CUP1 gene / reporter assay

[methionine]

Hi,
I have a question about the CUP1 gene... I read a paper about it, and I understand that it is a nonessential gene attached to the actin intron, which allows yeast to grow in otherwise lethal concentrations of copper. However, I don't understand precisely how it works. Can somebody help me, either by directing me to a webpage, or something like that? Thanks.

-M

[Jsnell]

Hi,
I'm glad to hear that you have been doing some background reading before jumping right into your science fair. This is very reassuring. To find out more about the CUP1 gene, I encourage you to ask your teacher about it. Not only might he/she be able to help explain it better to you, but he/she can also direct you to valuable resources in the school from which you might be able to find more information. Another option is looking on the internet.

"CUP1 encodes a metallothionein required to protect cells from the toxic effects of copper in the medium. Copper ions enter the cell and bind to the N-terminal domain of Ace1p, a transcriptional activator, resulting in the folding of this protein to form a DNA-binding domain that recognizes upstream activating sequences in the CUP1 promoter. " This quick definition is one of numerous ones that I found under 'CUP1 gene' on the google search engine. You can use this site to find more information on this specific gene, but be careful that the sites which you are recieving your information from are reliable.

Another site which gives a concise, yet helpful definition: http://www.ncbi.nlm.nih.gov/entrez/quer ... t=Abstract.

Good luck with your project and feel free to post any other inquiries you have. Best of wishes!
-jsnell

[carolinethorn]

Hi M,

The Saccharomyces genome database is a good place to find out about yeast geens and has selected papers that yeast biologists have chosen that describe the genes. See http://www.yeastgenome.org/

Also as well as the Pubmed link that jsnall gave you there are gene description resources at NCBI. If you go to the link jsnell gave and type CUP1 in the search box and select "gene" from the pulldown on the left instead of pubmed then it will give results for the page with basic gene information.

Th extract that jsnell gave you talks about the promoter part of the CUP1 gene - the part that controls when the gene is switched on. That is the part used for making reporter constructs. And they said how that works to switch on the gene when copper is present. In your question though you asked about how the whole gene works to help yeast grow in high concentration of copper which is a little different. The CUP1 protein is able to bind copper ions (read the paper on SGD that descirbes how many it can bind per protein molecule). An ion that is bound up into a protein is not polarizing and so not as potentially damaging as one that is free in the cell. Especially to a single celled organism, keeping a balence of ions can be very important because of how it effects osmosis (think about why that might be and post back if you need help).

Best of luck,
Caroline

[methionine]

So... CUP1 is a genetic switch sort of thing, in a way similar to the lac operon (that I learned about in biology last year)?
Thanks for answering that for me... it was really helpful.
I just want to clarify one thing: Is this gene inserted into the yeast via a plasmid?

-M

[carolinethorn]

That's right. The CUP1 promoter is acting as a switch for whatever the reporter is (you didn't mention what your reporter assay actaully was ). It is a similar principle to using the lac operon except with Cu ions rather than sugars. Good job for seeing the parallels.
I would expect that this was inserted using a plasmid. It is possible that they could have made a recombinant yeast and inserted it into the chromosome by homologous recombinantion but I would need to see more details of the system to tell.
Best of luck.
-Caroline

[methionine]

Thank you.

I know this might be getting annoying, but... I still have another question regarding this assay--
What is the relationship between the CUP1 gene and the ACT1 gene? I read something about it, but if the ACT1 intron is put into the CUP1 gene... how could this be used to test the expression of another (third) gene? I need help putting the pieces together.

-M

[carolinethorn]

Hi M,

Its not annoying - a whole big part of science is that answering one question always seems to bring up more questions!

But we need more information about the reporter and system that you are using in order to help you on this. If you can start from the beginning with a brief summary of your project and the materials then we should be able to help talk you through it and figure it out.

-Caroline

[methionine]

the gene does not contain an intron, but one can put an intron (and the one that's used is the ACT1 intron) into the CUP 1 gene, so that it needs to be spliced to be produced. The promoter is a strong one, so that it is transcribed at high frequency and all you are measuring is reporter gene expression (if the intron gets spliced well, more CUP 1 is expressed, if it is spliced poorly, less cup 1 is expressed


The above was something that my mentor wrote me... I want to make sure that I understand what she is saying. My project involves assessing the effect of intron length on the degree of gene expression. I was planning to alter the length of the intron through PCR, and then measure gene expression using the CUP1 reporter assay.

