The Sequence of 32 kb on the Left Arm of Yeast Chromosome XII Reveals Six Known Genes,a New Member of the Seripauperins Family and a New ABC Transporter Homologous to the Human Multidrug Resistance Protein

The analysis of a 32 kb DNA fragment from cosmid 2G12 on the left arm of chromosome XII identifies 14 open reading frames (ORFs) numbered L0948 to L1325, a new tRNA for proline, a delta remnant and two putative ARS. Six ORFs have been previously identified: HSP104, SSA2, SPA2, KNS1, DPS1/APS and SDC25. Three putative ORFs have significant homology with known proteins: L0968 is a new member of the very large ‘seripauperins’ family, comprising at least 20 yeast members; L1313 is a new ABC transporter highly homologous to the yeast cadmium resistance protein Ycf1p and to the human multidrug resistance protein hMRP1; the C-terminal part of L1325 present in our sequence is very homologous to the fruit fly abdominal segment formation protein Pumilio. Finally, two ORFs, L1201 and L1205, have weak homology with two yeast hypothetical proteins of unknown function identified by the yeast systematic sequencing genome. Since our nucleotide sequence overlaps by 11·6 kb the cosmid 2B18 sequenced by Miosga and Zimmerman (1996) on the right end, we have not reported here the analysis of the ORFs L1313, L1321 and L1325. The complete nucleotide sequence of 32,088 bp and the deduced ORFs were submitted to the EMBL database under Accession Number X97560.© 1997 John Wiley & Sons, Ltd.     

Sequence analysis of the CEN12 region of Saccharomyces cerevisiae on a 43·7 kb fragment of chromosome XII including an open reading frame homologous to the human cystic fibrosis transmembrane conductance regulator protein CFTR

In the framework of the European Union BIOTECH project for systematically sequencing the Saccharomyces cerevisiae genome, we determined the nucleotide sequence of a 43·7 kb DNA fragment spanning the centromeric region of chromosome XII. A novel approach was the distribution of sublibraries prepared by the DNA coordinator (J. Hoheisel, Heidelberg, FRG), using a new hybridization-based DNA mapping method, in order to facilitate ordered sequencing. The sequence contains 22 open reading frames (ORFs) longer than 299 bp, including the published sequences for ATS/DPS1, SCD25, SOF1, DRS1, MMM1, DNM1 and the centromeric region CEN12. Five putative ORF products show similarity to known proteins: the leucine zipper-containing ABC transporter L1313p to the yeast Ycf1p metal resistance protein, to the yeast putative ATP-dependent permease Yhd5p, to the yeast putative proteins Yk83p and Yk84p, to the human cystic fibrosis transmembrane conductance regulator protein (hCFTR) and to the human multidrug resistance-associated protein hMRP1; L1325p to the Drosophila melanogaster Pumilio protein, to the putative yeast regulatory protein Ygl3p and to the yeast protein Mpt5p/Htr1p; L1329p to human lipase A and gastric lipase, to rat lingual lipase and to the putative yeast triglyceride lipase Tgl1p; L1341p to the putative yeast protein Yhg4p; and the leucine zipper-containing L1361p to the two yeast proteins 00953p and Ym8156.08p and to the Arabidopsis thaliana protein HYP1. Eight ORFs show no homology to known sequences in the database, three small ORFs are internal and complementary to larger ones and L1301 is complementary overlapping the ATS/DPS1 gene. Additionally three equally spaced ARS consensus sequences were found. The nucleotide sequence reported here has been submitted to the EMBL data library under the accession number X91488.     

A Genetically Encodable System for Sequence‐Specific Detection of RNAs in Two Colors

Multicolor readout is an important feature of RNA detection techniques aiming at the investigation of RNA localization. Several detection methods have been developed, however they require either transfection of cells with the probe or prior tagging of the target RNA. We report a fully genetically encodable system for simultaneous detection of two RNAs by using green and yellow fluorescence based on tetramolecular fluorescence complementation (TetFC). To obtain yellow fluorescent protein (YFP), substitution T203Y was introduced into one of the three non‐fluorescent GFP fragments; this was fused to different variants of the Homo sapiens Pumilio homology domain. Using different sets of fusion proteins we were able to discriminate between two closely related target RNAs based on the fluorescence signals at the respective wavelengths.