development
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Development. Cell division. Neurobiology. Why do we work on yeast?. Yeast has a long history of serving mankind. Yeast satisfy important characteristics of a model organism. - PowerPoint PPT PresentationTRANSCRIPT
Development
Cell division
Neurobiology
Why do we work on yeast?
Yeast has a long history of serving mankind
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
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
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
Achieving accurate chromosome segregation in mitosis
Kinetochore
HeLa cell: metaphase
PtK1 cell: anaphase
Aberrations lead to chromosome loss associated with cancers and birth defects
EM by Lynne Cassimeris
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
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?
Cell division cycle mutants• Yeast morphology is an indicator of
position in the cell cycle
G1
S phase
metaphase
anaphase
cytokinesis
Lee Hartwell 2001 Nobel Prize
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 nucleardivision; lND, late nuclear division; BE, bud emergence; NM, nuclear migration; CK, cytokinesis;CS cell separation; MF mating factor. Reprinted from ref 7 with permission.
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
Other useful mutants
• Rad- (radiation sensitive)-DNA repair and recombination, DNA damage
check pointMad and Bub-(budding in presence of MT poison)mitotic checkpointSec-(cells became dense) secretory pathway,
protein sorting-GTPases, GAPs, adaptorsSwi/Snf- (growth defect on nonfermentable carbon
source) glucose derepressionChromatin remodeling factors
What if you are interested in chromosome organization?
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?
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.
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
Asymmetric plasmid segregation with just ARS
ARS
Symmetric segregation identified centromeres and telomeres
CEN
ARS
DIC Smc3GFP
Ndc80RFPSpc29RFP overlay
DIC Smc3GFP Ndc80RFP overlay
Kinetochores are clustered at the ends the cohesin cylinder
Intrastrand cohesin
Interstrand cohesin
C-loop
Inflectionpoint Kinetochore sleeve
Proposed Path of Centromere DNA in a Eukaryotic Kinetochore: C-loop
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
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