What is genetic material?What is genetic material?

Griffith experiment 1928Griffith experiment 1928

DNADNA

Watson-Crick model 1953Watson-Crick model 1953

DNA polymerase I and IIIDNA polymerase I and III DNA ligasesDNA ligases PrimasePrimase

DNA replication is semiconservative!DNA replication is semiconservative!

Meselson-Stahl experiment 1958Meselson-Stahl experiment 1958

oriC and dnaA BoxesoriC and dnaA Boxes

Cis acting sites: on Cis acting sites: on this side of (acting this side of (acting only on the DNA, only on the DNA, they made ofthey made of

Trans acting Trans acting (proteins) : on the (proteins) : on the other side of (acting other side of (acting on any DNA) on any DNA) dnaA, B, C, G, dnaA, B, C, G,

TerminationTermination

Bacterial genetic information:Bacterial genetic information: On bacterial DNAOn bacterial DNA On plasmidsOn plasmids On bacteriphagesOn bacteriphages On transposonsOn transposons

BActerial GenomeBActerial Genome-Usually 1 chromosome-Usually 1 chromosome

Circular or linearCircular or linear

No histon proteinsNo histon proteins

In circular bacterial DNA the replication In circular bacterial DNA the replication begins at the ori locus begins at the ori locus

Ends at ter locusEnds at ter locus

Plasmids:Plasmids:

ds DNA; circulards DNA; circular

Various copy numberVarious copy number

800-300 000 bp long800-300 000 bp long

Carry genes providing advantages for the Carry genes providing advantages for the bacteriumbacterium

Transposons (IS Transposons (IS seequences)seequences)

Can couple their replication Can couple their replication to the cell divisionto the cell division

- Their propagation Their propagation depends on the depends on the integration with the integration with the bacterial repliconbacterial replicon

- The insertion sites are not The insertion sites are not spesificspesific

BacteriophagesBacteriophages

Viruses of the bacteriaViruses of the bacteria

Ds/ss DNA,ds/ss RNADs/ss DNA,ds/ss RNA

Lytic or temperate phages (prophage)Lytic or temperate phages (prophage)

Different propagation strategiesDifferent propagation strategies

Gene transfer among bacteriaGene transfer among bacteria

Vertical transferVertical transfer Lateral or horizontal transferLateral or horizontal transfer

- conjugation- conjugation

- transduction- transduction

- transformation- transformation

ConjugationConjugation

Most frequently plasmids are transferred

Tra gene products are needed

F+ E. Coli Sex pilus Hfr

R plasmids

Interrupted MatingInterrupted Mating Chromosome transfer from the Chromosome transfer from the

Hfr into the F- is slow: it takes Hfr into the F- is slow: it takes about 100 minutes to transfer about 100 minutes to transfer the entire chromosome.the entire chromosome.

The conjugation process can The conjugation process can be interrupted using a kitchen be interrupted using a kitchen blender.blender.

By interrupting the mating at By interrupting the mating at various times you can various times you can determine the proportion of F- determine the proportion of F- cells that have received a cells that have received a given marker.given marker.

This technique can be used to This technique can be used to make a map of the circular E. make a map of the circular E. coli chromosome.coli chromosome.

TransductionTransduction

General Phage Life CycleGeneral Phage Life Cycle 1. Phage attaches to the cell 1. Phage attaches to the cell and injects its DNA.and injects its DNA.

2. Phage DNA replicates, and 2. Phage DNA replicates, and is transcribed into RNA, then is transcribed into RNA, then translated into new phage translated into new phage proteins.proteins.

3. New phage particles are 3. New phage particles are assembled.assembled.

4. Cell is lysed, releasing 4. Cell is lysed, releasing about 200 new phage about 200 new phage particles.particles.

Total time = about 15 minutes.Total time = about 15 minutes.

Generalized TransductionGeneralized Transduction Some phages, such as phage P1, break up the bacterial Some phages, such as phage P1, break up the bacterial

chromosome into small pieces, and then package it into chromosome into small pieces, and then package it into some phage particles instead of their own DNA.some phage particles instead of their own DNA.

These chromosomal pieces are quite small: about 1 1/2 These chromosomal pieces are quite small: about 1 1/2 minutes of the E. coli chromosome, which has a total minutes of the E. coli chromosome, which has a total length of 100 minutes.length of 100 minutes.

