The aim of this work is to clone, sequence and characterize the glnA gene from the Prochlorophyte Prochlorococcus, encoding for glutamine synthetase (GS) and to study the regulation of this enzyme. An heterologous probe will be used to screen a Prochlorococcus gene library already constructed, for glnA cloning. After sequencing, it will be characterized by comparison with glnA from other Cyanobacteria, organisms very related with Prochlorococcus. Experiments on GS regulation will focus on two aspects :
nitrogen source and light changes. The time course of GS activity, GS protein and glnA transcript levels will be measured by spectrophotometric assays, quantitative immunoelectrophoresis and Northern blotting (using the cloned gene as a probe), respectively. Nitrogen regulation will be studied using different nitrogen sources, including nitrate, nitrite, ammonium and nitrogen starvation. The effect of some inhibitors of enzymes involved in nitrogen assimulation (methionine sulfoximine, azaserine) will be analyzed as well. On the other hand, different experiments concerning regulation of GS by light will be performed, with particular attention to the effect of changes from high to low irradiance, and darkness, in the presence or absence of glucose.
The proposed project include the use of very different techniques, from classical biochemistry to molecular biology, which ensure the training in a wide range of modern laboratory methodology, to ensure a good background for future research.