molecular genetics 2010 welcome to the course!. molecular genetics 2008 welcome to the course!...
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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
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