ASSIGNING GENE FUNCTION BY EXPERIMENTAL ANALYSIS
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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. 5.21
“barcode tag” = 20-25 nt sequence that will uniquely identify deletion mutant is incorporated into construct
(so can detect by hybridization or PCR)
Fig. 5.30
Steinmetz Nature Rev. Genet. 5:190, 2004
Assaying molecular barcode tags in yeast pools
Microarray with complementarybarcode tag sequences for all yeast genes
In different environments (eg. drug D), which strains survive? competitive fitness in population?
Presence (abundance) of different mutant strains monitored by bar code tags
- yeast deletion strains with barcodes up & downstream of KanR gene
So if deletion of gene X is lethal under certain growth conditions … no PCR product
CP CP
Nature 418:387, 2002
- collection of 5916 gene deletion mutants
Only ~ 200 had lethal phenotypefor 6 growth conditions studiedGrowth properties on galactose
- most showed no major phenotypic effect
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
H. Dooner website, Waksman Institute of Microbiology, Rutgers U, New Jersey
“From 68 biolistic experiments, we produced 271 independent transgenic events…”
Bio-Rad “Biolistic particle delivery system”
(iii) RNA interference-short, antisense RNAs (21-25 nt length) in hybrid with specific mRNA triggers degradation
Fig. 5.23
“knock-down” of gene expression
Alberts Fig. 8-66
C.elegans
“Dicer” ribonuclease cleaves specific mRNA into short ds RNAs
T7
T7
Study of 2769 C. elegans genes on chromosome 1 (p.202-203)
- in 339 cases, saw detectable change in phenotype
Nature 408:325, 2000
Emb = embryonic lethal (226) Ste = sterile (96)Unc = uncoordinated (70)Pep = post-embryonic
Type of gene inactivated
2-cellstage
mature nematode
~ 660 genes required for early embryogenesis
2. Gene over-expression (gain-of-function)
- monitor phenotypic effect of high amount of protein
Fig. 5.24
- transgenic experiments using cDNA of protein of interest with strong promoter, high copy number vector…
Increased bone densityin opg transgenic mice
Simonet Cell 89:309, 1997
3. Gene alterationSite-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 mutagenesisusing 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
– galactosidase (blue colour)
Fig. 5.26
Use construct with regulatory sequences for “gene of interestupstream of reporter gene such as:
– green fluorescent protein (jellyfish)
lacZ
GFP
- can mutate regulatory sequences and monitor phenotypic effect…
- regulatory sequences for gene expressed in muscle precursor cells fused to lacZ reporter gene
Griffiths Fig. 14.27
Transgenic mouse embryo
2. Immunocytochemistry - fluorescently-tagged antibody directed against protein of interest to determine subcellular location
Fig. 5.27
Ab for mitochondrial DNA repair protein
Mol Biol Cell 16:997, 2005
HOW TO STUDY PATTERNS OF GENE EXPRESSION ON LARGE SCALE?
- to determine which sets of genes are transcribed incertain cell typedevelopmental stageenvironmental conditiondrug 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
Fig. 6.1
Ligate many fragments together & rapid sequencing of these concatemers
Ramskold PLoS Comp Biol 5:e1000598, 2009
Interpretation of these data?
see also Topic 6, slide 15
Example of “RNA-seq” data
TRANSCRIPT PROFILING WITH DNA MICROARRAYS
1. RNAs extracted from control and test cells (transcriptomes 1 & 2)
2. cDNA synthesis & labeling
3. Hybridize to microarray
4. Visualize hybrids
Fig. 6.3
5’cap AAAAAAAAAn
eg. for primer can use mixture of “anchored” oligo(dT)s with A, C or G in the 3’ position
3’ 5’
eg. laser scanning of fluorescence
DNA chip with genes of interest(eg. clones, PCR products, oligomer barcode tags …)
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
green = expressed at lower levels in testyellow = expressed at same level in both
red = expressed at higher levels in test than in control
Gibson & Muse Fig. 3.1
- laser scanning & ratio of fluorescence calculated
No drug present +drug
mRNAs for genes #1-3
AAAAn
AAAAn
AAAAn
AAAAn
AAAAn
AAAAn
AAAAn
RT
Red tag
TRANSCRIPT PROFILING WITH DNA MICROARRAYS
genes 1-3 on chip
- then cluster analysis to identify sets of co-regulated genes
“guilt-by-association”
- genes with related functions tend to have similar expression patterns
Transcriptome analysis during plant cell cycle
- examined 1340 cell-cycle modulated genes in tobacco
PNAS 99:14825, 2002
Some genes can give rise to more than one distinctive mRNA
Alternative splicing
mRNAs “SpliceArrays” (microarray)
- using junction-specific oligomers
Fig.6.5
Wang et al. Nature 456:470, 2008
Aside: How many human genes show alternatively splicing?
Some applications of DNA microarrays
2. Genotyping (SNPs)
1. Transcript profiling (expression analysis)
3. Drug discovery(eg identify potential drug targets by analyzingexpression profile in response to drug)