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Transcribed by Kevin Lin Lecture Date: July 22, 2014

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[General Pathology] [11] [Bacterial Genetics I] by [Dr. Boylan]

[1] [Bacterial Genetics lectures]
[Dr. Boylan] Okay well, as one of the faculty members of our department would
say, good morning or good afternoon or whatever time of the day it is, I dont know.
I guess its good afternoon. I guess I should mention his name. You had him last
year. But well talk today about bacterial genetics. We already talked about
cytology, bacteriophage, viruses, fungi. Going back down to bacteria, talk a little
about genetics. And one of the reasons, as well see more of this in the second hour
today, is that genetics or genes, especially gene transfer among bacteria, genes of
bacteria transfer amongst themselves over and over again. Just think of all the
billions of bacteria we have in our gut at this time. Living, growing, and then dying.
And when they die, they release their DNA, often picked up by other bacteria in the
gut. Or they have viruses. They infect them. Well see viruses go from one bacteria
to another and carry along with them genes, from the donor bacterium to the
recipient bacterium. And also bacteria in our gut and other places in the world,
conjugate, actually mate, pair and exchange genetic material, genes. And some of
these genes that are transferred among bacteria can lead to resistance to antibiotics.
Can lead to the emergence of new pathogenic strains of bacteria. So there really is
some importance in the real world about why were spending some time doing this.
Initially just the study of bacterial genetics is fairly simple, once again, compared to
eukaryotic genetics. So we learned of the simple fundamental facts about DNA
replication, transcription, translation, and protein synthesis in studying bacterial
genetics. So Ill review some of that, which youve already been exposed to in
Building Blocks, just to refresh your mind before we talk about gene transfer in
bacteria in the second hour.

[2] [Bacterial Genetics]
[Dr. Boylan] So we have bacterial genetics. Bacterial chromosomes, I mentioned,
about a thousand times longer than the bacterial cell itself. About a millimeter in
length, whereas a bacterial cell is only about one micrometer or one micron. Its
haploid. I mean one chromosome, double-helix, but its one chromosome DNA.
Circular. Genes in bacteria, from the smallest bacteria, you might have about 2,000
genes, to some larger bacteria, you have up to 6,000 or even more genes than that.
So large bacteria have large genome, small bacteria have smaller genome. And of
course the larger the bacteria, the larger the genome. The most genes they have, the
most properties they have or could express over a period of their lifetime.
Operon, as you know, is a group of genes that are coordinately expressed.
Okay. I thinkdo you have another example of this operon group of genes? All
bacteria, all of us our chromosomes, not all of them are turned on at the same time.
Transcribed by Kevin Lin Lecture Date: July 22, 2014

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Most of them actually are repressed. You dont have the gene active and
synthesizing the gene product. If you dont need the gene product, why actually use
that gene? So most genes in chromosomes of living cells are repressed, but those
that are not are activated, are active and lead to the production of gene products.
And the same thing in bacteria. The operon was actually discovered in bacteria. The
group of genes that regulate some particular function. A metabolic pathway, DNA
replication are often linked together, these genes. And an operon is repressed or
activated depending upon the need for that particular gene product or metabolic
pathway. The lac operon is one we know about, the genes for lactose metabolism,
and whether theyre on or off. If the bacteria are growing in the presence of lactose,
then they will turn on the genes for lactose utilization. If theyre growing in a broth,
medium, or rich medium even, without any lactose at all, they dont need to have
those genes for lactose metabolism, so they dont turn them on. They keep them
repressed. The example I tried to give this year, maybe I hope you have this one is,
for endospore production in bacteria, Bacillus and Clostridium. Those two genera of
bacteria, Gram positive. Bacillus being aerobic; Clostridium anaerobic. They always
have the genes for sporulation. And it might be 20 to 30 genes for sporulation, but
they dont turn them on. They have them repressed until they sense theyre running
out of nutrients in their environment, then they turn off their regular genes and turn
on the genes for sporulation, so they will survive. But they dont need them all the
time, so why activate them.
E. coli, I think it has when we talk about a lot has about 4,200 (roughly)
genes. Thats a lot of genes and gene products that can be formed from them. But
once again, most of them are repressed at any one particular time. Why use them if
you dont need them? Why waste energy?
Extrachromosal genes are found in plasmids and in bacteriophages as well.
Bacteriophages, bacterial viruses. We talked about how the temperate phages can
form the prophage. And the genes become a part of the bacterial chromosome.
Thats prophage. And some of those genes are involved with bacteriophage
replication, are turned off in the prophage stage. Other genes can be active. And
some of those genes as we saw can produce toxic products. Botulinum(?) toxin,
diptheria toxin. Those proteins are formed by genes in the bacteriophage and the
prophage. And well see also plasmids. Extra genes that are not found in the
bacterial chromosome. Extrachromosomal genetic elements, but can produce
have genes that give a lot of new properties to the bacteria that possess them.

