Concepts in modern genetics PDF

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Wintersemester, Dozenten: Yves Barral, Alex Hajnal, Daniel Bopp, Olivier Voinnet...

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CONCEPTS IN MODERN GENETICS HS 2019

Kashish Mallick [email protected] |

CONCEP TS IN Note: This summary covers quite a lot and is mainly based on the self-study chapters online. It should serve as a good and extensive repetition of the course material. However, for the exam I recommend you focus on concepts (rather than concrete examples), question sets and lecture material. I tried to include concepts from the lecture here as well, but I cannot assure completeness. Also, “s. workbook” refers to the scanned notes at the end of the summary.

The Power of Yeast 2 The Relationship between Mutation and Phenotype................................................................................................................4 Genetic Screening: Isolation of Yeast Mutants...........................................................................................................................8 Genetic Screening: Examples...................................................................................................................................................11 Gene Mapping......................................................................................................................................................................... 14 Genetic Identification..............................................................................................................................................................16 Gene Editing............................................................................................................................................................................ 18 Genetic Interactions................................................................................................................................................................ 19 Non-Mendelian Inheritance..................................................................................................................................................... 22

Classical Forward Genetic Screens in Invertebrates

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Screening of the F1 Generation...................................................................................................................................28 Revisiting Genetic Interactions.....................................................................................................................................32 Reconsidering Gene identification using Novel Mapping Strategies...............................................................35

Reverse Genetics Technologies Creating of Transgenics...................................................................................................................................................44 Reverse Genetics in Invertebrates..............................................................................................................................46 Gene Targeting in Mice....................................................................................................................................................50 Gene Editing.......................................................................................................................................................................53

Stepping into sRNA Biology

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Identification of miRNAs and Targets................................................................................................................................. 62 miRNAs in Animal Development and Disease..................................................................................................................64 miRNAs in Plants.................................................................................................................................................................. 66 siRNA-mediated Gene Silencing in Plants........................................................................................................................68 Beyond Mainstream Views..................................................................................................................................................70 Genome-scale, single-cell-type resolution of microRNA activities within a whole plant organ................................................71

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The Power of Yeast Why Yeast? technical advantages: o understand complex biological systems in a simple organism (simplest eukaryotic!) as model o cell division every 90 minutes; fast growth (24 hours in humans), no ethical dilemmas o robust towards environmental conditions; can be frozen / stored at room temperature for years o growth possible in liquid cultures as well as on plates o analysis possible of large-scale production as well as single clones o culture media defined so reproducibility of experiments higher than mice / humans o culture media defined so analysis of effects of depletion of certain media factors easy  creation of selective conditions to observe specific mutants o duplication easy  replica plating technique (s. below) biological advantages: o high rate of homologous recombination; can be hijacked to integrate exogenous DNA; study mutations, examine mutant dominance o

chromosome replication via autonomously replicating sequences (ARS) which contain

ORIs If ARS is present the DNA is replicated within the nucleus whether it is part of the chromosome or not. Plasmids carrying external DNA can be inserted into the yeast cell when technically created with ARS.  geneticists can easily mutate / manipulate gene expression and study resulting effects  Saccharomyces cerevisiae has the most advanced selection of genetic tools o reproduction possible asexually as well as sexually (s. below); haploid / diploid stages useful for different observations and experiments: haploid stage beneficial for genetic analyses because recessive traits always manifest, this means mutations can be isolated and result in phenotype, ergo recessive traits diploid stage beneficial for maintaining mutations and combine alleles and determine which traits are recessive / dominate or how they interact with each other o yeast shares a high degree of conservation on the level of the amino acid sequence & protein function with more complex eukaryotic organisms; can serve in screens for 2

CONCEP TS IN human diseases; (highly) applicable results especially because of separation of somatic and germline cells. Depending on environmental conditions and on details of genotype yeasts are in haploid or diploid state. If diploid, then reproduction can be mitotic or meiotic, the latter would lead to two haploid cells. If haploid, then reproduction can be mitotic or sexual (fusion), the latter would lead to one diploid cell. Meiosis is triggered by starvation and gives rise to spores / buds = haploid cells in a dormant state, which are resistant to harsh environmental conditions.

