QBM 1

Lecture 1

Molecular Biology Overview

Lecture Overview

  • History of Molecular Biology

  • DNA, RNA, and Proteins

  • Biomedical Research Project

  • Recombinant DNA Technology (Labs 9-11)

  • Protein Production, Purification, and Analysis (Labs 2-8)

Protein-DNA Relationship

  • Protein interacts with DNA and plays a critical role in gene expression and regulation.

History of Molecular Biology

Early Studies

  • Cells and small molecules have been investigated for centuries.

  • Experimental methods such as acid synthesis, distillation, crystallization, and quantitative analysis trace back to the 800s.

Advancements (1700-1900)

  • Major progress in chemistry, elements, molecular synthesis, and atomic theory occurred during this time.

Organism Studies (1900-1950)

  • Simple organisms like E. coli, yeast, and fruit flies were employed to understand genes and proteins.

  • New fields, including genetics, biochemistry, and microbiology, emerged.

  • 1944: Oswald Avery discovered that DNA was the basis for genes.

  • 1944: Erwin Schrödinger published "What Is Life? The Physical Aspect of the Living Cell."

Emergence of Molecular Biology (1950-1980)

  • Key developments included solving the structure of DNA, understanding DNA replication, genetic code, transcription, and translation, collectively known as the Central Dogma.

  • Contributions from geneticists, microbiologists, and biochemists were pivotal.

  • Max Delbrück, noted for work on viral replication and a member of the Phage Group, advocated for “quantitative biology.”

  • Delbrück trained many who progressed molecular biology during his Phage Course (1945-1970).

  • Tom Maniatis held a molecular cloning course at Cold Spring Harbor Laboratory in 1980 and published a manual on Molecular Cloning.

Prominent Figures E.g. Isaac Newton

  • Isaac Newton is quoted: "If I have seen further, it is by standing on the shoulders of giants."

    • Contributors to molecular biology listed:

    • Oswald Avery

    • Max Delbrück

    • Jabir ibn Hayyan

Significance of DNA

  • Watson and Crick elucidated the structure of DNA in 1953, marking the inception of molecular biology.

  • Following this, Crick introduced the Central Dogma:
    DNA→RNA→Protein\text{DNA} \rightarrow \text{RNA} \rightarrow \text{Protein}

  • RNA functions include gene regulation and catalysis.

  • Understanding protein structure and function is vital for comprehending molecular pathways.

  • Molecular biology techniques provide deeper insights into cell functions.

  • Recombinant DNA technology is crucial for biomedical research, allowing for cloning, PCR, DNA manipulation, plasmid generation, protein expression, and DNA sequencing.

Molecular Biology Defined

  • Molecular biology investigates molecular processes and pathways for insights into cell function.

  • Key areas include:

    • DNA storage, replication, repair, and recombination

    • RNA expression and regulation

    • Protein structure, function, and regulation

  • Arthur Kornberg's perspective: "Cell biologists look, geneticists count, biochemists clean."

Information Exchange in Molecular Biology

  • Impacts on chemotherapy and radiation therapy, focusing on cell signaling pathways:

    • p53 signaling

    • Cellular degradation mechanisms

    • Hypoxia and growth factor responses

    • Apoptosis (programmed cell death)

Pathways and Factors Included:

  • p53, JNK, ATM, EGFR, and more complexes influencing gene expression and survival pathways.

Recent Developments and New Disciplines

  • Advancements in recombinant DNA technology have led to:

    • Gene silencing, knockouts, gene chips, gene therapy, CRISPR.

  • Techniques like qPCR and RNA sequencing enhance understanding of RNA expression.

  • Protein purification and mutagenesis deepen functional insights and drug development.

  • The molecular biology landscape is interdisciplinary:

    • Fields include biochemistry, genetics, genomics, transcriptomics, proteomics, bioinformatics, metabolomics, molecular diagnostics, and systems biology.

Recent Discoveries in Molecular Biology

  • Neurons derived from ALS patients' skin cells.

  • Skin cells converted into stem cells, photoreceptors, and other types.

  • Rett Syndrome reversed in mice by inducing nerve cells to express MeCP2.

  • Gene therapy techniques utilized in various diseases, e.g., curing color blindness and spinal muscular atrophy.

Genetically Modified T-Cells for Cancer Treatment

  • Engineered T-cells show promise in treating leukemia, achieving remission in chronic lymphocytic leukemia patients with chimeric antigen receptors (CAR).

Techniques Involved:

  • Utilization of lentivirus vectors for cellular modifications

    • Methods include:

    • Spectrophotometry

    • DNA isolation

    • PCR, quantitative PCR, and flow cytometry among others.

Molecular Biology Components

DNA, RNA, and Proteins

  • Carbon's tetravalence enables structural complexity in biomolecules.

  • Elements forming the foundation: H2O, CO2, NH4+, NO3-, N2 resulting in nucleotides and amino acids.

Central Dogma of Molecular Biology

  • Details the flow of genetic information:
    DNA→RNA→Protein\text{DNA} \rightarrow \text{RNA} \rightarrow \text{Protein}

DNA Structure

  • Deoxyribonucleic Acid (DNA)

    • Double-stranded with a 5' to 3' orientation.

    • Phosphodiester backbone and complementary base pairing (A=T; G≡C).

RNA Structure

  • Ribonucleic Acid (RNA)

    • Types include mRNA, tRNA, rRNA, and various small non-coding RNAs (ncRNAs).

    • Base pairing abilities with DNA and other RNA; functional as enzymes.

    • Complementary base pairs: (A=U; G≡C).

Protein Structure

  • Polypeptides formed through peptide bonds between amino acids.

  • Protein synthesis proceeds from N-terminus to C-terminus.

Building Levels of Protein Structure Including:

  • Primary structure: Sequence of amino acids.

  • Secondary structure: Alpha helices and beta sheets; stabilized by hydrogen bonds.

  • Tertiary structure: Three-dimensional configuration of a protein.

  • Quaternary structure: Assembly of multiple polypeptides into a single functional unit.

Biomedical Research Projects

  • Examined the structure and activation of human vinculin through multiple studies.

Cell-Cell Interactions

  • Explores cell attachment methods (adhesion) using extracellular matrix and adhesion junctions.

Vinculin Discovery Timeline

  • 1979: Isolation of a 130 kDa protein from chicken gizzards named Vinculin.

  • Development from initial purification to understanding its full structure by 2004.

Sequence Alignment (Bioinformatics)

  • Illustrated differences in sequence alignment across species (e.g., human, mouse, chicken).

Molecular Techniques in Labs 9-11

Focus Areas:

  • Genetic predisposition testing through DNA sequencing, SNP analysis, and population genetics.

  • Techniques include reverse transcription, gene cloning, PCR, and agglomerative gene therapy methodologies.

RNA Extraction Methodology

  • Employs oligonucleotide primers for reverse transcription.

  • mRNA converted to cDNA for analysis.

Techniques Covered in Labs 9, 10, 11

  • Polymerase Chain Reaction: Amplification of DNA from a cDNA library.

  • Gel Electrophoresis: Used for analyzing and purifying DNA samples.

  • Restriction Digestion: Utilizing enzymes like EcoRI for DNA modification.

Conclusion: Molecular Cloning Key Techniques (Lab 10)

  • Recombinant DNA technology encompasses cloning and gene manipulation for studies across various cells and organisms.

  • Additionally focuses on protein purification techniques, enzyme activity, and post-translational modifications.