2581 Test 3

forward genetics approach

what genes are important for a process? phenotype to genotype approach

reverse genetics approach

what process is the gene important for. genotype to phenotype approach

forward genetics uses

mutagenesis to create a random pool of genome variants

two mechanisms of forward genetics

screen and selection

selection

takes cells mutagenized creating a random pool of gene variants and asks for the variant to survive under a set of conditions

screen

all variants in a random pool of genome variants potentially survive and identify a particular phenotype

genetic analysis requires

genetic variants

saturation screens

an attempt to identify as many genes whose products contribute to the process that you are studying as is statistically and technically possible

genetic screen for leucine auxotrophic yeast

haploid yeast cells, mutagenesis, random pool of dna sequence changes, compare leu+ and leu- media for which colonies grow

say i have 100 leu auxotrophic yeast mutants, does that mean there are 100 genes?

no, you can have multiple independent alleles in the same gene

complementation analysis

if two mutants alleles are independent mutations in the same gene, when making a diploid, are they complements

non-complementation

same gene, -

complementation

not in the same gene, +

haploid genetics

as soon as mutagenized are screened, important when allele is recessive, single generation, large number of variants to screen or select (10^6-10^8)

diploid genetics

mutagenized creates heterozygous diploid, need to create homozygous alleles for phenotype, several generations, small number of variants to screen or select (10^3-10^4)

maternal genetics

expressed until a later phase when information is expressed from the zygote

zygotic genetics

not transcribed until hrs to days after fertilization

maternal effect

maternally supplied transcripts program early development when the zygotic genome is not transcribed

maternal effect mutants

homozygous for lf allele, progeny shows the phenotype

zygotic screens

over three generations, dead embryo, 1/4 progeny homozygous for mutational change

maternal effect screens

homozygous mother, egg does not develop in fourth generation

mutational tagging

dna sequence variation tags the gene that you have identified as important

criteria for reverse genetics

need to be able to reintroduce dna into the organism

reintroducing dna mechanisms

transformation, injecting dna, transposon/viral mediated transformation, site specific recombination

transformation components

episome (plasmid), random insertion, homologous recombination

transformation steps

1. treat cells to make them competent to take up DNA
2. introduce episomes (plasmids)
3. random insertion into tissue culture cells
4. homologous recombination to incorporate marker with endogenous locus

episomes

dominant selectable markers and origin of replication

dominant selectable markers in bacteria

antibiotic resistance gene

dominant selectable markers in yeast

leu2 functional allele

injecting dna

inject dna into organisms with large cells generating random insertions

transposon mediated transformation

cut and paste mechanism, coding region is transcribed and translated, transposase protein binds to inverted repeats cutting it out of genome and moving it

components of transposon mediated transformation

transposase (trans acting) and inverted repeats (cis acting)

why separate the components of transposon mediated transformation

when together, transposons move

separated components of transposon mediated transformation

one encodes transposase with no inverted repeats, one is a plasmid with marker and gene you are trying to reintroduce flanked by inverted repeats

marker and gene only inserted when transposase is expressed

viral mediated transformation

incorporate gene and marker into viral genome, packed genome into viral particle, after viral infection gene is inserted into genome of the cell

site specific recombination

two recombination sites recognised by recombinase, recombination event, marker and gene inserted

uses of transformation/ transgenesis

functional complementation, misexpression and gain of function, knocking out genes

functional complementation

phenotype to gene, extract dna from wt yeast, fragment genome with restriction enzymes, clone them into yeast plasmid, transform yeast with episomes, look for particular phenotype

functional complementation (+/-)

\+ not essential for function

\- essential for function

misexpression and gain of function

requires ability to reintroduce dna, misexpression results in a phenotype

can use transposon mediated transformation for expression

knocking out genes

use homologous recombination, CRISPR

homologous recombination the marker

marker introduced into gene, marker selects for homologous recombination, insertion of the marker in the gene is the mutational event that disrupts the gene

knocking out genes reading it

screen strains for which gene is required for your process, each strain has a diff gene knocked out, see phenotype and know gene

how was RNAi discovered

in Petunia when they were trying to increase pigmentation, sectors of flower had no pigment

mechanism of RNAi

post transcriptional, ds RNA base pairs with mRNA which stops protein production

mechanism of RNAi using dicer

dsRNA to a mRNA rapidly digested by dicer into short 20 nt fragments, fragments recognised by AGO, creates RISC, RISC base pairs with mRNA and degrades it

lower level of protein expressed

major points of RNAi

1. specific mRNAs are targeted for degradation because of the complementarity of the RNA bound to AGO and the mRNA
2. RNAi mechanism is thought to have evolved as a mechanism that suppresses parasitic genetic elements (viruses, transposable elements)

discovery of microRNA (miRNA)

discovered in c. elegans because they are transparent and so divisions can be followed

