and "loosening" in the coleoptile compared to dry seed status.
Chromatin fractions and nuclear matrix
Tightly bound protein spectrum in organs of stage 07 and stage 10 shoots
Affinity of the TBP-bound DNA to nuclear proteins
Gene expression changes during seed development
In the present study we have revealed development-dependent changes of the TBP distribution in Amy32b and Bmy1 genes during transition of the barley seed from watery-ripe to middle milk ripe stage. Changes in TBP distribution in the genes were coupled to changes in their expression. Our RT-PCR data confirm earlier published data indicating that β-amylase expression is linked to starchy endosperm development, but α-amylase is not expressed on late seed development stages , moreover over-expression of α-amylase in developing seed leads to development defects .
TBP redistribution in the Amy32b gene
Our observation of a decrease in TBP interactions with promoter region of the Amy32b gene is in good agreement with a recently proposed hypothesis . According to this hypothesis in the dormant gene negative regulators bind to the corresponding cis-acting elements in the promoter and form a "repressosome," this diminishes the binding or transactivating activities of positive regulators to the promoter, thereby preventing Amy32b transcription. Induction of transcription is followed by binding of positive regulators to their respective DNA sequences and formation of the "enhanceosome," leading to a high level of Amy32b gene expression. Probably, formation of the "enhanceosome" is preceded by degradation of repressors. Taking into account that the RT-PCR data expression of the gene is observed exclusively in the watery ripeness stage, one can speculate that TBPs could stabilize "enhanceosome", release or degradation of TBPs could be one of the mechanisms of the promoter inactivation. Interestingly in the dormant Bmy 1gene the 300 bp region upstream the translation start codon (position 4) is neither TBP-enriched. The very organization of the Amy32b gene 5' area appears to be favourable for TBP binding, as it contains several inverted sequences , but it was shown that repeated sequences are often found in the TBP anchoring sites [3–6].
TBP redistribution in the Bmy1 gene
In Bmy 1 gene, besides the overall decrease of TBP binding coupled to up regulation of transcription we have also observed drastic changes of TBP binding in Exon 3 and Intron 3, changes 1.5 Kb upstream the Exon 1 were also well-pronounced. Interestingly, Intron 3 of this gene harbours a microsatellite, MITE element, several other repeats and a predicted MAR (Figure 3). Both microsatellites and mobile elements are often involved in tight DNA-protein complexes [30, 31] and hence these elements could also possess high affinity to TBPs. Indeed as position 10 of the microarray is very close to the microsatellite, sequences of the MITE element should hybridize with the oligonucleotide in the position 7. Enrichment of the mobile genomic elements in complexes with TBP was revealed also in our study on the characterization of the TBP-associated DNA sequences (unpublished results). On the other hand, the presence of a possible nuclear matrix attachment site (MAR) in Intron 3 of Bmy 1 gene and the absence of such sequences in other sites of the gene intensively binding TBPs indicate that TBP binding is not dependent on the presence of MAR.
TBP binding and transcription
The overall intensity of the DNA association with TBP appears to be independent of transcription, as in stage 10 seeds we observed an overall decrease in the R vs F ratio along both genes, although one of them became expressed on this stage, but the other – remained dormant. Triggering or cessation of transcription is rather accompanied by redistribution of TBPs along the gene, but not overall accumulation or release of these proteins from the whole gene sequence. The same conclusion was made after a similar study on the chicken produced alpha-globin gene domain . Comparison of TBP distribution in the domain in erythroid cells differing in the degree of alpha-globin gene transcription revealed changes in distribution of TBPs, however the TBPs were much more abundant along the domain in DNA from liver than in erythrocytes and erythroblasts. Thus, in animals overall enrichment in TBPs appears to be an organ-specific trait. Our results indicate that in plants it could be characteristic not only of the organ per se, but also of the development stage of an organ.
