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DNA and Chromosome Structure. Chromosomal Structure of the Genetic Material. The Essential Structure of DNA. The Replication Challenge. Size of an average human chromosome 130 million bp Rate of replication ~ 50 bp per sec Fidelity of replication. Replication of the Genetic Material.
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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
Replication of the Genetic Material Small chromosomes use a single origin Replication of large chromosomes requires multiple origins
Role of Recombinant DNA Analysis in the Study of Gene Structure/Function The essence of the problem: Human genome = 3 x 109 bp The b-globin gene = 3 x 103 bp Cloning of genes solves this problem and allows an analysis of function and the basis for mutation
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
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
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
Complexity of Gene Organization in Metazoans The b-globin locus
Unequal Crossing Over as a Mechanism for Gene Duplication and Gene Loss
The Impact of the Complexity of Gene Structure on Gene Expression
Formation of the mRNA 3’ Terminus Requires Specific Cleavage
Codon Recognition During Protein Synthesis Recognition of the initiating AUG
Elements of Transcriptional Control • Cis-acting regulatory sequences • Trans-acting regulatory proteins
Transcription Involves the Assembly of a Multi-Component Complex
Regulation of Transcription • Control of transcription initiation (major form of control) • Control of transcription elongation • Role of premature termination
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
Regulation of Transcription The b-Globin Gene Regulatory Sequence Transcription
Alterations in Transcriptional Control in Disease Activation of the c-myc gene by retrovirus mediated promoter insertion
Alterations in Transcriptional Control in Disease Activation of the c-myc gene by rearrangement in B cell lymphomas
Alterations in Transcriptional Control in Disease Creation of a chimeric transcription factor in AML