Neuronal responses in macaque area PEc to saccades and eye position.

Neurons in area PEc in the superior parietal cortex encode signals from different modalities, such as visual, extraretinal and somatosensory, probably combining them to encode spatial parameter of extrapersonal space to prepare body movements. This study reports the characterization of the functional properties of PEc non-visual neurons that showed saccade-related activity. We analyzed the pre- and post-saccadic firing activity in 189 neurons recorded in five hemispheres of three behaving monkeys. Spiking activity of PEc single neurons was recorded while the monkeys performed visually-guided saccades in a reaction time task. We found that 84% of neurons recorded from area PEc showed pre-saccadic activity with directional tuning. In 26% of neurons, we found inhibition of activity in the pre-saccadic period. The onset of this "pause" always started before the saccade and, in 51% of neurons, it was invariant among different gaze directions. The post-saccadic activity in these cells was either a phasic response with directional tuning (77%) and/or an eye position tuning (75%). The analysis of the preferred direction did not show hemispheric preference, however, for the majority of neurons, the angular difference in the preferred direction, in the pre- and post-saccadic period, was more than 60 degrees . By confirming, therefore, that PEc neurons carry information about eye position, these novel findings open new horizons on PEc function that, to date, is not well documented. The pre-saccadic activity may reflect an involvement in saccade control, whereas post-saccadic activity may indicate a role in informing on the new eye position. These novel results about saccade and eye position processing may imply a role of area PEc in gaze direction mechanisms and, possibly, in remapping visual space after eye movements.

tRNA-assisted overproduction of eukaryotic ribosomal proteins.

Structural studies of eukaryotic ribosomes are complicated by the tendency of their constituent proteins to be expressed at very low levels in Escherichia coli. We find that this is mainly due to their exceptionally high content of AGA/AGG arginine codons, which are poorly utilized by the bacterial translational machinery. In fact, we could overcome this limitation by the combined use of a T7 RNA polymerase expression vector and a plasmid carrying the E. coli gene argU, which encodes the minor tRNA(Arg) species that reads AGA/AGG codons. In this system, five cytoplasmic ribosomal proteins from three different eukaryotic lineages (Saccharomyces cerevisiae S8, L13, and L14; Arabidopsis thaliana L13; and Homo sapiens L7) could be overexpressed to up to 50% of total bacterial protein and were purified to homogeneity in tens of milligrams amounts. The purification procedure simply involved metal affinity chromatography followed, in some cases, by an additional heparin chromatography step. Recombinant polypeptides bound RNA with high affinity (K(d) between 50 and 300 nM). This novel overexpression/purification strategy will allow the production of high amounts of most eukaryotic ribosomal proteins in a form suitable for structural and functional studies. Coupled with recently completed and ongoing whole-genome sequencing projects, it will facilitate the molecular characterization of the eukaryotic ribosome.

High level expression in E. coli and purification of yeast transcription factor IIIA.

Saccharomyces cerevisiae transcription factor IIIA, a sequence-specific DNA binding protein that is required for transcription of 5S rRNA genes by RNA polymerase III, has been expressed in Escherichia coli in a full length, native form. High level expression was achieved through the combined use of a T7 RNA polymerase expression system and of a multicopy plasmid carrying an E. coli gene, argU, which codes for a minor Arg(AGA/AGG) tRNA species. Recombinant yeast transcription factor IIIA was purified to 95% homogeneity, at a final yield of 8 mg/liter of bacterial culture, by three chromatographic steps, and it was shown to be at least 55% active by quantitative in vitro transcription assays.