Genetic Analysis Genetic techniques to determine the role of genetics in phenotypic variation Mutational Dissection Ch 12 Regulation of Gene Transcription Ch 13 From Gene to Phenotype Ch 14 Example Genetic Analyses Regulation of Cell Number (Cancer) Ch 15 Genetic Basis of Development Ch 16

Mutational Dissection Ch 12 Make and study mutants in order to determine the role of wild type gene Mutagenesis random versus directed versus mutant phenocopies somatic versus germline genetic screens versus selections Mutant analysis/characterization mutant classifications inference of gene function 1. How to make and identify relevant mutants: A) What’s the biological process? Is it essential for viability or reproduction of the individual? - will you be able to study complete loss of function or dominant mutations? - how will you study partial loss of function mutations? - which experimental organism is the best choice for this study? B) Is the organisms haploid or diploid (polyploid???) - how will you detect recessive, loss of function mutations? - how do you keep stocks of strains carrying recessive lethal mutations? 2. What to do with the mutants to learn about gene function

Random (General) Mutagenesis How to make the relevant mutants Random Mutagenesis (general Mutagenesis): Which genes are important for a specific biological process? You want to know all the genes involved in a specific biological process Examples: Molecular Cell Biology: Which genes are involved in protein secretion and what does each gene do? Metabolism: Which genes are involved in nitric oxide synthesis and what does each gene do? Developmental Biology: Which genes are required for limb development and what does each gene do? Genomics: What is the function of each and every gene in a genome?

Natural or engineered Random Mutagenesis: What type of mutations best suits your purpose? Point Mutations: - includes all possible mutant classes - many mutations will not be detectable by phenotypic assays - good when you are looking for all types of mutations Insertions: - essentially always loss of function - good when you specifically want loss of function mutations or you need to tag the gene that is mutated to facilitate cloning - can you create TN insertion mutant s in the organism you are studying? Chromosomal rearrangements: - typically affects more than one gene - good for cytogenetic mapping and testing the function of chromosomal segments

When you want to detect every gene involved in a process, how do you know when you’re done looking? Saturation Mutagenesis - every gene identified by a mutation Some genes are smaller “targets” than others Target size - gene size (number of bps in regulatory, splicing, and coding sequences) - percentage of bps that are important for gene function - percentage of bps that can be changed in a way that makes the mutation detectable Saturation? - how many mutations have you identified in each gene that was detected?

X-rays are mutagenic Choose Bar eye female Assay for Absence of males H. Muller’s work to demonstrate that X-rays are mutagenic Devised a genetic screen to detect lethal mutations on the X-chromosome - recessive lethal X-linked mutations known - his screen identified recessive X-linked mutations by the absence of male progeny in the F2 generation after Mutagenesis of a male He constructed a Balancer Chromosome by standard crosses - X chromosome called ClB Cross-over suppressor ( two inversions) recessive Lethal mutation (loss of function of essential gene) Bar eye mutation (dominant mutation, easily observable phenotype) Inversions guaranteed that the original mutations induced in the X chromosome of the male fly stayed with THAT X chromosome in all crosses Recessive lethal mutation guaranteed that any viable male observed must be carrying the X chromosome originally subjected to Mutagenesis Bar eye allowed females that carried CIB to be detectable Assay for Absence of males Keep white eyed females as the stock

Directed (targeted) Mutagenesis How to make the relevant mutants Targeted Mutagenesis (directed Mutagenesis): What is the phenotype of individuals carrying specific mutations in a gene? You ask about the function of one or more specific genes Examples: - mouse gene is similar to human gene associated with a disease - is the mouse gene also involved in a similar process? - identify a protein associated with a process - does mutation of the gene encoding that protein affect the process - protein is present on ribosomes - is it important for translation? - protein binds to a hormone - is it required for hormone function? - functional Genomics - what is the function of every gene in a genome?

