Gene expression exhibits temporal and spatial patterns to response environmental changes and growth cycle. Gene expression is under strict control at different levels among which control at transcription level is the predominant mode, especially in prokaryotes. In this review, we summarized the new developments of methods used in transcriptional studies, including modifications and improvements of the classic methods, such as gel-shift assay, DNA foot printing, and in vivo reporter system. In addition, we introduced examples to apply new methods, such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize protein-DNA, ligand-protein, and ligand-protein-DNA interactions. The collection of these methods and their application could guide and accelerate relevant studies.
Calorimetry ; DNA Footprinting ; Gene Expression ; Ligands ; Proteins ; Surface Plasmon Resonance ; Transcription, Genetic

Polyamides, containing N-methylpyrrole (Py) and N-methyl-imidazole (Im) amino acids, are synthetic oligomers programmed to read the DNA double helix in the minor groove with high affinities and sequence specificities resulting in modulation of gene expression. They are cell permeable, stable and have no cytotoxicity, which provide a promising tool of gene regulation. We describe here recent advances in the field of DNA binding polyamides, including pairing rules, specifities and affinities to DNA, synthesis methods, cellular and nuclear uptake properties, gene regulation and effectiveness in vivo. The potential problems and difficulties in future research are also discussed.
Animals ; Base Pairing ; DNA ; chemistry ; genetics ; DNA Footprinting ; Gene Expression Regulation ; drug effects ; Imidazoles ; chemical synthesis ; chemistry ; metabolism ; pharmacology ; Nylons ; chemical synthesis ; chemistry ; metabolism ; pharmacology ; Pyrroles ; chemical synthesis ; chemistry ; metabolism ; pharmacology

PURPOSE: Androgen deprivation triggers a sequence of events that activates apoptotic cell death of the androgen-dependent epithelial cells within the rat ventral prostate, ultimately resulting in the involution of the gland. To investigate the mechanism of azaline B-dependent apoptosis in the rat ventral prostate, the regulation of apoptosis-related genes were examined. MATERIALS AND METHODS: Azaline B was subcutaneously injected in Sprague-Dawley rat. Fas receptor(Fas), Fas ligand(FasL), bcl-2 mRNA, and protein levels were detected by RT-PCR and Western blot. Azaline B-dependent apoptosis was determined by TUNEL and DNA fragmentation assay. Transacting factor of FasL promoter was identified by DNA footprinting and DNA mobility shift assay. RESULTS: The prostate regressed after azaline B treatment in rat, and the involuted ventral prostate regenerated after testosterone pretreatment. Apoptosis of the ventral prostate was detected by TUNEL assay and apoptotic DNA fragmentation assay after azaline B treatment. The levels of Fas and FasL mRNA and protein increased after azaline B treatment. In DNase I footprinting assay with FasL promoter using nuclear extract prepared from control prostate, at least two sites were protected: SP-1 binding site at -283bp and prostate-unidentified factor(P-UF) binding site at -247bp. SP-1 binding activity vanished in the nuclear extract prepared from azaline B-treated rats. In the DNA mobility shift assay, SP-1 binding activity decreased after azaline B treatment. Bcl-2 mRNA and protein were downregulated after azaline B treatment. CONCLUSIONS: These results suggest that Fas/FasL system and Bcl-2 are important to azaline B-dependent apoptosis in rat ventral prostate and that SP-1 is related to azaline B-dependent regulation of the FasL gene.
Animals ; Antigens, CD95 ; Apoptosis* ; Binding Sites ; Blotting, Western ; Cell Death ; Deoxyribonuclease I ; DNA ; DNA Footprinting ; DNA Fragmentation ; Electrophoretic Mobility Shift Assay ; Epithelial Cells ; Fas Ligand Protein ; Genes, bcl-2 ; In Situ Nick-End Labeling ; Prostate* ; Rats* ; Rats, Sprague-Dawley ; RNA, Messenger ; Testosterone

