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34 Cards in this Set

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What is most unique about sexual reproduction (i.e. what makessexual reproduction different from asexual reproduction)?

You produce individuals with new genetic combinations.


In both cases reproduction is happening, but it’s only with sexual reproduction that you are also making new combinations of alleles from two different sources.


Eukaryotes: sex and reproduction happen in one event (sexual reproduction)


Prokaryotes: sex (i.e. making new genetic combinations) and reproduction (making new cells) are separate events

How do cells divide in Prokaryotes?

By binary fission															
No meiosis in prokaryotes! (prokaryotes only have one circular chromosome, andsometimes some small circular plasmids, they aren’t diploid!)

By binary fission


No meiosis in prokaryotes! (prokaryotes only have one circular chromosome, and sometimes some small circular plasmids, they aren’t diploid!)

What are the three ways that a gene can be transferred unidirectionally?

1. Conjugation


2. Transformation


3. Transduction



What is Conjugation?

Conjugation = A “donor” cell forms a pilus that can attach it toa “recipient” cell. It can then transfer a copy of a plasmid (or evenparts of the chromosome) to the recipient cell.

Conjugation = A “donor” cell forms a pilus that can attach it to a “recipient” cell. It can then transfer a copy of a plasmid (or even parts of the chromosome) to the recipient cell.



What is Transformation?

Transformation = a recipient cell can pick up DNA fragments from the surrounding environment, and incorporate pieces of it into its genome through pairs of crossover events.

Transformation = a recipient cell can pick up DNA fragments from the surrounding environment, and incorporate pieces of it into its genome through pairs of crossover events.

What is Transduction?

Transduction = when a bacteriophage (a bacteria-infecting virus) transfers some DNA from it’s previous host into the recipient host cell(Normally, phages only hold phage DNA, but sometimes the new phage protein coats accidentally carry some bact...

Transduction = when a bacteriophage (a bacteria-infecting virus) transfers some DNA from it’s previous host into the recipient host cell(Normally, phages only hold phage DNA, but sometimes the new phage protein coats accidentally carry some bacterial DNA, instead.)



What are the advantages of working with bacteria?

1. Very fast generation time


2. Can work with huge numbers of cells in a very small space (small petri dish can grow 1000s of colonies in a day)


3. This means we can study very rare events

How can you test for and isolate antibiotic-resistance bacteria (E. Coli) when you only have wildtype bacteria in your lab?

Supplement the media with antibiotics, to eliminate the antibiotic-sensitive cells, and select for the resistant cells.


So grow the bacteria on complete media plus antibiotics or minimal media plus antibiotics.


If new antibiotic-resistant cell has second mutation that also makes it auxotrophic it’s better to use complete media, so we can isolate more antibiotic-resistant strains, even if they’re also auxotrophic.

What does growing bacteria on minimal media do?

- Minimal media eliminates auxotrophs (nutritional mutants) and selects for prototrophs (which can make all of their own nutrients)


- Screening based on nutrition

What is one way to isolate cells with particular mutations/alleles?

Add antibiotics (e.g. streptomycin) to the media. This eliminates the susceptible/sensitive cells (str-s) and selects for the resistant cells (str-r)


----> complete media plus antibiotics




(On the solid agar medium each str-r cell can grow into a visible colony of billions of genetically identical cells)

What is the other way to isolate certain types of bacteria?

Remove antibiotics (e.g. adenine) from the media. This eliminates the resistant cells (ad-) and selects for the susceptible/sensitive cells (ad+) ---> complete media minus adenine

What are selectable markers?

Alleles youcan select for, using different growing conditions.
- First three are vitamins/cofactors or amino acids:						- = the strain can’t grow unless the nutrient is provided in the media+ = the strain can make this nutrient (no supplement n...

Alleles you can select for, using different growing conditions.


- First three are vitamins/cofactors or amino acids: - = the strain can’t grow unless the nutrient is provided in the media+ = the strain can make this nutrient (no supplement needed)


Next two are types of sugar:- = the strain can’t use this sugar as its only carbon source.+ = the strain can use this sugar as its only carbon source.


Last two are antibiotics: r = the strain is resistant to this particular antibiotics = the strain is sensitive to this particular antibiotic



What's the simplest medium that can supportthe growth of a strain that is bio+ arg- met+ lac+ gal- strs?

Minimal media (which already contains glucose) plus arginine(It can use lactose too, but not needed if it already has glucose)



How would you isolate strains with this precise genotype? (bio+ arg- met+ lac+ gal- strs)

Treat each colony on an agar plate like a separatestrain/tube. Use a sterile velvet-covered “stamp” to transfercolonies from complete medium to (supplemented) minimal media.Then look for missing colonies to identify						auxotrophic colonies, ...

Treat each colony on an agar plate like a separate strain/tube. Use a sterile velvet-covered “stamp” to transfer colonies from complete medium to (supplemented) minimal media.Then look for missing colonies to identify auxotrophic colonies, etc.



What did Lenderberg and Tatum discover?

Discovered Conjugation:


They took two complementary auxotrophic strains (with different mutations), and grew them together in the same flask overnight.When plated on minimal media, found some new prototrophs



What is the difference between prototrophs and auxotrophs?

Prototrophs: wild-type bacteria that can synthesize all compounds needed for growth from simple ingredients.


Auxotrophs: mutant strains that lack one or more enzymes required for metabolizing nutrients and can only grow on supplemented media

What was wrong with Lenderberg and Tatum experiment and can it be fixed?