[3] [DNA Replication]
[Dr. Boylan] Replication of the chromosome. Bidirectional. Starts at the origin and
proceeds in both directions. So bidirectional replication of the whole chromosome,
not just a stand, but the whole chromosome replicates bidirectionally. From 12
Transcribed by Kevin Lin Lecture Date: July 22, 2014

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oclock to 6 oclock, and then the terminus there tells the chromosome that it has
replicated completely. And then the two daughter chromosomes separate and go to
daughter cells. Here it is after about three oclock and 9 oclock. And here it is at
5oclock, 7. 8 oclock . its progressing from the origin to the terminus. Anybody
figure out the question about how bacteria can still survive if they can replicate and
double in number. If they can double in number in 20 minutes. A generation time of
20 minutes. One cell goes to two. Two to four. Every 20 minutes. But the
replication of the chromosome always takes 40 minutes. Whether or not the
bacteria that can double in 20 minutes or bacteria that double in 20 hours, like
tuberculosis bacteria in our body takes about a day for them. And Syphilis bacteria
also replicates very, very slowly. But beginning of replication of DNA, begins and
always takes 40 minutes. So why dont bacteria that double in 20 minutes run out of
chromosome? Somebody actually emailed me--
[student] If you go from both directions, does the time cut in half?
[Dr. Boylan] This takes 40 minutes. Even though you go both directions, it still
takes 40 minutes to go from here, 12oclock, to 6 oclock. 40 minutes.
[student answering question, couldnt hear him]
[Dr. Boylan] Exactly right. It doesnt take 40 minutes, but this chromosome here
maybe at this point, replicated this far. But at the origin, maybe a second round of
replication occurs before the first one is complete. So in other words, a daughter
cell, when they get their new chromosomes, it might already be half way replicated.
And sometimes, thats called multiple forks. So point being, once again, these
bacteria can initiate rounds of replication of DNA even before the first round is
complete. And sometimes it initiated, threeyoure gonna have at least three
roundsof replication. And thats how they do it. So they dont run out of
chromosome even when they double in 20 minutes. They replicate in 40 minutes,
but they have--. Theyre kind of born partially pregnant in a way. Half way
replication of their genome. Replication forks are the active replications going on.
And theyre supercoiled. In order for the DNA to fit inside the bacterial cell, it has to
be tightly coiled, supercoiled. Thats critical in order to fit inside the cytosol. And an
enzyme that does this, that helps our the supercoiling of the genome is this enzyme
here, DNA gyrase. And I guess its kind of--, it works in both ways. It helps-- this
DNA gyrase is an essential enzyme for bacteria to replicate its DNA, both in the
supercoiling of it, like a squeezed rubber band over and over again, squeezed, twist
twist twist rubber band. But then when the genome has to be replicated, it has to
separate the two strands, the DNA gyrase also plays a role in that. So supercoiling
and uncoiling of the chromosome during replication. Done by this particular
enzyme, DNA gyrase. And well see that thats another point of attack of a certain
antibiotic. A drug that inhibits the DNA gyrase, thereby inhibiting replication of
chromosome of bacteria, thereby causing it to die because it cant replicate its
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genome. So another point of attack. And its supercoiling and uncoiling also, maybe
more the latter actually. Another name for it is helicase, needed for replication.
There are no introns in the chromosome of bacteria. No inter-mini-segments, where
theres really just DNA thats not used for anything at all, as we have in our
chromosome, at least we kind of figure, almost bit of DNA is like an exon. It really is
needed for some gene activity, either gene protein production or replication or
transcription of RNA from DNA. So no real introns baggage(?) DNA as far as we
know in chromosome of bacteria. Here we see nucleoid. Remember no nuclear
membrane. And one other point I should mention about the replicon genome of
bacteria is that, there are no histones. If you go back and look at replication of DNA
in our cells, we talk about histones. Well, bacteria dont have histones. But just
think, heres a genome here, and all those phosphate bonds they have in the
backbone of the DNA have a strong negative charge. There must be something to
help them neutralize that negative charge in the cytoplasm. Or else everything else
would sort of burn up in the cytoplasm in a way. So what do you use if you dont
have histones, which is thats what the rule of them is in eukaryotic cell DNA
replication. What you have are what they call histone-like proteins. That have a
lot of amino groups in them, NH
3+
. So they have a positive charge, whereas the DNA
has a negative charge. So it helps to neutralize the charge of the DNA, so that they
can function in the cytoplasm. And I just have a strong negative charge that might
do a lot of damage to other constituents inside the cell.

