Chapter 7

Question 1

Structure of Nucleic Acids:

Nucleotide Components

Answer 1

Sugar - Either ribose or deoxyribose (in RNA or DNA)
Nitrogenous base - Adenine, guanine, cytosine, thymine (only in DNA), uracil (only in RNA)
Phosphates - One to three, two of which are usually hydrolyzed to provide the energy to attach the nucleotide and form the phosphodiester bond

Question 2

What are the 4 possible bases that we see in DNA nucleotides

Answer 2

Thymine, adenine, guanine, and cytosine.

Question 3

Structure of Nucleic Acids:

Phosphate Sugar Backbone

Answer 3

Ribose in RNA and Deoxyribose in DNA

Question 4

DNA vs. RNA: Structural and Functional Differences

DNA

Answer 4

Double Stranded
Has one of five bases
Has the information to make the gene
Is the original blueprint

Question 5

DNA vs. RNA: Structural and Functional Differences

RNA

Answer 5

Single Stranded
Doesn’t have Thymines as a possible base, has Uracils
Is a copy of the DNA
Is the working blueprint

Question 6

The Structure of the Prokaryotic Genomes:

Prokaryotic Chromosomes

Answer 6

Consist of a circular molecule of DNA localized in a region of the cytoplasm called the nucleoid. The DNA of the circular chromosome is actually folded and compartmentalized with those little proteins.

Question 7

The Structure of the Prokaryotic Genomes:

Plasmids

Answer 7

In addition to chromosomes, many prokaryotic cells contain one or more plasmids, which are small molecules of DNA that replicate independently of the chromosome. Usually circular and 1-5% of the size of a prokaryotic chromosome.

Question 8

The Structure of the Prokaryotic Genomes:

Plasmids: Function

Answer 8

Each plasmid carries information required for its own replication, and often for one or more cellular traits.

Question 9

The Structure of the Prokaryotic Genomes:

Plasmids: Types of Genes

Answer 9

Typically, genes carried on plasmids are not essential for normal metabolism, for growth, or for cellular reproduction bur can confer advantages to the cells that carry them.
Fertility Plasmids: Carry instructions for conjugation, a process involved in transferring genes from one bacterial cell to another.
Resistance Plasmids: Carry genes for resistance to one or more antimicrobial drugs or heavy metals.
Bacteriocin Plasmids: Carry genes for proteinaceous toxins called bacteriocins, which kill bacterial cells of the same or similar species that lack the plasmid. In this way a bacterium containing this plasmid can kill its competitors.
Virulence Plasmids: Carry instructions for structures, enzymes, or toxins that enable a bacterium to become pathogenic.

Question 10

Regulation of Gene Function:

Control of Transcription

Answer 10

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Question 11

Regulation of Gene Function:

Prokaryotic Operons

Promotor

Answer 11

Remember, the promoter is the place where the RNA polymerase comes in and binds onto the DNA so they can start transcription.

Question 12

Regulation of Gene Function:

Prokaryotic Operons

Operator

Answer 12

There is also another stretch of DNA….notice this stretch is between the promoter and the structural genes. This structure of DNA is called the operator. The operator is the part where the regulatory protein, which in lab is that araC protein, it is where that regulatory protein is going to come in and attach on and that is going to keep RNA polymerase from being able to move down the tracks and transcribe the structural genes.

Question 13

Regulation of Gene Function:

Prokaryotic Operons

Structural Genes

Answer 13

The genes are what we are referring to here as structural genes….first, second, third, fourth….depends on what operon it is as to how many genes you are going to see on there. These genes encode for proteins that are related to a common function. Remember in the arabinose operon, these were the genes that encoded for the proteins that are needed to break down arabinose. Same idea here.

Question 14

Functional Classes of Operons

Inducible Operons

Answer 14

It can be turned on, which tells you that it is normally turned off.

