phosphorus-radioisotopes and dimethyl-sulfate

This unit provides thorough coverage of the most useful chemical and enzyme probes that can be used to examine RNA secondary and tertiary structure. Footprinting methods are presented using dimethyl sulfate, diethyl pyrocarbonate, ethylnitrosourea, kethoxal, CMCT, and nucleases. For chemical probes, both strand scission and primer extension detection protocols are included.

Topics: Aldehydes; Bacteriophage T4; Base Pairing; Biochemistry; Butanones; CME-Carbodiimide; Diethyl Pyrocarbonate; Electrophoresis; Endonucleases; Ethylnitrosourea; Indicators and Reagents; Nucleic Acid Conformation; Phosphorus Radioisotopes; Polynucleotide 5'-Hydroxyl-Kinase; Reference Standards; RNA; Sulfuric Acid Esters

Topics: Animals; Autoradiography; Base Sequence; Deoxycytosine Nucleotides; Deoxyribonuclease I; DNA; DNA Damage; DNA Footprinting; DNA Probes; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Humans; Indicators and Reagents; Lymphocytes; Phosphorus Radioisotopes; Polymerase Chain Reaction; Reproducibility of Results; Sequence Analysis, DNA; Sulfuric Acid Esters; Ultraviolet Rays

Cathepsin D is an estrogen (17 beta-estradiol, E2)-inducible lysosomal protease. A putative estrogen receptor (ER)-Sp1-like sequence (GGGCGG(n)23ACGGG) has been identified in the non-coding strand of the cathepsin D promoter (-199 to -165), and electromobility shift assays of nuclear extracts from MCF-7 and HeLa cells confirm that both the ER and Sp1 protein bind to 32P-labeled ER/Sp1 oligo. For example, nuclear extracts from MCF-7 cells bind to the 32P-labeled ER/Sp1 oligo; however, ER/Sp1 binding can be decreased by selective competition with excess unlabeled estrogen responsive element and Sp1 oligos, immunodepletion with ER or Sp1 antibodies, and by treating cells with ICI 164,384, an antiestrogen which inhibits formation of ER homodimer. Moreover, E2-induced chloramphenicol acetyltransferase (CAT) activity in MCF-7 cells cotransfected with a human estrogen receptor expression plasmid and a plasmid containing an ER/Sp1 sequence cloned upstream to a thymidine kinase promoter and a CAT reporter. In cotreatment studies, ICI 164,384 inhibited E2-induced CAT activity. In contrast, E2 did not induce CAT activity in MCF-7 cells transfected with plasmids containing mutations in the ER or Sp1 segments of the ER/Sp1 oligo, thus confirming that both cognate binding sites are required for estrogen responsiveness.

Topics: Adenosine Triphosphate; Base Sequence; Breast Neoplasms; Cathepsin D; Cell Line; Cell Nucleus; Chloramphenicol O-Acetyltransferase; Cloning, Molecular; DNA-Binding Proteins; Estradiol; Estrogen Antagonists; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Molecular Sequence Data; Nuclear Proteins; Oligonucleotides, Antisense; Phosphorus Radioisotopes; Polyunsaturated Alkamides; Receptors, Estrogen; Recombinant Fusion Proteins; Sp1 Transcription Factor; Sulfuric Acid Esters; Thymidine Kinase; Transfection; Tumor Cells, Cultured

The use of a postlabeling method to characterize and to detect infrequent base modifications in DNA is outlined. This method has the advantage that low levels of DNA modifications, approximately 1 modified base per 10(5) nucleotides, can be detected. Moreover, a broad spectrum of modification can be identified by using this methodology. The basis for the method involves transfer of a radioactive phosphate from the gamma position of ATP to the 5'-hydroxyl terminus of 3'-phosphoryl nucleotides that are derived from modified DNA by appropriate nuclease digestion. The second method involves use of a defined DNA sequence within human cells. The alpha sequence is used as a probe for DNA damage to specific nucleotides. The alpha DNA sequence is reiterated approximately 300,000 times in the human genome and exists in tandem arrays. It comprises approximately 1% of the entire genome. The reiterated sequence is sufficiently homogeneous to permit its use as a probe for a site specific in DNA damage. Examples of the application of both of these methodologies to DNA damage inflicted in human cells by chemicals and ultraviolet light are provided.

Topics: Alkylating Agents; Base Sequence; Cell Line; Chromatography, High Pressure Liquid; DNA; Humans; Mutagenicity Tests; Phosphorus Radioisotopes; Sulfuric Acid Esters; Ultraviolet Rays