Forward Genetics

Letting the Genome Tell You What Genes Are Required for the Biological

Process You are Studying

-Random screening as an unbiased appoach for gene discovery-Key entry point for determining molecular mechanisms

There is no substitute for loss-of function phenotype for finding out what your gene is doing!

A Little Genetics

Recessive allele: genotype needs to be homozygous mutant in order to see mutant phenotype

Dominant allele: have mutant phenotype even when heterozygous for mutant allele

Genotype: what alleles you have (heterozygous, homozygous, etc.)

Phenotype: what you look like (wild type or mutant for some trait)

Zygotic gene: genotype of embryo determines phenotype of embryo

Maternal effect gene: genotype of MOTHER determines phenotype of embryo

A-P polarity set up in egg chamber

bicoid mRNA oskar mRNA protein + nanos RNA

microtubule-based - +

bicoid, oskar and nanos are examples of genes acting in the OVARY that influence patterning of the EMBRYO

Therefore, the GENOTYPE of the MOTHER determines the PHENOTYPE of the EMBRYO

(Maternal Effect)

Examples:

Zygotic recessive (phenotype)m/+ X m/+: m/+ (wild type)

+/+ (wild type)m/m (mutant)

Zygotic dominantm/+ X m/+: m/+ (mutant)

+/+ (wild type)m/m (mutant)

Maternal effect recessiveMom Dad Embryo (phenotype)m/+ X m/+: m/+ (wild type)

+/+ (wild type)m/m (wild type)

Mom Dad Embryom/m X m/+: m/+ (mutant)

m/m (mutant)

Mom Dad Embryom/+ X m/m: m/+ (wild type)

m/m (wild type)

Types of MutantsHypomorph: Loss of function

Often recessive - genotype needs to be homozygous for mutation (m/m) to see phenotype

Amorph or Null: Complete loss of function(behaves like a deletion of gene)

Hypermorph: Gain of functionOften dominant - can see a phenotype even if genotype is only heterozygous (m/+)

Antimorph: Behaves stronger than null (e.g. dominant negative)

Neomorph: New function (e.g. gene now expressed in ectopic location)

Chemical Mutagenesis

EMS (Ethyl methane sulfonate): e.g. fliesENU (n-Ethyl-n-nitrosourea): e.g fish and mice

Chemically modify DNA bases to induce replication errorsPreferentially induce point mutations (but some small deletions)

Advantages:Most random of mutagensAlleles of different strengths

Disadvantages:Harder to identify lesion (clone gene)

Radiation Mutagenesis

X-rays and gamma rays most commonInduce double strand DNA breaks

-Deletions-Inversions-Translocations

Advantages:Often produce null mutationsEasy to identify lesion

(often just by looking at chromosomes)

Disadvantages:Usually take out multiple genes

Insertional Mutagenesis (Transposon or Retroviral)

Insertion of transposon or viral sequences affects gene functionCan control transposon jumping by separating transposase

from transposon

Advantages:Easy to identify lesion, clone gene

(gene is “transposon tagged”)

Disadvantages:Non-random integration/mutagenesis

-affects target distribution-affects allele strength

Currently being done in flies, fish and mouse

Traditional Screens for Recessive Mutations

Nusslein-Volhard and Weischaus Nobel Lectureshttp://www.nobel.se/medicine/laureates/1995/

EMS

Make mutant sperm

Make heterozygousmutant individuals

Make mutant brothers and sisters

Cross heterozygousbrothers and sisters

to make homozygousmutant offsprint

paired knirpsWT

Summary of X, 2 and 3

Nusslein-Volhard and Weischaus Nobel Lectureshttp://www.nobel.se/medicine/laureates/1995/

Large Scale Forward Mutagenesis in Zebrafish

Nusslein-Volhardand Dreiver Labs1996

Problems with traditional homozygous mutant screens:-Genes are not all equally mutable

-some are small targets or tough to induce loss of function(e.g. microRNAs)

-Genes can be redundant-knocking out one copy doesn’t always give phenotype

-Genes are pleiotropic-if embryo dies before your process “happens”, can’t tell if that gene is required

Sensitized Genetic Screens: the sevenlessTS screenSimon, Bowtell, Dodson, Laverty and Rubin, 1991

sevenless: receptor tyrosine kinase required for R7 specification

Problem: sev is specific to eye, but downstream RTK pathway common to all RTKs and therefore embryonic lethal

How do you identify sev pathway components (and therefore components of all RTK signaling)?

