Transcript of gene expression - Université d`Ottawa

ASSIGNING GENE FUNCTION BY EXPERIMENTAL ANALYSIS
1. Gene inactivation (loss-of-function)
- mutate gene (“knock-out”) and observe change in phenotype
(i)
Deletion mutagenesis - eg. by homologous recombination
Fig. 5.20
“Deletion cassette” vector – “substituted” DNA can have selectable
marker, restriction sites, “barcode tags”…
Fig. 5.30
“barcode tag” = 20-25 nt sequence that will
uniquely identify deletion mutant is
incorporated into construct
(so can detect by hybridization or PCR)
Fig. 5.21
Assaying molecular barcode tags in yeast pools
- yeast deletion strains with barcodes up & downstream of KanR gene
In different environments (eg.
drug D), which strains survive?
competitive fitness in
population?
CP
CP
Presence (abundance) of
different mutant strains
monitored by bar code tags
Microarray with
complementary
barcode tag
sequences
for all yeast genes
Steinmetz Nature Rev. Genet. 5:190, 2004
So if deletion of gene X is lethal under certain growth conditions … no PCR product
Nature 418:387, 2002
- collection of 5916 gene deletion mutants
- most showed no major phenotypic effect
Growth properties on galactose
Only ~ 200 had lethal phenotype
for 6 growth conditions studied
Aberrant cell morphology
(ii) Insertional mutagenesis
Griffiths Fig. 14.18
Transposon tagging - if transposon inserts into gene
(or into regulatory sequences) = gene inactivation
Transposon tagging is “random” form of mutagenesis
- so prior knowledge of gene location not required
- many different alleles can be generated
“From 68 biolistic experiments, we produced 271
independent transgenic events…”
H. Dooner website, Waksman Institute of Microbiology,
Rutgers U, New Jersey
Bio-Rad “Biolistic particle
delivery system”
(iii) RNA interference
-short, antisense RNAs (21-25 nt length) in hybrid with
specific mRNA triggers degradation
“knock-down” of gene expression
T7
T7
C.elegans
Alberts Fig. 8-66
“Dicer” ribonuclease cleaves specific
mRNA into short ds RNAs
Fig. 5.23
Study of 2769 C. elegans genes on chromosome 1 (p.202-203)
- in 339 cases, saw detectable change in phenotype
Type of gene inactivated
Emb = embryonic lethal (226)
Ste = sterile (96)
Unc = uncoordinated (70)
Pep = post-embryonic
2-cell
stage
mature
nematode
~ 660 genes required for early embryogenesis
Nature 408:325, 2000
2. Gene over-expression (gain-of-function)
- monitor phenotypic effect of high amount of protein
- transgenic experiments using cDNA of protein of interest
with strong promoter, high copy number vector…
Increased bone density
in opg transgenic mice
Simonet Cell 89:309, 1997
Fig. 5.24
3. Gene alteration
Site-directed mutagenesis
- introduce specific point mutation at pre-determined position
(Michael Smith UBC, Nobel prize)
5’ …. ATG …. AAA TGT CCA …. TAA 3’
How to change TGT (Cys) codon to GGT (Gly) codon?
Design oligomer with mismatch to original sequence
3’ … TTT CCA GGT …. 5’
Anneal to gene (ss form) & generate copies
- using M13 phage system (p.156)
- using two-step PCR (p.157)
Site-directed mutagenesis
using PCR
- use oligomer with
mismatch as PCR primer
to generate product
differing from template
sequence at desired site
Fig.T5.2
HOW TO DETERMINE WHERE AND WHEN GENE IS EXPRESSED?
1. Transformation of regulatory sequences + reporter gene
Fig. 5.26
Use construct with regulatory sequences for “gene of interest
upstream of reporter gene such as:
lacZ – b galactosidase (blue colour)
GFP – green fluorescent protein (jellyfish)
- can mutate regulatory sequences and monitor phenotypic effect…
Transgenic mouse embryo
Griffiths Fig. 14.27
- regulatory sequences for gene expressed in muscle
precursor cells fused to lacZ reporter gene
2. Immunocytochemistry
- fluorescently-tagged antibody directed against protein of
interest to determine subcellular location
Ab for mitochondrial DNA repair protein
Mol Biol Cell 16:997, 2005
Fig. 5.27
HOW TO STUDY PATTERNS OF GENE EXPRESSION ON LARGE SCALE?
- to determine which sets of genes are transcribed in
certain cell type
developmental stage
environmental condition
drug treatment…
1. RT-PCR differential display
2. SAGE – serial analysis of gene expression (Fig. 6.1)
3. DNA microarrays
4. RNA-seq “Deep sequencing”
SAGE – serial analysis of gene expression
Ligate many fragments together
& rapid sequencing of these
concatemers
Fig. 6.1
Example of “RNA-seq” data
Interpretation of these data?
see also Topic 6, slide 15
Ramskold PLoS Comp Biol 5:e1000598, 2009
TRANSCRIPT PROFILING WITH DNA MICROARRAYS
DNA chip with genes of interest
(eg. clones, PCR products, oligomer
barcode tags …)
1. RNAs extracted from control
and test cells (transcriptomes 1 & 2)
2. cDNA synthesis & labeling
5’cap
3’
AAAAAAAAAn
5’
eg. for primer can use mixture of
“anchored” oligo(dT)s with A, C or G
in the 3’ position
3. Hybridize to microarray
4. Visualize hybrids
eg. laser scanning of fluorescence
Fig. 6.3
Potential pitfalls with microarrays (see p.170-171)
- if target DNA is saturated with
probe, hybridization signal
strength will not reflect mRNA
abundance
Fig.6.4
- if comparing 2 transcriptomes using 2 microarrays, data must be
normalized to ensure equivalent amounts of DNA on array, same
efficiency of probe labelling, same effectiveness of hybridization
conditions....
so better to use 2 types of fluorescent probes on one microarray
More efficient if transcriptomes 1 & 2
are labeled with different
fluorescent tags (eg “red” Cy3-dUTP
& “green” Cy5-dUTP)
- then mix cDNAs and
hybridize to microarray
- laser scanning & ratio of
fluorescence calculated
red = expressed at higher levels in test than in control
green = expressed at lower levels in test
yellow = expressed at same level in both
Gibson & Muse Fig. 3.1
TRANSCRIPT PROFILING WITH DNA MICROARRAYS
+drug
No drug present
mRNAs for
genes #1-3
AAAAn
AAAAn
AAAAn
AAAAn
AAAAn
AAAAn
AAAAn
RT
Red tag
genes 1-3 on chip
- then cluster analysis to identify sets of co-regulated genes
- genes with related functions tend to have similar expression patterns
“guilt-by-association”
Transcriptome analysis during plant cell cycle
PNAS 99:14825, 2002
- examined 1340 cell-cycle
modulated genes in tobacco
Some genes can give rise to more than one distinctive mRNA
Alternative splicing
mRNAs
“SpliceArrays” (microarray)
- using junction-specific
oligomers
Fig.6.5
Aside: How many human genes show alternatively splicing?
Wang et al. Nature 456:470, 2008
Some applications of DNA microarrays
1. Transcript profiling (expression analysis)
2. Genotyping (SNPs)
3. Drug discovery
(eg identify potential drug targets by analyzing
expression profile in response to drug)