Seminar

Wednesday, May 15, Biology 212, 4 pm

"Genomic Consequences of Allopolyploidization"

Luca Comai, Ph.D., Department of Botany, University of Washington

5 points for attendance, 1-5 points each person for good questions during the Q/A period after the talk.

Mutation in Bacteria

…the ultimate source of variation in bacteria is spontaneous mutation,

– generally errors in DNA replication,

…mutations occur in specific genes at a rate of 1 in 106 to 1 in 107 cells,

…adaptive mutations are quickly replicated and adaptive colonies predominate.

Spontaneous Mutations

• DNA replication in E. coli occurs with an error every ~ 109 bases.

• The E. coli genome is 4.6 x 106 bases.

An error occurs once per ~ 2000 replications.

• If a single colony has 107 bacteria,

5,000 cells carry a mutation,

or, one mutation every ~ 1,000 bases (across a colony),

or, a mutation in about every gene.

Conjugation

… F+ cells donate genetic material via the F+ plasmid,

…Hfr cells; strains with a chromosome integrated F factor that is able to mobilize and transfer part of the chromosome to the F- cell.

F’Cells

• an F factor from an Hfr cell excises out of the bacterial genome and returns to plasmid form,

• often carries one or more bacterial genes along,

• F’cells behave like an F+ cells,

– merizygote: partially diploid for genes copied on the F’plasmid,

• F’plasmids can be easily constructed using molecular biology techniques.

Selective Media

• wild-type bacteria grow on minimal media,

• media supplemented with selected compounds supports growth of mutant strains,

– minimal media + leucine supports leu- cells,– minimal media + leucine + arginine supports leu- arg-

– etc.

• Selective Media: a media in which only the desired strain will grow.

Selection

...the process that establishes conditions in which only the desired mutant will grow.

Problem

Strain MatingType

Chromosome Genotype F factor Genotype

Strain 1 F' wild-type thr+ ; thi+

Strain 2 F' arg- thr+ ; thi+

Strain 3 F- thr- ; thi- ; met- -

You want to create cells that are only methionine auxitrophs.

The Wrong Answer

Strain MatingType

Chromosome Genotype F factor Genotype

Strain 1 F' wild-type thr+ ; thi+

Strain 2 F' arg- thr+ ; thi+

Strain 3 F- thr- ; thi- ; met- -

Strain 1 x Strain 3

How do you get rid of Strain 1?

You have a recombinant that is met- only.

Correct AnswerStrain Mating

TypeChromosome Genotype F factor Genotype

Strain 1 F' wild-type thr+ ; thi+

Strain 2 F' arg- thr+ ; thi+

Strain 3 F- thr- ; thi- ; met- -

Strain 2 x Strain 3

Grow on Minimal Media Plus Methionine

Strain 2 dies because there is no arginine.

Strain 3 dies because there is no threonine or thimine.

The new exconjugate lives.

High Frequency of Recombination(Hfr)

...bacteria exhibiting a high frequency of recombination,

…the F factor is integrated into the chromosomal genome.

F factor and Chromosomal DNA are Transferred

Recombination Requires Crossing over

Double Crossover

Incomplete Transfer of DNA

• Interrupted Mating: a break in the pilus during conjugation stops the transfer of DNA,

• Transfer occurs at a constant rate,

– provides a means to map bacterial genes.

How Do You Interrupt Bacterial Mating

spread on agar

mate for specified time

frappe

Hfr and MappingHfrH

strs (sensitive to streptomycin) thr+ (able to synthesize the amino acid threonine) azir (resistant to sodium azide)tonr (resistant to bacteriophage T1) lac+ (able to grow with lactose as sole source of carbon) gal+ (able to grow with galactose as sole source of carbon)

F-

strr (resistant to streptomycinthr- (threonine auxotroph)azis (sensitive to sodium azide) tons (sensitive to phage T1)lac- (unable to grow on lactose) gal- (unable to grow on galactose)

Hfr and MappingHfrH

strs (sensitive to streptomycin)thr+ (able to synthesize the amino acid threonine)

F-

strr (resistant to streptomycin)thr- (threonine auxotroph)

Streptomycin kills the HfrH cells in the mating mix.

No threonine kills the F- cells in the mating mix.

Hfr and Mapping

HfrH

azir (resistant to sodium azide)tonr (resistant to bacteriophage T1) lac+ (able to grow with lactose as sole source of carbon) gal+ (able to grow with galactose as sole source of carbon)

F-

azis (sensitive to sodium azide) tons (sensitive to phage T1)lac- (unable to grow on lactose) gal- (unable to grow on galactose)

Interrupting Bacterial Mating

spread on selective media

mate 9 min blend

Replica Plating

After 9 minutes, only azide resistant cells grow.

10 Minutes

Azide, and bacteriophage resistant cells grow.

15 Minutes

Azide, and bacteriophage resistant cells, and lactose utilizing cells.

18 Minutes

All recombinants grow.

% c

ells

wi t

h m

arke

rs

Bacterial Map Distances

units = minutes

F factor inserts in different regions of the bacterial chromosome,

Also inserts in different orientations.

Origin of Replication

Hfr Order of transferstrain

H thr azi ton lac pur gal his gly thi 1 thr thi gly his gal pur lac ton azi 2 lac pur gal his gly thi thr azi ton 3 gal pur lac ton azi thr thi gly his

F factor

Hfr F-

AA

a

Indicates direction of transfer.

F factor

Hfr F-

A

Leading Gene: the first gene transferred, it is determined empirically.

A

Hfr F-

A

A

A transfers first.

A transfers last.