Question 15

Functional Classes of Operons

Inducible Operons

Lactose (Lac) Operon

Answer 15

This being the lactose operon, these make the proteins, the enzymes that break down lactose. We don’t need them on so long as we have a plentiful supply of glucose in the cell. So let’s say the cell runs out of glucose, but let’s say there is some lactose around, so the cell wants to be able to use the lactose and get ATP from it and break it apart. On this little operator, there is a little spot that I didn’t draw before. So all of a sudden there is a bunch of lactose around and no glucose. Lactose can come in and it can bind to the bottom of the regulatory protein. When it binds on the bottom of the regulatory protein, the regulatory protein changes its shape. The regulatory protein closes off. It can’t fit over the operator. Since it can’t fit there and attach, it can no longer block this RNA polymerase, and so now RNA polymerase starts to move down the piece of DNA, and it starts reading these genes, and it starts transcribing them into mRNA. The cell takes the mRNA and translates that into proteins; specifically into enzymes that help us to break apart and metabolize lactose. Then what we have…we have all these enzymes running around now. The enzymes are taking the lactose, and they are digesting it, breaking it apart so that the cell can make ATP from it. Even the lactose attached to the regulatory protein…it gets pulled off and it gets broken down as well. Once those enzymes do their job, there is no more lactose around. Do we want to keep making those enzymes? We have no need for them anymore. What happens is when that lactose gets pulled off the regulatory protein, it opens back up like it was in the first place, and that allows it to bind back on to the operator, and that stops transcription and translation of these genes from going any further.

Question 16

Functional Classes of Operons

Repressible Operons

Tryptophan Operon

Answer 16

If you understand the lactose operon and how it is an inducible operon, then the tryptophan operon is really easy because it just kind of works everything sort of oppositely.

The basic structure is exactly the same. This is the tryp operon. The regulatory gene is being constantly transcribed, just like over there. We produce mRNA. That mRNA is constantly being translated and produced. Our repressor protein. When I say regulatory protein, it is also referred to as a repressor protein. Here’s the difference. In this regulatory gene, when it is transcribed and translated, here’s the repressor protein…this is the normal shape of it before you do anything to it (closed). In this position, what can happen? Nothing happens…no action. When it is closed up like that, it can’t on the operator. If it can’t bind on the operator, then RNA polymerase…there is nothing to block it, so it moves down and starts to transcribe our structural genes. Then the structural genes get translated into protein. Here’s the deal, though. These proteins that are made from the structural genes…their job is actually to help the cell synthesize the amino acid tryptophan. These proteins help make tryptophan. Tryptophan is one of those amino acids that—like all the other 20 amino acids that are really important. The cell has to have a constant supply of these to be able to make protein to sustain itself. This cell is basically constantly reading these genes, constantly churning out these proteins which are synthesizing tryptophan for it. The cell wants to always make tryptophan all the time…unless, somebody gives it a turkey sandwich. Unless it gets a big supply of tryptophan from the environment.

Question 17

Gene Mutation:
Types of Mutations

Point Mutation

Answer 17

. A point mutation is when you change just one nucleotide or 1 base of a DNA sequence. Point mutations include base pair …substitutions, insertions, and deletions.

Question 18

Gene Mutation:
Types of Mutations
Point Mutation

Insertion

Answer 18

Insertion or deletion of a letter. Insertions and deletions are also called frameshift mutations because nucleotide triplets following the mutation are displaced, creating new sequences of codons that result in vastly altered polypeptide sequences. Frameshift mutations affect proteins much more seriously than mere substitutions because a frame shift affects all codons subsequent to the mutation.

Question 19

Gene Mutation:
Types of Mutations
Point Mutation

Deletion

Answer 19

same as insertion

Question 20

Gene Mutation:
Types of Mutations
Point Mutation

Substitutions

Answer 20

The changing of a letter… does not change the sequence of codons but can still change the meaning.