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CONCEP TS IN About Yeast o o o o o o o o

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belong to the kingdom fungi, to the group ascomycetes have a cell wall for mechanical stability, protection (advantage: immune to viruses, disadvantage: cellular migration difficult) can easily be moved by water or wind (like most unicellular organisms) little modification by horizontal gene transfer; fairly stable (may be the reason why rate of homologous recombination is so high; low risk of foreign DNA inserted) the DNA of yeast is 50x denser than that of humans (1 gene every 2 kb storage) most of the in total 6600 genes do not contain introns, distributed among 16 chroms highly conserved genes and pathways (f.e. replication, recombination, metabolism, …) symbiosis: yeast agriculture, i.e. active cultivation of yeast cells, practiced by ants, beetles (in their guts probably to use an enzyme that helps digest plant cell wall), some insects – the latter play an important role as vectors for spread of yeast colonies by transporting them to fruits, and for humans; production of bread, alcohol, flavoring of some cheeses (fermentation) pathogenicity: can cause infections, f.e. Candida albicans (actually an endosymbiont) can invade and cause disease, if mucosal barriers are disrupted or the immune system is weak

Besonderes: C. albicans is a polymorphic fungus that can grow as yeast or as pseudohyphae (long branching filaments that resemble fungal hyphae) depending on its environment; at low pH (< 6), C. albicans cells predominantly grow in the yeast form, while at a high pH (> 7), hyphal growth is induced. Both growth forms are most likely important for pathogenicity, with the hyphal form being more invasive, while the smaller yeast form is probably primarily involved in dissemination (spreading). Temperature also plays a role.

Mating Type Determination in Yeast (S. cerevisiae) In other words, gender determination, but instead of male and female, they are either MAT-a or MATalpha and therefore either produce a-factors or alpha-factors which are mating / sex-specific pheromones that signal their presence to the opposite pheromone and mating type. More precisely, a-cells respond to alpha-factor by growing a mating projection, known as a shmoo, towards the source of the alpha-factor which is the alpha-cell. The alphacells respond to the a-factor in a similar manner. The difference between a- and alpha-cells are determined by activation & repression of different genes (s. workbook). These differences involve: 1. activation of the respective pheromone type 2. repression of the opposite pheromone type 3. activation of the receptor for the opposite ph. The genes present in the respective locus MAT encode regulators (TFs) of genes that effect production of the structures / factors mentioned (1-3). The two different MAT alleles differ by 700 base pairs of sequences. Not only do we have to think about the mating type, but also whether the yeast cell is in its haploid / diploid state; the n /2n determining genes are connected to mating type determinants; s. Table 1 4

CONCEP TS IN Btw: upon undergoing meiosis four haploid spores are produced and packaged together in an ascus (a kind of sac) which builds tetrad.

Switching of Mating Types (S. cerevisiae) Why? Colonies strive to become diploid because of the evolutionary advantage but problems could arise if there is only 1 mating type present in a certain colony. This explains the advantage of homothallic organisms (organism with both male and female reproductive structures in the same thallus [Pflanzenkörper]) where this is never the case. S. cerevisiae are able to switch their mating type such that the subsequent mating of cells to the opposite mating type enables yeast cells to selfdiploidize to MATa/MATalpha. (In research this is switched off for stable propagation of haploid cells.) How? Yeast cells have an additional silenced copy of MAT-a / MAT-alpha alleles at a different locus: HML = Hidden MAT Lefapproximately 200 kb away from the MAT gene loci HMR = Hidden MAT Right approximately 100 kb away from the MAT gene loci There is a region RE which promotes recombination with HML which is expressed in MATa (default). The same region is repressed in MAT-alpha by alpha2 (s. workbook). That is why MATalpha recombines with the next nearest homologous region which is HMR. This reinsures the switch to the opposite type. Copies silenced by protein interactions with sequences which lead to short regions of heterochromatin, which are not transcribed so they do not interfere with the allele present. HO gene encodes  DNA endonuclease The HO gene is switched off in research for hetero. DNA endonuclease cleaves specifically at  MAT locus cleaved MAT locus attracts  DNA exonucleases DNA exonucleases degrades DNA from both sides  gap formed at MAT allele gap needs to repaired  HMR / HML copy inserted to fill missing info (homologous recombination)

