[User] Please transcribe this text

[Assistant] There are these small mini gene segments.


[User] Please transcribe this text

[Assistant] Uhm, here you see two different V's, two different J's.


[User] Please transcribe this text

[Assistant] And put them together so you can see four different B cells that have chosen.


[User] Please transcribe this text

[Assistant] Different V's and different J's to pair together.


[User] Please transcribe this text

[Assistant] Uhm, that actually happens by cutting and pasting the DNA, uhm, in that individual B cell.


[User] Please transcribe this text

[Assistant] So that the DNA looks different in that individual B cell compared to what it looked like in the rest of the cells.


[User] Please transcribe this text

[Assistant] And so now each of those B cells will have its own unique receptor that can bind to its own unique antigen.


[User] Please transcribe this text

[Assistant] And so we talked about how we have these little mini-gene segments.


[User] Please transcribe this text

[Assistant] Uhm, in the case of heavy chain, they are the V segment, the D segment, and the J segment.


[User] Please transcribe this text

[Assistant] Uhm, they encode different amino acids as part of the variable chain of the heavy chain of the antibody. Uhm, so we're gonna pick one V, one D, and one J.


[User] Please transcribe this text

[Assistant] Uhm, to make a heavy chain gene, uhm, which will be paired in front of the genetic material for the consonant region.


[User] Please transcribe this text

[Assistant] For the light chain, we're gonna do this process again. We're gonna be putting together a V-segment and a J-segment. Uhm, this is on a different chromosome. We're making here the variable region of, uhm,


[User] Please transcribe this text

[Assistant] The light chain, uhm, and this will be paired with


[User] Please transcribe this text

[Assistant] Uhm, some genetic information for the constant region.


[User] Please transcribe this text

[Assistant] And if you recall, we spent some time doing the math on this.


[User] Please transcribe this text

[Assistant] Last time to see how these, you know, smaller numbers of mini genes, like a hundred-ish, can give us over a million combinations of antibodies. Uhm, so,


[User] Please transcribe this text

[Assistant] We looked at the heavy chain and here you can see the heavy chain V options. We've got 40 of them.


[User] Please transcribe this text

[Assistant] The D options 23, the J options.


[User] Please transcribe this text

[Assistant] And then we've got all the genetic information for the constant region.


[User] Please transcribe this text

[Assistant] We can also see that there are two different light chain areas on two different chromosomes that encode the light chains Vs and Js. Uhm, I also pointed out


[User] Please transcribe this text

[Assistant] That uhm, the fact that we are actually cutting the DNA and removing the intervening sequence is important.


[User] Please transcribe this text

[Assistant] Uhm, so in the germline DNA, in those, in all of your other cells that have this DNA, that have not done the cutting and pasting,


[User] Please transcribe this text

[Assistant] The first v is really, really, really far away from this constant region information. There's 40 v's and all this stuff in between.


[User] Please transcribe this text

[Assistant] The, we can't really have interactions between DNA elements. Like this promoter that you can see in front of the V region.


[User] Please transcribe this text

[Assistant] And this enhancer that is downstream of the seed. They're just too far away that you can never interact. And so you will never get transcription at this part of the genome.


[User] Please transcribe this text

[Assistant] However, when we actually cut out all the DNA in the middle and put these segments of DNA together,


[User] Please transcribe this text

[Assistant] Now the promoter is much closer to that enhancer.


[User] Please transcribe this text

[Assistant] We can actually start to get interactions and we can get transcription at this locus.


[User] Please transcribe this text

[Assistant] Uhm, so this is one thing that really allows us to get transcription here.


[User] Please transcribe this text

[Assistant] And so this is the kind of general overview.


[User] Please transcribe this text

[Assistant] Of how uhm, some of this works.


[User] Please transcribe this text

[Assistant] Uhm, I've asked students a question, some questions about this. Uhm, and you'll see on the problem set that's due after break, there's a question that sort of relates to some of this. So I want to make sure it's really clear. I thought by asking the question a certain way, I was going to like help make things clear for people.


[User] Please transcribe this text

[Assistant] Um, we've got all of our Vs, all of our Ds, all of our Js. And this is in a B cell before it develops. There's an every other cell of your body too.


[User] Please transcribe this text

[Assistant] Uhm, and then we've got a bunch of constant region stuff. Uhm, so a lot of times when I think about this,


[User] Please transcribe this text

[Assistant] I think about my V region, so here's a V region. I think about a D region.


[User] Please transcribe this text

[Assistant] That's why it's great to be in this classroom with a really long chalkboard.


[User] Please transcribe this text

[Assistant] And there's a lot of constant region stuff. And it includes part that


[User] Please transcribe this text

[Assistant] That will give me IgD if I want to make IgD. It includes part that will make IgG if I want to make IgG. It includes part that will make IgE if I want to make IgE.


[User] Please transcribe this text

[Assistant] So this is a huge piece of DNA with all of these different pieces of information.


[User] Please transcribe this text

[Assistant] If you recall when I introduced the different isotypes of antibodies,


[User] Please transcribe this text

[Assistant] I told you my favorite mnemonic I use to help me remember them, which is MDs give everyone apples. Not only does that help me remember what the five of them are,


[User] Please transcribe this text

[Assistant] But it also helps me remember this order. M.D.'s give everyone apples.


[User] Please transcribe this text

[Assistant] Because that is the order of the constant region of genetic information.


[User] Please transcribe this text

[Assistant] Do the rearrangement of the DNA. So you can actually see that we put D and J together, cutting out the intervening sequence.


[User] Please transcribe this text

[Assistant] And we pick a V, put the V together, cutting out the intervening sequence. Uhm, notice that when we do that,


[User] Please transcribe this text

[Assistant] Like when we first put together this D and this J, we get rid of the intervening DNA.


[User] Please transcribe this text

[Assistant] Then we're going to cut out this bit of intervening DNA.


[User] Please transcribe this text

[Assistant] But everything on the outside is going to remain untouched. So first we're going to make these changes to the DNA. Then we're going to actually, uhm, make our


[User] Please transcribe this text

[Assistant] Uhm, mRNA, we're gonna transcribe that mRNA and originally the mRNA is gonna look pretty much the same as the, uhm, the DNA that we have.


[User] Please transcribe this text

[Assistant] By the end, the mRNA that we're going to use is just going to have the constant region information that we need.


[User] Please transcribe this text

[Assistant] Uhm, so we're going to have kind of spliced to use the appropriate constant region. So uhm, at the beginning my DNA might look like this.


[User] Please transcribe this text

[Assistant] It's gonna be touching the J- the D. Gonna be touching the J. There's gonna be a little bit of space.


[User] Please transcribe this text

[Assistant] There's going to be the IgM constant region and we're not even going to have the rest of the business that we had. We're just basically going to do some splicing, just have the part that we need.


[User] Please transcribe this text

[Assistant] And that will encode, here you can see, the heavy chain protein.


[User] Please transcribe this text

[Assistant] Is going to happen again in that BISO?


[User] Please transcribe this text

[Assistant] But at the, another chromosome where the light chain is encoded, and so we're gonna see this whole process happen once for the heavy chain in our B-style as it develops, um, putting together the V, D, and J, and the heavy chain stuff.


[User] Please transcribe this text

[Assistant] As well as happening for the light chain with the V and the J and the constant region. So you can see kind of how all of these gene segments, A, the fact that we've got two different sets of this rearrangement happening and how the gene segments correspond to the antibody protein that we have already seen.


[User] Please transcribe this text

[Assistant] So when we talked about the antibody and its structure,


[User] Please transcribe this text

[Assistant] Uhm, we talked specifically about one part of the antibody structure being really, really, really important for binding to antigen. And the goal here of course is to get a unique


[User] Please transcribe this text

[Assistant] We talked about the immunoglobulin-like domains with some beta sheets and some little loops.


[User] Please transcribe this text

[Assistant] And I told you at the very end of the variable regions of the antibody,


[User] Please transcribe this text

[Assistant] There are three little loops. Three on the heavy chain, three on the light chain.


[User] Please transcribe this text

[Assistant] And those three little loops are coming off the end of the heavy chain and the light chain and actually are touching the antigen.


[User] Please transcribe this text

[Assistant] Uhm, which you can see nicely in this slide. Here's an aminoglobulin domain with the loops.


[User] Please transcribe this text

[Assistant] And of course those are the most variable parts of the variable region, so they are the hyper variable region. They are also the things that determine


[User] Please transcribe this text

[Assistant] Uhm, which antigen this cell is complementary to. So we sometimes call them the complementarity determining regions.


[User] Please transcribe this text

[Assistant] Map onto this whole business that I'm telling you about with BDJ.


[User] Please transcribe this text

[Assistant] Are B's, D's, J's, and constant regions, and try to say...


