Carga bacteriana de Azotobacter sp en los centros poblados de Camicachi, Santa

Figure 3.1 The Effect of Increasing Concentrations of Lambda DNA as Non-specific Competitor in the Electrophoretic Mobility Shift Assay.

A series of EMSA reactions containing the M probe were setup with a range of lambda DNA concentrations (0.1 to 200 nmol) as the non-specific competitor. From 10 nmol, the lambda began to effect the binding activity until at 200 nmol it was almost completly abohshed.

The sharp threshold was probably attributable to the lack o f excess probe at the low er • concentrations o f lambda D N A. The same effect was seen in EM SA reactions containing probe E (data not shown).

Lambda DNA (nmol)

4 -

Binding Activity -

Chapter 3 - lder)tification o f Promoter Binding Proteins Using EMSA 79

not seen over a wide concentration range using the Xsna probes. In fact, at 2.5 - 5.0 % the binding activity began to reduce. The wide range of pH in which the binding activity could still be detected was surprising as it indicates that the protein-DNA and any protein-protein interactions are unaffected by signiûcant changes in ionic charge o f the protein. The binding activities were unaffected by 1, 10, ortho-phenanthroline, a chelator o f divalent cations such as Zn^"”, suggesting that this metal was not required for the protein-DNA interaction.

Two major sources o f variability of the binding activity have been identified: the quality of the extracts obtained from the eggs of different frogs, and an unusual cold sensitivity o f the binding activity that these extracts exhibit. The biggest problem was the cold sensitivity o f the complex. This can be seen in figure 3.2, which shows an experiment where aliquots o f the same cell extract were stored at 20 °C, 4 °C, -20 °C and -70 °C overnight, after which they were incubated at either 20 °C or 37 °C for 1, 2 or 3 hours to see if any inactivation was reversible. Each aliquot was tested using probe M. Freezing the extract inactivates the capacity to form a DNA-protein complex. Storage at - 70 °C was less damaging than - 20 °C, possibly because the freezing process was quicker. This can be avoided to some extent by incubation at 20 °C or 37 °C, but the proteins are short lived probably due to the action of proteases. Similar results were obtained with the E probe (data not shown). In this experiment it appeared that storage of the extract at 4 °C had no adverse effect on the binding activities. However, subsequent experience showed that this was not always the case, and so the extracts were routinely kept at 15 °C for periods no longer than 24 hours.

Figure 3.2 also shows the two major binding activities observed with probe M. was the activity normally seen when the cell extract prepared was fresh. Mg was seen

Figure 3.2 The sensitivity of the binding activity to storage at low temperatures, seen with probe M. (See text for details).

C hap ter 3 - Identification of P rom oter Binding Proteins Using E M S A 81

P

\

I

I

w

after a period of storage probably resulting from degradation or dissociation of Ml, and occasionally appeared in fresh extracts from eggs that were presumably o f poor quality. In this experiment Mg became more apparent after the extract aliquots were incubated in the attempt to reactivate the binding activities. The binding activity seen with probe E was similar in size and intensity to Ml. A decrease in the size of this activity to one comparable with Mg was never seen.

Another possible source of the variability could have resulted from sequestration of the binding activities within the chromatin, which may have been removed by

centrifugation during extract preparation. To address this possibility, 1.0 M NaCl was added to some freshly prepared embryo extract to facilitate the extraction o f proteins from the chromatin, but this procedure did not improve the yield o f the binding proteins. In addition, any contaminating yolk that had not been removed from the extract prior to the NaCl treatment would have been solublised in the salt, possibly increasing the non-specific competition for the probe DNA.

It was apparent that the action o f proteases had to be minimised, and so during the course o f this work the panel of inhibitors added to the fresh extracts was expanded to included the seven described in chapter 2. Even so, in a typical extract stored at 15 °C, there was a significant reduction in binding activity only 48 hours after the cell extract was prepared (figure 3.3). This figure shows the two major binding activities seen with probe M, and also clearly demonstrates that a similar pattern is not found with probe E. | Although

in this experiment the probe was not in excess, it is unlikely that the appearance of Mg was solely attributable to a lack of probe saturation because in other experiments (see figure 3.2) it did appear in the presence of excess probe. However, this did not discount the

possibility that Mg has a lower binding affinity for this sequence than Ml- A larger binding activity associated with M, which only occasionally appeared, was also seen in the fresh cell extract. In later experiments, it was found that the stability of the extract could be improved by performing an ammonium sulphate fractionation with the extract (see chapter