DNA and Chromosome Structure
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Transcript of DNA and Chromosome Structure
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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 109 bp
The -globin gene = 3 x 103 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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Methods of Recombinant DNA Analysis
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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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Codon Recognition During Protein Synthesis
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Ribosome-Based Mechanismfor 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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Post-Transcriptional Gene Control Mechanisms
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Alteration of Post-Transcriptional Control Events
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Splice Site Mutations in Thalassemia
GCCAG GTTGGTATGCCAG TTGGTATA
GCCAG TTGGTATT
GCCAG GTTGTATT
GCCAG GTTGTATC
Exon 1
Splice site mutations in thalassemia
Normalo
o
+
+
1Intron
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Splice Site Mutations in Thalassemia
-AAA
-AAA
-AAA
Normal
Normal (10%)
Non-functional (90%)
+
Wild Type
1
1
2
2
3
3
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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