The Relationship between Mutation and Phenotype Forward Genetics: from mutant phenotype (abnormalities) to genes identification (classical approach) It’s about understanding the wild-type by observing mutant phenotypes and tracing back to the gene. Reverse Genetics: from gene (product) modification to identification of changes in phenotype

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CONCEP TS IN  Only possible since genome sequencing exists, so identification, cloning and mutating possible.

Types of Mutations Classification can either depend on the effect on 1. the DNA sequence; point mutation or chromosomal mutation (s. above and beside) 2. the function of a gene; conditional, loss/gain-of-function, null, dominant-negative, suppressive

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CONCEP TS IN Conditional Mutation mutation only phenotypical under certain conditions; restrictive conditions  phenotypical effect permissive conditions  no effect Examples:  If a mutation is temperature-sensitive the restrictive condition could be high temperature while the permissive condition is low temperature. This mutation generally leads to loss of function because the temperature-sensitive mutation destabilizes the fold of the mutant protein.  If a mutation that leads to the loss of ability to synthesis amino acids, whether the mutation has an effect on the phenotype or not depends on if the medium contains a source of amino acids or not. This is an example for an amino acid auxotrophy mutation. Other auxotrophs could for example be uracil auxotroph if a strain cannot synthesize this base of DNA / RNA and more. This mutation has a disadvantage compared to prototroph mutations, where only a carbon and nitrogen source Is required so that all required components for growth can be synthesized. In the wild, yeast strains are generally complete prototrophs. Loss-of-Function Mutation = reduction / abolition of respective gene activity, most common class of mutations, usually recessive* Null Mutation = complete abolition of respective gene activity (loss-of-function) Gain-of-Function (dominant-positive) = increase / activation of respective gene activity, usually dominant** (hence name) Dominant-negative Mutation = blockage of gene activity (loss-of-function / null mutation) but respective gene is normal (not mutated), this means the mutant gene product interferes with the function of a normal gene product… Suppressor Mutation = suppression of the phenotypic effect of another mutation (double mutation) so mutant normal - either the two mutations affect the same gene - or the second mutation is on a second gene and interacts with the product of the first gene Trends - deletions ofen cause lethal or null mutations - point mutations ofen cause conditional, dominant-negative or suppressor mutations - loss of function mutations are usually recessive because a diploid heterozygote organism will compensate the loss with the unmutated copy, and the recessive mutated copy is not expressed* - gain of function mutations are usually dominant because they change the expression pattern of one gene copy such that it is expressed at an altered rate, in a different tissue or at another time. If a gene with such a mutation determines a certain developmental process, its expression is sufficient to activate this process.**

From Mutation to Phenotype on a Molecular Level Example 1: Temperature sensitive (ts) Mutation Remember in this conditional mutation a rise in temperature quickly abolishes protein function. How can this destabilization and subsequent loss of protein function occur? There are several possibilities: - loss of a hydrophobic amino acid leads to decreased melting temperature - loss of an amino acid essential for a specific interaction with DNA / other proteins 7

