Einführung in die GenetikProf. Dr. Kay Schneitz (EBio Pflanzen)

http://plantdev.bio.wzw.tum.deschneitz@wzw.tum.deTwitter: @PlantDevTUM, #genetikTUMFB: Plant Development TUM

Prof. Dr. Claus Schwechheimer (PlaSysBiol)http://wzw.tum.de/sysbiolclaus.schwechheimer@wzw.tum.de

Einführung in die Genetik - InhalteEinführung in die Genetik - InhalteEinführung in die Genetik - Inhalte1 Einführung 16. 10. 12 KS2 Struktur von DNA und Chromosomen 23. 10. 12 KS3 Genfunktion 30. 10. 12 KS4 Transmission der DNA während der Zellteilung 06. 11. 12 KS5 Vererbung von Einzelgenveränderungen 13. 11. 12 KS6 Genetische Rekombination (Eukaryonten) 20. 11. 12 KS7 Genetische Rekombination (Bakterien/Viren) 27. 11. 12 KS8 Rekombinante DNA-Technologie 04. 12. 12 CS9 Kartierung/Charakterisierung ganzer Genome 11. 12. 12 CS

10 Genmutationen: Ursache und Reparatur 18. 12. 12 CS11 Veränderungen der Chromosomen 08. 01. 13 CS12 Genetische Analyse biologischer Prozesse 15. 01. 13 CS13 Transposons bei Eukaryonten 22. 01. 13 CS14 Regulation der Genexpression 29. 01. 13 KS15 Regulation der Zellzahl - Onkogene 05. 02. 13 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

• map distances are generally additive: assembly of genetic map by mapping many different loci

• gene distances vary between physical and genetic maps

• 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 basisof 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 - Requires methionine added as supplement to minimal medium

lac - Cannot utilize lactose as carbon source

gal - Cannot utilize galactose as carbon sourcestr

r Resistant to the antibiotic streptomycin

str s Sensitive to the antibiotic streptomycin

Mutant screens in E. coli

Screening for revertants

ConjugationBacterial sex

Observation

J. Lederberg and E. Tatum, 1946

strain A: met - bio

- thr + leu

+ thi +

strain B: met + bio

+ thr - leu

- thi -

Observation

J. Lederberg and E. Tatum, 1946

strain A: met - bio

- thr + leu

+ thi +

strain B: met + bio

+ thr - leu

- thi -

No cell contact - no recombinants

Bacteria conjugate by using pili

F plasmid transfer

W. Hayes, 1953

Observation

J. Lederberg and E. Tatum, 1946

strain A: met - bio

- thr + leu

+ thi +

strain B: met + bio

+ thr - leu

- thi -

High frequency of recombination (Hfr) strain

F plasmid integration occurs at insertion sequences (IS)

Hfr: donor chromosome transfer and crossover

Hfr: donor chromosome transfer and crossover

no reciprocal exchange products

Observation

J. Lederberg and E. Tatum, 1946

strain A: met - bio

- thr + leu

+ thi +

strain B: met + bio

+ thr - leu

- thi -

Observation

J. Lederberg and E. Tatum, 1946

strain A: met - bio

- thr + leu

+ thi +

strain B: met + bio

+ thr - leu

- thi -

Interrupted mating

Hfr azi r ton r lac

+ gal + str s F

- azi s ton s lac

- gal - str rx

Chromosome map: tracking time of marker entry

Simple genetic map

O Fa b c

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 %

4 %

9 %

4 %

9 %

4 %

9 %

87 %

4 %

9 %

87 %

very low

Genetic map of E. coli

Summary• Plasmids

• 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 F- crosses

• merozygote exconjugants where multiple crossovers can occur between exo- and endogenote

• Interrupted mating and recombination mapping

• circular genetic map of E. coli

TransformationTaking up DNA from the external environment

DNA: The genetic materialGriffith, 1928

Transformation

DNA: The genetic materialAvery, MacLeod, McCarty, 1944

Bacterial transformation

TransductionBacteriophages 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 cycleInfection

LysogenicLytic

Induction

Lytic vs lysogenic cycle

virulent phages

Infection

LysogenicLytic

Induction

Lytic vs lysogenic cycle

virulent phages

prophage

Infection

LysogenicLytic

Induction

Lytic vs lysogenic cycle

virulent phages temperate phages

prophage

Infection

LysogenicLytic

Induction

λ phage insertion

Lysis without transfer of phageparticles

Lysis without transfer of phageparticlesProphage transferthrough conjugation

General transduction

General transduction

virulent phages

Spezialized transduction

temperate 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