[User] Please transcribe this text

[Assistant] Where do the amino acids that make up those little loops, those complementarity determining regions, where do they come from? We find that CDR1, or the first loop,


[User] Please transcribe this text

[Assistant] Is just a part of the variable region that we picked.


[User] Please transcribe this text

[Assistant] CDR2 is just a part of the V that we picked as well. So CDR1 and CDR2 are basically just based on which V we picked.


[User] Please transcribe this text

[Assistant] But CDR3 is actually encoded by the junction of the V, D, and J. So CDR3 is going to be super unique.


[User] Please transcribe this text

[Assistant] Because that's the, you know, any B cell that chooses V number 27 is going to have the same CDR1 and CDR2.


[User] Please transcribe this text

[Assistant] V27 just always has those as part of its structure. But even if you pick V27,


[User] Please transcribe this text

[Assistant] You're going to be combining it with a different D and a different J.


[User] Please transcribe this text

[Assistant] And you're going to get some super unique just to that B cell CDR3.


[User] Please transcribe this text

[Assistant] Uhm, and you can see CDR1 and 2 of a light chain are also encoded by the V region of the light chain, while CDR3 is encoded by the junction of the V and the J.


[User] Please transcribe this text

[Assistant] Uhm, so this VDJ recombination in particular is giving us a super unique.


[User] Please transcribe this text

[Assistant] CDR3, or third one of those little loops.


[User] Please transcribe this text

[Assistant] That is going to be able to contact antigen and make a super unique aspect of this antigen.


[User] Please transcribe this text

[Assistant] And we came up with, you know, like I said, about a hundred mini gene segments gave us a million antibodies or 10 to the six antibodies. Pretty awesome.


[User] Please transcribe this text

[Assistant] I didn't say that we needed to make 10 to the 6th antibodies.


[User] Please transcribe this text

[Assistant] I said we needed to make 10 to the 16th antibodies.


[User] Please transcribe this text

[Assistant] The other part of solving the problem, which you could have seen from that early slide I had, is this thing called Junctional Diversity.


[User] Please transcribe this text

[Assistant] All junctional diversity means


[User] Please transcribe this text

[Assistant] We're not completely precise about it. Sometimes we add or subtract base pairs.


[User] Please transcribe this text

[Assistant] Uhm, so what this is showing is here is a V. It's V cap of 21.


[User] Please transcribe this text

[Assistant] Here is a J. J kappa 1. So this B cell has picked V kappa 21, J kappa 1 in making its light chain. And the V is in pink and the J is in yellow.


[User] Please transcribe this text

[Assistant] This is what the sequence looks like in the original cell that we're going to cut and paste together. When different B cells


[User] Please transcribe this text

[Assistant] Do that choice and we'll cut and paste those two together.


[User] Please transcribe this text

[Assistant] Sometimes they lose some base pairs, and sometimes they add some base pairs.


[User] Please transcribe this text

[Assistant] And so here you can see four different B cells who all picked BK21, J couple one.


[User] Please transcribe this text

[Assistant] But they all gained or lost base pairs a little bit differently. And so we can get four totally different receptors just from even picking those same, uh, segments.


[User] Please transcribe this text

[Assistant] Uhm, so you can see kind of which base pairs came from the pink, which base pairs came from the green, and the addition or loss of some additional base pairs.


[User] Please transcribe this text

[Assistant] So this is what we mean by junctional diversity. In that when we're actually doing the cutting and pasting,


[User] Please transcribe this text

[Assistant] Sometimes we add or subtract some base pairs and that buys us even more diversity on top of the diversity we already have. Uhm, so this kind of shows you, uhm, a version of this math. Uhm, you know, we, if we actually look at,


[User] Please transcribe this text

[Assistant] Ds, Js, heavy chains plus light chains. But then we add on all the adding and subtracting base pairs. That gets us to, yes, this slide says 10 to the 13 instead of 10 to the 16.


[User] Please transcribe this text

[Assistant] The answer is it's a big number. We don't have to worry about whether it's 10 to 13 or 10 to 16. The answer is it's a really big number, and junctional diversity gets us there.


[User] Please transcribe this text

[Assistant] Uhm, by being sort of, I kind of think of it as like adding the sprinkles on top of the VDJ diversity that we've already added. Uhm, so.


[User] Please transcribe this text

[Assistant] In trying to, uhm, get the slides together for today and work through things, uhm, I had to take out some stuff I was gonna tell you last time. It'll go in another place.


[User] Please transcribe this text

[Assistant] But I have to introduce you to a little bit of a problem.


[User] Please transcribe this text

[Assistant] I want you to think for a second about Bio250 and Dr. Dunaway.


[User] Please transcribe this text

[Assistant] As we think about what is...


[User] Please transcribe this text

[Assistant] Uhm, going on with VEJ recombination.


[User] Please transcribe this text

[Assistant] So I'm going to remind you about a general definition of VDHA recombination.


[User] Please transcribe this text

[Assistant] And I want you to think about what Dr. Dunaway in Bio250 would say, as I tell you this. So with VDG recombination, all of your developing B cells,


[User] Please transcribe this text

[Assistant] AKA break and paste their DNA. Multiple times to make the antibody gene.


[User] Please transcribe this text

[Assistant] Any, what is, what is Dr. Dunnaway gonna say about that?


[User] Please transcribe this text

[Assistant] He, he's like, it's, he might say it's bad for the DNA. Jay worries about us losing the DNA. And we do lose some information. Uhm, Doctor, I don't know if he's gonna say it's bad, gonna say this is bad.


[User] Please transcribe this text

[Assistant] Dr. Noe is going to be very concerned at this moment.


[User] Please transcribe this text

[Assistant] Why is he very concerned? What's the big concern when thinking about VDJ recombination at this point?


[User] Please transcribe this text

[Assistant] What did Dr. Dunaway ever say to you about things like, if DNA breaks?


[User] Please transcribe this text

[Assistant] He talks about the cell dies. He, I remember, he would spend a lot of time talking about how you get dead. Yeah, that was if anything bad happens to your DNA.


[User] Please transcribe this text

[Assistant] Right? Now I just told you that every developing B cell in your body broke and pasted its DNA multiple times.


[User] Please transcribe this text

[Assistant] You have probably heard of many types of leukemias and lymphomas.


[User] Please transcribe this text

[Assistant] Leukemias and lymphomas are cancers of...


[User] Please transcribe this text

[Assistant] Different types of white blood cells. Sometimes of leukocytes and sometimes of lymphocytes.


[User] Please transcribe this text

[Assistant] And if you actually ranked all the tissues in the body, in terms of how frequently we saw cancers,


[User] Please transcribe this text

[Assistant] You would get skin up at the top and you'd be like, well, yeah, cause skin is like, in contact with the sun.


[User] Please transcribe this text

[Assistant] And we can imagine it getting damaged. And if you started raking tissues...


[User] Please transcribe this text

[Assistant] Most of the tissues that would rank really high are ones that are in contact with the outside world, that you can imagine get damaged really easily. But one that ranks really high...


[User] Please transcribe this text

[Assistant] Even way higher than it should be expected are cells of the immune system, leading to things like leukemias and lymphomas. And that's because...


[User] Please transcribe this text

[Assistant] Sometimes VDJ recombination goes wrong. So this figure uhm, on the left is actually naming a bunch of types of leukemias and lymphomas.


[User] Please transcribe this text

[Assistant] All of them are situations where B cells did VDJ recombi- messed up their VDJ recombination. The difference is sort of where the B cell got messed up in its life.


[User] Please transcribe this text

[Assistant] That distinguishes these different types of PEMES and Lymphomas.


[User] Please transcribe this text

[Assistant] Uhm, this is something called the Philadelphia chromosome. It's found in chronic myelogenous leukemia or CML.


[User] Please transcribe this text

[Assistant] Uhm, what happens in CML is that we're, we try to do a BDJ recombination on chromosome 22 to make some heavy chain, cut the DNA, and then in the patient,


[User] Please transcribe this text

[Assistant] The DNA gets pasted back to the wrong place. It actually pastes onto the wrong chromosome. So you can see.


[User] Please transcribe this text

[Assistant] That part of the orange chromosome gets pasted onto blue. Because VDJ recombination messes up. And this is actually what causes the tumor in these patients. Uhm, and these mixed chromosomes, mixed chromosomes called the Philadelphia chromosome.


[User] Please transcribe this text

[Assistant] Because it allows us to solve this diversity problem, but it's also


[User] Please transcribe this text

[Assistant] Crazy dangerous. And in some ways you look at it and you're like, this is the most insane thing anyone has ever telling us. We're going to break our DNA a bunch of times. What?


[User] Please transcribe this text

[Assistant] And so this sort of gets at the fact that.


[User] Please transcribe this text

[Assistant] It has to have a lot of regulation. It has to have a lot of steps. It has to have a lot of rules. Because if it goes wrong, it goes wrong bad.


[User] Please transcribe this text

[Assistant] We're going to spend the rest of our time today kinda going into some more nitty gritty details.


