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Lecture 1: Introduction
Download the video from iTunes U or the Internet Archive.
Topics covered: Introduction
Instructors: Prof. Robert A. Weinberg
Lecture 10: Molecular Biolo.
Lecture 11: Molecular Biolo.
Lecture 12: Molecular Biolo.
Lecture 13: Gene Regulation
Lecture 14: Protein Localiz.
Lecture 15: Recombinant DNA 1
Lecture 16: Recombinant DNA 2
Lecture 17: Recombinant DNA 3
Lecture 18: Recombinant DNA 4
Lecture 19: Cell Cycle/Sign.
Lecture 26: Nervous System 1
Lecture 27: Nervous System 2
Lecture 28: Nervous System 3
Lecture 29: Stem Cells/Clon.
Lecture 30: Stem Cells/Clon.
Lecture 31: Molecular Medic.
Lecture 32: Molecular Evolu.
Lecture 33: Molecular Medic.
Lecture 34: Human Polymorph.
Lecture 35: Human Polymorph.
As I'm going to argue repeatedly today, biology has become a science over the last 50 years. And, as a consequence, we can talk about some basic principles. We can talk about some laws and then begin to apply them to very interesting biological problems.
And so our general strategy this semester, as it has been in the past, is to spend roughly the first half of the semester talking about the basic laws and rules that govern all forms of biological life on this planet. And you can see some of the specific kinds of problems, including the problem of cancer, how cancer cells begin to grow abnormally, how viruses proliferate, how the immune system functions, how the nervous system functions, stem cells and how they work and their impact on modern biology, molecular medicine, and finally perhaps the future of biology and even certain aspects of evolution. The fact of the matter is that we now understand lots of these things in ways that were inconceivable 50 years ago. And now we could begin to talk about things that 50 years ago people could not have dreamt of. When I took this course, and I did take it in 1961, we didn't know about 80% of what we now know. You cannot say that about mechanics in physics, you cannot say that about circuit theory in electronics, and you cannot say that, obviously, about chemistry.
And I'm mentioning that to you simply because this field has changed enormously over the ensuing four decades. I won't tell you what grade I got in 7. 1 because if I would, and you might pry it out of me later in the semester, you probably would never show up again in lecture.
But in any case, please know that this has been an area of enormous ferment. And the reason it's been in such enormous ferment is of the discovery in 1953 by Watson and Crick of the structure of the DNA double helix. Last year I said that we were so close to this discovery that both Watson and Crick are alive and with us and metabolically active, and more than 50 years, well, exactly 50 years after the discovery. Sadly, several months ago one of the two characters, Francis Crick died well into his eighties, and so he is no longer with us. But I want to impress on you the notion that 200 years from now, we will talk about Watson and Crick the same way that people talk about Isaac Newton in terms of physics. And that will be so because we are only beginning to perceive the ramifications of this enormous revolution that was triggered by their discovery. That is the field of molecular biology and genetics and biochemistry which has totally changed our perceptions of how life on Earth is actually organized.
Much of the biology to which you may have been exposed until now has been a highly descriptive science. That is you may have had courses in high school where you had to memorize the names of different organisms, where you had to understand how evolutionary phylogenies were organized, where you had to learn the names of different organelles, and that biology was, for you, a field of memorization. And one point we would like, hopefully successfully, to drive home this semester is the notion that biology has now achieved a logical and rational coherence that allows us to articulate a whole set of rules that explain how all life forms on this planet are organized. It's no longer just a collection of jumbled facts. Indeed, if one masters these molecular and genetic principles, one can understand in principle a large number of processes that exist in the biosphere and begin to apply one's molecular biology to solving new problems in this arena.
One of the important ideas that we'll refer to repeatedly this semester is the fact that many of the biological attributes that we posses now were already developed a very long time ago early in the inception of life on this planet. So if we look at the history of Earth, here the history of Earth is given as 5 billion years, this is in thousands obviously. The Earth is probably not that old.
It's probably 4.5 or 4 or 3 billion years but, anyhow, that's when the planet first aggregated, as far as we know.
One believes that no life existed for perhaps the first half billion years, but after half a billion years, which is a lot of time to be sure, there already begins to be traces of life forms on the surface of this planet. And that, itself, is an extraordinary testimonial, a testimonial to how evolutionary processes occur. We don't know how many planets there are in the universe where similar things happened.
And we don't know whether the solution that were arrived at by other life systems in other places in the universe, which we may or may not ever discover, were the similar solutions to the ones that have been arrived at here.
It's clear, for example, that to the extent that Darwinian Evolution governs the development of life forms on this planet that is not an artifact of the Earth. Darwinian Evolution is a logic which is applicable to all life forms and all biosystems that may exist in the universe, even the ones we have not discovered.
However, there are specific solutions that were arrived at during the development of life on Earth which may be peculiar to Earth.
The structure of the DNA double helix.
The use of ribose in deoxyribose. The choice of amino acids to make proteins. And those specific solutions may not be universal.
Whether they're universal in the sense of existing in all life forms across the planet, the fact is that many of the biochemical and molecular solutions that are represented in our own cells today, these solutions, these problems were solved already 2 and 3 billion years ago. And once they were solved they were kept and conserved almost unchanged for the intervening 2 or 3 billion years. And that strong degree of conservation means that we can begin to figure out what these principles were early on in evolution of life on this planet and begin to apply them to all modern life forms.
From the point of view of evolution, almost all animals are identical in terms of their biochemistry and in terms of their physiology.
The molecular biology of all eukaryotic cells, that is all cells that have nuclei in them, is almost the same.
And, therefore, we're not going to focus much in this course this semester on specific species but rather focus on general principles that would allow us to understand the cells and the tissues and the physiological processes that are applicable to all species on the surface of the planet. Let's just look here and get us some perspective on this because, the fact of the matter is, is that multicellular life forms, like ourselves, we have, the average human being has roughly three or four or five times ten to the thirteenth cells in the body. That's an interesting figure.
The average human being goes through roughly ten to the sixteenth cell divisions in a lifetime, i.e. ten to the sixteenth times in your body there will be cells that divide, grow and divide.
Every day in your body there are roughly ten to the eleventh cells that grow and divide. Think of that, ten to the eleventh.
And you can divide that by the number of minutes in a day and come up with an astounding degree of cellular replication going on.
All of these processes can be traceable back to solutions that were arrived at very early in the evolution of life on this planet, perhaps 550, 600 million years ago when the first multicellular life forms began to evolve. Before that time, that is to say before 500 to 600 million years ago, there were single-cell organisms.
For example, many of them survive to this day. There were yeast-like organisms. And there were bacteria. And we make one large and major distinction between the two major life forms on the planet in terms of cells. One are the prokaryotic cells. And these are the cells of bacteria, I'll show you an image of them shortly, which lack nuclei.
And the eukaryotic cells which poses nuclei and indeed have a highly complex cytoplasm and overall cellular architecture.
We think that the prokaryotic life forms on this planet evolved first probably on the order of 3 billion years ago, maybe 3.
billion years ago, and that about 1. billion years ago cells evolved that contained nuclei. Again, I'll show them to you shortly. And these nucleated cells, the eukaryotes then existed in single-cell form for perhaps the next 700 or 800 million years until multi-cellular aggregates of eukaryotic cells first assembled to become the ancestors of the multi-cellular plants and the multi-cellular animals that exist on the surface of the Earth today. To put that in perspective, our species has only been on the planet for about 150, 00 years. So we've all been here for that period of time.
And a 150,000 sounds like a long time, in one sense, but it's just a blink in the eye of the Lord as one says in terms of the history of life on this planet, and obviously the history of the universe which is somewhere between 13 and 15 billion years old.
You can begin to see that the appearance of humans represents a very small segment of the entire history of life on this planet.
And here you can roughly see the way that life has developed during this period of time from the fossil record. You see that many plants actually go back a reasonable length of time, but not more than maybe 300 or 400 million years. Here are the Metazoa.
And this represents -- Well, can you hear me? Wow, 614 came in handy.
OK. So if we talk about another major division, we talk about protozoa and metazoa. The suffix zoa refers to animals, as in a zoo. And the protozoa represents single-cell organisms.
The metazoa represent multi-cellular organisms. And we're going to be focusing largely on the biology of metazoan cells this semester, and we're going to be spending almost no time on plants.
It's not that plants aren't important. It's just that we don't have time to cover everything. And, indeed, the molecular biology that you learn this semester will ultimately enable you to understand much about the physiology of multi-cellular plants which happen to be called metaphyta, a term you may never hear again in your entire life after today. That reminds me, by the way, that both Dr. Lander and I sometimes use big words.
And people come up to me afterwards each semester each year and say Professor Weinberg, why don't you talk simple, why don't you talk the way we heard things in high school?
And please understand that if I use big words sometimes it's to broaden your vocabulary so you can learn big words.
One of the things you should be able, one of the big take-home lessons of this course should be that your vocabulary is expanded.
Not just your scientific vocabulary but your general working English vocabulary. Perhaps the biggest goal of this course, by the way, is not that you learn the names of all the organelles and cells but that you learn how to think in a scientific and rational way. Not just because of this course but that this course helps you to do so. And as such, we don't place that much emphasis on memorization but to be able to think logically about scientific problems. Here we can begin to see the different kinds of metazoa, the animals. Here are the metaphyta and here are the protozoa, different words for all of these.
And here we see our own phylum, the chordates. And, again, keep in mind that this line right down here is about 550 to 600 million years ago, just to give you a time scale for what's been going on, on this planet.
One point we'll return to repeatedly throughout the semester is that all life forms on this planet are related to one another.
It's not as if life was invented multiple times on this planet and that there are multiple independent inventions to the extent that life arose more than once on this planet, and it may have. The other alternative or competing life forms were soon wiped out by our ancestors, our single-cellular ancestors 3 billion years ago.
And, therefore, everything that exists today on this planet represents the descendents of that successful group of cells that existed a very long time ago. Here we have all this family tree of the different metazoan forms that have been created by the florid hand of evolution. And we're not going to study those phylogenies simply because we want to understand principles that explain all of them.
Not just how this or that particular organism is able to digest its food or is able to reproduce. Here's another thing we're not going to talk about. We're not going to talk about complicated life forms. We're not going to talk very much, in fact hardly at all, about ecology. This is just one such thing, the way that a parasite is able to, a tapeworm is able to infect people.
This is, again, I'm showing you this not to say this is what we're going to talk about, we're not going to talk about that.
We're not going to talk about that. There's a wealth of detail that's known about the way life exists in the biosphere that we're simply going to turn our backs on by focusing on some basic principles.
We're also not going to talk about anatomy. Here in quick order are some of the anatomies you may have learned about in high school, and I'm giving them to you each with a three-second minute, a three-second showing to say we're not going to do all this.
And rather just to reinforce our focus, we're going to limit ourselves to a very finite part of the biosphere.
And here is one way of depicting the biosphere. It's obviously an arbitrary way of doing so but it's quite illustrative.
Here we start from molecules. And, in fact, we will occasionally go down to submolecular atoms. And here's the next dimension of complexity, organelles. That is these specialized little organs within cells. We're going to focus on them as well. We're going to focus on cells. And when we start getting to tissues, we're going to start not talking so much about them.
And we're not going to talk about organisms and organs or entire organisms or higher complex ecological communities.
And the reason we're doing that is that for 40 years in this department, and increasingly in the rest of the world there is the acceptance of the notion that if we understand what goes on down here in these first three steps, we can understand almost everything else in principle.
Of course, in practice we may not be able to apply those principles to how an organism works or to how the human brain works yet.
Maybe we never will. But, in general, if one begins to understand these principles down here, one can understand much about how organismic embryologic develop occurs, one can understand a lot about a whole variety of disease processes, one can understand how one inherits disease susceptibilities, and one can understand why many organisms look the way they do, i.e. the process of developmental biology.
And so, keep in mind that if you came to hear about all of these things, we're going to let you down. That's not what this is going to be about. This also dictates the dimensions of the universe that we're going to talk about because we're going to limit ourselves to the very, very small and not to the microscopic. On some occasions we'll limit ourselves to items that are so small you cannot see them in the light microscope. On other occasions we may widen our gaze to look at things that are as large as a millimeter, but basically we're staying very, very small. Again, because we view, correctly or not, the fact that the big processes can be understood by delving into the molecular details of what happens invisibly and cannot be seen by most ways of visualizing things, including the light and often even the electron microscope. Keep in mind that 50 years ago we didn't know any of this, for all practical purposes, or very little of this. And keep in mind that we're so close to this revolution that we don't really understand its ramifications.
I imagine it will be another 50 years before we really begin to appreciate the fallout, the long-term consequences of this revolution in biology which began 51 years ago. And so you're part of that and you're going to experience it much more than my generation did.
And indeed one of the reasons why MIT decided about 10 or 12 years ago that every MIT undergraduate needed to have at least one semester of biology is that biology, in the same way as physics and chemistry and math, has become an integral part of every educated person's knowledge-base in terms of their ability to deal with the world in a rational way. In terms of public policy, in terms of all kinds of ethical issues, they need to understand what's really going on. Many of the issues that one talks about today about bioethics are articulated by people who haven't the vaguest idea about what we're talking about this semester.
You will know much more than they will, and hopefully some time down the road, when you become more and more influential voices in society, you'll be able to contribute what you understood here, what you learned here to that discussion.
Right now much of bioethical discussion is fueled by people who haven't the vaguest idea what a ribosome or mitochondrion or even a gene is, and therefore is often a discussion of mutually shared ignorance which you can diffuse by learning some basics, by learning some of the essentials. Here is the complexity of the cell we're going to focus on largely this semester, which is to say the eukaryotic rather than the prokaryotic cell.
And this is just to give you a feeling for the overall dimensions of the cell and refer to many of the landmarks that will repeatedly be brought up during the course of this semester.
Here is the nucleus. The term karion comes from the Greek meaning a seed or a kernel. And the nucleus is what gives the eukaryotic cell its name. Within the nucleus, although not shown here, are the chromosomes which carry DNA.
You may have learned that a long time ago. Outside of the nucleus is this entire vast array of organelles that goes from the nuclear membrane, and I'm point to it right here, all the way out to the outside of the cell. The outside limiting membrane, the outer membrane of the cell is called the plasma membrane.
And between the nucleus and the plasma membrane there is an enormous amount of biological and biochemical activity taking place.
Here are, for example, the mitochondria. And the mitochondria, as one has learned, are the sources of energy production in the cell. And, therefore, we'll touch on them very briefly. This is an artist's conception of what a mitochondrion looks like. Almost always artists' conceptions of these things have only vague resemblance to the reality.