heterochronic genes

control timing of developmental events

lin-14 lf

precocious (early) development of L2 lineage

lin-14 gf

suppression of L2 lineage and replacement with L1 lineage

lin-14

encodes a nuclear protein

lin-4

encodes a non-coding RNA, important for suppression of L1 lineage

lin-14gf mutants and lin-4

binding sites deleted

lin-4 RNA and lin-14

base pairs with lin-14 3’UTR

lin-14 mRNA and protein expression along lineages

mRNA expressed in all stages, protein only expressed at L1 stage

lin-4 miRNA expression across lineages

accumulates at L2

how are miRNAs made

transcribed by RNAPII, primary transcript requires processing to form pre-miRNA, pre-miRNA exported from nucleus and processed by dicer RNAse, miRNA associates with AGO, RISC forms

how many AGO members do humans have

8

AGO1

miRNA silencing

AGO2

RNAi mRNA cleavage

miRNA result

suppresses translation

RNAi result

degrades mRNA

tethering AGO to an RNA binding protein

Lambda N protein fused to AGO, control with mRNA no n protein binding site, experiment where N binds to b-box sequence

shows RNA components of AGO RISC complex guides proteins to specific RNA molecules

systematic RNAI screens in C. elegans

make copies of transcripts for each gene, two promoters surround the transcript and transcribe the cDNA into a dsRNA, c. elegans eats this e. coli, ds RNA is found in all cells, look for phenotype

defences against genomes

RNAi/miRNA, restriction modification, CRISPR

Bacteriophage

bacteriophage injects its DNA which takes over the host, all proteins make a virus particle which kills the cell by bursting it

restriction modification immunity

source of restriction enzymes, origin of recombinant dna technology

purpose of restriction modification immunity

protect from exogenous genomes

Restriction modification

phage injects dna, restriction enzyme chops phage dna, methylase methylates restriction enzyme recognition sites in genomic dna to protect it, unmethylated dna is digested

What does CRISPR stand for

clustered regularly interspaced short palindromic repeats

Cas

CRISPR associated

PAM stands for

protospacer adjacent motif

CRISPR array

gene that contains sequences that came from foreign invading genomes

CRISPR mechanism step: acquire

phage infects cell, cas binds to PAM adn cuts the genome releasing a fragment of DNA inserted into the genomic dna at a CRISPR array

CRISPR mechanism step: immunity

gene is transcribed to create pre-cr RNA and processed to release spacers homologous to past invading species, CRISPR forms hybrid with Tracer RNA, Cas9 recognises the complex and binds, if the phage comes again they can inactivate it

how does the cas9 complex inactivate phage genomes

finds PAM and unwinds DNA using guide RNA as a template and induces a double stranded break

why doesn’t cas9 cut the CRISPR array

there is no PAM

CRISPR guide RNA binds

20nt and 2nt in PAM, 44 bits of information

CRISPR reengineered

instead of a three component system (cas9+crRNA+trRNA), it is a 2 component system (cas9+chiRNA/gRNA)

CRISPR mechanism step: reengineered

cas9 binds to DNA and introduces a double stranded break which initiates mitotic recombination, at the same time dna with a selectable marker is introduced to create a mutation or bring another dna element, insertion of exogenous dna

how many areas in cas9 are important for ds breaks

2 areas

mutations in cas9

knock out ability to make a ds break

tethering with cas9

join other functions to a defective cas9 protein to inhibit transcription, activate transcription, or fluorescence

epistasis

assayed by comparing the phenotype of a double mutant organism with a single mutant organism

between phenotype a and b, if i see phenotype a

a is epistatic to b

epistasis: criteria for the two mutations

have related phenotypes, work on a pathway that makes a distinct decision, the two mutations have distinct/ opposite phenotypes

switch pathway

last step controls the phenotype since it is the most epistatic

negative interaction

flathead arrow

positive interaction

arrow

developmental genetics

application of genetics to understand/dissect the process of development

maternal information

egg, oogenesis

zygotic information

silent for a bit after fertilization, determines further stages of development

totipotent cell

can make any cell

outline of the stages of the choice (cells)

pluripotent cell, determination, determined cell, differentiation, differentiated cell

Christiane Nusslein-Volhard and Eric Wieschaus importance

where genetic dissection of Drosophila began, identified 120 genes important for determining the body plan (a-p axis)

body plan

where cells end up in the fully developed organism

cellular blastoderm

5000 cells, monolayer of cells at periphery of egg

genetic hierarchy regulating determination of the a-p axis

1. maternal coordinate genes (bicoid and nanos)
2. zygotic gap genes (hunchback)
3. pair-rule genes (fushi tarazu)
4. segment polarity genes
5. homeotic selector genes (antennapedia)

transition from maternal to zygotic in humans

first cleavage cycles