Transcribed by Kevin Lin Lecture Date: July 22, 2014

1
[General Pathology] [12] [Bacterial Genetics II] by [Dr. Boylan]

[17] [Transformation on the cellular level.]
[Dr. Boylan] I was just wondering, what if at those parties that my wife threw for
Barbara McClintock, what if I asked my wife to marry her to marry me in front of
Barbara McClintock, and Barbara McClintock said, Yes Bob, I will marry you. I
dont know. Where would I be today? I think she was happier with her corn than
with me, than I ever could have made her though. But actually, a few years ago, my
brother-in-law, whose name is also Bob, as is mine, was remarried and we were at
the ceremony. And the minister and Bob and his wife-to-be were, I would say, six
feet away from us. And the minister was going through all the prayer and finally
said, And Bob, do you take this women to be your lawfully wedded wife? And I
said, I do. And to my brother-in-law Bob, I said, Bob, I hate to say this, but I think
I might be married to your wife, too. You know, might be the two of us. But I guess
they didnt hear me say, I do. It just came out of nowhere. Haha okay.
Anyhow, Transformation. Of course today,we know now that this
transforming principle, the so-called secret of life. Responsible for heredity.
Change in the property at the gene level was DNA. That was the transforming
principle. That was being released from the dead smooth bacteria, being picked up
by the other strain of pneumococcus, the rough. Where recombination occurred,
that gene got into the chromosome of the rough strains. They were now able to
produce a capsule, which made them virulent. Its made them cause pneumonia.
Never underestimate the power of the capsule. And not just in this type of
pneumonia, pneumococcus, but also other types well talk about later. And also
some forms of meningitis. So bacteria cause a capsule. Once they form a capsule,
they are very hard to eradicate from the body. They slip away from our
macrophages and our phagocytes. Its a very important virulence factor.
But today we know we can do transformation experiments in the laboratory.
In a test tube. We have one strain of bacteria. Lets say theyre rough strain growing
in a test tube. And all we have to do is add some DNA from a smooth strain. Just
pure, naked DNA to that test tube, let it be filtered down into the broth that the
rough strains are growing in. And the rough bacteria pick up this piece of DNA and
some of them will be transformed from rough to smooth and are now able to
produce capsules and now become pathogenic. Its not just the capsule, but almost
any property of a bacterium can be transferred, gene transfer in this way. Any of the
genes on the chromosomal DNA. Today we do it in the lab. And heres a dead cell.
Lets say this is a cell in our gut right now. It dies. It releases its DNA. Other similar
bacteria are in the environment. And they go and they adhere to the surface of these
recipient cells. And these cellswell get back to that word competent againthese
recipient cells here, theyre going to pick this naked DNA, are called competent.
Transcribed by Kevin Lin Lecture Date: July 22, 2014

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That means they are able to be transformed by DNA. Remember that growth curve,
log phase and then stationary phase. Well usually if you do these experiments, you
do them in late log phase. Theyre in a competent stage. Theyre much more likely,
whether there are pores in their membrane or what, to receive foreign DNA. To pick
it up and let it get inside. So theyre competent. The stage which they are most
adequately transformed is called competency. So anyhow, they get insideyou
dont have to go through all these processes, but in essence, the piece of DNA, naked
DNA gets inside. You see it pairs up with this partner here or something that looks
like it. Sequence almost identical except for some new genes on the incoming
exogenote. And finally recombination occurs and the recipient is transformed. So
heres the red genes, DNA from the donor. So thats just transformation. Competent
bacteria are ones that are treated in such a way they are most likely to pick up the
DNA, a lot of it easily. So when you want to do these experiments, youll want the
recipients that pick up a lot of DNA. So competent stage, physiologically in a state to
pick up DNA from a donor. Naked DNA.

[18] [Transformation - notes]
[Dr. Boylan] And what got me interested in microbiology years ago when I was
taking the course, you could have these bacteria growing in a test tube, add a little
bit of inert, dead chemical to them. And these bacteria pick up this inert, dead
chemical and be transformed into something different. So I actually started my
career in research in studying bacteria genetics and in transformation as well, many,
many years ago. But transformation notes. In the mid-40s, they still were not
convinced that it was DNA that was the transforming principle. They thought it still
might be proteins. So Avery, McCarty, MacLeod were three scientists at Rockefeller
University. You know where that is? Upper East side, right across Sloan Kettering
Hospital. Beautiful campus. I spent some time there about four or five years ago in
a sabbatical, working in a lab there. And mid-1940s, these three scientists were
working on what actually was the transforming principle. Lets prove that its DNA
to the skeptical world out there that still thinks its proteins. DNA. So they did the
experiments with naked DNA. And they come in and Avery was the big shot. He
was the world renowned geneticist. McCarty, MacLeod were post-docs in his lab.
And I think they both went to NYU. And what they would do every day, they would
have these bacteria growing in the lab. And then they add DNA from some other
closely related strain to them. Maybe DNA one day, protein, RNA, different amino
acids, different nucleic acids, to RNA. They tried everything to transform these
bacteria to have them change their properties as a result of adding one of these
organic compounds, like nucleic acids or proteins. But only when they added DNA
that this process would occur. That transformation occur. That the recipients
picked up a new property, DNA. But there were still scientists who were still
Transcribed by Kevin Lin Lecture Date: July 22, 2014