Question 21

Effects of Mutations
Point Mutation

Silent Mutation

Answer 21

All the other amino acids are unchanged after that because we only affected that one base, so we still the other original amino acids. So it is a silent mutation: the protein is the same…we don’t see any effect from in how the protein functions. The only way you would tell there was a mutation is if you went in and sequenced the DNA.

Question 22

Effects of Mutations
Point Mutation

Missense

Answer 22

This missense mutation means that the protein is different at one amino acid position. Whether or not this is going to affect the overall function of the protein….that is still in question because let’s say that the original amino acid that should have been there was alanine but instead it got mutated and now we have glycine. Glycine and alanine are actually very similar, structurally they are very similar; their chemical properties are very similar. So this may not actually impact too severely the overall function of the protein.

Question 23

Effects of Mutations
Point Mutation

Nonsense

Answer 23

Here’s another example of this. This is what is called a nonsense mutation. We are going to mutate. We are going to change this base right here, this nucleotide, so that the mRNA has an A there. If you read this, UUU, phenylanine…we’re good there. Now we need to read the next one. UAA. That is changed from what it was before. When you read that, instead of reading, some of the ribosomes saying, we should put tyrosine there, the ribosome thinks that that means stop. Remember how we had the stop codons like UAA and UGA…do you remember all that from biology? Now, the ribosome says, oh, that’s where we stop, and so it stops right there, and it will not read anything further down, which means our protein is now truncated, it is now shorter than it should have been, losing all the stuff afterward. If you take your protein chain and you chop off the end of it…is that going to affect the function of it? Yes, probably. Especially the further up your stopping it, the greater the effect it is going to have on that protein. If it is the very last amino acid…maybe it will change it and mess it up…maybe it won’t do that. If it is right at the beginning…you have a protein that has 1 amino acid in it and there is nothing else after that and that is really bad.

Question 24

Effects of Mutations
Frameshift Mutations

Insertions, Deletions, Effects:

Answer 24

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Question 25

Causes of Mutations: Mutagens
Radiation
Ionizing

Answer 25

Ionizing radiating is the strongest kind of radiation. It has the most energy in it. I want you to imagine a strand of DNA. Remember how DNA has a phosphate, a sugar, another phosphate, and a sugar. Remember it has that sugar-phosphate backbone and it has the bases that come out. Ionizing radiation includes things like gamma radiation, x-rays, and that radiation is so strong that it can come in and it hits the bonds between phosphates and sugars and knocks the electrons out of those bonds. When the electrons get knocked out of the bonds, the bonds break. What we end up doing is basically chopping our DNA up into pieces, just obliterating the DNA when we expose it to ionizing radiation.

Question 26

Causes of Mutations: Mutagens
Radiation
Non-Ionizing

Answer 26

This is UV light. People talk about UV light and limiting your exposure to UV light. This is the reason. UV light, nonionizing radiation, doesn’t have enough energy to go in and break the bonds in the sugar-phosphate backbone. So it can’t chop your DNA up into pieces. What nonionizing radiation—UV light—does is if say you have a strand of DNA. This strand has 2 T’s that are next to each other. This light is enough to take the two T’s and get them to chemically react with each other so that they actually form covalent bonds with each other. When these 2 T’s are forming covalent bonds with each other, they can’t base pair with A’s on the complementary strand. The result of that is that you get this little kink in your DNA.

Question 27

Nucleotide Analogs:

Answer 27

Are compounds that are structurally similar to normal nucleotides. When nucleotide analogs are available to replicating cells, they may be incorporated into DNA in a place of normal nucleotides, where their structural differences either inhibit nucleic acid polymerases or result in mismatched base pairing.

Question 28

Nucleotide Altering Chemicals:

Answer 28

Some chemical mutagens alter the structure of nucleotides. ex: A group of nucleotide-altering chemicals, called aflatoxins, are produced by Aspergillus molds growing on grains and nuts. Aflatoxins catabolized in the liver can convert guanine nucleotides into thymine nucleotides, so that a GC base pair is converted to a TA base pair, resulting in missense mutations and possibly cancer.

