Rapid simultaneous measurement of rat alpha- and thyrotropin (TSH) beta-subunit messenger ribonucleic acids (mRNAs) by solution hybridization: regulation of TSH subunit mRNAs by thyroid hormones.
Using a sensitive method for the simultaneous measurement of TSH subunit mRNAs, we have investigated their hormonal regulation in the pituitary glands of normal and hypothyroid rats. Oligodeoxyribonucleotides (probes) complementary to coding regions of rat alpha- and TSH beta-subunit mRNAs were synthesized. These probes were 5'-end labeled with gamma-[32P] ATP and hybridized with total pituitary RNA obtained from T3-treated and untreated normal and hypothyroid rats. The samples were then exposed to S1 nuclease to digest single stranded nucleic acids. Specific hybridization of probes to the TSH subunit mRNAs would yield double stranded structures resistant to this enzyme. Measurement of the amount of undigested probes by denaturing polyacrylamide gel electrophoresis, autoradiography, and densitometry permits quantification of these mRNAs. Both rat alpha and TSH beta mRNAs were detected with as little as 0.1 microgram total pituitary RNA, representing a more than 10-fold increase in sensitivity compared to a standard RNA blot hybridization assay. Thyroidectomy resulted in a 3- to 5-fold increase, whereas T3 treatment caused a significant decrease in the subunit mRNAs in both normal and hypothyroid animals. However, in all treatment groups, the TSH beta mRNA was affected to a greater extent than the alpha mRNA by the changes in thyroid status. The ratio of alpha- to beta-subunit mRNAs was decreased with hypothyroidism and increased with T3 treatment. This assay allows simultaneous quantification of multiple mRNAs from a single pituitary gland within 48 h and should facilitate studies of the regulation of mRNAs encoding TSH subunits specifically and other pituitary proteins in general.
Carr
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D000818:Animals; D004720:Endonucleases / Q000378:metabolism; D015292:Glycoprotein Hormones, alpha Subunit; D007037:Hypothyroidism / Q000378:metabolism; D008297:Male; D008722:Methods; D009693:Nucleic Acid Hybridization; D010446:Peptide Fragments / Q000235:genetics*; D010908:Pituitary Hormones, Anterior / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013963:Thyroid Hormones / Q000494:pharmacology*; D013972:Thyrotropin / Q000097:blood / Q000235:genetics*; D014284:Triiodothyronine / Q000097:blood
D015292:Glycoprotein Hormones, alpha Subunit; D010446:Peptide Fragments; D010908:Pituitary Hormones, Anterior; D012333:RNA, Messenger; D013963:Thyroid Hormones; D014284:Triiodothyronine; D013972:Thyrotropin; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases