Chapter 1. A PRIMER ON TRANSCRIPTIONAL REGULATION IN MAMMALIAN CELLS
Section 1. INTRODUCTION
1. A general model for regulation of a gene
2. Activating a gene
3. Inactivating a gene
Section 2. CONCEPTS AND STRATEGIES
1. Core promoter architecture
2. The general transcription machinery
3. The holoenzyme and mediators
4. Regulatory promoters and enhancers
5. Transcriptional activators
6. Repressors and corepressors
7.Chromatin
8. ATP-dependent remodeling complexes
9. Acetylation of chromatin
10. Histone deacetylation, transcriptional repression, and silencing
11. Locus control regions, insulators, and matrix attachment regions
12. The enhanceosome theory
Chapter 2. INITIAL STRATEGIC ISSUES
Section 1. The initial steps in a gene regulation analysis
Section 2. Consider the time commitment and resources needed
1. Two general strategies that provide preliminary albeit superficial insight into transcriptional regulation mechanisms
2. An example of a rigorous, yet incomplete gene regulation analysis
3. Defining the project goals
Section 3. Evaluate the feasibility of the analysis
1. Appropriate source of cells for functional studies
2. Source of cells for protein extract preparation
3. Success in developing an appropriate functional assay
Section 4. Initiate an analysis of transcriptional regulation
Chapter 3. MODES OF REGULATION mRNA ABUNDANCE
Section 1. Transcription initiation versus mRNA stability
Section 2. Transcription elongation
Section3. Differential pre-mRNA splicing, mRNA transport, and polyadenylation
Chapter 4. TRANSCRIPTION INITIATION SITE MAPPING
Section 1. Initial considerations
Section 2. Primer extension
Section 3.RNase protection
Section 4. S1 nuclease analysis
Section 5. Rapid amplification of cDNA ends
Chapter 5. FUNCTIONAL ASSAYS FOR PROMOTER ANALYSIS
Section 1. Choosing an assay: Advantages and disadvantages of each assay
Section 2. Transient transfection assays
Section 3. Stable transfection assays by chromosomal integration
Chapter 6. IDENTIFICATION AND ANALYSIS OF DISTANT CONTROL REGIONS
Section 1. DNase 1 hypersensitivity
Section 2. Identification of matrix attachment regions
Section3. Functional approaches for the identification of distant control regions
Section 4. Functional assays for the characterization of distant control regions
Chapter7. IDENTIFYING cis-ACTING ELEMENTS WITHIN A CONTROL REGION
Section 1. Identification of control elements by comprehensive mutant analysis
Section 2. Identification of control elements using in vivo or in vitro
Section 3. Identification of control elements by database analysis
Section 4. Mutagenesis techniques
Chapter 8. IDENTIFICATION OF DNA-BINDING PROTEINS AND ISLATION OF THEIR GENES
Section 1. Database methods
Section 2. Development of a protein-DNA interaction assay for crude cell- lysates
Section 3. Cloning by protein purification and peptide sequence analysis
Section 4. Cloning by methods that do not require an initial
Chapter 9. CONFIRMING THE FUNCTIONAL IMPORTANCE OF A PROTEIN-DNA INTERACTION
Section 1. Abundance of a protein-DNA complex in vitro
Section 2. Relative expression patterns of the DNA-binding protein and target gene
Section 3. Correlation between nucleotides required for protein binding and those required for activity of the control element
Section 4. Relative affinity of a protein-DNA interaction
Section 5. In vivo protein-DNA cross-linking
Chapter 10. IN VIVO ANALYSIS OF AN ENDOGENOUS CONTROL REGION