But, in any case, you can begin to get a feeling for what one thinks about their appearance. Here are mitochondria sliced open by the hand of the artist. And, interestingly, mitochondria have their own DNA in them. One now accepts the fact that mitochondria are the descendents of bacteria which insinuated themselves into the cytoplasms of larger cells, roughly 1.5 billion years ago, and began to do a specialized job which increasingly became the job of energy production within cells. To this day, mitochondria retain some vestigial attributes of the bacterial ancestors which initially colonized or parasitized the cytoplasm of the cell.
When I say parasitized, you might imagine that the mitochondria are taking advantage of the cell.
But, in fact, the mitochondria represent the essential sources of energy production in the cell. Without our mitochondria, as you might learn by taking cyanide, for example, you don't live for very many minutes. And the vestiges of bacterial origins of mitochondria are still apparent in the fact that mitochondria still have their own DNA molecule, their own chromosome. They still have their own ribosomes and protein synthetic apparatus, even though the vast majority of the proteins inside mitochondria are imported from the cytoplasm, i.e., these vestigial bacteria now rely on proteins made by the cell at large that are imported into the mitochondrion to supplement the small number of vestigial bacterial proteins which are still made here inside the mitochondrion and used for essential function in energy production. Here is the Golgi apparatus.
And the Golgi apparatus up here is used for the production of membranes.
As one will learn throughout the semester, the membranes of a cell are in constant flux and are being pulled in and remodeled and regenerated. The Golgi apparatus is very important for that.
Here's the rough endoplasmic reticulum. That's important for the synthesis of proteins which are going to be displayed on the surface of cells, you don't see them depicted here, or are going to be secreted into the extracellular space.
Here are the ribosomes, which I might have mentioned briefly before. And these ribosomes are the factories where proteins are made.
Again, we're going to talk a lot about them. And, finally, several other aspects, the cytoskeleton. The physical integrity, the architecture of the cell is maintained by a complex network of proteins which together are considered to be the cytoskeleton. And they enable the cell to have some rigidity, to resist tensile forces, and actually to move.
Cells can actually move from one place to the other.
They have motile properties. They're able to move from one location to another. The process of cell motility, if that's a word you'd like to learn.
Here is what a prokaryotic cell looks like by contrast.
And I just want to give you a feeling. First of all, it looks roughly like a mitochondrion that I discussed before. But you see that there is the absence of a nuclear membrane. There's the absence of the highly complex cytoarchitecture. Cyto always refers to cells.
There's the absence of the complex cytoarchitecture that one associates with eukaryotic cells. In fact, all that a bacterium has is this area in the middle. It's called the nucleoid, a term which you also will probably never hear in your lifetime.
And it represents simply an aggregate of the DNA of the chromosomes of the bacterium. And, in most bacteria, the DNA consists of only a single molecule of DNA which is responsible for carrying the genetic information of the bacteria. There's no membrane around this nucleoid. And outside of this area where the DNA is kept are largely ribosomes which are important for protein synthesis. There's a membrane on the outside of this called the plasma membrane, very similar to the plasma membrane of eukaryotic cells. And outside of that is a meshwork that's called the outer membrane, it's sometimes called the cell wall of the bacterium, which is simply there to impart structural rigidity to the bacterium making sure that it doesn't explode and holding it together. And then there are other versions of eukaryotic cells. Here's what a plant cell looks like.
And it's almost identical to the cells in our body, except for two major features. For one thing, it has chloroplasts in it which are also, one believes now, the vestiges of parasitic bacteria that invade into the cytoplasm of eukaryotic cells. So, in addition to mitochondria which are responsible for energy production in all eukaryotic cells, we have here the chloroplasts which are responsible for harvesting light and converting it into energy in plant cells. The rest of the cytoplasm of a plant cell looks pretty much the same.
One feature that I didn't really mention when I talked about an animal cell is in the middle of the nucleus, here you can see, is a structure called a nucleolus. And a nucleolus, or the nucleolus in the eukaryotic cell is responsible for making the large number of ribosomes which are exported from the nucleus into the cytoplasm. And, as I mentioned just before, the ribosomes are responsible for protein synthesis.
It turns out this is a major synthetic effort on the part of most cells. Cells, like our own, have between 5 and 10 million ribosomes in the cytoplasm. So it's an enormous amount of biomass in the cytoplasm whose sole function is to synthesize proteins.
As we will learn also, proteins that are synthesized by the ribosomes don't sit around forever. Some proteins have long lives.
Some proteins have lifetimes of 15 minutes before they're degraded, before they're turned over. One other distinction between our cells, that is the cells of metazoa and metaphyta, are the cell walls, analogous to the cell walls of bacteria, this green thing on the outside. As I said before, we do not have cell walls around our cells. And we will, as the semester goes on, go into more and more details about different aspects of this cytoarchitecture during the first half of the semester. Here, for example, is an artist's depiction of the endoplasmic reticulum.
Why it has such a complex name, I cannot tell you, but it does.
It's called the ER in the patois of the street. The ER.
And the endoplasmic reticulum is a series of membranes.
Keep in mind, not the only membrane in the cell is the plasma membrane.
Within the cytoplasm there are literally hundreds of membranes which are folded up in different ways.
Here you see them depicted. And one set of these membranes, often they're organized much like tubes, represents the membranes of the endoplasmic reticulum which either lacks ribosomes attached to it or has these ribosomes attached to it which cause this to be called the rough endoplasmic reticulum to refer to its rough structure which is created by the studding of ribosomes on the surface.
As we will learn, just trying to give you a feeling for the geography of what we're going to talk about this semester, these ribosomes on the surface of the endoplasmic reticulum are dedicated to the task of making highly specialized proteins which are either going to be dispatched to the surface of the cell where they will be displayed on the cell's surface or actually secreted into the extracellular space. Many of the proteins that are destined for our body are not kept within cells but are released into the extracellular space where they serve important functions, and so we're going to focus very much on them.
Here's actually what some of these things look like in the electron microscope to see whether we can either believe or fully discredit the imaginations of the artists. Here's the rough endoplasmic reticulum I showed you in schematic form before. And you can see why it's called rough. All these black dots are ribosomes attached on the outside. Here's the Golgi apparatus.
You see these vesicles indicated here. And a vesicle, just to use a new word, is simply a membranous bag.
And keep in mind, by the way, that we're not going to spend the semester with these highly descriptive discussions.
Our intent today is to get a lot of these descriptive discussions out of the way so that we can begin to talk in a common parlance about many of the parts, the molecular parts of cells and organisms.
Here is the mitochondrion which we saw depicted before.
It looks similar to, but not identical to the artist's description of that. And keep in mind that the mitochondrion in our cells, as I said before, are the descendents of parasitic bacteria. Here's the endoplasmic reticulum, and the way it would look, as it does in certain parts of the cell when it doesn't have all of these ribosomes studded on the surface. The endoplasmic reticulum here is involved in making membranes.
The endoplasmic reticulum here is involved in the synthesis and export of proteins to the cell's surface and for secretion, as I mentioned before. Much of what we're going to talk about over the next days is going to be focused on the nucleus of the cell, that is on the chromosomes on the cell and on the material which is called chromatin which carries the genetic material.
So the term chromatin is used in biology to refer simply to the mixture of DNA and proteins, which together constitutes the chromosomes. So chromatin has within it DNA, it has protein, and it has a little bit of RNA in it.
And we're going to focus mostly on the DNA in the chromatin, because if we can begin to understand the way the DNA works and functions many other aspects will flow from that.
I mentioned the cell's surface, and I just want to impress on you the fact that the plasma membrane of a cell is much more complicated than was depicted in these drawings that I showed you just before.
If we had a way of visualizing the plasma membrane of a cell, we would discover that it's formed from lipids. We see such lipids there, phospholipids, many of them. We'll talk about them shortly. That the outside of the cell, there are many proteins, you see them here, which thread their way through the plasma membrane, have an extracellular and intracellular part.
And these transmembrane proteins, which reach from outside to inside, represent a very important way by which the cell senses its environment. This plasma membrane, as we'll return to, represents a very effective barrier to segregate what's inside the cell from what's outside of the cell to increase concentrations of certain biochemical entities.
But at the same time it creates a barrier to communication.
And one of the things that cells have had to solve over the last 700 to 800 million years is ways by which the exterior of the cell is able to send certain signals and transmit that information to the interior of the cell. At the same time, cells have had to use a number of different, invent a number of different proteins, some of them indicated here, which are able to transport materials from the outside of the cell into the cell, or visa versa. So the existence of the plasma membrane represents a boon to the cell in the sense that it's able to segregate what's on the inside from what's on the outside.
But it represents an impediment to communication which had to be solved, as well as an impediment to transport. And many of these transmembrane proteins are dedicated to solving those particular problems.
Here you see, once again an artist's depiction form, aspects of the cytoskeleton of the cell. And when we talk about the cytoskeleton we talk about this network of proteins which, as I said before, gives the cell rigidity.
Keep in mind that the prefix cyto or the suffix cyt refers always to cells. Allows the cell to have shape. And here you can see this network as depicted in one way, but here it's depicted actually much more dramatically. And here you begin to see the complexity of what exists inside the cell. Here are these proteins.
These are polymers of proteins called vimentin which are present in very many mesenchymal cells. Here are microtubules made from another kind of protein. Here are microfilaments, in this case made of the molecule actin. And if we looked at individual molecules of actin they would be invisible.
This is end-to-end polymerization of many actin molecules.
And we're looking here under the microscope from one end of the cell to the other end of the cell. And you can see how these molecules, they create stiffness, and they also enable the cell to contract and to move. Some people might think that the interior of the cell is just water with some molecules floating around them. But if you actually look at what's present in the cell, more than 50% of the volume is taken up by proteins.
It's not simply an aqueous solvent where everything moves around freely.
It's a very viscous slush, a mush. And it's quite difficult there for many cells to move around from one part of the cell to the other. Here you begin to get a feeling now for how the connection, which we'll reinforce shortly in great detail, between individual molecules and the cytoskeleton.
And here you see these actin fibers. I showed them to you just moments ago stretching from one end of the cell to the other.
And each of these little globules is a single actin monomer which polymerize end-to-end and then form multi-strand aggregates to create the actin cytoskeleton. Here's an intermediate filament and here's the microtubules that are formed, once again giving us this impression that the cell is actually highly organized and that that high degree of organization is able to give it some physical structure and shape and form. I think we're going to end today two minutes early. You probably won't object.
Lecture 30: Stem Cells/Cloning 2
Download the video from iTunes U or the Internet Archive.
Topics covered: Stem Cells/Cloning 2
Instructors: Prof. Robert A. Weinberg
Lecture 10: Molecular Biolo.
Lecture 11: Molecular Biolo.
Lecture 12: Molecular Biolo.
Lecture 13: Gene Regulation
Lecture 14: Protein Localiz.
Lecture 15: Recombinant DNA 1
Lecture 16: Recombinant DNA 2
Lecture 17: Recombinant DNA 3
Lecture 18: Recombinant DNA 4
Lecture 19: Cell Cycle/Sign.
Lecture 26: Nervous System 1
Lecture 27: Nervous System 2
Lecture 28: Nervous System 3
Lecture 29: Stem Cells/Clon.
Lecture 30: Stem Cells/Clon.
Lecture 31: Molecular Medic.
Lecture 32: Molecular Evolu.
Lecture 33: Molecular Medic.
Lecture 34: Human Polymorph.
Lecture 35: Human Polymorph.
Good morning, class. Nice to see you here, you loyal holdouts, the stalwarts who haven't gone home early for Thanksgiving. You recall that last time we were talking about the Matevoidic system, and much of the rationale for studying it stems from two reasons. First of all, it recapitulates in a formal sense what happens during embryogenesis, i.e. one has relatively undifferentiated stem cells which are able to differentiate into a number of different directions by committing themselves to either the myeloid or lymphoid compartment, and then going down yet other pathways, more detailed pathways to generate a whole variety of cell types.
Secondly, we really understand the differentiation pathways of Matevoisis better than we understand any tissue in the body, in no small part because it's much easier to study the soluble cells in the blood and in the immune system than it is to study how these processes happen in normal tissues. But having said that, I want to emphasize the fact that in each of our tissues there are oligopotential stem cells. When I say oligopotential I mean they can go down several different pathways. Recall up there on that diagram we talked about pluripotential which means multiple, and today we're going to talk a bit about todipotential stem cells, which are able to disperse descendants into all the different differentiation lineages in the body.
At the end of our last lecture, we were focusing on the red blood cells. And this is sometimes called erythropoiesis, which is to say the process by which red blood cells are generated.
We mentioned the concept of homeostasis, and homeostasis just refers to the fact that all of these systems are in very delicate balance so that the body can respond to the physiologic needs of the organism at any one point in time. We talked about the fact that for example when there's a massive infection in the body, then the homeostatic mechanisms allow an increase in these kinds of immune cells in order to encounter the infection.
And at the end of our last lecture, we were talking about this specific branch, and how in fact homeostasis is maintained there.
And what we see here is a series of committed progenitors.
So when I talk about committed progenitors I'm referring to cells that have already made the commitment to go down one or another pathway. They're not yet fully differentiated.
As you can see here, we have first forming cells and colony forming cells. We don't need to remember all the different abbreviations except to say that these cells here are in a relative undifferentiated state. And the only end stage differentiation comes at the very end here when we get to red blood cells. We said in general that it's the case that most highly differentiated cells are post-mitotic, which is to say they're never going to reenter into the growth and division cycle of the cell that we talked about earlier in the semester.
And that's obviously dictated here by the fact that this erythrocyte lacks a nucleus, i.e. during the final stage of differentiation, in addition to accumulating large amounts of hemoglobin in its cytoplasm, this cell actually pops out its nucleus, and that obviously represents an irrevocable change in that cell can never again enter into growth and division cycle. The immediate precursor of an erythrocyte is often called an erythroblast. And the term blast here refers to a cell of embryonic appearance. Blast is used often to indicate, we'll mention that again shortly, a cell which looks very primitive, and embryonic, and undifferentiated. And that ends up going into an erythrocyte, which we said is actually a synonym for a red blood cell, an RBC, a red blood cell.
And we talked about the fact that this progression is actually maintained and furthered by the stimulus of the compound called erythropoietin. So, we're using some of the same words over and over again. And erythropoietin is essentially a growth factor which stimulates the end stage differentiation of the erythroblast into the erythrocyte.
Epo, as erythropoietin's often abbreviated, is actually made in the kidneys. And it's made in the kidneys in response to the physiological stimulus of hypoxia. Hypoxia means inadequate oxygenation of the tissues. You might ask, well, why is red blood cell contractions controlled, as they are, in the kidney?
