1.2 Genotype to phenotype

Question 1

This lecture will focus on advances in transgenic plant work in detail: gene structure, how to build a new plant gene, which genes are used commercially, use of reporter genes to study link between geno and pheno.

Question 2

Give 2 issues which must be resolved in order to design synthetic genes.

Answer 2

**1. Transcriptional initiation requires interaction with distal promoter elements. It’s not enough to just stick a bit of DNA into a plant genome, since
*** Specific control sequences are necessary for the proper expression/regulation of synthetic genes, via interaction with transcription factors, RNA polymerase & other regulatory proteins
* Host sequences flanking the inserted gene influence its expression too
So, when we insert foreign coding DNA into the host plant, we need to also make sure that appropriate regulatory sequences are positioned adjacent to it.

Question 3

Name 2 things you have to do to make synthetic genes work.

It’s not enough to just stick a bit of DNA into a plant genome

Answer 3

• Appropriate regulatory sequences must be positioned adjacent to the synthetic gene once it is inserted, since:
  
  • transcriptional initiation requires interaction with distal promoter elements
  • host sequences flanking the inserted gene influence its expression too
• Synthetic genes must include sequences which mediate the interaction between host machinery & nucleic acid sequences, in order to allow proper mRNA translation

Question 4

Explain why transcriptional initiation requires interaction with distal promoter elements.

Answer 4

• Core protein elements of RNA Pol II bind the TATA box sequence (green) ~25 bp upstream of the start site (red)
• But the complex formed is insufficient for specific initiation
• So transcription factors are required: distal promoter elements (enhancers) contain binding sites for transcriptional activators or inhibitors, which then regulate initiation via making contact with the core RNA Pol II via mediator/regulatory proteins

Question 5

Standard promoter notation…what does +35S::GFP mean?

Answer 5

35S is the promoter & GFP is the open reading frame.

Question 6

Describe how post-transcriptional modification occurs in eukaryotes.

Answer 6

• 7-methylguanylate cap is added to 5’ end of pre-mRNA, and a polyadenylate cap to 3’ end
• Most plant pre-mRNA contains introns which are removed:
• Spliceosomes (ribonucleoprotein complexes) detect intron-exon junctions & branch sites, then excise the introns
• mRNA is stitched back up via transesterification reactions mediated by the interaction between host machinery & nucleic acid sequences

Question 7

Explain why synthetic genes must include sequences which mediate the interaction between host machinery & nucleic acid sequences.

Answer 7

We need to account for post-transcriptional processing in order to allow proper mRNA translation & avoid aberrant cryptic sites.

Question 8

In order to make synthetic genes work, it must contain sequences which mediate the interaction between host machinery & nucleic acid sequences, and appropriate regulatory sequences must be positioned adjacent to the synthetic gene once it is inserted.
What is the solution to this? Especially the 2nd thing…how do you control something external to what you are inserting?

Answer 8

We profit from the fact that basic DNA structure is quite conserved.
* Gene architecture can be organised into functional regions, which are conserved and therefore modular, meaning they can be exchanged between genes (provided the order and position is maintained) e.g. one jack to jack can be exchanged with another, same with one mic, but you can’t replace a mic with a jack
* A syntax has been developed to describe plant DNA parts, by defining arbitrary but standardised boundaries between domains (and made compatible with schemes for gene assembly via type II restriction enzyme

Question 9

Engineering single-gene traits into crops

The first wave of GM crops were given single genes. Name 3 examples of single-gene traits.

Answer 9

• Pest/insect resistance (eg using the Bt toxin)
• Herbicide resistance
• Virus resistance

Question 10

Name 3 reasons why you would include introns in synthetic genes which you are delivering to plants via bacteria.

Answer 10

1. to mimic the fact that introns are found in plants, so that the synthetic gene will transcribe better
2. introns might be regulatory and important for the expression of the synthetic genes in plants
3. often, plasmids inserted into bacteria will express the dna you’ve inserted via a plasmid – which might kill the bacterium eg an enzyme. so insert intron to prevent expression in bacterium

Question 11

What is the mechanism of Bt toxin action in insects?

Answer 11

• Insect ingests toxin
• As a result of proteases & low pH in the gut, toxin is activated & binds membrane receptors
• Causes formation of pores, triggering uncontrolled leakage of water & ions across epithelial membranes

Question 12

• What strain of bacteria produces the Bt toxin?
• Why is the Bt toxin suitable for use in insecticides, and latterly transgenic, pest-resistant crops?

