Archive for the ‘Biology’ Category

Lecture 13

January 18, 2009

1) Variations in bacteria, viruses and eukaryotes – bacteria have circular chromosomes, eukaryotes have linear chromosomes. Today we’ll talk about variation but not between organisms, but within organism from time to time and place to place. How some gene activities are turned on on some occasions and turned off on other occasions. This is very important for a multicellular organism like you, since it makes sure the same basic code is doing different things in different cells. Its also important for a bacteria to make sure its doing different things at different times depending upon its environment.

2) Which are the steps in the central dogma where you would like to regulate the activity of a gene. Could you regulate the activity of a gene by changing the DNA encoded in the genome? It does happen, not very common though.

3) Levels of regulation – DNA rearrangement. Your immune system creates new genes by rearranging locally some pieces of DNA. Some bacteria decide whether a gene should be on or off, by actually flipping around a segmenet of DNA in their chromosome.

4) The most common form is at the level of transcriptional regulation, whether or not a transcript gets made or how its processed is different. a) Initiation of mRNA – RNA polymerase should sit down at this gene, on this occasion, is a regulatable step. You only turn on alpha globin and beta globin used to make haemoglobin in red blood cells. b) Splicing choices that you make wrt your message – skip an exon say. c) regulate at the level of mRNA stability, persistence of the message, degradation of the message. In some cells, the message is protected so that it hangs around longer.

5) Regulation at the level of translation – If I give you an mRNA, is it automatically going to be translated? May be the cell has a way to sequester (wrap it up) the mRNA in some way so that it doesn’t get to the ribosome in some conditions. There are an exciting new set of genes called microRNAs, that encode 21-22 base pair segments, that are able to pair with a mRNA and interfere in some ways partially with its translatability. By the no of microRNAs, an organism can control how actively a mRNA is translated.

6) Post translational control – once a protein is made, the protein is modified in some way – the protein is inactive unless you put a phosphate group on it, and some enzyme comes along and puts a phosphate group on it. Or its inactive until you take off the phosphate group.

7) Every step can regulate whether you have a biochemical activity of a certain amount at a certain time, and every one of them is used. What we think is the most important, is initiation of mRNA. The fundamental place where you are going to regulate whether you are going to have the product of a gene, is whether or not you bother to transcribe its RNA.

8′) The amount of protein that a cell might make varies wildly. Your red blood cells, 80% of the protein they make is alpha or beta globin. That’s not true about any other cell.

9) Simplest case – gene regulation in bacteria – Lac operon in EColi. EColi likes carbon, it likes sugar, and its favorite sugar is glucose – for glycolysis. If there is no glucose, it can work on other sugars. It has a variety of pathways, that will shunt other sugars to glucose, which will allow you to go through glycolysis. Suppose you give it lactose, its a disaccharide, where you have got a glucose and a galactose. EColi is able to break lactose into glucose and galactose. It does that by a particular enzyme called beta-galactocytase, which breaks down galactocides, and it will give you glucose + galactose. How much beta-galactocytase does a Ecoli cell have around? A lot, when there’s only lactose around. 10% of protein can be beta-gal,when you have lactose but no glucose. When you have glucose around, you have almost none.

10) Why have this complex mechanism? Why not always have 1%, like a insurance policy, so what if your digestion is slow when there’s lactose around, life’s long. Competition. If the other bacteria has a mutation that allows it to make 4 times beta-gal, it will suck all the lactose. So if Ecoli has 0% when glucose, and 10% when none, you can conclude that it has worked that out through pretty rigorous competition. It doesn’t waste energy when you don’t need it, and when you do need it, it can compete hard.

11) How does it get the lactose in the cell? It has another protein which is a lactose permease, that makes the cell permeable to lactose, then the beta-gal can break the lactose down. Both these proteins, beta-gal and lactose permease get regulated.

12) How does it work? Structure of Lac Operon: What’s an operon? In bacteria you often made a transcript, that had multiple proteins encoded on it. A single mRNA could be made, and multiple starts for translation could be present, and this would be a good thing, if you wanted to make multiple proteins that were part of the same biochemical pathway. Such an object, a regulated piece of DNA, that makes a transcript encoding multiple polypeptides, is called an operon, because they are operated together.

13) The lac operon has a promoter, and we’ll call it p-lac. The first gene is given the name lacZ, it happens to encode beta-galactocytase enzyme. Remember, they did mutant hunt, and they didn’t know what each gene was for, so they gave them names as letters. Next is lacY, that encodes the permease. There is also a lacA, and that encodes transacetylase. There’s another gene, before the promoter that’s called lacI, and it too has a promoter, which we can call pI, and this lacI encodes a very interesting protein. So this is monocistronic, the p-lac region is polycistronic, cistrons is the name for these regions that were translated into distinct proteins.

14) lacI encodes a very interesting protein which is called the lac repressor. The lac repressor is not an enzyme like beta-galactase, its not a self-service channel for putting in lactose, it is a DNA binding protein. It binds to DNA. It doesn’t bind to any DNA, it has a sequence preference. It particularly likes to recognize a specific sequence of nucleotides, and binds there. What is that sequence where this likes to bind? It binds after the p-lac promoter, and this is called the operator sequence, or the operator site. It overlaps the p-lac promoter site. Who likes to bind at the promoter site? RNA polymerase. What’s going to happen if the lac repressor protein is sitting there? RNA polymerase cannot bind.

15) What will happen in terms of the transcription of the lac operon? No mRNA. Are we done? No, sometimes we want to make beta-galactocytase, so we want to get the repressor off from there. When do we want it to come out? When there’s lactose present. So somehow, we need to build a sensory mechanism, that is able to tell if lactose is present, and send a signal to the repressor protein, and it comes off. What signal?

16) How about using lactose itself? If lactose binds to the repressor, it will fall off, because its more interested in the lactose, than in the DNA. How does this interest come about? The liking for lactose, may change some energy, some shape, so that it bends around in ways so that it can no longer bind to the DNA. What happens is allo steric change, which means other shape, so it just changes its shape on binding of lactose, and falls off. So in the presence of lactose, lacI does not bind, and the lac operon is transcribed.

17) But how is lactose going to get in, because the lactose permease is made by the same operon? Instead of having this strong repressor, what if build some sloppy repressor that occasionally falls off, and occasionally allowes transcription of the lac operon, then we’ll have some trace quantities of permease around. And even if we have a little lactose around, it will shift the equilibrium so that the repressor is off more, that will make more permease and so on. So instead of having no mRNA when repressor binds, have very little mRNA. So we have lacON and lacMostlyOff.