And the fact is, we don't really know why evolution has chosen the kidney as the site of monitoring the degree of oxygenation of the blood. And in response to hypoxia, it begins to crank out erythropoietin, or Epo. You can think of erythropoietin as an extracellular liggon just like a growth factor.
It has its own cognate receptor on the surface of the erythroblast, and when Epo released by the kidney hits an erythroblast in the context of the bone marrow, it actually has two effects.
It happens to be the case that roughly even 95% of the erythroblast that are made routinely are forced to go into apitosis under routine conditions. So, this is an enormously wasteful system, i.e. as every moment we speak, 90 or 95% of the erythroblast that have come into existence in your bone marrow apitose.
They never go into end stage differentiation.
But when Epo is around, Epo provides a strong anti-apoptotic signal to the red blood saves some and maybe even all of the erythroblasts from their normal fate of undergoing apitosis.
So here, if we imagine there are actually two fates, one is to become an erythrocyte, and the other is to apitose, where the aptisosis is paradoxically enough the dominant fate of the cell, the moment that an Epo comes on the scene, it blocks this alternative fate, allowing these cells to mature. Epo at the same time stimulates the erythroblast to differentiate. Now, you might as yourself the question, why is there this enormously inefficient process?
An enormous effort is made to crank out large, astronomical numbers of erythroblasts, and yet most of them are wasted even before they've had a chance to undergo end stage differentiation.
And the rationale here is as follows. This is a terrific system for rapidly ramping up the level of red blood cells in your circulation because here, within a matter of a day or two, one can crank up, actually in a matter of hours, you can crank up the rate of production of red blood cells by maybe even a factor of ten.
Instead of having 90% of the erythroblast apitose, let's say 0% of them do so, and therefore, instead of having 10% of the erythroblasts becoming red blood cells, 100% of them will do so.
And therefore, you have the virtually miraculous response that if you go from here high up in the rocky mountains at ten or 12,000 feet, within a matter of two or three days, your red blood cell concentration actually has compensated, has risen up to create the oxygen caring capacity that enables you to deal with the thin oxygen, with the low oxygen tension that's present at high altitudes. Now, having said that, the fact is that there is an Epo receptor on the surface of the erythroblast, and what we see there is the following.
Let's talk about the erythroblast and just blow it up a little bit.
So, here's the erythroblast. That's the undifferentiated precursor. And by the way, the erythroblast is actually still a white blood cell. Often we call a white blood cell a leukocyte. You may know that gluco means white. So, a leukocyte, it's still white. And after the erythropotent impinges on it, one of the things it starts doing is to make the hemoglobin, which turns it into a red blood cell.
At this stage, it's still white. On the surface of the erythroblast are these Epo receptors. I'll just abbreviate them like this, Epo receptor, and once it binds the liggon Epo just like the growth factor receptors, we talked early in the receptors signals are sent into the erythroblast to stimulate both differentiation and to prevent the initiation of the cell suicide program that we call apitosis. Interestingly, one of the things that happens normally is the following, that when these signals come in, there is an enzyme called a phosphotase which is attracted to the receptor. The Epo receptor works like a tyrosine kinase growth factor receptor that we talked about earlier in the semester. And here, we have an enzyme, a phosphotase, which actually counteracts the function of the tyrosine kinases. So, after the Epo receptor has bound its liggon, here's the plasma membrane, it has a whole series of I'll draw Y here for tyrosine.
It has a whole series of phosphates attached to it because of the actions of tyrosine kinase enzymes that are associated with its cytoplasmic domain indirect analogy to what we talked about in the case of growth factor receptors. But, one of the things that happens is that this phosphotase, which removes phosphates, then gloms onto the receptor like this. It grabs hold of some of these tyrosine kinases. And what this phosphotase does is reach around. It reaches around and it begins to prune off all of these phosphates because that's what a phosphate does.
It cuts away all the phosphates, thereby directly reversing the previous actions of the tyrosine kinase that led to the formation of these phosphates, and that in turn allows downstream signaling to occur. This is obviously a functional negative feedback loop, i.e. whenever there is an agonist you want an antagonist. Whenever there's a stimulus which is induced in the body, there has to be an inhibitory signal, and this is part of the whole issue of homeostasis, the balance between forward and backward. Interestingly enough, there's a family in Finland, I believe, which has a mutant receptor.
And their mutant receptor lacks this tyrosine.
And what happens as a consequence is that that particular tyrosine doesn't get phosphorolated. Because that tyrosine doesn't get phosphorolated, the phosphotase cannot be attracted to the receptor because there isn't a tyrosine there.
There's some other amino acid residue. I don't know what it is.
It's not important, but it's not a tyrosine. And this cannot happen because they don't have this tyrosine. This phosphotase could not be attracted to the receptor to shut it down as it normally would be. So normally homeostasis is imbalanced, and several members of this family have become Olympic cross-country ski winners. They've become Olympic champions.
Why? Because their Epo receptor's hyperactive. Because the Epo receptor's hyperactive, they have higher than normal levels of red blood cells in the circulation, and this clearly allows them to function better in cross country skiing, which as you know is a really physically demanding task.
Again, I'm not saying this is a good thing for them necessarily.
There are other things in life besides, believe it or not, winning cross country Olympic competitions because as I mentioned last time, having too many red blood cells in your circulation, there's a downside to it which is that you have a much greater tendency to have occlusions, to have blood clots in your circulation which obviously is not a very good thing to have.
Oh, so is there a threshold of Epo receptor activation before phosphotase shuts it down?
These things are not really well understood, are not well studied.
The fact is, you might be able to say we should make a mathematical model of all of these different circuitry. But the fact is if you want to make a mathematical model, you have to know some of the constants. You have to know some of the parameters, the binding constants. And in fact, for most of the signaling interactions, no one's ever really studied them in such great detail. So, one really doesn't know how much phosphate you need here before the phosphotase becomes really active. And so, there's not a really good mathematical model of this feedback loop, even though we know without any doubt that it exists. So, I want to get into other issues that are related to the whole issue of accumulated differentiation traits as one moves down this pathway.
Again, we've used this as a model for how differentiation takes place in the entire body. The faith that's been implicit in this kind of scheme for the last 20 or 30 years is that this acquisition of different kinds of phenotypes is not accompanied by genetic changes, that is, in the genomes of these cells. I.e. one can accomplish these different kinds of differentiation not by rearranging genes but just by rearranging transcriptional programs, and that the DNA sequence of these cells as they proliferate and differentiate is fully unchanged. And that's a matter of faith because you could say to me, how do you know that it's really true. The fact is that people have looked at genes in many kinds of cell types, but it's essentially impossible, or it has been at least until recently, to preclude the possibility that as cells move down these differentiation pathways, they begin to change the nucleotide sequences of different ones of their genes. In fact, I've already told you about one instance where that's clearly the case. And that is in the differentiation of the B cells of the immune system, which happen to be right up here on this chart, because as you recall from our discussion vis-à-vis immunology, the B cells actually do rearrange their genes in order to cobble together DNA sequences that together are able to enable them to make antibodies that are able to react to specific antigens. So there, there's no doubt at all that there's a somatic rearrangement of the genes, somatic meaning it's not a germ line change. It's happening in the soma outside of the germ line. There's a somatic mutation.
It's not a mutation that's deleterious, but rather is directed towards a physiologically normal and desirable end point.
But for example, how do you know that when you remember things in the brain, part of the memory does not derive from changing the DNA sequence and different neurons in the brain?
What's the molecular basis of memory? Could it be that each time we learn some things that there are different nucleotide sequences, critical nucleotide sequences, that are changed in neurons in the brain, and that those nucleotide sequence changes represent an important basis for ensuring that memory is retained over decades of time. Or, rather than having genetic changes in the brain, might it all be epigenetic, i. . all the other changes that happen to the cell besides changing DNA sequences in the chromosomal DNA.
So, here we're dealing with the dialectic between epigenetic and genetic. And, have we talked about DNA methylation here? Yes, so we talked about DNA methylation, and do you recall or having discussed the fact that when DNA gets methylated, that suppresses the transcription of a gene.
But that doesn't change the nucleotide sequence, and that methylation configuration of a gene can be passed to one cell generation to the next. It's heritable, but it's not genetic in the strictest sense of the term, i.e.
it doesn't involve a change in nucleotide sequence, which is what we want to limit this term to referring.
So, epigenic can represent all the changes in the cell including DNA methylation, alterations in transcription, and all other downstream events that result in changes in the cell.
And how can one address this? Well, there are different ways of addressing this question or addressing the possibility that in fact there are changes in the nucleotide sequence of the gene.
One way to do this is the following. And that is to take cells from an early embryo, and here we see an early vertebrate embryo.
This looks really more like a frog embryo or a slightly different shape, and here we see an early embryo. It's after a blastula. It's called a blastocyst. Here again we have the word blast.
How about one question per lecture? We have to have some equity here.
Other people can ask questions. It's good to ask questions, but how about one per lecture that's fair, equitable.
All right, so here's an early vertebrate embryo.
Here we see the blastocyst. This comes after the earlier stages in the embryo, and here we see the inner cell mass.
And as it turns out, the inner cell mass is going to be the precursor of many of the tissues of the ultimately arising embryo.
And here, one can do an interesting experiment. One can take cells out of the inner cell mass. And one can begin to propagate them in culture. And what one ends up with is embryonic stem cells.
And the intrinsic interest of embryonic stem cells is manifold.
For one thing, you can take embryonic stem cells and you can genetically alter them. You can put a new gene in, in the case of a mouse, or you can take another gene out.
And then what you can do is you can inject the genetically altered embryonic stem cell into the blastocyst of another embryo.
So let's say we take the cells out of the inner cell mass.
We develop embryonic stem cells. We can call them ES cells. That's what they're called in the trade, ES cells. We take them out. We can propagate them in culture. And then, what we can find is we'll put a genetic marker in those ES cells. Let's say we put in those embryonic stem cells the marker for the gene beta-galactosidase.
And beta-galactosidase in the presence of a proper indicator, if you put a proper indicator and make a cell turn blue.
So now we have an ES cell line that produces the beta-galactosidase enzyme. The beta-galactosidase enzyme beta-gal itself has no effect on the biology of the cells. It's only a marker. And now, we take those ES cells, and we inject them into another embryo, a wild type embryo that lacks this beta-gal marker.
And what we can see is that we inject the ES cells into this blastocyst. The injected ES cells will now insinuate themselves, will now intrude into the massive cells in this embryo into which we injected the ES cells, and they will become part of the entire embryo genesis that follows. I.e. soon these foreign ES cells will weasel their way into this inner cell mass.
And they will become established and become functionally equivalent to the inner cell mass cells that were resident there prior to this injection. And what you can do then is follow the subsequent fate of, in this case, a mouse. And what will happen often is that you can find blue cells all over the mouse sometimes in the paws, sometimes in the coat. Let's imagine that the hair would turn blue, which in fact is not the case. But let's imagine the hair would turn blue. So here's the mouse, happy because it's part of an important experiment.
And what you'll sometimes see is that, well, remember that art was not my forte. Anyhow, here you might see stripes of blue cells on the skin. The hair won't turn blue actually, but the skin may if you give it the proper indicator.
And what this indicates is that in this case, the cells that were injected into the blastocyst could become part of lineages which committed themselves to becoming skin cells.
Or, the cells in the brain might be blue. Or, the cells in the gut might be blue. Or under certain conditions, the cells in the intestine might be blue. In telling you that, I mean to indicate that the cells that we injected into this blastocyst, which carry beta-gal were totipotent.
They could create all the tissues of the mouse under the proper conditions. The proper conditions are obviously being put into this very special environment in which all kinds of differentiation inducing signals, which we don't really understand, can induce this cell to commit itself to enter into one or another differentiation lineage. And in principal, you can make a whole organism out of an ES cell. ES cell has as much plasticity, as much flexibility, as a fertilized egg.
It has not yet lost the ability to make all the parts of the body.
On some occasions, the ES cell will even get into the gonads of the mouse, which are down here somewhere. And if that's so, if the ES cell which you injected has been able to seed the formation of these cells down here, then what will happen is that either the sperm or the egg coming from this mouse will now transmit the blue gene. And now, in the next generation, all of the mice will inherit the blue beta-galactosidase gene in all of their cells because now this will have entered into the germ line.
If these blue cells happen to colonize the testes, the ovary, or the testes, then these blue cells will become ancestors to the sperm or the egg. And now, in the next generation, mice will inherit a blue gene in all of their cells.
And now this mouse is really happy because it's now part of an extremely important experiment because now all of its cells will become blue, having inherited them as part of the oocyte which led to its formation. In this kind of an animal, we call this animal a kind of a chimera. Chimera is a mythical beast which is, let's say, half human and half horse or something like that. Or a chimera means it has genetically different parts in it. That is not to say that these parts carrying the blue gene are necessarily defective, they're just genetically different, one from the other. But they can participate in embryogenesis in a fashion that's indistinguishable from the non-blue cells. They just do everything they're supposed to do, and they pretend as if they were in this embryo from the get go, from the very beginning, from the moment of fertilization. So they are totipotent.
There's an alternative experiment you can do, and you can take the ES cells, and you can inject them under the skin of a mouse, let's say. So now, you're putting them in a very unfamiliar environment. And what you see then on many occasions is you can actually get a tumor. You can get what's called an embryonal carcinoma.
Now you'll say, well, so what? That's not so interesting. But it's very interesting. Why?
Because if you look at the genome of those embryonal carcinoma cells which we can call EC cells if you want, those cells are genetically full wild type. And yet, we're getting a tumor here.
So, it means that these cells, which have been placed in a fully unfamiliar environment under the skin or in the belly of a mouse will begin to form a tumor. And in fact, they represent the only type of cell that we know about where a cell having a wild type genome can actually give you a tumor.
As you sensed from our previous discussions, all other kinds of human cancer cells we know about have to have mutant genes in order for them to grow as a malignancy. These cells are fully wild type and can grow as an embryonal carcinoma. They are very primitive. These cells have quite a bit of autonomy. They're not so responsive to all the growth factors that normally are required by many cells throughout the soma of an animal throughout the tissues.
So this allows us to begin to move on and ask other kinds of questions.
For example, you can take these embryonal carcinoma cells.
You put them in a Petri dish, and you can actually induce them to differentiate into different cell types in vitro.
How can you do that? Well, we're just beginning to learn how to do that. We don't really know how to do that.
But, if you give them the right cocktail of growth factors, they might begin to form muscle cells. If you give them another cocktail of growth factors, they might begin to give pancreatic eyelid cells that form insulin, or in this case cartilage cells.
