plant genomes and their manipulation

Question 1

transformation and its two forms

Answer 1

when we introduce DNA into plants

1. transient - electrocute a cell you can get it to take up DNA or inject it with DNA. this DNA will be expressed but is lost as it is not copied when the cell divides.
2. stable - DNA is copied into host chromosome so every time the cell divides it copies the DNA and the daughters of those cells will ave the trans genes inserted.

Question 2

what is crown gall disease and why is it called that?

Answer 2

tutor like growths occurring on plants.

crown - where root meets stem

gall - tumour like growth caused by insects.

Question 3

what causes crown gall disease and how was this discovered?

Answer 3

agrobacterium tumefaciens.

bacterium extracted from galls on daisies were inoculated onto another plant. this resulted in the growth of a gall on the new plant showing that bacterium causes gall growth.

Question 4

how do plants grow tumours even after the agrobacterium is dead.

Answer 4

agrobacterium transfers a tumor inducing plasmid to the plant. a small bit of this plasmid (tDNA) ends up in the plant cell and this creates tumours.

Question 5

what did tumor tissue culture show?

Answer 5

cells will proliferate as callus if given auxin and cytokinin. callus will develop shoots and roots. showed that you could eventually regenerate a plant.

high auxin and low cytokinin - will produce roots

low auxin high cytokinin - will produce shoots.

Question 6

callus

Answer 6

early stage plant tissue. rapidly dividing and hasn’t differentiated yet (gone green for example.)

Question 7

opines

Answer 7

1. used by agrobacterium as food.
2. can’t be made or used by plant cells.

Question 8

transfer DNA

how does agrobacterium section off this bit?

Answer 8

this is the section of agrobacterium plasmid which ends up in the plant cell.

this section of plasmid is bordered by small sequences and is cut at these points.

Question 9

virulence gene

Answer 9

important for infection of the plant and getting the DNA into the plant. don’t end up in the plant themselves.

Question 10

how does agrobacterium sense plants and move towards them?

Answer 10

1. wounded plants produce small molecule called acetosyringone.
2. agrobacterium receptor smells this chemical it binds it and switches on its virulence genes.

Question 11

type four secretion system

Answer 11

1. found in agrobacterium membranes.
2. transfer DNA goes through it and gets transferred into plant cell wall.

Question 12

how are opines made?

Answer 12

1. tDNA goes into plant cell.
2. this stimulates plant to make metabolites which are a nutrient source for bacterium.

Question 13

how does tDNA promote growth of a tumour?

Answer 13

hacks the plant to make auxins and cytokinins that then get these cells to divide and make a tumour.

Question 14

oncogenes

Answer 14

two kinds of genes encode enzymes promoting cytokinin and auxin production respectively.

Question 15

what happens to a tumour if there is no tmr gene?

Answer 15

mutants make tumours with low cytokinin concentration and high auxin promoting root formation.

Question 16

what happens to a tumour if there is no tms gene?

Answer 16

mutants make tumours with high cytokinin and low auxin promoting formation of shoots.

Question 17

what happens if there are no oncogenes?

Answer 17

no tumour will form.

bacteria infects but no proliferation of cells occurs.

Question 18

how can modification of tDNA be beneficial?

Answer 18

can replace tutor inducing and opine producing genes with beneficial genes of interest.

Question 19

binary vector system

Answer 19

splitting ti plasmid in two.

virulence genes on one and tDNA in the other.

Answer 20

1. need to get the DNA into plant cells, ideally in chromosomes.
2. select transformed cells.
3. need to regenerate a new plant by applying auxin and cytokinin to tissue culture. all cells in this plant will be transgenic.

Question 21

forward genetics

Answer 21

mutagenesis - break up DNA with X-ray.

screen (look at) and identify mutant plants.

check if mutant phenotype is heritable.

see if the phenotype identifies a single gene.

map the gene, identify DNA sequence, protein etc

[phenotype — gene — DNA sequence]

Question 22

what is the consequence of random tDNA insertions?

Answer 22

will insert anywhere, if a tDNA is inserted into the coding region of plant chromosomes, it usually causes a disruption.

Question 23

reverse genetics

Answer 23

start from genome sequence

determine the function of this gene.

inactivate the gene

make multiple mutants if there are closely related genes

use trans genes to make over active version of gene of interest

use trans genes to find out where in cells your gene acts.

Question 24

promotor region of a gene

Answer 24

tells the gene when and where to be active

Question 25

transcribed region of a gene

Answer 25

encodes for the protein

Question 26

chimeric genes

Answer 26

promotor regions are swapped

Question 27

ectopic expression

Answer 27

driven by a promoter from a different gene

Question 28

over/consituitive expression

Answer 28

driven by promoter elements from pathogens
designed to express at high levels in all plant cells.

Question 29

wild-type gfp

Answer 29

put it into a plant, mRNA is translated, the protein folds to make the barrel.

in the middle of this barrel is the fluorofore.

fluorofore absorbs blue light but emits green.

Question 30

advantages of GFP

Answer 30

1. no need for substrate, active once protein has folded.
2. illuminate cells with UV using fluorescent microscopy
3. live, cell specific imaging
4. small - can be fused to other proteins to visualise their location without affecting their ability to move or function.

Question 31

disadvantages of gfp

Answer 31

not really quantifiable - hangs around in cells for a long time.