Chapter 16 – The Molecular Basis of Inheritance

The Griffith Experiment

The Griffith experiment demonstrated the phenomenon of transformation in bacteria, providing early evidence that genetic material could be transferred between organisms.

• Transformation: The process by which one strain of bacteria is changed by a gene or genes from another strain.

• Key finding: Harmless (R) bacteria became pathogenic when mixed with heat-killed pathogenic (S) bacteria, indicating transfer of genetic material.

• Example: Streptococcus pneumoniae strains in mice.

The Hershey-Chase Experiment

This experiment confirmed that DNA, not protein, is the genetic material in viruses.

• Method: Used radioactive isotopes to label DNA (with phosphorus-32) and protein (with sulfur-35) in bacteriophages.

• Result: Only DNA entered bacterial cells and directed viral reproduction.

Chargaff's Rules

Erwin Chargaff discovered that DNA from any cell has a 1:1 ratio of pyrimidine and purine bases.

• Rule: %A = %T and %G = %C in DNA.

• Implication: Base pairing is specific (A with T, G with C).

Discovering the Structure of DNA

James Watson and Francis Crick, with data from Rosalind Franklin and Maurice Wilkins, determined the double helix structure of DNA.

• Double helix: Two antiparallel strands held together by hydrogen bonds between complementary bases.

• Base pairing: A–T (2 H-bonds), G–C (3 H-bonds).

Meselson-Stahl Experiment

This experiment demonstrated that DNA replication is semiconservative.

• Semiconservative replication: Each new DNA molecule consists of one old strand and one new strand.

• Method: Used isotopes of nitrogen (N-15 and N-14) to distinguish old and new DNA strands.

Introduction to DNA Replication

DNA replication is the process by which a cell copies its DNA before cell division.

• Origin of replication: Specific sequence where replication begins.

• Replication fork: Y-shaped region where new DNA strands are synthesized.

DNA Polymerases

DNA polymerases are enzymes that synthesize new DNA strands by adding nucleotides to a pre-existing chain.

• Direction: DNA polymerases add nucleotides in the 5' to 3' direction.

• Proofreading: Many DNA polymerases have proofreading activity to correct errors.

Leading & Lagging DNA Strands

During replication, the two new DNA strands are synthesized differently due to the antiparallel nature of DNA.

• Leading strand: Synthesized continuously toward the replication fork.

• Lagging strand: Synthesized discontinuously away from the fork in short segments called Okazaki fragments.

Steps of DNA Replication

• Initiation at origins of replication

• Unwinding by helicase

• Primase synthesizes RNA primers

• DNA polymerase extends new DNA strands

• DNA ligase joins Okazaki fragments

DNA Repair

Cells have mechanisms to repair DNA damage and maintain genetic integrity.

• Mismatch repair: Corrects errors missed by DNA polymerase.

• Nucleotide excision repair: Removes and replaces damaged DNA segments.

Telomeres

Telomeres are repetitive DNA sequences at the ends of eukaryotic chromosomes that protect them from deterioration.

• Function: Prevent loss of genetic information during replication.

• Enzyme: Telomerase extends telomeres in germ cells and some stem cells.

Chapter 17 – Gene Expression: From Gene to Protein

Central Dogma

The central dogma of molecular biology describes the flow of genetic information: DNA → RNA → Protein.

• Transcription: DNA is transcribed into RNA.

• Translation: RNA is translated into protein.

Introduction to Transcription

Transcription is the synthesis of RNA from a DNA template.

• Enzyme: RNA polymerase

• Product: Messenger RNA (mRNA) in eukaryotes

Steps of Transcription

• Initiation: RNA polymerase binds to promoter region.

• Elongation: RNA strand is synthesized in the 5' to 3' direction.

• Termination: RNA polymerase detaches at terminator sequence.

Eukaryotic RNA Processing and Splicing

In eukaryotes, pre-mRNA undergoes processing before becoming mature mRNA.

• 5' cap: Modified guanine nucleotide added to 5' end.

• Poly-A tail: Series of adenine nucleotides added to 3' end.

• Splicing: Removal of introns and joining of exons by spliceosome.

Introduction to Types of RNA

• mRNA (messenger RNA): Carries genetic code from DNA to ribosome.

• tRNA (transfer RNA): Brings amino acids to ribosome during translation.

• rRNA (ribosomal RNA): Structural and catalytic component of ribosomes.

Genetic Code

The genetic code is a set of rules by which information encoded in mRNA is translated into proteins.

• Codon: Three-nucleotide sequence on mRNA that specifies an amino acid.

• Redundancy: Multiple codons can code for the same amino acid.

• Start codon: AUG (methionine)

• Stop codons: UAA, UAG, UGA

Introduction to Translation

Translation is the process by which ribosomes synthesize proteins using mRNA as a template.

• Occurs in: Cytoplasm (eukaryotes and prokaryotes)

• Key players: mRNA, tRNA, ribosomes, amino acids

Steps of Translation

• Initiation: Ribosome assembles at start codon on mRNA.

• Elongation: tRNAs bring amino acids; peptide bonds form.

