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DNA and Chromosome Structure. Chromosomal Structure of the Genetic Material.

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Presentation on theme: "DNA and Chromosome Structure. Chromosomal Structure of the Genetic Material."— Presentation transcript:

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 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

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

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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  -Globin Gene

20 Complexity of Gene Organization in Metazoans The  -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

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32 Ribosome-Based Mechanism for 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  -Globin Gene Transcription Regulatory Sequence

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

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49 Alteration of Post-Transcriptional Control Events

50 Splice Site Mutations in Thalassemia

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52 AAUAAA AACAAA Normal +  Thalassemia Mutations That Affect Polyadenylation

53 Nucleic Acid Hybridization

54 Hybridization of Complementary DNA Sequences Allows Detection of Specific DNAs


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