Characterization of TASK-4, a novel member of the pH-sensitive, two-pore domain potassium channel family.

We report the primary sequence of TASK-4, a novel member of the acid-sensitive subfamily of tandem pore K(+) channels. TASK-4 transcripts are widely expressed in humans, with highest levels in liver, lung, pancreas, placenta, aorta and heart. In Xenopus oocytes TASK-4 generated K(+) currents displaying a marked outward rectification which was lost by elevation of extracellular K(+). TASK-4 currents were efficiently blocked by barium (83% inhibition at 2 mM), only weakly inhibited by 1 mM concentrations of quinine, bupivacaine and lidocaine, but not blocked by tetraethylammonium, 4-aminopyridine and Cs(+). TASK-4 was sensitive to extracellular pH, but in contrast to other TASK channels, pH sensitivity was shifted to more alkaline pH. Thus, TASK-4 in concert with other TASK channels might regulate cellular membrane potential over a wide range of extracellular pH.

Sequence analysis of a 78.6 kb segment of the left end of Saccharomyces cerevisiae chromosome II.

We report the sequence analysis of a 78,601 bp DNA segment on the left arm of chromosome II of Saccharomyces cerevisiae. This 78.6 kb segment spans the region from the start of a subtelomeric Y' element up to the ILS1 gene. It contains 49 open reading frames (ORFs) with more than 100 amino acids length including 14 internal and five overlapping ORFs. The gene density, excluding the internal ORFs, was calculated as one ORF per 2.2 kb. Eight ORFs (PKC1, TyA, TyB, ATP1, ROX3, RPL17a, PET112 and ILS1) correspond to previously characterized genes. ORF YBL0718 was identified as CDC27; YBL0706 as TEL1. Four other ORFs show strong similarities to already known genes. The gene product of YBL0838 is 60% identical to the ribosomal protein RPL32 from rat, mouse and man. YBL0701 encodes a protein with significant similarity to the initiation factor eIF2 associated p67 glycoprotein from rat. Eight ORFs were disrupted and the resulting yeast strains analysed with respect to their phenotype.

Cloning and characterization of the ALG3 gene of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae alg3-1 mutant is described as defective in the biosynthesis of dolichol-linked oligosaccharides (Huffaker and Robbins, Proc. Natl. Acad. Sci. USA, 80, 7466-7470, 1983). Man5GlcNAc2-PP-Dol accumulates in alg3 cells and Endo H resistant carbohydrates are transferred to protein by the oligosaccharyltransferase complex. In this study, we describe the cloning of the ALG3 locus by complementation of the temperature sensitive growth defect of the alg3 stt3 double mutant. The isolated ALG3 gene complements both the defect in the biosynthesis of lipid-linked oligosaccharides of the alg3-mutant and the under-glycosylation of secretory proteins. The inactivation of the nonessential ALG3 gene results in the accumulation of lipid-linked Man5GlcNac2 and protein-bound carbohydrates which are completely Endo H resistant. The ALG3 locus encodes a potential ER-transmembrane protein of 458 amino acids (53 kDa) with a C-terminal KKXX-retrieval sequence.

TEL1, a gene involved in controlling telomere length in S. cerevisiae, is homologous to the human ataxia telangiectasia gene.

Yeast chromosomes terminate in tracts of simple repetitive DNA (poly[G1-3T]). Mutations in the gene TEL1 result in shortened telomeres. Sequence analysis of TEL1 indicates that it encodes a very large (322 kDa) protein with amino acid motifs found in phosphatidylinositol/protein kinases. The closest homolog to TEL1 is the human ataxia telangiectasia gene.

Toward a catalog of human genes and proteins: sequencing and analysis of 500 novel complete protein coding human cDNAs.

With the complete human genomic sequence being unraveled, the focus will shift to gene identification and to the functional analysis of gene products. The generation of a set of cDNAs, both sequences and physical clones, which contains the complete and noninterrupted protein coding regions of all human genes will provide the indispensable tools for the systematic and comprehensive analysis of protein function to eventually understand the molecular basis of man. Here we report the sequencing and analysis of 500 novel human cDNAs containing the complete protein coding frame. Assignment to functional categories was possible for 52% (259) of the encoded proteins, the remaining fraction having no similarities with known proteins. By aligning the cDNA sequences with the sequences of the finished chromosomes 21 and 22 we identified a number of genes that either had been completely missed in the analysis of the genomic sequences or had been wrongly predicted. Three of these genes appear to be present in several copies. We conclude that full-length cDNA sequencing continues to be crucial also for the accurate identification of genes. The set of 500 novel cDNAs, and another 1000 full-coding cDNAs of known transcripts we have identified, adds up to cDNA representations covering 2%--5 % of all human genes. We thus substantially contribute to the generation of a gene catalog, consisting of both full-coding cDNA sequences and clones, which should be made freely available and will become an invaluable tool for detailed functional studies.