CONTENTS. LIST OF FIGURES... i. LIST OF TABLES... iii. SUMMARY OF THE WORK DONE... vi

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1 CONTENTS SECTION ~ LIST OF FIGURES... i LIST OF TABLES... iii LIST OF ABBREVIATIONS USED... ~... iv SUMMARY OF THE WORK DONE... vi 1. INTROD UCTION... : AN OVERVIEW OF TRANSCRIPTIONAL REGULATION... 2 II Introduction to the general transcription machinery RNA Polymerase II and its subunits Eukaryotic class II promoters General Transcription Factors TFIID TFIIB TFIIF TFIIE TFIIH TFIIA TFIIJ Initiation in TATA-Iess promoters General repression of transcription by Dr Tissue specific transcription factors Activation domains and mode of activation CHROMATIN AND TRANSCRIPTION Chromatin in the interphase nuclei Status of the transcriptionally active and inactive chromatin 10

2 1.3. STRESS RESPONSE- AN OVERVIEW Various forms of stress induce the same response The heat shock proteins Functions of stress proteins Organization of heat shock inducible promoters Nature of heat shock elements General features 'and organization of heat shock factors HSF1 is responsible for the stress inducible expression of heat shock proteins HSF2 is involved in developmental induction of hsp genes A model for the autoregulation of stress response Cooperativity in HSF-HSE interactions _11. Heat shock factor independent pathways in stress response CHROMATIN AND HEAT SHOCK GENE EXPRESSION The GAGA factor Role of GAGA factor in presetting the promoters Importance of (CT)n or (GA)n repeats in the heat shock promoters Role of GAGA factor in countering histone H 1 mediated repression Mammalian GAGA factors DEVELOPMENT OF LIVER Rodent liver organogenesis Regulation of liver specific gene expression The HNF1 family The HNF3 family The HNF4 family The CCAAT I enhancer binding protein (C/EBP) family Transcriptional regulation of albumin gene in rat liver BACKGROUND AND RATIONALE FOR THE PRESENT STUDY II. MATERIALS AND METHODS Bacterial strains Plasmids and rat liver edna library Laboratory animals and cell lines Bacterial media, antibiotics and chemicals H411E-C3 cell culture and heat shock conditions Source of chemicals used

3 2. 7. Sterilization Siliconization Preparation of competent cells Transformation Precipitation of DNA Large scale isolation of supercoiled plas!'ylid DNA Small scale isolation of plasmid DNA Purification of plasmid on density gradient Estimation of nucleic acids concentration Restriction enzyme digestion Purification of insert DNA from agarose gels by electroelution Kinase reaction Dephosphorylation of the vector DNA Ligation Selection of recombinant clones Nick translation Random primer labeling of DNA End labeling of the DNA Sephadex G-25 column chromatography Measurement of radioactivity in nucleic acids..., Plating the library and primary screening Prehybridization and hybridization Secondary screening Tertiary screening Excision of the plasmid pbs containing the insert of choice from positive lambda Zap phages Preparation of genomic DNA Agarose gel electrophoresis and Southern transfer Southern hybridization Isolation of total RNA Northern blotting and hybridization RNA dot blots Preparation of plasmids for sequencing Denaturing the DNA for sequencing Sequencing reaction Oligonucleotide synthesis and annealing

4 2.42 Crush and Soak Method of purification of oligonucleotides Polymerase chain reaction Preparation of whole cell extract for use in electrophoretic mobility shift.\ assays Isolation of nuclei Preparation of nuclear extract - High salt extraction procedure Ouantitation of proteins SDS-polyacrylamide gels Staining with Coomassie brilliant Blue Western blotting of proteins onto nitrocellulose lmmuno blotting South-western blot technique Electrophoretic Mobility shift assays (EMSA) or Gel retardation assays Electroporation for transient transfection Chloramphenicol Acetyl Transferase (CAT) assays Densitometric scanning CLONING AND CHARACTERIZATION OF RAT HEAT SHOCK TRANSCRIPTION FACTOR a. RESULTS a.1. Identification of the presence of an HSE binding activity in the rat liver a.2. Screening of rat liver edna library using South-western technique a.3. Isolation of the clone encoding rat heat shock transcription factor a.4. Mapping the restriction sites on the rat HSF1 clone a.5. Subcloning the rat HSF1 clone a.6. Sequencing the rat HSF1 clone a.7. Analysis of the rat HSF1 sequence a.8. Southern blot hybridization analysis with rat HSF a.9. Northern dot blot analysis of the expression of rat HSF1 in different tissues of an adult rat b. DISCUSSION b.1. Rat HSF1 is very similar to mmhsf1 and HsHSF

5 b.2. Presence of variations in the sequence of rhsf1 as compared to mmhsf1 and HsHSF b.3. Is HSF1 alternatively spliced? b.4. Southern blot analysis with rat HSF b.5. Northern dot blot analysis shows that the rhsf1 is ubiquitou c. DIRECTIONS FOR FUTURE WORK d. A MODEL FOR INTERACTION OF HSFs WITH HSEs d.1. Steric problem associated with HSF-HSE:interaction d.2. Importance of HSF mediated bend in the DNA d.3. A model for interaction between HSFs and HSEs: Both DNA bending and a flexibility in the HSF are required for binding HSEs CHARACTERIZATION OF PROMOTER ELEMENTS RESPONSIBLE FOR HEAT INDUCIBILITY OF THE RAT ALBUMIN PROMOTER a. RESULTS a. 1. Choice of cell line for further studies a.2. Induction of albumin in H411E-C3 cells upon heat shock a.3. Amplification of rat albumin promoter using Polymerase Chain Reaction a.4. Construction of expression vectors with cholramphenicol acetyl transferase gene under the control of rat albumin promoter a.5. Identification of the orientation of the albumin promoter in the paib 440 -CATconstructs a.6. Albumin promoter activity is modulated by temperature a. 7. Presence of sequences resembling heat shock elements in the rat albumin promoter a.8. Rat liver contains factors capable of binding the HSEs present in rat albumin promoter... :..., a.9. H411E-C3 cells also contain HSFs capable of binding the rat albumin promoter elements a.1 0. Presence of GAGA repeats in the rat albumin promoter a.11. Presence of GAGA binding factors in the rat liver a.12. Presence of GAGA factor(s) in H411E-C3 cells a.13. Effect of heat shock on the expression of albumin in other tissues of an adult rat

6 3.2b DISCUSSION b.1. Albumin promoter activity is modulated by heat shock b.2. Regulation of albumin promoter is mediated through the action of heat shock transcription factor b.3. Heat shock transcription factors bind the rat albumin promoter in a sequence specific and reversible manner b.4. Which of the HSFs is involved in the regulation of albumin gene? b.5. GAGA factor binds the rat albumin promoter in a sequence specific and reversible manner..., b.6. Are there multiple forms of GAGA factor? b. 7. Heat shock factor induces the expression of rat albumin gene in concert with and not independent of other liver specific factors b.8. What is the significance of the induction of albumin gene in the embryonic stage under stressful conditions? b.9. Is this the end, or are we still beginning? b. 10. Future directions for further work REFERENCES... 85