chapter 8: microbial genetics
Chapter eight, Microbial Genetics. A few terminologies that we need to know, the definitions of genetics, which is the science of heredity, studying what genes are, how they're transmitted, whether the products and and the molecular at the molecular level, genes are segments of DNA. As you know, DNA is a nucleic acid, which is composed of nucleotides, subunits and nucleotide subunits are composed of nitrogenous bases and phosphate and sugar. And so the nitrogen containing bases are in DNA there at any time in cytosine. Gordin So those segments. Of DNA molecule are composed of different numbers of those nucleotide bases. So that's what genes are, chromosomes are the actual physical structures that contain DNA molecule. Genom is the entire genetic genes of an organism that organism can be a single celled organism or multicellular. So includes all the genes in the DNA in addition to if there's a plasmid, what's in the plasmid. Those are all part of the genome of that organism's genome. Genomics is the study of genome. Genotype is a genetic make up of an organism, and phenotype is the expression of the genes and the expression of the genes. Typically, when genes are expressed, the products are, of course, protein. But also, they can be transparent, as they can be ribosomal already. What we're looking at here is a double stranded DNA molecule. It looks like a twisted ladder and this is an electron micrograph. So it's a real picture of DNA and. The sugar phosphates for their phosphate are in the backbone and the nitrogen containing base bases are facing one another and we're looking at the ecological as well. But it's not as small as one of the small bacteria that you have seen in the lab. And bacteria, as you know, typically they have a circular one, only one chromosome, which is which contains a double stranded DNA. But it's not linear. It's circular as opposed to eukaryotic DNA, which is always linear. So you see that this entire DNA molecule that you're looking at is just one and it's a circular one, but double standard and it fits right in that small typically about one mushroom's long. That's how small these bacteria are. Whereas the ecology's whereas the the DNA, if it's DNA, if you were to unravel it, it would be about 1000 times longer. But it fits in there because of all the twists and supercold.
As early as 1869, actually, the existence of DNA was known it.
It but it was discovered by Yohannes Meisha, who was a physician, but because he was deaf, he could not practice his profession. So instead he was involved in research and and he noticed something in the nucleus and that wasn't seen before. And it and he knew that that was not protein. So what he was actually had discovered was DNA, and it wasn't until the 1950s, early 1950s, where, as you know, Watson and Crick, they they built a model of how DNA molecule actually looks like composed of double sided DNA. And they also got a Nobel Prize for it. But they couldn't have deciphered the structure of DNA without Rosalind Franklin crystallography pictures which were using X-ray machines, X-ray to take images of the nucleus and the DNA in it. So this is actually the DNA and what he was what she was able to do was to she was able to calculate the distance between the two strands and also the distance between those bases from one another. So unfortunately, she died of cancer because of all that radiation exposure.
And did not show the.
Nobel Prize Human Genomics Project, which its goal was to sequence the entire human DNA, that project was it started in 1990, and they thought that it would take years, but they they completed it ahead of time. It actually was done in 2003. So Francis Collins and the. Craig Venter were the leads, but this was a multi national project, so sequencing, meaning that they determined the sequence of bases in human DNA, the colors that you see, each one is representing a different base. So therefore, you know, one is for every one is for thymine, one is for coding and the other for cytosine. So DNA sequencing, meaning that the sequence of bases of human DNA, that that's what the goal was and they were able to finish ahead of time because they you know, there were machines that were invented and used to do this a lot faster. So it was automated. And then that's how they could determine that. Human genome contains more than three billion bases. Right now, about 1000 species
genome is already sequenced. So these are these are some of those species. You can see the number of genes near that same equal I've been talking about has only only has about 4200 genes as opposed to a typical virus. Nine. All right. So going back to the DNA molecule and the fact that before a cell divides first, it has to multiply its DNA, it has to duplicate, I should say, duplicate its DNA. So DNA replication.
Well, before I talk about that, let me just show you this double stranded DNA where the fact that the the two strands are about parallel and so the end that has phosphate attached to carbon number five is facing the and that has an O h group attached to carbon number three. So that's why this is called five and this is called three prime. And so there are these the two strands are.