Gene knock-out or knock-in experiments Knock-out as before in mouse knock-out experiment - mutant allele contains selectable maker - double cross-overs in yeast and other fungi common enough to detect by screening transformants - organisms like mouse need a second selectable marker to select for double crossovers Knock-in experiments create strains with specific mutant alleles (not loss of function) - done in two steps - create a knock-out strain using a selectable marker that can be selected FOR and selected AGAINST - yeast example is URA3 select for: uracil prototrophy (transform to URA3+) select for 5-flouro-orotic acid resistance (transform to ura3-) - transform a ura3 strain with knock-out construct by selecting for URA3+ - transform knock-out strain with knock-in construct by selecting for ura3-

Creation of specific mutant alleles in vitro (to be used in knock-in experiments) In vitro, site-directed Mutagenesis Wild type gene cloned in plasmid that can produce ssDNA Oligonucleotide sequence matches wild type except for desired mutation DNA synthesis and ligation creates a dsDNA heteroduplex Transformation and replication in E. coli produces colonies containing wild type or mutant copies of gene Selection against wild type – make DNA in mutant strain that Makes excess UTP and reduced levels of dTTP lacks uracil deglycosylase ssDNA template contains U instead of D Transform DNA after synthesis into wild type E. coli Destroys UTP containing strand and uses the mutant stand to make dsDNA Only mutant strand is replicated and present in transformants

Other in vitro site directed mutations PCR procedure creates mutations in two steps: 1st step is related to oligonucleotide-mediated site-directed Mutagenesis PCR product contains mutation at one end Use this product as a the primer for a second PCR reaction (“long primer”) Complete PCR product includes entire gene carrying the desired mutation

Mutant Phenocopies: Complete loss of function of the GENE PRODUCT without mutation of the GFNE! Antisense RNA and dsRNAi (RNAi) principle: introduce RNA into cell/organism that inhibits the function of one specific mRNA Lack of mRNA = lack of gene product = loss of function of gene product but the gene is intact Antisense RNA is complementary to the mRNA strand Hybridization lead to either inhibition of translation or degradation - Antisense RNA is thought to be needed in a one to one stochiometry with mRNA (not catalytic, and not persistent) dsRNAi = mechanism is both catalytic and persistent! Introduction of dsRNAi into one cell can prevent expression of a gene in its daughter cells (and their daughter cells…)

Mechanisms for introduction of dsRNAi into cells/organisms: Synthesize in vitro and inject or transform Introduce transgene with the gene sequence present as an inverted repeat - “spacer” DNA in between repeats - Stem loop RNA forms, converted into linear dsRNA in vivo Introduce transgene with promoter on both ends - Make both strands, they hybridize to form dsRNAi

More dsRNAi produced by Products of digestion, or By RNA-dependent RNA Polymerase Formation of small inhibitory RNAs by RISC = RNA-induced silencing complex Both sense and Antisense RNAs inhibit gene expression Ds RNA works best! Worms can be fed bacterial expressing dsRNAi and all cells in the worm are silenced and even F1 progeny are silenced!!! RISC is an endogenous complex involved in gene silencing in invertebrates and plants (and probably more organisms) Targeting complex helps hybridization of small Antisense RNAs to mRNA Exonuclease destroys mRNA More dsRNAi produced somehow…

Mutant Phenocopies Use “chemical genetics” by identifying chemicals that bind and inhibit the protein of interest Study chemical protein interaction in vitro or in vivo In vivo: express proteins in yeast bacteria where activity can be assayed and add chemical to yeast/bacterial cultures In vitro: use protein in biochemical reactions and add chemicals to reactions Use chemical identified above to treat cells/organisms to phenocopy loss of function mutation

Genetic Selections versus Genetic Screens Selection: only the mutants you are interested in survive Screen: all survivors of Mutagenesis survive and then you do experiments to determine which carries mutations interesting to you

