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SouthCity High School: Student BC -  25th April 2pm

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Tangaroa College: Karen, Bonnie and Ms. Folkard 28 August 2009
We were wondering how we could apply this knowledge in a scholarship context? We were looking at the Scholarship exam paper from 2005, and we found that Question 1 could be altered to plants. Is there a way we could write a question similar to this about plants and transgenics, as we believe it would help us greatly during our revision?


Rosemary Bellamy (Tutor) Plant Sciences Group SBS Uof A 2nd September

Well, given the importance of plants to horticulture and agriculture in New Zealand one would like to think that could happen. There has been a lot of selective breeding and marker assisted breeding done with economically important crops. Apples are one example. You might like to look up what the apple group is doing at Plant & Food (previously Hort Research). The techniques used to develop transgenic plants are a little different from those used with animals – some might say more effective - and of course the public are not ready for transgenic crops. However a lot of selective breeding with genetic markers has been done and this involves molecular skills that could be discussed. Rather than transgenes, there is increasing interest in transforming plants with genes from within the same species or genus e.g. from apple to apple or from a member of the solanaceae to potato. This is feasible with plants because there are so many varieties available.


Selective Breeding vs Transgenics Jacquie Bay and Helen Mora 3rd September

A possible question for discussion in class around this would be to consider the difference between a "transgenic organism" that uses genes from the same genus vs a transgenic organism that introduces genes from another genus. Compare and contrast same-genus 'transgenics' , inter-genus transgenics, and selective breeding. In your comparison think about the how same-genus and selective breeding are similar and/or different to inter-genus selective breeding.  Don't forget to think about the vector that is being used to insert the new gene if transgenics is being used.  





Transgenic Arabidopsis ABP1 inactivated



The scientific research team developed understanding of the role of Auxin Binding Protein 1 (ABP1) in auxin action using two key biotechnologies:

  • Transgenic plants
  • Polymerase Chain Reaction

By creating a transgenic plant where a gene / protein could be turned on or off, the scientific team worked out what ABP1 did in the cell and therefore the plant.

(a)        Explain how a transgenic plant is created and what biotechnologies are required to do this.

(b)        Describe what mechanism the team used to turn the gene on and off

(c)        Explain how PCR technologies are used to measure whether the gene is turned on or off

(d)        Why did they also need to make observations of the plant using microscopes and simple measurements of leaves and roots?          

                             


JB, MW, & DW From Sacred Heart College Auckland 21/8

1a. A transgenic plant is created through the process of transgenesis. This starts with the use of restriction enzymes cutting the desired gene from the genome of another organism. The same restriction enzymes are used to cut the plasmid vector to ensure the sticky ends are the same before ligation. The gene is then inserted into the plasmid and introduced back into the bacteria. This bacteria is then cultured in desirable conditions to produce many bacterium with the foreign DNA. This specialised bacterium is called Agrobacterium tumefacins and is crucial in the process of creating transgenic plants. The plant is then infected by the bacterium with the foreign gene.

1b. Repressable receptors were attached to the beginning of the pre-existing ABP1 gene allowing its expression to be turned off at will with the judicial application of ethanol.

1c. mRNA is extracted from the cells and reverse transciptase is initiated on the mRNA to produce cDNA with flourescent labels. This sample is subjected to PCR with only the primer neccesary for the gene in question allowing the final product to be analysed for the presence of that gene. If the gene is turned on its mRNA product will be synthesized and amplified during PCR. If the gene is turned off there will be no mRNA product and thus no amplification. This will be shown when run through agarose gel (gel electrophoresis).

1d. Microscopic measurements taken of the plants leaves and roots allowed the extent of the effect of the absence of ABP1 in the plant to be analysed ie. cell size.


Nathan Deed (PhD Student), Rosemary Bellamy (Plant Science Group) 25 August

Thanks for your prompt answer Sacred Heart! 

You have given a very good start to part (a) giving a clear description of how the gene would be inserted into the plants. 

How would you know (as the scientist) that you had been successful in inserting the gene?  This is an important step.

For part (b), think about which gene was being turned on and off by the ethanol.  Look back at the seminar question that came in from Waimea College.

For part (c), remember PCR amplifies DNA, not RNA.  Have another look at your answer and see if you need to change anything.

Well done, we look forward to your further responses. 


MW Sacred Heart College Auckland 27/08

Thanks for your response! Been thinking about our 1c answer and realize how we got that wrong...but cannot work out how to use PCR to measure gene expression. I understand how microarrays could be used...but not PCR...is there a direction someone could point me in?

