Transcript Budding Technologies and Budding Yeast

BUDDING TECHNOLOGIES
AND BUDDING YEAST
2012 HHMI Summer Workshop for High School
Science Teachers
The Genomics of S.cerevisiae
GOALS
Introduction to the
Genomics of Yeast
Sequencing Technologies
and how they are evolving
Introduction to Systems
Biology and modern Yeast
Genetics
Genetics and Genomics

GENETICS is the science of genes, heredity and
variation.
 Genetic
studies typically focus on a single gene.
 Experiments typically involve mutation of the model
organism, then looking to figure out what went wrong.

GENOMICS is a discipline of systems biology that
focuses on the genome.
 Genomic
studies typically study all genes at once
Basic Yeast Statistics

16 chromosomes
Genomic Organization &
Nomenclature

16 Chromosomes.
 Range
from 230kbp –
1.5Mbp
Basic Yeast Statistics
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16 chromosomes
13.1 Mbp of sequence
E.coli:
4.6 Mbp
Yeast:
13.1 Mbp
Drosophila:
122 Mbp
Zebrafish:
1.2 Gbp
Human:
3.3 Gbp
Basic Yeast Statistics
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16 chromosomes
13.1 Mbp of sequence
6,183 open reading frames
73% of the genome codes for genes
E.coli:
4,377
Yeast:
6,183
Drosophila:
17,000
Zebrafish:
15,800
Human:
23,000
Basic Yeast Statistics
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Crick Strand
Left arm
16 chromosomes
Y A L 014 C
13.1 Mbp of sequence
6,183 open reading frames
Chromosome I
14th gene from
the centromere
73% of the genome codes for genes
Genes are named by position.
Where to learn more:

Saccharomyces Genome Database
Where to learn more: Browser

Saccharomyces Genome Database
Yeast as a Model System
Yeast share most basic
systems with human.
-
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Polymerases
Nucleosomes
Translation
Splicing
Stress response
DNA damage response
Cell Cycle
Mitotic mechanisms
Meiosis
More about Yeast

About75% of yeast
genes have something
known about them.
More about Yeast


About75% of yeast
genes have known
functions.
Many genes serve
to regulate other
genes.
More about Yeast



About75% of yeast
genes have known
functions.
Many genes serve
to regulate other
genes.
About 1/3 of
proteins are in the
nucleus.
GOALS
Introduction to the
Genomics of Yeast
Sequencing Technologies
and how they are evolving
Introduction to Systems
Biology and modern Yeast
Genetics
Sequencing the First Eukaryote
•
•
•
600 Scientists
>100 labs
World wide
effort
Sanger Sequencing
Sanger Sequencing
So… How do you sequence a Genome?

Walking
So… How do you sequence a Genome?

Walking
So… How do you sequence a Genome?

Walking
 Types
of vectors
Type
Host
Amount of DNA
plasmid
E.Coli
1-20 kb
cosmid
E.Coli / phage
37-52 kb
fosmid
E.Coli – F’ element
40 kb 1/cell
BAC
E.coli
150-350 kb
YAC
Yeast
100 – 3,000 kb
So… How do you sequence a Genome?
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Walking
Shotgunning
Completely sequence
Randomly fragment
~1-2kb
Reassemble
So… How do you sequence a Genome?
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Walking
Shotgunning
Mixed Approach
 Prescaffolding
markers
Large
vectors
So… How do you sequence a Genome?



