1. 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

2. “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

3. Steinmetz Nature Rev. Genet. 5:190, 2004 Assaying molecular barcode tags in yeast pools Microarray with complementary barcode 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 Kan R gene So if deletion of gene X is lethal under certain growth conditions … no PCR product CP

4. Nature 418:387, 2002 - collection of 5916 gene deletion mutants Only ~ 200 had lethal phenotype for 6 growth conditions studied Growth properties on galactose - most showed no major phenotypic effect Aberrant cell morphology

5. (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”

6. (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

7. 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-cell stage mature nematode ~ 660 genes required for early embryogenesis

8. 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 density in opg transgenic mice Simonet Cell 89:309, 1997

9. 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)

10. 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

11. 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 interest upstream of reporter gene such as: – green fluorescent protein (jellyfish) lacZ GFP

12. - 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

13. 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

14. 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”

15. SAGE – serial analysis of gene expression Fig. 6.1 Ligate many fragments together & rapid sequencing of these concatemers

16. Ramskold PLoS Comp Biol 5:e1000598, 2009 Interpretation of these data? see also Topic 6, slide 15 Example of “RNA-seq” data

17. 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 …)

18. 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

19. 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 test yellow = 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

20. No drug present +drug mRNAs for genes #1-3 AAAA n RT Red tag TRANSCRIPT PROFILING WITH DNA MICROARRAYS genes 1-3 on chip

21. - 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

22. 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?

23. Some applications of DNA microarrays 2. Genotyping (SNPs) 1. Transcript profiling (expression analysis) 3. Drug discovery (eg identify potential drug targets by analyzing expression profile in response to drug)