W10 Functional Genomics

what does the term functional genomics apply to

• the genome, transcriptome, or proteome

• the use of high throughput screens

• the perturbation of gene function

• the complex relationship of genotype and phenotype

forward genetics

• observable phenotype to gene

• based on searching for mutants with altered phenotypes

reverse genetics

• from gene to function

• mutate gene then observe phenotype

• used to assign function to unknown genes

importance of budding yeast to functional genomics

• have many genes that are orthologs of human disease

• genes that are essential for viability in yeast, in particular those lacking human homologs, have been proposed to be the targets for antifurncal drug development

homologous recombination

• occurs between segments of DNA molecules that have extensive sequence homology

site specific recombination

• occurs between DNA molecules that have very short regions of sequence similarity

how can gene inactivation be achieved

• disrupting a gene with an unrelated segment of DNA

• achieved via homologs recombination bw chromosomal copy of the gene and a second piece of DNA that has sequence similarity wth target gene

explain gene inactivation via deletion cassette

• deletion cassettes is made of a selective marker which is usually antibiotic resistance gene, a promoter sew, and 2 RE sites for molecular cloning of target seq

• can tell cells where disruption occurs bc they now express the antibiotic resistance gene so it will grow on agar medium with the antibiotics

PCR based deletion cassette

• doesn't need RE recognition sites bc PCR is used to add homologous sequence to the deletion cassette

• short regions of budding yeast sequence that are identical to those found upstream and downstream of the targeted gene are placed on each end of the marker gene through PCR

• this allows the gene from PCR product to be introduced into yeast cells and replace the targeted gene via recombination

selection vs screen

selection: growth condition that allows for selective propagation of genetically marked cells

screen: growth condition where both WT and mutant can grow but can be distinguished by phenotype

G418/ Geneticin

• amino glycoside antibiotic

• blocks polypeptide synthesis by inhibiting the elongation step in both prokaryotic and eukaryotic cells

• resistance to G418 is conferred by the Neo gene from Tn903 encoding an amino glycoside 3’-phosphotransferase

KanMX selection marker

• hybrid gene consisting of amino glycoside phosphotransferase from Tn903 under the control of transcription elongation factor

2 applications of the non-essential deletion library

1. the utility of barcoded deletion mutant

2. response to chemicals in growth medium

deletion cassette used in barcode strategy

• modified deletion cassette with 20 nucleotide barcode seq added to any as a tag for deletion mutant

• barcode flanked by a sequence that can be amplified via PCR

• this allows different deletion mutant to be grown together and their phenotypes can be screened in the same experiment

MMS (methyl methanesulfonate)

• a DNA alkylating agene

• carcinogen

• inhibits DNA synthesis at the N3-deoxyadenine location

transposon mutagenesis

• inactivation is achieved by insertion of a transposon into the gene

• normally transposition is rare but can use recombinant engineered transposon systems that change their position in response to external stimulus

cons of transposition strategy

• transposition is not sequence specific bur random

• if you need to disrupt a gene of interest you must screen through many candidates

pros of transposition strategy

• genes underlying interesting phenotypes can be clones easily using the flanking transposon as probe

synthesis of siRNA

obtained by cleave of a linear dsRNA precursor

synthesis of miRNA

obtained by cleavage of the dsRNA portion of a ssRNA that has formed a stem loop

processing pre-miRNAs

• pro-miRNA is a transcript with a 5’ CAP and a 3’ poly A tail (hairpin structure)

• pro-miRNA recognized by nuclear protein DGCR8

• DGCR8 associates with Drosha RNase to free the hairpin and form pre-miRNA

• this is exported from nucleus via exportin 5 ran

• in cytoplasm, dicer RNase processes pre-miRNA to make miRNA, and affects gene expression by pairing with mRNA which is cleaved via argonaute

what is RNA interference (RNAi) used to perform

gene inactivation

RNAi

series of natural processes by which short RNA molecules influence gene expression in living cells

what does RNAi do in genomics research

provides a means to silence expression of specific gene by targeting mRNA but not disrupting gene locus

drawbacks of RNAi

• does not completely eliminate gene function and most often yields a knockdown rather than a knock out - may not see phenotype

• siRNA has off target effects

• not permanent and most be maintained by siRNA

• in mammals artificial induction of siRNA results to interferon activation (signalling proteins that stimulate antiviral defence) which makes it difficult to differentiate phenotype

difference between RNA interference and miRNA

• miRNA pathway regulates endogenous protein coding gene expression

• RNAi serves as a form of innate immunity targeting viruses and mobile elements

gene inactivation via programmable nuclease

• nuclease is directed to a specific site and is programmed to make a double stranded DNA break of a gene of interest

• cut stimulates DNA repair mechanism innate to host cell name non-homologous end joint pathway (NHEJ)

• this is error prone

• the inactive is permanent, thus, true knockout

cleavage of DNA by Cas9 endonuclease

• cas9 nuclease is directed via gRNA

• gRNA binding site is upstream of 5’ NGG or '5’ NAG seq

• target sequence must be precise known (need to sequence)

• cuts are make, and this Cas9 is a component of the prokaryotic immune system (CRISPR)

genetic interaction

sometimes mutation in two genes produce a phenotype that is surprising in light of each mutants individual affect

epistatic miniarray profiles (EMAP)

• quantitate measurement of phenotype

• includes hypomorphic alleles

SGA

• systematic cataloguing of the phenotypes of yeast double mutants / genetic interactions

EMAP

• Bioinformatic analysis of genetic interaction data sets obtained form SGA

• Hierarchal clustering of genes based on the similar patterns of genetic interactions