And so in order for replication to occur first, there are there are multiple enzymes involved. So an enzyme Taupo is omurice or DNA, Giris or involved with relaxing the coil. You know how sparklingly not so relaxation of the code ahead of the replication fork, which is the point where the two strands are separated from one another. So relaxation of the super is done by this these enzymes. And then the application form is right here where DNA in any case separates the DNA strands from one another. And then DNA polymerase is another enzyme that its function is to its function is to bring nucleotide subunits, new nucleotides, subunits based on base pairing Ruth, they are deposited. And what I mean by that is that the original strand is used as a template so that because there's a timing there, then the nucleotide sovereignly that has adenine would fit right there and so on. And then at the end, DNA Loynes is the enzyme that will bind the sugar phosphate butterballs and so that you get a continuous strength and notice that the same thing is going on on the other side. So at the end of that replication process, there will be two molecules of DNA where each one has the. Each one of those molecules has one of the original strands on the new strands, so that's why the DNA replication process is called CIMMYT conservative DNA replication. By the way, the is the energy coming from for the DNA polymerase enzyme, you know, to deposit the new nucleotide subunits during DNA replication. It comes from here. As you know, each nucleotide is is composed of sugar, phosphate and nitrogen containing bits. So it has just one phosphate. However, when the nucleotide subunits are brought in to be to be inserted to make the new strain, this is a nucleoside triphosphate. So they come in as nucleus like triphosphate three Fusi AB and then these molecules. Then each one loses the last two phosphate. That's what the energy is coming from. OK, so the energy was these to these bonds are broken, their potential energy and the chemical bonds. Right. So once these are broken, these energy in the bonds here, they are used in order to insert this in place. And then, of course, this carries Thymine because the template there's an adult in the original template. So that's why then it will become a nucleotide instead of a nuclear site triphosphate. And so if you look at. What we're looking at, the DNA replication on the enzymes again. So here's a replication fork and what this slide is showing you is this, that the DNA polymerase enzyme can only bring and attach a new subunit to the three primum. So therefore, the new strand that is being synthesized can only be synthesized in the five primed to three prime direction, five prime the three prime direction. And so if you look at the two strands of DNA and you're looking at this astron being synthesized continuously, that's what is called the leading strand. But on the other side, there's a problem because the DNA from
the new strain is supposed to be made from can only be made in five one, two, three prime direction. But in order for that to happen on this side, first
enzyme, primate's will the blue oval shape here that you're looking at will make a primary RNA primer, which is another short strand of RNA here. And then after that, then DNA polymerase can use that exposed three prime and to bring new nucleotide subunits there and attach it there. So once that's that happens, this RNA primer disappears and the premise is gone. So if you look at this strand, this is the new strand here is being synthesized in fragments, in pieces. Because of that, those fragments are called Kazuki Fragments, named after the scientists who discovered them. And so was this. Fragments are made then DNA Ligeti's will join the sugar phosphate, that backbone so that it will seal the gaps here. OK, so this strand is called lagging strand because it's made in fragments, whereas this one is called a leading stem because it's made in a continuous manner.
But now we're going to talk about the process of transcription.
The genetic information that is in the DNA molecule, it's transcribed and the transcription is an RNA molecule, the genetic information that is in the DNA is transcribed. The transcription is an important molecule. So RNA molecule stands for messenger RNA and we're looking at the process of the synthesis of tomorrow night. So the green strands are supposed to be Amarone strands and noticed that the enzyme that is involved here is RNA polymerase. So RNA polymerase is the enzyme that is bringing RNA nucleotide subunits. And again, one of the strands is going to be used as a template. So if you look at the Ducros here, here's a strand that is this one is used as a template and the green snot is the marinade as being synthesized. I noticed that RNA polymerase brings RNA nucleotides any they would be inserted by some base on a base, on base pairing roles where adenine always points uracil and timing always points agree. And so then you get. I mean, not binding, but it's it's complementary to it, so eventually, again, you're going to get an Amarone molecule released. So Amarone is short, the molecules are short because they're only complementary to the gene of interest. And so, again, the enzymes, the enzyme involved here is RNA polymerase or what I forgot to mention earlier is this, is that the RNA polymerase starts the process of the synthesis of Amarone at what's called promotor region. And then here's a read through the report here, Promotor Region. And this is going to continue until it reaches the end of the gene. And the end of the gene is the is the Terminator region. So promotor regions are thought that's where RNA polymerase starts the process of transcription and it will end at the Terminator regia. This actual image is actually of the actual thing is showing you the RNA polymerase, two of them here, enzymes and then DNA. So these are already attached. And there, you know, go through the entire gene, that one gene that at the time that they are making the Amarone.
Complementary to it and and, of course, are much eukaryotic, the prokaryotic process of DNA replication happens in the cytoplasm because there's a nucleus transcription is also in the second and then followed by translation.
One thing that is different in the eukaryotic organisms is, is this, that, first of all, there is a nucleus, so the process of DNA replication takes place in the nucleus and then the process of Amarone synthesis is also taking place in the nucleus. So here's and and then another difference is that in the eukaryotic organisms, the DNA is composed of both genes. And then in between there are nucleotide nucleotide subunits where the sequence of bases do not code for any functional products. So those are called entrants. So in eukaryotic organisms, the DNA is composed of eggs and those are the genes, you know, that will be expressed. Followed by entrance where those are they don't quote for any functional products and we don't have that ID in the prokaryotic in the prokaryotic DNA, there are no entrance. So therefore, in the process of transcription, first, the irony that is synthesized has the bases that are complementary to all the bases here in the original DNA molecule. So these are complementary to exons and insurance, but then later there will be an orany plus protein together called Snopes. This is what is used by the cell to cut out the entrance out of that or a and splice back together the exons. So then the outcome is called amorally. This all happens in the nucleus, marinate them, leaves the nucleus through these nuclear membrane port and goes into the cytoplasm where ribosomes are for the process of translation.
This table is showing the genetic code. Genetic code is already deciphered, and it's showing you that every triplet of bases on Amarone. Calls for.
Which have been lot. So, for example, you're looking at Lucene, the distributor that that ship had called for, you see, but also all of these could follow. These are amino acids, you know that we only have 20 amino acids. And and then as opposed to AOG that Triplette calls for Mytilene in in the eukaryotic, but in prokaryotic, it calls for forming an entire line. So it's a little different, but it's also called a start code on all of these, Jupitus, are called codons. So codons are the triplets of. Bases on Amarone. And so, again, this table shows you which codons code for which amino acids and as you. As you notice, as you can see here, that for almost all of the exception of just a few
were of maybe this only, there's only one quote unquote. And for this there's only one, quote unquote for mutiny. So with the exception of a few, most of these amino acids have more than one combination of codons coding for that amino acid. This is called the degeneracy of the genetic code, meaning that that the that the genetic code is redundant and the reason for it is to minimize the possibility of a disastrous.
