Analysis of glucocorticoid unresponsive cell variants using a mouse glucocorticoid receptor complementary DNA clone.
We have used a glucocorticoid receptor cDNA isolated from a mouse lymphoma cell line to characterize receptor mRNA and genomic sequences present in wild type and mutant rat hepatoma (HTC) and mouse thymoma (S49 and WEHI7) cells. Wild type rat and mouse cell lines contain two receptor mRNAs, 5 and 7 kilobase pairs (kb) in length, which differ in the length of their 3'-untranslated regions. Levels of receptor mRNA present in mutant HTC, WEHI7, and S49 cells of the r- (receptorless) phenotype are decreased compared to wild type cells. This decreased level of receptor mRNA parallels the decreased level of total immunoreactive receptor protein found in these cells. S49 nt- (nuclear transfer minus) cells contain receptor mRNA levels which parallel their hormone binding and immunoreactive receptor levels. Cells of the r- and nt- phenotype contain no detectable deletions or rearrangements of the receptor gene. We conclude that r- cells have lesions which affect the expression of receptor mRNA. Surprisingly, HTC cells of the r- phenotype differ from WEHI7 and S49 r- cells in that HTC r- cells contain a lower level of receptor DNA than does their parental wild type cell line. Although these cells may contain multiple lesions, it appears that loss of receptor genomic sequences is responsible, in part, for the phenotype of the HTC r- cells. The S49 nti (nuclear transfer increase) cells produce glucocorticoid receptors of molecular weights 40,000 and 94,000. These cells produce, in addition to the wild type 5- and 7-kb receptor mRNAs, two other receptor messages 5.5 and 3.5 kb in length. RNA blot analysis using various portions of our receptor cDNA indicates that these are 5' truncated messages and suggests that the 40-kDa nti receptor is truncated at its NH2-terminal end. These data also indicate that the hormone and DNA-binding regions of the receptor are located in the COOH-terminal half of the protein.
D000483:Alleles; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000235:genetics; D008223:Lymphoma / Q000235:genetics; D051379:Mice; D008970:Molecular Weight; D010641:Phenotype; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D013945:Thymoma / Q000235:genetics; D013953:Thymus Neoplasms / Q000235:genetics; D014158:Transcription, Genetic
D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D004247:DNA; D004262:DNA Restriction Enzymes