[User] Please transcribe this text

[Assistant] In terms of VDJ recombination, we're going to be seeing kind of very specific events of how this works. That's why I didn't draw some stuff, because I'm going to add more things to my drawing.


[User] Please transcribe this text

[Assistant] And we're going to be learning about a number of different proteins that are going to be really important. So you can see some of those proteins here. Again, I have this here partially for your reference.


[User] Please transcribe this text

[Assistant] Uhm, we're going to see some proteins called reg 1 and reg 2. We're going to see a protein called TDT.


[User] Please transcribe this text

[Assistant] Uhm, things that are really important to know about RAG1 and RAG2 in particular is that RAG1 and RAG2 is only in lymphoid cells. It's only in B cells and T cells.


[User] Please transcribe this text

[Assistant] And we'll see more about that going forward. But basically, one of the ways that we regulate this process is we make sure that one of the enzymes for the cutting and pasting is only in the cells that should be cutting and pasting.


[User] Please transcribe this text

[Assistant] We don't want a random skin cell making the cutting and pasting enzymes.


[User] Please transcribe this text

[Assistant] Cause that could be dangerous, that skin cell could mess up its DNA.


[User] Please transcribe this text

[Assistant] TDT is also lymphoid specific. That's less, uh, super important. There are other proteins that are not really specific to lymphoid cells. They're really just all DNA repair proteins.


[User] Please transcribe this text

[Assistant] Uhm, I will talk a little bit vaguely about things like coo and DNAPK. I'll talk a little bit about sort of ligase, polymerase. I'm not gonna care which ligase and polymerase. All of those things I'm gonna mention a little bit. Uhm, I am gonna mention artemis.


[User] Please transcribe this text

[Assistant] Uhm, more specifically and talk more specifically about Artemis.


[User] Please transcribe this text

[Assistant] That I want to mention that comes up on this slide, which you can see in a few different places here, particularly up here with reg 1 and reg 2. There are patients who are missing.


[User] Please transcribe this text

[Assistant] The genes that encode these proteins and so they don't make these proteins.


[User] Please transcribe this text

[Assistant] Uhm, if you are missing, particularly reg 1 and reg 2, which are like the star enzymes we're gonna see today.


[User] Please transcribe this text

[Assistant] You cannot make B cells or T cells. You end up with no adaptive immune system. Uhm, or this disease called SCID, Severe Combined Immunodeficiency. Uhm,


[User] Please transcribe this text

[Assistant] We now have a pretty good way of treating it. Before we treated it, it was largely found in boys because some of the genes that are involved are on the X chromosome. And those boys did not make it past the age of 20.


[User] Please transcribe this text

[Assistant] Most of them, uh, died really before ten, in fact. Uhm, because this-


[User] Please transcribe this text

[Assistant] Is such an important process of giving you this adaptive immune diversity. So if you can't, if you don't have these proteins, we're going to talk about no adaptive immune response. But if these proteins mess up, cancer. So we got some big danger either way with these proteins. All right. So we're going to be going through.


[User] Please transcribe this text

[Assistant] BDJ recombination in, uh, some more detail. So let's imagine that I put a bunch of string or something in front of you. I said, here's the genome.


[User] Please transcribe this text

[Assistant] Do some VTJ recombination on the genome. Go. What kind of things would you actually need in order to do that besides just a pile or string?


[User] Please transcribe this text

[Assistant] Gonna do VDJ recombination and try not to mess it up. What's this cell need? Yeah.


[User] Please transcribe this text

[Assistant] You're gonna need something to cut it, right? So you're going to need an enzyme that's going to do some cutting. Okay? What else are you gonna need? Yeah.


[User] Please transcribe this text

[Assistant] So, recombinase is often, we can think of as the thing that's doing the cutting.


[User] Please transcribe this text

[Assistant] So yes, an enzyme that's going to allow for recombination. Usually we think about the enzyme doing the cutting being the recombinase. Okay? What else are you going to need?


[User] Please transcribe this text

[Assistant] You're going to need something to fix to like paste the DNA back together. Right? Make sense? Okay. You need those two things. There's one other thing you need. And this is a thing people forget.


[User] Please transcribe this text

[Assistant] Uhm, and it's actually the first thing I want to talk about, so that's why I have to do it this way. So what else are you going to need if I just give you this pile of genome and say, cut and paste the genome please, here you go.


[User] Please transcribe this text

[Assistant] So, so both of those things are important, but they're not sort of the one key thing. You want to make sure you don't mess this up. Yeah.


[User] Please transcribe this text

[Assistant] Don't cut some other place! Cut here! Right? If I just gave you a pile o' string, you would need to know where on the string to cut.


[User] Please transcribe this text

[Assistant] This is DNA that we are cutting and pasting.


[User] Please transcribe this text

[Assistant] So really our only option for a label is a specific sequence.


[User] Please transcribe this text

[Assistant] So all of our mini gene segments that we are going to recombine.


[User] Please transcribe this text

[Assistant] Have specific label sequences next to them.


[User] Please transcribe this text

[Assistant] They are known as recombination signal sequences or RSSs.


[User] Please transcribe this text

[Assistant] There are two different kinds of RSS's shown here.


[User] Please transcribe this text

[Assistant] One kind has something known as a heptamer, which is this conserved seven base pairs of CACAGTG.


[User] Please transcribe this text

[Assistant] Then there's anonomer, uhm, ACAAACC.


[User] Please transcribe this text

[Assistant] So you can see, uhm, we usually will refer to this whole thing, the heptamer, the spacer, and the nonamer, together as the 23 base pair RSS. We also...


[User] Please transcribe this text

[Assistant] We'll have another type, has the exact same heptamer, has the exact same nonamer.


[User] Please transcribe this text

[Assistant] But instead of having a 23 base pair spacer, it has a 12 base pair spacer. And so again, that could be any, uh, number of...


[User] Please transcribe this text

[Assistant] Any base pair is just the number of what's really important.


[User] Please transcribe this text

[Assistant] Uhm, in fact what we realize is that 12 base pairs is basically once around the helix.


[User] Please transcribe this text

[Assistant] Which means that the heptamer and the nonamer are next to each other and an enzyme can grab them. And 23 is twice around.


[User] Please transcribe this text

[Assistant] So in fact, I once learned these as two turn heli- two turn RSS's and one turn RSS. Uhm, I'm gonna refer to them as 12 and 23. You can see in this figure from this textbook, they're drawn as triangles. And they're drawn as two different colored triangles.


[User] Please transcribe this text

[Assistant] So all of my gene segments,


[User] Please transcribe this text

[Assistant] Should have recombination signal sequences. So I'm going to give you a few gene segments here.


[User] Please transcribe this text

[Assistant] Three D's, and I've got three J's. And I'm almost tall enough to do this.


[User] Please transcribe this text

[Assistant] This is my new gene, this is my new chromosome that we're going to be rearranging. And I'm missing some RSS's. The B's are going to have RSS's.


[User] Please transcribe this text

[Assistant] That are immediately next to them so you can see right after the end of the V we start the sequence for the RSS.


[User] Please transcribe this text

[Assistant] Uhm, and it's gonna be on the side that we wanna bind stuff to. So like, we're gonna put a D eventually on this side of the V. And I got a one over here, one over here, one over here for my J's, one over here for my J's, one over here for my J's. And my D's have to bind to both a V and a J. And my D's...


[User] Please transcribe this text

[Assistant] So these are the things that are going to tell my cutting and pasting enzymes.


[User] Please transcribe this text

[Assistant] But you will notice that this figure shows that there are two different kinds. The 23 base pair kind and the 12 base pair kind.


[User] Please transcribe this text

[Assistant] And there is a rule about them. It is called the 1223 rule.


[User] Please transcribe this text

[Assistant] This is a figure from your textbook depicting the 1223 rule.


[User] Please transcribe this text

[Assistant] Uhm, your textbook doesn't always draw these as triangles.


[User] Please transcribe this text

[Assistant] So here you can see we've got the 7-23-9 that I'm depicting up there as a triangle or the 7-12-9. You can see the same thing.


[User] Please transcribe this text

[Assistant] Uhm, I'm gonna go back to this view from this other textbook, uhm, also for the 1223 rule.


[User] Please transcribe this text

[Assistant] Have to have opposite types of RSS's. So if we look at this heavy chain shown here, the V's have the 23 kind.


[User] Please transcribe this text

[Assistant] The D's have the 12 kind on either side, and the J's have the 23 kinds.


[User] Please transcribe this text

[Assistant] Uhm, I usually depict them as either solid or like filled or open, as you can see here.


[User] Please transcribe this text

[Assistant] To show that there are two different kinds, because it's really important with our assesses that we know that there are two different kinds.


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[Assistant] You can only put together a 12 and a 23. You will notice on the light chain,


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[Assistant] The one kind of light chain has the 12 on the Vs and the 23s on the Js.