Sensitized Genetic Screens: the sevenlessTS screenSimon, Bowtell, Dodson, Laverty and Rubin, 1991

sevTS: Making flies “on the edge”22.7°C R7 mostly present--screen for dominant enhancers of sev

-R7 now lost24.3°C R7 mostly absent--screen for dominant suppressors of sev

-R7 now restored

e.g. rasras-/ras+ = wild typeras-/ras- = uninformative dead embryo

sevTS 24.3°C with ras-/+ = no R7 cellNormally ras is recessive but now behaves as a dominant enhancer of sevTS

Sensitized Genetic Screens: generic eye screens

Express programmed celldeath gene in eye

Look for suppressors/enhancers of programmed cell death pathway

Mosaic ScreensGenetic mosaics can be created by mitotic recombinationinduced by X-rays or a site-specific recombinase

FLP/FRT in Drosophila: Golic and Lindquist, 1989Essential reagents: Xu and Rubin, Chou and Perrimon

Treisman Lab

Forward/Reverse Genetics: Whole genome RNAi Screens

Cell Culture: Transfect or bathe cells with dsRNACell Culture: Lentiviral vectors expressing dsRNAC. elegans: Feed worms bacteria expressing dsRNAFlies: Inject embryos with dsRNAFlies: UAS-shmiRNAs for every gene in genome

Advantages: -Save gene identification step

Disadvantages: -Depends on genome sequence/annotation-Efficiency of RNAi is variable-Issues with delivery of dsRNA trigger

Positional CloningFinding a needle in a haystack

First fly gene: Ultrabithorax 1979

First human disease genes: chronic granulomatous disease 1986Duchenne muscular dystrophy 1987Cystic fibrosis 1989

(approx. 1200 disease genes now cloned)

First fish gene: one-eyed pinhead 1998

Finding a single bp change in 3.6 x 108 (Fly)3.4 x 109 (Fish)6.0 x 109 (Human/Mouse)

How do you find your gene?

Identify a transposon-induced allele of your gene:transposon then “tags” genomic region of interest

Find a genomic lesion (deletion/inversion/transposition) allele of your gene:

breakpoints in genome identify region of interest

For point mutants: meiotic mapping and positional cloning

Genetics 101

aa

bb

; ++

++;X

a+

b+;

aa

bb

;X

aa

bb

; +a

bb

; aa

+b

;+a

+b

;

1 : 1 : 1 : 1

Mendelian Inheritance

Parentals = Recombinants

Linkage

a ba b

+ ++ +X

a b+ +

a ba b

X

a ba b a b

+ + + ba b

a +a b

Parentals > Recombinants

RecombinationFrequency

# RecombinantsTotal X 100 =

% RecombMap UnitsCentiMorgans (cM)

=

160 40

40/200 x 100 = 20 cM

Meiotic MappingThree genes: a, b, c

a ca c

+ ++ +X

a c+ +

a ca c

X

a ca c a c

+ + + ca c

a +a c

Parentals Recombinants

c bc b

+ ++ +X

c b+ +

c bc b

X

c bc b c b

+ + + bc b

c +c b

Parentals Recombinants

190 10

10/200 x 100 = 5 cM

170 30

30/200 x 100 = 15 cM

a c b

5 15

a c b

To Clone Gene C

1) Link meiotic map to physical map (DNA)2) Identify markers and map crossovers to define limits of C3) Identify genes within this region4) Determine which gene is C

Markers for Meiotic/Physical Mapping“Classically” done using visible dominant and recessive mutations

-Low density of useful markers-Less rooted in physical map

Can improve the density of visible markers using transgenes e.g. w+ transposons in flies

Modern methods directly assess DNA polymorphismsRandom markers

e.g. Randomly Amplified Polymorphic DNA (RAPDs)PCR w/ primers of random sequence, get few random productsPresence or absence of product can depend on as little as single bp changeDon’t require prior knowledge of genome sequenceAllows “entry” into physical map (identifies STS near gene of interest)

Simple sequence length polymorphisms (microsatellite DNA, e.g. CA repeats)PCR shows small polymorphic changes in repeat numberAdvantage: easy to analyzeDisadvantage: Not enough (low density)

Single nucleotide polymorphisms (SNPs)Advantage: Maximum possible densityDisadvantage: Can be difficult to assay

SNPs alter oligo annealingSuitable for microarrays

Single nucleotide “mini-sequencing”Suitable for microarrays

SNPs alter oligo annealing+/- PCR product

Syvanen, Nat Rev Gen 2001

Afymetrix offers SNP Chips that can genotype 10-50,000 SNPs

Also,-Single strand conformation polymorphisms (detected in gels)-Denaturation HPLC-Mass-spec DNA sequencing

Sounds easy but…-Compare mutagenized chromosome with interesting phenotype to control, parental chromosome that was isogenized before screen

-Found 165 sequence changes on third chromosome.

-Could only verify 103 (some false positives). Others likely not found (false negatives) since not all regions have good sequence.

-Of these, 11 made changes to ORFs. Therefore, still some work to figure out correct gene.