Relation between TBP and nuclear matrix attachment sites
Application of the microsatellite analysis tool developed for the needs of marker-assisted selection during analysis of long-range DNA-protein interactions proved its utility especially for work with a partly sequenced genome like the barley genome. It should be mentioned that using the conventional PCR protocol we could observe only the most drastic changes in the sequence distribution of fractions; a quantitative real-time PCR technique would substantially increase the sensitivity of the approach. Nevertheless the assay enabled us to obtain interesting results. For example, the relation between TBP and nuclear matrix binding sites has been discussed for a long time and in some reports these are claimed to be identical structures [32–34], others consider TBP-DNA complexes to be specific structures, not related to nuclear matrix attachment sites [2, 35]. Comparative study of the changes in distribution of complexes with TBPs and the nuclear matrix along Chromosomes 1H and 7H in the first leaf of the barley shoots indicates detachment of several sites in both chromosomes of the nuclear matrix during transition from watery to milky ripeness, at the same time the number of interaction with TBPs is increased (Figures 4 and 5). Thus, TBPs appear to form complexes different from the nuclear matrix association sites to DNA. Data from nucleoprotein celite chromatography are coherent with results obtained with salt-extracted nuclear matrices, but reveal the same process in more detail (Figures 5 and 6). The presence of a possible MAR only in one site of TBP accumulation also indicates the different nature of the two types of DNA-protein complexes.
Individual peptides of TBP fraction
Detailed characterization of the TBP spectrum by means of 2D electrophoresis and mass-spectrometry are currently ongoing. However our preliminary results on development-dependent changes in TBP spectrum reported here are in good agreement with microsatellite analysis data. Indeed, we observe some increase in the TBP-binding sites during the transition of the first leaf from stage 07 to stage 10, the number of polypeptides in the TBP preparation increases, while in coleoptile we observed a decrease in the TBP binding sites and the peptide spectrum of TBPs is also simplified (Figures 7 and 8). Protein blot assays confirmed the same trend: in nuclear extracts of stage 07 coleoptiles several peptides bind TBP-associated DNA (Figure 8), in stage 10 coleoptiles the only low-molecular peptide, also prominent on electropherograms (Figure 7) retains its ability to form tight complexes with DNA.
Our data indicate the existence of development-dependent redistribution of the TBP along Amy32b and Bmy1 and along studied stretches of Chromosomes 1H and 7H in barley. Sites of the TBP binding do not co-localize with the nuclear matrix attachment regions. The TBP spectrum and affinity to DNA change during plant development.
Seeds of the barley cultivar Balga were obtained from the Latvian State Stende Cereal Breeding Institute and Latvian State Priekuli Crop Breeding Institute. Etiolated shoots were grown for 3 – 5 days at 30° in darkness. Coleoptiles, first leaves and roots were dissected from shoots of Zadoks 07 (coleoptile emerged stage, classification according ) and Zadoks 10 (first leaf through coleoptile) development stages. Dissected coleoptile and first leaf tissue from 50–100 shoots were combined in one sample for each tissue at both developmental stages. Ten embryos per sample from dry grains (Zadoks 0) and after 20 h of imbibition (Zadoks 1) were used in some experiments. Coleoptiles, first leaf, and embryo samples were subsequently used for bulk DNA extraction. Seeds of watery ripe (Zadoks 71) and medium milk development (Zadoks 75) stages were collected in the field of the Latvian State Priekuli Crop Breeding Institute. 70 – 100 mg of seed tissue was used for bulk DNA and RNA isolation.
GenBank accession X05166 sequence information was used to develop primers and the microarray and to analyze data on the barley Amy32b gene . GenBank accession AF061203 sequence information deposited for Adorra cultivar was used in this study for Bmy1 gene. The previously reported data  indicate the identity of the selected gene sequences in the Adorra and Balga cultivars used in this study.
