ABSTRACT
This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.
Subject(s)
Genome , Mice/genetics , Terminator Regions, Genetic , Transcription Initiation Site , Transcription, Genetic , 3' Untranslated Regions , Animals , Base Sequence , Conserved Sequence , DNA, Complementary/chemistry , Genome, Human , Genomics , Humans , Promoter Regions, Genetic , Proteins/genetics , RNA/chemistry , RNA/classification , RNA Splicing , RNA, Untranslated/chemistry , Regulatory Sequences, Ribonucleic AcidABSTRACT
We have produced a plasmid designed for the expression of heterologous G protein alpha subunits in the yeast Saccharomyces cerevisiae. Introduction of these genes is by simple cassette replacement using unique restriction sites, and their expression is controlled by the regulatory sequences of the S. cerevisiae GPA1 gene. Levels of expression are therefore suitable for interaction of these heterologous proteins with elements of the yeast pheromone response pathway. We believe that this plasmid will facilitate the coupling of more members of the seven transmembrane domain superfamily of receptors, through their native G protein alpha subunit, to the yeast pheromone response pathway. The plasmid pRGP, is a stable centromeric shuttle vector with a HIS3-selectable marker. We have demonstrated that production of GPA1 from this plasmid functionally complements a gpal1- null mutation. A similar response is obtained when an alternative G protein alpha subunit, G(olf), is introduced using pRGP. We believe that this is the first example of a heterologous G protein shown to couple to a yeast pheromone receptor.
Subject(s)
GTP-Binding Protein alpha Subunits , GTP-Binding Proteins/physiology , Heterotrimeric GTP-Binding Proteins/genetics , Receptors, Peptide/physiology , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Transcription Factors , GTP-Binding Protein alpha Subunits, Gq-G11 , Heterotrimeric GTP-Binding Proteins/physiology , Plasmids , Receptors, Mating FactorABSTRACT
A putative olfactory receptor-encoding gene was cloned from human genomic DNA and shown to be expressed by isolation of a full-length cDNA from olfactory tissue. A second cDNA clone was found to encode an olfactory receptor pseudogene. The expression of a pseudogene from the olfactory gene repertoire, in neurons which express only a single receptor type, implies that many neurons will be non-functional.