Outcome from mutations, that does happen spontaneously. What I mean is that, for example, if one in the original Amerine is if one adenine is by mistake, is replaced by a guanine still that that replaced the newly formed chip that still codes for. See, those are other alternatives that still could for for fear of losing. All right, so here we're looking at the process of looking at the process of translation and notice that ribosomal subunits only joined together during the process of translation. You're looking at the Triplette AOG, which is the code on that is also signaling the start of translation. And so that's a signal for the word translation should start. And so here's the Amarone, so notice that there is another orany molecule transfer already that is more on one at one end, it brings the specific nucleic specific amino acid. In this case, of course, is Methionine because a huge equals form with and at and at the other end, it has its own triplet of bases, which are those bases are complementary to the codons. So triplets are bases on Emilian. They are called codons, bases on transfer. O'Neill called anticoagulants. And why is it that McEneaney is brought here? Because a EUGE can only bind UAC, so this codon can only bind this particular. And any transparency with this specific article, Don, always has me standing at the other end. So it will depositors' methionine and it leaves and then the next one, based on the next cold, on the second coat, on the transparency with the specific complementary antico that can be found here and that would always have Lucene with it. So this is how, you know, the ribosomal subunit wall along the length of that Amerine and Dusko translocation in order for the genetic code to be translated to the language of protein proto language, a protein would be alphabet's or the amino acids. So. The enzyme that binds these amino acids together is ribosomal RNA, remember that enzymes are protein, but there are exceptions. Here's one exception. Ribosomal RNA is RNA molecule, but it's an enzyme and its function is to make peptide bonds between the adjacent amino acids to make it polypeptide chains. And once was ribosomal subunit Ritsch one of the top cooldowns. That's what I was the one subunits would associate from one another. And that polypeptide chain then will undergo, you know, it's in the same is in the primary shape, but it's going to undergo secondary, tertiary or quaternary depending on what that protein is supposed to be. So here's UJA, which is one article on excuse me, Onestop codon and I forgot to talk about here. There are other than we have three of these codes that signal the start of the translation process. OK, any of these, if they are encountered by the ribosomal seven, is the translation process will stop.
And again, as I mentioned, in bacteria in prokaryotic or prokaryotic, the MRSA is synthesized right in the cytoplasm and the DNA is replicated right in the cytoplasm and, of course, protein of the at. The ribosomes in inside have risen again, so therefore, as soon as Monday, you know, Amarna is not even finished being synthesized. Translation We're already stuck. So here's here's a here's what I mean. And notice that we're looking at DNA, right? It's in the cytoplasm because this is bacteria. And then we're looking at RNA polymerase, which is making that green strand. The green strand is the emotively. So we're looking at the process of transcription. But as soon as there's exposed part part of that RNA molecule, ribosomal subsidies are already attached and the process of translation is going on. And so this is electron micrograph of what is really going on. And again, you're looking at Amerine is in the background. You're looking at all the ribosomes attached, and then you're looking at a polypeptide chains. Those are individual amino acids. Ulufa'alu together, OK? All right. Geez, gene expression in what, eukaryotic in prokaryotic is regulated.
Meaning that not all of the products of all the genes in the DNA are produced in a cell at all times. Right, that would be a waste. Not all the products are produced at all times. And so we're looking at and we're not going to talk about the regulation of gene expression in UK, I think we're going to just talk we're going to only talk about the prokaryotic once. And the genes that are their products are the fixed rate are being produced, they're called constitutive genes. So those would be, for example, genes that are involved in making the enzymes that are involved with glycolysis. Right. They don't stop mixing, doesn't make a start. That doesn't stop making them. They're made produced in a cell at a fixed rate. But other genes, they are turned on and off depending on whether or not the products are needed in the cell. So there's a regulatory mechanism going on. Some genes are induced and some are repressed. And there is in in bacteria, we have
different genes involved with the production of a specific product. There is a model called offering that offer. A model of gene expression was first discovered and described by
one, not Jacob French by chemists in the 1960s. And what they said is that all parents are, you know, looking at DNA. So they are
each of is composed of genes that are involved with a product of that. So what what does each operand composer here's hit from here to here? Is the opera, right is composed of a promoter region. We're looking at Disney promoter region. These are US bases, right? Sequence of nucleotide nitrogenous bases promotor region followed by operator region, followed by what's called a structure of genes that in this case there are three genes involved. It could be one. So the promoter region is the same place where the
start of transcription is a sort of transcription, remember that when we talked about DNA transcription that we took with it, we said that the start is promotor Terminator, the end of it. This is the same promotor region. But after that, there's the operator region. So this this is what an opera is composed of. And before prior the basis of nostalgia's business, before that opera, this is a gene that is called regulatory gene, which its product is a regulatory protein. OK, so we're looking at over here. We're looking at an opera, right? We're looking at a promoter region where RNA polymerase wants to in order for Amarone to be synthesized. Then we have operator Ridgen. Then we have structural genes. So wants to promote a must see RNA polymerase binds to the promoter region that is going to start transcribing until it reaches the end, which is the end of the gene determinative region. OK, so let's look at the different types of operands that exist. So some operands are known as a as the crucible. Oberon's irrepressible offramp is always on meaning its product is being synthesized, the genes in there are expressed and the products are made. OK, so why is it that they are always on because of this? Because what's happening is that, yes, you have the operator from promoter operator, the genes in this example. The it's always on because there is nothing preventing you from that RNA polymerase to bind to the promotor region and then going ahead and make the, you know, make the Amoretti, which the transcription has happened, then you get translation and the product is made. Why is it that irrepressible everyone is on, because that's regulatory g, which always calls for a repressor protein or a regulatory protein, the regulatory protein is not active. So in irrepressible operation, the regulatory protein, even though is synthesized but is not active, that's why there is nothing preventing from this RNA polymerase to bind to the border region and therefore express those genes. But irrepressible upper arm can be turned off. Remember, it's always on its own because inactive repressor protein, but it can be turned off. How is it turned off like this? So here's an example. Example is cheap. Uppal cheap is for tryptophan and amino acid that can be used for Rochet as well in bacteria. So notice how this is turned off. How is this off? Like this here is a regulatory you, same regulatory protein, but then when there is enough of tryptophan already producing a cell.