And presumably, the cocktail of growth factors you're providing each one of these cells with in vitro, i.e. in the Petri dish, is mimicking the growth factor environment that each of these cell types is experiencing within the embryo. In other words, cells in different parts of the embryo experience different combinations of growth factors that persuade them to commit themselves to becoming these kind of cells, these kind of cells, and these kind of cells. And therefore, one of the promises of embryonic stem cell research is the possibility of being able to regenerate different kinds of tissues in a fashion that I just showed you here. But this whole experiment in the case of human beings is ethically extremely controversial.
Why? Because the experiment starts out making these ES cells here, and if we want to start out with an early embryo like this, start out with a blastocyst, in the case of a human blastocyst, this human blastocyst has the potential under the proper conditions of becoming a newborn human being. And therefore, we have this enormous ethical conflict in this country.
Is this blastocyst already a human being? Can you already afford to truncate the life of this blastocyst at this stage of development, and in so doing, are you actually extinguishing human life, or is this organism, if you want to call it that, already still much too primitive to consider it to be equal to human life?
And here, I would not, unlike my political views, be forward enough to venture an opinion because it's really something that no one really can argue about in any objective way.
It's all a matter of opinion. Is this a human being already, or is it simply an inanimate cluster, a clump of cells?
Now, in principal, how could we do this?
How could we actually create this kind of tissue therapy?
Because the fact is, as you get older, your tissues start falling apart. You haven't experienced that.
But I have. And the fact is that even if you try to stay in shape, things just start falling apart. And the older you get, the more they fall apart. Even people who eat well, which I do, and exercise well, which I don't, even they fall apart.
And so the question is, are there way of replacing and repairing tissue? And this would, in principal, represent one such strategy because it means that you could possibly inject replacement cells into an agent tissue and generate cells which could then restore and regeneration function which has somehow inevitably deteriorated over the decades. Well, that raises the question of how you can actually get a blastocyst, how you can make a blastocyst like this. To state an obvious thing which you might already have intuited, let's say you had such cells differentiated from various cell types that you want to inject into somebody's muscle or into their liver if they had diabetes and had lost their beta cells, or into their cartilage if they banged up their knee during basketball practice or something like that, or jogging, which is allegedly good for you.
Who knows? How could you deal with that? Well, the fact is, let's imagine there were such a blastocyst which we'd produce in this fashion that we differentiated like this.
OK, this is now the sequence of events. There's an important consideration we have to take into account, and that is if this blastocyst came from a different person than you, and we induced these cells to differentiate, and we injected those differentiation cells into your muscle, things wouldn't work. Why? Because these cells, if the blastocyst originated in a different person than yourself would be genetically different from you, and would be recognized as foreign tissue by your immune system. So even though you were getting an injection of cells which could regenerate your muscle perfectly well, those cells would never be given a chance to establish themselves and to thrive, and to reconstruct the tissue simple because the immune system would regard those cells as being foreigners and would go after them hammer and tongs trying to get rid of them in the same way it tries to get rid of all kinds of foreign invaders. I.e. the only way you could avoid it is if this blastocyst was genetically identical to you.
But how can you make a blastocyst which is genetically identical to you? Well, I'm glad I asked that question. That's really the big challenge we have here because we don't want to create a situation where we have to restore somebody's tissues, but the only way we can restore them is to leave them immunosuppressed for the rest of their lives. When I say immunosuppressed I mean we have to prevent their immune system from attacking all of these cells that we've injected in them, these foreign cells, in the same way that we have to suppress the immune system of any person who has received a graft from another individual including often bone marrow transplants. In all cases, we have at least for a while to prevent their immune system from attacking and eliminating these engrafted cells. And this is where the whole strategy comes for the whole process of cloning. You may recall the case of Dolly about five years ago, and let's remember what happened here because this would a momentous experiment in mammalian biology.
It asked the question, really, if you take cells from a somatic tissue, from here, or here, or here, are those cells, in principal, still totipotent, i.e. is the nucleus, is the genome of those cells totipotent, or has the genome, the chromosomal complement of cells in their cells undergone some kind of irrevocable, irreversible change, which precludes those cells from ever becoming totipotent? Well, in fact, if you take mammary epithelial cells from the breast of a human being or from the breast of a ewe and you put them into the blastocyst, nothing's going to happen. Those introduced mammary epithelial cells will not be able to establish themselves in the blastocyst.
And, we will not be able to insinuate themselves amidst the inner cell mass, and they will not be able to participate in embryogenesis. So therefore, the epigenetic program in these somatic cells seems to be irrevocably set to preclude the participation of the already differentiated mammary epithelial cells in subsequent embryogenesis. Therefore, you could not do this experiment all over again of introducing cells into the inner cell mass as I just described over here, injecting them into this.
But still, that doesn't answer the question. The issue is not whether the mammary epithelial cell is irrevocably committed to being a mammary epithelial cell. The issue: is its genome capable under the proper circumstances of becoming an early embryonic cell.
And therefore, what was done is the following. One took mammary epithelial cells, in this case from Dolly's quote unquote "mother, one prepared nuclei from these cells, taking them out of the cytoplasm, and then one got fertilized eggs or eggs that have been induced to become.
So here's an oocyte. An oocyte is an unfertilized egg.
In principle, you can activate an oocyte by putting a sperm in, or in fact it's actually better if you take the oocyte and you fool it into thinking it's become fertilized by treating it with different salts, high potassium concentration, and so forth.
And that will induce the egg to say I've been fertilized.
I better start embryogenesis. But what you do in this case is the following. The egg has its own haploid nucleus here, and you can take a little needle. And, you suck that nucleus right out of the egg. So, you've enucleated it.
That's what you've done, and now the egg is enucleate.
It doesn't have a nucleus in it. But keep in mind, much of what happens during early embryogenesis is programmed not only by the genes but by all array of cytoplasmic proteins that are present throughout the egg, and which play critical roles in determining the subsequent course of embryogenesis.
So now what you can do is you inject into this enucleate oocyte the nucleus of a mammary epithelial cell.
The mammary epithelial cell is obviously highly differentiated.
It's there to make milk. We'll call it an MEC if you want, and you put that in there, and under certain circumstances, and then you can treat this with a little bit of salt to mimic the physiological stimulus that comes after the sperm hits the egg.
And now this egg will think it's been fertilized.
And now it will begin to divide. But keep in mind, the genome of this quote unquote "unfertilized egg" has come not from the sperm and the preexisting nucleus of the egg. It's come because the nucleus has been injected from a mammary epithelial cell.
An experience over the last 30 years had indicated that this will never work. But finally somebody in Scotland, a man named Ian Wilmouth tinkered enough with the conditions of these cells that he could actually get it to work not so often, maybe one, or two, or three times out of 100 tries. But on those conditions, this thing would begin to divide. The nucleus would begin to divide its diploid. Keep in mind that when a sperm comes into an egg, the egg is haploid. The sperm is haploid. Together they make a diploid genome. This introduced genomus diploid, and the question is, the critical question is, can the genes in this introduced nucleus totally rearrange their transcriptional program so that even though these genes might all be intact in terms of nucleotide sequence, can the entire infinitely complex array of DNA associated proteins, I.e. the proteins that constitute the chromatin which is not only the histones but also the transcription factors, the TF's, can they all jump on and jump off as they should to mimic and replicate the spectrum of transcription factors that is normally present shortly after an egg is fertilized?
If they can do that, then this embryo can begin to replicate, and can ultimately develop into a complete embryo.
If they can't, then embryogenesis is going to be truncated shortly thereafter maybe at the two cell stage, at the four cell stage, at the 16 cell stage, but shortly thereafter, not because of the DNA sequences being defective, but because the spectrum of transcription factors is up and down regulates certain genes is in fact not been able to re-assort themselves in response to what?
Initially, in response to the signals coming from the cytoplasm because one might imagine, correctly so, that the nucleus in here is getting signals from the cytoplasm telling it, in effect, telling this nucleus, you should behave functionally as if you were the nucleus of a fertilized egg. In other words, the environment of proteins here is influencing the behavior of this nucleus. That goes backwards to our normal way of thinking because keep in mind our normal vectoral way of thinking is that the nucleus is influencing the cytoplasm.
That's the direction of information flow. But here, we're having a different situation. Here, the cytoplasm is telling this injected nucleus, well, you used to be a mammary epithelial cell nucleus, but now you've got to take on a different job. And we're going to force you to do so. And to the extent that happens, then in principle, one can end up having a normal embryo.
And, it happened actually on rare occasion that this worked.
Here they used actual electrical stimulus rather than salt to get the nucleus to divide. This electrical stimulus, again, was to mimic the stimulus that the sperm entering the egg normally provides, thereby activating the egg and forcing the entire fertilized egg to proliferate.
And so, once this starts developing, let's say, the blastocyst stage, here we have a blastocyst. You can see the inner cell mass once again here. This can be transferred into a pseudo-pregnant ewe. Pseudo-pregnant means you take a female ewe and you inject it with a series of hormones that persuade her reproductive system including prolactin, and progesterone, or estrogen, persuade her reproductive system, her uterus, that she's pregnant. You inject this early embryo into her, and this early embryo will then implant into the wall of her uterus and begin to develop. And if it all works well, you get a Dolly is born. You get a new sheep coming out of this.
It doesn't work so often, one, two, three, four times after out of a hundred, and very often in the great majority of cases, there are mis-births, mis-carriages, which happen in the middle of embryogenesis. So, almost in the great majority of cases, this fails. Somehow, the reprogramming of this nucleus, which is what we're talking about, reprogramming it in terms of its transcriptional program, goes awry. And therefore, bad things happen. The fact that on a rare occasion gets and succeeds here already is extremely interesting because it proves irrevocably that the genome of a mammary epithelial cell is in principle competent to program entire embryonic development.
And that means that during the development of Dolly's mother, we'll put her up here, as she developed from one cell into 1, 00 or 10,000 billion cells, as that development occurred the DNA sequences that went from the fertilized egg to her didn't really change. I.e. the DNA sequences that were in one of her mammary epithelial cells were intact, and as capable in principle of launching the full-fledged development as would be a fertilized egg. And that is one of the proofs, by the way, that in fact differentiation does not involve, with some rare exceptions, alterations in DNA sequence.
This, in turn, ends up being connected with the whole issue of embryonic stem cells. Let's say that I wanted to have my muscles regenerated, although they're still pretty good.
So, I take a skin cell of mine, and I inject the skin cell.
I take the nucleus out, and I inject it into an oocyte.
And then I let the oocyte develop up to this stage.
And I don't put the oocyte back into a sheep or another woman, although I could in principle. I actually take the cells out of the inner cell mass. Those are ES cells, and I begin to use them to regenerate my muscles to do this strategy. So, the cells are, in this case, not used for reproductive cloning, which is what this is here.
They're used for therapeutic cloning, where instead of taking these cells and the ES cells and allowing them to form a whole embryo, they're used to form a cell line of ES cells from the blastocyst from the inner cell mass. What we talked about before, here you see the blastocyst with the inner cell mass here.
You see it again. But now, rather than allowing this blastocyst to continue development, we simply extract cells from it and again create ES cells. I could create therefore in principle, ES cells, which are genetically identical to all the cells in my body, and any one of you could as well.
And here, there's not only one, but there's two ethical complications.
First of all, here we're starting human life with the intent of truncating it very early, and secondly, where are the oocytes going to come from? Well, you could say you can get them from some women, but producing oocytes from a human female isn't so easy. You have to inject her with all kinds of stimulatory hormones, choreogramatatrophic hormones. It's an unpleasant procedure. Usually women are paid $5, 00 or $10,000 to produce some oocytes. Well, you say, that's OK, but is that OK? I don't know.
Is it OK to pay a woman to donate her oocytes to make herself into an oocyte factory? I don't know. You have to judge.
I think there's arguments both for and against it.
Clearly, any one of us would be extraordinarily naïve if we thought that this was a procedure which had no ethical encumbrances in it.
And, you have to think about them for yourself. Still, the potentials are enormous, and therefore the question exists.
Will there be ways in the future of taking differentiated cells from one's tissue, and in fact using them in these ways to make ES cells without having to go through an oocyte, and without having the potential of creating human life. The alternative to this has been to do the following, to go into our normal tissues and pull out adult stem cells. What do I mean by adult stem cells?
These are not stem cells that are totipotent. These are stem cells which are in my muscles and regenerating muscle mass, which happens believe it or not. These are stem cells which might be in my skin and are continually regenerating skin cells.
Keep in mind that in the maintenance of all our normal tissues there are stem cells whose configuration can formally be depicted like this with the transit amplifying cells we talked about before.
And maybe, if one took the stem cells out of an adult tissue right here, if we had a way of extracting them, those could be propagated in vitro, and then injected back in. Those are so-called adult stem cells. And the individuals who are against this kind of manipulation of human embryos and so forth say that adult stem cells are really the solution. You take stem cells out of a person's tissue, you expand them. Ex vivo means out of the body, in vitro, and then you use them. You inject them into somebody's tissue to regenerate their tissue.
There's only one problem with that. It's ethically far less encumbered obviously, but it doesn't work that well. In fact, some people think it hardly works at all, that the exceptions are really rather far and few between. And so, this issue will long be or continue to be debated. But it obviously represents a very new and exciting area of biomedical research. And interestingly enough, it impinges as well in a fully unexpected way on cancer because this whole paradigm of stem cells, it turns out, also applies to cancer cells. If you were to have asked me two or three years ago, what did the cells in the tumor look like? I would draw a picture like this, that these are a series of exponentially growing cells so that all the cancer cells, all the neoplastic cells in the tumor mass are biologically equivalent to one another. They all have the same mutant genome, and they all are capable of multiplying exponentially.
But it turns out that work in the Matavoidic system on Matevoidic tumors like leukemias, and now on breast cancers, yields a very different results, because it turns out that the way that the tumors are organized looks like this. The tumors also are organized in this hierarchical array just like normal tissue.
How do we know that? Again, I'm glad I asked that question.
Because if you take these cells out of the tumor and put them in another mouse, let's say, you get a new tumor.
These cells are tumorogenic, I.e. they concede a new tumor.
If you take these cells out of the tumor, they have the same mutant genome. They constitute the bulk, the vast mass of the cancer cells in a tumor. You put these into a mouse, and they're non-tumorogenic.
And, in some kinds of tumors, the tumorogenic cells can represent only 1 or 2% of the total mass of cancer cells in the tumor.
And from this, we begin to realize that you look inside tumors: the tumors deviate minimally from the organization of normal tissue. They also depend on self-renewing stem cells which can make transit amplifying cells and can give end stage cells, which although they're neoplastic, have many of the differentiated characteristics of the normal tissue from which they arose. And this has enormous implications for, for example, therapies against tumors.
If you ask somebody, how do you develop and how you judge the success of an anticancer treatment? You talk to somebody like from the pharmaceutical industry. And let's say that's easy.
