MCB Lecture [In-person]-20250228_090315-Meeting Recording

Understanding Yeast and Complementation

• Yeast Genetic States: Yeast can exist in two forms: haploid (one genome copy) or diploid (two genome copies).

  • Haploid: Can fuse to form diploids.

  • Diploid: Capable of undergoing meiosis and sporulation to create haploid offspring.

Cell Cycle and Mutation Analysis in Yeast

• The study of yeast cell division processes has importance for higher eukaryotes, as many cellular mechanisms are conserved.

• Mutants: Yeast mutants can be created using random mutagenesis techniques (e.g., chemical mutagens, radiation).

• Screening Mutants: Focus on mutants that show interrupted cell cycles or growth issues, often indicating essential gene mutations.

Temperature Sensitive Mutants

• Temperature Sensitivity: To study essential genes, temperature sensitive mutants are used:

  • At permissive temperatures: Organs grow normally (wild-type).

  • At non-permissive temperatures: The mutants exhibit the phenotype of interest (e.g., failure to grow).

Complementation Test

• Purpose: To determine if mutations occur in the same gene or in different genes.

  • Mutants are crossed to create diploids and tested at different temperatures.

• Outcome: If growth occurs at non-permissive temperatures, the mutations are in different genes (complementation); if no growth, then they are likely in the same gene.

Gene Discovery Strategies

• Cloning Techniques: Shuttle vectors allow navigation between bacteria (e.g., E. coli) and yeast for genetic manipulation.

  • Library Creation: A genomic library containing thousands of yeast genomic fragments is created for screening.

  • Yeast cells are transformed with this library to rescue phenotypes of temperature sensitive mutants, aiding gene identification.

Challenges with Eukaryotic Systems

• Complexity of Eukaryotic Genomes: Eukaryotic genomes (like humans) are significantly larger and contain many introns.

  • Exonic sequences form only 5-10% of gene structures, making them harder to manipulate.

• BACs (Bacterial Artificial Chromosomes): Used to store larger DNA fragments (up to millions of base pairs), but come with challenges in manipulation.

Reverse Transcription and cDNA Construction

• Reverse Transcription Process: Converts RNA into complementary DNA (cDNA) using the enzyme reverse transcriptase, obtained from retroviruses.

  • Only mRNAs with poly A tails are used for cDNA synthesis, eliminating intronic sequences.

• PCR Amplification: Necessary for creating enough copies of a specific DNA sequence for further study.

  • The process involves denaturation, annealing of primers, and extension through polymerase action.

DNA Cloning Considerations

• Restriction Enzymes and Adapters: Used to create specific sticky ends allowing precise insertion into vectors.

• Library Representation: cDNA libraries only represent coding sequences, excluding non-coding RNAs.

Next-Generation Sequencing (NGS)

• Massive Parallel Sequencing: Ability to conduct millions of sequencing reactions simultaneously, overcoming earlier technical limitations.

• Methods: Sequencing involves attaching DNA fragments to surfaces with complementary primers for amplification.

  • Fluorescent nucleotides signal addition during sequencing, allowing for real-time data collection.

Human Genome Sequencing Approaches

• Traditional Mapping: Involves creating overlapping libraries to establish a complete map of the genome.

• Shotgun Sequencing: Proposed alternative by Craig Venter; random sequencing of fragments without prior mapping relies on computational alignment of sequences.

• Outcome and Efficiency: The shotgun approach reduces the initial workload at the expense of increasing reliance on computational analysis.