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Transcriptional Regulation of the Human C3 Gene
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
Gretchen J. Darlington, Deborah R. Wilson, Todd S.-C. Juan
Transcriptional analysis by nuclear run-on was done according to the method previously published.35 Hybridization was carried out using 5 × 106 to 107 cpms per reaction. The following plasmids containing human cDNA segments were immobilized on filters for transcriptional analysis: albumin, F47 (Alb), C3, pHC3.11 (C3), α1-acid glycoprotein, pAGP-1 (AGP),36 and α-fibrinogen, p115.6 (Fib).
Changes in Gene Expression During Aging of Mammals
Published in Alvaro Macieira-Coelho, Molecular Basis of Aging, 2017
Certain genes that have low activity in an adult tissue become increasingly active in older tissue, and certain genes get expressed in nontarget tissues in old age. Jaiswal and Kanungo83 measured the level of α-skeletal actin (α-SKA) mRNA in the heart of young and old rats by RNA-DNA slot-blot hybridization using a cDNA probe for α-SKA. The level was found to be higher in the heart of old rats (Figure 8). However, nuclear run-on transcription was not found to be different. α-SKA is the major isoform of skeletal actin of the fetal cardiac ventricle, whereas a different isoform is predominant in the adult heart.84 Under experimental aortic coarctation in the adult, α-SKA gene is rapidly reexpressed followed by hypertrophy of the heart. Hypertrophy occurs due to an increase in cell size without cell division. The continuous pressure overload of the heart throughout the life span may stimulate increased expression of the α-SKA gene which, in turn, may lead to hypertrophy of the heart. This may lead to lower contractibility of the heart and affect its function. This is consistent with the finding that c-myc, c-fos, and hsp-70 genes are activated first as an early response to pressure overload, and this is followed by overexpression of the α-SKA gene.84
The Use of Molecular Hybridization Techniques as Tools to Evaluate Hepatic Fibrogenesis
Published in Marcos Rojkind, Connective Tissue in Health and Disease, 2017
Mark Α. Zern, Mark J. Czaja, Francis R. Weiner
We have adapted the nuclear run-on assay and have begun to apply it to the evaluation of human liver samples. While we initially employed this technique to evaluate the regulation of the ceruloplasmin gene in Wilson's disease,49 the technique can readily be used to study matrix gene regulation. In an attempt to better delineate the level of gene expression responsible for a decrease in the steady-state levels of ceruloplasmin mRNA in Wilson's disease patients, the nuclear run-on assay was used to analyze transcriptional rates by modifying previously described techniques.50 Nuclei were extracted from portions of human livers that had been harvested prior to transplantation, then frozen at — 70°C for periods of 1 year or more. Despite this long period of freezing, the nuclei were still able to actively transcribe hnRNA (an example of an assay is shown in Figure 8). The amount of ceruloplasmin gene transcription in four Wilson's patients was decreased to 44% that of three controls. These results indicate that nuclear run-on assays can be readily done in livers that have been frozen for prolonged periods, thus making analysis of human hepatic collagen gene transcription a realistic possibility.
Inhibitory effect of chlormethiazole on the toxicokinetics of diethylnitrosamine in normal and hepatofibrotic rats
Published in Drug and Chemical Toxicology, 2019
Gaoju Wang, Kang Xiao, Jie Gao, Shan Jiang, Shang Wang, Shijia Weng, Chen Xu, Tong Wang, Hai-Ling Qiao
Previously, it has been reported that CMZ-mediated inhibition of CYP2E1 activity was present in human beings even when CMZ was undetectable in the blood (Gebhardt et al.1997), which implied that this inhibition cannot be explained by inhibition of the enzyme alone. The CYP2El gene is constitutively expressed and regulated at both the transcriptional and posttranscriptional levels (Simi and Ingelman-Sundberg 1999). From nuclear run-off experiments, it was apparent that CMZ specifically inhibited the elevation of CYP2El messenger ribonucleic acid by decreasing the rate of the gene transcription (Hu et al.1994). Our results show that the t1/2 and Vd of DEN increased with multiple dosing of CMZ, compared with single dosing of CMZ. A possible explanation was that CMZ inhibited the synthesis of CYP2E1 at the gene level and then slowed the metabolism and prolonged the residual time of DEN in normal rats.
AID Biology: A pathological and clinical perspective
Published in International Reviews of Immunology, 2018
Meenal Choudhary, Anubhav Tamrakar, Amit Kumar Singh, Monika Jain, Ankit Jaiswal, Prashant Kodgire
AID off-targets in the B-cell genome has been uncovered by large-scale techniques like Translocation Capture Sequencing (TC-Seq) [109] and High-Throughput Genome-wide Translocation Sequencing (HTGTS) [110]. The Alt group had previously identified 15 non-Ig genes which are the frequent targets of AID for initiating DNA double-strand breaks (DSBs) [110]. Further, detailed analysis of activated B-cells extended this range to 51 AID mistargeted genes [111]. All of these off-target genes were highly transcriptionally active; a feature shared with Ig genes and AID action was limited to narrow regions within these off-target genes. Another large-scale approach used to unravel AID off-targets is genome-wide nuclear run-on sequencing (GRO-seq) which showed that the aberrant AID activity in non-Ig genes was localized in regions where sense and antisense transcription overlaps denoted as convergent transcription, though most of the convergently transcribed genomic regions are not targeted by AID [112]. Another study identified that AID off-targets are situated within super-enhancers (SEs) [113]; a cluster of multiple enhancers having high transcriptional activity. GRO-seq analysis done in the setting of lymphoid and non-lymphoid cells described a connection between AID off-target activity and SEs with almost all of the AID targets were localized inside intragenic SEs. Regardless of this association, in CSR-activated B-cells, out of the 448 SEs analyzed only about 10% were off-targeted by AID [112].