PubMed · 1821649
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J W Hawkins. 1991. Antisense technology: commercial implications.. https://pubmed.ncbi.nlm.nih.gov/1821649/
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We have employed antisense methods to study the transcriptional functions of c-fos protein (Fos). Clones expressing inducible anti-fos RNA have been employed to inhibit c-fos expression, resulting in activation of c-fos transcription by inhibiting its normal repressor function. The sites of negative regulation by Fos have been mapped using this antisense mapping method which demonstrates that the serum response element represents the major site of repression by endogenous c-fos protein. A similar strategy (antisense cloning) has been employed to clone four target genes that are Fos dependent. These cDNAs encode mRNAs that are rapidly induced by serum (although this induction is blocked by cycloheximide) but are blocked by induction of anti-fos RNA. These inhibitory methods of studying transcription factor function are extremely useful for transcription factors (like Fos) that require cooperation with other factors to modulate gene transcription.
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.
Considerable progress in tomato molecular biology has been made over the past five years. At least 19 different mRNAs which increase in amount during tomato fruit ripening have been cloned and genes for enzymes involved in cell wall degradation (polygalacturonase and pectinesterase) and ethylene synthesis (ACC synthase) have been identified by conventional procedures. Transgenic plants have been used to identify regions of DNA flanking fruit-specific, ripening-related and ethylene-regulated genes and trans-acting factors which bind to these promoters have also been identified. Antisense genes expressed in transgenic plants have proved to be highly effective for inhibiting the specific expression of ripening-related genes. These experiments have changed our understanding of how softening occurs in tomato fruit. Antisense techniques have also been used to identify genes encoding enzymes for carotenoid biosynthesis (phytoene synthase) and ethylene biosynthesis (the ethylene-forming enzyme). The altered characteristics of fruit transformed with specific antisense genes, such as retarded ripening and resistance to splitting, may prove to be of value to fruit growers, processors and ultimately the consumer.