Heterogeneity of Messenger RNA That Encodes the Rat Insulin Receptor Is Limited to the Domain of Exon 11: Analysis by RNA Heteroduplex Mapping, Amplification of cDNA, and In Vitro Translation
| Autor: | Alana L. Dudley, Barry J. Goldstein |
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| Rok vydání: | 1992 |
| Předmět: |
Male
Endocrinology Diabetes and Metabolism Molecular Sequence Data Restriction Mapping Polymerase Chain Reaction Rats Sprague-Dawley Exon Complementary DNA Internal Medicine Animals Coding region RNA Messenger Messenger RNA Base Sequence biology Alternative splicing Nucleic Acid Heteroduplexes Genetic Variation RNA DNA Exons RNA Probes Molecular biology Receptor Insulin Rats Molecular Weight Insulin receptor Antisense Elements (Genetics) Oligodeoxyribonucleotides Protein Biosynthesis biology.protein Heteroduplex |
| Zdroj: | Diabetes. 41:1293-1300 |
| ISSN: | 1939-327X 0012-1797 |
| Popis: | Structural isoforms of the insulin receptor that occur in various tissues have been postulated to be involved in certain actions of insulin in target cells. To determine whether these insulin-receptor subtypes are caused by alterations in the receptor primary structure, we used RNA heteroduplex mapping and amplification of cDNA to detect variation in the coding region of insulin-receptor mRNA from 5 rat tissues. A complete series of overlapping antisense [32P]RNA probes was prepared from plasmids containing segments of a full-length rat insulin-receptor cDNA, and probes were hybridized individually in solution with polyadenylated RNA from rat brain, kidney, liver, skeletal muscle, and spleen. After ribonuclease digestion, probe fragments were analyzed by denaturing gel electrophoresis. Tissue-specific cleavage of the mRNA:RNA probe heteroduplex, attributable to sequence mismatch, was detected only for a single probe covering the distal α-subunit, as expected for the known alternative splicing of rat insulin-receptor mRNA in this region. No evidence for additional heterogeneity of the receptor mRNA coding region was observed in the 5 tissues studied either by RNA heteroduplex mapping or, in some areas, by regional amplification of insulin-receptor cDNA. Cell-free translation of size-fractionated polyadenylated RNA was used to further demonstrate that each of the major insulin-receptor mRNA size classes in rat liver contained both forms of the alternatively spliced mRNA transcripts and produced two insulin-proreceptor polypeptides. These results suggest that heterogeneity of the insulin-receptor mRNA coding region affecting the receptor primary structure is limited to the distal α-subunit near the subunit cleavage site. Many of the reported structural isoforms of the mature insulin receptor are therefore likely to occur by specific posttranslational processing events that may influence the transmission of the insulin signal in various tissues. |
| Databáze: | OpenAIRE |
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