Molecular Characterization of the Novel Basic Helix–Loop–Helix Protein DEC1 Expressed in Differentiated Human Embryo Chondrocytes
Ming ShenTakeshi KawamotoWeiqun YanKazuko NakamasuMami TamagamiYasuhiko KoyanoMitsuhide NoshiroYukio Kato
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Basic helix-loop-helix
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Changes of expression in cold-regulated mRNA levels in potato (Solanum tuberosum L. cv. Superior) were analyzed. A total of 12,000 cDNAs was subjected to reverse Northern blot analysis using ^(32)P-dCTP- labeled first strand cDNA generated from the total RNA isolated from 25℃- and 4℃-treated potato plants. A total of 245 cDNA clones were sequenced from a cDNA library constructed from the cold treatment. Based on the BLAST results, 103 cDNA clones were found to be redundant. The analyzed genes were classified into 12 groups according to their putative functions, where the 20.2 % of group I was associated with energy metabolism, 13.1％ of group XI with cell rescue and defense, 2.6％ of group Ⅷwith signal transduction. Among the cDNA clones up-regulated by cold treatment from the results of the reverse Northern blot analysis, 32 were used for the Northern blot analysis. Most of the cold-treated clones showed overexpression compared with the control, while some showed down-regulation. In general, it was found that cold stress related genes were overexpressed more than two-folds at 4℃ treatment.
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Summary We used an in situ hybridization technique using single‐stranded RNA probes to study the expression pattern of γ‐glutamyl transpeptidase (GGT) in rat liver and kidney during development. The results were compared to those obtained with an immunoperoxidase technique and with Northern blot analysis of GGT mRNA. In the kidney, northern blot revealed a 20‐fold increase of GGT mRNA between day 18 of gestation and adulthood. Protein and mRNA localization clearly identified the proximal tubules as the site of synthesis of GGT. In the liver, the expression was lower than in the kidney and Northern blot showed a dramatic decrease of expression after birth. Using immunohistochemistry, the protein was detected within parenchymal cells in embryo and hepatocyte membranes and bile ducts in adults. Using in situ hybridization, GGT mRNA was only detected on days I and 2 after birth and exclusively in hepatocytes. Immunoperoxidase may be more sensitive than in situ hybridization to study the expression of minor liver protein such as GGT. However, the study of GGT expression using in situ hybridization is possible in cases of increased expression such as alcoholism, cholestasis, and carcinogenesis.
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Using bovine brains of adult and developmental stages, the time and place of the appearance of mRNA for glial fibrillary acidic protein (GFA-protein) were studied by in situ- and Northern blot-hybridization. Double-stranded cDNA labeled with [3H]dCTP or [32P]dCTP was used as the probe for this mRNA. To compare the location of GFA-protein mRNA and GFA-protein itself on serial sections. GFA-protein immunohistochemistry was used. By in situ hybridization with adult bovine brain. GFA-protein mRNA was detected in astroglia, most of which were in the white matter. The distribution of these astroglia by in situ hybridization was consistent with the findings by GFA-protein immunohistochemistry and Northern blot hybridization, indicating that each techniques were specific. Concerning fetal stages, GFA-protein mRNA could be detected in the brain of a fetal calf with a body length of 28 cm by in situ hybridization using the 32P-labeled probe, and the mRNA was localized in the subpial area and the fornix. These results indicated that glial maturation first became recognizable at least in the subpial area and the fornix in the brain of a fetal calf measuring 28 cm. In this fetal brain, GFA-protein mRNA was almost undetectable by Northern blot hybridization. This suggested that in situ hybridization was more sensitive and useful for the analysis of gene expression than Northern blot hybridization, when the target mRNA is present in only a limited area, such as in the brain.
