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 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
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 b-Globin Gene
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
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 Mechanism
for 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 b-Globin Gene
Regulatory Sequence
Transcription
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
Splice site mutations in b thalassemia
Exon 1
Intron 1
GCCAG GTTGGTAT
GCCAG ATTGGTAT
GCCAG TTTGGTAT
Normal
GCCAG GTTGTTAT
b
GCCAG GTTGCTAT
b+
o
b
o
b
+
Splice Site Mutations in Thalassemia
1
2
3
Wild Type
-AAA
1
2
Normal
3
b+
-AAA
Normal (10%)
-AAA
Non-functional (90%)
Thalassemia Mutations That
Affect Polyadenylation
AAUAAA
Normal
AACAAA
b+
Nucleic Acid Hybridization
Hybridization of Complementary DNA
Sequences Allows Detection of Specific DNAs