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Molecular Genetics 2010 Welcome to the course!

Molecular Genetics 2010 Welcome to the course!. Molecular Genetics 2008 Welcome to the course!. Describes the use of Molecular Genetics to study a range of different topics

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Molecular Genetics 2010 Welcome to the course!

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  1. Molecular Genetics 2010Welcome to the course!

  2. Molecular Genetics 2008Welcome to the course! • Describes the use of Molecular Genetics to study a range of different topics • We don’t have time to tell you EVERYTHING about how Molecular Genetics has been/is being used, as the study of many different areas now involves molecular genetic techniques • So: • On this course we have 3 lecturers, and we will each tell you about how to use molecular genetics to study different areas of biology/biochemistry/genetics/biotechnology • This means that the topics covered by the 3 lecturers will probably not be linked in terms, other than that they all involve Molecular Genetics

  3. Lecturers and their favourite topics! • Felicity Watts (8 lectures) • Yeast as a model system • Homologous recombination, mating type switching, cell cycle control, DNA integrity checkpoints • Majid Hafezparast (8 lectures) • Human and mouse • Gene cloning in mouse, complex traits and the HapMap project, Functional genomics • Neil Crickmore (4 lectures) • Application of Molecular Genetics to the Biotechnology Industry

  4. What is the difference between classical and molecular genetics? • Classical genetics • Isolation of mutants • analysis of the nature of the mutants • e.g. dominant/recessive -look in diploid m/M • pathways • A B C D E or • extragenic suppressors A B E C D

  5. Molecular genetics • identify genes by complementation • genome sequencing projects • clone by Email! • clone gene by homology • used to use hybridisation • PCR • Create new mutants • e.g. delete a whole gene • make point mutations • knockout expression with antisense RNA • add a tag to a protein • microarray analysis

  6. Why do we use model systems and why don’t we all study humans? • Classical genetics • Isolation of mutants • analysis of the nature of the mutants • e.g. dominant/recessive -look in diploid m/M • pathways • extragenic suppressors • Molecular genetics • identify genes by complementation • genome sequencing projects • clone by Email! • clone gene by homology • used to use hybridisation • PCR • Create new mutants • e.g. delete a whole gene • make point mutations • knockout expression with antisense RNA • add a tag to a protein • microarray analysis

  7. Yeasts as model organisms Eukaryotes Prokaryotes S. pombe 4,900 E. coli 4,286 S. cerevisiae 5,570 Streptomyces >8,000 Drosophila 13,919 Nematode 19,622 Arabidopsis 25,498 Human 37,000 S. pombe: 3281 have homology with genes in S. cerevisiae/nematode 145 have homology with genes in nematode 769 have homolgy with genes in S. cerevisiae 681 are unique to S. pombe

  8. Why analyse 2 yeasts: S. pombe and S. cerevisiae • Both have small genomes • Both easy to grow • Doubling time 2-3 hours • Both easy to use for classical and molecular genetics • Many mutants • Both have haploid and diploid forms • Many cloning vectors and reagents available • Both genomes totally sequenced • So why use both?

  9. S. cerevisiae and S. pombe are as related to each other as each is to humans! Humans (mice) S. pombe S. cerevisiae So: if we find processes that are common to both yeasts, they may also occur in humans

  10. S. pombe and S. cerevisiae Fission yeast Budding yeast

  11. Genetic recombination • Homologous recombination • site-specific recombination • transposition • illegitimate recombination/non-homologous end joining

  12. Homologous recombination • involved in meiosis • repair of DNA double strand breaks (DSBs) during the mitotic cycle homologous recombination between sister chromatids to repair the break S. pombe cell in G2 with DSB

  13. Homologous recombination (HR) • 3 stages • pairing • formation of an intermediate • resolution • a number of models proposed as to how recombination occurs • these must take into account the experimental evidence • Many HR proteins now identified and their functions are being characterised

  14. The sort of evidence that needs to be considered Neurospora Comes from analysing the products of meiosis From: Fincham, Genetis (1983) Pub John Wright

  15. The sort of evidence that needs to be considered Non-Mendelian inheritance not common due to gene conversion or post-meiotic segregation How does this occur? Its due to heteroduplex DNA From: Fincham, Genetis (1983) Pub John Wright

  16. Aberrant segregation Recombination events can result in mismatches Mismatches might be repaired to give 2:4 or 1:3 segregation or might not be repaired, in which case they will give 3:5 Will explain in more detail later X T Y G

  17. Pairing (meiosis) • In eukaryotes this results in a synaptonemal complex DNA seems too far apart for recombination to occur but can in some cases see ‘recombination nodules’ Unknown how homologous sequences identify one another possibly there is single stranded DNA search for homology From: M Westergaard

  18. How does Pairing occur? Possibly by ‘horsetail’ Movement From Chikashige et al., Science (1994) 264, 270

  19. Timing of events

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