Question 29

Frame-shifting chemicals:

Answer 29

Other mutagenic chemical agents insert or delete nucleotide base pairs, resulting in frameshift mutations. Ex: Benzopyrene, which is found in smoke, ethidium bromide, which is used to stain DNA and acridine, one of a class of dyes commonly used as mutagens in genetic research.

Question 30

Repairing Mutations

Repair of Thymine-dimers:

Answer 30

The first one is what we call light repair. Interestingly enough, it’s the UV light that triggers the formation of thymine dimer in the first place. Light repair uses an enzyme, photolyase, which is actually activated by exposure to UV light. This enzyme can go it, and it can take a thymine dimer, and it can break the covalent bond between the thymines, so that those thymines can base pair correctly with their complementary strand. This only works, though, in the presence of light when that photolyase enzyme can be activated.

Question 31

Repairing Mutations

Base-Excision Repair:

Answer 31

. Base excision kind of works like the dark repair that we just saw for the thymine dimers. So what happens in base excision. You have an incorrect base pair in a DNA strand. So we need to take that out. A base excision will go in and will cut out the nucleotide but it also might take a little bit of what’s on the either side of it. So cut that out, and then we have to go back and fill in with DNA polymerase. So it is just like dark repair, except it is not for thymine dimers.

Question 32

Other Repair Mechanisms

Mismatch repair:

Answer 32

Then we could use a different type of repair mechanism called mismatch repair, and this enzyme is much more specific in how it cuts out mismatched bases. It can come in and it can take out only that one little base and fill in with the correct base. The advantage to that is that we don’t have to cut out a big piece of the DNA, and we don’t have to synthesize extra nucleotides…there’s less chance for errors. A less complicated, better way to go. It is just that this only works when the DNA is newly synthesized and fresh and young.

Question 33

Other Repair Mechanisms

SOS Response:

Answer 33

SOS repair is not a specific kind of enzyme or specific technique. SOS repair is sort of a general name for a bunch of different potential processes that can be activated when DNA damage is really severe. That’s a fancy way of saying…. Let’s say you have big, long chromosome. We’re not talking about 1 or 2 bases…we’re talking about this big chromosome. Let’s say that a chunk of it is wiped out. That is potentially really bad. The loss of ____ in your chromosome. Chances are this cell is going to die. But to sort of give it one last gasp, one last shot at it….there are these SOS repair processes that can come and say, well, we lost this but let’s just try to fill it in as best we can. It doesn’t necessarily know everything that missing there. Sometimes there is no complementary strand to go by. It just fills in randomly.

Question 34

Finding the Mutants

Positive Selection:

Answer 34

The first way that I am going to show you is what is called positive selection. Positive selection is used to find cells that have a particular mutation that confers additional trait or ability on that mutated cell in a big population. I’ll rephrase it in a different way. For those of you who are into superhero movies, like the X-men movies…. Do you remember that Dr. Xavier had this machine called Cerebro. He goes into it, and it has this thing that goes on his head because he’s psychic or he has mental abilities. With this machine, he is able to look at all the living people in the whole world and pinpoint which ones are mutants. That’s what this is. That’s positive selection. Let’s say you have this culture—which represents the world in a culture tube—and all these little cells represent all the different people. Now within that population of cells, just like in the population of the people at large, the cells are going to be different. Even if we are all the same species, we still different in lots of different ways. So somewhere in this population there is going to be a cell in there that has a special mutation that gives it the ability to do something that the other cells can’t. Maybe the ability to be really smart, or the ability to fly, or the ability to make ice come out of your hand or whatever all the X-men superpowers are. In this case, it is going to give you the ability to be resistant to a certain antibiotic, like penicillin.