I would like to know what the relationship between the actin intron (ACT1) and the CUP1 gene is, and how I will be able to use this to study the intron length of a third gene...
... you see, what I had originally planned to do was alter the intron of a specific gene (srb2), but now I don't know if using the CUP1 assay will even involve the srb2 gene/intron, if my mentor meant that we would test the actin intron instead.


Thank you!

[carolinethorn]

Thanks, now I understand what you are doing it's easier to help. This is a very challenging project with lots to think about, it sounds very interesting.

So it sounds like your system has the CUP1 promoter and the CUP1 gene and the ACT1 intron has been engineered into the CUP1 gene, which does not normally have an intron, so that you can measure splicing efficiency. Yes, you are correct that your mentor means you to test the actin intron rather than srb2 (at least at this point). I expect your mentor chose the actin intron because actin is a very well known gene or maybe because they had already made the plasmid (making plasmids can be tricky so it saves time and money to use one your mentor already has). It is not particularly related to CUP1. You could look up information about ACT1 on SGD ( the saccharomyces genome database) like I suggested doing for CUP1. It tells you the length of the intron.

You will need to talk more with your mentor about if when you look at intron length you will use different lengths of the actin intron by making deletions using PCR or if you will use an intron from a different/third gene that is a different length. Perhaps you can think about what are the pros and cons of those different methods (hint, think about how the sequence might be different and might have "enhancer" or "repressor" elements in it. - if you cannot find information on what these elements are, post back and I will help you)

Best of luck,
Caroline

[methionine]

Thank you!

That is true-- my mentor's lab specializes in the ACT1 intron, which is probably why she suggested it.
I just want to know one thing --- would it be bad if I simply did my experiment on this gene "just because" my mentor already had those plasmids? I'm speaking in terms of the science fair now. (I know this is slightly irrelevant, but since it still pertains to me using the same assay...) I understand that judges like seeing that the student has really made the effort and not simply had the mentor set up everything for them. ... I mean, this is not the case here, since I truly am trying to learn the concepts behind the procedures and I will be doing the procedures myself under my mentor's supervision. But would that still be looked upon negatively? I want to learn a lot from this project, but I also am looking forward to doing the best I can in my school's research fair.

On the other hand, I know of an experiment that also altered the ACT1 intron and asessed the splicing efficiency, etc. ... it might be interesting to compare my experiment with that experiment (done around... 1985, I think it was), because it was focused on ACT1 as well, but with different assays-- I believe they used Northern Blot (which might be too advanced for me at the moment anyway, although it also probably is a better(?) way of measuring gene expression in this case). ... Since this is my first research project, I suppose it could be alright if I did a similar experiment with a different and somewhat simpler assay. ... I might even come out with different results. ... Is this correct?

-M

[carolinethorn]

Hi M,

Its perfectly valid to use existing plasmids, people do it all of the time. When someone publishes a paper about a new plasmid, other scientists can write to them and ask from some of it so they don't have to make it again so using existing plasmids is an accepted practise as long as you acknowledge who made it.

You are right that what counts is finding out what the concepts are and doing the procedure yourself and trying to figure out what the results mean.

Doing experiments that ask the same question but in a different way or with a different procedure is an important part of science. Its called validation. It a result was only seen with one type of assay it might be due to some kind of artifact in the procedure. So i think its great that you have the other data to compare your results to. I would agree that you are probably better off doing a different assay to the Northern blot - using radioactivity requires training and careful monitoring and most investigators try to use alternative methods where possible. What is the assay that you will be doing? I am interested to here some more...

Best wishes,
Caroline

[methionine]

Hi,

From the information I have read and what you've told me, the way I will be really doing this is by plasmid construction. primers will be designed to insert introns of varying lengths into the naturally intronless CUP1 gene, and then external primers will be used to insert the CUP1 gene into the primer. Plasmids will... be amplified in E. coli, and then the yeast will be transformed. After, a yeast culture will be performed on Petri dishes with a copper-containing medium.

So the bottom line is that I will be observing how well the yeast grow on the copper-containing medium; I will be measuring the reporter gene expression (as opposed to the gene I had thought I was specifically studying). I suppose this could be good, because I think (please correct me if I'm wrong) that I might be able to really graph/get a good idea of what the relationship is... of the effect of intron length on gene expression, especially if I do enough petri dishes with different concentrations... because the question dwells on searching for a relationship between intron length and the degree of gene expression, I don't think it matters as much that I am studying any specific gene. My experiement is actually pretty simple, I guess.