[4] [Bacterial variation and Inheritance]
[Dr. Boylan] Bacterial variation and inheritance. Mutation. Well, were all here,
were all different because I think over the eons of time, mutations occurred in life
forms to lead to the tremendous number of species on the Earth today. And we
study what happens in DNA and replication of DNA and genes by looking at mutants.
But as mutation, of course, is a change in the DNA. Some change in the base pair of
the DNA. And since the bacteria have a half way chromosome, its much easier to
follow what the result of a mutation is in the chromosome of bacteria, than it is in a
eukaryotic cell because the function of a gene activity will be expressed or not in a
mutation almost right away. So consequences of mutation. Well as you know, some
mutations are non-sense mutations because basically if a mutation changes the
sequence of amino acids in a protein, thats a mutation that has had some effect. A
new amino acid in the protein. But as you know, because of triplet codon, there is
more than one codon for an amino acid. So some mutations, changes in DNA, wont
have any effect on the cell at all because of the triplet codon, same amino acid will be
put in place after a certain mutation. Other mutation may lead to the death of the
cell. Amino acid put in the wrong place, so the enzyme wont function and the cell
will die. Or sometimes mutations cause the bacteria to have new properties. Once
Transcribed by Kevin Lin Lecture Date: July 22, 2014

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again, often if there are virulence factors like capsule production or toxin production
result in mutation, that can happen a lot, too. So there are different outcomes of
mutations. And the genotype refers to all the genes of an organism. The phenotype
is the properties that we can visualize, the observable properties of a bacteria or any
organism. I mean, it is a large bacterium? And when it grows on a Petri plate, does
it form large colonies, red colonies, colonies that are nice and smooth, colonies that
have a irregular border outline to them or flack? All these different properties are
part of the phenotype. And we use these characteristics to help identify bacterium
in the lab, of course, in addition to biochemical tests. But sometimes the bacteria
can change their properties without a mutation. Thats the point I wanna make
here. Sometimes the environment itself can affect the phenotype of an organism,
while the genome stays exactly the same. An example I always give of the
environmental influence on the phenotype is this bacterium, Serratia marcescens.
Example of a variation. We know that when we grow this bacterium at about 20
degrees, roughly the temperature in the environment were in now, room
temperature, it grows on a Petri plate and forms a nice red pigment. The whole
colony you see this plate with all these colonies of bacteria and theyre all red. If you
then put the same bacterium on the same Petri plate. Put them in an incubator at 37
degrees, say body temperature, they form colonies that grow, but lose the red
pigment. They cannot produce the red pigment in different environmental
conditions, that is an elevated temperature. Drop it back down to 20 degrees, it will
produce the red pigment. So thats the change in the phenotype without a change in
the genotype. Why does that happen? Well probably when you raise the
temperature, some enzymes that helped produce that red pigment loses its
structure and cant be involved in the pigment production cant work that way.
When you drop it back down to 20 degrees, that same enzyme now regains its
function and plays a role in the red pigment production. Thats called a change in
the phenotype without a change in the genotype.

[5] [Mutations]
[Dr. Boylan] Mutations. Spontaneous mutations do occur. And thats why there
are so many different bacteria, mainly because of spontaneous mutations over the
years. Bacteria are ideal organisms to study genetics. We use mutagenic agents in
the lab. Many chemicals can be used to induce mutations in bacteria. In other
words, people devote their lives to these studies. To study bacteria, learn about
metabolic pathways, learn about how they synthesis their cell walls, cell membrane,
by studying mutants. Mutants are bacteria that are somehow defective in these
processes. You dont learn how anything works from A to B to C to D to E unless
you have mutants, bacteria that dont carry out that process. They dont carry out A
to B to C to D to, say F. Something is wrong. So then you say, okay, this bacterium
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does not carry out all these steps some mutant what is the step thats wrong and
the pathway. Why cant they function as the other bacteria do, that can produce this
product? And then you piece this information together. So various mutations in
bacteria, piece them together, and then learn how the mutations, how the mutants
work together, and how normal systems eventually work by studying mutants in
which something has gone wrong and finding out why. Piecing information together
and then learning about these pathways.
Some chemicals can be used. Ultraviolet light. Some base-analogues. These
are bases like purines and pyrimidines that look like the bases in DNA. Adenine,
thymine, guanine, cytosine. They look like it, but there is something unusual about
them. Some unusual side group like a methyl group or something additional. So
often these base analogues that look like the four bases in DNA. They fool the
mechanism of DNA replication and theyre inserted incorrectly into replicating DNA.
They stop the replication. They cause some mutation in the chromosome thats
being replicated. So base-analogues they use a lot and this type will work to induce
mutations. Bases that look like the four purines and pyrimidines, but are different
and interrupt and cause mutations in the DNA. Many type--. Okay we already went
through that, already. So heres one uhh. We use UV a lot. Although not high dose. I
mean, true, the UV is a very good uhh, well Its a good disinfecting agent, I would
say. UV is even a good sterilizing agent. However, it does not penetrate well. UV
will--. If you had a nice flat Petri plate, just flat bacteria, and you shone some UV
light on them, it will kill all of them. But we know that bacteria grow in colonies.
Billions of cells. Or we know that some of them even live on water surface. So the
top of the Petri plate. You shone UV light on that, it wouldnt penetrate it. So UV
light is good for use of certain purposes to cut down on number of bacteria, but its
not a good sterilizing agent. We use it a lot to cause mutations in bacteria. How
does it work to cause mutations? Well, we see here on the top on my right here,
heres normal DNA. You shine some UV light on it. And wherever there are sites in
one of the two strands of DNA, where there are thymines adjacent to each other, as
shown here. Thymine bases. Here you have T, C, G, A. So here two thymines next to
each other. What UV light does is cause a formation of thymine dimers. So this
shouldnt occur here. This bonds between the two thymines doesnt occur naturally.
So when that does occur, when you take UV light and shine on the DNA, those two
thymines often are removed from that strand of DNA. And some other bases are put
in, A, C, or G. So the difference between the sequence of bases in DNA as relative to
UV light causing thymine dimers that are lost from the DNA, replaced by any others
of the bases in DNA. New sequence of bases. New mutant has risen.