Thanks in advance 

 

Jacquie Bay 27th August
You are not totally wrong. Your statement in the 1st sentence is correct. Where you go wrong is when you say that if the gene is turned on its mRNA product will be synthesised and amplified. If the gene is turned on, the tissue extraction will contain mRNA for that gene. Once the mRNA is isolated and the reverse transcriptase reaction applied, cDNA for that gene will be produced and amplified. In real time PCR, that would show up in the curve as the reaction proceeds (due to the fluorescent labels). In normal PCR, you would as you say run a gel and a band of DNA the size of your target would show up, telling you that this gene was present.

Pat & Mike Horowhenua college 27/8

1b) A gene that represses the gene expression for producing ABP1 is inserted into the plants DNA. To turn the repressor gene on ethanol is used as the trigger. This enables the production of repressor molecules which binds to the ABP1 gene. This slows down the rate of transcription of the ABP1 gene thus less ABP1 is produced. However if ethanol is absent then the repressor gene is turned off so ABP1 is produced. The results expected from the scientist's experiment would be that the transgenic plant with ethanol would have abnormal leaves and shoot growth in comparison with wild plants. The transgenic plant without ethanol would have leaves and shoot would grow quite normally. For scientists this shows that ABP1 helps regulate cell growth because when the repressor gene is active then less ABP1 is produced thus the plant will develop abnormally. If the repressor gene is not active then ABP1 is produced normally and normal plant growth occurs.This also shows if the gene was properly inserted or not.


Rosemary Bellamy (Tutor) Plant Sciences Group SBS Uof A 2nd September

b) A good answer and shows that you understand well how a repressor that prevents the expression of a gene would have an effect on the presence or absence of the protein that gene codes for. You also describe well how a plant designed to have an extra gene that controlled the expression of the repressor, and if that gene could be induced with ethanol, this would enable some external control of gene expression. However that is not quite what Karine et al actually did. If you go back to the beginning of the seminar you might pick up that knocking out the gene for ABP1 was not useful because the embryo did not grow – a knockout was lethal. Instead they produced an antibody to the ABP1 protein (probably in a rabbit). Check out what happens when a protein and its specific antibody meet. Then they inserted the gene (a mini-gene) for the small part of the antibody that interacts with ABP1 together with the promoter that could be induced by ethanol into a plasmid and transformed plants with this. When these plants were exposed to ethanol (vapour) the gene for the antibody was turned on, the antibody inactivated any ABP1 protein present in the cells and for a while the cells would have no functional ABP1 even though the gene for ABP1 had not been turned off. Very elegant. So going back to the question what mechanism did the team use to turn the gene on and off – you have to be careful which gene you are talking about. The gene for ABP1 – was it turned off? What mechanism was used?


Tangaroa College: Karen and Bonnie 28 August 2009

a) A transgenic plant is one with genes added or removed to give new traits to a plant e.g. resistance to pesticides. Karine was able to choose a gene that would repress the ABP1 gene in Arabidopsis, which is a model plant and has had it's genome sequenced and mapped. DNA is collected from the source plant, and the desired gene cut out using restriction enzymes. A plasmid from Agrobacterium tumefacins is removed from the bacterium, and using restriction enzymes again, the plasmid is cut so that the ABP1 repressor gene can be spliced by ligation to form a recombinant plasmid. This plasmid is then introduced to Arabidopsis plant cells, where the part of the plasmid containing the ABP1 repressor gene is integrated into the plants chromosomal DNA. These cells are then grown using tissue culture within ABP1 repressor gene.

b) As plants do not develop when ABP1 is not present, down-regulation after early development is the only way to see the effect of ABP1 on a plant. In transgenic Arabidopsis plants, exposure to Ethanol vapour would turn on the ABP1 repressor gene, stopping ABP1 from being produced. In theory, the gene could also be turned off by removing the plant from the ethanol vapour, allowing ABP1 to be produced.

c) qRT-PCR was used to amplify and analyse target DNA molecules, to identify whether the ABP1 gene was being expressed or not. This is done by removing RNA from the plant cells, and using the reverse transcriptase reaction to produce complementary DNA (cDNA). This gives an idea as to what genes are being expressed in the plant. Then PCR can be done, using specific primers to target the ABP1 gene, the DNA is also fluorescently labelled so that a quantitative analysis of the PCR product can be undertaken. This product is then analysed using Gel Electrophoresis, and we can find out whether the ABP1 gene is being expressed or not.

d) The measurements of leaves and roots provides a quantitative comparison of the different plants. This allows scientists to prove that ABP1 has a definite effect on the cells of a plant, in particular their size and division. It also allows for other scientists to analyse the work of Karine and her team without the need for photographs.