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Human IL-1 alpha, IL-1 beta, and TNF-alpha mRNA expression was examined in peripheral blood monocytes (PBM) from normal individuals and in primary synoviocytes isolated from patients with rheumatoid arthritis by Northern blot and in situ hybridization. Cells cultured in the presence or absence of LPS were analyzed using in vitro synthesized 35S-labeled sense or antisense RNA probes to determine the relative abundance and the cell type expressing each of the mRNA for these potent inflammatory mediators. The results indicated that 72% of the LPS-stimulated PBM expressed detectable levels of IL-1 alpha mRNA, 89% IL-1 beta mRNA, and 10% TNF-alpha transcripts. Thus, the majority of activated PBM produced both IL-1 alpha and IL-1 beta. Experiments combining immunofluorescence for IL-1 beta protein with in situ hybridization for TNF-alpha mRNA demonstrated that monocytes expressing TNF-alpha mRNA also produced IL-1 beta. Primary synoviocytes from four patients with RA were also examined for the mRNA expression of each cytokine. Northern blot analyses of total RNA isolated from 0 to 72 h after LPS- or mock-stimulation showed that IL-1 beta mRNA was the most abundantly expressed, followed by TNF-alpha. In situ hybridization revealed that IL-1 beta and TNF-alpha transcripts were detected exclusively in synovial tissue macrophages. IL-1 alpha mRNA was not detected in these cultures by either method.
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The cellular localization of angiotensinogen messenger RNA (mRNA) in rat atria, aorta, and mesentery was studied using hybridization in situ. Angiotensinogen mRNA was identified in periatrial and periaortic brown adipocytes and in fibroblast-like cells of periaortic connective tissue and mesentery. Angiotensinogen gene expression in brown adipose tissue was confirmed by both Northern blot analysis and cell-free translation of RNA extracted from brown adipose tissue. For control rats, the level of angiotensinogen mRNA of brown adipose tissue, quantitated by Northern blot analysis, was 5% of the level in liver and showed a 12-fold increase in response to treatment with the combination of dexamethasone, ethynylestradiol, and T3. Comparison of the relative amounts of angiotensinogen mRNA detected by hybridization in situ in liver and brown adipose tissue, with the amounts determined by Northern blot analysis, revealed that hybridization in situ detected mRNA in brown adipose tissue with higher efficiency than in liver. These results suggest a role for angiotensinogen gene expression in brown adipose tissue function. In addition, these studies show that tissue-specific factors may modify the efficiency of detection of tissue mRNA using hybridization in situ. (Endocrinology121: 1616–1626, 1987)
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Abstract Quantitative, very sensitive estimates of gene expression may be achieved by Northern blot analysis and solution hybridization, particularly if many cells are making few copies of a specific mRNA. In situ hybridization histochemistry, on the other hand, is well suited to studies of the spatial and temporal distribution of mRNA expression in the central nervous system (CNS), wherein phenotypically heterogeneous neuronal and neuroglial populations may be present at very different development stages at any given point in time. The mRNA in question may be present in high abundance in only a few cells or only at a specific stage of their development, in which case it might otherwise be diluted out in Northern blots or solution hybridization by the large number of cells in which gene transcription is not apparent.
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We used an in situ hybridization technique using single-stranded RNA probes to study the expression pattern of gamma-glutamyl transpeptidase (GGT) in rat liver and kidney during development. The results were compared to those obtained with an immunoperoxidase technique and with Northern blot analysis of GGT mRNA. In the kidney, northern blot revealed a 20-fold increase of GGT mRNA between day 18 of gestation and adulthood. Protein and mRNA localization clearly identified the proximal tubules as the site of synthesis of GGT. In the liver, the expression was lower than in the kidney and Northern blot showed a dramatic decrease of expression after birth. Using immunohistochemistry, the protein was detected within parenchymal cells in embryo and hepatocyte membranes and bile ducts in adults. Using in situ hybridization, GGT mRNA was only detected on days 1 and 2 after birth and exclusively in hepatocytes. Immunoperoxidase may be more sensitive than in situ hybridization to study the expression of minor liver protein such as GGT. However, the study of GGT expression using in situ hybridization is possible in cases of increased expression such as alcoholism, cholestasis, and carcinogenesis.
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