18′) How do we know this? Mutants at lac operon: Remember this is before the times of DNA sequencing, they wanted to collect mutants, that affected this process. In order to collect mutants that screwed up the regulation, they knew that beta-galactocytase was produced in much higher quantity if lactose was around.

19) Wild type Ecoli, when you had no lactose would produce very little beta-galactocytase, say 1 unit, and in the presence of lactose, would produce a lot, say a 1000 units of ß-galactocytase. The problem with playing around with this is, lactose is playing two different roles. Lactose is both the inducer of the expression of the gene, by virtue of binding to the repressor, but it is also the substrate of the enzyme, as beta-galactocytase gets made, it breaks down the lactose, so there is less lactose. If you wanted to really study the regulatory controls, you have the problem that the thing that is inducing the gene by binding to the repressor, is the thing that’s getting destroyed, by the product of the gene. So its going to make the kinetics of studying such a process really messy. It would be very nice if you could make a form of lactose, that could induce ß-galactocytase by inducing to the repressor, but wasn’t itself digested. Chemically, you can do that. You can make a molecule called IPTG. This can induce ßgal, but its not a substrate, it won’t get digested. It is also convenient to use a molecule called XGAL, this is not an inducer, but it is a substrate, it will be broken down by the enzyme, and when its broken down it turns blue. These two chemicals turned out to be very handy in studying the expression of the lac operon.

20) Instead of adding lactose, if I add IPTG, I’m going to get ßgal, when I don’t have IPTG, I won’t get ßgal, but then I don’t have the problem of this getting used up. What kind of a mutant might I look for? I’d look for a mutant, that in the absence of the inducer, IPTG, still produces a lot of ßgal. I can also look for mutants that no matter what do not produce ßgal – what would they likely be? They would be structural mutations affecting the coding sequence  of ßgal. But that’s not as interesting as mutations that block repression – that cause ßgal to be produced all of the time. How would I find such a mutant? I want to find a mutant that makes a lot of ßgal, even when there’s no IPTG.

21) Put Ecoli on a plate, and don’t put IPTG. How do I tell if anybody is producing lot of ßgal? Put XGAL, and if anybody is producing a lot of ßgal, they turn blue. So mutants were found that were constitutive mutants – means they were expressing all the time, no longer regulated.

22) Characterising these constitutive mutants: They fell into two different classes. Mutant no 1 are operator constitutive – they have defective operator sequence. Mutations have occurred at the operator site. Mutant no. 2 have a defective repressor protein, the gene for the repressor protein.

23) How could I tell the difference? – some mutation in the operator site causes the repressor protein not to bind there anymore, defective repressor also doesn’t bind even if the operator site is good. One way to tell the difference is by crossing them to wild type, and asking whether they are dominant or recessive and things like that. The problem is EColi is not a diploid. So you cannot cross two EColi and make a diploid EColi, its a prokaryote, it has only one genome. But turns out you can make temporary diploids, you can make temporary diploids, partial diploids out of EColi because turns out you can make bacteria, which have a bacterilal chromosome, also engage in sex, and in the course of bacterial sex, plasmids can be transferred, for eg – an F-factor is able to be transferred from another bacteria, and through the wonders of partial mero diploidy, you can temporarily get Ecolis that are partially diploid.

24) Suppose the repressor is wild type, the operator is wild type, and the lacZ gene is wild type, and suppose I have no IPTG, then I’ll have 1 unit of ßgal. When I add inducer, I get 1000 units of ßgal. Suppose I had a operator constitutive mutation, then the operator site is defective, ßgal is going to be expressed all the time, even in the absence. Suppose I made the following diploid – I+O+Z+ over I+OconstitutiveZ+. What would be the phenotype? In the absence of IPTG, 1001 ßgal, and in the presence of IPTG, it would be 2000 ßgal. So the operator constitutive site looked like it was dominant.

25) Lets try I+O+Z+ over I+OconstitutiveZ-.  In the absence of IPTG, 1 unit, but in the presence, 1000 units. What if I reverse, I+O+Z- over I+O-Z+, then in the absence, 1000 units, in the presence 1000 units. From this experiment, you can tell, that the operator site is only operating the chromosome it is physically on, that it doesn’t make a protein that floats around, it works in cis (cis means on the same chromosome).

26) Lets look at the lack of the repressor mutants – I+O+Z+ over IconstitutiveO+Z+, in the absence, 2 units, in the presence, 2000 units. note this. If I give you I+O+Z- over IconstitutiveO+Z+, then in the absence, 1 unit, in the presence 1000 units. As long as I have a working copy of repressor, it will work on both chromosomes. It makes a product that diffuses around, and it is said to work in trans i.e. across.

27) Operator works in cis, mutation on the operator only affects the chromosome it lives on, whereas a functional copy of the lac repressor will float around because its a protein, that’s how they knew the difference. They proved their model by proving that these 2 kinds of mutations had very different properties. Operator chromosome only affected the physical chromosome on which they occurred, whereas a functional copy of the repressor could act on any chromosome.

28′) What about glucose? Glucose Control: When lactose its present, operator comes off, and RNAP sits, but wait! RNAP is not supposed to sit down if glucose is present – we need another sensor to tell if glucose is present or not, or if there is low glucose. There is another site before the promoter, on which a completely different protein binds, and this protein is the cyclic AMP regulatory protein. In the cell, when there is low amounts of glucose, we have high amounts of cAMP. Whereas lactose is used directly as the signal, cAMP is used as the signal here. cAMP helps RNAP, instead of being a repressor, its an activator, it makes it more attractive for RNAP to bind. Just like the repressor wasn’t perfect, the promoter isn’t perfect, and unless RNAP gets help from the cAMP, it doesn’t work. wow

29) We have 2 controls – a negative regulator responding to an environmental cue, a positive activator responding to an environmental cue, helping polymerase decide whether to transcribe or not, basically that’s how a human egg goes to an adult and lives its entire life, minus a few other details. That’s a sketch of how you turn genes on and off.

Lecture 4

January 16, 2009

1) Proteins can penetrate and arrange themselves to lie in and out of phospholipid bilayers by having hydrophobic molecules in the region between the membrane and hydrophilic ones on either side. You could also have a positive charged and a negatively charged amino acid in between the membrane, although these are hydrophilic, they satisfy each other’s requirements being oppositely charged.

