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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
Small chromosomes use a single origin
Replication of large chromosomes requires multiple origins
The Mammalian DNA Replication Apparatus
The Importance of Molecular Cloning
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
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
Methods of Recombinant DNA Analysis
Methods of Recombinant DNA Analysis
Methods of Recombinant DNA Analysis
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
Methods of Recombinant DNA Analysis
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
Isolation of a Gene and Gene Structure
Isolation of a Gene
Discontinuous Nature of a Eukaryotic Gene
Structure of a Typical Eukaryotic Gene – the -Globin Gene
Complexity of Gene Organization in Metazoans
The -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
Gene Expression
The Complexity of Gene Expression
Gene Expression Requires Splicing of Primary Transcripts
Conservation of Sequences at Splice Sites
Splicing Involves the Assembly of a Multi-Component Complex
Formation of the mRNA 3’ Terminus Requires Specific Cleavage
Codon Recognition During Protein Synthesis
Recognition of the initiating AUG
Codon Recognition During Protein Synthesis
Codon Recognition During Protein Synthesis
Ribosome-Based Mechanismfor Translation
Transcription
Elements of Transcriptional Control
• Cis-acting regulatory sequences
• Trans-acting regulatory proteins
Transcriptional Control Sequences
Transcription Involves the Assembly of a Multi-Component Complex
Regulation of Gene Expression
Measuring Gene Expression - Recognizing the Complexity
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 -Globin Gene
Transcription
Regulatory Sequence
Thalassemia Mutations That Alter Transcription Regulation
Regulation of Transcription – Examples from the Myc Gene
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
Post-Transcriptional Gene Control Mechanisms
Post-Transcriptional Gene Control Mechanisms
Alteration of Post-Transcriptional Control Events
Splice Site Mutations in Thalassemia
GCCAG GTTGGTATGCCAG TTGGTATA
GCCAG TTGGTATT
GCCAG GTTGTATT
GCCAG GTTGTATC
Exon 1
Splice site mutations in thalassemia
Normalo
o
+
+
1Intron
Splice Site Mutations in Thalassemia
-AAA
-AAA
-AAA
Normal
Normal (10%)
Non-functional (90%)
+
Wild Type
1
1
2
2
3
3
AAUAAA
AACAAA
Normal
+
Thalassemia Mutations That Affect Polyadenylation
Nucleic Acid Hybridization
Hybridization of Complementary DNA Sequences Allows Detection of Specific DNAs
