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