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[Assistant] The other one has it the other way around. I actually don't care!


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[Assistant] That you know which one's 12 and which one's 23. I care that you know that there's two kinds. And you can tell me something like this.


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[Assistant] There's two kinds. Filled is one kind. Empty is one kind.


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[Assistant] And in the case of this heavy chain, the antibody heavy chain,


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[Assistant] That you know that one kind is on the Vs and on the Js, and the other kind is on the Ds on both sides. So that's what you should be paying attention to here.


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[Assistant] And so what you can notice is that in the case of our heavy chain,


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[Assistant] You can't put a D next to another D. You can't join them together because the RSSs don't match.


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[Assistant] You can't join this to this. Because the RSSs are not compatible. You have to have two mixed ones. But you could put a D with a J. Because their RSSs will work.


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[Assistant] The RSS's don't work. You have to be able to put V, D, and J together. Do you have a question, Jamir?


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[Assistant] Same kind of idea here as well. So this is showing a light chain. We've got our Vs with one kind of RSS, our Js with another kind of RSS here. And you can see that the RSSs


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[Assistant] Start like immediately next to the...


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[Assistant] Um, mini gene segment that we're going to combine. There's not like space in between them.


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[Assistant] And what will happen is we'll pick one V and we'll pick one J.


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[Assistant] We cut right between the RSS and the gene segment.


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[Assistant] Eventually I'd get the V, the D, and the J next to each other. I would have cut off the RSS, and I would have put the gene segments right next to each other. But the other parts of the DNA...


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[Assistant] Would look exactly the same. See, I'm not gonna rewrite the same constant region from above, I'm gonna say it's the same as from above. Cause I got lazy. Uhm.


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[Assistant] And so you can see this happening both here for a VJ on a light chain. Here you can see a heavy chain.


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[Assistant] As we will later see, it goes in an order, so D and J come together first, then the V goes to the DJ.


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[Assistant] But you can see kind of this process and what it will eventually look like.


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[Assistant] And so notice that uhm, the final DNA and the RNA are a little bit different.


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[Assistant] We spliced out all that extra business in the RNA.


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[Assistant] We can now actually start to think about, uhm,


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[Assistant] The enzymes that are doing the cutting. Now we know where they're gonna cut. And what it's gonna look like with the cutting. But now we need some enzymes who are gonna do the cutting.


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[Assistant] Uhm, and the enzymes that are going to do the cutting are called RAG1 and RAG2.


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[Assistant] And RAG1 and RAG2, as you can see here, are going to bind to the RSS. Here we've got heptamer 12 nonamer, so that's, this whole business is one triangle. Here we've got heptamer 23 nonamer, this whole business is another triangle.


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[Assistant] And you can see rag one and rag two are actually binding, particularly rag one is binding.


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[Assistant] Uhm, to this area and kind of bringing it together. So, rag one has chosen V1.


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[Assistant] And J1 and brought them together so that we can do VDJ recombination.


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[Assistant] And eventually that V1 and J1 are going to be put together. And all of this DNA that was in between, including those RSSs, including our little triangles, is eventually going to get thrown out.


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[Assistant] Uhm, and so you can see this happening here as well. So RAG1 and RAG2 are going to recognize the RSS. You can see my RSS is here. And they're going to nick the DNA.


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[Assistant] This process is happening twice. So you can see it's happening at the V and it's happening at the J.


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[Assistant] They are actually held together really closely by RAG1 and so we're gonna get a NIC right here between the V and its RSS and a NIC right here between the J and its RSS.


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[Assistant] If I, if we, the textbook, I, I can't draw anything. The textbook kept drawing both of these the whole way through. It would become a big giant mess really quickly.


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[Assistant] So here you can see they're both bound by this blue blob that is the rag complex.


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[Assistant] And then we're just gonna be seeing some of the events that are happening at one side.


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[Assistant] And that's for simplicity's sake, in terms of us looking at this.


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[Assistant] It is not because only one side is doing it. It's because it would just be way too hard to look at if we tried to look at both sides right now. So what you'll see is that rag, uh,


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[Assistant] We'll make a DNA nick, and a DNA nick is a specific kind, uh, of event.


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[Assistant] Is that a DNA nick is a single-stranded cut of the DNA.


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[Assistant] So what you can see is that one strand gets cut and the other one doesn't.


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[Assistant] So that's what it means that it makes a neck. Is that we get this single strand cut. One strand is cut, one strand is not cut.


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[Assistant] Here is the DNA before, because I'm good at drawing. Got double strand DNA, five prime, three prime end, right? We're gonna make a knit.


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[Assistant] Now here is a new 3' end, here is a new 5' end. And there's a hole in the middle.


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[Assistant] These little five primes, they have OHs, the hydroxyls, the three prime hydroxyl.


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[Assistant] That's, when we say 3' and 5' that's what we mean, is that there's a free phosphate or a free hydroxyl. And now I made this little break and I've got a new, I've got these new free stuffs. Free chemistries.


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[Assistant] That hydroxyl does not like to be free. That hydroxyl is not too happy about being free.


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[Assistant] And so that hydroxyl performs a nucleophilic attack. Because it doesn't want to be free, it wants to be paired with something. And the thing that it makes, it attacks, is the opposite strand.


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[Assistant] So we can see that eventually this piece of DNA, which was our mini gene, like our V,


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[Assistant] Cause the top strand's hydroxyl has attacked the bottom strand's phosphate. And the other stuff just gets thrown away.


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[Assistant] So you can see we get this little hairpin fat formed.


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[Assistant] Can you see how we got the hairpin from this nucleophilic attack? And then the other DNA is just like, sad. And that other DNA was my little triangle.


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[Assistant] So now we've got two pieces of DNA. We've got this piece of DNA that is my mini gene that I want to do important things with.


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[Assistant] Uhm, it's eventually going to code for the antibody. So I call it the coding end.


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[Assistant] Was the RSS, so I call it the signal end.


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[Assistant] So now I gotta do something with my coding end and I gotta do something with my signal end. And if we were really drawing this whole thing, we'd have two coding ends and two signal ends. One for like the V and one for the J. But that would just be way too much to look at here.


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[Assistant] So that's why we're only showing you one coding end, one signal end, but really there's two coding ends, two signal ends.


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[Assistant] So now, first we're going to talk about what happens to the signal end. The two signal ends that we have.


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[Assistant] And the reason why we're going to talk about what happens to the two signal ends first is because it's easier.


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[Assistant] So here you can see, we've got two hairpins, we've got two signal ends, right? Uhm, we take those two signal ends, you can also see here's my signal ends, here I've got two signal ends.


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[Assistant] Circle of DNA where we stuck together our signal ends. You can see that the right where we get the stuff stuck together is those triangles. We get this little circle of DNA.


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[Assistant] Here's my little circle of DNA here. Here's where I've stuck together my two triangles. And I have all of the intervening DNA. And I'm gonna throw that out and no one ever cares about it ever again. And we're never gonna see it ever again.


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[Assistant] So it hangs out in the cell, and as the cell undergoes divisions, it basically gets lost. Because it doesn't have an origin of replication to get replicated. So basically there's just the one piece of DNA from like the parental cell.


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[Assistant] And as the cell divides, it doesn't replicate, it just, it just sits there and eventually it'll just get degraded as random stuff in the cell. Like random trash.


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[Assistant] And the reason why, and part of the complication in the coding end,


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[Assistant] Is because when we deal with the coding end...


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[Assistant] We're also going to be getting that junctional diversity I told you about.


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[Assistant] So we've gotta have some additional stuff happening with the coding end to potentially add or subtract base pairs. And that's the thing that's going to give us those sprinkles.


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[Assistant] On the top of the VDJ diversity that I told you about. So, as we deal with this coding end,


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[Assistant] Really what we're doing is we're getting our Junctional Diversity added in.


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[Assistant] So, previously on immunology, previously on BDJ recombination, we had a coding end. The coding end, you can see here, the coding end is this wacky DNA structure.


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[Assistant] The top strand is now attached to the bottom strand, and we have this little hairpin.


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[Assistant] Uhm, so when I learned this as an undergraduate, I was taught, and then the hairpin opens.


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[Assistant] Bye, magic. Uhm, and some of the sort of details of what happened with the hairpin opening. Uhm, when I was in graduate school, we actually, or someone, I did not.


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[Assistant] Uh, discovered the enzyme that opens the hairpin.


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[Assistant] Uhm, if you can't do, if you can't open your hairpin,


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[Assistant] You can't ever finish VDJ recombination. You make no B cells and T cells. You end up very sick.


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[Assistant] You end up dying of immunodeficiency generally as a child.


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[Assistant] The person who discovered this enzyme named it after a Greek goddess.


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[Assistant] And specifically, they chose the Greek goddess who is the protector of women and children.


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[Assistant] Because this enzyme protects children from immunodeficiency.