The First Association Between the Meiotic and Physical Maps

http://avery.rutgers.edu/WSSP/StudentScholars/project/archives/onions/rapd.html

RAPDs

Allele 1: products A and B

Allele 2: Change in site 2No product A

AB

Bulk Segregant Analysis: Look for Linkage (lack of recombination)

Mutant = mPolymorphic Markers = a1, a2, b1, b2

Start with mutation heterozygous in strain 1Use strain 2 as polymorphic mapping strain

a1

m

b1

a1

+

b1

a2

+

b2

a2

+

b2

Strain 1: m/+ Strain 2: +/+

X

a1

m

b1

a2

+

b2

X

a1

m

b1

a1

+

b1

X

Sort mutant vs. wt ‘brosMake DNA from pools

Mutant ‘bros

a1

m

b1

a1

m

b1

a1

m

b2

a1

m

b1

wt ‘bros

a1

+

b1

Any combo

a1

m

b1

a1

m

b1

a2

+

b2

a2

+

b1

b is mixed 1 and 2, therefore unlinked to ma is always (or mostly) 1, therefore linked to m (few recombinations)

Backcross to Parental Strain 1

-Bulk segregant analysis identifies 15AH and 20K as “close” to oep

1) Identify closely linked polymorphic markers

oep

Now look for recombinants between closely linked marker and gene

Mutant = mMarkers (alleles) = a1, a2, b1, b2

a1

m

b1

a1

+

b1

a2

+

b2

a2

+

b2

Strain 1: m/+ Strain 2: +/+

X

a1

m

b1

a2

+

b2

X

a1

m

b1

a1

+

b1

Backcross to Parental Strain 1

X

Examine INDIVIDUAL offspring

= individual meioses (gametes) of parentsa2

m

b2

a1

m

b1

-Look for recombination between close marker and gene -Screen 3100 “meioses” to find rare recombinants-Save Recombinants: can go back and analyze later with new

markers to further define WHERE recombination took place and therefore limits of WHERE oep can be!

-Bulk segregant analysis identifies 15AH and 20K as “close” to oep-Analyze DNA from 3122 INDIVIDUAL mutant ‘bros

-find 1 recombinant b/w 15AH and oep0.03 cM = 18 kb (IF 600 kb/cM)

-find 5 recombinants b/w 20K and oep0.16 cM = 96 kb

Now close enough to go after DNA in region(link meiotic map to physical map)

1) Identify closely linked polymorphic markers

oep

Use 15AH and 20K markers to gain “entry” into genomic region-Make probes using RAPD PCR bands from each-Probe genomic library to isolate clones 134 and 32-Use ends of these to isolate contiguous clones (“walk”)-Stop when two directions of walk meet

2) Create Physical Map of Region (Genomic “walk”)oep

If genome sequence is available, don’t have to walk since you know sequence of interval between markers

Clone 134F10 failed the “deletion test”-see if all of clone is missing in deletion of oep genomic region-if not missing, then some of clone’s DNA is from other region-suggests clone is chimeric (contains different parts of genome)-would be disaster to continue “walking” from chimeric clone

could jump to entire new (irrelevant) region or new chromosome

2) Create Physical Map of Region (Genomic “walk”)oep

3) Recombinant Fine Mapping

-Subclone 14 and 240 into cosmids-Use ends to make STS’s (need spaced sequence info)-Use sequence to identify additional polymorphic markers

SSCPs and CAPS-Go back to previously identified recombinants (1 on left, 5 on right)-Use new markers to map recombination eventse.g. 46T7 has allele of “strain 2”, so recombination is b/w 46T7 and oep

-Therefore oep is between 46T7 and 32T7

oep

4) Identify the Gene

-Use genomic DNA to probe cDNA library from stage of interest (223 cDNAs!)-Find out that these represent 13 “classes” of cDNA-Use in situ hybridization to see if any expression patterns fit predictions

oep

-Sequence mutant genomic DNA to identify potential bp changes that are responsible for mutant phenotype-Rescue with in vitro synthesized RNA

-Gold Standard for Gene ID: sequence point mutations and rescue mutant defect with transcript

How would things be changed today?

-High density polymorphism map produced so don’t need to search for polymorphic markers-Genome sequence being completed so don’t need to walk-Large scale EST (cDNA) sequencing so know transcript distribution and candidate genes (at least those that are correctly annotated!)-Can use morpholinos (RNAi in other species) to test candidate transcripts-Whole genome sequencing becoming helpful for identifying mutations

http://www.hapmap.org/

Goal: Determine the existing human haplotypes with a defined set of SNPsLong Term Goal: Associate haplotypes with phenotypes for

-cloning disease genes-understanding genetics of complex traits-pharmacogenomics