2) Once a protein has been polymerized, it is not the last thing. Proteins undergo posttranslational modifications. Process of synthesizing a protein is called translation. Further chemical modifications can be imposed on the sidechain to further modify the protein. eg- proteolytic degradation, proteolysis is the degradation of a protein. Or, one part of the protein may simply be clipped off. In many proteins which protrude into the extracellular space, there is another kind of covalent modification, the process of glycosylation, in which a no of sugars is covalently attached to the polypeptide chain, using the hydroxyl of the sidechain of serines for eg. These modify the extracellular domain of the proteins.

3) The hydroxyl chains of carbohydrates offer numerous opportunities for using dehydration reactions or condensation reactions, you remove a water, to attach different things. There are 4 different hydroxyls in ribose that you can use to do that – 1′, 2′, 3′, and 5′. In truth, the 2′ hydroxyl is rarely used. What we have is a glycosytic bond, that is a bond between a sugar and a non-sugar entity. Here a bond is formed between a base and a 1′ hydroxyl of the ribose. At the 5′ hydroxyl, another condensation reaction, called esterification reaction (where acid and base react together, and through a condensation reaction, cause the removal of water.) There are 3 phosphate groups, nearest called alpha, farthest called gamma. This chain of phosphates has very important consequences for energy metabolism and biosynthesis. Because all 3 have negative charge, and hence it takes potential energy to keep them together. And they cannot break apart as long as they are in this triphosphate configuration. But once they are broken apart, the energy released can be used for various purposes.

4) Two basic kind of bases – nitrogenous bases. They aren’t just aromatic rings, infact all of them have nitrogen in the rings. Pyrimidines have 1 six-membered ring, purines have 2 rings, a 5 and a 6 membered ring. What distinguishes one from another is these sidechains.

5) The fact that T has a methyl group doesn’t matter as far as the coding is concerned.

6) Once bases are attached to the sugars, they change their names slightly. The lowest nitrogen participates in the formation of a glycocytic bond. The base + the sugar is called the nucleoside. If on top of that, we add one or more phosphates, it is called nucleotide.

7) Uracil changes its name to uradine when it attaches to the sugar, cytosine changes to cytodine, thymidine, adenicine.

8) Nucleic acid syntesis always occurs in a certain polarity, it goes in a certain direction. You can’t add nucleotides on either end, you can only add them to the 3′ end. The energy of the triphosphate is used to form this bond. The resulting linkage is called phosphodiester linkage. (because there are 2 esterifications) Therefore, polymerization doesn’t take place instantaneously, it requires energy investment of a high energy molecule. This can be repeated. A human chromosome contains 10 mega bases of DNA, million nucleotides.

9) These different bases have complementarity to each other i.e. like to be together with one another. One purine opposite one pyrimidine. If we have two pyrimidines, they are not enough close to each other, whereas if we have two purines they are too close to each other. Polarity of the two chains the constitute the double helix. One runs in 3′ – 5′ direction, other in the opposite direction. We speak of the double helix as being antiparallel.

10) There is specificity here, any purine doesn’t pair up with any pyrimidine. The way they associate with one another is via hydrogen bonds. Therefore, by boiling, we break those hydrogen bonds, remember they have only 8kcal of energy per mole. DNA strands come apart, and the DNA ends up being denatured. If there were a covalent link (instead of a hydrogen bond) between the DNA strands, that’s a bad news for a cell carrying such a DNA molecule, it often means the cell should go off and die. Because the cell has to some day pull apart these strands, and it will have a hard time doing that. So, this association should be tight enough so that its stable at body temperature, but not so tight that it can’t be pulled apart, when certain biological conditions call for it.

11) In C-G, there are 3 hydrogen bonds, in A-T there are 2. The third bond in A-T between H and O cannot be formed because they are very far apart. Each of these base pairs is 3.4Angstroms apart. (distance between two bases on the same strand, so 10 of them make 34 Angstroms) and 10 base pairs is roughly 1 turn of the alpha helix.

12) Information is encoded in two strands, information is redundant. One thing we appreciate is that the phosphates are on the outside, and the bases are on the inside. The bases are protected from the outside world, because the information content in DNA must be held very stable and constant, else we have things like cancer. That’s the reason why the DNA remains stable for 30 thousand years.

13) The structure of the DNA allows it to be copied. Replication. Remember we start out as a fertilized cell with one genome, and throughout our lifestyle we produce, 10^16 cells. We pull apart the two strands, not by putting them in boiling water, but by enzymes whose dedicated function is to put separate those two strands,

14) T and U are from an information point of view, equivalent. We could make a DNA-RNA hybrid helix. The RNA molecule could extract information out, and then leave elsewhere. So extracting information doesn’t mean to destroy it.

15) Often RNA molecules can form intra-molecular double helices. i.e.RNA double strand. This is called a hair pin. They will hydrogen bond to themselves using the complementary sequences. This confers on them very specific structure.

16) Another aspect of the 2 vs 3 hydrogen bonds is the following – if the double helix has many G’s and Cs, then it has more hydrogen bonds holding it together, and you need to put in more energy to denature the double helix.

17) The presence of hydroxyl in RNA has important consequences on the stability of the RNA and the DNA. When hydroxyl ion attacks a phosphodiester bond in an RNA, the phosphodiester bond will try to cyclise producing a 5 membered ring, and ultimately that will resolve and break, causing the cleavage of the RNA chain. That means that if you put RNA molecules in alkali, they will fall apart quickly. What happens to DNA molecules? Nothing, they are alkali resistant, because there isn’t a hydroxyl there to form this 5 membered ring, and alkali cannot cleave apart the DNA. This is another reason why DNA is more stable and lasts for years.

Lecture 12

January 16, 2009

1) We have a messenger RNA that has been transcribed from a specific region of the chromosome starting from a promoter and going to a stop signal and that mRNA will include some particular sequence. Ribosome comes and starts scanning this message for the place to start. It finds the first AUG. There are messages that don’t start at AUG, and there are also messages that don’t start at the first AUG, because the ribosome is looking for something very special.

2) How does it accomplish this matching b/w codons and amino acids? When it reaches UAG, its a signal for stop, don’t put anymore amino acids. There are 3 stop signals. How does the right amino acid get put on the right tRNA? There’s a dedicated enzyme for that. Its aminoacetyl synthetase.

3) How does this happen physically? It happens in this vast machine called the ribosome. In the ribosome, there’s a cavity for each tRNA-codon-amino acid. When the right tRNA fits in the cavity, the ribosome catalyzes a peptide bond between the amino acids. Then it moves the sequence by one step, until stop. Is there a tRNA for stop? No, there’s some protein factor. This is the smallest two tape turing machine.