Could have been revertants (a mutant that has reverted to its former genotype or to the original phenotype by means of a suppressor mutation, or else by compensatory mutation somewhere in the gene)


Can be fixed by adding controls (suggest that no revertants appeared in this time frame)


- Genes are being exchanged


- Besides conjugation it can also be transformation

In Lenderberg and Tatum experiment how do we know if its conjugation or transformation?

Growing the two strains in one flask, but separated by a membrane that allows media, but not cells, to pass through ---> No growth on MM agar plates


So it is not transformation because direct contact between cells is needed for genetic exchange

What are other discoveries about conjugation?

- Genetic exchange is unidirectional, from donors (F+ or Hfr cells)to recipients (F- cells). (Note: F = “fertility”)


- After donation, the recipient cells become donor cells too!


- Donor cells rarely lose the ability to donate.





How does conjugation work/what's it's process?

F+ cells have an F plasmid (aka an F factor) containing genes for recognizing F- cells and for building pili to join with them


During conjugation, one strand of the plasmid is peeled off and transferred into the F- cell.


DNA replication makes each single strand into a double-helix again.(i.e. this is a replicative transfer)


Now, both cells are F+ cells because they both have F plasmids!

What happens to DNA during conjugation?

- A single strand of DNA enters the recipient as it separates form the rest of the original F-factor


- Both single strands are copied into Double Stranded DNA, as one strand passes through the pilus and the other stays behind


- The ds DNA then circularizes to make a new circular F-factor in the recipient


- The plasmids are thenreplicated, along with the mainbacterial chromosome, everytime the cell divides.

What happens when there is a single crossover between thebacterial chromosome and the F plasmid, within an F+ cell and how does it work?

- They join together to make one big piece of circular DNA double-helix


- This integration can happen at various locations in the bacterial 48 chromosome, because there is an Insertion Sequence (IS) in the F plasmid, and several homologous insertion sequences found in the bacterial chromosome.


- The relative orientation with which an F plasmid is integrated can also vary

What happens once the F factor is integrated into the bacterial chromosome?

The F+ cell becomes an Hfr (high frequency recombination) cell and they can still donate to F-/recipient cells because Hfr cells still have the F plasmid’s genes for forming pili and donating genes

Are Hfr cells more or less likely to donate chromosomal genesto recipient cells, when compared to F+ cells?

Much more!


Within an F plasmid, the Origin of Transfer (OriT) specifies where a donor cell should start transferring DNA to the F-cell. For the F+ cell, the transfer starts at OriT and stops when replicative transfer of the entire F plasmid is complete (so F- cell turns into F+ cell)

What does the order of the genes transferred depend on?

On the location and orientation in which the F plasmid was integrated.


(Because this integrated chromosome is much larger than theF plasmid alone, conjugation is usually interrupted before transfer is complete, so we very rarely see every gene transferred)

In most Hfr x F- crosses, would you expect the F- cells to becomedonor cells?

No, because the F factor genes are the last to be transferred, and conjugation is usually interrupted before they are transferred.

If the recipient cell doesn't receive the F factor, what other genes are transferred?

Once the donor fragment of DNA enters the recipient cell, pairs of crossovers can recombine alleles between the donor fragment and the recipient cell’s bacterial chromosome.


(appearance of wildtype prototrophs)

How do we map bacterial genes using conjugation?


( Ex: Hfr strain = strs thr+ leu+ azir gal+ lac+ tonr F- strain = strr thr- leu- azis gal- lac- tons)

1. Isolate two bacterial strains that we will "mate" together


2. Then make a rough map of the 4 underlined genes:


- mix two strains in liquid complete media


- allow to "mate" for some minutes


- blend to separate sells and interrupt mating


- plate on complete solid media but with missing threonine and leucine, and added streptomycin




What’s left are F- recipient cells that successfully conjugated with the Hfr strains (to get thr+ and leu+)

How do you determine gene order based on time?

Genes further from OriT require longer conjugation to bebe transferred to recipient cell

Why can't we use a single Hfr strain to map the whole E. Coli genome when using interrupted mating experiments?

We can map the chromosomal genes that are transferred right after OriT, but conjugation ends spontaneously before the later genes get transferred.


To map remaining genes: we isolate more Hfr Strains in which the F factors are integrated in different locations and orientations

How do we map the whole bacterial chromosome for E. Coli?

1. We make many Hfr strains for E. Coli


2. We do interrupted mating experiments with each Hfr strain to map the few genes that are transferred soon after the OriT


3. We use the overlap between these separate maps to assemble them into a crude but complete genome map

Difference between mapping with conjugation versus transformation

Conjugation:


- need to carefully isolate an F- strain and many Hfr strains


- we need antibiotics to isolate the F- cells from the Hfr cells


Transformation:


- No need to make many Hfr strains (no f factor needed at all)


- We'll kill the donor cells first (no need to exclude them later)


- Can even use donor and recipient cells of different species

How does transformation work in more detail?

1. To get donor DNA fragments, kill donor cells (e.g. strr cells),extract their DNA, and randomly break it into pieces.


2. Mix these donor fragments with competent recipient cells(cells that can transform themselves with DNA fragments takendirectly from the surrounding environment): e.g. strs cells. We can induce competency in the lab (e.g. via heat shock in high [Ca2+])


3. The fragments enter some recipient cells (as single strands?)


4. Pairs of crossovers may integrate new alleles into chromosome


5. Use selective media (e.g. str+ media) to select for transformants

Imagine that you exposed competent a- b- recipient cells toDNA fragments from destroyed a+ b+ donor cells.If the a and b genes are closely linked, what would you expect?

Some recipient cells will be a+ b+




Only some recipient cells were transformed, and only a few of those cells received a fragment with both a+ and b+ on it, and also recombined both alleles into its chromosome.




(if distantly liked we would see much fewer a+ b+ recipient cells)