Section 1. In vivo analysis of sequence-specific protein-DNA interactions
Section 2. Nucleosome positioning and remodeling
Section 3. DNA methylation
Section 4. Subnuclear localization of a gene
Chapter11. APPROACHES FOR THE SYNTHESIS OF RECOMBINANT TRANSCRIPTION FACTORS
Section 1. Prokaryotic expression systems
Section 2. Strategies for overcoming expression problems in E.coli
Section 3. Synthesizing large regulatory proteins
Section 4. Synthesizing small quantities of crude protein
Section 5. Synthesis and purification of macromolecular complexes
Section 6. Choosing an appropriate system
Chapter 12. IDENTIFYING AND CHARACTERIZING TRANSCRIPTION FACTOR DOMAINS
Section 1. Basic mutagenesis principles
Section 2. Domains of a gene activator
Section 3. Separating DNA-binding and activation domains of an activator
Section 4. Subdividing DNA-recognition and oligomerization
Section 5. Interaction of activation domains with coactivators and general factors
Section 6. Affinity chromatography
Section 7. Altered specificity genetic systems
Section 8. Structure-function analysis of the general transcriptional machinery
Chapter 13. THEORY, CHARACTERZATION, AND MODELING OF DNA BINDING BY REGULATORY TRNSCRIPTION FACTORS
Section 1. General theory and examples of DNA-protein interactions
Section 2. Analysis and modeling of DNA-protein interactions
Section 3. Analysis of promoter-specific multicomponent nucleoprotein complexes
Chapter 14. CRUDE AND FRACTIONATED SYSTEMS FOR IN VITRO TRNSCRIPTION
Section 1. Preparation of extract
Section 2. Transcription assays
Section 3. Fractionated systems
Chapter15. APPROACHES FOR STUDYING TRANSCRIPTION COMPLEX ASSEMBLY
Section 1. Formation of the basal preinitiation complex
Section 2. Open complex formation, initiation, and promoter escape
Section 3. Assembly of activated complexes at a promoter
1．The total period of this course is 36 hours.
Assign below:
Chapter 1. A PRIMER ON THANSCRIPTIONAL
REGULATION IN MAMMALIAN CELLS…………………………………………………4 hours
Chapter 2. INITIAL STRATEGIC ISSUES ………………………………………….3 hours
Chapter 3. MODES OF REGULATION mRNA ABUNDANCE …………………………….3 hours
Chapter 4. TRANSCRIPTION INITIATION SITE MAPPING ………………………..3 hours
Chapter 5. FUNCTIONAL ASSAYS FOR PROMOTER ANALYSIS ………………………2 hours
Chapter 6. IDENTIFICATION AND ANALYSIS OF DISTANT
CONTROREGIONS……………………………………………………………………..3 hours
Chapter 7. IDENTIFYING cis-ACTING ELEMENTS WITHIN
A CONTROL REGION………………………………………………………………… 2 hours
Chapter 8. IDENTIFICATION OF DNA-BINDING PROTEINS
AND ISLATION OF THEIR GENES……………………………………………………2 hours
Chapter 9. CONFIRMING THE FUNCTIONAL IMPORTANCE
OF A PROTEIN-DNA INTERACTION………………………………………………… 2 hours
Chapter 10. IN VIVO ANALYSIS OF AN ENDOGENOUS
CONTROL REGION…………………………………………………………………….2 hours
Chapter 11. APPROACHES FOR THE SYNTHESIS
OF RECOMBINANT TRANSCRIPTION FACTORS……………………………………….2 hours
Chapter 12. IDENTIFYING AND CHARACTERIZING
TRANSCRIPTION FACTOR DOMAINS……………………………………………….. 2 hours
Chapter 13. THEORY, CHARACTERZATION, AND MODELING OF DNA
BINDING BY REGULATORY TRNSCRIPTION FACTORS……………………………… 2 hours
Chapter 14. CRUDE AND FRACTIONATED SYSTEMS FOR IN
VITRO TRNSCRIPTION……………………………………………………………….2 hours
Chapter 15. APPROACHES FOR STUDYING TRANSCRIPTION
COMPLEX ASSEMBLY …………………………………………………………………2 hours
2． Teach and examination method
Teach method
The course adopts both Chinese and English
Examination method
Oral and written examination
