Lecture 22 Flashcards Preview

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Flashcards in Lecture 22 Deck (25):
1

Creating a complete loss of function in yeast - double cross over:

- Insert a selectable marker (eg. LEU2+) in the middle of the gene, destroying the function of the gene
- Transform into a recipient
- A double recombination event will cause the construct of interest to be inserted and this can be selected for by selecting for the selectable marker
- The LEU2+, Gene X- is generated

2

Creating a complete loss of function in yeast - single cross over:

- Insert a small portion - internal regions only of gene into YIp vector, by delete the 5' and 3' end.
- A single recombination event will integrate (by homology) the entire construct
- The genomic copy of our gene will lack a functional copy, one copy will lack the 5' end the other will lack the 3' end

3

What will happen if your gene is essential?

- The inactivation will be lethal
- It can be hard to know WHY you are not getting transformation (is this lethal, for example?)
- Yeast is haploid but can be diploid
- a type cells and alpha type cells can mate to form an a/alpha haploid, which replicate.
- Under stress (nitrogen limitations) meiosis of diploid yeast is induced, and a, a, alpha and alpha will be produced, inside an ascus.

4

Gene inactivation in a diploid recipient:

- Create a ura3- which is viable, and transform the inactivation construct into a diploid containing a WT copy Y+ and an inactive copy Y-.
- Conditions can be shifted to turn it back into a haploid state.
- Two haploids will be derived from the WT version of the gene and two will be inactive, so we can expect 2 Ura+ and 2 Ura-.
- If you see this ratio you know that this gene is not essential as everything is retrieved
- If it is an essential gene you will only get WT (Ura+) haploid yeast

5

Two step gene replacement strategy uses:

- Introduce small changes in the DNA, such as a couple of bases changed

6

Two step gene replacement strategy using a YIp: Step 1:

- Into the YIp, introduce a YIp with a selectable marker and an altered version of your gene
- Select for the selectable marker and introduce the whole plasmid in yeast
- Finish with the (new) altered version of your gene present in the genome and the old gene

7

Two step gene replacement strategy: Step 2:

- Expose the effect of this mutation
- Looking for intra-chromosomal duplication event between the sequences 'old' and 'new'
- A recombination event could occur and create copies with ONLY the old copy and the other ONLY the new copy
- This gives us a yeast genome with only the new copy of the gene (no selectable marker or WT copy), determining through RE analysis

8

Uses of two step gene replacement in HIS4 mutants:

- HIS4 gene is regulated by GCN4, but there are no mutants with altered control regions (cis) isolated
- Create a set of deletions for HIS4, taking out different parts of the promoter
- These were individually introduce back into yeast using 2 step gene replacement

9

List of steps:

1. Take a YIp with selectable marker and the construct containing a deletion in the 5' region of HIS4
2. Introduce into yeast
3. Select for URA3+
4. Finish up with a yeast transformatnt containing the old and new version and the selectable marker

10

Second half of steps:

5. Remove the selectable marker and the old copy
6. Regions of recombination with the 2 regions of homology produces the WT and the mutated copy of HIS4

11

HIS4 two step gene replacement results:

4 distinct classes:
- 1. No effect
- 2. Expression of HIS4- dropped, but was still regulated
- 3. Regulation of HIS4 was lost, even though the gene was still expressed
- 4. Loss of expression altogether

12

Loss of regulation of HIS4:

- Regulation by GCN4 must occur in the -138 region of the DNA. This is the promoter
- It was found that there were two other copies of the same sequence, but it was only when you mutate the last site
- There are three copies, so if only one is mutated there is no effect, but if you delete all other copies it is detectable

13

Further steps:

- Took the -136 strain which has lost regulation and selected for revertants
- The -136 cuts through the sequence and introduces new sequences
- The revertants convert the base pair change back to the original nucleotide

14

Different types of yeast plasmids and their uses:

- Clone by complementation (YRp/YEp)
- Regulation of expression (YRp/YEp or YCp)
- Over expression (YRp/Yep or YIp)
- Gene inactivation/replacement (YIp)

15

Aspergillus nidulans:

- Can be haploid or diploid
- Have asexual and sexual cycles
- Has multinucleate hyphae
- Easy to quickly generate protoplasts

16

Generating protoplasts/recombinants:

- Cell wall degrading enzyme in the presence of CA2+ (facilitate uptake) and PEG (make cell membrane penetrable)
- Good for drug resistance selectable markers:

17

Difference between asp and yeast:

- Integration in yeast is exclusively by homology
- In Asp there are more non-homolgous events than homologous

18

Advantages of usually being non-homologous

- Can introduce almost any gene (there is no homology)
- Can leave the native gene intact
- Can generate multiple copies of the gene

19

Disadvantages of usually being non-homologous:

- Possible positions effects on expression
- Homologous events are more difficult to isolate

20

Strategies developed to identify homologous integrations to be gene specific include:

- Targeting to the argB locus
- Transform an argB- mutant recipient with an argB- plasmid (different mutations to produce argB-) select for argB+, which will occur when the construct enters the argB- locus

21

Aspergillus conidiation:

- Ordered developmental program leading to the production of conidia
- Conidia are asexual spores
- This requires the brlA gene. The bristleA mutant cannot make the transition to producing conidia
- brlA is expressed at the time of conidiation in a WT asp.

22

Does brlA cause conidiation, or does conidiation cause the production of brlA?

- Expressing brlA in the mycelia (not during conidiation)..
- brlA coding region under the control of alcA promoter, intorduced into brlA- protplasts
- Transformants that are argB+ with an alcA::brlA fusion showed that in the presence of ethanol (in response to the expression of brlA) results in conidiation

23

Aspergillus transformation:

- High frequency transformation can be cloned by complementation
- DNA must be integrated

24

nkuA gene in asp is the equivalent to Ku70 in most eukaryotes:

- Non-homologous end joining DNA repair occurs with the use of Ku70/Ku80
- In asp, nku mutants of asp will behave like yeast, in that plasmids are targeted to a region of homology

25

Differences between Asp and yeast:

- Simple, high efficiency transformation system
- Cloning by complementation
- Gene inactivation/replacement
- Difference in DNA repair affects the way these two fungi handle exogenous DNA