[6] [DNA repair]
Transcribed by Kevin Lin Lecture Date: July 22, 2014

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[Dr. Boylan] There are repair mechanisms in bacteria. Some of these DNA
polymerases, and even RNA polymerases, that are used for DNA replication and RNA
transcription, are very faulty enzymes. They make a lot of mistakes. And because of
mistakes, there may be mutations after mutations if the mistakes that they made
werent repaired during replication or transcription almost right away. So these
enzymes, and well see the worst one of all, I think when we talk about HIV, the
faultiest enzyme of all time is the reverse transcriptase found in HIV. But most of
these enzymes, too. DNA polymerase, RNA polymerase. They make mistakes when
theyre replicating. Theyre trying to replicate faithfully the message in the template
of one strand or the other. And when these mistakes are made, bacteria we know
for sure, and our cells, too, have genes for repair of the DNA. Damage occurs. Its
called the SOS response. Up to 30 different genes, 30 different enzymes are there
watching the DNA polymerase do its work. And saying, uh-oh, T was put there
instead of C. Across an A-A-T, C was put instead of T, whatever. A mistake was
made. Right away get rid of that wrong base, pair it up with its correct base and
dont allow a mutation. Or repair that mutation before it gets the chance to exist and
stay there and cause serious damage to the cell. So damage, repair, and remove
bases that should not be there right away, so mutations do not occur. And prevent
mutations. SOS system.

[7] [Plasmids]
[Dr. Boylan] Plasmids. Extrachromosomal. Once again, bacteria do not need them
to survive. They have the chromosome, thats all they really need to grow, enlarge,
to divide. Theyre happy. But if they have extrachromosomal genes as some
plasmids and some bacteriophage, theyll have additional properties because they
have these DNA-containing extra genomes. Extrachromosomal genetic activity.
Dispensable. Copy number refers to the number of plasmids, here maybe it would
be three. 20. How many plasmids does a cell have? Often its characteristic of
particular bacterium. Autonomous replication, in other words, the plasmids
replicate independently of the chromosome. They may be at the same time as the
chromosomes, plasmids replicate. Yeah, they do that, but not as a part of the
chromosome. However, later on well see an exception to that rule when we talk
about the F factor. A particular type of plasmid known as an episome. They can do
either. They can replicate autonomously or as a part of the chromosome. Well talk
about that later. Give the host new properties. Drug resistance genes, that are being
transmitted throughout the world now, are often results of plasmids that have these
genes for uhh, for example, well one of the most prevalent is the one plasmid found
in Staph. aureus. Staph. aureus has a plasmidit can grow without a plasmid, of
course it has a chromosomebut if Staph. aureus is infected with a particular
plasmid that has a gene for production of the enzyme, penicillinase. In many case,
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that enzyme will hydrolyze penicillin. Why are there so many Staph aureus today
resistant to penicillin? Its because penicillinase. Why does bacteria produce
penicillinase? Because they have their own plasmid in them. Theyre infected with
a plasmid that has a gene that codes for penicillinase. 25 years ago, 30 years ago,
Tisch Hospital, which is Langone Medical Center now, I think about every hundred
patients that come in that have a staph infection. 95% of them could be treated with
penicillin. Today, only about 5% of people in Langone hospital come by with staph
infection can be treated with penicillin because of the emergence and transmission
of some resistant staphylococci. Because of plasmids that they harbor have these
genes. Thats a new property.