DNA and RNA isolation
Plant tissues were frozen in liquid nitrogen and ground in a mortar or in TissueLyser (Qiagen) to a fine powder. DNA from plant material was extracted according to the previously described protocol using chloroform-isoamyl alcohol extraction  with some modifications. Taking into account our previous observations on partial degradation of TBPs by proteases and the formation of artificial DNA-protein complexes due to the RNaseA treatment (reported also in ), we excluded the exogenous enzymes from DNA extraction protocols. RNA was precipitated with concentrated LiCl solution (up to 4 M) with subsequent 1 h incubation on ice  and removed as a pellet by centrifugation. DNA was precipitated with 1 volume of butoxyethanol. Nucleic acids from the NPC-chromatography fractions were concentrated by absorption on hydroxyapatite and elution with 1 ml of 0.24 M phosphate buffer. DNA was purified using a Wizard DNA Clean-Up System kit (Promega).
Total RNA was isolated according to the RNA extraction protocol of Rneasy Plant Mini Kit (Qiaqen). Samples of 2 μg – 3 μg each were stored at -20°C until use.
Isolation of TBP-DNA complexes by means of fractionation on nitrocellulose
DNA was digested with HindIII and Pst1 restrictases or alternatively with AluI restrictase (1 U/1 μg of DNA) at 37°C overnight in appropriate buffer. For microarray experiments DNA was fragmented by sonication in a SONICS Vibra Cell™ device (Sonics & Materials, 53 Newtown, USA) with 60% output for a period of time ranging from 30 seconds to 4 minutes. Completeness of DNA digestion or extent of fragmentation was tested by gel electrophoresis. DNA samples with average fragment sizes of about 500 bp were diluted in 3 ml of filtration buffer (0.5 M KCl, 5 mM EDTA, 10 mM Tris- HCl, pH 7.4) to a final concentration of 25–70 μg/ml. The solution was pressed through a nitrocellulose filter (HAWP 025 00, HA 0.45 μ, 25 mm, Millipore or NC 45, 0.45 μm, 25 mm, Whatman (Schleicher & Schuell) pre-soaked with filtration buffer and supported in a Swinnex holder. The filter was washed up to five times with the same 3 ml volume of filtration buffer to avoid contamination between fractions. Filtration buffer wash off resulted in filtered DNA fraction (F). The filter retained a DNA fraction enriched in the tightly bound proteins; it was eluted with five washes of 3 ml each of 5 mM EDTA, 10 mM Tris HCl, pH 7.4 (low-ionic strength eluted fraction R1) and 3 ml of 50 mM NaOH (alkali-eluted fraction R2) in sequence . The DNA content in the F, R1 and R2 fractions was measured spectrophotometrically.
Chromatin fractionation and nuclear matrix isolation
A protocol of conventional extraction of nuclei with high salt solutions modified for monocots  was used for chromatin fractionation and nuclear matrix isolation. Briefly, nuclei isolated using 1% Triton X100 extraction were consecutively extracted with:10 mM Tris HCl, pH 7.5; 10 mM NaCl, 3 mM MgCl2; 20 mM NH4Cl to obtain the nucleoplasm fraction; with 10 mM Tris HCl, pH 7.5; 0.2 mM MgCl2 to obtain the fraction of soluble chromatin; and with 2 M NaCl, 10 mM Tris HCl, pH 7.5; 0.2 mM MgCl2 to obtain the fraction of insoluble chromatin. The residual fraction contained nuclear matrix.