Answer 12

• Bacillus thuringiensis (Bt) bacteria produce the Bt toxin. It produces a variety of strains specific to different classes of insect
• None of the toxin strains are toxic to mammals, so the crops are safe to eat

Question 13

Describe how we would produce transgenic, insecticide-resistant plants which produce the Bt toxin.

Answer 13

• We develop a synthetic gene which we insert into agrobacterium, for delivery into plants
• Must contain: promoter, intron, Bt coding sequence, transcription terminator
• Eg MON810 is a synthetic gene used commercially in GM maize, which contains the P-e35S promoter (from a virus which normally infects plants), hsp70 intron, cry1AB (i.e. Bt) coding sequence, plus T-nos (nopaline synthase transcription terminator)

(however, pests can develop resistance)

Question 14

Name a common herbicide.

Answer 14

Glyphosate is a common herbicide which has attracted controversy, but it’s effective & non-toxic to animals.

Question 15

Explain the mechanism of glyphosate action & lethality.

Answer 15

• Glyphosate inhibits EPSPSase, an enzyme involved in the shikimate pathway which synthesises aromatic amino acids (amongst other things)
• Glyphosate travels quickly to apical plant regions, inhibiting protein synthesis & rapidly stopping growth
• Plant tissue degrades gradually. Chlorosis, yellowing & necrosis develop until the plant dies from dessication & dehydration

Question 16

How would we engineer glyphosate resistance into plants?

Answer 16

• Glyphosate inhibits EPSPSase, an enzyme involved in the shikimate pathway which synthesises aromatic amino acids
• So we achieve glyphosate resistance by transgenically expressing a glyphosate-resistant enzyme, i.e. one which complements (compensates for) the defect in aromatic amino acid synthesis

Question 17

How can we combat herbicide-resistant weeds?

Answer 17

Stacking multiple transgenic herbicide-resistance traits, for better robustness.
* Stacking of traits is becoming more common, eg in maize/soybean/cotton – had lead to systematic naming systems, eg G11UF8-3111A

Question 18

1. What are reporter/marker genes used for?
2. Discuss the advantages of using a reporter gene compared to RNA extraction & analysis.

Answer 18

1. Deciphering/analysing the in situ activity of a single gene, when it’s expressed in a genome along with many other genes — ie allows whole-plant analysis.
2. Allows visualisation of where a gene is expressed, without requiring molecular analysis. Avoiding dismemberment (studying intact plants) avoids potential stress responses which may affect gene expression.

Question 19

How do reporter genes work?

Answer 19

• The promoter of the gene of interest is fused to a marker/reporter gene, which encodes an easily measurable enzyme which isn’t encoded anywhere else in the genome
  
  • E.g. a reporter gene could code for fluorescent proteins, luminescent proteins, or enzymes which can be histochemically localised
• Because the reporter gene is under the control of the promoter which it is fused to, it is expressed in only the cell types in which the gene of interest is expressed
• So the presence of the marker allows visualisation of where a gene is expressed, without requiring molecular analysis – i.e. allows you to analyse intact plants

Question 20

Name 5 common reporter genes.

Answer 20

• Beta-galactosidase (encoded by lacZ)
• Beta-glucuronidase, i.e. GUS (encoded by uidA)
• Chloramphenicol acetyltransferase
• Luciferase (luc)
• GFP

Question 21

Name 3 reporter genes which can be analysed by histochemical localisation.

Answer 21

GUS, luciferase, GFP

Question 22

1. What is GUS otherwise known as?
2. What gene encodes GUS?

Answer 22

1. Beta-glucuronidase
2. uidA

Question 23

Why is uidA a good plant reporter gene?

Answer 23

• GUS is a bacterial enzyme which has low endogenous activity in plants (ie few plants have a counterpart)
• GUS is stable and will hydrolyse a range of beta-glucuronides
• GUS can be easily assayed for histochemical analysis using X-gluc. Histochemical localisation allows sensitive detection of gene expression

Question 24

Name 2 methods of assaying GUS.