Walking
Shotgunning
Mixed Approach
 Prescaffolding
 Shotgunning
markers
Large
vectors
Small
plasmids
the fragments
Yeast to Human….
A new revolution
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454
Solexa
ABI
How NGS works
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Fundamentally different
from Sanger
Detect each base
individually, then
extend
Watch as polymerase
moves along the chain
Each molecule is read
multiple times
How NGS works
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Illumina Sequencing
uses “Sequencing by
Synthesis
Adaptors added to
DNA to make them bind
the flowcell.
In situ, the DNA is
amplified into a cluster
How NGS works

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Primer then binds to the
sequence.
Bases are flowed over
the cluster and
nucleotides are read.
How NGS works
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Primer then binds to the
sequence.
Bases are flowed over
the cluster and
nucleotides are read.
Billions of reads are
happening at once.
A new revolution

Sequencing costs are
plummeting.
A new revolution

Sequencing costs are
plummeting.
 Cut
in half every year.
A new revolution

Sequencing costs are
plummeting.
 Cut

in half every year.
Yields are sky
rocketing.
Applications
gDNA
mRNA
miRNA
IP
Re-Sequencing
De Novo Sequencing
SNP Discovery
Transcript Discovery
Expression Analysis
miRNA Analysis
Allelic Expression
ChIP-Seq
Nuclear run-on
Copy Number Variation
… and more
Applications: Genetics
Mutation in alk in 224A/+
R>H
D>N homozygous
GOALS
Introduction to the
Genomics of Yeast
Sequencing Technologies
and how they are evolving
Introduction to Systems
Biology and modern Yeast
Genetics
Systems Biology

Most molecular biology has
been carried out with a
reductionist point of view
 Look
at one gene or one protein
or a class of genes

Systems Biology attempts to
look at organisms holistically
 “OMICS”
(genomics, proteomics,
metabolomics, transcriptomics,
etc.)
Systems Biology: Beginnings

First whole genome experiments
were done with microarrays.
 Surface
of the microarray is
spotted with DNA reflecting
every gene in the genome
 Total RNA is hybridized to the
surface
 Amount of material can be
measured by intensity
Forward Genetics v Reverse Genetics

Forward genetics is the
classical method for doing
screens.
 1)
Find a phenotype.
 2) Find out why it happens.

Reverse genetics mutates a
gene, then sees what it
does.
 This
defined genetic
alteration makes it
amenable to systems biology
approaches.
Functional Screen: Two-Hybrid
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Screen genome wide for
protein interaction partners.
A “prey” library requires
every protein to be fused to
a transcription activation
domain.
Screen with a bait protein
that binds to the DNA.
Functional Screen: Two-Hybrid


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Screen genome wide for
protein interaction partners.
A “prey” library requires
every protein to be fused to
a transcription activation
domain.
Screen with a bait protein
that binds to the DNA.
Create large networks.
The Modern Yeast Toolkit
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Two-Hybrid
Knockout library
GFP Fusion library
Overexpression library
 High
Copy
 Low Copy
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GST fusion library
Screening GFP Libraries
GFP Library
STRESS
Cntl -factor HU
MMS
Protein: RNR4
Control
-factor
HU
FIX and STAIN
IMAGE
Quantify changes in intensity
and location
Data from Samson Lab
Knockout Library and “BARseq”


Knock out strains have
unique molecular
barcodes that act as
finger prints.
By pooling all the strains
together, frequency of
each strain can be
determined by the
frequency of the barcode
in NGS experiments
Knockout Library and “BARseq”
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Experiments can be
done by looking at the
variations in frequency
of the pool after
changing the
environment of the
library.
ALL STRAINS
RICH MEDIA MINIMAL MINIMAL + AAs
SEQUENCE AND LOOK FOR
CHANGES IN FREQUENCY
The Future – Synthetic Biology

Key limitations of current
toolset
 Have
to create each strain
separately.
 Finite number of mutations
being created.
The Future – Synthetic Biology

Assembly of chromosomes
in vitro.
 Can
add any mutation
anywhere by replacing a
segment and reintroducing.
 Can create designer
chromosomes with complex
and unusual traits
 Do not require “carrier
markers”
Craig Venter, 2010
The End
Introduction to the
Genomics of Yeast
Sequencing Technologies
and how they are evolving
Introduction to Systems
Biology and modern Yeast
Genetics