BACKGROUNDS: Castration-induced androgen deprivation triggers a sequence of events, which activates apoptotic cell death of the androgen-dependent epithelial cells within the rat ventral prostate. To investigate the mechanism of castration-dependent apoptosis in the rat ventral prostate, the regulation of apoptosis-related genes was been investigated. METHODS: Azaline B was subcutaneously injected into Sprague-Dawley rat. The Fas receptor (Fas), Fas ligand (FasL) and bcl-2 mRNA, as well as the protein levels were detected by RT-PCR and Western blot analyses. Azaline B-dependent apoptosis was determined using TUNEL and a DNA fragmentation assay. The transacting factor of the FasL promoter was identified by DNA footprinting and a DNA mobility shift assay. RESULTS: The rat prostate was regressed after castration, with and the involuted ventral prostate regenerated by testosterone pretreatment, but not by that with FSH. Apoptosis of the ventral prostate was detected, after castration, using toluidine blue staining, a TUNEL assay and an apoptotic DNA fragmentation assay. The levels of Fas, FasL mRNA and protein were increased after castration. In the DNase I footprinting assay, using the FasL promoter and a nuclear extract prepared from a control prostate, at least two sites were protected: the SP-1 binding site at -283 bp and the prostate-unidentified factor(P-UF) binding site at -247 bp. The SP-1 binding activity vanished in the nuclear extract prepared from castrated rats. In the DNA mobility shift assay, the SP-1 binding activity was slightly decreased after castration. Both the Bcl-2 mRNA and Bcl-2 protein were downregulated after castration. CONCLUSION: These results suggest that the Fas/FasL system and Bcl-2 may be important to castrationdependent apoptosis in the rat ventral prostate, with SP-1 related to the castration-dependent regulation of the FasL gene
Animals ; Antigens, CD95 ; Apoptosis* ; Binding Sites ; Blotting, Western ; Castration ; Cell Death ; Deoxyribonuclease I ; DNA ; DNA Footprinting ; DNA Fragmentation ; Electrophoretic Mobility Shift Assay ; Epithelial Cells ; Fas Ligand Protein ; In Situ Nick-End Labeling ; Prostate* ; Rats* ; Rats, Sprague-Dawley ; RNA, Messenger ; Testosterone ; Tolonium Chloride

BACKGROUNDS: Castration-induced androgen deprivation triggers a sequence of events, which activates apoptotic cell death of the androgen-dependent epithelial cells within the rat ventral prostate. To investigate the mechanism of castration-dependent apoptosis in the rat ventral prostate, the regulation of apoptosis-related genes was been investigated. METHODS: Azaline B was subcutaneously injected into Sprague-Dawley rat. The Fas receptor (Fas), Fas ligand (FasL) and bcl-2 mRNA, as well as the protein levels were detected by RT-PCR and Western blot analyses. Azaline B-dependent apoptosis was determined using TUNEL and a DNA fragmentation assay. The transacting factor of the FasL promoter was identified by DNA footprinting and a DNA mobility shift assay. RESULTS: The rat prostate was regressed after castration, with and the involuted ventral prostate regenerated by testosterone pretreatment, but not by that with FSH. Apoptosis of the ventral prostate was detected, after castration, using toluidine blue staining, a TUNEL assay and an apoptotic DNA fragmentation assay. The levels of Fas, FasL mRNA and protein were increased after castration. In the DNase I footprinting assay, using the FasL promoter and a nuclear extract prepared from a control prostate, at least two sites were protected: the SP-1 binding site at -283 bp and the prostate-unidentified factor(P-UF) binding site at -247 bp. The SP-1 binding activity vanished in the nuclear extract prepared from castrated rats. In the DNA mobility shift assay, the SP-1 binding activity was slightly decreased after castration. Both the Bcl-2 mRNA and Bcl-2 protein were downregulated after castration. CONCLUSION: These results suggest that the Fas/FasL system and Bcl-2 may be important to castrationdependent apoptosis in the rat ventral prostate, with SP-1 related to the castration-dependent regulation of the FasL gene
Animals ; Antigens, CD95 ; Apoptosis* ; Binding Sites ; Blotting, Western ; Castration ; Cell Death ; Deoxyribonuclease I ; DNA ; DNA Footprinting ; DNA Fragmentation ; Electrophoretic Mobility Shift Assay ; Epithelial Cells ; Fas Ligand Protein ; In Situ Nick-End Labeling ; Prostate* ; Rats* ; Rats, Sprague-Dawley ; RNA, Messenger ; Testosterone ; Tolonium Chloride