Conjugative and non-conjugative plasmids. Well see that conjugative ones
later on well talk about their role in bacterial conjugation. Some plasmids enable
bacteria to mate with other bacteria. And well talk about later also about the role of
plasmids in recombinant DNA technology. You have the same slide? No? Sorry. I
know I changed it around a little bit, but okay. Uhh what else? Oh, discovery of
plasmids. Well, what happened was back in the 1950s in Tokyo, there was an
outbreak of dysentery. Bacterial dysentery. People were getting very sick. And it
was an outbreak, quite a few people in this one part of Tokyo. They said, Okay well,
we know what causes bacterial dysentery. Another bacterium well talk about
later, Shigella dysenteriae. Doesnt matter. Dysentery outbreak. Lets go, lets get
the antibiotic that we use to treat people who are infected by the bacteria that cause
dysentery. So we have about 6 of them we can use actually in our refrigerator so
lets try, well. Lets say they tried chloramphenicol. And it didnt work. Well, thats
odd. These people are not getting better when treating with chloramphenicol. They
said, lets try tetracycline. That works, too. Well, that didnt work in these cases.
People who had dysentery. And they tried about half a dozen different antibiotics
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that previously were very effective in treating dysentery. Killing the bacteria that
caused dysentery in these people in Japan. So they thought, What happened? How
could all of these bacteria all of a sudden become resistant to 6 different drugs?
They thought, Is it some mutation in their chromosome, chromosome of the
bacteria? Six different mutations? That enabled them to somehow resist these
drugs? They said, No, there must be something else. And indeed there was
something else. And it was the fact that these bacteria that caused dysentery were
harboring, infected by plasmids. For some reason, these bacteria that caused
dysentery at that time in the 1950s in Tokyo had picked up plasmids from some
source, were not sure where. But these plasmids infected these bacteria and they
already had the genes for multiple drug resistance in them. So, thats how they said
theres something else in these bacteria, not just their chromosome, but they must
be harboring something else and indeed they did find that it was plasmids with the
genes for resistance to multiple drugs, so they gotta find something else to treat the
infections of those people, those patients.

[8] [Transposons - 1]
[Dr. Boylan] Transposons are another way we can cause mutations in bacteria.
And these are also known fondly as jumping genes. Jumping genes. Stretches of
DNA. Transposon, they have different components, were not gonna be too worried
about those. But these are stretches of DNA. Transposon, well call them. They
hopscotch. The stretches of DNA that can go back and forth between the
chromosome of a bacterial cell and the plasmid. They can jump back and forth from
here to there. They can then go from the plasmid to the chromosome, back and
forth. They can move from one side of the chromosome to another. And they can do
the same thing in a plasmid. So these pieces, little stretches of DNA called
transposons hopscotch, jumping genes, jump around and then insert themselves,
they kind of bully their way into the chromosome of the bacteria or to the plasmid
DNA. And when they do that, they cause mutations. They change the DNA sequence
in the genome of that plasmid of that bacterium. They also harbor genes for drug
resistance. Heres another way drug resistance occurs in bacteria. Transposons,
youll see in Dr. Tiernos lectures coming up, the role they play in drug resistance
among bacteria. But the important thing is right now, is that DNA jump around from
one side of the chromosome to another, they release themselves, and they really
upset the functioning of the genome or their plasmid. When they bully their way
into the chromosome at different sites, causing mutations. Often leading to the
death of the cell, but often giving these cells new properties as well. So the genes for
drug resistance can be found on them. They cause mutations. They cannot
independent--. No independent replication. How do they differ therefore from
plasmids? Well, plasmids can replicate autonomously. Separately from the
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chromosome. The important thing about transposons, how to distinguish between
these two, the plasmids and the transposons. Transposons only replicate as part of
the chromosome or as part of the plasmid. They cannot be out there in the
cytoplasm and replicate on their own, as plasmids do. So no independent
replication. More in the function of transposons and drug resistance when Dr.
Tierno comes over the next few lectures.
Does anyone know who discovered transposons? You probably had this
information in Building Blocks, right? Anybody wanna venture a guess? Her name
is Barbara something. Barbara McClintock, exactly. Barbara McClintock. And she
worked with corn, really. And one of the things I guess to me is simple, I think, is
she knows that little kernels on the cob of corn, they come off to be different shades
of yellow or brown or have different features of them. And so whats going on here,
how can all these different kernel, how do these difference in the phenotype occur?
And she said, it cant just be the chromosome itself having mutations of all these
different kernels on the corn. So anyhow, she studied these transposons and found
out there were jumping genes there. And jumping around caused these mutations
within the cells of the kernels of corn. And of course her results when listed, they
were scoffed at. She was ridiculed. They said how can--. This was of course before
they knew about transposons, what she was discovering. And finally her wisdom
and her research was so fantastic and wonderful and true that she won the Nobel
Prize for it. And I actually met her one time. The story, I met her a long time ago.
She worked at the Cold Spring Harbor Labs on Long Island. James Watson is still in
charge, I believe, or is still there anyhow. Every summer, she would go to spend
about a month or so at Cornell in upstate Ithica. Well, my wife was a graduate
student at the time. And she would go there and go talk to graduates students, she
loved graduate students. So she talked about her work and talked about their work.
And my wife would often, every time for three or four years in a row, she threw a
party in her apartment for Barbara McClintock. And I thought, thats pretty good,
you know? Youre a graduate student and youre inviting this Nobel Prize winner
(not yet) to your apartment for a party? I was really impressed, so I asked her to
marry me. I mean, my wife, not Barbara McClintock. I dont think she ever married.
She couldnt get away from her corn. My wife said she never served corn at these
parties that she had for her. But I thought that was pretty impressive. Of course she
had stature even then. Almost 40 years ago now.

