Utilization of the gel shift assay to determine DNA binding by Spl
| العنوان: | Utilization of the gel shift assay to determine DNA binding by Spl |
|---|---|
| المؤلفون: | Swatt, John M. |
| وصف مادي: | 50 leaves : illustrations ; 29 cm |
| Supplemental Data: | System requirements: PC and World Wide Web browser. |
| مستخلص: | In eukaryotic cells the genetic message that is stored in DNA is expressed by transcribing the code from the DNA sequence into shorter segments of messenger RNA, these, in turn, serve as a template for protein synthesis in the presence of ribosomes. Control of gene expression in these cells can be carried out by blocking protein synthesis, destroying the messenger RNA or regulating the transcription of DNA into messenger RNA. One mechanism for transcriptional regulation occurs when a regulatory protein binds to a specific site on the DNA molecule. When the protein is bound it can either assist or interfere with the attachment and activation of the enzymes responsible for transcription. Several DNA binding proteins, or transcription factors, are known and are actively being investigated. The development of assays that measure DNA binding ability and transcriptional activity will aid in understanding mechanism by which cells control the expression of their genetic messages. These developments may lead to a better understanding and possible treatments for diseases caused by inadequate gene regulation, including cancer, viral infections and many genetic disorders. Exposure to certain metals, such as nickel or cadmium, can result in damage to the genetic mechanisms of both somatic and germ line cells, which can lead to cancer, birth defects or teratogenesis. Since these metals do not appear to interact directly with DNA, as other genotoxic agents can, it has been proposed that they may interact with the proteins that regulate gene transcription. Many regulatory proteins contain zinc, and it is possible that displacement of zinc by nickel or other trace metals may affect their binding to DNA. The goal of this project was to set up an assay that will measure the DNA binding capability of a specific transcription regulating protein, namely Sp1, that may be implicated in the etiology of certain genetic disorders. Sp1 is a transcription factor that was isolated from the nuclear extract of the human cell culture line, HeLa. Initially it was found to bind to and initiate transcription of the Simian virus (SV40) early promoter, but it also has been shown to regulate mammalian (human metallothionein, mouse dihydrofolate reductase) and viral (HIV, Herpes Simplex virus) transcription, including genes known to be altered during carcinogenesis (H-ras). The region of Sp1 responsible for DNA binding contains three zinc finger loop structures. The zinc helps to stabilize the 3-dimensional configuration of the amino acids which bind to the DNA. A specific sequence of DNA bases that is rich in guanine and cytosine nucleotides, which is called a GC box, serves as the binding site of Sp1. The effect of Sp1 binding to DNA can be inhibitory as well as initiatory, so Sp1 may be one of many regulatory proteins involved in the transcription of distinct genes and its function may be governed by the location of the binding site within the promoter region of the gene. The assay that is used to detect DNA binding is called the gel shift assay or gel retardation assay. When a DNA sample is placed in a gel matrix and an electric current is applied the DNA migrates through the gel towards the anode. When a protein is bound to the DNA the migration through the gel will be retarded and, thus, its final position will be shifted from the position of the unbound DNA. The DNA can be visualized by using samples that are linked to radioactive phosphorus (P-32_ and developing an autoradiograph on X-ray film. In this project, a working procedure for the gel shift assay has been developed for use with agarose gel. The new procedure was created by modifying a procedure intended for a commercially prepared kit that utilized polyacrylamide gels. Several successful gel shift assays were obtained by performing experiments such as a test of the usefulness of bulk carrier nucleic acids and a titration of Sp1 against a constant amount of DNA to demonstrate a protein-DNA ladder effect. The procedure can now be used to perform other experiments such as determination of Sp1 DNA-binding activity in Xenopus embryo extracts or determination of trace metal interference with Sp1's DNA-binding ability, either in vitro or in vivo. |
| الموضوعات: | DNA polymerases., RNA polymerases., Radioligand assay., Protein binding., ADN polymérases., ARN polymérases., Dosage radiocompétitif., Protéines Fixation., DNA polymerases., Protein binding., Radioligand assay., RNA polymerases. |
| مصطلحات الفهرس: | Academic theses., Academic theses., Thèses et écrits académiques. |
| ملاحظة: | "Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Chemistry." Thesis advisor: Carol A. Jones. Includes bibliographical references (leaves 43-46). Also available via the World Wide Web. |
| أرقام أخرى: | CTB 26944995 |
| المصدر المساهم: | From OAIster®, provided by the OCLC Cooperative. |
| رقم الأكسشن: | edsoai.ocm26944995 |
| قاعدة البيانات: | OAIster |
| الوصف غير متاح. |