ATP-citrate lyase (ACL), an enzyme catalyzing the first step in biosynthesis of fatty acids, is induced during the lipogenesis and cholesterologenesis. We demonstrate that the region -213 to -128 of human ACL promoter is responsible for conferring glucose-mediated transcription. This region in the ACL promoter contains Sp1 binding sites determined by DNase I foot-printing assay. Gel retardation assay using oligonucleotides from -179 to -141 and -140 to -110 showed two specific DNA-protein complexes postulated to be formed by transcription factor Sp1. Competition gel shift and supershift assays have confirmed that these DNA-protein complexes were the result of induced Sp1 as well as another Sp1-related proteins. Western blot analysis also demonstrated that transcription factor Sp1 was slightly increased in the nuclear proteins extracted from Alexander cells following supplementation of glucose. In addition, expression of 110 kDa protein reacting with antibody against Sp3 was dramatically increased by glucose supplementation, while isoforms of Sp3, about 80 kDa in size was decreased in its amounts. Our results suggest that changes in the expression of Sp1 family proteins play an important role in activation of the ACL promoter by glucose.
ATP Citrate (pro-S)-Lyase/metabolism ; ATP Citrate (pro-S)-Lyase/genetics* ; Binding Sites ; Cells, Cultured ; Chloramphenicol O-Acetyltransferase/genetics ; DNA Footprinting/methods ; Deoxyribonuclease I/metabolism ; Electrophoresis, Polyacrylamide Gel ; Gene Expression Regulation, Enzymologic* ; Glucose/pharmacology ; Glucose/metabolism* ; Human ; Immunoblotting ; Promoter Regions (Genetics)* ; Transcription Factor, Sp1/metabolism* ; Transcription, Genetic* ; Transfection

DNase I footprinting assay using liver nuclear extracts revealed six protected regions between nucleotide -600 and +110 and hence named Box I-VI. Upstream promoter element (UPE), a DNA element playing crucial role in transcriptional control of the tissue specific expression of pancreatic beta-cell, has been detected within the proximal region of rat GLUT2 promoter. This region is included in Box VI. The protein-DNA interaction in this region (Box VI) was confirmed by mobility shift assay using liver nuclear extracts. Deletion of the region between -585 bp and -146 bp resulted in dramatic changes in promoter activity when they were expressed in liver and beta-cell derived cell line. When -585/-146 construct was expressed in liver, the activity was decreased to 46%, whereas the activity in beta-cell line, HIT-T15 cell, was increased by 84% when compared to -146/+190 construct. These opposing phenomena can be explained by the fact that beta-cell specifically expresses the UPE binding protein. Assuming that there may be Box VI-binding protein playing negative roles both in hepatocyte and beta-cell, and that the protein acts as a negative regulator of GLUT2 gene, the UPE binding protein in the beta-cell may overcome the inhibition by binding to the protein.
Animal ; Binding Sites ; Cell Line ; Comparative Study ; DNA Footprinting ; Deoxyribonuclease I ; Gene Expression Regulation ; Islets of Langerhans/metabolism* ; Islets of Langerhans/cytology ; Liver/metabolism* ; Liver/cytology ; Monosaccharide Transport Proteins/genetics ; Monosaccharide Transport Proteins/biosynthesis* ; Promoter Regions (Genetics)* ; Protein Binding ; Rats ; Transcription Factor AP-1

OBJECTIVETo screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function.

METHODSThe screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells, and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins.

RESULTSTwo polymorphisms, C (-106) T and C (-12) G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C (-12) G and WT/C (-106) T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C (-106) T were 31.5% and 17.5% (P < 0.05) respectively, and the frequencies of WT/C (-12) G were 10.5% and 2.5% (P > 0.05) respectively. The total frequency of WT/C (-12) G and WT/C (-106) T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P < 0.025). The relative transcription activities of the wild-type, the C (-12) G and the C (-106) T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors.

CONCLUSIONThe polymorphisms C (-12) G and C (-106) T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.

5' Flanking Region ; genetics ; Adult ; Aldehyde Reductase ; genetics ; metabolism ; Binding Sites ; genetics ; China ; Chloramphenicol O-Acetyltransferase ; genetics ; metabolism ; DNA ; chemistry ; genetics ; DNA Footprinting ; Diabetes Mellitus, Type 2 ; enzymology ; genetics ; Electrophoretic Mobility Shift Assay ; Female ; HeLa Cells ; Humans ; Male ; Middle Aged ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Single-Stranded Conformational ; Recombinant Fusion Proteins ; genetics ; metabolism ; Regulatory Sequences, Nucleic Acid ; genetics ; Sequence Analysis, DNA ; Transcription, Genetic