Rosemary Bellamy (Tutor) Plant Sciences Group SBS Uof A 2nd September

a) You have described the big picture well. You do not explain how the recombinant plasmid is introduced into the cells however. Have you heard of floral dipping? This is the usual way with Arabidopsis. Plant cell cultures on the other hand are transformed in a number of ways (heat shock, electric shock etc) that make them more permeable to the DNA. Plant cells are nice little things because they are totipotent (check that word out) hence a single transformed cell can (ultimately) be grown into a whole plant. You do not mention how the scientist would know if the plant or the cells had been successfully transformed. How might they do that? I think this part of the question was quite general but you do refer to what Karine and her group did. Check back to the response I gave to the students from Horowhenua College. She did not insert a gene that repressed the gene for ABP1 but instead in a round about way inactivated the ABP1 (protein) which of course has the same effect.

b) And that last statement is one of the beauties of the method. The scientist can take measurements without ABP1 and then stop the expression of the antibody(and hence prevent any more inactivation of ABP1 and follow what happens when there is active ABP1 again

c) Again a good response, well done, just one thing to correct. The result of a simple PCR is an amount of amplified DNA of a certain size (determined by where the primers were designed to anneal) and to show that you have a product, gel electrophoresis is done. If you get a band of the expected size, you are entitled to say that the DNA of interest was present in the sample. With qRT-PCR however the result (the fluorescence) is recorded over the whole series of cycles, recorded automatically on a computer and displayed as a graph. So no gel is needed. You clearly understand the connection between mRNA/gene expression and cDNA which is great. Simple PCR is not only used for gene expression studies. Do you know of other times is it used?

d) Yes, the purpose of the science is to identify what ABP1 does – its function – so if the hypothesis is that it has a role in moderating auxin response and one effect of auxin is to increase cell size by expansion and cell division, the measurement of plant size would give some indication of that. Tables can show that comparison between the treated and untreated plants but photographs are more visual and would also be given in the results. You might want to consider why the scientist would then go the extra step and bother to take photomicrographs of the root, leaf and stems. Look at the photographs supplied with Challenge Two.







Challenge 2










The scientific team found that as well as affecting shoots and leaves, inhibiting Auxin Binding Protein affected the growth of roots. As with the shoots they ran tests with and without ethylene to check that the gene was being turned off.The photomicrographs on the right shows what the cells looked like in the roots. The white arrows show how extensive the meristematic tissue was in the roots.
Meristematic tissue is the tissue in plants that can still divide—it is undifferentiated. We know that when ABP1 was inactive the roots did not grow.
What does the information in the micrographs above tell us about the role of ABP1 in root growth?
Why did having ABP1 turned off stop the roots from growing?


Pat & Mike Horowhenua college 27/8

Hypothesis: ABP1 controls the rate at which cell’s divide; however wouldn’t low ABP1 would mean less cyclin regulating cell division and comparing with cancer cells wouldn’t that imply uncontrolled cell division in cells? This can't be so, can it? as the decrease in ABP1 would imply fewer signals to growth genes and a decrease in transcription in cells thus the cell would grow very slowly. Therefore since the meristematic zone is where cell division occurs, this is like the main growth area, so if there is low ABP1 less growth occurs and therefore the meristematic zone is smaller this is vice versa for high ABP1.

"Stop the roots from growing?" is a bit wrong isn’t it? ABP1 is not the only Auxin receptor,there could also be many others growth receptors according to Dharmasiri et al and other scientists. We also have the TIR1 protein which with Auxin forms a complex and turns on the growth gene by breaking the repressor binded to the gene. Therefore i conclude that growth does not stop but would occur at a slower rate. Assumable Auxin is present.

problems we face: does ABP1 affect growth gene which affects elongation? and if so by what way? when we talk about growth response, does it mean cell division, elongation and differentiation of cells?