[9] [Transposons - 2]
[Dr. Boylan] Transposons, discrete mobile genetic elements that possess the follow
common features. Incapable of autonomous replication. We already went through
these. The way they do this, they insert themselves with the enzyme, transposase.
And may carry genes for drug resistance. So thats a recap. But the one important
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enzyme they code for among other gene activity they have, is the enzyme
transposase, which enables it to both get out, excise itself from one part of the
chromosome or plasmid and insert itself somewhere else. Lots of destruction
occurs as a result.

[10] [The central dogma]
[Dr. Boylan] central dogma, of course, of molecular biology is you go from DNA to
RNA to protein. DNA to RNA, transcription. RNA to protein, translation. You form a
polypeptide and the ribosome, messenger RNA. Transfer RNA brings the amino
acids down. Okay.

[11] [untitled TRANSCRIPTION]
[Dr. Boylan] And dont forget though, many students do forget, that the genes on
the DNA also codes for information of the rRNA as well. Not just for mRNA, but
transfer and ribosomal RNA are also replicated from segments of the DNA. Not just
the mRNA that carries information out, but the other two types of RNA, too, are
encoded by genes, by DNA material.


[12] [Gene transfer in bacteria]
[Dr. Boylan] Gene transfer in bacteria. Let me hop on ahead here, it looks like
Umm. Okay. Horizontal gene transfer. Horizontal, from mother to daughters going
down--. No wait a minute, thats vertical.
Vertical transfer. Lets take a look first at vertical. From mother to daughter.
Vertical, going down. By binary fission. So heres the mother cell. Heres a
chromosome. Heres a segment of the chromosome. When it divides into two cells
by binary fission, both the daughter cells have the same chromosome, same genes.
Replicate again, second generation, three. Binary fission. One to two to four cells,
but the chromosomes, all the same. Thats the way the genes are transmitted
vertically from mother cell to daughters.
In bacteria however, we also have horizontal gene transfer, in which you go
from one cell--, shown here. Heres a donor cell. The donor cell, we use that term, it
means, its going to give up its DNA or contribute its DNA somehow to a recipient
cell. So donor cell contributes its DNA in one way or another, well talk about three
different ways this is going to happen, to a recipient cell that picks up the DNA from
the donor. How do we know this happens? We know this happens all the time, but
how did we figure out that this happened. Well, its only when DNA from a donor
goes to a recipient, the DNA from the donor has to have a slightly different sequence
of bases than the bases in the recipient. I mean, if its exactly the same donor DNA
getting to a recipient and changing genes in a recipient, we wouldnt know it
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happened. But maybe just one property, one gene might be different. For example,
well talk about a gene later on for capsule production. Lets say a recipient cell can
grow very well on media, but it never forms a capsule. And we get the DNA from a
close relative of it that does form a capsule. We get that DNA from the donor cell,
has a gene for capsule production, and we introduce that gene into the bacteria that
cannot produce a capsule. And its only when that gene replaces the faulty gene in
the recipient, that now that the recipient gene can produce a capsule. A new
property. So we know this process goes on, the way we figure it out and determine
it is by the new properties that can be picked up by recipients, a result of getting a
new piece of DNA from a donor because, once again, some new property. It may be
a virulence factor, it may difference in the cell wall. It can be almost anything, any
property, any phenotype. But capsule production is one of them. So, something has
to be different about the genes its picked up in a recipient cell that gives it a new
property. So we know this process, whatever it may be, went on, this gene transfer
went on.
When a piece of DNA is picked up from a donor, its picked up by a recipient
cell and replaces some DNA in the recipient cell, its called recombination. Its a very
complicated process. We could spend hours talking about recombination, but for us,
I think our purpose will be suitable by saying a piece of DNA (a small piece of DNA,
often), picked up by a recipient cell, finds its way to the chromosome of the recipient
cell, and then matches up with the sequence of bases that are very similar to it, and
then replaces that sequence. Recombination. It kind of kicks out the segment that
was there and puts in a new donor piece of DNA. Recombination, well call that. So
heres your donor cell. Heres your recipient cell. This, we see here, actually, two
different kinds of the three kinds of gene transfer that occur in bacteria. And this
process here, a donor cell, recipient cell. In this case here, only the DNA, shown by
the red A, those genes, sequence of bases in DNA from the donor are released free,
called naked DNA. DNA only. Pure DNA. Lost from that cell, probably when it died,
all this chromosome chopped up into different smaller fragments. And a piece of
DNA was picked up by a recipient cell. And eventually, this piece of DNA will come
over, match up with the chromosome of the recipient cell and replace a stretch of
DNA, about the same size as it. But when there are new properties on this A gene
was not found in the recipient cell chromosome, a new property was picked up by
the recipient cell. Heres another way of horizontal gene transfer among bacteria,
where the bacteria here we see are actually combining with each other. They
actually conjugate, or mate. They form a bridge between them and by this way, well
talk more about that soon. The donor cell gives up some of its DNA to the recipient
cell by this process, here we call conjugation. So here we see two of the three major
ways by which horizontal gene transfer occurs in bacteria. Genes from one
bacterium get into another bacterium. From a donor to a recipient.
Transcribed by Kevin Lin Lecture Date: July 22, 2014