4) Variations on the theme: How does this central dogma vary among different kind of organisms? Eukaryotes, prokaryotes, viruses. Prokaryote – E.Coli. Prokaryote has no distinct nucleus. Do they all do the same?

5) DNA Replication: a) Eukaryotes – What’s the structure of our chromosomes? Its a long linear chromosome. We have 23 chromosomes and together they make up 3e9 nucleotides. A typical chromosome is 150e6 bases long, its a single connected molecule. One tricky bit of replicating DNA. In replicating the lagging DNA, since we go reverse, the chance that we will get a primer at the end is pretty low, and if we don’t get it right, it’ll be short a little. That’s nothing here, but it’ll again be short in the next cell division and so on. Its tricky to replicate linear chromosome on a lagging strand. A special little solution is used. The ends of the chromosomes are called telomeres. These have very specific structures. In the humans they repeat- TTAGGG again and again and again. There’s a special enzyme that’ll come along and add some extra telomere to the chromosome. What’s the enzyme that adds telomere, telomerase. What cells need to have telomerase – rapidly dividing cells. Cells that have stop dividing can shut off their telomerase. Cells that need to divide lots and lots of times, need to tidy up its telomerase. What cells care about having telomerase on? Cancer cells.

6) Prokaryotes differ because their genomes are not linear chromosomes. The typical prokaryote chromosome is a double stranded circular DNA. It doesn’t have this problem of telomeres, you just keep replicating around and you get to the end. You have much smaller genomes too – few million bases. Humans also have 1 circular chromosome, the mitochondria have their own chromosome, its a circle. The mitochondria arose as a symbiolic bacterium, that became a symbiot of eukaryotic cell. Because it lives in your cell, it has thrown out all its genes that it doesn’t need anymore, so the mitochondria genome is 16000 bases long.

7) What do viruses have? Some viruses have double stranded linear, some have double stranded circular. Some have single stranded circular DNA. They inject this into the cell. As soon as it gets into the cell however, its replicated to make a double stranded DNA which can be transcribed and so on. But it travels around as a single stranded DNA.

‘8) Some viruses actually consist not of DNA at all but of RNA, single stranded RNA. When it gets into the cell, what does it do? How does it convert to DNA. Reverse transcriptase. Where does it get this reverse transcriptase from? You yourself encode it on your RNA. You might decide to put in the genetic code for reverse transcriptase, and when that message gets into the cell, it will first act as an mRNA translate the reverse transcriptase enzyme, which will then reverse transcribe the the RNA into DNA. This is a +-strand virus, it encodes its own reverse transcriptase instructions on its RNA. There are minus strand viruses, which don’t have this, what they do is, in their own package bring in your own reverse transcriptase. RNA thus converted to DNA, then to double stranded DNA, which can then be slammed into and inserted into your own chromosomes. What virus does this? HIV. More generally, retro viruses are the viruses that can run this process of converting from RNA to DNA, and install DNA copies into your genome. How do you then get the DNA copy out of your genome? You don’t, it doesn’t come out. We have to make sure that the virus is shut down by other ways by inhibiting its products, etc. but you can’t get its DNA copy out of your genome.

9) So you want to inhibit AIDS virus, you need to make inhibitors of this aspect of replication. Inhibitors of reverse transcriptase.

10) Transcription Prokaryote genome has some kind of a promoter that tells RNA polymerase to come sit down here. RNA polymerase starts copying, eventually it hits a signal that says stop transcribing. Note this is not a stop codon, that is for translation. This mRNA then goes off. Nothing special about proks.

11) Eukaryotes are different. It starts the same, there’s a promoter. RNA polymerase sits there, it starts transcribing. It stops. Then this RNA gets processed in interesting ways. At the 5′ end a funny modification is put on. A methyl G triphosphate is put on backwards. Gppp is put on at the 5′ end. This is called a cap. This is a signal for the cell saying this is a messenger RNA, get the ribosome on it, and so on. At the other end of the message, a long string of A’s is added. Its called a poly(A) tail. If you want to purify mRNA’s you can do so by using poly(T), because the poly(A) binds to it. The function of this A’s is to regulate the stability of the messages. If you don’t have the poly A tail, the message will be degraded rather rapidly. This poly A tail also acts like a clock that tells how long that mRNA sticks along.

12) But these are small modifications – The major difference is – only a small portion of the gene matters in order to make the protein. The mRNA gets made, it includes the whole sequence, and then the cell comes along and splices this message together. It is processed by clipping the unnecessary portions. That’s a mature RNA. This splicing is a remarkable phenomenon. Its a complex phenomenon. There’s a big body of enzymes that help accomplish that. Hence it’s not splicase, its splicosome. How does the splicosome know where to do this? There is information encoded in these messages. The sequence just at the end is a GU, and the sequence at each start is AG, but that’s obviously not enough information. We don’t have the complete information.

13) These bits that stay in are called exons, the bits that go out are called introns. This is confusing terminology. The introns are so named because they are intervening sequences. For a typical human gene, the length of the gene itself might be 30 thousand bases, but the mature mRNA might be 15 hundred bases. The clotting factor VIII gene, the gene that is mutated in individuals with haemophilia, that gene is 200 thousand bases long, and it gets spliced down to a mere 10 thousand bases. That’s nothing compared to Duschan muscular distrophy, that gene makes an immature mRNA of 2 million bases, its at it for hours. This gene is spliced down to 16 thousand messages.

14) Splicing mutations could be a problem, some errors could occur from errors in splicing. That does happen. There could be mutations that change a splicing, or create a new splicing, and all of that could screw up the gene.

15) Why do this? Why waste this energy? I might be able to encode multiple proteins with the same gene. One type of cell might splice up the gene one way, a different one might splice it another way, to produce a different protein. Alternative splicing could create multiple proteins. A typical gene in the human, has atleast two different splicings on average. How does the cell know to splice it one way or the other? We don’t fully know.

16) This is a huge overhead to do that, is it justified. There are other reasons- an evolutionary reason. Suppose a random event happens and a chromosome breaks. And suppose it sticks one part of the chromosome, to some other random part. If it lies in the code, that’s trouble, but if it lies in the intron, it created a new gene, that could still work. By having a random break b/w two genes in their introns, and slamming them up, you could make a gene that had a bunch of exons from one gene, and a bunch of exons from another gene, and it would get spliced up. Evolution might like that. Because it would be a very easy way to build new genes that would have a portion of one protein, and a portion of another protein. This kind of mix and match domain could be very useful. And when we look at a lots and lots of genes, we see genes that have similar first half but a different second half.

