Cloning the transcribed portion of the genome
Farzin Farzaneh, David N. Cooper in Functional Analysis of the Human Genome, 2020
Some sequences, known as promoters, are able to bind to transcription factors in the upstream portion of transcription units of the DNA and give rise to RNA via transcription. The most important of these is RNA polymerase which reads the antisense strand of the gene and produces a sense-encoding RNA copy using nucleotide triphosphates of the bases adenine, uracil, cytosine and guanine. Capping is ultimately necessary for recognition of the mRNA by the small ribosomal subunit, to enable translation, whereas polyadenylation stabilizes the transcript in the cytoplasm. The clones which have been identified and isolated can then be sequenced and the amino acid sequence of the encoded protein deduced. This DNA can be further manipulated and constructs prepared from which functional proteins can be isolated and characterized to show that the gene of interest has indeed been isolated. The most effective method for the isolation of RNA from a range of tissues uses guanidinium thiocyanate and acidic phenol.
The Process of Transcription
David S. Latchman in Gene Control, 2020
In prokaryotes a single RNA polymerase enzyme is responsible for the transcription of DNA into RNA. RNA polymerase I is responsible for the transcription of the tandem arrays of genes encoding ribosomal RNA, such transcription constituting about one-half of total cellular transcription. The involvement of a specific transcription factor which acts to recruit the RNA polymerase as well as of other factors which recruit the specific factor is also illustrated by RNA polymerase III. The recruitment of TFIIH plays a critical role in allowing the RNA polymerase to initiate transcription. TFIIH is a multicomponent complex whose molecular structure has been determined and which plays a key role in both transcription and the repair of damaged DNA. Transcriptionally inactive genes lacked polymerase while actively transcribed genes had polymerase molecules distributed over their entire length, with slight peaks near the 5' and 3' ends of the gene.
DNA-Dependent RNA Polymerases of Plants and Lower Eukaryotes
Samson T. Jacob in Enzymes of Nucleic Acid Synthesis and Modification, 2018
This chapter discusses nuclear and organellar RNA polymerases from plants and lower eukaryotes in terms of sources of enzymes, purification methodology, catalytic properties, molecular structures, immunological relationships, transcriptional functions, selectivity of transcription in nuclear and reconstituted systems, and modulation during cell cycle and growth transitions. Nuclear RNA polymerases which have been solubilized from chromatin templates by sonication in the presence of high salt or those enzymes recovered in the soluble fraction of total tissue extracts can be separated into three distinct classes by chromatography on either anionic or cationic exchange resins. Relatively large quantities of homogeneous enzyme are required for studies on RNA polymerase subunit structures, reconstitution of active enzyme from separated subunits, and in vitro transcription with reconstructed systems. The development of plant and lower eukaryotic soluble transcription systems is certainly required for future advancement in the understanding of plant and lower eukaryotic RNA polymerases and transcriptional regulation.
Characterization of cells expressing RNA polymerase II tagged with green fluorescent protein: Effect of ionizing irradiation on RNA synthesis
Published in International Journal of Radiation Biology, 2008
Etsuko Hongo, Yoshie Ishihara, Keiko Sugaya, Kimihiko Sugaya
Purpose: To isolate and to characterize cells expressing RNA polymerase II tagged with green fluorescent protein for analyses of the effects of ionizing radiation on transcription in living cells. Materials and methods: We introduced an α-amanitin-resistant mutation into a vector encoding the largest subunit of RNA polymerase II tagged with green fluorescent protein (GFP-pol). Cell lines stably expressing functional GFP-pol were isolated under selection with α-amanitin from a Chinese hamster cell line, CHO-K1, and a radiation-sensitive mutant CHO cell line, XR-1. Results: We tested the functionality of the fusion protein in vivo by determining RNA synthesis activity by incorporation of nucleoside analogues. Both CHO-K1 and XR-1 cells expressing GFP-pol had properties similar to those of their respective parental cell lines, indicating that GFP-pol is functional. Conclusions: These stable lines might prove useful for analyses of the roles of transcription after ionizing radiation.
A PCR-Derived, non-isotopic labeled prolactin cRNA probe suitable for in situ hybridization
Published in Endocrine Research, 1995
Hong Wu, Dian Wang, William B. Malarkey
We have developed a novel vector-free method for the synthesis of non-isotopic (digoxigenin) labeled prolactin (PRL)-gene specific cRNA probe based on the direct in vitro transcription of DNA template amplified by polymerase chain reaction (PCR). The T7 and T3 RNA polymerase promoters were incorporated into the amplified DNA by including the promoter sequences in the 5′ end of the oligonucleotides used to prime the PCR. These promoters allowed the subsequent transcription of digoxigenin labeled antisense and sense cRNA probes from the amplified DNA. We successfully utilized these probes to detect specific PRL mRNA in human pituitary and decidua tissues by in situ hybridization (ISH) which can provide identification and localization of PRL-gene expression at a single cell level. This approach avoided time consuming steps which required subcloning of target DNA into the vectors that contains bacteriophage RNA polymerase promoter as well as the need for radioactive materials. This non-isotopic ISH procedure takes less than 72 h from specimen preparation to microscopic analysis and should prove to be useful for molecular biological studies of hormones and clinical diagnosis.
Expression and function of Set7/9 in pancreatic islets
Published in Islets, 2009
Takeshi Ogihara, Nathan L. Vanderford, Bernhard Maier, Roland W. Stein, Raghavendra G. Mirmira
Histone tail acetylation and methylation are known to enhance accessibility of islet genes to transcription factors and the basal transcriptional machinery. In this brief report, we follow up on a recent study in which we identified the islet enriched factor Set7/9 as a potentially important histone methyltransferase in β-cells (Deering, et al. Diabetes 2009; 58:185-93). We had suggested that the methylation of H3-Lys4 by Set7/9 enhances accessibility of the insulin gene to the basal transcriptional machinery. Consistent with this hypothesis, we show here that RNA polymerase II occupancy at the insulin and IAPP genes is considerably enhanced in β-cells compared to α cells (or NIH3T3 cells), and that the converse is true for RNA polymerase II occupancy at the glucagon gene. The enrichment of Set7/9 in β-cells appears to be dependent upon Pdx1, as knockdown of Pdx1 in INS-1 β-cells using small hairpin RNAs almost completely abolishes Set7/9 expression. A LacZ expression vector driven by the -6.5 kilobase pair Set7/9 promoter that contains putative Pdx1 binding sites shows β-cell-line-specific expression. Taken together, our data support further the hypothesis that Pdx1-dependent Set7/9 expression may be crucial to enhancing chromatin accessibility and transcription of β-cell genes.