I'm honestly unsure if this is completely correct, but my mentor was busy this week, so she was unable to meet up with me.

[carolinethorn]

Hi M,

It sounds like you have a pretty good grasp of the concepts what you are doing and the terminology, and should be able to convey that to the judges. I agres that you should be able to get a quantitative measure of numbers of colonies per concentration of Cu per intron size that you can graph. Make sure you have replicates as well as different concentrations so you can measure the mean number of colonies for each condition in case for some random reason one plate grew better than another. Have you thought about what controls your will use? Thats an important part of experiment design - if you havent already discussed it with your mentor i bet it would impress her if you can think it through on your own and come up with some.

I agree that its doesn't matter so much which gene it is that you study but CUP1 one does bring up a question to me - if the gene does not normally have any introns, how did you and your mentor decide where to insert the ACT1 intron into it? This might be important and is worth thinking about how it might have an additional effect on your results.

This is a really cool project and I am interested to hear how it goes.
Best wishes,
Caroline

[methionine]

Hi,

Yes, I have started thinking about my experiment design... I've actually been looking at the science fair rubrics so I will be able to make my project the best that it can be.
A control is something that doesn't change in an experiment; a constant. I understand that there are things like positive controls and negative controls, but I'm not a hundred percent sure on what would be considered "positive" and "negative."
Either way, the most obvious controls are that I will probably be inserting the intron in the same place (into the ACT1 gene). I will grow the yeast in the same environment/temperature/lighting conditions.
... I have to go now (on a school computer), but I'll think about it.

-M

[paulogoeb]

Some more sites to look at:
http://clarklab.nichd.nih.gov/rem_tar_ace_spt10p.htm

http://www.pubmedcentral.nih.gov/articl ... id=1398063

http://www.ncbi.nlm.nih.gov/entrez/quer ... ds=1427032


...this seems to be a very difficult project, but look at it this way--you'll be examining genes that even professional scientists are just beginning to examine!!

Paulo

[methionine]

Thank you, Paulo.

...I was thinking about controls, and I was wondering how I would be able to control/keep constant the inserts (of artificial introns) .... because the inserts will be of different lengths, of course they are going to have different sequences in some parts. That would mean that... not only will be variable be intron LENGTH, but it will also be intron SEQUENCE. I know, the sequences within the splicing signals haven't really been noted as having great importance concerning splicing efficiency/expression... but either way, it would still be considered a varible, is this correct?
I think that maybe just keeping the GC content within each insert constant will be the best that I can do.

Do you have any pointers for me, to help me in the right direction?
Also, can somebody help me understand what a positive and negative control is?

thanks, and happy holidays!

-M

[carolinethorn]

Hi M,

Happy holidays.

The positive controls are a situation or situations that have worked before with that procedure and should yield measurable results. It shows that the procedure worked and any change in results from other treatments are due to the treatments. The negative controls are where the procedure was not performed to show that the results would not have happened by chance. I think wikipedia probably has some definitions for this too, or any other type of encylcopedia. Maybe its easier to understand with an example...
Lets say you have a plasmid that has a kanamycin resistance gene that allows bacteria to grow on kanamycin antibiotic and you are going to delete parts of the kanamycin resistance gene to see which part of the gene is most important. The positive control would be the full plasmid and the plates of bacteria should grow as they had done before on the kanamycin containing plates, because they have the full resistnace gene. The test plates would have plasmids with parts of the kanamycin resistance gene deleted and they may or may not grow. Then the negative controls would have no kanamycin resistance gene plasmid or the plasmid with no kanamycin gene in it. Without the kanamycin resistance gene the bacteria should not grow at all on kanamycin containing plates showing that any loss of growth is due to loss of activity of the gene.
Does that make sense? Can you apply that logic to your experiment? We can help you talk through your ideas. And you might want more than one positive and negative controls for different parts of your procedure.

Good job for considering the GC content and that sequence motifs. What do you think about using the same intron sequence just cutting it into pieces to test shorter introns or adding extra pieces of the same sequence in tandem? What do you think about using the same intron sequence but inserting it into the plasmid n the opposite direction so that it "reads" backwards? Remember that DNA has direction and there are 2 strands so there are a number of ways of looking at the same piece of sequence. Of course some sequence motifs are "palindromic" (look that up if you don't know the word) which may effect tteh results too. POst some ideas and how you think they might help you determine information about your experiment, and we can help you think about the strengths and weaknesses of the different possibilities.

