RESUMO
Tyrosine aminotransferase (TyrATase; L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) from rat liver is subject to glucocorticoid and cAMP as well as developmental control. To isolate DNA sequences encoding TyrATase, we constructed a cDNA library from rat liver poly(A)+RNA enriched for TyrATase mRNA. Recombinant plasmids were screened by differential colony hybridization to poly(A)+RNA isolated from adrenalectomized and dexamethasone-treated animals. Differentially hybridizing plasmids were then shown to contain TyrATase cDNA sequences by their ability to select a mRNA whose in vitro translation product is immunoprecipitable with antiserum against TyrATase. In confirmation, we detect mRNA homologous to TyrATase cDNA sequences in hepatoma cell lines known to contain TyrATase activity but not in a cell line lacking this activity. We show that treatment of rats with dexamethasone or N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate leads to a 5- to 10-fold increase in the amount of TyrATase mRNA.
Assuntos
Tirosina Transaminase/genética , Animais , Bucladesina/farmacologia , Clonagem Molecular , DNA/genética , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/fisiologia , Masculino , RNA Mensageiro/genética , RatosRESUMO
Tryptophan oxygenase (TO, EC 1.13.1.12) from rat liver is subject to glucocorticoid and developmental control. To study the mechanism of regulation, TO mRNA sequences and the chromosomal TO gene were cloned. From a cDNA library prepared from rat liver poly(A)+ RNA enriched for TO mRNA, a recombinant plasmid containing TO cDNA sequences was identified by translation of hybrid-selected RNA and immunoprecipitation with antibodies directed against TO. This cDNA clone hybridizes to a mRNA 2000 bases long that is inducible by dexamethasone. With this clone as probe we isolated from a bacteriophage lambda rat DNA library genomic clones which together span a region of 32 kilobase pairs (kb). Heteroduplex analysis revealed that the gene extends over 19 kb and is interrupted by at least 11 introns. To characterize the presumptive control region the DNA sequence around the 5' end of the TO gene was determined. S1 nuclease protection experiments revealed two separate start sites for TO mRNA transcription within this region.
