Download lecture1a - The School of Life Sciences at Sussex

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
What is the difference between
classical and molecular
genetics?
• Classical genetics
– Isolation of mutants
– analysis of the nature of the mutants
• e.g. dominant/recessive -look in diploid m/M
– pathways
A B
• A B C D E or
– extragenic suppressors
E
C 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
Molecular genetics
•Isolation of mutants
•identify genes by
•analysis of the nature
complementation
of the mutants
•genome sequencing projects
•e.g.
•clone by Email!
dominant/recessive
•clone gene by homology
-look in diploid m/M
•used to use hybridisation
•pathways
•PCR
•extragenic suppressors
•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
S. pombe
S. cerevisiae
Drosophila
Nematode
Arabidopsis
Human
Prokaryotes
4,900
5,570
13,919
19,622
25,498
37,000
E. coli
Streptomyces
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
4,286
>8,000
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/nonhomologous end joining
Homologous recombination
• involved in meiosis
• repair of DNA double strand breaks
(DSBs) during the mitotic cycle
S. pombe cell
in G2
with DSB
homologous recombination
between sister chromatids
to 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
From:
Fincham, Genetis
(1983)
Pub John Wright
Neurospora
The sort of evidence that needs
to be considered
Non-Mendelian inheritance
From:
Fincham, Genetis
(1983)
Pub John Wright
not common
due to gene conversion or
post-meiotic segregation
How does this occur?
Its due to heteroduplex DNA
Aberrant segregation
Recombination events can result in mismatches
X
T
Y
G
Mismatches might be repaired to give 2:4 or 1:3 segregation
or 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 occur
but 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 does
Pairing occur?
Possibly by
‘horsetail’
Movement
From Chikashige et al.,
Science (1994) 264,
270
Timing of events