Chromatography of nucleoproteins on celite (NPC-chromatography)
The NPC-chromatography was performed as described [28, 29]. Barley embryos or organs taken from 5 shoots were homogenized in a tightly-fit Dounce homogenizer in 10 mM Tris-HCl, pH7.6; 5 mM MgCl2, 1% Triton X100. The lysate was directly applied on a precooled (0°C) water-coated column of Celite R-630 (Fluka). The column was rinsed with 50 ml of 5 mM MgCl2, 10 mM Tris HCl, pH 7.4 (breakthrough fraction) at 0°C and 80 ml of NaCl in linearly increasing concentration (0 → 2 M) and the same temperature was pumped through the column. The fraction eluted in NaCl gradient (DNA 0) was considered to be unbound to the nuclear matrix. Then a gradient of LiCl – urea (0 → 4 M; 8 M) was applied in the same manner. Finally the column was gradually heated from 0°C to 100°C under a constant flow of 4 M LiCl, 8 M urea solution. Fractions eluted in LiCl-urea gradient and at a temperature below 70°C were combined (DNA I) and was considered to represent a fraction loosely bound to the nuclear matrix in contrast to the DNA II fraction eluted at high temperature (70° → 100°C) and considered to be tightly attached to the nuclear matrix.
TBPs sample preparation and electrophoresis
Protein samples were obtained using the method of exhaustive DNase digestion . The DNA was digested with DnaseI (Fermentas) (1 U/100 μg, room temperature, overnight) in 10 mM Tris-HCl, pH 7.6; 5 mM MgCl2. Completeness of digestion was monitored by gel electrophoresis. Proteins were concentrated by evaporation of excess water. The DNA digest was mixed with the sample buffer, heated to 100°C for 5 minutes and cooled. Electrophoresis was performed in 10% or 15% PAAG gels using the conventional protocol for Laemmli system. Gels were silver stained according to .
PCR based marker chromosome profiling
Twenty two and twenty four primer pairs representing barley markers mapped along chromosomes 1H and 7H correspondingly were chosen for analysis (Figures 5, 6, 7). The primer sequences, genetic map position and microsatellite motif of HvALAAT, HVHVAI, Bmac, EBmac, Bmag and EBmag markers were previously published  and are publicly available http://bioinf.scri.ac.uk/ssr/barley_s.html. GBMS markers were developed and mapped in IPK . The complete sequences of GBMS as well as WMCIE8, cMWG 773, AF022725A, cMWG 725, cMWG 728, ABG 461 markers are available from IPK on request. Markers of cMWG, ABC, and ABG series are primers of sequenced genomic RFLP clones , other markers are microsatellites.
To equalize the DNA content in different samples after fractionation on nitrocellulose, an aliquot of the F fraction was diluted 5 times, and aliquots of the R1 and R2 fractions were diluted twice. 5 μl of the diluted solution was taken for the amplification reaction. The final PCR reaction mix contained: about 100 ng of genomic DNA; 1.5 mM MgCl2, 40 μM of dCTP, dGTP, dTTP, dATP; 0.4 μM of each primer; and 1U of Taq polymerase in a 25 μl reaction mixture. The PCR conditions were: 3 min at 94°C, followed by 45 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min with chain elongation of 7 min . PCR was performed on a Perkin Elmer PCR machine in 96-well format. Fragment length analysis was performed on an ALF II Express automated laser fluorescence sequencer (Pharmacia) using a short gel cassette. Denaturing polyacrylamide gels were prepared following the manufacturer's protocol (Pharmacia). The sample standard mix was heat denatured and snap cooled before loading. The gels were run in 0.5 × TBE buffer with 600 V, 50 mA and 50 W and gel temperature 50°C. Fragment sizes were calculated using the Fragment Analyser 1.02 computer program (Pharmacia) via comparison with the internal size standards (73, 122, 196, and 231 bp). Results were scored as presence/absence of amplification.
DNA-binding protein blot assay
The assay was performed according to . Nuclear proteins were electrophoretically separated in one dimensional 12% SDS-polyacrylamide gel. Proteins were electroblotted from the gel onto 0.45 μm nitrocellulose filters (Bio-Rad) in 19 mM glycine, 25 mM Tris, 0.1% SDS, and 20% methanol. Protein blots were blocked overnight at room temperature in blocking buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM EDTA, 1% blocking reagent (BSA) and subsequently preincubated for 1 hour in binding buffer (10 mM Tris-HCI pH 7.4, 50 mM NaCl, 2 mM EDTA, 0.05% BSA). Later, the membranes were transferred into a fresh portion of the binding buffer supplemented with 10 ng/ml of radioactively labelled DNA probe (10,000–70,000 cpm/ml) and 100 ng/ml of competitor DNA (Eco R1 digested plasmid pUC19). TBP-bound DNA fragments obtained according to the exhaustive DNaseI digestion procedure were used as probes. Binding was carried out overnight at 37°C in a hybridization oven under gentle agitation. The membranes were washed three times for 15 min in 100 ml of binding buffer and autoradiographed.
