Qualitative gene profiling: a novel tool in genomics and in pharmacogenomics that deciphers messenger RNA isoforms diversity.

RNA splicing is a tightly regulated process. It is essential for gene expression and, therefore, intervenes in every biological phenomenon in mammals. RNA splicing regulation is cell type-specific in such a way that a cellular situation can be characterised by its repertoire of spliced events, the spliceome. Comparison of the splicing repertoire of two situations identifies alternative exons and introns. This regulation involves cis-acting sequences and transacting factors. Mutations, as well as modifications of signalling pathways, can alter the accuracy of splicing. Since deletion of exons or retention of introns within coding sequences modifies the corresponding proteins and functional domains of proteins are encoded by contiguous exons, identifying changes in the spliceome pinpoints functional domains, which are specifically regulated at the level of RNA splicing. We have developed a new method of gene profiling, qualitative gene profiling, that allows the comparative study of the repertoires of spliced events that characterise distinct physiopathological situations. We present in this review the different uses of this new genomic technique that can help each step of the R&D process in the pharmaceutical industry, and that represents a short cut towards functional genomics and pharmacogenomics.

Structural aspects of the gastric H,K ATPase: the M5/M6 domain and alpha beta association.

This review summarizes some of the structural information that has been obtained on the gastric H,K ATPase. Methods such as tryptic digestion, site specific labeling and in vitro translation combine to provide a ten membrane segment model with however reservations as to the full transmembrane nature of M5 or M6. Labeling this region with the thiophilic luminal face reagent omeprazole provided cogent evidence that cys 813 but not cys 822 was labeled. On the other hand, cysteine mutagenesis provided evidence that removal of cys 813 did not affect inhibition of Rb transport by omeprazole whereas removal of cys 822 although not affecting ATPase activity abolished omeprazole inhibition of transport. A model to reconcile these data is presented where M5 and M6 although intramembranal are not transmembrane hairpin structures. Analysis of the region of alpha beta interaction by tryptic digestion and WGA chromatography to define those fragments of alpha that remain beta associated shows that leu 853 to arg 922 in the TM7-loop are a major region of association with the beta subunit. Yeast two hybrid analysis, when combined with these data and those from a chimeric construct, indicates that the sequence Q 907 to R 922 is the important element of interaction in the alpha subunit and no other extracytoplasmic domain was found to interact. Two regions of the beta subunit interact with this region of the alpha subunit between Q64 and N130 as well as A156 and R188. Apparently the beta subunit is folded around a small region of the large extracytoplasmic loop between TM7 and TM8, closer to TM8.

Regions of association between the alpha and the beta subunit of the gastric H,K-ATPase.

A binding and a yeast two-hybrid analysis were carried out on the gastric H,K-ATPase to determine interactive regions of the extracytoplasmic domains of the alpha and beta subunits of this P type ATPase. Wheat germ agglutinin fractionation of fluorescein 5-maleimide-labeled tryptic fragments of detergent-solubilized H, K-ATPase showed that a fragment Leu855 to Arg922 of the alpha subunit was bound to the beta subunit. The yeast two-hybrid system showed that the region containing only a part of the seventh transmembrane segment, the loop, and part of the eighth transmembrane segment was capable of giving positive interaction signals with the ectodomain of the beta subunit. The sequence in the extracytoplasmic loop close to the eighth transmembrane segment, namely Arg898 to Thr928, was identified as being the site of interaction using this method. We deduced that the sequence Arg898 to Arg922 in the alpha subunit has strong interaction with the extracytoplasmic domain of the beta subunit. Again, using yeast two-hybrid analysis, two different sequences in the beta subunit Gln64 to Asn130 and Ala156 to Arg188 were identified as association domains in the extracytoplasmic sequence of the beta subunit. These data enable identification of major associative regions of the alpha-beta subunits of the H,K-ATPase.