• Termination: Ribosome reaches stop codon; polypeptide released.

Post-Translational Modification

After translation, proteins may undergo modifications that affect their function.

• Examples: Phosphorylation, glycosylation, cleavage of signal peptides

Review of Transcription vs. Translation

Mutations

Mutations are changes in the DNA sequence that can affect gene expression and protein function.

• Point mutations: Single nucleotide changes (substitution, insertion, deletion)

• Frameshift mutations: Insertions or deletions that alter the reading frame

• Effects: Silent, missense, nonsense mutations

Chapter 18 – Regulation of Gene Expression

Introduction to Regulation of Gene Expression

Gene expression is regulated to ensure that genes are expressed at the right time, place, and amount.

• Importance: Allows cells to respond to environmental changes and differentiate.

Prokaryotic Gene Regulation via Operons

In prokaryotes, genes are often organized into operons, which are clusters of genes under the control of a single promoter.

• Operon: Includes promoter, operator, and structural genes.

• Regulation: Repressors or activators bind to operator to control transcription.

The Lac Operon

The lac operon in Escherichia coli controls the metabolism of lactose.

• Inducible operon: Normally off; turned on in presence of lactose.

• Key components: lacZ, lacY, lacA genes; operator; repressor protein

Glucose's Impact on Lac Operon

Glucose availability affects lac operon expression via catabolite repression.

• cAMP-CAP complex: Activates transcription when glucose is low.

• High glucose: Low cAMP; lac operon not activated even if lactose is present.

The Trp Operon

The trp operon in E. coli is a repressible operon that controls tryptophan synthesis.

• Repressible operon: Normally on; turned off when tryptophan is abundant.

• Feedback inhibition: Tryptophan acts as a corepressor.

Review of the Lac Operon & Trp Operon

Introduction to Eukaryotic Gene Regulation

Eukaryotic gene expression is regulated at multiple levels, including chromatin structure, transcription, and post-transcriptional processes.

• Complexity: Involves enhancers, silencers, transcription factors.

Eukaryotic Chromatin Modifications

• Histone acetylation: Loosens chromatin, promotes transcription.

• DNA methylation: Silences genes by adding methyl groups to DNA.

Eukaryotic Transcriptional Control

• Transcription factors: Proteins that bind DNA and regulate transcription.

• Enhancers: DNA elements that increase transcription from a distance.

Eukaryotic Post-Transcriptional Regulation

• Alternative splicing: Produces different mRNAs from the same gene.

• mRNA stability: Influences how long mRNA is available for translation.

Eukaryotic Post-Translational Regulation

• Protein modification: Phosphorylation, ubiquitination, etc.

• Protein degradation: Proteins marked for destruction by ubiquitin are degraded by proteasomes.

Chapter 20 – DNA Tools and Biotechnology

Introduction to DNA-Based Technology

DNA technology involves the manipulation and analysis of genetic material for research, medicine, and industry.

• Applications: Genetic engineering, forensics, gene therapy

Introduction to DNA Cloning

DNA cloning is the process of making multiple, identical copies of a DNA fragment.

• Vectors: Plasmids commonly used to carry foreign DNA into host cells.

Steps to DNA Cloning

• Isolate DNA of interest

• Insert DNA into vector (e.g., plasmid)

• Introduce recombinant DNA into host cell

• Grow host cells to amplify DNA

• Screen for cells with recombinant DNA

Introduction to Polymerase Chain Reaction (PCR)

PCR is a technique to amplify specific DNA sequences in vitro.

• Key enzyme: Taq DNA polymerase

• Applications: Diagnostics, forensics, research

The Steps of PCR

• Denaturation: DNA strands separated by heating

• Annealing: Primers bind to target sequence

• Extension: DNA polymerase synthesizes new DNA

Gel Electrophoresis

Gel electrophoresis separates DNA fragments by size using an electric field.

• Smaller fragments: Move faster and farther through the gel

• Visualization: DNA stained with dyes or labeled probes

Southern Blotting

Southern blotting detects specific DNA sequences in a sample after gel electrophoresis.

• Steps: Transfer DNA to membrane, hybridize with labeled probe

DNA Fingerprinting

DNA fingerprinting identifies individuals based on unique patterns in their DNA.

• Uses: Forensics, paternity testing

• Technique: Analysis of variable number tandem repeats (VNTRs) or short tandem repeats (STRs)

Introduction to DNA Sequencing

DNA sequencing determines the precise order of nucleotides in a DNA molecule.

• Applications: Genomics, evolutionary biology, medicine

Dideoxy Sequencing

Dideoxy (Sanger) sequencing uses chain-terminating nucleotides to generate DNA fragments of varying lengths for sequence determination.

• Key feature: Incorporation of dideoxynucleotides (ddNTPs) stops DNA synthesis at specific bases.

Chapter 21 – Genomes and Their Evolution

Genomes

A genome is the complete set of genetic material in an organism.

• Genomics: The study of whole genomes, including gene mapping, sequencing, and analysis.

• Comparative genomics: Comparing genomes across species to understand evolution and function.