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Einführung in die Genetik Prof. Dr. Kay Schneitz (EBio Pflanzen) http://plantdev.bio.wzw.tum.de [email protected] Twitter: @PlantDevTUM, #genetikTUM FB: Plant Development TUM Prof. Dr. Claus Schwechheimer (PlaSysBiol) http://wzw.tum.de/sysbiol [email protected] Einführung in die Genetik - Inhalte 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Einführung Struktur von Genen und Chromosomen Genfunktion Transmission der DNA während der Zellteilung Vererbung von Einzelgenveränderungen Genetische Rekombination (Eukaryonten) Genetische Rekombination (Bakterien/Viren) Rekombinante DNA-Technologie Kartierung/Charakterisierung ganzer Genome Genmutationen: Ursache und Reparatur Veränderungen der Chromosomen Genetische Analyse biologischer Prozesse Transposons bei Eukaryonten Regulation der Genexpression Regulation der Zellzahl - Onkogene 18. 10. 11 25. 10. 11 08. 11. 11 15. 11. 11 22. 11. 11 29. 11. 11 06. 12. 11 13. 12. 11 20. 12. 11 10. 01. 12 17. 01. 12 24. 01. 12 31. 01. 12 07. 02. 12 14. 02. 12 KS KS KS KS KS KS KS CS CS CS CS CS CS KS CS Genetic Recombination in Eukaryotes Genetics 06 Summary • • Dihybrid inheritance • 2. Mendel’s law: gene pairs on different chromosomes assort independently in gamete formation • segregation of phenotypes in F2 obtained from inbreeding: 9:3:3:1 • segregation of phenotypes in test cross progeny: 1:1:1:1 Recombination • • • generation of new allele combinations • • interchromosomal recombination intrachromosomal recombination 2 gene pairs: 50% recombinant gametes to detect recombinants in diploid organisms: use a test cross • Summary • • • Linkage and crossover (CO) • if two genes do not assort independently: linkage (located on same chromosome) • • recombination still possible through CO RF < 50% Genetic maps • • RF an estimate of distance between two linked genes • gene distances vary between physical and genetic maps map distances are generally additive: assembly of genetic map by mapping many different loci • • suppression of recombination (e.g., CEN) CO hotspots Meiotic recombination • DSB (Spo11), trimming, 3’ ssDNA ends, homology search, strand invasion, heteroduplex formation, HJs • • resolution of HJs: CO or no CO gene conversion: one allele turned into the homologous allele (mismatch repair at heteroduplex) Mitotic recombination Genetic Recombination in Bacteria and their Viruses Genetics 07 Topics Conjugation Transformation Transduction Bacterial genetics: the basis of modern DNA technology Is there genetic exchange in asexually growing bacteria? Yes Bacteria exchange DNA by several processes Lactococcus lactis Escherichia coli (E. coli) Bacterial colonies, each derived from a single cell Detection of mutants lac - lac + lac + lac + Some genetic symbols used in bacterial genetics Symbol Character or phenotype associated with symbol bio - Requires biotin added as supplement to minimal medium arg - Requires arginine added as supplement to minimal medium met lac gal str r Requires methionine added as supplement to minimal medium Cannot utilize lactose as carbon source Cannot utilize galactose as carbon source Resistant to the antibiotic streptomycin str s Sensitive to the antibiotic streptomycin Mutant screens in E. coli Screening for revertants Conjugation Bacterial sex Observation strain A: met - bio - thr + leu + thi + strain B: met + bio + thr - leu - thi - J. Lederberg and E. Tatum, 1946 No cell contact - no recombinants Bacteria conjugate by using pili F plasmid transfer W. Hayes, 1953 Observation strain A: met - bio - thr + leu + thi + strain B: met + bio + thr - leu - thi - J. Lederberg and E. Tatum, 1946 High frequency of recombination (Hfr) strain F plasmid integration occurs at insertion sequences (IS) Hfr: donor chromosome transfer and crossover no reciprocal exchange products Observation strain A: met - bio - thr + leu + thi + strain B: met + bio + thr - leu - thi - J. Lederberg and E. Tatum, 1946 Interrupted mating Hfr azi r ton r lac + gal + str s x F - azi s ton s lac - gal - str r Chromosome map: tracking time of marker entry Simple genetic map O a b c F F plasmid insertion site and the order of gene transfer 1 2 3 Conjugation: two types of DNA transfer Recombination mapping in E. coli Bacterial genetics is merozygote genetics 4% 4% 9% 4% 9% 87 % 4% 9% 87 % very low Genetic map of E. coli • Plasmids • Summary • • • small DNA circles (1-2% of bacterial DNA), replicate autonomously in bacterial cell contain additional genes (e.g, resistance genes, F genes) Conjugation • directional transfer of DNA from a donor to a recipient cell, requires physical contact • F plasmid confers “maleness” Hfr strains • copy of F plasmid integrated somewhere in bacterial chromosome • produces high number of recombinants in Hfr x Fcrosses • • merozygote exconjugants where multiple crossovers can occur between exo- and endogenote Interrupted mating and recombination mapping • circular genetic map of E. coli Transformation Taking up DNA from the external environment DNA: The genetic material Griffith, 1928 Transformation DNA: The genetic material Avery, MacLeod, McCarty, 1944 Bacterial transformation Transduction Bacteriophages transfer bacterial DNA from one bacterial cell to another Bacteriophages Phage T4 Phage infection Lytic cycle Plaques Phage cross by double infection Lytic vs lysogenic cycle Infection Lytic Lysogenic prophage Induction virulent phages temperate phages λ phage insertion Lysis without transfer of phage particles Prophage transfer through conjugation General transduction virulent phages Spezialized transduction temperate phages • Summary • • • • • Bacteriophages • bacterial viruses Virulent phages • • immediately lyse and kill their host bacterium e.g., bacteriophages P1, T4 Temperate phage • maintained in host bacterium without immediately killing the host • e.g., bacteriophage λ Prophage • • phage genome that is integrated into the host chromosome lysogenic bacterium carries a prophage General transduction • Phage transfers any piece of bacterial genomic DNA between cells Special transduction • Prophage integrated at a single, specific site in bacterial chromsome (e.g., λ attachment site) • transfers only genes located close by the attachment site THE END