Best of luck,
Caroline

[methionine]

Hi,

Yes, I had discussed that with my mentor. I understand how DNA is double stranded, has complimentary strands, etc.. we had discussed something about how the DNA in the plasmid construct could be "flipped" and ... something about how we wouldn't be able to tell, and so that would be a variable / weakness... I need to clear that up.

I looked it up in Wikipedia, and it had this basic definition:
"The positive control in experiments reveals that a certain procedure works according to expectations and actually gives you the expected results. It demonstrates that the experiment is functioning as it should."
The negative control was a bit unclear to me... What I think a negative control is is a part of an experiment in which you measure things just to prove that it isn't affecting the variable you're testing? Please correct me if I am wrong about this.

So if I apply this to my experiment, following your example, the test/variable would be the different intron lengths inserted, and the positive control would be the complete CUP1 gene containing the entire ACT1 intron without any alterations? No, that would not be it because that would just be "another length," if you know what I mean. I'm honestly not sure what the positive control would be in this case.
The negative control would be ... I want to say that it is the CUP1 containing plasmid without any intron at all, but that doesn't seem right because the yeast would therefore grow anyway. So would it (the negative control) simply be the plasmid without any CUP1 gene or ACT1 intron, to show that it is the CUP1 which allows the yeast to survive on the copper-containing medium. We would expect that the yeast containing this plasmid to not survive on the copper containing medium.

Other parts of my experiment:
-intron inserts
* sequence
* length
* location in which the intron is inserted/elongated/shortened
* GC content
* method of insertion? I'm using overlapping PCR for all of them, I don't know if this even really counts as a control, but I'm keeping it ... constant..
-conditions in which to grow yeast in
* varying concentrations of copper when I grow the yeast (I don't know the ranges for these copper concentrations; I'll have to look it up.)
* I need to keep the temperature constant..

I have another question about the CUP1 assay - would I just be measuring gene expression by counting the number of surviving colonies to assess how well the yeast grow on the copper-containing medium? I don't know how else to put it, but counting the number of surviving colonies seems so... unscientific and prone to error; unreliable.
So, how is measuring gene expression in the CUP1 assay done? Do we really just "count" the surviving colonies, or do we look for certain characteristics of the growing colonies to see if they are struggling to survive, etc.? Is there any place where I could learn more about this?

Thank you.
-M

[carolinethorn]

Hi M,

I think your general understanding of positive and negative controls is good now. One thing to remember is that you can have more than one of each to address different aspects of your procedure.

You can use your plasmid with the complete CUP1 gene containing the entire ACT1 intron without any alterations as a positive control since your mentor has used this before and knows what to expect from it. Another positive control would be using the complete CUP1 with no insertion to show how mant colonies a CUP1 plasmid will generate. Your idea for no CUP1 as a negative control is a good one. Also people often use a no plasmid control to show that the yeast have to be trasnformed with the plasmid.

I am not a yeast person so do not know how standard the CUP1 assay is. I would think that someone has already demonstrated how the number of colonies for a specific amount of plasmid, at a specific copper concentration, is proportional to the activity of the CUP1 gene. So counting colonies would be scientific but you would want to have several plates in order to take a mean number of colonies per plate to control for minor differences between plates and check if it is statistically different between treatments. If no one has done this before you might need a pilot experiment to show how number of colonies relates to CUP1 activity. You would want it to be as linear relationship as possible so that any change in activity will effect the number of colonies in a proportional manner. If your plate is too saturated with colonies for the full length ACT1 intron then you wouldn't be able to see any enhancement effects of changing the intron size you would only be able to see a decrease in activity if one occurred - and vice versa, if you have too few colonies on your positive control it would be hard to see a significant decrease in activity. Does that make sense?

I don't know of a good resource to point you to about the assay. Hopefully you can get hold of your mentor to talk through this. Now that you have a good idea of what kinds of controls should be done and are up to speed on the lingo, you can have a good discussion about it and get their feedback. Since they know the assay and have a outline of how they see this experiment going you can get better advice than the general kinds of advice that I can give.

Best of luck,
Caroline

[methionine]

Hi,

Thanks!

I met up with my mentor today, and it turns out that I had the wrong idea-- it isn't about the NUMBER of yeast on the plates; it's about the CONCENTRATION of copper that the yeast are able to grow on. Now that makes sense. (Just thought that you'd like to know.)

-M