Use of a multiple S1 nuclease protection assay to monitor changes in RNA levels for type 1 phosphatase and several proto-oncogenes in response to insulin.
Changes in insulin-regulated gene expression occur in a time- and tissue-dependent fashion. To monitor these changes we have adapted the S1 nuclease protection assay to allow simultaneous estimation of multiple RNA species in a single sample by using synthetic oligonucleotides of various lengths as probes for specific RNA species, which can then be resolved by electrophoresis. The multiple S1 nuclease protection assay was used to assess the influence of insulin on the RNA concentrations of 12 different genes in human skeletal muscle. Estimates obtained by this assay were comparable with those obtained by Northern analysis. RNA levels for proto-oncogene c-src displayed a transient 4-fold increase, whereas RNA levels for type 1 protein phosphatase were suppressed by 50% during the same time period. RNAs corresponding to known insulin-responsive genes such as c-fos, c-myc, c-Ha-ras, and c-src displayed rapid and transient 2-4-fold increases between 30 and 60 min as detected by either Northern analysis or the multiple S1 nuclease protection assay. In addition, RNA levels for the insulin receptor, Glut-4, Glut-3, and c-jun were apparently unaffected by exposure of the cells to insulin.
Clinical Diabetes and Nutrition Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Phoenix, Arizona 85016.
D B
DB
Thompson
J
J
Sommercorn
D016375:Antisense Elements (Genetics); D001483:Base Sequence; D015152:Blotting, Northern; D005786:Gene Expression Regulation / Q000187:drug effects*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D016376:Oligonucleotides, Antisense; D009994:Osmolar Concentration; D010749:Phosphoprotein Phosphatases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D000090063:Proto-Oncogene Mas; D011519:Proto-Oncogenes / Q000187:drug effects*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D011972:Receptor, Insulin / Q000235:genetics; D012208:Rhabdomyosarcoma / Q000235:genetics / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D016375:Antisense Elements (Genetics); D007328:Insulin; C000715470:MAS1 protein, human; D016376:Oligonucleotides, Antisense; D000090063:Proto-Oncogene Mas; D012334:RNA, Neoplasm; D012313:RNA; D011972:Receptor, Insulin; D010749:Phosphoprotein Phosphatases; D015719:Single-Strand Specific DNA and RNA Endonucleases