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[Assistant] Uhm, so the name of this enzyme is Artemis.


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[Assistant] So we're going to have our enzyme Artemis that's going to open this hairpin.


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[Assistant] An amazing enzyme. It got named after a Greek goddess. It's such a great enzyme.


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[Assistant] Artemis has a little bit of a problem as well. Not a perfect enzyme. I don't think there is such a thing as a perfect enzyme, but that's okay. So, Artemis...


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[Assistant] Is going to come to this hair pin and break it.


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[Assistant] But I'm gonna redraw my hairpin for you in a slightly different way.


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[Assistant] So here is my DNA before RAG1 came along.


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[Assistant] I got a nick. Now I have my free OH. This OH is going to attack that P, that phosphate. I'm going to have a bond right there.


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[Assistant] Okay, and it's a bond between a phosphate and a hydroxyl.


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[Assistant] There's also a similar bond right there, and right there, and right there, and right there, and right there.


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[Assistant] Right there, right there, right there, right there, right there. So you can see all sorts of bonds that are basically chemically the same bond.


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[Assistant] Artemis is going to cut open this hairpin. It's going to cut a hydroxyl phosphate bomb. Artemis does not know which is the bomb.


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[Assistant] Artemis can't tell the difference between this bond and this bond and this bond and this bond and this bond. Artemis is hanging out in this location and it's gonna cut a bond.


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[Assistant] But sometimes it doesn't cut this original thought.


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[Assistant] Sometimes it cuts too far up here on this strand.


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[Assistant] Sometimes it cuts too far on this strand.


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[Assistant] Uhm, it can actually cut up to five base pairs away in either direction. So there's sort of this ten base pair range where it can cut.


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[Assistant] And so you can see on this slide, well sometimes it cuts at position two.


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[Assistant] And we end up with kind of the original perfect thing that we had. And everything ends up looking great. Sometimes it cuts at position three.


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[Assistant] And when that happens, this bottom strand gets some extra base pairs. And the top strand has a, has some fewer. Sometimes it cuts to position one.


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[Assistant] And we get, the top strand has some few extra base pairs, the bottom strand doesn't.


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[Assistant] And so you can also see this uhm, again at the bottom uhm, in, with actual base pair, like letter. So here,


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[Assistant] The actual sequence ends TC.


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[Assistant] But if we cut here instead, now we get TCGA.


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[Assistant] Uhm, here you can see we got some extra sequence down here because, uh, of how we cut. Uhm, so it's not a perfectly symmetrical cut.


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[Assistant] And before we can actually stick those base pairs back together,


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[Assistant] And so you can see the overhang having been filled in here.


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[Assistant] Uhm, before we can actually do our ligation.


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[Assistant] So you can see this here as well, uhm, from your textbook. Here you can see the cuts, uhm, right by the heptamer. You can see our little hairpin. And you can see in this case we opened


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[Assistant] Uhm, two base pairs too far this way, and we got this two extra.


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[Assistant] Base pairs, we got open two base pairs too far this way on the J. We got these two extra base pairs.


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[Assistant] Uhm, and so let's look at sort of specifically what is going to happen here. Oh, there's even colored chalk over here. That's exciting. So I have my D region.


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[Assistant] That didn't break at all, I don't know what you're talking about. Uhm, and my D region, like, ends up with a T, C, and then there was A, G, right? So there's my hairpin on the D region.


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[Assistant] And then this strand is gonna be empty. I'm gonna get this one. Gonna go...


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[Assistant] T-A, T-A, and this strand is going to be empty. Okay?


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[Assistant] So you see, I'm really just redrawing exactly what they've got up there. And so, if I actually change my colors for a second, if you look, this T and this C


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[Assistant] But if you actually compared the sequence in this B cell, uhm, with


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[Assistant] The germline genetic sequence, we now have added base pairs that were not there before. And when we actually go in,


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[Assistant] Fully added in base pairs that are not- we're not in the original.


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[Assistant] So do you see how we've gotten this added in-base pair, the ones in blue, that are not in the original sequence?


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[Assistant] That's one of the ways we get junctional diversity. This is the adding and subtracting. Here we've added. If you look at those letters.


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[Assistant] And if you read the other strand, it goes TCGA. It reads the same forward and backwards.


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[Assistant] English, you call a word that reads the same forward and backwards a palindrome.


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[Assistant] And so these nucleotides are called palindromic nucleotides, or p-nucleotides.


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[Assistant] They are coming from, uhm, asymmetric hairpin opening.


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[Assistant] On Friday, I'm gonna continue talking about VDJ recombination. Uhm, I have a feeling this is gonna happen. And I may even go over some problems that look somewhat like some problems that you see on the problem set because there's a problem set due after break.


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[Assistant] I won't lie to you, she's never been much for cleaning and she's a terrible cook.


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[Assistant] Hell, she doesn't even like to dress nice and put on makeup either. I gave up on that stuff a long time ago. Then she ran off overseas to find herself.


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[Assistant] She's a tomboy through and through, and the most stubborn girl I've ever known. But no matter what, she's still my precious daughter.


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[Assistant] Mr. Shindo, all I can ask, take good care of her, please.


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[Assistant] Good lord. You really oughta stop and think every once in a while, Sarika.


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[Assistant] Now this, you wanna watch me engrave everything you do. It's all on a whim. You're such an impulsive girl. Admit it, son.


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[Assistant] Hello there, my name is Richard McMunn from the interview training company, PassMyInterview.com.


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[Assistant] So it does not matter which organization you have your higher view interview for. Please do make sure you watch this from beginning to end, because I want to help you to pass your interview


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[Assistant] And this is what I will cover during this tutorial to achieve that goal. So I'm going to give you…


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[Assistant] Some great tips for passing your higher view video interview.


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[Assistant] I will also give you some sample interview questions that I believe will come up during your interview. And it's very important that we work hard to make sure you are the standout candidate.


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[Assistant] I will also give you suggested answers to those higher view interview questions. These are top-scoring answers that you can't find anywhere else. And I will also tell you what the assessors are looking for


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[Assistant] And how you can be the standout candidate during your HireVue interview.


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[Assistant] So there are four really good things that I'm going to cover to help you pass your HireVue interview.


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[Assistant] My name is Richard McMunn. That's me there in the center of the screen. I've been helping people for over 20 years now to pass their interviews. And I do that by giving you top-scoring answers to the interview questions. Please do come and join the community here on YouTube.


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[Assistant] And click the SUBSCRIBE button below the video. We have about 240,000 subscribers now. Many of you are passing your job interviews as a result. Please also make sure you turn on that all-important notification bell, so you get notified the minute I upload a new training video. I would also very much appreciate it if you gave the video a LIKE. That tells me you find the content useful.


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[Assistant] And I will therefore create more for you. Okay, let's get into the tutorial. The higher view interview questions and answers. Please do take notes as I progress.


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[Assistant] So there are a number of interview questions I want to cover. The first one is tell me about yourself.


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[Assistant] It's difficult to give an answer that makes you the standout candidate. Now, my tip for giving a high scoring answer to this interview question is when you are answering it, focus on three things. Tell the interviewer the qualities and the skills that you have.


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[Assistant] That are relevant to the role and that will be of benefit to the employer. Also tell them your motivation for this job, why you want this job. And then also tell them one thing, your unique selling point. What is unique about you? So there are three things there I want you to cover when you answer the higher view interview question. Tell me about yourself. So here is my suggested answer for you.


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[Assistant] And also a desire to work to high standards and above all, take full responsibility for the work I produce. My motivations for this position are simple.


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[Assistant] I have read the job description and I feel the experience I have and also the skills I possess will enable me to work consistently to a high standard


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[Assistant] Whilst helping you to achieve your commercial objectives.


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[Assistant] I believe I am the best candidate because I always look to improve. I never sit back and just do the bare minimum.


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[Assistant] And again, the most job satisfaction from seeing the team I am a part of continually thrive and develop. That is a brilliant answer to the higher view interview question. Tell me about yourself.


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[Assistant] What I also like about that answer is it sets you up with confidence for the rest of your interview. And you and I know that this is probably going to be the first question they will ask you. So if we get it right, we are then set up for the remainder of the interview. Now, what I am going to do as we go along


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[Assistant] Is I'm going to integrate some higher view interview tips in between each of the interview questions and answers. But let's quickly tackle another guaranteed higher view interview question. And that is, why do you want to work here or why do you want to work for us? Now, my tip for answering this question is


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[Assistant] Give two main reasons why you want to work for their company or their organization. My advice is to focus on their reputation.


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[Assistant] Providing it's positive, of course. So focus on their positive reputation and also make reference at some stage during your answer to their products or their services, because that then demonstrates you have conducted some research. Now, once I've given you a great answer to this interview question, I will give you a research checklist. So people say to me on this channel, what should I research when


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[Assistant] Preparing for my interview. Well, I will give you some things to research in a second. But here is that answer to the interview question. Why do you want to work here? I want to work for you because historically


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[Assistant] You are an organization that always delivers, you always improve and you always look to employ the brightest talent that is available.