CONCEP TS IN These unfolded proteins are eventually degraded and recycled. Remember the Replica Plating Technique; (s. pg 2) – here an application example based on example 1: 1. mutagenize a cell population (= treatment of a cell with mutagenic agents which can cause a mutation by damaging DNA f.e. through lesioning) and keep cells under non-selective conditions (more on mutagenesis in ‘Genetic Screening’)  all cells, both normal and mutated, would grow 2. replication of cells onto two plates; a) permissive conditions (23 degrees), b) restrictive (37) 3. comparison of the two cell populations a) and b)  the ts mutants should only be visible on the first plate so see missing spots on the second Example 2: Dominant-negative (dn) Mutation Remember two loci involved here; a mutant product interferes with the function of a normal protein. There are several possibilities of how this looks like on a molecular level: - dn mutation affects an upregulating transcription factor; no transactivation however, if the dn mutant protein is still able to build a complex with DNA it will permanently block the binding site for wilt-type (wt) functioning transcription factors; displacement - dn mutation effects a subunit of an oligomeric protein complex; inactivity of the entire complex

The Concept of Epistasis - operation of genes rarely in isolation - Epistasis: Gene A is epistatic over Gene B  phenotype of gene A mutant = phenotype of a double mutant (A- & B-mutation) ergo B needs A in order to work properly; Why? – possible scenarios (s. workbook): - 2 or more loci interact to create a new phenotype - Gene A masks / modifies the effects of Gene B and maybe also other Genes Example 1: Genes involved act in the same biological pathway: Adenine Biosynthesis Pathway (very roughly speaking): 1. The precursor of adenine needs to be converted to AIR (Zwischenprodukt) 2. Ade3 enzyme (encoded by gene ade3) required for AIR formation (red) 3. Ade2 enzyme (encoded by gene ade2) required for AIR carboxylation (white) 4. Afer carboxylation AIR can be converted to adenine - a mutation in ade2 leads to adenine auxotrophs and a red population because without carboxylation the red pigment of AIR accumulates, and the biosynthesis of adenine is not possible - a double mutation in ade2 and ade3 leads to adenine auxotrophs and a white population Conclusion: Ade3 is epistatic over ade2. (s. definition of epistasis above) Explanation: Ade3 is upstream in the pathway; its effects come in before those of ade2, so if AIR is never formed, there is no substrate for ade2, so this protein becomes irrelevant and its mutation has no consequence. The red pigments never accumulate because AIR was never formed. The other effect of the mutation in ade2 however, is not reversed by a second mutation of ade3 because both genes act in the same pathway to produce adenine. Example 2: Genes involved act in different biological pathways: The Secretory Pathway and ERAD The secretory pathway: 1. translocation of secretory proteins into the ER (endoplasmic reticulum) 8

CONCEP TS IN 2. maturation of secretory proteins (folding into 3D conformation) 3. packaging into transport vesicles that go from ER to the Golgi apparatus (further processing) Notice how proteins that fail to fold in the ER are retained in the ER. Most of these proteins are subsequently exported to the cytosol of the cell and degraded by proteasomes. This is ERAD (= ER-associated degradation); Pathways connect… Also see the translocator SEC61 at the membrane of the ER. For the SEC61 gene there is a temperature-sensitive mutated allele sec61-1. However, it is only a part of the protein that is misfolded at 37 degrees and the full protein would actually still show limited activity. Nevertheless, the misfolded domain is recognized and degraded by ERAD machinery, so these mutant yeast cells cannot grow – not because of the mutation itself but because of the pathway that is induced by the mutation upon recognition of the mutant protein because it is a sign of stress. Another gene UBC6 encodes a ubiquitin-conjugating enzyme that functions in the ERAD pathway. If this is mutated (double mutation), the first mutation is reversed because Sec61-1 is no longer degraded. Example 3: Synthetic Lethality In General: This is when the combination of mutations in two or more genes leads to cell death, whereas a mutation in only one of these genes would not. Also, here the genes can function in the same biochemical process or in pathways that appear to be unr...