Animalism getable is already there, Sarah doesn't need any more, this tryptophan works as a core repressor by binding to the repressor protein, causing it to be active, activating it together. They bind the operator region, preventing the problem, preventing the already polymerase physically, preventing it from actually moving ahead and transcribing the genes. So no more tryptophan will be made. OK, so that's an example of a repressive opera.
What about them in usable, upright and usable front is one that is always off, it's always off in the.
Why is it always off? Because this is happening. So here's again, regulatory gene coding for regulatory protein, the regulatory protein always is binding, has already attached to the operator region, preventing the RNA polymerase from expressing the genes. So the product of this operation is not made. Meaning is of an example is like opera, like is for lectors, like opera for metabolising lactose in a bacteria, there are actually three different enzymes that are involved. That's why you have the three genes here. OK, so when is it that I'm in useable operand, which is always off, what is it? I can be turned on. It can be turned on when the inducer is there. So you're growing a bacteria in it. In the media, the inducer would be lactose if lactose is present in the media. That's an end user for turning this operation on because it's always off. Right. So how is it going to be turned on? So here's how it's going to be turned on, lactose is going to be converted to a low lactose adult lactose is inducer and I originally said lactose is inducer and I'm correct because if lactose is not there, there's not going to be adult lactose. OK, so you can you can you can definitely say that lactose is a lactose, however, will be converted to a lower left us alone lactose is going to bind to that repressor protein in activating it, preventing it from bonding to the operator region. Therefore, that RNA polymerase, which was already attached here, there is nothing to prevent it from moving ahead and express those genes, the products or transcription. But here's the commodity and then the products after translation. These are three different. Enzymes that are involved with metabolizing lactose, so Larry O'Brien is an example of an invisible opera. Now, what we have here is that we have a flask, suppose we have a flask here where
the bacteria you're looking at growth of here, a number of cells, a number of bacteria, and then time passed and it shows that bacteria will grow a much better exponential growth on glucose if they were to use glucose as food as opposed to lactose. Right. So increasing a number a lot faster as opposed to growing a lactose. We know that already that glucose is always preferred then over here. What they've done is that they have put both glucose and lactose in in a in the media and they have the bacteria in there. So obviously they're going to use glucose first. So you are seeing a nice exponential growth, very rapidly growing, using glucose as food and then stopping because glucose is gone and they just stop. There's a lag time, you know, deciphering what to do. And then they find the lactose and they start growing on lactose. And lactose, obviously is the one that is showing you the graph. The curve that you see is just like this. Still growing, but not as exponentially sharp exponential growth as if they were using glucose, so. So notice that. The question here is that if you have what glucose levels in this amedia and we know they're going to use glucose first, but is the lack of ground on then? Because we said that lactose is the inducer for lack of. Right. So if that's inducer for the like Brown, then the lack of brown should be on. And so therefore that bacteria should produce those enzymes to also metabolize lactose. But it doesn't. It doesn't until the glucoses gone. So far, the lack of rain to be turned on, not only lactose, should be present because it's the inducer, but also glucose should be absent. That's another of another criteria that has to be has to be met for the lack of rain to be turned on. And so what is happening is this. Well, lactose is their.
I think this. I have to correct this, because, as you can see, a. And B is telling you the exact same thing, whereas it's not the same thing that's happening.
Hmmm, OK, I'll fix that in the PowerPoint, so. Notice what's going on is that you have.
If glucose is a scarce. Well, let's start here when glucose is present, right, glucose is present and they're going to use glucose, right? Yeah, see, I have corrected it here, but it's not covering this. The this should be lactose present and glucose prison. OK, but they're going to start using glucose because glucose is present. And and there is an alarm hormone called cyclic amp, cyclic AMP is produced when the bacteria find themselves in unsuitable situation for, for example, no food. So there is no alarm hormone being produced when glucose is there, so therefore they're not using glucose and are growing nicely on it and like operand is not on. But what happens when glucose is gone? Right. Glucose is scarce. That's when the bacteria start to make lots of a lot of more. The allowable hormone cyclic amp is going to be produced, which is then going to bind the receptor together. They're going to bind the.
Promote region, and that's going to induce the turn on this witness and initiate, I should say, initiate the attachment of RNA polymerase and transcribing the genes that are necessary to use lactose as nutrient. OK, so it all has so it's describing that as soon as glucoses gone, that's when the cyclic AMP is going to reproduce, is going to burn, the receptor scepter is going to bind the promoter region, not the not the operator region is binding the promoter region, promoting transcription. And so so, in other words, is causing the is leading to the production of those enzymes. All right. And then on top of everything else, we have epigenetic control of gene expression also. So epigenetic control is also seen in both bacterial and eukaryotic organisms, and that's expressed by DNA methylation and meaning that adding methyl group to certain nucleotides and Bethwaite inactivating them prevent their expression. And that is not considered mutation because it's DNA methylation is not changing the sequence of bases in DNA. That's why it's not considered a mutation. It's just adding methyl group to some to certain nucleotides and therefore turning them off. However, this can be transmitted or next generation and also it can happen. You know,
it can also be turned down in even not only next generation, but generation after that. But yet this is not a secret. It's not any change in the sequence of bases and it's not necessarily permanent. And so that's what epigenetic control is. A mutation. However, is any change in the sequence of bases that's called mutation. That's permanent, that's permanent, and that can also be transmitted to next generation and mutations can happen spontaneously. They do happen spontaneously, but at a very low rate. However, the presence of a mutagen, a mutagen would be anything into anything in the environment, you know, a chemical or radiation that will increase the rate of mutation.
