Nuclear run

The methods used for the evaluation of regulation of gene expression are too numerous to be described in detail here. They include Northern analysis to determine levels of a particular mRNA, nuclear run on to determine whether an increase in mRNA is due to an increase in the rate of transcription, and promoter deletion analysis to identify specific elements in the promoter region responsible for the control of expression. Of much current interest is the use of microarrays that permit the study of the expression of hundreds to thousands of genes at the same time. Reverse transcriptase-polymerase chain reaction and RNase protection assay techniques are used to amplify and quantitate mRNAs, while the electrophoretic mobility shift assay is used to measure binding of a transcription factor to its specific DNA consensus sequence. 

Fig. 2C. Time course of calbindin-D,8K gene transcription in chick intestine following a dose of l,25(OH)2D, to vitamin D-deficient chicks. Transcription was measured by nuclear run-off assay.

Okamoto, T., Mitsuhashi, M., andKikkawa, Y. (1994) Fluorometric nuclear run-on assay with oligonucleotide probe immobilized on plastic plates. Anal. 

Nuclear run-off assays have been carried out on splenic B cells [116,119,154], In contrast to the findings in cell lines, the RNA polymerase density on the fx gene was found to increase when resting B cells were stimulated with bacterial lipopo-lysaccharide, in one case by as much as 8-10-fold [116]. However, it should be remembered that lipopolysaccharide treatment not only causes B cells to differentiate but also causes them to leave the resting state and start proliferating, thus making it difficult to draw direct comparisons with cell lines. 

The soybean GH3 clone was initially isolated by differential hybridization screening as an auxin-induced cDNA clone from etiolated hypocotyls [9]. Nuclear run-on experiments have shown that transcription of the GH3 gene is induced within 5 minutes after auxin treatment of excised soybean plumules [20]. The soybean GH3 gene is specifically induced by biologically active auxins. Unlike SAURs and Aux/IAA mRNAs, GH3 mRNAs do not accumulate in response to protein synthesis inhibitors like cycloheximide... 

The relative transcript levels and transcription rates of the polymerase gene during the cell cycle in HeLa cells were determined using cells synchronized by a double thymidine block. Total RNA as well as nuclei from cells were isolated at hourly intervals during the progression of S phase and examined either by nuclear run-off transcription or Northern analysis (10). 

Fig. 5. Replication-dependence of histone, nonhistones and poly(ADP-ribose) polymerase mRNA transcription rates. HeLa cells were synchronized and S phase cells (0-Tlme) were incubated in the presence or absence of 0.1 mM hydroxyurea for 2 hr (B, D) or 5 hr (A, C). Nuclei were prepared and assayed for nuclear run-off transcription. (Taken fixnn Ref. 10).

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