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)