If America is around, it's going to increase the rate of mutation and drastically so the two types of mutations that are discussed in your textbook, one is based substitution, where one base is replaced by another one and the other one is called Tradeshift mutation, where we're going to talk about it. But notice that this is. Normal.
Normal event, as opposed to when mutation happens in the DNA, that mutation, that's change, that mistake is going to continue all the way to where the protein that is made is not is altered. That might not even be functional. OK, OK. So Besançon solution and so fashionable to talk about this substitution. Here's normal again in what normally should happen. So it's just a piece of one part of DNA you're looking at and then that's the that is.
That is synthesized complementary to this, and that is showing your translation, which, again, you know, each triplet codes for a certain amino acids that are put together onto, you know, that reaches the end of the gene and then therefore. So what happens in base substitution? In the base substitution, one base is replaced by another one like you. Say, in this Triplette, I mean, this Triplette.
Instead of AC, instead of instead of see that cytosine is replaced by a thymine. In DNA, so there's a mistake right here in the DNA molecule, therefore there's a mistake here because this is not cytosine. So that's why he's not claiming that timing. Now, this is Avani. So this new Triplette code on that is created that has been created a G, C they now form does not code for glycine is supposed to be glycine here in this polypeptide. But this code on is not coding for it. It calls for certain, which is a different amino acid. That's exactly the case that takes place. It is not the same amino acid, but it's the same situation where it's a base substitution. An example would be sickle cell anemia and sickle cell anemia is so as a result of a base substitution instead of a glutamic acid, there's a wailin inserted in the in the polypeptide chain. And and that's why all the problems that patients with the same you have with multiple organs in their body and and the same thing. OK, so what I should also mention is that based substitution, which is the replacement of one base by another. There are two types of base substitutions also. One is called me says. The other one is called nonsense MS. Sense is this example I just gave you that. Yes. You got one best replaced by another one, but you got the new triplet that now calls for a different amino acids for what it should be. So the example of a disease, genetic disease, that is because of. Mrs. Mutation, which is a type of substitution, is an any other. Just explain another one. The second type of base substitution is called nonsense mutation because yes, there is one base replaced by another one. Like in this case is this thymine, it is replaced by an adenine, therefore now you don't you don't have other other than guanine, you have you agea that's a stock. Whether it's a stock, whether that's created, is being created, is formed. And so what does that mean? That means the translation is going to stop right here. Whereas, you know, are there other cooldowns that need to be translated, but because the ribosomal soberness encountered the stop chodak they're going to stop. And so that's you can see how you know, this is.
It's going to be the outcome is going to be disastrous because this is not going to be a functional protein, an example of a genetic disorder that is due to that is thalassemia, which is another blood disorder
in case of Mrs. Mutation, because of the degeneracy of the genetic code. It's possible that even though one base is replaced by another, it's possible that you still end up with a cordon that codes for the same amino acid, or maybe the amino acid change would be a similar one. So or in case of serious anemia, cell anemia, that would be it leads to a lot of problems for multiple organs. But in case of in the case of nonsense, mutation stuck sarcoidosis created. You can see that none of the amino acids after that are going to be served and this is going to be a non-functional polypeptide chain or an enzyme. This is just showing you that sickle cell anemia and multiple organs that are involved, the problems that are involved all has to do with one base being replaced by another one. All right, frame shift mutation is the second type that is discussed in your textbooks. So we're looking at a normal situation here where we're looking at DNA and not only on the amino acids, but what is happening is that in the frame shift mutation, one or more. Of bases are the bases in DNA are added or depleted. Deleted, others were deleted. Look here, one of the other needs is gone. Deleted. So you can see how that the the frame of translation is shifted because instead of you, you you being the Triplette that would have called it forfeited any now because the other team is gone here now, you don't have that other one there. So this is the new Triplette. This new report calls for a new and different amino acid or all the rest of these triplets are changed. So that's what I mean by the frame of translation is shifted, so every one of those amino acids could be the wrong amino acid.
Huntington's disease is one that is due to Frenchified mutation is it is a degenerative neurological disorder, and then cystic fibrosis is also due to fractured mutation. Some chemicals in the environment, Inspiron causes mutations that say it's a chemical insert. And then aflatoxin, which is produced by
some fungi, is a toxin that also causes SIFF mutation or at some of those drugs that are anti microbial, the Orantes microbial, because they are the cause mutation, such as those that are analogs of those nucleotide subunits. You know, like Asakawa we talked about in Chapter 20, which is an analogue. They create mistakes because they insert themselves instead of the inside of one of the bases and in the DNA of the bacteria, but because mistakes, mutations, right. Nitrous acid is something that we are exposed to because every day, because this is what's produced as a result of fossil fuel burning,
it's in the few. And that causes mutation and the.
Because here's here's another example that is showing you that because this chemical normally it would be Gaspare by.