17) Why don’t bacteria have this? The metabolic cost is too high, it has to replicate every 20 mins, and all these extra bases would be bad news. Bacteria are more sophisticated than us because they are under rigorous evolutionary selection than we are. You might imagine that early life all had introns, and bacteria, competing to be more efficient got rid of their introns.

18) Small eukaryotes like yeast have introns, but very small in number. Only 5% of the genes have an intron. The bigger the genome size, the more the introns. The more pressure you are under to replicate rapidly, the less you can tolerate this interesting and complicated innovation.

19) Viruses depending on whether they are eukaryotic of prokaryotic do or do not have splicing.

20) Translation: Eukaryotes are simple. You get a gene, you get an mRNA, the ribosome goes to the mRNA, and it starts turning out one protein as it chugs along.

21) Prokaryotes: differ in an interesting way. I get a promoter that is transcribed into my mRNA. The mRNA can encode multiple independent proteins. Protein 1, 2, 3 simultaneously, and you have a poly cistronic message. Why have a single mRNA that encodes multiple distinct proteins each starting with its own ribosome start site? Efficiency. How about, make them multiple steps in a biochemical pathway? Have them encoded on a single messenger, so you’d have to only worry about regulating that once, you will make all the enzymes for the pathway. That’s exactly what bacteria do, they put all enzymes for a single pathway on a single message, so when they say, lets digest hexose this morning, they have all the ingredients on a single RNA. That’s because they are small genomes, they are pressed for space. This single unit, that has multiple genes encoded in a single message, is called an operon.

22) Viruses: Viruses have very little room, their genomes are tiny. A typical virus might have a genome of 5000 bases to 10000 bases, to 200 thousand bases. It wants to pack a lot of protein coding information in, and so viruses have come up with the most extraordinary way of doing that. Some viruses have gone to the extreme of having RNA’s that get made from them, and they can use the same sequence in 3 different reading frames, and get different proteins. In a prokaryotic or eukaryotic, only one of the reading frames is used. This is an extraordinary packing of information density.

Lecture 11

January 14, 2009

1) Central Dogma: DNA is replicated(replication) to make copies of DNA. It is read out(transcription) into the intermediate RNA, and then it is translated into protein.

2) DNA Replication: How do you demonstrate DNA replication? Take a DNA w/o bacteria, and show that you can copy it. Crack open the cell and purify an enzyme that can copy the DNA. Which cell? E Coli, its a bacteria, therefore simple.

3) What do we do? Add materials to testtube containing DNA? What else should we add? Nucleotides, since we know it is made of nucleotides. We’ll add some dATP, we’ll add some dGTP, dCTP, dTTP, all together known as dNTPs.

4) What else? We want to copy DNA, so put in a DNA template strand. We add enzymes and we hope that that is going to copy the DNA. But that’s too optimistic. In order to copy the DNA, its helpful to give it a start. So we also add a short complementary primer strand, with the hope that he would be able to purify an enzyme, which may not be able to start the synthesis of DNA, but would be able to extend the synthesis of DNA.

5) The primer strand is 5′-TpApCpGpTpA. The template strand is 3′-ApTpGpCpApTpTpApGpGpC – 5′

6) What is the enzyme expected to do – catalyze the addition of a triphosphate to the growing end of the DNA chain, the 3′ end. Where is it going to get the energy from? From the dehydration synthesis and breaking of the triphosphate bond. This hypothetical enzyme that can polymerize DNA like that is called polymerase. Note that the replication always goes 5′ – 3′. No one has ever found a DNA polymerization system where it goes the other way.

7) In the other case, the triphosphate would have to be on the long growing chain. Why do you care if the triphosphate is on the long grown chain or the monomer? The high energy triphosphate bonds are unstable. What if they should just spontaneously hydrolize? Its no big deal if one of the monomers hydrolizes from a triphosphate to a monophosphate, you can always find another.

8) What happens in an organism? It has a long chromosome and DNA replication is going along on this chromosome. Btw, where does the primer come from? What enzyme makes it? Primase.

9) For one strand its fine. But for the other strand, since replication can occur only one way, how does it replicate? Since the DNA is opening in the wrong direction for it. What it does is it creates new primers as the DNA opens, and polymerizes them little till it reaches the next primase. But how does it covalently ligate (join) them together. Turns out this is done by an enzyme called Ligase. This model has been experimentally proven. The slower strand is called the lagging strand, and the other is called the leading strand. The lagging strand tries to catch up with the leading strand. The fragments are called Okuzaki fragments.

10) Take a long chromosome. Even assume its circular, like bacteria have, imagine trying to replicate this. We are going to have interlaced double helices. There’s no way w/o cutting to separate them. It cuts it. You’ll need to cut the DNA, pass it through the other side. They are topoisomers. What enzyme does that? Topoisomerase. Drugs that inhibit topoisomerase are therefore good anti-cancer drugs.

11) Fidelity of DNA replication: Why don’t we put any other base, why just the right one? Because there is an energetic difference, between the right base and any base. If I know delta G, I know equilibrium constant, so I know how often DNA polymerase makes a mistake. Keq = 1000. So DNA polymerase gets it right 99.9% of the time. This is terrible. A typical gene is more than a 1000 letters. So we are going to make a mistake on an average on every gene.

12) So there’s proof reading. Two kinds of DNA proof reading that go on. DNA polymerase itself has a proof reading activity. Whenever DNA polymerase adds a base, it also has an activity that’ll remove a base. It doesn’t just add bases forward, it has exonuclease activity that removes bases backward. It adds more than it subtracts, and if there is a mismatched base, its much more likely to be subtract then add. Presence of a mismatch induces the enzyme to do its removal more than if it was a match. Therefore one error in 10^6.

13) Then there are DNA mismatch and repair enzymes that come after polymerase has done its job. They feel the DNA, any mismatch creates funny structures, they chop out some sequence, and that is then replaced. Now you can get down to one error in 10^8 bases.

14) How does it know which strand is wrong and which is correct? You leave some mark on the old strand. Bacteria do that. Methylation enzymes mark the old strands, and it takes time for the methylation enzymes to mark the new strands, and that leaves some time between.

15) About 1 person in 400, is heterozygous for the mismatch repair enzyme genes i.e. genes that encode the mismatch repair enzymes. What if you lost one of your copy of the repair enzyme gene? No problem, other will do the work. What if you lost both? High mutation rate, and cancer. A colon cancer is caused by mutations in the gene encoding the mismatch repair enzyme. Speed of a DNA polymerase is 2000 nucleotides/sec.