If you have a new drug, and that drug reduces the mass of a tumor by 50%, that means that you've done something really good.
But let's look what's going on here. If these cells are 99% of the tumor in terms of the mass and these cells are 1% of the tumor, let's say you've invented a new drug which wipes out all of these cells but doesn't touch these cells. The bulk of the tumor has shrunk and everybody will say, eureka, we've succeeded in curing cancer. But keep in mind that the self-renewing capacity of the tumor rests in these cells. And if these cells are allowed to survive, then they'll start proliferating again and regenerate the entire tumor mass. And you won't really know that you had any success because these cells look like all the other tumor cells under the microscope. But biologically, they're very different. And therefore, the future of cancer therapy, and it will take five or ten years to do this, has to begin to focus on getting rid of these self-renewing stem cells which create this enormous regenerative capacity on the part of tumors.
Lecture 17: Genomes and DNA Sequencing
Professor Martin talks about DNA sequencing and why it is helpful to know the DNA sequence, followed by linkage mapping and then the different methods of sequencing DNA.
Instructor: Adam Martin
Lecture 1: Welcome Introdu.
Lecture 2: Chemical Bonding.
Lecture 3: Structures of Am.
Lecture 4: Enzymes and Meta.
Lecture 5: Carbohydrates an.
Lecture 9: Chromatin Remode.
Lecture 11:Cells, The Simpl.
Lecture 16: Recombinant DNA.
Lecture 17: Genomes and DNA.
Lecture 18: SNPs and Human .
Lecture 19: Cell Traffickin.
Lecture 20: Cell Signaling .
Lecture 21: Cell Signaling .
Lecture 22: Neurons, Action.
Lecture 23: Cell Cycle and .
Lecture 24: Stem Cells, Apo.
Lecture 27: Visualizing Lif.
Lecture 28: Visualizing Lif.
Lecture 29: Cell Imaging Te.
Lecture 32: Infectious Dise.
Lecture 33: Bacteria and An.
Lecture 34: Viruses and Ant.
Lecture 35: Reproductive Cl.
PROFESSOR: And today I'm going to talk about DNA sequencing. And I want to start by just sort of illustrating an example of how knowing the DNA sequence can be helpful. So you remember in the last lecture, we talked about how one might identify a gene through functional complementation. And this process involved making a DNA library that had different fragments of DNA cloned into different plasmids and then involved finding the needle in the haystack where you find the gene that can rescue a defect in a mutant that you have.
So if this line that I'm drawing here is genomic DNA, and it could be genomic DNA from, let's say, a prototroph for LEU2, the leucine gene. So this is from a prototroph.
Then you could cut up the DNA with EcoRI. And if there is not a restriction site in this LEU2 gene, you get a fragment that contains the LEU2 gene. And then you could clone this into some type of plasmid that replicates in the organism that you're introducing it and propagating it in.
And so that would allow you to then test whether or not this piece of DNA that you have compliments a LEU2 auxotroph, OK?
Now one thing I want to point out is that because these EcoR1 sites, these sticky ends, would recognize this EcoR1 one end or this EcoR1 end, you can imagine that this gene-- if the gene reads this way to this way-- it could insert this way into the plasmid. Or it could insert in the opposite direction. So it could be inverted. So this would have some sort of origin of replication and some type of selectable marker.
But if you have the same restriction site it can insert one way or the opposite way. That's just one thing I wanted to point out.
Now let's say rather than leucine, you're interested in cycling dependent kinase, and you had a mutant end CDK and you had this sequence of your yeast CDK gene. Well, rather than having to dig through a whole library of pieces of DNA for the CDK gene, basically you're sort of fishing for that needle in the haystack. If you knew the sequence of the human genome, you'd be able to identify similar genes by sequence homology.
And you could then take a more direct approach, where you take-- let's say you have a piece of human DNA now, double stranded DNA, and it has the CDK gene. You could take human DNA with this CDK gene. And you have unique sequence around the CDK gene, which would allow you to denature this DNA. And if you denature the DNA, you'd get two single strands of DNA. And you could then design primers that recognize unique sequences flanking the CDK gene.
So you could imagine you'd have a primer here and a primer here. And then you could use PCR to amplify specifically CDK gene from, it could be the genome or from some library. And then you get this fragment here, which includes CDK. So knowing the sequence of the genome would allow you to more rapidly go from maybe a gene that you've identified as being important in one organism, and find the human equivalent that might be doing something similar in humans. So this step here is basically PCR.
And let's say the CDK gene had restriction sites. Let's see, we'll say restriction site K and A here. Then if you have these restriction sites in your fragment of DNA, you can then digest or cut that piece of DNA with these restriction endonucleases. And then you'd get a fragment of CDK that has K and A sticky ends. We'll pretend that both of these have sticky ends.
And now you have unique sticky ends between K and A. And you might have a vector that also has these two sites. And you could digest this vector with these two enzymes. And that would allow you to insert the specific gene in this plasmid.
And if you have two unique sites, because K only recognizes K here and A only recognizes A, then it will ligate in. But you can do it with a specific orientation because you have two different restriction sites. So I hope you all see how it's with one restriction site versus two.
All right. Now let's say you want to do something more complicated than this. Let's say rather than just identifying the gene that's involved in cell division, you want to engineer a new gene, in order to determine where this particular protein, CDK, localizes in the cell. So we have CDK, which could be from yeast or human, it doesn't matter. And you want to engineer a new protein, basically, that you can see.
So remember Professor Imperiali introduced green fluorescent protein earlier in the year. And this green fluorescent protein is from a gene from jellyfish. So now we could, using what I've told you, reconstruct or engineer a gene that has DNA from three different organisms, in order to make a CDK variant that we are able to see in the cell.
So remember, a green fluorescent protein is like a beacon, if it's attached to a protein. If you shine blue light on it, it emits green light. And so you can use a fluorescent microscope in order to see it.
In this case, let's say there's also another restriction site here, R. And let's say you have a fragment of GFP that has two restriction sites, A and R. You could then cut this fragment and this fragment with these restriction enzymes A and R. And you could insert GFP at the C terminus of the CDK gene. So you could go and have a gene that has CDK GFP inserted inside a bacterial vector.
Now which one of these junction sites do you think would be most sensitive in doing this type of experiment? So there are three junction sites. There's this one, this one, and this one. Which is the one you're probably going to put the most thought into when you're doing this experiment?
ADAM MARTIN: The A site. Miles is exactly right. This one is going to be important. And why did you choose that site?
AUDIENCE: Of the three sites, two are half insert, half originals [INAUDIBLE]. But at A, both sides of it are inserts. So [INAUDIBLE] carefully.
ADAM MARTIN: And if you're trying to make a fusion protein, what's going to be an important quality of this? Malik, DID you have a point?
AUDIENCE: Well, they try to [INAUDIBLE] we'd have to make sure that the [INAUDIBLE].
ADAM MARTIN: Excellent job. So Malik just pointed out two really important things. To make this a fusion protein, you have two different open reading frames. These two open reading frames have to be in frame with each other.
So this junction here has to be in frame where GFP is in frame with CDK, meaning that you're reading the same triplet codons for GFP, there in the same frame as CDK. Also, you want to make sure there's no stop codon here. Because if you had a stop codon here, you're just going to make a CDK protein. And then it's going to stop and then you won't have it fused to GFP.
And you guys will work through more of these in the homework. So you'll be able to get a sense of it.
So now for the remainder of this lecture and also for Monday's lecture, I want to go through a problem with you. Basically, if you have a given disease that's heritable, how might you go from knowing that disease is heritable to finding out what gene is responsible for that given disease? And this is going to involve thinking about different levels of resolution, in terms of maps.
So the highest resolution map you can have for a genome is the sequence. You can have the full nucleotide sequence of a genome. And that's the highest possible resolution because you have single nucleotide resolution as to what every single base pair is. But that's like knowing like your apartment number and your street number and basically knowing everything. But starting out, you might want to know what continent it's on, or what country is it in.
And so you first have to narrow down the possible locations for a given disease gene. And that will, at first, involve establishing what chromosome and what region of a chromosome a given disease allele is linked to. And that involves making essentially a linkage map, where you establish where a disease gene is located based on its linkage to known markers that are present in the genome.
Now this is going to require that you remember back two weeks ago, to when we talked about linkage and recombination. And you'll recall that we were looking at the linkage between genes and flies and genes and yeast. One difference between that type of linkage mapping and human linkage mapping is we don't have really clear traits that are defined by single genes. You can't just take hair color and map the hair color gene to link it to a disease gene. Because hair color is determined by many, many different genes.
So in fruit flies, you can take white eyes and see if it's connected with yellow body color because both of those are determined by single genes. So we need something other than just having phenotypic traits that we can track. We need what are known as molecular markers to be able to perform linkage mapping.
And so what we need in these molecular markers-- well, if we just think about if we wanted to determine the linkage between the A and B genes. And if you did this cross, would you be able to determine linkage?
Georgia, you made a motion that was correct. Tell me. Why did you shake your head no?
AUDIENCE: They'd all be heterozygous.
ADAM MARTIN: Yeah they'd all be heterozygous. Because this individual has the same allele on both chromosomes, you're not going to be able to differentiate one chromosome from the other. And so the point I want to make is that in order to see linkage, what you need is variation.
So we need to have variation. And another term for genetic variation is polymorphism. So we need polymorphism, or genetic variation, between these molecular markers.
We also need genetic variation in the disease. But we have that. We have individuals that are affected by a disease and individuals that are not affected by a disease. So we have variation in alleles there. But in order to map it with a molecular marker, to map linkage to a molecular marker, you also need variation here. So the problem with this cross is here you need to have heterozygote. There needs to be variation in this individual, where both of these alleles are heterozygous.
So now I want to talk about some of these molecular markers that we can use, and how they vary between individuals and between chromosomes. Now this is going to be maybe the lowest resolution map. But I'm talking about this linkage map here. And you can see highlighted that the bottom here are various types of polymorphisms that we can use to link a given disease allele to a specific chromosome and a specific place on chromosome.
So I'll start with the first one, which is a simple sequence repeat. It goes by many names. But I will stick with what's on the slide.
So a simple sequence repeat is also known as a microsatellite. So you might see that term floating around, if you're reading about this. And what a simple sequence repeat is, as the name implies, it's a simple sequence. It could be a dinucleotide, like CA. And it's just a dinucleotide that's repeated over and over again.
So on a chromosome, you might have a unique sequence, which I'll just draw as a line. , And then you could have a CA dinucleotide that's repeated some number of times, N. And then that's followed by another unique sequence. And that's what's present in it.
So that would be one strand. And then in the opposite strand, you'd have a unique sequence, the complement of CA, which is GT, and then, again, unique sequence. And so there's variation in the number of repeats of the CA. And so there's polymorphism. So we can use this to establish linkage between this marker and a phenotype, like a disease phenotype.
So how might you detect the number of repeats that are present here? Anyone have an idea of a tool that we've discussed that could be used here? So one hint that I gave you is that the sequence here is unique and the sequence here is unique. So is there a way we can leverage that unique sequence to determine whether there's a difference in the number of repeats?
What's a technique we discussed that involves some component of the technique recognizing a unique sequence? Yeah, Natalie?
ADAM MARTIN: Well, CRISPR Cas9 is a possibility. Jeremy, did you have an idea?
ADAM MARTIN: PCR-- so it's true. You could get it to recognize that. But then you have to detect it, somehow. So what's more commonly used is PCR. Those are both good ideas. But using PCR, you could design a primer here and a primer here. And you could amplify this repeat sequence. And the number of repeats would determine the size of your PCR fragment.
So if you did PCR, then you'd get a PCR fragment that has the primers on each end, but then has this certain size based on the number of repeats. So in that case, we need some sort of tool that enables us to determine the size of a particular DNA fragment. And so I'm going to just introduce to you one such tool, which is gel electrophoresis.
And gel electrophoresis involves taking DNA that you've generated, by either PCR or by cutting up DNA with a restriction enzyme, and loading it in a gel that has agarose. Maybe it's composed of agarose. It could be composed of polyacrylamide. And then because DNA is negatively charged, the backbone, if you run a current through it, such as the positive electrode is at the bottom, then the DNA is going to snake through this gel.
Now we'll do a quick demonstration, if you two could come up. I need one volunteer. Ori, find 10 of your friends and bring them down. All right. That's probably good. Yeah.
All right, Hannah, why don't you-- you guys have to link up, OK? Stay over here. We'll start at this end. This is the negative electrode over here. The positive electrode is going to be down there. And Jackie is going to be our single nucleotide. You guys link like-- yeah. You don't have to do-si-do, or anything like that.
All right. Now what I want you guys to do is I want you to slalom through these cones like it's all agarose gel. So that you're going towards the other side. And I'm going to turn on the current now. So go. All right, stop.
All right. See how the shorter DNA fragment is able to more easily navigate through the cones and get farther. So it was somewhat rigged. I know. But I just needed some way to make sure you always remember that the shorter nucleotide, or the shorter fragment, is going to migrate faster.
You guys can go back up. Thank you for your participation. Let's give them round of applause.
All right. So what you just saw is that the longer DNA fragments, they're going to be more inhibited by moving through the gel. And so they're going to move slower and thus, not move as far in the gel. Whereas, the small fragments are going to move much faster because they're able to maneuver their way through this gel much more quickly. So there's going to be an inverse proportionality between the size of the DNA chain and its rate of movement. You're always going to see the shorter DNA fragment moving faster.
So what one of these gels actually looks like is shown here. So this is a DNA gel that's agarose. And DNA has been run in these different samples. And what you're seeing is this gel is subsequently stained with a dye, like ethidium bromide, which allows you to visualize the individual DNA fragments. And so a band on this gel indicates a whole bunch of DNA fragments that are all roughly the same length. So essentially, you can measure DNA length using this technique.
What's over here at the end of the gel, this is probably some sort of DNA ladder, where you have DNA fragments of known length that you can use to calibrate the length of these bands over here. So this is how you measure DNA length. And we're going to use it over and over again, as we talk about DNA and sequencing.
So now, let's think about how this is going to help us establish linkage between a particular marker in the genome and a genetic disease. So if we think about these microsatellite repeats, I told you they're polymorphic. They exhibit a lot of variation in size. And so here's an example showing you a female who has two intermediate sized microsatellites. And if you look at this-- if you did PCR and measured the size of these, you get two different bands because there are two different alleles of different length here.
So you can see this individual has two intermediate length repeats. And this person has had children with an individual that has a short and a long microsatellite. And you can see that on the gel, here.
Now this female is affected by some disease. And these two individuals have children. And you can see that a number of those children are affected by the disease. So what mode of inheritance does this look like? If you had your choice between autosomal recessive, autosomal dominant, sex linked dominant, and sex linked recessive, what mode of inheritance is this looking like? Oh, Carmen.