How do we find that one cell, wherever it is? Grow it on penicillin. Penicillin will kill all the other cells. The cells that have the mutation that allow them to resist the penicillin will survive. So yes, that is exactly what we do. We make a medium—just like we did for the pGLO lab. It has the antibiotic built in to the agar, and we grow that population of cells on their, and we can grow some on agar that doesn’t have any penicillin, and we can look at the difference between the 2 of them. We see that one growing there…that’s our 1 mutant out of that big population that has that naturally occurring resistance to penicillin. Then what we can do is now take that, pick it off there, and inoculate a fresh culture, and now we will have a pure culture of antibiotic-resistant cells. All of them will have antibiotic resistance. That’s how positive selection works.

Question 35

Finding the Mutants

Calculating Mutation Rate:

Answer 35

There is a whole formula that we can use to calculate. The formula says you take the number of colonies that you count with the mutagen added minus the number of colonies without the mutagen divided by the number of original colonies times 100%.

[# of colonies with mutagen - # of colonies without mutagen]
------------------------------------------------------------------------------	X 100%
		# of colonies without mutagen

So the mutagen induces mutations at a rate of X% greater than normal.

Question 36

Finding the Mutants

Negative (Indirect) Selection:

Answer 36

This is the way it works. Here’s your culture. In this culture, they are adding a mutagen in there to create an auxotroph. Add the mutagen, take the cells, and you incubate them on—in this case, it is a medium that contain tryptophan. Which cells are going to grow…which cells are not going to grow? Shouldn’t all of them grow? Because the agar medium has tryptophan built into it, so both wild-type cells will grow as well as tryptophan auxotrophs. Here’s what you do then. Once you have this grown out and you have those little colonies on here. Just by looking at them, you have no way of knowing if that is deficient in making tryptophan or it that one is…which one is which. What you do is you take your plate, make a little mark on the side of the plate for orientation or reference. Then you take a piece of sterile velvet. The velvet is stretched out around a dish or something that is the exact size of the inside of your Petri plate. Then you take the velvet, and then you make a little mark on the side of it…and you bring it and you set it down so the velvet is right on top of your colonies, so that the marks on the plate and velvet are lined up together. While that is happening, those little cells, colonies get stuck to the velvet and then you pull the velvet up, and it has bacteria on it in the same pattern that was on this plate. Then you take 2 sterile plates, put a little mark on each one. One of these plates has tryptophan in it. One of them doesn’t have tryptophan. You take your velvet and you stamp onto both plates in the exact same position, and then you incubate both of the plates. When you grow this up, and you look at the 2 plates, you expect everything to grow on the one plate. On the other you expect cells that are wild-type to grow but not the auxotroph. So you compare the pattern of the colonies, and you look at this and say, “This one is missing a colony right here that that one has.” Why is here but not over there? Because it is an auxotroph, and it can’t make its own tryptophan. It can’t grow on media without tryptophan. Then you say, all right, that’s my mutant right there. You take it in your loop, and you inoculate it another media and then you have a pure culture of your auxotroph.

Question 37

Finding the Mutants

Ames Test for Identifying Carcinogens
-uses Salmonella his (histidine auxotrophs)