Example Genetic Selections: Select for Revertants to wild type phenotype Original mutation was a forward mutation (abnormal gene function), reversion mutation is a back mutation (to a more normal gene function) Easiest when forward mutation inhibits growth (auxotrophic or lethal) Types of revertants: - Intragenic revertants: back mutation is in same gene as forward mutation. Cross revertant to wild type: all progeny are wild type - Extragenic revertants: back mutation is in a different gene. - informational suppressor: - nonsense suppressor: in tRNA gene that allows amino acid to be inserted at the site of a nonsense codon - suppressor mutation in interacting protein - proteins A and B form AB in order to function - forward mutation of gene A to A*; A* can’t bind B - suppressor mutation in B to B*; B* can now bind A*; A*B* functional, wild type phenotype - cross of revertant due to extragenic suppressor to wild type: ¼ progeny are mutant

Genetic Screens for Germline Mutations: In haploid microbes, all nuclei are part of germ line. In haploids, recessive and dominant mutations are expressed immediately Both types of mutations evident in decedents from mutagenized strain

Genetic Screens of Germline mutations In diploids, mutation is not expressed until at least F1 progeny X-linked recessive and autosomal dominant evident in F1

Genetic Screens of Germline mutations In diploids that self-fertilize (plants), recessive mutations are detectable in F2

Genetic Screens of Germline mutations In diploids that cannot self cross, an extra cross is necessary, mutations not evident until F3 generation F1 get progeny heterozygous for different induced mutations Each F1 is crossed to WT to generate male and female F2 individuals heterozygous for same recessive mutation F2 siblings crossed to make homozygous recessives (1/4 of progeny, if non-lethal)

Specific Types of Genetic Screens: For auxotrophic mutants in bacteria or fungi Assay for mutations by looking for inability to grow on certain media.. Purpose: - to study microbial physiology to look for microbial enzymes or pathways that are relevant to other organism (humans) example: human genetic disorder Alkaptonuria (AKU) by Garrod First heritable disease identified (1908), but gene only recently cloned (1995) Human gene was cloned by identification of Aspergillus nidulans gene, then by homology of Aspergillus gene to human DNA sequences Example procedure: Isolate appropriate auxotrophic mutant Clone gene from genomic or cDNA library by complementation of auxotrophy Sequence fungal gene Search human sequence database for related gene

Genetic Screen for developmental abnormalities (not lethalities) Morphological Screens If mutations are lethal, have to keep them heterozygous, mate two heterozygotes, and observe early stages of development in homozygous progeny to determine phenotype Or, for lethal mutations, can use modifier mutation strategy to identify genes involved in a process…

Example of modifier mutant strategy: screen for genes important for eye development in Drosophila 800 facets with multiple cells per facet Mutations can effect size of eye…

Modifier Screen starts with a mutant that reduces eye size Mutate and look for individuals (F1 for dominant; F3 for recessive) that have either smaller or larger eyes Can find mutations in genes that are important for eye and embryonic development Mutations must be other than complete loss of function Recessive, hypomorphic mutations (partial loss of function) due to haplosufficiency Dominant: hypermorphic, null, or neomorphic mutations

Mutations used to distinguish between loss or gain of function mutations Cross new mutant to strains carrying deletion or duplication of the same gene Dominant hypermorphic mutations that cause increase in gene activity (MUT/+) tell hypermorphic mutations become more severe when increase copy number of the gene (MUT/+/Dp+) phenotype become less severe when copy number decreases (MUT/Df) Dominant null (loss of function) mutations – due to haploinsufficiency (mut/+) Tell that mutation is null because phenotype is less severe when increase copy number of the gene (mut/+/Dp+)… opposite if (mut/Df) Dominant neomorphic mutation cause gene to have new activity (new biochemical activity or expressed in cell types not normally expressed (Ectopic expression) Tell neomorphs because phenotype doesn’t change with gene dosage

Complementation analysis. How many different genes did you identify in your screen/selection? Genes referred to as complementation groups Recombinational mapping of recessive mutations cross mutants to each other For haploids: If in the same gene or tightly linked genes, few if any wild type progeny Make diploid, if wild type = different genes; if mutant = same gene If in different genes, ¼ of progeny = wild type For diploids, cross heterozygotes: For mutations in same gene, ¼ of progeny = mutant For mutation is different genes, all progeny = wild type For dominant mutations – map the mutations, determine if any are linked to each other, eventually have to clone the mutant alleles to see if they are same or different