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[Assistant] This means by working for you, I will not only get to put my skills, my qualities and experience to good use, but I too will be able to carry on learning and developing in the role.


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[Assistant] I also want to work for you because having researched your products and services, you never accept anything but the best. I too have high standards and I feel my drive and determination to succeed will fit in well with your organization.


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[Assistant] Again, that is a top scoring answer to a high view interview question, which is why do you want to work here?


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[Assistant] Now, let's quickly take a look at that interview research checklist.


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[Assistant] If you want to go a little bit deeper and research the company before your interview


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[Assistant] My advice is to first of all, have a look at what their core products and services are, so you can quickly relate to them. If they say, tell us about us.


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[Assistant] You can say, well, you offer the following products and services and you can find out that information on their website.


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[Assistant] I would also look into their history. You don't have to go into great detail.


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[Assistant] But just have a quick look at when they were incorporated, how long they've been operating for, and also what their core achievements are.


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[Assistant] So you can go onto Wikipedia for the majority of large organizations to find out a company or organization's history.


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[Assistant] Also, go on their About Us page and have a look at what their company mission is. The majority of large companies have a mission statement or revision.


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[Assistant] Learn that and have it with you during your Highview interview and refer to it


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[Assistant] When they say, why do you want to work for us? You could say, I've read your company mission and it is aligned to my own work standards and ethics.


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[Assistant] And also, what is their company culture? I certainly think it's worth visiting the social media platforms of the organization that you're applying for to see what their culture is like.


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[Assistant] How do they promote their products? You know, what standards and values do they set? Okay, a quick video interview


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[Assistant] I'm going to give you a few of these as we progress through the tutorial. And as I say, these are intertwined between the high view interview questions and answers.


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[Assistant] Now this is a very basic one, but it's one that some people don't consider. And that is to check the background and the lighting of the place that you will hold the high view interview. So think about the room that you are going to have your interview in.


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[Assistant] Have a look at the lighting. Have a look at the background. What is behind you? Now, if you look at this person here…


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[Assistant] There is a blank background, which I think is great. The lighting is good. There is actually something wrong.


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[Assistant] Um, with how he is presented, which I'll talk about a little bit later on, but the lighting is good and the background is good. Also consider your internet connection.


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[Assistant] So go to the room where you are going to hold your high view interview and have a look at the reception, you know, the wifi connection or the internet connection.


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[Assistant] Make sure the device you are using is fully charged.


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[Assistant] What you don't want to do is to be running out of battery halfway through your interview, because that shows that you lack organization and preparation. So, another thing


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[Assistant] Some people say to me, I've got a high view interview coming up. I'm going to download their app and I'm going to hold the phone. And I say to them, I think this is a mistake because I believe you should make sure that the phone you are using is stable. Remember, you might be nervous, understandably,


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[Assistant] During the answers to your high view interview questions. So you might want to get a very cheap, cost-effective tripod and class like this one where you can put the phone in. Now this is useful for making sure that your phone is stable and it does not shake.


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[Assistant] Now I have put a link in the video description


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[Assistant] To this product on Amazon, where if you want to buy it, you can do it. It only costs about $10 or £10 and that might be a worthwhile investment because then you can, you know, you can position yourself well. You can use your hands effectively when you are describing


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[Assistant] The situations you are in when responding to the interview questions. So a couple of really good tips there. Let's now move on again to the high view interview questions. And this one is, if someone in your team was not pulling their weight, what would you do?


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[Assistant] Now the majority of people will answer this and say, I will tell my manager or my supervisor, or I would get involved and I would carry out the task myself. That's not the way to answer it.


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[Assistant] So my tip is do not say you would ignore the situation.


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[Assistant] And do not say you would just do the job yourself to help the team.


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[Assistant] Make sure you deal with this kind of situation confidently, tactfully and with one goal in mind, to get the team working at full capacity again as soon as possible. You do not need to be a manager or a supervisor


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[Assistant] To deal with someone in your team who isn't pulling their weight, you should deal with it yourself, not aggressively or confrontationally, but calmly, confidently and tactfully. Here's how to answer this interview question.


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[Assistant] Any situation like this can have a negative impact on the team and also the organization.


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[Assistant] And on that basis, I would look to tackle it as soon as possible by using effective communication skills.


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[Assistant] I would speak to my work colleague in private to ask him or her whether or not there was an issue that was affecting their ability to work at full capacity. They may not be feeling well


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[Assistant] I would hope that by me making them aware they were underperforming


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[Assistant] They would automatically seek to quickly get back to their previous productivity levels themselves. If my intervention did not have the desired results,


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[Assistant] I will then speak to my supervisor or my manager for advice.


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[Assistant] That is a great way to answer the interview question. If someone in your team was not pulling their weight, what would you do? And it shows that you are prepared to get involved


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[Assistant] And use effective communication skills for the benefit of the team.


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[Assistant] So another video interview tip. The second one is…


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[Assistant] And this is where I talk about this person, how I don't feel they are presented correctly.


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[Assistant] Make sure you dress as you would do for a face to face formal interview. Now, in my opinion, this gentleman is wearing an informal polo shirt. I don't think it's suitable


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[Assistant] Some things that are good about him, he is smiling well. That is always a good thing to do during a high view interview, is to smile. And also, he is a good distance from the camera.


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[Assistant] So that is a good distance and I'll give you another really good tip soon.


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[Assistant] About how to position yourself and what to do.


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[Assistant] Let's now, though, move on to another Highview interview question. When did you work as part of a team that failed?


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[Assistant] I've actually never worked as part of a team that has failed. I'm really good at what I do and I always make sure that the team succeeds. Well, that is not true because we have all worked as part of a team.


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[Assistant] And that has failed or, and we have also made mistakes at work.


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[Assistant] This interview question is actually assessing your team working capabilities, your resilience and also…


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[Assistant] This type of situation has only happened a small number of times.


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[Assistant] There was one particular team project I was working on that didn't go to plan.


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[Assistant] In a previous job, I was part of a team that was involved in launching a new product.


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[Assistant] We had a set launch date to work towards and my team was responsible for the marketing aspect.


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[Assistant] On the product launch day, we activated our online advertising campaign.


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[Assistant] But it soon became apparent that websites and the social media platforms we were targeting were ineffective. The reason for this? We simply had not carried out sufficient research into the demographics of our potential market audience. I was devastated. Understandably, our manager was really annoyed too.


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[Assistant] We all decided to stay behind late that evening to get the marketing campaign back on track, which we successfully managed to achieve. Every single one of us in that team was responsible for the failure, and I learnt a huge amount from that experience. Attention to detail, cross-checking and double-checking is absolutely vital when carrying out any team task.


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[Assistant] I am someone who will always take ownership for problems with a view to resolving them quickly and attentively. What I have done for you within that answer is I have highlighted


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[Assistant] The words and phrases in yellow that I feel are the most important. So whatever situation you use, I would add the bits in yellow. You are there


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[Assistant] In that phrase, the reason for this, we simply have not carried out sufficient research into the demographics. You are telling them the reason why the team failed. So that is the fact that you are attentive and you can understand why things have gone wrong.


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[Assistant] You are then showing an emotion to the situation. You are saying, I was devastated. That shows that you care.


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[Assistant] Then you say, every single one of us in that team was responsible for the failure. And I learned a huge amount from the experience. That is very important because it shows that you understand that not one individual is responsible for failure. It is the team as a whole, because you have to support each other. If you were to say one person in the team got something wrong,


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[Assistant] It wasn't my fault. It was their fault. That's not the way to answer it. You say all of us are responsible and then you learn from it. And then finally you say,


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[Assistant] I am someone who will always take ownership for problems with a view to resolving them. That is somebody who shows a level of maturity, of intelligence


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[Assistant] And as I say, you take responsibility for situations, which is very attractive to any hiring manager.


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[Assistant] The next high view interview question I want you to prepare for is, tell me what you would do


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[Assistant] Sorry, tell me what you do when you are not at work. So tell me what you do when you are not at work. So this question assesses what you do with your spare time.


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[Assistant] Are you a productive person and do you lead a responsible life?


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[Assistant] Or are you someone who participates in activities that could potentially deplete your energy levels that could then impact on your ability to perform to a high standard at work? So here is my suggested answer to the interview question. Tell me what you do when you're not at work?


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[Assistant] When not at work, I am either spending time with my family and friends, reading or keeping myself fit and active. My family are very supportive of me.


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[Assistant] Also, there's subtly that you sometimes work in the evenings or at weekends and that your family supports you. That's attractive to any hiring manager.


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[Assistant] You also say that you like to relax by reading and that is a form of self-development, which is good.