AUDIENCE: Autosomal recessive.
ADAM MARTIN: Autosomal recessive? Why do you go with recessive? Yeah, go ahead.
AUDIENCE: Because there is a male that's affected. But not both of the parents are affected. So it seems like the father is heterozygous and the mother is homozygous recessive.
ADAM MARTIN: That's possible. That's exactly the logic I want to see. Is there another possibility? Yeah, Jeremy.
AUDIENCE: Autosomal dominant.
ADAM MARTIN: It could also be autosomal dominant. So you're right. You're right. If this was not a rare disease, then that male could care be a carrier and could be passing it on to half the children. So that's good. You'd essentially need more information to differentiate between autosomal recessive and autosomal dominant.
For the purposes of this, we're going to go with autosomal dominant. And what you see is that you want to look at the affected individuals and see if the disease phenotype is linked, or connected, with one of these microsatellite alleles. So if we look at-- we basically PCR DNA from all these individuals. And if you look at who is affected, each one of the individuals has this M double prime band. And none of the unaffected individuals has it.
So obviously, it would be better to have more pedigrees and more data to really establish significance between this linkage. But this is just a simple example, showing what you could possibly see if you have one of these molecular markers linked to a particular disease allele. So that kind of establishes the principle.
Now let's think about what are some other molecular markers that are possible? So another type of marker, and this is one that's the most common one, if I go here. So here, you see here's is a linkage map, here. And you see most of these bands are green. And the green markers, here, are what are known as Single Nucleotide Polymorphisms, or SNPs.
So single nucleotide polymorphisms-- and this is abbreviated SNP. And what a single nucleotide polymorphism is, is it's a variation of a nucleotide at a single position in the genome. So it's just a one base pair difference at a position. So there's variation of single nucleotide at a given position, at a position in the genome. And because that's a pretty general definition, there are tons of these in the genome.
Now one thing to think about is you could have a mutation in an individual that creates a SNP. So you could have a de novo formation of a SNP. But if you have a SNP and it gets incorporated to the gametes of an individual, then that variant is going to be passed on to the next generation. So this is something that could occur de novo. But it is also heritable. And if it's heritable, then you can follow it and use it to determine if a given variant is linked to a given phenotype, like a disease.
So to identify a single nucleotide polymorphism, it's helpful to be able to sequence the DNA. And I'll talk about how we could do that in just a minute. But before I go on, I just want to point out a subclass of SNPs that can be visualized without sequencing. And these are called restriction fragment length polymorphisms.
So restriction fragment-- so it's going to involve some type of restriction enzyme digest length polymorphism. It's a long word. But it's abbreviated RFLP. And what this is, is it's a variation of a single nucleotide. But this is a subclass of SNP. Because this is when the variation occurs in a restriction site for a restriction enzyme.
So if you remember your good friend EcoR1, EcoR1 recognizes the nucleotide sequence GAATTC. And EcoR1 only cleaves DNA sequence that has GAATTC. So if there was a single nucleotide variation in the sequence, such that it's now GATTTC, or something like that, that destroys the EcoR1 site. And so EcoR1 will no longer be able to recognize this site in the genome and cut it.
So you could imagine that if you had one individual in the genome having three EcoR1 sites, if you digest this region, you'd get two fragments. But if you destroyed the one in the middle, then if you digested this piece of DNA, then you'd only get one fragment. And that's something. Because it results in different sizes of fragments, that's something you can see just by doing DNA electrophoresis. And maybe you would use some method to detect this specific region, so that you're not looking at all the DNA in the genome, but you're establishing linkage to this specific area.
You could use PCR. You can have PCR primers here and here. And you could then cut with EcoR1. In one case, you'd get two fragments. In this case, you'd get two fragments. In this case, if you amplified this region of the genome and cut with EcoR1, you'd only get one fragment. So you'd be able to differentiate between those possibilities.
AUDIENCE: When you use PCR, are there [INAUDIBLE]?
AUDIENCE: Are there [INAUDIBLE]?
ADAM MARTIN: Oh. You're saying what causes it to stop? That's a great question, Malik. Yeah.
So initially, it's not going to stop. That's absolutely right. But because every step, each time you replicate, it's then primed with another primer. So you'd replicate something like this that's too long. But then the reverse primer would replicate like this. And it would stop.
So if you go back to my slide from last lecture, look through that and see if it makes sense how it's ending. Because if you do this 30 times, you really will enrich for a fragment that stops and ends at the two primers, or begins and ends at the two primers, I should say. Good question. Thank you.
All right. Now, let's talk about DNA sequencing. Because as I showed you, obviously, these SNPs, because there are so many of them, are probably the most useful of these markers to narrow in on where your disease gene is. And to detect a SNP, we need to be able to sequence DNA.
So I'm going to start with an older method for DNA sequencing, which conceptually, is very similar to how we do DNA sequencing today. And so it will illustrate my point. And then at the end, I'll talk about more modern techniques to sequencing.
So the technique I'm going to introduce to you is called Sanger sequencing. And that's because it was identified by an individual named Fred Sanger. And I'm going to just take a very simple DNA sequence, in order to illustrate how Sanger sequencing works.
So let's take a sequence that's really simple. This is very, very simple, and then more sequence here. So let's say we want to determine the nucleotide that's at every position of this DNA fragment. So one way we could maybe conceptually think about doing this, is to try to let DNA polymerase tell us where given nucleotides are. And if we're going to use DNA polymerase, what are we going to need, in order to facilitate this process? Yes, Rachel.
ADAM MARTIN: You're going to need nucleotides, definitely. So we're going to need nucleotides. What else? To start, what are you going to need? Miles?
ADAM MARTIN: You're going to need a primer, exactly. Good job. So you need a primer. So here's a primer.
And now, we're going to try to get DNA polymerase to tell us whenever there is a given nucleotide in this DNA sequence. And so think with me. Let's say we were able to get DNA polymerase to stop whenever there was a certain nucleotide.
So if we go through just a couple nucleotides, let's say, at first, we want DNA polymerase to stop whenever there's an A. So let's say there was a possibility it would stop at this A. If it's stopped at this A, you'd generate a fragment of this length. But if it read on through that A, there's another possibility that it would stop at this A.
So we're kind of looking at when these are stopping. And the final possibility is it goes on and stops at this A. So if this DNA polymerase stopped only at As, you'd get fragments that are these three discrete lengths.
Now let's consider another possibility. So pink here is stop at A. And in blue, I'm going to draw what would happen if it stopped at T. So they all start from the same place. If it stopped at T, it would just stop one nucleotide beyond this A in this simple sequence. So in blue here, this is stop at T.
But if it's just a possibility, it stops. And some of the polymerases could go beyond this T and go to the next T and stop here. And again, this would be one nucleotide length longer than this pink one, here. And the final one would-- I'll just draw it down here-- would get out to this last T, here.
So what you see is if we could get DNA polymerase to stop at these discrete positions, we'd get a different sized fragments, whether it was stopping at one nucleotide versus the other nucleotide. You all see how this is resulting in different fragment lengths. Yes, Andrew.
AUDIENCE: How would you create a pattern [INAUDIBLE]?
ADAM MARTIN: There are companies now. You can basically take nucleotides and synthesize these primers chemically, not using DNA polymerase.
AUDIENCE: I'm saying how would you know what primer to use, if you don't know the sequence?
ADAM MARTIN: Oh, in this case, you'd have to start with some sequence that you know. So in most sequencing technologies, you kind of make a DNA library, where you know the sequence of the vector. And then you'd use the vector sequence as a primer to sequence into the unknown sequence. Great question. Good job.
All right. So what we need now then is some sort of tool or ability to stop DNA polymerase when there's a certain nucleotide base. And to do that, we can use this type of molecule, here, which is known as a dideoxynucleotide. Remember, for DNA polymerase to elongate a chain, it requires that the last base have a three prime hydroxyl.
And so what this dideoxynucleoside triphosphate is, is it's a nucleoside triphosphate that lacks a three prime hydroxyl. Here, I'll highlight that.
So you see this guy? You see it bolt the highlight H? There's a hydrogen there on the three prime carbon, rather than the normal hydroxyl group. So if this base gets incorporated into a elongating chain, DNA polymerase is not going to be able to move on.
So this method where you can add a certain dideoxynucleoside triphosphate to stop chain elongation is known as a chain termination method. So you're getting chain termination. And you're getting this chain termination with a specific dideoxynucleoside triphosphate. So these dideoxynucleotide triphosphates, if they get incorporated into the DNA, are going to halt the synthesis of that DNA strand.
So if we take our example, here, this might be a reaction that has dideoxythymidine triphosphate. So if we had dideoxythymidine triphosphate in this sample and it's elongating, then when the polymerase reaches this point, there's a possibility that it will incorporate the dideoxynucleoside triphosphate. And if this is a dideoxynucleoside triphosphate, then there won't be a three prime hydroxyl.
And DNA polymerase will just be like, oh, I can't go on! Because it's not going to have a three prime hydroxyl. So it's not going to be able to continue with the next nucleotide. So this is known as chain termination.
So let me take you through an example, here. All right. So here's an example that you have a slide of. And again, there's a template strand, which is the top strand. And this method requires that you have a primer. And what's often done is you label the primer.
So the first step is you have to denature your DNA. So you have to go from double stranded DNA to a single stranded DNA. And then you mix the double stranded DNA with first, this labeled primer, such that the primer can then yield to the single stranded DNA. You need DNA polymerase, as I've mentioned. And as, I believe, Rachel mentioned before, you need the building blocks of DNA. So you need the four dideoxynucleoside triphosphates.
So you always have the four dideoxynucleotide triphosphates. But what's special here is you're going to spike several reactions with one of the dideoxynucleoside triphosphates. So you spike the reaction with a tiny amount of one of your dideoxynucleoside triphosphates.
So let's say you have a reaction, here. And this this one here has dideoxyadenosine triphosphate. Then polymerase will along get this strand until there's a thymidine on the template. And then there's a possibility that it will incorporate this dideoxy NTP. And if it does, then you get chain termination. And you get a fragment of this length.
But the other possibility, because there is still the deoxy form of the NTP present, it's possible that it incorporates a deoxyadenosine triphosphate there. And keeps going, and then incorporates a dideoxy ATP later on, where you have another T. And so the polymerase will essentially randomly stop at these different thymidine residues, depending on whether or not a dideoxynucleoside triphosphate is incorporated. And that means for a given reaction, one in which you have dideoxy ATP, you get a certain pattern of bands that represent the length of fragments, where you have, in this case, a thymidine base.
And then you do this for all four bases, where you have four reactions, each with a different base that's dideoxy. So when you're adding these, you're going to do four reactions, one with dideoxy ATP spiked in, one with dideoxy TTP, one with dideoxy CTP, and the last with dideoxy GTP. And because these nucleotides are present in different positions along the sequence, you're going to get distinct banding pattern for each of these reactions. But using that banding pattern, you can then read off the sequence of DNA that's present on the template strand.
So this is how sequencing was done for many, many years. These days, it's been made cheaper and faster. And now what's often used is next generation sequencing. And one the pain in the ass about sequencing before is you'd use a lot of radioactivity. Your primer would be radioactive, so that you could detect these bands. Right now, everything's done using fluorescence, which makes it much nicer, I think.
And so in next generation sequencing, your template DNA is attached to a solid substrate, such that it's immobilized on some type of substrate. And then you add each of the four nucleoside triphosphates. In this case, they're labeled with a dye, such that each one is a different color. But the dye also functions to prevent elongation, such that, again, it's this chain termination. When you incorporate one of these, the polymerase just can't run along the DNA. It incorporates one and then stops.
So if you get your first nucleotide incorporated, it will incorporate one of these four. And it will be fluorescent at a certain wavelength, which you can see using a device or microscope. And then what you then do is chemically modify this base, such that you remove the dye and allow it to extend one more base pair. And so you go one nucleotide at a time. And you read out the pattern of fluorescence that appears. And that gives you the sequence of DNA on this molecule that's stuck to your substrate.
And you can do this in parallel. You can have tons, many different strands of DNA. And you can be reading out the sequence of each one of these strands in parallel.
Great. Any questions about DNA sequencing? OK. Very good. I will see you on Monday. Have a great weekend.
Why Do Cells Divide?
Cells divide for many reasons. For example, when you skin your knee, cells divide to replace old, dead, or damaged cells. Cells also divide so living things can grow. When organisms grow, it isn't because cells are getting larger. Organisms grow because cells are dividing to produce more and more cells. In human bodies, nearly two trillion cells divide every day.
Watch cells divide in this time lapse video of an animal cell (top) and an E. coli bacteria cell (bottom). The video compresses 30 hours of mitotic cell division into a few seconds. (Video by the National Institute of Genetics)
Types of Cell Division
Prokaryotic Cell Division
Prokaryotes replicate through a type of cell division known as binary fission. Prokaryotes are simple organism, with only one membrane and no division internally. Thus, when a prokaryote divides, it simply replicates the DNA and splits in half. The process is a little more complicated than this, as DNA must first be unwound by special proteins. Although the DNA in prokaryotes usually exists in a ring, it can get quite tangled when it is being used by the cell. To copy the DNA efficiently, it must be stretched out. This also allows the two new rings of DNA created to be separated after they are produced. The two strands of DNA separate into two different sides of the prokaryote cell. The cell then gets longer, and divides in the middle. The process can be seen in the image below.
The DNA is the tangled line. The other components are labeled. Plasmids are small rings of DNA that also get copied during binary fission and can be picked up in the environment, from dead cells that break apart. These plasmids can then be further replicated. If a plasmid is beneficial, it will increase in a population. This is in part how antibiotic resistance in bacteria happens. The ribosomes are small protein structures that help produce proteins. They are also replicated so each cell can have enough to function.
Eukaryotic Cell Division: Mitosis
Eukaryotic organisms have membrane bound organelles and DNA that exists on chromosomes, which makes cell division harder. Eukaryotes must replicate their DNA, organelles, and cell mechanisms before dividing. Many of the organelles divide using a process that is essentially binary fission, leading scientist to believe that eukaryotes were formed by prokaryotes living inside of other prokaryotes.
After the DNA and organelles are replicated during interphase of the cell cycle, the eukaryote can begin the process of mitosis. The process begins during prophase, when the chromosomes condense. If mitosis proceeded without the chromosomes condensing, the DNA would become tangled and break. Eukaryotic DNA is associated with many proteins which can fold it into complex structures. As mitosis proceeds to metaphase the chromosomes are lined up in the middle of the cell. Each half of a chromosome, known as sister chromatids because they are replicated copies of each other, gets separated into each half of the cell as mitosis proceeds. At the end of mitosis, another process called cytokinesis divides the cell into two new daughter cells.