Rosemary Bellamy (Tutor) Plant Sciences Group SBS Uof A 2nd September

Some good comments there in your response to Challenge Two. But it is not quite clear to me what you thinking when you look at the photomicrographs. You have done some good work finding out about growth, cell division and receptors but be a little careful extrapolating what is known about animal cells (cancer )to plant regulation. You are correct ABP1 appears to be important for cell division and also for the expansion of cell walls after cell division. ABP1 is sometimes described as a mediator of auxin responses. ABP1 itself may not be a signal to growth genes – as the name implies it binds to auxin but what happens next is still unresolved. What the photomicrographs show is the arrangement of cells, shape, size, number in the root apical meristem in a plant with inactive ABP1 and in the normal (control) plant. Ask yourself again your own questions –but think, is there any evidence from these images that suggest that the absence/inactivation of ABP1 is interrupting the normal auxin mediated response of cell division and/or cell elongation? What would you expect to see if it was. (Cell differentiation is not part of growth per se but is when the fully grown cells become altered to serve a special role in the plant – packing tissue, conducting tissue, epidermal tissue support tissue etc). Hope that helps you are asking very good questions but may be the data is what you should look at. I will check out if auxin has been connected with cyclin – don’t think so. There ARE a lot of factors that affect important cell processes.



Tangaroa College: Karen and Bonnie  28 August 2009

The plant that had ABP1 production inactivated had little root growth, and also had a very small meristematic tissue zone. This tells us that ABP1 is very important in root growth, and that meristematic tissue is also important in root growth. We know that ABP1 is involved in cell expansion and that it also controls cell division, and that the meristematic tissue is the tissue in the plants that can still divide. Without ABP1, cell expansion and division do not occur, and this is shown by the smaller meristematic zone in the ABP1 inactive plant, and the lack of root growth.


Rosemary Bellamy (Tutor) Plant Sciences Group SBS Uof A 2nd September

Meristematic tissue continues to divide throughout the life of the plant. The shoot apical meristem (SAM) and the root apical meristem (RAM) continue to produce new cells that then elongate/expand then differentiate into mature cells in specialised tissues. You are correct in your observations but can you say which process is most affected in the ABP1 inactivated plant? It is pretty difficult from these images to be sure – what do you think and what could you suggest would be the next step to answer that problem.


Pat and Mike Horowhenua college 28 august

Ok cell expansion, can we see a difference between active ABP1 root and not active ABP1? From the diagram is doesn’t look like it. If there was a difference then we would expect the cells on the active ABP1 root to be larger and thus a smaller root cell to surface area ratio compared with the inactive ABP1 diagram. I do agree that ABP1 is involved in cell expansion as seen in the leaf examples. Question: does “cell expansion” occur in roots I do believe that it should but in the diagrams I do not see cell expansion occurring. I do see the zone of elongation though.

Rosemary Bellamy (Tutor) Plant Sciences Group SBS Uof A 2nd September

When looking at cells would you expect the difference to be huge? What might the scientist do to statistically state the inactivation of ABP1 had affected (a) cell division (b) elongation of cells. Unfortunately the diagram does not show the size of the cells in the control plant beyond the meristematic region to compare with the experimental plant. But what can you say about the size and number of the cells in the actual meristem? Roots certainly grow and they do it through the root apical meristem. Lateral roots are formed from mature tissue in a region called the pericycle, and lateral roots will also have an apical meristem.


Campbell Horowhenua College 1st September

ABP1 is the auxin receptor and this provides the stimulus. In the ABP1 inactivated roots, it is likely that most but not all ABP1 are inactive, there is less meristematic tissue as the detected level of auxin is lower than the detected level of auxin in the ABP1 active plants. The cells are generally larger in the ABP1 plants and there is less meristematic tissue, which suggests that ABP1 is responsible for sending the stimulus for cell division in response to auxin concentration. The root width is greater in the ABP1 active plants as they are growing more actively in response to auxin. Having ABP1 turned off caused the root growth to be stopped/slowed, as auxin is an important plant growth hormone that, when detected in low concentrations, causes elongation and division of root cells. It is interesting to see that, on average, the transgenic plants that were not subjected to ethanol grew longer than the wild type plants. This may be because a little of the ABP1 was repressed by the new gene (which may have been expressed in low levels as eukaryotic gene expression is like an equilibrium, right?) which would have lowered the detected auxin level and therefore promoted it more than the wild type plants (because if the detected auxin concentration was too high, root growth would be inhibited).


Rosemary Bellamy (Tutor) Plant Sciences Group SBS Uof A 2nd September

Do you know that there will be” less auxin” in the inactive ABP1 plants? Check what the letters ABP1 stand for (Hint:Auxin - -). You have given some really good comments and have looked well at the diagrams, and well done to note that the concentration of hormones( like auxin) are very important because each part of the plant will respond differently to high or low concentrations. Plants have to be tricky this way. I am not sure if equilibrium is quite the way I would express gene expression. Certainly the processes by which genes are turned on/off are reversible but there are so many factors involved I would be cautious about saying that it is a state of equilibrium. Gene expression can be altered at many places – at the time of transcription, during translation, and also post translation – regulation of the protein.