development

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