Hfr Order of transferstrain

H thr azi ton lac pur gal his gly thi 1 thr thi gly his gal pur lac ton azi 2 lac pur gal his gly thi thr azi ton 3 gal pur lac ton azi thr thi gly his

E. coli Map

• 0 minutes is at the threonine,

• 100 minutes is required to transfer complete genome,

Typical Problem

combine

combine

+

Join MapsRefer to partial maps for map distances.

11.5 minutes 26 minutes

Practice

• Insights and Solutions, #2,

• Problem 8.17, 8.18, 8.19.

Transformation

• heritable exchange brought about by the incorporation of exogenous DNA,

– usually DNA from same, or similar species.

Donor and Recipient

Not all cells are competent to receive DNA.

Competence

…a transient state or condition in which a cell can bind and internalize exogenous DNA molecules,

…often a result of severe conditions,

– heat/cold,– starvation, etc.

Competent Cell

Genes are expressed that produce proteins that, in turn, span the cell membrane.

Exogenous DNA Binds Receptor

Complementary Strand Degraded

...one strand of the exogenous DNA is degraded also.

Exogenous DNA Incorporated

Heteroduplex

Cell Divides

Transformation and Mapping

• transformed DNA is generally 10,000 - 20,000 base pairs in length,

– carries more than one gene,

• When two or more genes are received from the same transformation event, they are said to be co-transformed.

Linkage in Bacteria

• genes that are closer together, have a higher probability of being cotransformed,

– higher probability of being on same donor DNA,

– lower chance of crossover event between genes,

• probability of transformation by two separate events is low,

• linkage in bacteria refers to proximity.

F factor Review

Now F+ Now F+

Transduction

…virally mediated gene transfer from one bacterium to another,

…bacteria viruses are termed bacteriophages.

T4 Bacteriophage

…infects E. coli,

Two Bacteriophage Strategies

• Lytic,

– a type of viral life cycle resulting in the release of new phages by death and lysis of the host cell,

• Lysogenic,

– a type of viral life cycle in which the visus becomes incorporated into the host cell’s chromosome.

Lytic Cyclespecific transmembrane phage/bacteria binding sites,

virus DNA inserted into host cell,

1. host cell physiology is shut down,

2. host cell physiology is used for phage work,

3. phage DNA replicated,capsule parts made,

4. phage reassemble with repackaged DNA,

5. host cell is degraded and lyses.

Generalized Transduction

…enzymatic process which can result in the transfer of any bacterial gene between related strains of bacteria.

Phage Infects Host

Specific Binding Sites,

Phage DNA inserted,

Upon infection, host cell physiology is shut down,

we’ll follow gene C+.

Phage Hijacks the Host Cell’s Transcription/Translation

Machinery

Host cell degraded, the host chromosome is cut, Phage replicates own DNA,

makes protein head etc.,

gene C+ is present on a DNA fragment.

Cell Lyses, Phage Move On

C+ is packaged instead of phage DNA in one of thousands of new phages,

phage particle with C+ moves to another host cell.

End of the RouteHost Chromosome,

Phage DNA,

inserted in Genome,via double crossover.

packaged host DNA,

packaged host DNA,

inserted in cell,

Virulent Phages

…reproduce via the lytic cycle only.

Two Bacteriophage Strategies

• Lytic,

– a type of viral life cycle resulting in the release of new phages by death and lysis of the host cell,

• Lysogenic,

– a type of viral life cycle in which the visus becomes incorporated into the host cell’s chromosome.

Lytic vs Lysogenic

viral DNA is incorporated into the host genome.

Lysogeny

…the integration of viral DNA into the bacterial genome,

– a virus that can integrate into the genome is termed temperate,

– an integrated phage is termed a prophage.

Prophage

…non-virulent units that are inserted in the host chromosome, and multiply via binary fission along with the host DNA,

…prophage can re-enter the lytic cycle to complete the virus life cycle.

Phage Induction

…prophage express a repressor protein that inhibits further infection,

– also inhibits prophage DNA excision genes, and genes used during the lytic cycle,

…environmental cues (especially events that damage DNA) block the expression of the repressor protein,

– prophage excises and enters a lytic cycle.

Specialized Transduction

…upon excision of the prophage, adjacent host DNA is taken along,

…the completion of the lytic cycle and subsequent infection of another host moves the flanking DNA to another bacterium.

Normal Excision

Abnormal Excision

flanking DNA is removed.

Transfer to Other Cells

Biotechnology

• Bacteria: again with the gene therapy,

– here they are harnessing another organism to do their dirty work,

• Humans: use phages to do a variety of molecular biology work,

– also use virus to deliver genes in new gene therapy technologies.

T Plasmids

…bacteria also have plasmids that they transfer to other organisms,

…upon infection, the T plasmid enters the host cell, becomes incorporated in the host genome, and the T plasmid genes become expressed,

…Agrobacterium tumefaceins transfers genes that force plants to make strange sugars, that only the Agrobacterium can digest.

Transposable Elements

…a segment of DNA that can move to, or move a copy of itself to another locus on the same or a different chromosome (hopping DNA),

…may be a single insertion sequence, or a more complex structure (transposon) consisting of two insertion sequences and one or more intervening genes.

Bacteria are Geniuses

• Cloning: identical copies,

• Gene therapy: insertion of a healthy, or functional gene into a organism lacking a good gene,

• Defense: develop genes to ward off poisons, predators, etc.

• Genetic engineering: inserting DNA into another organism to do your bidding,

• Harness Mutation: to speed evolution.

Weds.

• Work the Assigned Problems.

• Study the Benzer Experiment.