Molecular cloning of cDNA corresponding to mRNA species whose steady state levels in the thyroid are enhanced by thyrotropin. Homology of one of these sequences with ferritin H.
A lambda gt11 cDNA library was constructed using poly(A)+ mRNA from thyrotropin (TSH)-stimulated Fisher rat thyroid (FRTL5) cells. The library was screened for nonthyroglobulin cDNA sequences by differential plaque filter hybridization using single-stranded cDNA probes synthesized from mRNA prepared from quiescent and TSH-stimulated FRTL5 cells. Thyroglobulin cDNA-containing recombinants in the library were avoided by prehybridizing the TSH probe to excess thyroglobulin cDNA. Of 48,000 clones screened, 60 were chosen as representing mRNA species whose abundance was increased in TSH-stimulated versus quiescent cultures. Southern blot analysis of 9 clones confirmed that the TSH-cDNA probe hybridized to a greater extent to the cDNA inserts than did the control probe. cDNA insert sizes varied between 0.3 kilobase (kb) and 1.0 kb. Northern slot blot analysis using as probes the cDNA of four of these clones (FC4, FC26, FC29, and FC43) demonstrated that TSH stimulation of FRTL5 cells increased the steady state levels of the respective mRNA species by 4-12-fold. For all 4 clones, increases in mRNA levels were apparent within approximately 1 h and were maximal after 14-18 h of TSH stimulation. Determination of the partial nucleotide sequence of these 4 clones confirmed that none was thyroglobulin, thyroid peroxidase, or any other gene previously reported to be stimulated by TSH. Three of the clones bore no homology to any known nucleotide sequence, but FC26 was 85% homologous with human ferritin H. Northern blot analysis using the FC26 cDNA insert as a probe confirmed hybridization to an mRNA species of 1 kb, the known size of ferritin H mRNA. In summary, using the technique of differential plaque filter hybridization, we have identified 4 new genes whose mRNA levels are increased by TSH stimulation of thyroid cells. One of these genes is homologous to human ferritin H.
Cox
F
F
Department of Medicine, Veterans Administration Medical Center, San Francisco, California.
Gestautas
J
J
Rapoport
B
B
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D005293:Ferritins / Q000235:genetics*; D005796:Genes / Q000187:drug effects; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics*; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013954:Thyroglobulin / Q000235:genetics*; D013961:Thyroid Gland / Q000187:drug effects / Q000378:metabolism*; D013972:Thyrotropin / Q000494:pharmacology*
D012333:RNA, Messenger; D013972:Thyrotropin; D004247:DNA; D005293:Ferritins; D013954:Thyroglobulin