Within weeks, I mean, but it doesn't, so it's altered, but it's not in a way that it's not going to behave the same way. So we're looking at the normal case and we're looking at altered because adenine is not, you know, this not by good timing, but it's to the cytosine that mistake is going to carry over to replication and then transcription and then faulty product. That's how it kills those bacteria.
And this is just another example, but. Simply, this is an analog that could be used this is actually used for cancer treatment. If I'm not mistaken, yeah, anti cancer. And that's just because it represents I mean, under-represent resembles a thymine nucleoside, but it doesn't behave like thymine, meaning that it's not going to add is not going to be complementary to everything. And that's how it creates mistakes in the cancer cells and killing them. All right. Other other mutagens. In addition to analogs radiation, there are different kinds of radiation, ionizing radiation or X-rays or gamma rays that are called ionizing because they actually produce ions and free radicals, which free radicals
physically break the backbone of DNA. So you're looking at chromosomes. Broken in different parts of also that cause base substitution mutation. And. Whereas ultraviolet radiation is a non-ionizing radiation and what it does is that it causes the anywhere that there are adjacent primitives, mostly when their time means it will cause them to. But to bind one another is to the binding adoni on the opposite strength and so is responsible for what's called thymine dimmers and bacteria. And similarly, we also have repair mechanisms, but they have repair mechanisms. Also, one repair mechanisms called light repair mechanism, which is in the presence of light. There are enzymes that are activated, are fertilisers, and what fertilisers do is that they break the bonds between the adjacent timings. And so therefore time is go back to binding the the other on the opposite side. But there is also one that is called excision repair mechanism and in this case, extensive repair mechanism, what's happening is that the enzyme. And the nucleus cuts the DNA and then an external nucleus removes the damaged portion, which is double damaged portion DNA polymerase, then, you know, brings new nucleotide subunits instead of some. They're based on base theory. And DNA like this will seal the backbone of sugar and phosphate. But there is a this is also a genetic disorder called xeroderma pigmentosa, where
those babies are born with no repair mechanism. We are exposed to UV all the time and we do have repair mechanism, but they don't have a repair mechanism. So therefore they're susceptible to develop skin cancer very early in their lifetime.
And that's what that case is about. All right. How do we identify carcinogens, carcinogens, carcinogens or anything that causes anything that causes cancer is a carcinogen. We order this test to Dr. Bruce Abes. Of UC Berkeley, and there are EAMS kits that you can actually purchase in order to test a substance to see if it's a mutagen, if it's a mutagen, more than 90 percent of the mutagens are also. Are proven to be carcinogens. So what this test is about is this, that in his test?
He's assuming that once you mutate a bacterium.
If you have a mutated bacteria and then you mated, it mutated again, it will go back to its pre mutated state. So here's here's how it's happening. So you have a suspected mutagen say this is coffee. And you're adding you can purchase actually his in negative salmonella, hisss independent salmonella, but it means that this is a submarine that is has undergone mutation, therefore it cannot make his Sedin. Samila, normal son in law, also called wild type, they can make history, but once they're mutated, they cannot make history. Therefore, they would die unless you put history in the media. That's why they called up his independent. So you have a history of negative bacteria in here, you're adding your suspect mutagen, you're adding rathgeber extract to to mimic what really happens. For example. Say, in our body, when we consume something, they are going to be exposed in our GeoEye digestive system. They're going to be exposed to our liver enzymes. So here's the difference. And so this medium does not have any history in it.
Lacking history doesn't have any meaning, so. If you put your battery didn't wasn't able to make his city, so that means they should be able to grow in here. Right. But after intubation, if you see all this growth. Then that means that whatever substance there was in that sample, which, you know, like coffee, I said it, that substance has caused another mutation in the salmonella. And so they all reverted back to being his Sedin positive. So meaning now they can make history. So even though the back the media doesn't have any, but because they can make their own history and they are growing nicely here. So that means they're back to being something positive. So that means that that substance was a mutagen. My example was coffee and actually coffee is a mutagen.
Now, control is when you don't have your sample, the suspected mutagen. You only have your resident negative the would extract you put it on the native plate after incubation. There is nothing you find one or two colonies. That's a spontaneous mutation. There are still undergoing mutation. That's what they're growing in is the negative point. But that must be just spontaneously happening. So in our lab also, we do test for we will do this. There are two ways to look for Mutantes bacteria that have undergone mutation. Those are also called also Trott's, as opposed to a wild type. There are two ways that can be done. One is called positive or direct selection. Positive of the next election would be, for example.