16) Cornberg’s enzyme, is it actually the right enzyme? Is it the enzyme the cells use to copy the DNA? A biochemist would say yes. A genetist would say, take out the component, and demonstrate the cell can’t replicate. 

17) They took many mutant bacteria, one at a time, they grew them up, and they did Cornberg’s purification to purify DNA polymerase. This is unbelievably tedious. Take each one, purify it, get DNA polymerase, ok its there, next one, and so on. Suppose you found a mutant that couldn’t make Cornberg’s DNA polymerase, but still grew? That would prove Cornberg’s enzyme is not necessary.

18) Cornberg’s enzyme although it can replicate DNA is not the enzyme that cells use to replicate their DNA. It turns out to be a relatively minor enzyme used to fill in gaps. The actual enzyme is DNA polymerase III. So this duality b/w biochemistry and genetics is very important.

19) Transcription: Where do we start on the DNA? Somewhere on the DNA there is some information, we want to make a copy of that information. Where to start? There’s a sign that says start. Such a thing is called a promoter. The promoter says here is the place to start copying the DNA into RNA. It gets an enzyme that starts at the promoter. What’s the difference b/w DNA and RNA? deoxy. That deoxy is important, otherwise the hydroxyl would interfere with the making of the double helix in the DNA. RNA doesn’t make the double helix.

20) What’s the difference b/w T and U? T has a  methyl group.

21) DNA is used as a template to copy a strand of RNA. Some important names – the strand that is being transcribed is called the transcribed strand. The other is called the non-transcribed strand. The transcribed strand is also called the non-coding strand, because the RNA contains the code of the non-transcribed strand.

22) How does it know where to stop? Stop signal, that says stop of transcription.

23) Orientation of genes along the chromosome, which way you read, is not a fixed thing across the entire length of the chromosome. So when I say transcribed strand, that’s just a local definition, that says wrt that gene, this strand is coding and this strand is non-coding. How does RNA polymerase know when to turn on a gene? How does it turn on the right genes on the right tissues? That’s gene regulation.

24) Translation: We take our RNA, what’s the direction its been copied? 5′-3′. Single stranded molecule. How is this RNA interpreted? On an abstract sense, it is interpreted by a triplet code – 3 letter codons. Does it start anywhere? It always starts on the same codon. It always starts at AUG, this is the initiator codon, and it encodes a methionine. The interesting challenge in the world is how do you get from a sequence of nucleotides to a sequence of amino acids?

25) Transcription is easy due to matching. How are we going to get amino acids to match specific RNA sequences? Initial ideas were physical. The RNA message would fold into some kind of a funny shape, that would just happen to match a lysine – Amino acids would be directly read off the RNA message. But amino acids have so many different properties, it just didn’t make sense.

26) Francis Crick said when I want a certain amino acid into a growing protein chain, I’m going to build an adaptor molecule b/w amino acid and the codon. Turned out there was an adaptor molecule which was made itself out of RNA, called transfer RNA, and tRNA matched up by base pairing to each codon, and had amino acids attached to it. How do the amino acids get stuck onto the right transfer RNAs? There’s a bunch of enzymes that do precisely that job, they look at the tRNA, attach amino acid.

Lecture 10

January 12, 2009

1) Do the biochemistry of genes.

2) Discovery of the Transforming Principle: Griffiths. He wanted to understand bacteria – pnumecoccus which could infect and kill mice. They came in two different types, one produced a glistening shiny colony, and they were virulent, these would kill the mouse, smooth because of the polysaccharide coat. Other were rough bacteria, these were non-virulent. Mouse immune system was able to f ight them. Griffiths injects virulent bacteria into mouse, mouse dies.  Take rough bacteria, mouse lives. Take smooth bacteria, autoclave them, heat them to very high temperature so they are dead, mouse lives. Rough bacteria + heat killed smooth bacteria, mouse dies.

3) When you autopsy the mouse you could recover from the mouse smooth virulent live bacteria. Rough + heat killed smooth = live smooth. Take dead virulent bacteria, and start fractionating it biochemically and asking what fraction of the material from the dead bacteria gives this property. This is a painful process. He didn’t succeed, so people say there’s some transforming principle.

4) Avary Mccardy Mclow, same experiment, but no mice. Take dead bacteria, combine with live rough bacteria, and on a petri plate you would see the smooth bacteria come out. They began purifying. They knew they were transforming the heredity of the bacteria. They found that consistently the fraction that contained heredity was the fraction that contained DNA. The reaction to that was – mostly it was that must have goofed. Most smart people knew that DNA was an absolutely boring molecule. The interesting molecules was proteins.

5) Why was DNA boring? Structure of DNA: DNA has 3 components – sugar 2′ deoxyribose, its a pentose. The base can be adenine thymine cytosine guanine. To make up the monomers, we need triphosphate. When you combine nucleotides to create a DNA strand, you do so to create a sugar-phosphate backbone. The phosphate is always attached to the 3′ carbon of the preceding sugar, and 5′ carbon of the next sugar. We often speak of chains of DNA growing from the 5′ end to the 3′ end.

6) Bases come in two types – purines – adenine and guanine, they are 6 membered rings with a 5 membered ring and pyrimidines – thymine and cytosine – they have 6 membered rings. Compared to proteins this is pretty boring.

7) Bacterial Viruses: Instead of using bacteria to infect mice, Herschey and Chase used viruses to infect bacteria. These were called bacteriophage. All they had is some DNA in the capcid and some protein. They could attach to a bacteria and cause it to burst open and produce lots of daughter bacteriophage, it could replicate within this bacteria. When people discovered bacteriophage, people thought we should drink them.

8) How do these viruses kill the bacteria? They inject something into the bacteria, which causes virus particles to be made. What goes in? The thought was if this thing is injecting its DNA, then the DNA must be carrying the instructions to make phage, and this would be hereditary material.  How would you find out what goes in? Radioactive labelling. Label DNA with one label and the protein with another. What do you label the DNA with? Phosphorus – none of the 2o amino acids has phosphorus. P-32 label the DNA. How do you make them? Just allow them to grow in P-32 medium. Label proteins with? Sulphur S-35.

