Subtractive hybridization cloning of a tissue-specific extinguisher: TSE1 encodes a regulatory subunit of protein kinase A.
Tissue-specific extinguisher 1 (TSE1) is a trans-acting locus on human chromosome 17 that down-regulates expression of seven liver genes in hepatoma x fibroblast hybrids. To study the mechanism by which TSE1 functions, we used subtractive cDNA hybridization to clone transcripts encoded within a 2-4 Mb segment of chromosome 17 that includes TSE1. High resolution mapping within this region indicated that 8 of 9 different human cDNAs so obtained were distinct from TSE1. The remaining cDNA clone mapped concordantly with TSE1 in a panel of fragment-containing hybrids. DNA sequencing indicated that this cDNA encoded regulatory subunit RI alpha of cAMP-dependent protein kinase, and RI alpha mRNA levels correlated with TSE1 activity in various hybrid lines. Stable transfection of wild-type or cAMP-binding mutant RI alpha alleles into hepatoma recipients produced an extinction phenotype indistinguishable from that encoded by human TSE1. We conclude that TSE1 encodes a regulatory subunit of protein kinase A whose activity differs in different cell types.
Jones
K W
KW
Department of Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Shapero
M H
MH
Chevrette
M
M
Fournier
R E
RE
D001483:Base Sequence; D002886:Chromosomes, Human, Pair 17; D003001:Cloning, Molecular; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases; D004247:DNA / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000235:genetics*; D011506:Proteins / Q000235:genetics*; D011953:Receptors, Cyclic AMP / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D009841:Oligonucleotides; C517834:PRKAR1A protein, human; D011506:Proteins; D011953:Receptors, Cyclic AMP; D004247:DNA; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases