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Lecture 14

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University of Toronto St. George
Jennifer Harris

Lecture 2: Transcriptome & Chromatin - Last Lecture: Central dogma: DNA  RNA  Protein – showed that this was an overly simplified, and not surprisingly dogmatic way of looking at the way in which molecular mechanisms function in our cells – had very elaborate view of the central dogma & we focused in on the interplay b/w DNA organization. - See DNA wrapping around the core histone octamer – going to bend back. The interaction b/w DNA & histone proteins & indeed, non-histone proteins (not going to talk about those) – the building blocks of chromosomes. - If you were take all 23 pairs (all 46 chromosomes) in each of your cells, stretch them out so that they’re a linear piece of DNA & put those end to end together, they would be 2 nm long. - Realized that this poses a particular problem for cells – how does the cell access that highly condensed, highly compressed chromatin structure in your chromosomes to actually do things like replicate or transcribe? Slide 1 - Consider that you are a walking disco because you think about a disco at a night club, the lights are flashing on and off, in your cell genes are being turned on and off all the time - You are like a little disco - Now we’re going to talk about what genes are transcribed Slide 2 - What is it that makes the differences between each of us, unless we have an identical twin beside us, we look different and indeed if you look at your hand, the cells within that hand must be different - What are the things that define those differences, what are the molecular mechanisms in this course that define those differences? - Today: going to take a look at the regulation of that process & how it plays against the regulation of transcription. Consider going from the genome (info content) to the transcriptome (info content made available for doing things like building cells). Slide 3 - One thing that distinguishes the cells of two different organisms is that the genes are transcribed at different levels so you can imagine Gene A in human are transcribed in high amounts versus in the chimpanzee, less so that means less protein is made as well - So one of the key features is that the differences in transcription will define our cell types, tissue types and what kind of organism we are - In each case we have different amounts of transcription - Consider what it is that makes the differences b/w each one of you? What are the things that define those differences? - Let’s consider a primate and compare it to another primate (he’s showing the PowerPoint slide of him and a chimp). There must be things that define the differences between a chimpanzee and the professor just as there are differences between the person sitting beside you and yourself. - Let’s look at a model cell with only 3 chromosomes, both chimps and professors have more than just the 3 chromosomes, more like 46, just like the rest of us. We are just going to look at the 3 idealized chromosomes in these two idealized eucaryotic cells. We see the nucleus and the three representative chromosomes and on each of those chromosomes we see representative genes A B C and D. It turns out that these genes between these two chimps are very similar – but there must be something different about the way in which those genes are used that defines the differences b/w those 2 chimps – transcription happens & what happens as a consequence of transcription is that not all genes are used equally depending on the tissue, depending on the individual, depending on the species in this particular case – so different genes get used differently that is why we’re a walking disco because in each cell with its 46 chromosomes and round numbers 26,000 genes in duplicate one pair inherited from mom and one from dad so 26,000 x 2 are used differently. Slide 4 - Here is the simplified version of the transcriptome of two cells, all the mRNA of one cell versus the transcriptome of the other cell - How do these transcriptome differ, one way is by using microarrays - Large amount of messenger RNA will cause greater amounts of Protein A and C but small amount of protein B so there will be a different phenotype as a result and in the chimpanzee a different combination of protein amounts will results in another phenotype - One way to test this is through using a microarray - You can think of the genes being used differently as lights going on and off - So genes flipping on and off is the walking disco and it’s those genes flipping on and off and being differentially transcribed that makes up those differences because when we take a look at those transcripts what’s going to happen is on the basis of the quantity of transcripts that we have of each of those different genes, we’re going to end up with different amounts of protein made from them. - So we see lots of protein A very little protein B and intermediate amount of protein C but no protein D in the first slide because there are no transcript made for D. As a consequence you have phenotype 1 – odd looking middle age chimp 1. - On the right side you get a small amount of protein A, intermediate amount of protein C and a lot of protein D but no protein B made because there is no transcript made for it. We end up with phenotype 2 as a consequence due to the difference in amounts of protein being made. - How do we know this occurs? As biologists we know it occurs because of experiments and today we will focus on microarray analysis. Slide 5 - What you do is level the mRNA, the cDNA made from the mRNA, you take the mRNA and make cDNA and label it with two fluorescent dyes, one would be green while the other will be red. - Then you can apply it to a microarray which can be a glass slide on which dotted are different genes so each dot is a different gene so you can array them out so that you know exactly what DNA has been placed on what dot - You see 4 dots in the slide and they represent parts of Gene A B C and D - Now we are going to do something like a reverse northern blot - WE are going to put mRNA or cDNA on that plot so you can imagine different scenarios that you would see in some cases with gene A there are a lot of mRNA for gene A so we would expect to see more greenish, for B we have more in humans so its greenish, for yellow equal amount of transcription in both animals so it will be a blend of the two colours and lastly, there are occasions when the chimpanzee expresses the gene but not humans - We can compare the relative amounts of the two samples - Usually we use the colours red and green but we can use others - In microarray analysis what you’re doing is you’re measuring transcript abundance by comparing it between two cell types or two organisms. - How do we do that? We start by labeling transcripts, it's explained a bit in the textbook but **all we need to know for the exam** is that the transcripts are differentially labeled. - On the one hand from chimp 1, we will label with green fluorescent dye and from chimp 2 we label with red fluorescent dye and the amount of fluorescence that gets incorporated for each transcript will reflect the amount of the given transcript that is present within the cell. - Somehow what we want to do is capture each of the individual transcripts and measure the corresponding fluorescence. The way we do this is that we capture them using hybridization. Through process of hybridization those transcripts are captured individually on the surface of a solid support, on the surface normally of a glass slide. On a glass slide you can imagine seeing 26,000 spots each one corresponding to one of the genes in your cells/chromosomes, and each spot is actually a piece of DNA – it is DNA in excess to how much transcript should be there so there an awful lot there and it's spotted on that glass slide. - What happens is that they will adhere to those labeled transcripts and will hybridize to those labeled transcripts so it will function to capture the transcripts and so we have spots corresponding to genes A B C and D. Each of them is a DNA spot but it’s important since it is a single stranded DNA and it is basically waving saying ‘yo RNA molecule I’m ready to hybridize’ so it will grab onto that RNA molecule and as a consequence you’ll get a corresponding amount of fluorescence there which is relative to the amount in the sample. - What happens though in the old school way of doing this is that you hybridize the two pools of DNA simultaneously so that the relative amount of red versus green fluorescence will tell you there is more transcript in chimp one versus chimp two or vice versa. - Basically once you hybridize all those spots, for the gene A spot recall we got lots of transcript from chimp 1 so lots of green fluorescence but much less red fluorescence from chimp 2 so we will have a greenish spot. For transcript B, there is only transcript that is green labeled and no red labeled so we will have a very bright green spot because there is nothing to compete with it. Transcript C we can see an equal amount form chimp 1 and 2 so this spot on the slide would appear yellow. We have a lot of transcript D from chimp 2 and none from chimp 1 so we only see red fluorescence so this indicates how much transcript there is from one organism relative to the other or one cell type relative to the other or one tissue, whatever you want to compare. - This sort of analysis using microarrays is called transcript profiling. Slide 6 - Here this is the principle from the textbook in greater detail so in this example on the left its from our textbook and the right one is from another textbook that’s better - You isolate the mRNA you reverse transcribe it and label it with green and red dye and hybridize it onto the microscope slide that can have thousands of genes, and the DNA (cDNAs) will hybridize with the DNA on the blot on the slide and the condition is that the DNA can be single stranded so you can end up with variation of colours telling you what gene is expressed when serum is present versus not present - In the left sample it’s the same idea and each block is a different DNA and you can see green yellow or red, some cases you have black so that means no hybridization meaning that particular gene is not expressed - This tells you that some aren’t expressed, some are elevated in some condition etc - The serum has growth factors - Robots shoot the DNA onto the plate so we know that on this position we have specific DNA - This is shown in our textbook, it's effectively what he described earlier, organism all labeled red and organism 2 labeled green. - The transcripts are then hybridized to the microarray & we compare the red versus green fluorescence versus yellow fluorescence which indicates you’ve got a lot of transcript from sample 1 versus sample 2 vs. an equal amount in both. - It is also shown nicely in his other example because it provides more detail shown in pictorial form what he described in words – take a look at it and it explains what he said once again. Slide 7 - Instead of looking for one mRNA you’re looking for thousands of mRNA at once and the probe are on the slide so you have thousands of copy of that DNA on that slide and at another location you have another DNA - The transcripts are labeled with red or green and the brighter green there is the more of that transcript there is - You can look at the amount of fluorescence and it will tell you approximately how much mRNA there was - The way to think about this is that given what you know what northern blot is, think of it as a reverse northern blot where instead of looking at one gene at a time, you’re looking at potentially 26,000 genes at a time and what you have as opposed to having your transcript fixed on a solid support and hybridizing a DNA probe, you have reversed it where you have your DNA probe on the slide in excess and it functions to capture the transcripts that are labeled and the amount of fluorescence label indicates the amount of transcript made. - This is actually old school – we don’t do things this way any more. Slide 8 - The reason why was people weren’t very good at microarray experimentation, the reproducibility is not as good as they are today - Today they do two microarrays that are identical and they compare the two - IN this case what you do now is that you have one sample with cDNAs and you apply it to a microarray and then you apply the other messenger RNA cDNA to another slide and because these slides are so well made, they have identical amounts of DNA you can compare the two together - By the computer - In these days, the way we analyze transcript abundance is that instead of comparing transcripts on one slide, you compare them from two different slides. The reason why people compare them on one slide originally is because there was very high slide to slide variability so the best way to compare two samples was to compare them on the same slide. Now we don’t worry about that, quality control is so great that one slide will work as well as the next slide. - The way we do it is we use one fluorescent dye and use two different slides and now what we end up with is amount of abundance on the slide reflecting how much transcript there was. So we can see (for organism 1) lots of transcript A so lots of fluorescence, small amount of transcript B so there’s a lower amount of fluorescence, intermediate amount of transcript C so an intermediate amount of fluorescence and no transcript D so no fluorescence and you can do it for organism 2 as well: a little bit for gene A, none for gene B, an intermediate amount for gene C and none for gene D. Slide 9 - The computer will take the two separate information and create the 3 thing rd that shows the comparison of the two showing which gene is off or on - The red is that the sample in the chimp is only in the chip but not sure why the gene c is black but maybe because the sample amounts are exactly equal - This is called gene chip transcript profiling and looks like the thing the hand is holding - The company made these gene trips and making lots of dough - Now how do we make the comparison between the two organisms to say how much transcript is there? This is even more intuitive because all you need to do is to ask is there more fluorescence for this gene on this slide compared to the same gene on the other slide? (Another benefit he doesn’t mention is that you have a standardized fluorescence so you can use one of these slides to compare with all the other slides you make instead of doing a double hybridization for each pair of organism). - The comparison is all made by a computer in silico (on silicon chip). The output you get from the computer looks exactly the same as the original output described earlier where you have more of gene A in the first organism, a lot of green for gene B compared to the second organism as well so greenish, the comparable amounts shown in black and a lot of expression in the second organism shown in red. This is arbitrarily done so that one is assigned green color the other is assigned red. - This is known as gene chip profiling because it's done on a gene chip, an Affymetrix (company name) gene chip on the slide. The little square lets us make very accurate and controlled measurements – there aren’t 26,000 spot but there are hundreds of thousands of spots that allow us to have this level of high quality control on the chip. Slide 10 - Now that you have this information you have to manipulate it in different ways, if you are comparing organism to cell conditions what’s the difference - You can plot every single dot and say whether a transcript is found in relatively the same amount or in one condition you have high amounts of one and low amounts of the other - So for the green there was a lot of that particular gene but in condition 2 there was a lower amount so there was a decrease in the transcription of the gene in condition than in condition 2 - You are looking for the ones that are outliers, the ones that lie around the line are the ones that are identical in both conditions so we want to find the odd ones - How do we make the decision where the amount of transcript in one organism is actually greater than that in the other organism? - This is done using statistics, in most instances, the amount of transcript that is expressed in one cell versus another is going to be pretty equivalent because most genes are known as housekeeping genes so they’re just doing normal cellular processes. - If we were to compare transcript abundance between them, so comparing transcript abundance in condition 2 (chimp 2) compared to transcript abundance in condition 1 (chimp 1), and we ask what’s the relative transcript abundance between the two, we would find normally there is a one to one correspondence. For one transcript in one organism, there is one transcript in the other organism because those genes function in an equivalent fashion and therefore they have equivalent abundance, 1:1 and it's always equivalent and that is what that red line is meant to represent up the middle. - There will be some scatter around that, close to 1:1 correspondence and most genes will look like that so we can establish a statistical boundary indicated by the black line that says anything within these bounds we should consider as equivalent abundance but anything outside those bounds, we should consider statistically as being different in one organism vs. to the other so there is higher transcription in one organism as vs. to the other organism. - So something that is of higher transcript in condition 1 (chimp one) would appear outside those bounds and be colored green as a consequence, it gets that artificial colour. By contrast if there is one transcript more abundant in condition 2 and you see there are over – 3 transcripts on average relative to on average one transcript in condition one that ends up getting a red colour so 3 versus 1 there and 3 versus one there – a computer makes those decisions and on the basis of that, statistically here are the subset of genes that show a difference in transcript abundance from one condition to another. Slide 11 - You can use that information to create heat maps called clusterograms Clusterogram Heat map - In this experiment what it is showing is 5 different conditions compared to a reference condition and each of the thin, thin lines represent a single gene and it shows whether a gene is higher or lower under specific condition and are grouped together, lower condition are up at the top and the higher transcription are found at the bottom - You can see these groups are clustered together at the top because they’re all
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