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– 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

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

• 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

What is the difference between classical and molecular genetics?

A B EC D

• 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

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

Yeasts as model organisms

Eukaryotes ProkaryotesS. pombe 4,900 E. coli 4,286S. cerevisiae 5,570 Streptomyces >8,000Drosophila 13,919Nematode 19,622Arabidopsis 25,498Human 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

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?

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

S. pombe and S. cerevisiae

Fission yeast Budding yeast

Genetic recombination

• Homologous recombination• site-specific recombination• transposition• illegitimate recombination/non-

homologous end joining

Homologous recombination

• involved in meiosis• repair of DNA double strand breaks

(DSBs) during the mitotic cycle

S. pombe cellin G2

with DSB

homologous recombination between sister chromatidsto repair the break

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

The sort of evidence that needs to be considered

Comes from analysing the products of meiosis

Neurospora

From: Fincham, Genetis(1983)Pub John Wright

The sort of evidence that needs to be considered

Non-Mendelian inheritance

not commondue to gene conversion or post-meiotic segregationHow does this occur?Its due to heteroduplex DNA

From: Fincham, Genetis(1983)Pub John Wright

Aberrant segregation

X T Y

G

Recombination events can result in mismatches

Mismatches might be repaired to give 2:4 or 1:3 segregationor might not be repaired, in which case they will give 3:5

Will explain in more detail later

Pairing (meiosis)

• In eukaryotes this results in a synaptonemal complex

DNA seems too far apart for recombination to occurbut 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

How doesPairing occur?

Possibly by ‘horsetail’ Movement

From Chikashige et al., Science (1994) 264,270

Timing of events