Efecto biofertilizante de la bacteria Azotobacter sp en plántulas de trigo

Previous work by Mayor et al. (1993) identified a region within the Xsna promoter, -45 to -112 base pairs from the translational start site, that is responsible for the spatial and temporal expression o f Xsna. Deletion analysis of this region suggested two adjacent sequences could control mesodermal and ectodermal expression, possibly as the binding sites o f distinct DNA transcription factors (Mayor et al., 1993). Within these two regions o f the promoter there are no obvious sequence homologies with known transcription factor binding sites. It was expected that the identification of the precise limits o f these binding sites, together with an investigation into the effect of base substitutions on their binding capacity, would confirm and extend the initial observations. Therefore, the main aim was to identify individual bases that, when substituted, would inhibit either o f the binding activities. With this data, DNA affinity chromatography ligands could then be constructed which would bind the sequence specific proteins, or remove non-specific proteins from an extract that might otherwise be able to recognise aspects o f the sequence, and thereby provide a powerful means of discriminating in favour of the specific proteins.

Throughout this work the significance of the data depended on the integrity of the probes used. Early in the investigation it was discovered that there were ssDNA binding proteins in the extract that could bind to probes that did not form a proper duplex, and which would cause a non-specific shift in the EMSA similar in mobility to the one that would be expected by a properly formed probe. To overcome this potential problem great care was taken to provide the best conditions for duplex formation. Gradual cooling fi’om 90 °C to room temperature, whilst in the T4 kinase buffer, was sufficient for the probes based on the ectodermal region, which is GC rich, to form correctly. This was generally true for the other probes used, however, shorter mesodermal probes (set M; appendix A),

Chapter 4 - Investigating the Binding Site_______________________________________________ W

which are AT rich, required the addition of 0.1 M NaCl for satisfactory duplex formation. Even so, one probe would not anneal correctly, Ms2 (appendix A), despite repeated attempts. After the annealing reaction was completed the probes were purified on a 12 % (w/v) PAG, to remove the excess labelling reagents. Incomplete duplex formation could be identified by anomalous migration of the labelled probes. Only when it was clear that the probes had formed correctly were they used experimentally.

4.2 Using EMSA to Investigate the Binding Sequence

The specificity o f the binding activities visuahsed as retarded bands in the EMSA was assessed by titrating the radioactive probe against increasing concentrations of specific and non-specific unlabelled probes. A specific effect will occur at a much lower concentration than the non-specific. Two non-overlapping probes derived from the mesodermal and ectodermal regions (Msl and Esl), were used together with a set o f oligonucleotides containing four adjacent base substitutions (appendix A), designed to test their involvement in the binding activity. The substitutions were in general the alternative Watson and Crick base pair (1953), but with the purine in place of the pyrimidine. The prediction was that an alteration of an important part of the binding site would reduce its capacity to interact with the binding proteins, and that at least one o f each set would show reduced binding.

Each o f the probes within set E was used in a normal EMSA reaction to assess whether they contained the sequence necessary for the binding proteins to bind. These reactions were repeated in the presence of an increasing amount of specific competitor, unlabelled E sl, to estimate the relative affinities of the protein for the probes, from which their specificity profile could be assessed. Probe Es2 was bound less strongly than Es3

and Es4, suggesting that the ^ ggag^’ is the most significant part of the sequence. Es2 and Es4, in which the adjacent 4 bases were changed, seemed to have a greater binding activity. | Probes Es2, Es3 and Es4 were not in excess in all reactions, and therefore this data should be treated as preliminary (figure 4.1).

A similar analysis o f the M region was conducted using the oligonucleotides Msl - 4, although no useful information could be obtained using Ms2 as it could not be annealed satisfactorily. The shorter exposure of the autoradiograph (figure 4.2) indicated that Ms4 had a greatly reduced binding capacity, suggesting that the ^ a t t a ^ sequence was

particularly significant. The probe excess qualification mentioned above also applies to this data.

These data suggested distinct sequences within the mesodermal and ectodermal regions mediate the interaction of the binding proteins with the promoter. However, it seems unlikely that single or even double base substitutions will abolish binding

completely. While the binding sites appeared to extend over a number of bases, the binding capacity seemed to be tolerant to gross substitutions.

An alternative approach to using the radioactive probes above, is to assess the binding specificity of the radiolabelled probes Msl and Esl in the presence of increasing amounts o f unlabelled duplexes containing the substituted bases. Uncertainty about the integrity o f the unlabelled duplexes suggested this may be unreliable. However,

subsequent work indicates that similar results are obtained (M. F. Bennett, personal communication).

4.3 DNA Footprinting

DNA footprinting is another powerful method that has been developed to identify sequence specific contacts between DNA and binding proteins. The principle is that sequence specific proteins protect the DNA to which they are bound fi*om enzymic or