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DNA and Chromosome Structure
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Chromosomal Structure of the Genetic Material
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The Essential Structure of DNA
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The Replication Challenge Size of an average human chromosome 130 million bp Rate of replication ~ 50 bp per sec Fidelity of replication
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Replication of the Genetic Material Small chromosomes use a single origin Replication of large chromosomes requires multiple origins
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The Mammalian DNA Replication Apparatus
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The Importance of Molecular Cloning
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Role of Recombinant DNA Analysis in the Study of Gene Structure/Function The essence of the problem: Human genome = 3 x 10 9 bp The -globin gene = 3 x 10 3 bp Cloning of genes solves this problem and allows an analysis of function and the basis for mutation
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Two Critical Components for Cloning Recombinant DNA Utility of restriction enzymes for precise manipulation of DNA molecules Use of DNA vectors that can replicate and also accept foreign DNA sequence
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Methods of Recombinant DNA Analysis
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Requirements for Cloning Recombinant DNA The conditions under which the population of recombinant DNAs is mixed with a population of recipient cells must favor the introduction of a single recombinant molecule into a recipient cell. This results in the separation of each recombinant from all the others Each recipient cell must be separated from all the others in the population to permit isolation of a clone of cells or viruses containing a unique recombinant Cells or viruses that receive recombinant DNAs must be distinguishable from those that do not so that they can be selected or identified by screening Cells that receive the desired recombinant must be distinguishable by screening or selection from those that contain other recombinant DNA molecules
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Methods of Recombinant DNA Analysis
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Generation and Use of Recombinant DNA Libraries Sequence libraries Genomic or cDNA sequences that represent all possible sequences from the source Expression libraries Library constructed in a specialized vector that allows expression of the insert sequence to generate protein
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Isolation of a Gene and Gene Structure
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Isolation of a Gene
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Discontinuous Nature of a Eukaryotic Gene
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Structure of a Typical Eukaryotic Gene – the -Globin Gene
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Complexity of Gene Organization in Metazoans The -globin locus
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Unequal Crossing Over as a Mechanism for Gene Duplication and Gene Loss
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The Impact of the Complexity of Gene Structure on Gene Expression
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Gene Expression
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The Complexity of Gene Expression
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Gene Expression Requires Splicing of Primary Transcripts
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Conservation of Sequences at Splice Sites
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Splicing Involves the Assembly of a Multi-Component Complex
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Formation of the mRNA 3’ Terminus Requires Specific Cleavage
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Codon Recognition During Protein Synthesis Recognition of the initiating AUG
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Codon Recognition During Protein Synthesis
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Ribosome-Based Mechanism for Translation
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Transcription
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Elements of Transcriptional Control Cis-acting regulatory sequences Trans-acting regulatory proteins
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Transcriptional Control Sequences
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Transcription Involves the Assembly of a Multi-Component Complex
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Regulation of Gene Expression
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Measuring Gene Expression - Recognizing the Complexity
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Regulation of Transcription Control of transcription initiation (major form of control) Control of transcription elongation –Role of premature termination
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Mechanisms Regulating Transcription Initiation Control of synthesis of transcription factors Control of DNA binding activity of the factor Control of transcriptional function of the factor
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Regulation of Transcription The -Globin Gene Transcription Regulatory Sequence
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Thalassemia Mutations That Alter Transcription Regulation
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Regulation of Transcription – Examples from the Myc Gene
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Alterations in Transcriptional Control in Disease Activation of the c-myc gene by retrovirus mediated promoter insertion
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Alterations in Transcriptional Control in Disease Activation of the c-myc gene by rearrangement in B cell lymphomas
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Alterations in Transcriptional Control in Disease Creation of a chimeric transcription factor in AML
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Post-Transcriptional Gene Control Mechanisms
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Alteration of Post-Transcriptional Control Events
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Splice Site Mutations in Thalassemia
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AAUAAA AACAAA Normal + Thalassemia Mutations That Affect Polyadenylation
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Nucleic Acid Hybridization
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Hybridization of Complementary DNA Sequences Allows Detection of Specific DNAs
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