13

[13] [3 types of gene transfer in bacteria]
[Dr. Boylan] three types of gene transfer in bacteria are called--. And were gonna
go through these in order. Theyre not the same order, but well go through them.
Conjugation. Transfer process. Conjugation, transduction, or transformation. The
three main types of gene transfer in which recombination occurs in recipient cells.
The process of conjugation is that DNA is transferred from one bacterium to
another. How? Well talk, itll make more sense later. But either chromosomal or
plasmid transfer. Transduction, transfer by a virus, by a bacteriophage, from one
cell to another. Any gene, and well talk about this--. Transduction basically means
gene transfer between bacteria via a phage. A phage picks up a gene from a donor,
infects recipient cell, takes that gene along with it, and the gene goes into the
chromosome of the bacterial cell eventually. The third type is transformation.
Purified DNA taken up by a cell. Any DNA. And this can also happen with eukaryotic
cells. We can put DNA into our cells as well, but were gonna transformation that
occurs in bacteria.

[14] [untitled A = transformation; B = transduction; C = conjugation]
[Dr. Boylan] Let me just take a look ahead here. Okay. Here they are again. Heres
transformation. Heres naked DNA. Taken up by a recipient cell. Here you see here,
some of that blue DNA from competent, donor cell is taken up by the recipient cell.
Integrated by recombination. So competent bacterium is a donor bacterium that
can pick up--. Im sorry lets go back.
Heres the DNA from a donor cell. Heres a recipient cell. Its called a
competent bacterium because its competent, its able to pick up DNA. Well talk
about competency in a minute, too. So heres a recipient cell, picks up naked DNA,
takes it inside. Some of the DNA integrates into chromosome, so now we have
transfer of DNA by transformation.
In transduction, we have a phage, a bacteriophage, infecting a cell. In this
case here, its a lytic phage. Remember what happens with lytic phage? They infect
the cell, they replicate inside of the bacterial cell and they kill the cell. And after the
mature virus is produced, mature virus is produced, mature phage inside the
bacterial cell, they are released. But look what happens here. The case here,
sometimes, this phage will pick up some bacterial DNA by a mistake. Well talk now
about gene transfer among bacteria. Certain phage, well talk a little more about
when they replicate inside of a bacterial cell, they make mistakes. Imagine the chaos
thats going on inside a bacterial cell infected by a phage. And 20 minutes later,
theres gonna be a hundred new phage. 20 minutes later. They synthesize their
DNA, their capsule proteins, their envelope proteins. They have, not phage, but
capsid proteins and their nucleic acid. In 20 minutes, hundreds of progeny. It must
Transcribed by Kevin Lin Lecture Date: July 22, 2014

14
be chaos inside that bacterial cell. So sometimes, these phage capsids, by a mistake,
pick up bacterial chromosomal DNA by a mistake. And sometimes, they pick up
bacterial DNA thats attached to the viral DNA. And hook it up with them. And when
they infect other cells now, they take along the bacterial information with them,
those genes.
The third type of conjugation [gene transfer] is conjugation, where theres
mating between cells. But lets spend a little more time on each of these.

[15] [Possible fates of DNA introduced into a bacterial cell by horizontal gene
transfer]
[Dr. Boylan] And then the piece of DNA that is picked up by a cell is called the
exogenote. And the chromosome or the plasmid--. And well later on [inaudible]
can also pick up this new DNA. Well call it the endogenote. Recombination.
Transfer. Heres the DNA, gets into a cell, by one of those three ways. Is picked up
by a plasmid or the chromosome. Integrates into chromosome, recombination. So,
two pieces of DNA, exogenote gets added to the recipient, and the endogenote thats
already inside the bacterial cell. Homologous regions pair up and exchange pieces
by the process of breakage and reunion, that means recombination. So, they cant
linger outside in the cytoplasm. Its gotta somehow bump out, integrate into the
chromosome or plasmid, and replace the DNA that was there. And now a new
property has been picked up by the bacterial cell.