Eukaryotic Cell Division: Meiosis
In sexually reproducing animals, it is usually necessary to reduce the genetic information before fertilization. Some plants can exist with too many copies of the genetic code, but in most organisms it is highly detrimental to have too many copies. Humans with even one extra copy of one chromosome can experience detrimental changes to their body. To counteract this, sexually reproducing organisms undergo a type of cell division known as meiosis. As before mitosis, the DNA and organelles are replicated. The process of meiosis contains two different cell divisions, which happen back-to-back. The first meiosis, meiosis I, separates homologous chromosomes. The homologous chromosomes present in a cell represent the two alleles of each gene an organism has. These alleles are recombined and separated, so the resulting daughter cells have only one allele for each gene, and no homologous pairs of chromosomes. The second division, meiosis II, separated the two copies of DNA, much like in mitosis. The end result of meiosis in one cell is 4 cells, each with only one copy of the genome, which is half the normal number.
Organisms typically package these cells into gametes, which can travel into the environment to find other gametes. When two gametes of the right type meet, one will fertilize the other and produce a zygote. The zygote is a single cell that will undergo mitosis to produce the millions of cells necessary for a large organism. Thus, most eukaryotes use both mitosis and meiosis, but at different stages of their lifecycle.
CH 16: The Cell Cycle - (Key Terms) Flashcards Preview
Phase of Mitosis during which Sister Chromatids SEPARATE + MOVE to OPPOSITE Poles of the Spindle.
a Ubiquitin-Ligase that Triggers Progression from Metaphase to Anaphase by Signaling the Degradation of Cyclin B + Cohesins.
Protein Kinase that RECOGNIZES Damaged DNA + LEADS TO Cell Cycle ARREST.
Protein Kinase related to ATM that leads to Cell Cycle ARREST in Response to DNA damage.
Family of Protein Kinase involved in Mitotic Spindle formation, Kinetochore Function,+ Cytokinesis.
A member of a family of cyclin-dependent protein kinases that control the cell cycle of eukaryotes.
Member of a family of proteins that bind Cdk’s and inhibit their activity.
A protein-serine/threonine kinase that is a key regulator of mitosis in eukaryotic cells.
cell cycle checkpoint
A regulatory point that prevents entry into the next phase of the cell cycle until the events of the preceding phase have been completed.
A specialized chromosomal region that connects sister chromatids and attaches them to the mitotic spindle.
The microtubule-organizing center in animal cells.
Protein Kinase (Chk1 or Chk2) that BRINGS ABOUT Cell Cycle ARREST in response to Damaged DNA.
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Cleavage and Blastula Stage
The development of multi-cellular organisms begins from a single-celled zygote, which undergoes rapid cell division to form the blastula. The rapid, multiple rounds of cell division are termed cleavage. Cleavage is illustrated in (Figure 24.24 a ). After the cleavage has produced over 100 cells, the embryo is called a blastula. The blastula is usually a spherical layer of cells (the blastoderm) surrounding a fluid-filled or yolk-filled cavity (the blastocoel). Mammals at this stage form a structure called the blastocyst, characterized by an inner cell mass that is distinct from the surrounding blastula, shown in Figure 24.24 b . During cleavage, the cells divide without an increase in mass that is, one large single-celled zygote divides into multiple smaller cells. Each cell within the blastula is called a blastomere.
Figure 24.24. (a) During cleavage, the zygote rapidly divides into multiple cells without increasing in size. (b) The cells rearrange themselves to form a hollow ball with a fluid-filled or yolk-filled cavity called the blastula. (credit a: modification of work by Gray’s Anatomy credit b: modification of work by Pearson Scott Foresman, donated to the Wikimedia Foundation)
Cleavage can take place in two ways: holoblastic (total) cleavage or meroblastic (partial) cleavage. The type of cleavage depends on the amount of yolk in the eggs. In placental mammals (including humans) where nourishment is provided by the mother’s body, the eggs have a very small amount of yolk and undergo holoblastic cleavage. Other species, such as birds, with a lot of yolk in the egg to nourish the embryo during development, undergo meroblastic cleavage.
In mammals, the blastula forms the blastocyst in the next stage of development. Here the cells in the blastula arrange themselves in two layers: the inner cell mass, and an outer layer called the trophoblast. The inner cell mass is also known as the embryoblast and this mass of cells will go on to form the embryo. At this stage of development, illustrated in Figure 24.25 the inner cell mass consists of embryonic stem cells that will differentiate into the different cell types needed by the organism. The trophoblast will contribute to the placenta and nourish the embryo.
Figure 24.25. The rearrangement of the cells in the mammalian blastula to two layers—the inner cell mass and the trophoblast—results in the formation of the blastocyst.
Epidemics in Western Society Since 1600
Chapter 1. Malaria: Relationships between Diseases and Genetics [00:00:00]
Professor Frank Snowden: I’d like to welcome you back, and hope that you all had really good breaks. We’ll be talking this week about malaria, both today and on Wednesday. And I think I may be saying a couple of things that you may find surprising. I suppose — this may be an assumption — that when I mention malaria, that most of you think of it as an exotic tropical disease, and probably think that it’s very distant from us, and has relatively little to do with the modern world, or with modern history, and probably nothing at all to do with the United States and with those of us here. But the point I’m wanting to make, this time and next, is that the reality of malaria is quite different from that that when we’ve come to talk about this disease, I could say that of all human diseases it’s one of the oldest and some question, a question that some of you might ask me, is of all the diseases in our course, which is the one that, in the aggregate, has caused the most human suffering and death? And I think that of all of the diseases in our course, it’s probably malaria which has that primacy.
The relationship of human beings to malaria has been so close, and so extensive, that we could also say that we, as a species, and malaria, as a disease, have evolved together, and that the human genome bears the imprint of our experience with malaria. Good examples of that are a couple of genetic diseases that I’d like to mention, one that may be familiar to you also, one being sickle-cell anemia, a second being thalassemia, and a third being what’s called Duffy negativity. Now, all of these are genetic diseases, and they have in common the feature that there’s a difference between the trait which — it’s a recessive in all three cases, the recessive features.
And, so, a heterozygote — that is, a person who has just one X chromosome, rather than X and Y, expressing the trait for the disease — enjoys a protection from malaria. But if, in fact, you are a homozygote and have it on both X and Y chromosomes, and you develop the syndrome for sickle-cell anemia, instead you have a terrible disease that leads to — it causes sickle cell — it affects the quality of the hemoglobin and the red cells, and therefore it leads to anemia, occlusion of blood vessels, sometimes hypoxia, and serious respiratory diseases. So, it’s a fatal illness in many cases. But if you have the trait, then in a highly malarial area you have a great selective advantage in enjoying a great deal of resistance or immunity.
In terms of human evolution, in intensely malarial areas there was a strong Darwinian pressure for the retention of the sickle cell trait. The same would be true of thalassemia, which affects not the quality of the red cells but rather their quantity, and leads to again terrible complications in homozygotes, and with asthma and terrible anemia, or Duffy negativity, which is another genetic disease that leads again to terrible diseases.
Chapter 2. Scope [00:04:45]
Another feature of malaria, apart from the fact that our genome has actually been strongly influenced by our history with malaria, is that until very recently it extended more or less across the globe, affecting not only the tropics in Africa, Asia and South America, but also the north, including most of Europe and North America. So, I thought I’d show you a map of malaria in the United States in the nineteenth century, just to give you an idea — this is 1882 — to show how extensively prevalent it was in this country, as recently as the late nineteenth century. And indeed it comes up — Oliver Wendell Holmes wrote a book on malaria in New England.
This is a map of 1912, when it’s receded a great deal and is primarily a disease of the South. But remember that it had a major impact on public health in this country, until the end of the Second World War, which is when it was eradicated in the United States. It’s sobering to remember, for example, that the Centers for Disease Control, the CDC, in Atlanta, was originally founded as an anti-malarial agency, and that the disease played a major role in the settlement of the West, and in the economic and social development of the South. And if you read Mark Twain, for example, you can read a great deal about shivering along the banks of the Mississippi River. In terms of our discussion of relationship of diseases to the big picture of history, malaria is also one of the diseases that I think helps us to make the case most effectively.
Malaria, it’s now believed, played a major part in the fall, for example, of the Roman Empire, when an epidemic offalciparum malaria led to the disruption of agriculture and the Roman Legions. And I’m not trying to say that malaria caused the downfall of the Roman Empire. I’m saying that it was a major factor, leading to the kinds of military problems and social dislocation that we can’t ignore in discussing that major series of events. It’s led to major impacts on the outcome and on the conduct of warfare. One need go back only to the Second World War, when malaria was a major preoccupation of the armies on both sides of the conflict. It had an enormous role and impact on European expansion and colonization, and one of the great challenges to colonial expansion, in the Indian subcontinent, and in Africa, was what to do about the problem of malaria.
It’s affected the pattern of human habitation and settlement, the way that cities and towns are built across the landscape, where human habitation often was a form of prophylaxis for malaria, and people lived far from swampy areas, and often on high ground, above dangerous wetlands low down. Malaria is also a major factor in economic development and under-development today, in ways that we’ll be discussing. A decisive factor in its prevalence, and in its history, has always been the relationship of human beings to agriculture and the environment that is to say that intensive forms of agriculture, with modern livestock raising practices, modern crop rotations, water management, ecological sanitation, with improved housing and conditions of diet, wages and clothing, have led, wherever they’ve been introduced, to a spontaneous recession of malaria and much better health outcomes.
And this has been a major factor in the divergence between the global North and the global South the North with modernized intensive systems of agriculture, and in the South instead the persistence of extensive forms of practice that are conducive, for reasons we’ll be examining, to the transmission of malaria. So, malaria both reflects and reinforces developmental differences and disparities. Now, let’s remember that malaria is vitally important in our world today. Let me give you an example of some of the pictures that represent the global burden of malaria, and let me show you a map of where malaria is prevalent today that is, in the twenty-first century. Indeed, at the moment, one can say the statistics are extraordinary.
About 500,000 people become seriously ill of malaria every year. Approximately a million people die of it, the majority of them being children under five, and pregnant women, concentrated particularly in Sub-Saharan Africa. In fact, one can say that a child dies of malaria, in Africa today, every thirty seconds, making this disease a global public health emergency, along with HIV/AIDS and tuberculosis. Let’s look at more particularly the areas. This is the epicenter, if you like, of the present day resurgence of malaria as a major, major public health problem. And there are — malaria kills 3,000 children every day of the year. The burden of malaria though is greater than statistics for mortality and morbidity suggest. It is, for example, one of the worst possible complications of pregnancy. It leads to high rates of miscarriage to maternal death through hemorrhaging and severe anemia, and all of the sequelae that follow from severe low birth rate.
Malaria also can be transmitted vertically that is, trans-plancentally, from mother to fetus, and can lead to the birth of infants who are congenitally infected. We also need to remember, as we’ll say in a moment, that malaria is a major immunosuppressive disease, and its victims therefore are highly susceptible to other opportunistic infections especially respiratory infections, tuberculosis, influenza, pneumonia. In those areas of the tropical world where malaria is hyper-endemic, and transmission continues throughout the year, the population at risk can be infected, re-infected and super-infected every single year. If the victims of malaria survive, they possess a painfully acquired immunity. But it comes at a terrible price, because repeated bouts of malaria lead to severe neurological deficit and cognitive impairment. The results are ineradicable poverty, illiteracy and compromised economic growth, a stunted development of civil society and political instability.
We’ll be talking in a moment about Ronald Ross, who was the Nobel Laureate, who was one of two people who discovered the mosquito theory of transmission for disease. And he wrote, quite movingly, that those malaria doesn’t — in areas where malaria is prevalent — “those it doesn’t kill it enslaves.” Malaria, in other words, in our present world, is a major contributor to inequalities between North and South, and to the economic and political international dependency of third-world countries in the tropical world. Well, let’s talk about the — malaria also will be of interest to us because it’s an extremely complex disease, and so we need to spend a little bit of time talking about how it’s transmitted, and about its effects on the human body, in order, after that, to talk about how the discoveries that led to our understanding of it, and to the impact on society and on history.
Chapter 3. Etiology [00:15:03]
Until the end of the nineteenth century, malaria was thought to be explained by a theory that you already know, by miasmatism. In other words, malaria was a form of bad air. In fact, that’s what the word means, from the Italian mala, bad, and aria, air. So, malaria was bad air that somehow a susceptible person inhaled, and it got in his or her body, or was absorbed, perhaps, through the pores of the skin, and led, in susceptible people, to this terrible fever. It was also sometimes called paludisme, from the word for swamp so it was swamp fever. So, the disease then was absorbed or breathed in, in some way.
In fact, malaria isn’t one disease. It’s a family of four different diseases, caused by a parasite, with an extremely complex lifecycle. The parasite is known as a plasmodium, and there are four species of plasmodium that cause human malaria. And I have them on your handout. They’re Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, andPlasmodium ovale. For the purposes here, the first two, Plasmodium falciparum and Plasmodium vivax, are the most important, medically and historically, and the ones we’ll be talking about mostly. Now, the plasmodia differ fundamentally from the other microbial pathogens we’ve examined so far in the course — bacteria, for example, and viruses — in that they’re much more complex life forms, with complex lifecycles that we need to unravel.
Plasmodia were discovered in 1884 by Alphonse Laveran — there he is — a French Army doctor working in Algeria. It turns out that the plasmodia don’t exist free in the environment at any stage of their lives. Instead, they’re adapted to live either in the body of human beings, or in the gut of certain species of mosquitoes. And — there we are — this is an anopheles mosquito doing its thing that is to say, having a blood meal, which is the way that malaria is transmitted from person to person. The plasmodia — this is again, in a more schematic way, it gets the point across about the relationship of human beings and the mosquito.
Plasmodia migrate in the body of the insect to the salivary glands in the biting apparatus. So, a biting mosquito, like this one, is in effect an extremely efficient vector. It’s sometimes referred to as a flying syringe, because what it does, the mosquito does, is to inoculate the plasmodia directly into the bloodstream of the host. At this stage the parasite — we’ll move to look at what happens next. The first thing we have is the mosquito taking a blood meal and inoculating the plasmodia directly into the bloodstream of the unfortunate victim.
At that stage — and here we’ll see one of the points about the plasmodia, is that it undergoes a series of morphological changes in becoming distinct stages in its life, both in the human body and in the body of the insect. Initially when it’s injected, it’s known as a sporazoite, and what it does next is it migrates, after just a few hours after inoculation — say you were bitten right now, within a few hours the plasmodia in your bloodstream would have migrated to your liver. And this begins the incubation period in which the plasmodium reproduces — that is, asexually — in the liver. And you see its reproduction. And then after just a number of days or weeks, it’s released again now in a new phase, this time known as a merozoite, into the bloodstream. So, it returns at that point to the bloodstream.