Answer 37

The way that the Ames test works. It is a test to determine if a chemical is likely to cause cancer in human. What it does is instead of using human as test subject—which wouldn’t be very ethical—we are going to use bacteria because their DNA functions just like our DNA. Their DNA mutates just the same as our DNA mutates. The way that this works is we actually use salmonella bacteria, but it is a special strain Salmonella, and it is an auxotroph, which means it can’t make a certain nutrient. This is a histidine- auxotroph. Histidine is one of the amino acids that cells need to make. So this type of Salmonella can’t make its own histidine. Here’s the way that they do this test. They take the culture tube that has a bunch of this Salmonella ___, and then they dump in a bunch of the chemical that they are trying to test to see if it is a carcinogen. Then they also take some liver extract. Liver extract….they usually take liver cells from a rat and grind them up in a blender and dump it in. The reason for this is having a liver extract in there simulates conditions inside your body because a lot of the toxins, the mutagens and chemicals that flow through your body end up going through your liver, and your liver processes them. Your liver is trying to detoxify and make things less harmful to your body, but sometimes the liver in that detoxification process…sometimes it actually does the opposite. Sometimes it can take something that is not toxic or that is not carcinogenic, and as it tries to manipulate it and change it around a little bit, it may turn it into something more dangerous. We need to account for that. That’s why we are adding the liver extract. It has all the enzymes and things that are normally present inside liver cells. Mix everything together, and then grow these cells on a plate that doesn’t have histidine. Normally, if we took this culture grown on this plate, we wouldn’t expect to see any growth, or very little growth. We look at this plate and all of a sudden there is a bunch of growth…what does that mean? This organism that couldn’t make histidine is all of a sudden is making histidine. Otherwise, it wouldn’t be able to grow. It has done what we call a reversion. It has reverted back to histidine+ and it has done that because of the mutagen that we added. The thing about mutagens is they can cause mutations and knock out abilities, but they could also mutate genes and reinstate them or cause them to become functional again. That is what is happening here. The mutagen is mutating this gene that was already knocked out and causing it become functional again by changing the base pairs. So we look at this and we say, wow, 5 colonies here. We do a control tube as well, and we don’t see 5 colonies on the control plate. What can we say about that chemical? That chemical was changing the genes of this organism and mutating it, so it is likely to be mutating other things. It is likely to cause cancer.

Question 38

Transfer and Recombination of Genes

Vertical Transfer:

Answer 38

Both prokaryotes and eukaryotes replicate their genomes and supply copies to their descendants. This is known as Vertical-Gene Transfer, the passing of genes to the next generation.

Question 39

Transfer and Recombination of Genes

Horizontal Transfer:

Answer 39

You’ve got this other bacteria over here and this other one over here…and it is sharing genes with those guys that aren’t even its offspring. This is an example of what we call horizontal gene transfer. Hopefully, the horizontal versus vertical part makes sense, because vertical is through generations…horizontal is within the same generation.

Question 40

Transfer and Recombination of Genes
Horizontal Transfer
Transformation:

Answer 40

A recipient cell takes up DNA from the environment, such as DNA that might be released by dead organisms. Fredrick Griffith discovered this process in 1928.

Question 41

Transfer and Recombination of Genes
Horizontal Transfer
Transduction:

Answer 41

Transduction is really cool because it uses intracellular parasites to carry the gene from one cell to another. So intracellular parasites is code for viruses. Bacteria, one of their biggest fears in life or their biggest challenges or their biggest obstacle is viruses that will infect them. Viruses that infect bacteria we call bacteriophages (or phages for short).

Question 42

Transfer and Recombination of Genes
Horizontal Transfer
Conjugation:

Answer 42

Unlike the typical donor cells in transformation and transduction, a donor cell in conjugation remains alive. Conjugation requires physical contact between donor and recipient cells. Conjugation is mediated by conjugation pili, also called sex pili, which are thin, proteinaceous tubes extending from the surface of a cell. The gene coding for conjugation pili is located on a plasmid called an F (fertility) Plasmid.

Question 43

Transfer and Recombination of Genes
Horizontal Transfer
Genetic Maps:

Answer 43

not on test!!!!!!

Question 44

Transfer and Recombination of Genes
Horizontal Transfer
Transposons:

Answer 44

Transposons are segments of DNA that transpose (move) themselves from one location in a DNA molecule to another location in the same or a different molecule. American geneticist Barbara McClintock discovered these “jumping genes” through a painstaking analysis of the colors of the kernels of corn. She discovered that the genes for kernel color were turned on and off by the insertion of transposons. Subsequent research has shown that transposons are found in many, if not all, prokaryotes and eukaryotes and in many viruses.

Question 45

Transposons

Structure:

Answer 45

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Question 46

Transposons

Function:

Answer 46

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Question 47

Transposons

Effect:

Answer 47

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