For example, you have intermixture, you have some bacteria that are resistant to penicillin. Resistant to penicillin in a mixture, not all of them with some resistance. So you want to you want to isolate those and meaning you're looking for them. You're looking for those mutants or groups that are resistant to penicillin. So what you do is that you're going to get a plate in it. You have penicillin and then you culture your mixture onto that plate and and then anything that would grow on the plate. Obviously, they are the ones that are resistant to penicillin. Otherwise they wouldn't be growing their. So that's how you are directly. Was finding them and growing them. OK, so that's one, but then there's also a negative selection or indirect selection, also known as replicability. That's also another method for finding Mutant's or the so that this is the one we're going to be doing in the lab. So going back to this was about massively with new content. You know, this is about. Here's here's a plate. Which has a medium that has history in it. There is a medium that has this attitude, and you have in our case, we're going to use a wooden block that has a velvet on the surface that is going to be so nice. This is going to be pressed on the colonies that are growing in the plate in this plate, by the way, that you don't have to. I mean, you do have to use a plate. There is history in here in your mixture that you grow on the plate. That mixture contained both. So the native and his deposit back to you. So it had both Mutantes and the wild type. So after growing up here, you don't know which is which, right, you want to look, you look for your rocks and the plate has already instituted. So after incubation, then you use this velvet and you're just going to stop the surface and by stabbing, you're going to transfer these colonies onto the surface of this vervet and notice that their reference marks on both of these plates and also right here on this vervet. How are these plates different? One is just like that one. It has something in it. This one doesn't have history. So then you're transferring these colonies onto these two plates. So you're going to stop this and then a start up and then incubate again after a period of incubation. You'll have history in here. So all types were growing here, right? The ones that are his negative meaning, they can't make it. They are going to grow here because you've provided this in here. And there was I can make history in the wild type. They are also able to grow here. And however, this plate that there is no history, only the mutant, only the ones that. Are able to make his city can grow here. So then you're going to superimpose this plate on top of this and look for colonies that are present here but absent here. So if you look at all of these, they're here also. But this is only one that does not exist here. So that means that most of the of the truth or the mutant. Back to you. Hey, so you go back to the split, if you were to take that and then do further experiments, that's what you would have done.
All right. Bacteria are not only a production, you know, binary decision dividing into are, then the progeny will have the exact same genetic information as the one parent or cell that's called vertical transfer of genes. But bacteria also, I should have done I should have made this horizontal and vertical that should be horizontal genetic transfer in bacteria. They also undergo horizontal. Gene transfer in bacterial.
That was first discovered by Frederick Griffith in 1928. So here's what he did. He had he inoculated the mouse with encapsulated streptococcus pneumoniae and the mouse obviously died of pneumonia over the bacteria. You're looking up in the microscope. They all had capsule. Then he got some non encapsulated sort of streptococcus pneumonia with no capsules, they didn't have capsules. So they he not really the mouse. The mouse did not die because did not contract under the plate. When he grew, the he grew the bacteria that were isolated from the mouse. They did not have capsules. And that's to be expected. Then he.
Killed those Bisio that have capsule's and then inoculated the mouse and obviously the mouse survived because the the those were the right to where did. And then he got alive non encapsulated live bacteria that did not have capsule, he mixed them in, mix them with dead encapsulated bacteria and injected both into together to into a mass. And the mouse died of pneumonia.
And then he hit. He cultured the bacteria isolated from the mouse into a plate. He observed that they all had capsule. They all had jobs. So here's these are life, but don't have capsule's, these have capsule's, but they're dead. So these by themselves should not cause pneumonia and the encapsulated ones when their deaths should not cause pneumonia, but the mixture. Caused the mass to develop pneumonia and die from it. Not only that, all the progeny. Or that were isolated from the mass, they all had capsules, so meaning that. The life bacteria that did not have capsules, they were able to become tabulated, how did does happen? Right. How did that happen? That was his experiment. And again, it should the previous slide should be horizontal gene transfer like here, gene transfer between bacteria can also be transferred. Genes can be transferred from one bacterium to another one within the same generation. That's called horizontal gene transfer. So. One is transformation, which is what happening in here. So what is transformation? Transformation is when is when a bacterium can pick up what's called naked DNA, what is it called naked DNA? Because it's not in the bacterium anymore. You know, also by Theodores, one bacterium dies. The genetic information, the DNA. Fragments are in the media and some bacteria are not all in nature, they can actually pick up those fragments from media. That's what they're called naked because they're not in the cell. And when they pick them up, then those genes are become part of the DNA of the bacteria. And now that Buffalo has transformed whatever those genes were, their products will be produced just like any other gene that this bacterium has. So in this case, the gene was for making capsule. These bacteria were dead, so naked DNA were picked up by streptococcus streptococcus, by the way, is a bacterium that does in nature, naturally, it's capable of picking up naked DNA from the environment and become transformed. That's called competency. So they are competent. And then and then so all their progeny had the same genes now, and that's why all the progeny had the genes would make capsule. That's that's called transformation in it, again, in the real life equality, which is the microbiology pet used in all experiments, naturally cannot be it doesn't have that capability to not pick up naked DNA, but we can make them competent by using calcium chloride.
Chemical and then after mixing them together, a multi treatment market shock. All right, so next is another way that bacteria can get new genes from another body in the same direction is what conjugation that we've already talked about. And that's this one. Look at conjugation. You've seen this slide before. We're through Pillar's bacteria can exchange genes. And so in Grandpaw gram, negative bacteria make these Peli Peli is proto gram positive. Bacteria don't make polite, but they make a sticky surface molecule sify sort of a substance that through that they exchange genes. Contact is needed. Right, for for this to happen. So the donor cell is called a plus cell and the one that doesn't have that particular plasmid that's called F minus cell. So you're looking at. And the plasma that is being.
To answer to the other one that's called effectors, so that's what you're seeing happening here.
And then so these are plasmids. So if you remember plasmids or extra chromosome of DNA that contain genes like a few hundred genes and some of the genes for resistance to bacteria, to antibiotics, you're actually on the plasmid. So that's conjugation. There are different kinds of plasmids, so some are for those antibiotic resistance, drug resistance, some are for producing toxin.
Some are called conjugating plasmids, and so that's conjugation and then transduction is another way of transfer of genes horizontally among bacteria, and that's why that death transfer is through viruses. Viruses that infect bacteria are called bacteriophage or phage and noticed that when a phage effects a bacterium, it introduces its nucleic acid into the bacterium. Notice that nucleic acid in pieces is going to
be during.
