Molecular analysis of cDNA clones and the corresponding genomic coding sequences of the Drosophila dunce' gene, the structural gene for cAMP phosphodiesterase

We have isolated and sequenced cDNA clones representing portions of the polyadenylylated transcripts of the dunce+ gene. These define an open reading frame of 1086 bases and some of the 5'- and 3'-untranslated regions of the transcripts. The deduced amino acid sequence is strikingly homologous to the amino acid sequence of a Ca^2+/calmodulin-dependent cyclic nucleotide phosphodiesterase isolated from bovine brain and more weakly related to the predicted amino acid sequence of a yeast cAMP phosphodiesterase. These homologies, together with prior genetic and biochemical studies, provide unambiguous evidence that dunce^+ codes for a phosphodiesterase. In addition, the dunce^+ gene product shares a seven-amino acid sequence with a regulatory subunit of cAMP-dependent protein kinase that is predicted to be part of the cAMP binding site. We also identify a weak homology between a region of the dunce+ gene product and the egg-laying hormone precursor of Aplysia californica. The open reading frame is divided in the genome by four introns

Cellular/Molecular Differential Ubiquitination and Proteasome Regulation of Ca V 2.2 N-Type Channel Splice Isoforms

Ca V 2.2 (N-type) calcium channels control the entry of calcium into neurons to regulate essential functions but most notably presynaptic transmitter release. Ca V 2.2 channel expression levels are precisely controlled, but we know little of the cellular mechanisms involved. The ubiquitin proteasome system (UPS) is known to regulate expression of many synaptic proteins, including presynaptic elements, to optimize synaptic efficiency. However, we have limited information about ubiquitination of Ca V 2 channels. Here we show that Ca V 2.2 proteins are ubiquitinated, and that elements in the proximal C terminus of Ca V 2.2 encoded by exon 37b of the mouse Cacna1b gene predispose cloned and native channels to downregulation by the UPS. Ca V 2.2 channels containing e37b are expressed throughout the mammalian nervous system, but in some cells, notably nociceptors, sometimes e37a-not e37b-is selected during alternative splicing of Ca V 2.2 pre-mRNA. By a combination of biochemical and functional analyses we show e37b promotes a form of ubiquitination that is coupled to reduced Ca V 2.2 current density and increased sensitivity to the UPS. Cell-specific alternative splicing of e37a in nociceptors reduces Ca V 2.2 channel ubiquitination and sensitivity to the UPS, suggesting a role in pain processing

A Novel Plasmid-Based Microarray Screen Identifies Suppressors of rrp6Δ in Saccharomyces cerevisiae

Genetic screens in Saccharomyces cerevisiae provide novel information about interacting genes and pathways. We screened for high-copy-number suppressors of a strain with the gene encoding the nuclear exosome component Rrp6p deleted, with either a traditional plate screen for suppressors of rrp6Δ temperature sensitivity or a novel microarray enhancer/suppressor screening (MES) strategy. MES combines DNA microarray technology with high-copy-number plasmid expression in liquid media. The plate screen and MES identified overlapping, but also different, suppressor genes. Only MES identified the novel mRNP protein Nab6p and the tRNA transporter Los1p, which could not have been identified in a traditional plate screen; both genes are toxic when overexpressed in rrp6Δ strains at 37°C. Nab6p binds poly(A)(+) RNA, and the functions of Nab6p and Los1p suggest that mRNA metabolism and/or protein synthesis are growth rate limiting in rrp6Δ strains. Microarray analyses of gene expression in rrp6Δ strains and a number of suppressor strains support this hypothesis

CACNA1B mutation is linked to unique myoclonus-dystonia syndrome

Using exome sequencing and linkage analysis in a three-generation family with a unique dominant myoclonusdystonia-like syndrome with cardiac arrhythmias, we identified a mutation in the CACNA1B gene, coding for neuronal voltage-gated calcium channels Ca(V)2.2. This mutation (c.4166G>A; p.Arg1389His) is a disruptive-missense mutation in the outer region of the ion pore. The functional consequences of the identified mutation were studied using whole-cell and single-channel patch recordings. High-resolution analyses at the single-channel level showed that, when open, R1389H Ca(V)2.2 channels carried less current compared with WT channels. Other biophysical channel properties were unaltered in R1389H channels including ion selectivity, voltage-dependent activation or voltage-dependent inactivation. Ca(V)2.2 channels regulate transmitter release at inhibitory and excitatory synapses. Functional changes could be consistent with a gain-of-function causing the observed hyperexcitability characteristic of this unique myoclonus-dystonia-like syndrome associated with cardiac arrhythmias