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Development

Development

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Development

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  1. Development Cell division Neurobiology

  2. Why do we work on yeast?

  3. Yeast has a long history of serving mankind

  4. Yeast satisfy important characteristics of a model organism • Genetics: introduce mutations (UV, chemical, Xray) and design screens to identify mutations in process you are interested in • Isolate the products of meiosis • Recover mutations: stable haploid and diploid lifecycles. • Easy molecular manipulations

  5. Versatile transformation system Maintains circular plasmids and mini chromosomes Homologous (integrative) transformation very efficient • Tag proteins with GFP to determine function in vivo. • Integrate specific DNA sequences to follow chromosomes • Tolerate large amounts of DNA

  6. Budding yeast (Saccharomyces cerevisiae) • Similarity to higher eukaryotes • Great for genetics (tagging, deleting, controlling transcription of genes; genome sequenced) • Haploid and diploid • Fast growth (~90 minute doubling time) • Morphology reflects cell cycle stage

  7. Achieving accurate chromosome segregation in mitosis Kinetochore HeLa cell: metaphase PtK1 cell: anaphase Aberrations lead to chromosome loss associated with cancers and birth defects

  8. Electron Micrograph of a PtK1 Chromosome Kinetochores are essential for chromosome segregation Kinetochores- protein-DNA complex built at the centromeric region of the chromosome Function- Physical linkage between DNA and microtubules Structure- Integrate DNA binding and microtubule binding proteins EM by Lynne Cassimeris

  9. What can budding yeast tell us about the process of mitosis? • Take an historical perspective to illustrate the importance of yeast as a model organism for understanding the regulation and mechanism of cell division. • How do you clone regions of DNA that do not encode genes i.e. centromeres, telomeres and origins of replication?

  10. Cell division cycle mutants • Yeast morphology is an indicator of position in the cell cycle G1 anaphase Sphase cytokinesis metaphase Lee Hartwell 2001 Nobel Prize

  11. Figure 6. A pathway of gene controlled events in the S. cerevisiae cell cycle. Numbers refer to cdc genes. Abbreviations are: iDS, initiation of DNA synthesis, DS, DNA synthesis, mND, medial nuclear division; lND, late nuclear division; BE, bud emergence; NM, nuclear migration; CK, cytokinesis; CS cell separation; MF mating factor. Reprinted from ref 7 with permission.

  12. 2001 Nobel Prize in Medicine • Cdc28 required to initial the cell cycle -Lee Hartwell • Cdc2 in S. pombe controlled rate limiting step in mitosis- Sir Paul Nurse • Cyclin dependent kinase (CDK) in sea urchins -Tim Hunt

  13. Other useful mutants • Rad- (radiation sensitive) -DNA repair and recombination, DNA damage check point Mad and Bub-(budding in presence of MT poison) mitotic checkpoint Sec-(cells became dense) secretory pathway, protein sorting -GTPases, GAPs, adaptors Swi/Snf- (growth defect on nonfermentable carbon source) glucose derepression Chromatin remodeling factors

  14. What if you are interested in chromosome organization?

  15. Three chromosomal elements essential for chromosome segregation are: • Origin of replication ( ARS) • Centromere (CEN) • Telomere (TEL) • None encode proteins. • How do you clone these sequences?

  16. Yeast Transformation Vectors circa 1980 • YIp-integration vectors: E coli sequences for replication, selection, yeast gene for selection like Leu2, Ura3, Trp1. Single copy • YEp-yeast episomal vector. 2u endogenous plasmid, high copy in all the cells, stable • YRp-yeast ARS (autonomously replicating sequence), high copy in some of the cells, unstable without selection. First origin of replication cloned because ARS1 was linked to TRP1.

  17. What are the hallmarks of chromosome segregation? • Sister chromatids separate to mother and daughter cells • Highly accurate • Linear DNA is stable and not recombinogenic i.e. telomeres protect ends

  18. Asymmetric plasmid segregation with just ARS ARS

  19. Symmetric segregation identified centromeres and telomeres CEN ARS

  20. Ndc80RFP Spc29RFP overlay Smc3GFP DIC Ndc80RFP overlay Smc3GFP DIC Kinetochores are clustered at the ends the cohesin cylinder

  21. Interstrand cohesin Inflection point Kinetochore sleeve Intrastrand cohesin C-loop Proposed Path of Centromere DNA in a Eukaryotic Kinetochore: C-loop

  22. Yeast in Biotechnology • The analysis of eukaryotic DNA sequence has been facilitated by cloning the genes in prokaryotes. But some functions such as glycosylation, mitosis, meiosis, etc. are absent in prokaryotes. When genes functionally related to such a function are to be analysed, those genes have to be cloned in a eukaryotic system. • Expression of mammalian genes in yeast exploited for drug discovery. • Identify protein partners

  23. Yeast as a model organism for human disease: • ~20% human disease genes have counterparts in yeast • Cancer-chromosome loss, DNA and mitotic checkpoints, DNA repair • Alzheimer's and Parkinson's diseases-chaperones involved in protein misfolding • Aging-sir genes, telomere loss • Mitochondrial disorders