A phage containing E. coli DNA can infect a fresh host, A phage containing E. coli DNA can infect a fresh host, because the binding to the cell surface and injection of because the binding to the cell surface and injection of DNA is caused by the phage proteins.DNA is caused by the phage proteins.

After infection by such a phage, the cell contains an After infection by such a phage, the cell contains an exogenote (linear DNA injected by the phage) and an exogenote (linear DNA injected by the phage) and an endogenote (circular DNA that is the host’s endogenote (circular DNA that is the host’s chromosome).chromosome).

A double crossover event puts the exogenote’s genes A double crossover event puts the exogenote’s genes onto the chromosome, allowing them to be propagated.onto the chromosome, allowing them to be propagated.

Transduction MappingTransduction Mapping

Only a small amount of chromosome, a few Only a small amount of chromosome, a few genes, can be transferred by transduction. The genes, can be transferred by transduction. The closer 2 genes are to each other, the more likely closer 2 genes are to each other, the more likely they are to be transduced by the same phage. they are to be transduced by the same phage. Thus, “co-transduction frequency” is the key Thus, “co-transduction frequency” is the key parameter used in mapping genes by parameter used in mapping genes by transduction.transduction.

Transduction mapping is for fine-scale mapping Transduction mapping is for fine-scale mapping only. Conjugation mapping is used for mapping only. Conjugation mapping is used for mapping the major features of the entire chromosome.the major features of the entire chromosome.

Specialized TransductionSpecialized Transduction

Some phages can transfer only particular genes Some phages can transfer only particular genes to other bacteria.to other bacteria.

Phage lambda (λ) has this property. To Phage lambda (λ) has this property. To understand specialized transduction, we need to understand specialized transduction, we need to examine the phage lambda life cycle.examine the phage lambda life cycle.

lambda has 2 distinct phases of its life cycle. lambda has 2 distinct phases of its life cycle. The “The “lyticlytic” phase is the same as we saw with the ” phase is the same as we saw with the general phage life cycle: the phage infects the general phage life cycle: the phage infects the cell, makes more copies of itself, then lyses the cell, makes more copies of itself, then lyses the cell to release the new phage.cell to release the new phage.

Lysogenic PhaseLysogenic Phase The “The “lysogeniclysogenic” phase of the lambda life cycle starts the same way: ” phase of the lambda life cycle starts the same way:

the lambda phage binds to the bacterial cell and injects its DNA. the lambda phage binds to the bacterial cell and injects its DNA. Once inside the cell, the lambda DNA circularizes, then incorporates Once inside the cell, the lambda DNA circularizes, then incorporates into the bacterial chromosome by a crossover, similar to the into the bacterial chromosome by a crossover, similar to the conversion of an F plasmid into an Hfr.conversion of an F plasmid into an Hfr.

Once incorporated into the chromosome, the lambda DNA becomes Once incorporated into the chromosome, the lambda DNA becomes quiescent: its genes are not expressed and it remains a passive quiescent: its genes are not expressed and it remains a passive element on the chromosome, being replicated along with the rest of element on the chromosome, being replicated along with the rest of the chromosome. The lambda DNA in this condition is called the the chromosome. The lambda DNA in this condition is called the “prophage”.“prophage”.

After many generations of the cell, conditions might get harsh. For After many generations of the cell, conditions might get harsh. For lambda, bad conditions are signaled when DNA damage occurs.lambda, bad conditions are signaled when DNA damage occurs.

When the lambda prophage receives the DNA damage signal, it When the lambda prophage receives the DNA damage signal, it loops out and has a crossover, removing itself from the loops out and has a crossover, removing itself from the chromosome. Then the lambda genes become active and it goes chromosome. Then the lambda genes become active and it goes into the lytic phase, reproducing itself, then lysing the cell. into the lytic phase, reproducing itself, then lysing the cell.

TransductionTransduction

Phage mediated Phage mediated recombinationrecombination

TransformationTransformation

Competent cells

Artificially (forced) with CaCl

or temperature shock

Genetıc MappingGenetıc Mapping

Sequencing of the geneSequencing of the gene Cloning the geneCloning the gene Gene labeled Gene labeled Hybridization Hybridization

Localization of the gene on the bacterial Localization of the gene on the bacterial genomegenome

Nucleic acid amplificationNucleic acid amplification

- PCR and other technologiesPCR and other technologies- Real time PCRReal time PCR