[16] [Transformation initial experiments]
[Dr. Boylan] Well talk about this and well take a break. Here is actually I think a
good representation of the first experiments that were done to show the
importance, to really show the importance of actually nucleic acids in genetics. Up
until 1940s worldwide, 1940s, they were still a mystery as to what exactly
constituted heredity. What was it that was responsible for the differences in species
worldwide, in animals, plants? And they thought, well, the differences could only be
accounted by the differences in the makeup of proteins. Its gotta be it. Protein
gotta be the key to genetic variation, to heredity because there are 20 different
amino acids. It cant be nucleic acid, it cant be DNA because there are only four
bases. Can there be a variation in the world in DNA in just four bases to account for
in genetic differences? Well they began to study this actually in a roundabout way.
What happened back in 1920s, this fellow named Griffith over in London, he and,
uhh, this is before the pre-antibiotic era, its before we knew about nucleic acids as
being involved in genetics. He did these studies where he, one of many labs in the
world studying pneumonia, Streptococcal pneumonia, lobar pneumonia, a deadly,
deadly killer. Even today, its a killer, but back then in the pre-antibiotic era, it was
lethal. Had a very high mortality rate. So, many labs working on the pneumococcus.
Transcribed by Kevin Lin Lecture Date: July 22, 2014

15
Streptococcus pneumonia bacteria were gonna talk about here to see how did it
cause pneumonia? What are its virulence characteristics? How can we maybe find
other ways to treat it in people who have pneumococcal pneumonia? So they were
studying it just to learn a lot about it because they could. So he was doing it. He was
doing mouse studies with a pneumococcus. The bacterium, Streptococcus
pneumonia, that causes bacterial lobar pneumonia. And he was working with two
different types of strains of Streptococcus pneumonia. Strains. One strain, he called
the S strain, for smooth, and its shown here in the red colonies. And the other strain
is called R for rough, and theyre shown as these blue bacteria here. Blue bacteria or
blue colonies. So, he knows that the smooth strains of pneumococcus had a capsule,
theyre encapsulated. And they were lethal. Whereas, the pneumococcus, the same,
exact same pneumococcus, exact same except for the fact they could not form a
capsule. Could not produce a capsule. Every other property, they had in common
with the smooth, but no capsule, non-pathogenic. So he said, okay, whats going on
here? What is it about the smooth, whats happening? Why is one so lethal and the
other avirulent? And so he started to inject mice with these different strains, either
smooth or rough. And as you would probably imagine, when he injected the mice
with the smooth, the encapsulated pneumococci. Next day, come back, and the
mouse would be dead. And they isolated from the body of the dead mouse: all
smooth colonies of the pneumococcus. So smooth, encapsulated. These were nice,
rounddidnt look red, but they were big, smooth colonies because they produced
this mucoid capsule. Kind of like mozzarella cheese, you know.
When he injected the blue, the rough strains of the pneumococcus in the
mice, they would not die. The next day, theyd still be alive. And he would recover
from them the rough variant, the rough strains of pneumococcus. Harmless bacteria
were found on the Petri plates. In the third experiment, he injected mice with same
bacterium, only what he would do is, before injecting the bacteria is he would killed
them. He would heat-kill them. He would destroy the smooth bacteria by heating
them. So dead smooth bacteria injected into the mice. What happens? No
pneumonia. Actually no pneumococci from the mice. The mice would live.
But here was the interesting phenomenon that he observed. When he
injected into the mice harmless bacteria, such as shown here. And the combination
of the two types. So the second and third experiments. A combination of the dead
smooth and the living rough, neither of which by themselves would kill the mouse.
The next day, come back, and the mouse would be dead. And he could isolate from
the body of the dead mouse both smooth and rough colonies. So once again, the key
being here, neither one of these, harmless bacteria, the R strain nor the killed
pathogenic strain could by themselves kill the mice. A combination of the two of
them as shown in the last column here killed the mice. So what was going on?
Griffith injected this mixture of rough and smooth, the mice died. When Griffith
Transcribed by Kevin Lin Lecture Date: July 22, 2014

16
cultivated blue bacteria found in the blood, he found live S strains. So there were no
live S strains up here [in the needle], but indeed, he could isolate from a mouse
living smooth strains. So what happened, something must be passing from the dead
smooth, some component of the dead smooth must be being picked up by the living
rough. Changing the living rough, from rough to smooth. That was the key
hereditary material. Whatever it was, being picked up by these rough, dry cells from
the smooth cells, even though they were dead. Some component of them was being
transformed. Transformed the recipient bacteria, transformed them from rough to
smooth. And for years they thought, okay, that must be the protein. That must be
the protein. They didnt know. Okay so lets stop there. And well find out it was
when we resume in about 7 or 8 minutes, okay?

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