9) We infect bacteria with them. We need to knock of the bacteriophage from the bacteria. Specialized devices were used to create intense agitation. Kitchen blenders. Called the wearing blender experiment. Virus particles are much lighter than the bacteria, so we can separate them using centrifuge. We measure radioactivity in the palette and the supernatent (stuff that remains above) What shows in the palette – P32. In reality there will also be some S35 in the pellet, but it was less than 1%. Most of the S35 stays in the supernatent. Does all of the phosphorus go in? No, some viruses didn’t even attach.

10) We can conclude more DNA went in than protein. Suppose that 1% sulphur is tracking the minor protein that is the secret, its very hard to rule out that. Infact, Avery’s experiment was pure than this experiment, but by this point thinking had started to shift.

11) What was it about the DNA that conferred these properties? Crick and Watson: DNA Structure On the basis of a lot of modelling and using the X ray diffraction pictures, made a model. One chain is 5′-3′, and another antiparallel chain is 3′-5′. The X ray diffraction pictures told you it had to be helical. Key to the model was recognition of base pairing. The distance between adenine and thymine, and cytosine and guanine, would be the same.

12) There was an observation – the amount of A close to amount of  T, amount of C close to amount of G. This model also explains how a DNA molecule can replicate. All it takes is for those 2 strands to come apart, and each can serve as a template for the other. What’s mutation? It sometimes gets it wrong.

13) The above replication model is called semiconservative replication. In theory, DNA replication could occur by old strands staying together, and serving as a template for making a new double helix. Semi-conservative implies each DNA has one old and one new. Radioactive labelling. Grow DNA initially in normal nitrogen and then shift it to radioactive nitrogen. How do I prove that these were 50-50. What property do I test? Density. The molecules have intermediate density between all heavy and all light. They had to work out a centrifugation technique.

Lecture 3

January 11, 2009

1) Simple sugars differ in 3 respects – a) the location of the carbonyl group. b) the number of carbon atoms present c) the spatial arrangement of the atoms – particularly the relative positions of the hydroxyl groups.

2) Glycogen is used to store glucose. Its branched because it has to store glucose in an inactive form.

3) At neutral pH, a amino acid won’t remain unionized. Its amino group would attact a proton, causing it to positively charged, and its carboxyl group would release a proton causing it to be negatively charged. At very low pH, due to the large availability of protons, the inclination would be to attaching the proton to the carboxyl group. At very high pH, where the hydroxyl group is in predominance, they scavenge protons, and hence the amine group would be in its original state.

4) The amino acids exist in a  very specific 3d configuration. Mirror images called chirals

5) Amino acids polymerize through peptide bonds, hence proteins called polypeptides. If these chain lengths are small, we call them oligopeptides. Here again, we have the possibility of extending them infinitely. The side chains can be anything out of 20 R molecules.

6) There is a polarity. The new amino acid is always added on the carboxyl end. Hence, there is a N end and there is a C end. Things are growing at the C terminal end progressively.

7) Virtually every biochemical reaction is reversible. If one is able to form peptide bonds, one is able to break them as well, through hydrolysis. There are 20 R’s for proteins. 99.99% of all the proteins that is created is through the synthesis of these amino acids.

8) Since there are 4 distinct side chains around the central carbon, you’d expect to have handedness, but the glycine amino acid doesn’t exhibit chirality.

9) Side chains have quite distinct biochemical properties, proteins and then biochemical attributes can be dictated by the amino acids that are used to construct them. We can talk about non-polar vs. polar amino acids. Amino acids which have a poor affinity for water, hydrophobic. You would wonder how can they be hydrophobic, because the amine and carboxyl group is charged. But I am talking about them not as single form, but when they have polymerized. So when we talk about polar and non-polar amino acids, we are concentrating upon the side chain.

10) Polar side chains – serine with a hydroxyl group that can form hydrogen bonds with water. Threonine can form hydrogen bonds through hydroxyl, asparagine can form hydrogen bonds through carbonyl and amine group, as can glutamine.

11) When the side chains are charged, strongly hydrophilic.

12) Tyrosine has the strongly hydrophobic benzene ring, and the hydroxyl group which loves water. Cysteine has SH group which can form bonds with other SH groups from other cysteines, through a disulphide bond. This is a very strong bond in the absence of reducing agents. Thus polypeptide links can be covalently crosslinked. Conversely, if you add reducing agent, that will add protons back, and reduce the oxidation state of the sulphurs, causing the disulphide bonds to break apart. The disulphide bonds could be used to link two proteins together, but more often than not, there are intramolecular bonds, bonds from one domain of the protein to another. Why do we have these disulphide bonds? Because a protein can only function when it has a certain 3dimensional stereochemical configuration. This structural rigidity is maintained by these disulphide bonds, which link neighboring regions of a polypeptide chain, these intramolecular links.

13) There can also be intermolecular links between two polypeptide chains that are mediated by the disulphide bond.

14) In proline the side chain is covalent bonded to the amine group, it creates a 5 member ring. This amino acid when occurs in a polypeptide doesn’t have the flexibility of assuming certain configurations that the other ones have.

15) After we wrestle with the 3dimensional structure of the chain, we realize that after the initial chain is synthesized, its initially chaotic, and as it extends, it begins to assume a 3d molecular configuration.

16) If you knew the primary structure, the sequence of amino acids, you should be able to develop a computer algorithm that can predict the 3d configuration. This has not been possible yet because of the infinite no of intermolecular interactions that greatly complicate how the protein assumes its structure. And if this is the native state of the protein, there are ways of disrupting that, because much of this native state is created because of intramolecular hydrogen bonds. Hydrogen bonds are weak and we break the 3d structure when we heat. Some molecules upon cooling down will reassume their native structure. Most proteins however will not do so.

17) Primary structure is the amino acid sequence. Secondary structure represens structures containing the hydrogen bonds. (eg helix structure)

18) Prolene is known as a helix breaker, because it cannot twist itself around to form an alpha helix. So locally where there is a prolene, there won’t be a helix structure. So, secondary structure means, a certain segment can form alpha helix, another segment of the protein will form beta pleated sheets.

19) Tertiary structure – How the alpha helixes are disposed wrt one another.

20) Proteins act as catalysts in cells, as enzymes. Almost all biochemical reactions require an enzyme to propel them forward. Catalytic cleft – active site of the enzyme where the substrates are pulled in, and are manipulated and changed by the actions of the enzyme.

21) Proteins also have another function – to create a biochemical structures. Complex structures are formed out of proteins.

22) Hemoglobin is a tetromer. Two alpha helices and two beta pleated sheets, there are combined together through hydrogen bonds. If you break it apart into 4 pieces the individual parts are useless.