One of the points of the migration to the liver is that when it’s in the liver, it’s safely beyond the detection of the human immune system, and so it reproduces safely in the liver and then emerges, much more numerous, into the bloodstream in a new phase in its lifecycle. At this point in the bloodstream what happens is that the merozoites — that’s what they’re now called, the new name of the parasite for that phase — it enters into — it attaches itself to and enters into red blood cells or erythrocytes. Once safely inside the red blood cell — again it’s not detected by the immune system — it reproduces asexually — and you can see it doing so — until at a certain point it has destroyed the red blood cell and ruptures the red blood cell, and the parasites return once again to the bloodstream. I have — this is a picture.
These are of the — by electron microscope — of the actual rupturing of red blood cells, and the emergence, once gain, of the parasites that have just reproduced, returning to the open bloodstream. At that point, the much more numerous merozoites keep repeating this process of invading red cells, reproducing, destroying the red blood cells, and then bursting them at periodic intervals. The interval of time that it takes is determined by the species of plasmodium. ForPlasmodium falciparum and vivax, it’s every forty-eight hours. And so for Plasmodium malariae, it’s every seventy-two.
Now, eventually, among the brood of merozoites — that is, let’s see, again — after it’s gone through a number of cycles, they produce among their offspring what are called — it’s a new morphologically different stage in the lifecycle, and that is gametocytes, that are male and female and these are in the open bloodstream. And then the next anopheline mosquito that takes a blood meal, as it does so it sucks up the male and female gametocytes that reproduce sexually this time — we see them here — in the gut, in the body of the female mosquito, and then that begins the phase of life in the body of the mosquito where once again we find it reproduces and eventually it leads to the production of sporozoites that migrate, once again, to the biting apparatus, the salivary glands of the mosquito.
The mosquito again takes an infective bite and then — in its next blood meal — and the whole cycle, this complex cycle of both asexual reproduction in the body of man, and sexual reproduction in the body of the mosquito, the cycle is then complete. Now, let’s return for a second to the plasmodia and to the insect. And let me just deal with the fact that in order — the reason that the female — and it’s only female anophelines that take blood meals on human beings — and the reason that the female anopheline does that is that it needs blood in order to mature its eggs and to lay them. Having taken a blood meal, she’s able to mature her eggs, and at that point lays them in water, and they pass through the cycle of larvae, pupae, and then the adult mosquito known as an imago.
Now, what happens at that point? It takes about a week for the eggs to develop as larvae, pupae and then adult mosquitoes, and then the mosquito is ready to take flight and to visit you and me. From the breeding site to the blood meal, the anopheles mosquitoes have delicate wings and are normally weak flyers. So, normally she flies about no more than three kilometers or so, from her birthplace. Most species of anopheles avoid sunlight, that dries up their wings, and they avoid strong winds. But as they take flight, they’re able to orient themselves to places of human settlement. Why? Because on their antennae there are sensors that are highly stimulated by carbon dioxide in the air. And, so, this — the carbon dioxide plume arising from human settlements and human bodies, enables the mosquitoes to be attracted to them.
Having arrived at closer range, the mosquitoes then detect, with other sensors, odors emanating from sweat. They’re also attracted by light. And then at close range they finally use their vision to settle on the site of the body most suitable for their feast. And human beings cooperate in this enterprise in that anopheline mosquitoes feast between dusk and dawn, and they thereby attack sleeping bodies, lying mostly motionless. Now, when you hear the buzz of the harmless culex mosquito that buzzes noisily around you and attracts your attention, you can be happy, because most anophelines that transmit malaria are silent and therefore don’t disturb their hosts.
You’re probably wondering, how is it that transmission is maintained if the vast majority of mosquitoes don’t transmit malaria? Only the females of certain species of anophelines — and I’ve included two on your handout as being most important to us: Anopheles gambiae, which would be my candidate for the most deadly insect for human beings on our planet, and Anopheles labranchiae, which was one of the most important vectors of malaria in Europe and in parts of Africa. You’re probably wondering if a female anopheline mosquito lives on average just a few weeks, and needs to be infected herself before transmitting the disease, how can transmission be maintained?
Well, there’s a couple of facts that we need to remember. First, is the vast numbers of mosquitoes involved in areas where malaria is endemic. In most areas of high endemicity, no more than two percent or so of female mosquitoes are infected at any given time. But on average, a human being can be bitten thousands of time in a year. And it’s also true that an insect like Anopheles gambiae is famished and doesn’t feed a single time, but having entered a place of human settlement feasts repeatedly, moving from one body to another, thereby ensuring that in crowded conditions one malarial patient is a major source of danger to all of those around him or her.
Chapter 4. Symptomatology and Relationship to Poverty [00:30:17]
Well, that’s the story from the standpoint of the mosquito. What happens to the human victim? What are the symptoms of malaria? How does the disease have its impact on the human body? After the incubation period, symptoms begin when the plasmodia have achieved a critical threshold number in the bloodstream. It’s then that the classical symptoms of malaria begin, with their onset. Now, let’s remember — return to our diagram. This process of reproduction, of entering — that is to say the parasite enters the blood cell, reproduces, bursts the blood cell and returns to the bloodstream — occurs simultaneously for an entire brood throughout the bloodstream. In other words, this is happening at the same time throughout the body. And it’s when there are sufficient numbers of the parasite in the open bloodstream that the immune system of the body can detect the parasite, and it’s then that symptoms begin.
The term for malaria also — it has many names, this disease. It was often called intermittent fever. This process of synchronicity was known as Golgi’s law, after the malariologist Camillo Golgi, who discovered it and he talked about the different timings of fever. Tertian fever, that is to say, every forty-eight hours or Quartan fever, every seventy-two hours or Quotidian fever. You can also have a bout of intense fever every twenty-four hours, and that means that you have more than one species of plasmodium in your bloodstream. You don’t have to choose just one, you can have several species and several types in your bloodstream at once. If that occurs, you can have fever, intermittent fever, every twenty-four hours.
The recurring classic symptoms then are this intermittent fever, recurring at regular intervals, like a train schedule. You have recurring paroxysms of high temperature, plus chills, profuse sweating, headache, general malaise, exhaustion, and with it often nausea, vomiting and diarrhea. The precise symptoms depend on the species of plasmodium, and the most virulent is Plasmodium falciparum, which causes the most frequent life-threatening complications, and Plasmodium malariae and ovale are the most mild. You see that by entering the red and attacking red blood cells, the parasite initiates a cascade of consequences. The red cells can become misshapen, and they adhere to one another in clumps, thereby causing blockages in blood cells- blood vessels, that can be rapidly fatal, depending on the organ that’s affected.
A frequent cause of mortality is cerebral malaria, in which there are blockages in the brain. But the heart can also be affected, or the gastrointestinal system and if malaria attacks the gastrointestinal in particular, it mimics the symptoms of Asiatic cholera. Destruction of the red blood cells also is a cause of profound anemia. Another important symptom of the disease is — and this is a child who’s a malaria patient, and what you see is a painful and pronounced swelling of the spleen. This is one of the classic signs of malarial infection. As I’ve said, also malaria is terrible in its effects on pregnant women, leading to hemorrhaging and miscarriage, and also to congenital malaria with infants born with the disease.
Malaria also is a disease that’s a major immunosuppressive disease. It suppresses the immune system of the body, and therefore gives rise to complications, especially respiratory diseases as I mentioned earlier, pneumonia, influenza and tuberculosis. So, we should say that the tuberculosis emergency in the present day, and the malaria emergency, are inter-locking and inter-related malaria provides the substratum for rampaging re-emerging tuberculosis. I also said that recurring bouts of malaria lead to neurological damage, and in the worst cases to a state known as cachexia, in which a person is indifferent to his or her surroundings is unable to learn to be productive, to take part in civil society.
Another feature of malaria is that it can lead to relapses that is, with Plasmodium vivax. You remember that after the plasmodium is injected into the bloodstream, it migrates to the liver. Well in Plasmodium vivax, the parasite does emerge, but not all of the parasites. They continue to nestle in the liver, and they’re beyond the detection of the immune system. And they can then — even after the patient thinks that he or she has recovered, there can be a relapse when the plasmodia, the parasites, re-emerge from the liver into the bloodstream. This can be months later, or even years later, after the initial infection. Immunity — that is, once you’ve had lots of bouts of malaria, and you survive, you develop a partial immunity, an acquired immunity. But it is short-term, and it’s also at considerable cost in terms of neurological damage to the body.
Well, the impact on society, as you can imagine, is severe and we’ll be talking about that next time. But it leads, this disease — the symptoms, listing the symptoms, helps us to understand that someone like this as an adult, who’s anemic, who has perhaps respiratory diseases, moves painfully and slowly, and is therefore not a productive worker in agriculture or in industry. So, malaria leads to backward systems of cultivation, low productivity, and lack of investment in agriculture. It leads to the desertion of whole- of some of the most fertile areas, land, because it’s particularly dangerous, and known to be so. It leads then to poverty, to illiteracy.
Indeed, malaria and poverty are mutually reinforcing in a kind of vicious downward spiral. Poverty makes people vulnerable to the disease. Poverty, that is, makes people vulnerable because it causes them to live in poor housing, overcrowded housing housing that’s porous and vulnerable to flying insects. It also leads them to occupational hazards in having to work in areas where the disease is prevalent. It leads to poor diet, which makes people more vulnerable to inadequate clothing, which makes them more vulnerable to biting insects. But malaria, in turn, then leads to further poverty. The burden of looking after the ill, that falls on families and communities low productivity low wages limited education. This was what Ross meant when he says, “Those malaria doesn’t kill, it enslaves.”
Chapter 5. Mosquito Theory of Transmission [00:40:14]
Well, when was the mosquito theory of transmission unraveled, and how so? The idea that mosquitoes were involved in this disease wasn’t at all obvious. It wasn’t obvious because, well, first of all scientists and physicians knew that there are lots of places where there are gazillions of mosquitoes and no malaria. It was also clear that mosquitoes — there was no clear correlation between being bitten by mosquitoes and developing the disease. And so the dominant theory in the nineteenth century was of miasmatism as the cause of the disease. The unraveling of the disease — you’ll remember this man, Patrick Manson, the father of tropical medicine. He was also one of the figures who was most closely associated in the development of tropical medicine, and of the mosquito theory of transmission, which he discovered for a different disease called filaria. And then he had the idea that perhaps if filaria could be transmitted by mosquitoes, possibly malaria could as well. And so he joined forces — he worked, Manson, in London.
This is Ronald Ross, who was a British military physician working in India. Now, India was a tremendously important place in terms of malaria. Let me just — there was a book that you might be interested in, and that I would recommend to those of you who are, which is the correspondence between Ronald Ross and Patrick Manson, that led them to the discovery of the mosquito theory of transmission. Now Ross worked in India, and he noted that malaria, amongst the general population of India in the 1890s, led to 5,000,000 deaths. And he said that it was the greatest problem of public health in India. “I think on the whole,” he wrote, “that the Indian population of 400,000,000, it causes directly or indirectly 10,000 deaths a day. And apart from this amount of sickness, malaria is, of all diseases, the most important in political, agricultural and military affairs,” he wrote, “since it renders large tracts of fertile land uninhabitable, impedes cultivation, planting and public works, and is the most fierce, vicious enemy that armies in the field have to contend against. On the whole I think we’re justified in claiming that the malaria question is as important as famine or bubonic plague.”
In India, what Ross and Manson did was to discover — let’s go back to our picture of the lifecycle of malaria. They traced — through microscopy, were able to detect in the body of the mosquito after it had bitten, and they did experiments in which they — among not human beings but birds — and they discovered that it was possible to detect the plasmodia responsible for avian malaria, under the microscope, in the body of the mosquito. That was a first major insight, that the mosquito was in some way implicated. But then they went further and they followed the process by which it changed various phases in the body of the insect and reproduced, and they traced the migration of the parasite to the biting apparatus of the mosquito. And then they were able to take healthy birds and have mosquitoes, who were known to be infected, feast on them, and to produce malaria experimentally on birds.
And, so, in 1898, if you were reading this correspondence, there’s a eureka moment in which Ross announces that he’s discovered the mosquito theory of transmission, and proved it. And he claims that he feels like Captain Cook, the explorer, or possibly like Napoleon. And he was not, however, a naturalist, and he didn’t know about the speciation of mosquitoes, and the mosquitoes he described, he described as dappled, brindled or light brown. He didn’t know about the species of Anopheles mosquitoes.
It’s at this point that we should mention then a second major figure — oops, anyway we’ll see — it doesn’t matter —Giovanni Battista Grassi, who was the next figure in the development of the mosquito theory of transmission, and he does so for human beings. And what he does is he discovers that it’s possible — in a place called Capaccio he takes railroad workers, in the midst of a major malaria outbreak in the summer, and he introduces one variable in their lives, from a control group of railroad workers and the surrounding peasantry and that is that one group he has living from dusk until dawn in well-screened houses and this difference protecting them from the one factor, which is from the bites of mosquitoes, prevents their being contracting malaria. This was one place in which he did that.
And then he also did a different experiment, which was to use quinine, which kills malaria parasites in the open bloodstream. He gave it prophylactically to a series of workers, as a control group, in a place like Ostia, during the summer malaria season, and found that he could protect them as well from malaria by establishing a chemical barrier between the mosquitoes and human beings. And then he took the further step of actually taking mosquitoes who were known to have feasted on people ill of malaria, and took them to a hospital in Rome known as the Santo Spirito Hospital, where he had a volunteer on the second floor who was in a room where at night they released hundreds of intentionally infected mosquitoes, and a couple of weeks later they had their eureka moment when he had a spike and a temperature of 104, and they knew that they had successfully transmitted malaria by human experimentation to someone who had been healthy until mosquitoes infected with the disease had been allowed to feast upon him.
So, this then happens between 1898 and 1901. And this then is a powerful factor in the development of tropical medicine. But it also leads to programs to combat malaria. Having discovered the pathogen responsible to it, the plasmodia, and the vector, female anopheles mosquitoes, we see the development of public health programs to destroy the disease — either by attacking the plasmodia with chemical therapy, that is, through quinine or by killing mosquitoes, that is, vector control — the idea of possibly being able to eradicate malaria. And next time what I’d like to do is to follow the practical application then of the discoveries we’ve talked about today, about the life cycle of plasmodia and of anopheline mosquitoes, and see how that leads to the development of public health strategies. And I’d like to talk about how those strategies are being used in the real world today to combat this crisis of this dreadful vector-borne disease.