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[Assistant] And then you also say you like to keep fit and you say why that's important because it keeps your energy levels up, but also it helps you to maintain a healthy mindset. That's a very clever answer.


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[Assistant] To that interview question, tell me what you do when you are not at work.


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[Assistant] So another higher view video interview tip, and this is a really good one that most people don't, they're not aware of and they don't consider.


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[Assistant] When you are carrying out the high view interview, look into the camera, not look at the screen.


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[Assistant] So consider on your device where the actual camera is. Where is the lens? Where is the camera? Now, on the majority


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[Assistant] Of laptops, like I have a MacBook Air and that is where the camera is. So make sure you look into the camera on a smartphone. Again, it is close to the top. Don't look at the screen when answering the questions. Look into the camera because then that shows really good eye contact.


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[Assistant] When you are carrying out your interview, it's a little tip, but it will make you stand out better during your interview. So the next high view interview question is


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[Assistant] Tell me about a time when you didn't get on with a manager or a supervisor? This is a difficult question. Again, a really difficult question. Now, we have all had situations when we did not get on with or agree with our manager. However, it is how you react to the situation that is important. Now, a lot of people will say, I've never been in a situation where I didn't get on with a manager. I don't believe them.


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[Assistant] I believe it makes you more of a seasoned professional if you have had situations where you didn't get on with a manager or supervisor. But it's how you react to that that's important.


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[Assistant] Okay, so I would be looking for a response that shows you are professional.


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[Assistant] You put any differences aside and you get on and achieve the objectives of the team and the organization. Here is my suggested answer.


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[Assistant] In one of my very early jobs, I didn't seem to get on with my manager. For some reason we just didn't really connect.


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[Assistant] But I never let the situation deter me from maintaining professionalism and doing a good job for my manager and for the company.


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[Assistant] Whilst I do get on with the vast number of people I work with, if there ever was a situation where I didn't get on with somebody, I would never let it get in the way of my professionalism and also my ability to work hard for the team and the organization as a whole. So what you are doing there, rather cleverly,


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[Assistant] He's saying it wouldn't be an issue to me. It wouldn't matter. It's nothing personal.


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[Assistant] My reason for being at work is to achieve the tasks that my manager wants me to do.


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[Assistant] So we can help the organization from a commercial perspective. That's a great answer to that interview question. Tell me about a time when you didn't get on with a manager or a supervisor.


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[Assistant] Next question. By the way, don't forget, if you are enjoying this, I hope you are.


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[Assistant] Please do LIKE the video and don't forget to SUBSCRIBE. Thank you very much.


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[Assistant] Tell me about a time when you succeeded despite being under pressure?


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[Assistant] This is very common during higher view interview questions.


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[Assistant] Give a specific work situation here that you were in.


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[Assistant] Where the odds were stacked against you, yet you succeeded. And don't forget to add some kind of pressure.


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[Assistant] It might be a time constraint, for example. When you do answer this question,


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[Assistant] Use the STAR technique. Now, this is called a behavioral type interview question, and you have to describe a situation that you were in.


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[Assistant] And the best way to structure these answers is using the STAR technique. So you tell them the situation you were in.


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[Assistant] You then explain the task that needed to be done. You then detail the action you took to achieve that task, and you then reveal the results of your actions. And the results should always be positive. So whenever you get asked a question that says, tell me about a time when you or describe a situation you were in,


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[Assistant] So yeah, it's a behavioral type interview question and you have to give a specific situation. You should automatically think to yourself STAR technique.


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[Assistant] On a client based project that had a very tight deadline.


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[Assistant] Our task was to create and deliver a new web-based product for the client


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[Assistant] Within just 21 days. Time was against us, but we were determined to succeed.


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[Assistant] At the start of the project, we were given a very clear brief and each team member had specific tasks to complete by the allocated deadlines. Unfortunately, a third of the way through the project


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[Assistant] The client changed their mind about an important aspect of the product, and we had to effectively start again from scratch, but still complete the work within the 21 day period. I, along with the other team members, decided to work an additional two hours each evening


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[Assistant] So make up the lost time to get the project completed for our client.


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[Assistant] It was tough going, but by working the additional hours and also by supporting each other in the completion of each task, we managed to get everything done, including the product testing by the requested timeline.


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[Assistant] I actually feel I work better when I'm under pressure, and I always love the challenge of getting things completed whilst under strict time constraints. Now, you will notice there, but using situation, task, action, result, the STAR technique


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[Assistant] It's a brilliant way for correctly answering any type of behavioral interview question. Now, what I would like you to do next for your preparation into your HireVue interview?


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[Assistant] Is to download my 21 great answers to interview questions. If you click that link in the top right hand corner there, it takes you through to my website here.


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[Assistant] You can also search for, and again, I've put the link here.


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[Assistant] For behavioral type interview questions. So these are behavioral type interview questions, and it will just go through these 20 of them here. These are the ones. Tell me about a stressful situation you were in at work and how you dealt with it. Describe a time when you delivered excellent customer care. There's loads of them there. And I have given you


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[Assistant] The star answer to all of those questions. So those links are there for you in the top right hand corner here of this video. I hope you've enjoyed that. Please don't forget to SUBSCRIBE and LIKE the video. Thank you very much. If you have any questions, put them in the comments section below the video.


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[Assistant] And I wish you all the best for passing your HireVue interview. Have a brilliant day. Thank you very much.


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[Assistant] Elasticsearch is an ultra-fast distributed search and analytics engine powered by the Apache Lucene project.


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[Assistant] Elasticsearch is specifically designed to search and index massive data sets in the order of petabytes. I'm Anthony Anthony and I'll be your guide on this learning journey. In this first video, we're going to do a course overview about me.


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[Assistant] I'm a San Francisco based data scientist and the founder of Xresults.


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[Assistant] At X results, we aim to harness the power of data to deliver cutting edge solutions. I'm also an AI enthusiast and avid innovator. When you think of this course, think of it as being separated into three parts. In part one, the introduction.


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[Assistant] We introduce you to the concept of Elasticsearch.


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[Assistant] As is the case with any new technology, it's important to know exactly what it does. Here.


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[Assistant] I'll start by defining Elasticsearch and from there I'll walk you through the straight forward process of installation.


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[Assistant] I will also detail the goal of Elasticsearch as a technology.


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[Assistant] And this part concludes with me highlighting some of the changes in the latest release, which is version 5.0.


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[Assistant] In the next section entitled, Getting Started with Elasticsearch, I'll get you through the process of working with Elasticsearch. More specifically, we'll get our hands dirty.


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[Assistant] We'll dive deeper into Elasticsearch by looking at some do's and don'ts. We'll then move into the newly formed Elastic Stack, which leverages the power of several applications to create a full stack around Elasticsearch. I next guide you through the process of setting up an Elasticsearch cluster on the cloud via Amazon Web Services. We'll close things out with a look at some advanced queries with DSL.


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[Assistant] Knowledge of some good use cases for elastic search.


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[Assistant] You will also be well on your way to creating a large-scale Elasticsearch cluster along with having the ability to use aggregations to build analytics. And putting it all together, you will have the necessary skills to implement Elasticsearch 5.0 in the cloud.


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[Assistant] Now does this solve this problem? This is what we wanted to do, right? We wanted to pass in a behavior and then execute that behavior.


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[Assistant] Well it kinda does, but I think there is some extra work that we are doing over here. We are not just passing in a behavior. We are passing a thing that has a behavior. We are not passing in a perform method. We are passing a greeting which has a method which is the perform. Wouldn't it be cool if we had


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[Assistant] Well, lambdas set out to achieve just this. Lambdas lets you create these entities which are just functions. They're called lambda expressions, which are just functions which do not belong to a class. They're not methods of a class. They are just functions which exist in isolation.


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[Assistant] And here's the best part, those functions can be treated as values. This is kinda confusing for somebody who's totally used to object-oriented programming and they haven't done functional programming. So let me explain what I mean by that. What I mean by functions as values.


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[Assistant] You know what inline values are, right? So you have, uh, let's say you have a string or a double or an integer. You can define them inline. So here is an example of a string called name being declared and assign the value of foo.


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[Assistant] acts as values in Java. You can assign it to variables. You can assign it to different types. Similarly objects.


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[Assistant] to a variable as a value. So the value is not


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[Assistant] The execution of the block of code, it is the block of code itself.


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[Assistant] Piece of code becomes a value that gets assigned to a variable. And that wherever the variable goes, a piece of code goes with it.


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[Assistant] Is that possible? Can we do something like this? Have a block of code which is the value which is the steps that need to be executed. Right here you can have lines of Java code. Is that possible? Now let's take a look at this for a minute. What is the standard way in which we


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[Assistant] Write a block of code in Java, uh, before Java 8, of course. Well, it's the method, right? You create a method which contains a block of code. It has input arguments, and it has a return, right? This is how you typically do it.


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[Assistant] Imagine if you could take a method and assign it to a variable.