23) The hydrophobic proteins do not like water, therefore they are tucked inside the protein far away from the surfaces. They don’t have contact with water. The hydrophilic amino acids are tucking out from the surface.

24) Summary – there are disulphide bonds, there are hydrogen bonds, there are these hydrophobic and hydrophylic interactions, and there are also some VanderWaals interactions.

25) In carbohydrate, you have the same monomer in 100 or 500 stretches. Protein is much more interesting.

26) If the 3d structure is disrupted (denature) by heating, it loses its function irreversibly.

27) Nucleic acids – We start with two pentoses, recall they have 4 carbon atoms. Two basic kinds of pentose molecules that are present in nucleic acids. Nucleic acids are polymers just like proteins are polymers, but instead of being made of monomers called amino acids, they are made of nucleotides. A nucleotide contains a phosphate group, a sugar, and a nitrogenous base.

28) The two kinds of sugars define the essential difference between DNA and RNA. – ribose and deoxyribose. In carbohydrates, the hydroxyl groups represent opportunities for all kinds of dehydration reactions, which can enable one to build much more complex molecules. The nitrogenous base is attached to the sugar by a dehydration reaction. One of the things you are going to have to memorize is the numbering system here.

Lecture 2

January 11, 2009

1) Covalent bonds have 80kcal/mole

2) Polar molecules are able to dissolve certain kinds of compounds.

3) Water has energy 5kcal/mole. Thermal energy has 0.6kcal/mole.

4) micell – lipids form in water

5) lipid bilayer – is the way most biological membranes are organized, because biological membranes separate two hydrophilic spaces. In eukaryotic  cells, you want to separate two aqueous layers. When you see a membrane, implicit is the fact that it’s a bilayer.

6) Esterification. Reverse is hydrolysis, reintroduce water.

7) Selective permeability of membranes. Polar molecules cannot go through. Most of our energy is spent in running pumps that keep these concentrations intact inside and outside of the cell.

8) Carbohydrates – one carbon atom roughly per water molecule. Glucose has 6 carbon atoms. Called a hexose. Pentose has 5. Glycerol is also considered a carbohydrate, but its a triose.There are mechanisms to join two glycerol molecules to form a hexose.

9) Hexose isn’t linear. Because of thermodynamic reasons, it likes to cyclise.

10) Stereochemistry refers to the 3d structure of a molecule. Dictated by which atoms are present (tetravalent, trivalent, etc.) as well as thermodynamic considerations. One of the 6 atoms in the cycle is a oxygen, not a carbon, and a CH2OH is relegated to the outside – called extracyclic.

11) What’s the difference between alpha glucose and beta glucose? Whether the hydroxyl is above the plane or below the plane.

12) Glucose and galactose differ in the spatial arrangement of the atoms – particularly the relative positions of the hydroxyl groups. There exist catalysts, called enzymes that can convert one to another.

13) Monosaccharides converted to disaccharides through esterification reaction.

Mendel and the Gene

January 11, 2009

1) Wild type – Individuals with the most common phenotype.

2) Meiosis

3) Proposing that a gene is on the X chromosome is called X-linkage. Proposing that a gene is on the Y chromosome is called Y-linkage. Proposing that a gene is on either sex chromosome is called sex-linkage.

4) The + symbol always stands for the wild type trait.

5) Non-sex chromosomes are called autosomes. Genes on non-sex chromosomes are said to show autosomal inheritance.

6) Studying the organism yeast. Yeast is a fungus, it is a single celled eukaryote, it has a nucleus, it has chromosomes that pair up, its cells to a first order approximation are a lot like your cells. How do you grow yeast? Takes growth medium that has a lot of rich medium. Put yeast cell in the broth. I get culture of yeast.

6) put them on a petri plate that again has nutrients. Dilute it, take a little of the broth, and spread it, so that there are individual cells scattered randomly. and I get a colony all of which descend from a single cell. Called colonies.

7) Life cycle of yeast – has a diploid stage, it can undergo mitosis. Yeast has 16 chromosomes. Humans have 23. yeast also undersgoes meiosis to produce spores. They come in 2 flavors in A and alpha, like male and female. This is identical to the human genetic cycle. turns out that, yeast can also undergo mitosis as a haploid, the haploid cells of yeast, they can continue to grow indefinitely. Your gametes cannot. This is very convenient for geniticists.

8) Yeast can grow on minimal media. with very few macromolecules. It needs a carbon source, which is some sugar that it can ferment, it needs a source of nitrogen, phosphorus, salts and water. Why is it able to grow? It makes them. It is extraordinarily self reliant. You are not.

9) Yeast is not stupid, if you give it amino acids, it will use it. yeast is able to use rich media. Why not always manufacture? Save ATP, = energy. How does it make argenine?

10) Biochemist will find an enzyme that makes argenine. Geniticist will find one that cannot make it. Geneticists wants mutants. Find them by going on a mutant hunt. Grow yeast on a medium that has argenine, and pour them on a plate that has minimal medium w/o argenine. Those that can grow are the ones we are not interested in.

11) Yeasts that are able to grow on minimal media are called prototrophs. Yeasts that need help are called oxotrophs. One way, find those that grow on minimal + argenine, but cannot grow on minimal. We will end up with argenine auxotrophs.

12) The yeast cells we plated, were they haploid or diploid. – haploid. Because, we’ll need a mutation in both copies for the diploid, assuming oxytrophy for argenine is a recessive trait.

13) Suppose we get n colonies, arg1, arg2, … arg n that show oxotrophy for argenine. How many distinct genes does this affect? Are these mutants all in the same gene? A geneticist wants to know what are all the ways to disrupt a cell so that it cannot make argenine.

14) Geneticist do a variety of tests. Tests of recissivity or dominance. Whether the oxotrophy for argenine is dominant or recessive for each colony. Make a cross with wild type. Test each from 1 to n.

15) Lets assume all are recessive. How do I tell if they are on the same gene or not? This is called Test of Complementation. If two mutations complement each other’s effect, they are on different genes. Suppose arg 1 and arg 2 are on different genes. Cross them.

13) So we can make a complementation table. Rows contain arg1 to arg n, and so do colums.The groups that fail to complement define mutations in the same gene. These are called complementation groups because they don’t complement each other. Actually it should be called failure to complement groups.

14) Tests of Epistasis – Suppose a biochemist wanted to test his hypothesis about manufacturing of argenine. A goes to B goes to C goes to argenine. Suppose specific genes were necessary to catalyze specific steps of this biochemical process. The geneticist and biochemist could collaborte with each other to study whether these mutants affected each step of this pathway.