DNA probe labelling
Different DNA fractions were labelled with [α32P]dATP (Hartmann Analytic, Germany) using a HexaLabel™ DNA Labeling Kit (UAB Fermentas, Lithuania). Unincorporated nucleotides were removed by selective precipitation of DNA with ethanol in the presence of ammonium acetate.
Amy32b array oligonucleotide sequences.
Bmy1 array oligonucleotide sequences.
RNA samples of 2 – 3 μg were pretreated with DNaseI using DNA-free kit (Ambion) and used for cDNA synthesis with Revert Aid™ H Minus First Strand cDNA Synthesis Kit (Fermentas) with unspecific primers according to the manufacturer's protocol. Aliquots containing 100 – 150 ng of the first strand cDNA were used as a template for PCR amplification performed in 30 μl of the reaction mixture with a final concentration of 1× PCR buffer (Fermentas), 3 mM of MgCl2, 40 μM of each dNTP, 1 μM of each forward and reverse sequence-specific primers and 0.612 U of Taq polymerase (Fermentas) per sample. Cycling parameters were as follows: denaturation/RT inactivation step at 94°C during 3 min was followed by 30 cycles of three steps; denaturation 45 sec at 94°C, annealing 45 sec at 60°C, and extension 1 min at 72°C, and final extension 5 min at 72°C. Primer design was performed by the Primer 3.0 program using coding regions of the genes. Bmy1 cDNA fragment of 178 bp in size was amplified with forward 5'-TGCCGTCCAGATGTATGC-3' and reverse 5'-GCAGATGAATTCTCCGATGC-3' primers to encompass part of exon II, the whole of exon III and part of exon IV. Amy32b cDNA fragment of 447 bp in size was amplified with forward 5'-TAAGCCCAACTACGATCAGGA-3' and reverse 5'-ACGTAGGCGTCTCCTTCGTG-3' primers to encompass partly exon III and exon IV. Sequences of primers for the alpha tubulin (Tub) reference gene, a ubiquitous and stably expressed gene in barley, have been published . Amplification resulted in the 248 bp product of expected size.
Data management and analysis
Numbering of any loci in the genes is given from the first ATG of the corresponding genomic sequence. Identification of transcription factor binding sites and clusters, analysis of similarities between the two genes and prediction of nuclear matrix attachment regions (MAR) was performed by Genomatix software (DiAlign TF, Release 3.1, and MatInspector, Release 7.4, Smartest Release 2.2) tools at http://www.genomatix.de/).
tightly bound protein
tightly bound proteins
The work was funded in part by the grant No 05.1401 (T. Sjakste) from the Latvian Council of Science, the Deutsche Forschungsgemeinschaft grant 436 LET 17/1/05, and the Lithuanian State Science and Studies Foundation grant No T-109/07. Collaboration between French, Latvian and Lithuanian teams was supported by ECO-NET program 2006–2008, project "Development of an integrated genomic DNA-array based approach for the epigenetic study of the genome". Network between Latvian and Lithuanian colleagues was also supported by NordPlus Neigbour 2007 – 2008 project "Genomics for all: bringing new tools to breeders" and NordPlus Horizontal 2008 – 2011 project "Implantation of genomics and bioinformatics in agricultural practice". We thank Ina Belicka, Mara Bleidere (Latvian State Stende Cereal Breeding Institute) and Linda Legzdina (Latvian State Priekuli Crop Breeding Institute) for providing plant material.
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