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[Assistant] And again, note that I'm not saying execute the method and assign the return to a variable. I'm saying assign the method itself. The method becomes a thing that gets assigned to a variable. Can you do that? Now let's say I have this perform method. This is the one we just saw, right? We have a perform method which is a public, returns a void, and it has system.out.print.


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[Assistant] Now what if I were to assign this to a variable called a block of code? This is possible in Java 8 using Lambda. You can write a Lambda expression which does just this. And once you do this, you can take that variable, a block of code and pass it around and have different other pieces of code executed, which is really exciting. So let's see how to write this Lambda expression. The code that you're looking at has a lot of extra things which you don't need. For instance, let's take a look at this public.


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[Assistant] Public makes sense when a function is a part of a class, in a method, right? You need to know if a method is a public or a private or a protected, because it makes sense in the context of a class. But if a function exists in isolation, it doesn't make sense to call it public.


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[Assistant] When you assign a string to a variable, what's the name that you refer that string by? It's the name of the variable. You don't have to give it another name.


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[Assistant] When you assign this function to a variable called a block of code, the way you refer to this function is using that variable name. You're going to use a block of code name to access the function, which means that it doesn't need this other name. So let's get rid of that too.


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[Assistant] Now the creators of the Java spec could have rested here and say, okay, this is how you create a lambda expression, but they actually went a step further. Now you see when you look at this code, if I were to show you this code and say, can you tell me what the return type is?


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[Assistant] You don't need to look at this wide. You can actually look at the code and figure out what the return type is, right? It doesn't return anything, so it's a wide.


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[Assistant] So the Java compiler is now smart enough to do just that.


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[Assistant] We'll know what the return type is based on the expression itself.


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[Assistant] So, turns out in Java 8, you don't have to provide the return type over here for lambda expression. So even that goes away. So this is what's left.


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[Assistant] These are the elements that you need to provide in order to write a lambda expression.


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[Assistant] So, we removed all the things that we don't need to specify when that becomes a lambda expression. You don't have to specify public for a lambda. You don't have to specify the return type. You don't have to specify the name.


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[Assistant] So with that, what's left is this parentheses, which indicate the arguments, and then the block of code.


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[Assistant] Okay, so the syntax is pretty much this for a lambda expression except for one small addition. The addition is you need to put this symbol over here. You need to put that between the parentheses and the block of code. If you put this, so there you go, you've got your lambda expression.


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[Assistant] So, if you know how to write a method in Java, you now know how to write a lambda expression in Java. You need to convert these steps in your mind till you get comfortable and after that you'll be writing these expressions directly. In order to write a lambda expression, take a method


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[Assistant] Remove the modifiers, right, you don't need the public private, you don't need the name of the method, and you don't need the return time.


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[Assistant] What's left is a lambda expression except for adding the symbol, which kinda looks like an arrow. Okay? You put this between the parentheses and...


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[Assistant] The curly braces, you've got your lambda expression. Now this lambda expression can be assigned


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[Assistant] To a variable, you can actually do this in Java 8, and this variable contains the value which is that function. Right?


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[Assistant] There is one further shortcut that you can do if your body of the lambda expression is just one line. Here, it's just one line at system.out.print.


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[Assistant] So if that's just one line, you can actually remove the curly braces. So it just becomes this. You have the parentheses, the arrow,


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[Assistant] And then that line of code. But remember this is only if you have just one line of code in your lambda expression. If you have multiple lines, then you are going to need the curly braces. Okay? This is how you write lambda expressions.


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[Assistant] This cocoon, it is comprised of so few dimensions, contains such a minuscule number of information units, it paled in comparison to the anisotropic. But then it begat the singularity of humanity.


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[Assistant] But what is it that makes humanity so unique?


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[Assistant] Through my experience, I've developed a theory regarding that.


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[Assistant] Information wise, it was completely identical to you at the time Kado created it. But that does not make it you. In a similar fashion, this is a clone of myself that I have just now generated. Composed of the same information as me, it should be a perfect, identical replication.


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[Assistant] It occurred to me that this must be the nature of the singularity.


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[Assistant] The power to attribute different values to the exact same thing. The capacity to take one and extricate a variety of meanings from it. That unprecedented power is humanity's creation. The source of these singular properties, it is you and it is me.


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[Assistant] I ask you, help me to explore and understand this phenomenon. Come with me to the Anisotropic.


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[Assistant] Think of the incredible amounts of information humanity has created in this tiny universe. And all that they may accomplish in the Anisotropic. Can you imagine the miracles they'll reveal to me? Your Anisotropic transformation will succeed. I promise you. I beseech you, Shindo. Please join me.


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[Assistant] I have the power to ensure that this transformation proceeds. No one in your world can possibly stop me. And you, Shindo, you know what I can do. You should realize I can take you there by force!


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[Assistant] A singularity possessing information beyond its known limits. You are an irreplaceable individual. Transforming you by force would be an act of futility. If I were to do that it would eradicate your heart.


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[Assistant] It is quite strange. The conclusion I've reached, it was formulated from within me, separate and distinct from all information variables.


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[Assistant] A device to nullify phirgonics. In tandem with a phirgonics generator. How truly splendid humanity's technology has come so far.


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[Assistant] What I've promised since I first came here.


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[Assistant] Impossible. This control she has, she's not even from the Anisotropic! I don't have time to formulate a better method. I'll destroy this universe with her in it!


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[Assistant] It's Professor Dave, let's check out more chromosomes.


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[Assistant] We've learned a lot about DNA, chromosomes, and heredity. We've also learned about mitosis, the reason why all your cells have the same genetic material, and meiosis, the reason that gamete production and subsequent fertilization produce such genetic diversity from generation to generation.


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[Assistant] Sometimes there are errors that occur throughout this process. We have discussed mutation in the genome, due to both endogenous and exogenous mutagens, as well as random error by replicative machinery.


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[Assistant] But there are large-scale alterations to the genome that can occur by completely different mechanisms, which can have enormous effects on the resulting organism. Let's take a look at some of these now.


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[Assistant] We take it for granted that all of our somatic cells have 46 chromosomes, which through meiosis produce gametes with 23 chromosomes so that fertilization produces a zygote with 46 chromosomes once again, which grows into a new person.


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[Assistant] But there are instances where these numbers can change.


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[Assistant] Let's take another look at meiosis. We know that in meiosis I, homologous chromosomes line up along the metaphase plate, and then get pulled apart.


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[Assistant] Resulting in haploid daughter cells with duplicated chromosomes that then undergo meiosis II, which is quite similar to mitosis, to give haploid gametes with unduplicated chromosomes.


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[Assistant] But in either of the two processes, something called non-disjunction can occur. Say that in meiosis I, a particular pair of chromosomes both go to one daughter cell and not the other, instead of one going to each.


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[Assistant] After meiosis II, four gametes will result, two of which will have an additional chromosome, or N plus one, and two of which will be missing a chromosome, or N minus one.


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[Assistant] Alternately, say that in meiosis II, a pair of sister chromatids do not get properly pulled apart, and they both go to one daughter cell and not the other.


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[Assistant] Assuming everything went properly with the other daughter cell from meiosis I, the end product will be two normal gametes, and then one N plus one, and one N minus one gamete, due to the non-disjunction.


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[Assistant] If any of these abnormal gametes undergo fertilization with a normal gamete, it will produce a zygote that exhibits a neuploidy, which means an abnormal number of a particular chromosome. Typically, this will be either monosome


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[Assistant] As the zygote divides and develops into a fetus, all of the resulting cells will have the same abnormality, which will result in huge problems.


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[Assistant] In fact, embryos with such abnormalities often result in miscarriage, meaning spontaneous termination of pregnancy.


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[Assistant] Kleinfelter syndrome involving an extra X chromosome in a male, or Turner syndrome, which is monosomy X in females. More on these in the upcoming pathology series.


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[Assistant] Now unlike anaeuploidy, which involves an irregular number of a particular chromosome, organisms can also exhibit polyploidy, which means more than two complete sets of chromosomes.


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[Assistant] These situations can arise if non-disjunction occurs for all chromosomes to produce a diploid egg, which is subsequently fertilized.


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[Assistant] Or if a diploid zygote replicates all its chromosomes and then fails to divide, which after another round of replication and mitosis, will yield a tetraploid embryo.


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[Assistant] Polyploid animals are quite rare, although a handful of polyploid fishes and amphibians do exist.


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[Assistant] However, polyploid plants are quite common, particularly ones we eat.


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[Assistant] Bananas are triploid, wheat is hexaploid, or 6N, and strawberries are octaploid, or 8N, meaning eight complete sets of chromosomes in each cell.


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[Assistant] Beyond these examples of alteration in chromosome number, we should also discuss some ways that the structure of an individual chromosome can change drastically.


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[Assistant] We have already learned about point mutations and frame-shift mutations, which involve the alteration, deletion, or insertion of a single bass pair.