Answer 24

• specific histochemical staining using X-gluc (a beta-glucuronide)
• a sensitive fluorimetric assay using MUG (a different beta-glucuronide)

Question 25

Explain how GUS can be easily located via specific histochemical staining.

Answer 25

• GUS cleaves beta-linked glucuronide groups from many substrates, one of which is X-gluc, which is inactive by default
• GUS cleaves a sugar (an indoxyl monomer) from X-gluc, which is then oxidised, and then it dimerises to form an indigo precipitate
• Therefore, a blue stain indicates the presence of GUS, and therefore indicates expression of the gene of interest

Question 26

Histochemical localisation allows sensitive detection of gene expression. What is its disadvantage?

Answer 26

the process is usually lethal & the gene is hard to localise at subcellular resolution

Question 27

Why is the gene encoding luciferase a good reporter gene? 4 reasons

Answer 27

• Luc is an enzyme found in luminescent organisms, ie is not endogenous to plants
• Luc produces flashes of light in the presence of its substrate (luciferin or coelenterazine) and ATP. This means it can be used for histochemical localisation, allowing sensitive detection of gene expression
• Luc can be detected both in plant tissue extracts, and in intact plants (you just water the plant with Luc’s substrate), allowing non-destructive imaging
• Luc is single-use, in that once it has emitted light, it won’t do it again. Benefits:
  
  • Allows real-time visualisation of protein synthesis
  • Allows experiments to be automated, ie you can plot activity over time

Question 28

What 2 molecules are substrates of luciferase?

Answer 28

luciferin & coelenterazine

Question 29

How does GFP work?

Answer 29

• GFP is barrel-shaped, consisting of beta sheets surrounding a central alpha helix. Barrel is capped by alpha helical segments. Effective solvent cage
• Chromophore is produced via autocatalytic cyclisation & oxidation of the tripeptide Ser-Tyr-Gly, forming a multi-ring aromatic group on the central alpha helix (rather than a conjugated system)

Question 30

Why is the gene encoding GFP a good plant reporter gene? 6 reasons

Answer 30

• GFP comes from jellyfish, ie is not endogenous to plants
• The jellyfish GFP gene has been modified to allow efficient expression in plants
• GFP does not require the addition of a substrate in the way that Luc does (since the reaction which makes it fluorescent occurs in all tissues)
• GFP allows non-destructive imaging & can be localised on the subcellular level using microscopy (confocal, not fluorescence)
• A palette of different protein colours has been created via mutating GFP’s chromophore & surrounding amino acids
• Can be used to map whole-plant gene expression

Question 31

Fill out this table.

Answer 31

Question 32

Explain how fluorescence microscopy works.

Answer 32

• A molecule absorbs then emits light, but the excitation is shorter-wavelength
• Difference in wavelengths allows selective blocking of excitation light using optical filtration, in order to only detect fluorescence emissions of a chosen wavelength
• Excitation light is directed at sample by reflection from a chromatic beam splitter / dichroic filter
• Microscope objective condenses & focuses light onto sample
• Light from sample is collected by objective, then directed through the dichroic filter, then hits the detector. Allows it to be detected sensitively without being drowned out by excitation light

Question 33

Name an advantage & disadvantage of fluorescence microscopy.

Answer 33

Produces widefield images, meaning:
+ wide visual field
- but it has mediocre resolution and depth is hard to make out

Question 34

How does confocal microscopy work?

Answer 34

• Laser beam illuminates sample & scans along it, building up an image
• Confocal microscopes can differentiate between different wavelengths (other microscopes can’t), and can emit any desired wavelength
• Fluorescent signals from the laser excitation pass through the confocal pinhole, then are focused at the back plane
• Emission light from above or below the plane of focus is blocked by the pinhole in order to largely prevent its detection (eliminates out of focus blur, making it high res)

Question 35

Give 2 advantages of confocal microscopes.

Answer 35

• Can differentiate between different wavelengths (other microscopes can’t), and can emit any desired wavelength
• High resolution, such that subcellular features can be resolved down to less than a micron

Question 36

What type of microscopy would be used to obtain a GFP image which shows subcellular features?

Answer 36

must be confocal microscopy rather than fluorescence

Question 37

Name 2 disadvantages of confocal microscopes.

Answer 37

• More expensive than fluorescent microscopes
• Lasers shine intense light onto sample, which can damage tissue. Can’t examine the specimen for very long before damage is incurred