I'm not going to talk about the details in between and it's not mentioned here either because in Chapter 13, we don't talk about it more. The chapter is about viruses. But just I just mentioned that once they inject their nucleic acid into the bacterium, the bacterial DNA itself is also broken into pieces. So when
the virus genes are forcing the bacteria to make vital products, what components? When the viral components are put together, some of these protein coats of viruses, as you can see, they're carrying bacterial genes. So instead of, you know, the original VIAJES, so during the packaging of oral components together, some of these viruses will have these in there instead of packaging, they're so after they're released from this bacterium, then they're going to infect another bacteria. And so injecting it's nucleic acid into the second bacterium. I notice that this is really a serious a gene that belonged to this bacteria. So the new bacterium did not have that gene, but now it does. And some new properties, it's conferring new properties. This is called general or generalized transduction. We also have specialized transduction, but we'll talk about that in the Interruptive 13. So this is showing you the transfer of genes from one bacterium to another, one by viruses, so that's called transduction. And then also there's something else called Transposon Transposons, or they used to be called jumping genes, meaning these are genes that the removeable there's sort of sickness or DNA. Right. But they there is a piece of it that called for for an enzyme that enzymes called Transposes Transposes says that there are different ones, but then Transposes says enzyme will. It has make it all make that transposon to court itself, detach itself from that piece from where it is in the DNA and. Attach itself into another even Crosio another day and another also. Or on the same chromosome, but at a different site that's called that's called that's what a transposon is able to do. This was discovered by Barbara McClintock
and Transposon is about 700 to 40000 base pairs. Long transposons, therefore, because they can also jump from the chromosome onto plasmids. As you know, plasmids can be transferred from another bite from one bacterium to another. Therefore, you can see that this is another way of genes to be transferred from one person to another. Right. And this is actually exactly how Staphylococcus aureus became resistant to vancomycin Staphylococcus aureus today. It's resistant, does not respond to vancomycin. And that's because it got this resistant genes. It was a transposon on a transport that came from originated from Enterococcus Bechler's, which is. No more microbiota in order to attract. I should say, Colin. So so that's another way that genes can be transferred from one back to into another one. That was the last one, you guys. We'll talk to you later, bye.
Chapter eight, Microbial Genetics.
Terminology
Genetics: The science of heredity focusing on what genes are, how they are transmitted, and their molecular products.
Genes: Segments of DNA that code for proteins or functional RNA.
DNA: A nucleic acid made of nucleotide subunits comprising nitrogenous bases, phosphate, and sugar.
Nitrogenous Bases in DNA: Adenine (A), Thymine (T), Cytosine (C), and Guanine (G).
Chromosomes: Physical structures that contain DNA.
Genome: The complete set of genes in an organism, including plasmids.
Genomics: The study of genomes.
Genotype: The genetic makeup of an organism.
Phenotype: The observable expression of the genotype.
DNA Structure
Double-stranded DNA appears like a twisted ladder.
Backbone: Comprised of sugar-phosphate.
The nitrogenous bases are bonded in the middle.
Bacterial DNA typically has a circular chromosome, while eukaryotic DNA is linear.
Discovery of DNA
1869: DNA's existence recognized by Johann Friedrich Miescher, who discovered it in the nucleus.
1950s: James Watson and Francis Crick proposed the double helix model of DNA based on Rosalind Franklin's X-ray diffraction images.
Human Genome Project
Began in 1990; completed ahead of schedule in 2003.
A multinational effort aimed to sequence the entire human genome, revealing over three billion bases.
DNA Replication
Crucial before cell division; involves several enzymes:
Topoisomerase: Relaxes the DNA coil.
DNA helicase: Separates the DNA strands.
DNA polymerase: Synthesizes new DNA strands based on the template strand.
DNA ligase: Joins Okazaki fragments on the lagging strand.
DNA replication is semi-conservative, meaning each new DNA molecule has one original and one new strand.
Transcription
The process of copying genetic information from DNA to RNA.
RNA polymerase synthesizes messenger RNA (mRNA) using one strand of DNA as a template, starting at the promoter and ending at the terminator.
Translation
The process by which ribosomes use mRNA to synthesize proteins.
Transfer RNA (tRNA) brings specific amino acids to ribosomes based on codons.
The genetic code is parsed in triplets (codons), which correspond to specific amino acids.
Gene Regulation
Bacterial gene expression can be constitutive (always on) or regulated (turned on/off as needed).
Operon Model: Describes how genes are coordinated in bacteria.
Promoter: Sequence where RNA polymerase binds to initiate transcription.
Operator: Regulatory region that can block transcription.
Structural Genes: Genes coding for proteins.
Types of operons:
Repressible Operons: Typically on; can be turned off by a corepressor (e.g., trp operon for tryptophan synthesis).
Inducible Operons: Typically off; can be turned on by an inducer (e.g., lac operon for lactose metabolism).
Mutations
Changes in DNA sequences that can affect protein function.
Base Substitution: One base is replaced by another.
Can be missense (changes one amino acid) or nonsense (creates a stop codon).
Frame Shift Mutation: Addition or deletion of bases alters the reading frame, affecting all downstream amino acids.
Mutagens: Environmental agents that increase mutation rates.
Horizontal Gene Transfer
Bacteria can exchange genes via transformation, conjugation, or transduction:
Transformation: Uptake of naked DNA from the environment.
Conjugation: Transfer of genetic material via direct contact.
Transduction: Gene transfer via bacteriophages.
This structured overview provides detailed insights into microbial genetics, focusing on definitions, processes, and key discoveries continuously throughout the chapter.