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DNA and Chromosome Structure

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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DNA and Chromosome Structure

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  1. DNA and Chromosome Structure

  2. Chromosomal Structure of the Genetic Material

  3. The Essential Structure of DNA

  4. The Replication Challenge • Size of an average human chromosome 130 million bp • Rate of replication ~ 50 bp per sec • Fidelity of replication

  5. Replication of the Genetic Material Small chromosomes use a single origin Replication of large chromosomes requires multiple origins

  6. The Mammalian DNA Replication Apparatus

  7. The Importance of Molecular Cloning

  8. 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

  9. 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

  10. Methods of Recombinant DNA Analysis

  11. Methods of Recombinant DNA Analysis

  12. Methods of Recombinant DNA Analysis

  13. 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

  14. Methods of Recombinant DNA Analysis

  15. 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

  16. Isolation of a Gene and Gene Structure

  17. Isolation of a Gene

  18. Discontinuous Nature of a Eukaryotic Gene

  19. Structure of a Typical Eukaryotic Gene – the b-Globin Gene

  20. Complexity of Gene Organization in Metazoans The b-globin locus

  21. Unequal Crossing Over as a Mechanism for Gene Duplication and Gene Loss

  22. The Impact of the Complexity of Gene Structure on Gene Expression

  23. Gene Expression

  24. The Complexity of Gene Expression

  25. Gene Expression Requires Splicing of Primary Transcripts

  26. Conservation of Sequences at Splice Sites

  27. Splicing Involves the Assembly of a Multi-Component Complex

  28. Formation of the mRNA 3’ Terminus Requires Specific Cleavage

  29. Codon Recognition During Protein Synthesis Recognition of the initiating AUG

  30. Codon Recognition During Protein Synthesis

  31. Codon Recognition During Protein Synthesis

  32. Ribosome-Based Mechanismfor Translation

  33. Transcription

  34. Elements of Transcriptional Control • Cis-acting regulatory sequences • Trans-acting regulatory proteins

  35. Transcriptional Control Sequences

  36. Transcription Involves the Assembly of a Multi-Component Complex

  37. Regulation of Gene Expression

  38. Measuring Gene Expression - Recognizing the Complexity

  39. Regulation of Transcription • Control of transcription initiation (major form of control) • Control of transcription elongation • Role of premature termination

  40. 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

  41. Regulation of Transcription The b-Globin Gene Regulatory Sequence Transcription

  42. Thalassemia Mutations That Alter Transcription Regulation

  43. Regulation of Transcription – Examples from the Myc Gene

  44. Alterations in Transcriptional Control in Disease Activation of the c-myc gene by retrovirus mediated promoter insertion

  45. Alterations in Transcriptional Control in Disease Activation of the c-myc gene by rearrangement in B cell lymphomas

  46. Alterations in Transcriptional Control in Disease Creation of a chimeric transcription factor in AML

  47. Post-Transcriptional Gene Control Mechanisms

  48. Post-Transcriptional Gene Control Mechanisms

  49. Alteration of Post-Transcriptional Control Events

  50. Splice Site Mutations in Thalassemia

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