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skeptical. They [skeptics] said maybe it was contaminated with some protein.
Maybe there were some protein thats there. Maybe a small amount that you can
barely detect it when you measure how much DNA you have. But thats
contaminating your DNA and its really the protein, not DNA causing transformation
to occur. So the convincing experiment that they did was add DNA to the bacteria in
a test tube, but also at the same time add the enzyme deoxyribosenuclease. As
simple as that. Thats the enzyme that cleaves DNA. And when they added that
enzyme that cleaved DNA, no transformation occurred. DNA without the enzyme,
transformation occurred. So basically that was it to prove that DNA was the
hereditary material of life. Many bacteria, many genes can be transformed, naked
DNA. So I would say that these scientists--. Well, one more story about Avery,
McCarty, Macleod. Avery was the big shot and McCarty and Macleod, they did the
bulk of the work. Every day, I mean they knew they were onto something very
impotratnt in the work they were doing. But they had a pact that they do
experiment and the next day they went and look at the results. Open up the
incubator door and look at the test tubes and see what grew, what didnt grow. And
they could tell almost immediately if the experiment was successful. Every day,
protein, RNA. But they had a pact, that they would not open the door, look at an
incubator where the test tubes were growing or not growing, unless they were all
together, the three of them. So McCarty and MacLeod would get in every day very
early, and they would just looking at the incubator, waiting for results. They
couldnt open that door. They were so anxious to see what happened. Until Avery
came in, once again. Hed stroll in about 10 oclock or after. And then theyd jump
up and open the door and see what the results were. And its interesting how they
found the answers leading to prove that DNA was the transforming principle. And
the same thing happens today in labs throughout the world. I wanna find out the
results, dont find out before I do. Lets do it at the same time. And MacLeod
actually died just a few years ago. And one more point about this, he, actually when
I was up there, Rockefeller, I happened to be working on the same floor, same
building he was just a couple labs away. And he had died about a year or two before
I moved up there. And I got to use his printer. I used it once or twice, it was an old,
chunky one that I couldnt use, but I thought it was kind of neat that I was able to go
into the room that he did most of this work and take his printer and take it back and
found out that it didnt work too well. The secret of life, DNA. Microbiologists
discovered that. The secret of survival, we had discovered that as well. The
immune system. Okay? We wouldnt survive without our immune system. So
immunology. Dont tell Dr. McCutcheon I said that.
[Dr. Boylans McCutcheon voice]: Microbiologists discovered the immune system?
Oh no.
Transcribed by Kevin Lin Lecture Date: July 22, 2014

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But Mechnikov and those others way back when, they were microbiologists. And
weve also discovered the secret of financial success. Microbiologists. Thats
available--, actually I have discovered the secret of financial success. And I was just
wondering if you would all like to know what that is? You would? I dont blame
you. Heres what you do. Next time we meet, I think its on Thursday, Ill introduce
the speaker. Every one of you, give me $1,000. Thats a small down payment,
alright? I will tell you the secret of financial success. The next time you come to
lecture, I will tell you. If I dont show up, you will know my secret of financial
success. And you can try it yourself some other way. Okay.

[19] [Transduction]
[Dr. Boylan] Okay so Transformation, naked DNA. Transfers gene. Gene transfer
among bacteria. Happens in our gut all the time. Transfer of genes.
Transduction. Remember temperate phages? Those are not the lytic, but the
other type of phage that injects its DNA into a bacterial cell and then the DNA inserts
itself into the bacterial chromosome as a prophage. Prophage DNA. So these are
temperate phages. They are involved in what we call transduction. But we also
have the lysogenic phage that become lytic right away. So remember there are two
type of bacteriophage, lytic that begin the replication of new viral phage right away
after they inject themselves into the bacterial cell; whereas lysogenic temperate
phage become prophage and dont replicate right way. But both of them are nucleic
acid DNA that are found injecting themselves into the bacterial host cell to replicate.
Uhh. Make more phage DNA... Make mistakes Now, when these
bacteriophage, whether lytic or temperate, are replicating inside a bacterial cell,
they often make mistakes. Thats the key, they make mistakes. In other words,
when they form the new viral particles, new phage particles inside these cells. At
lytic phage or when temperate phage begins to replicate and form new phages,
sometimes they make mistakes, meaning sometimes instead of being pure phage
DNA inside the capsids of these viruses, often they pick up by mistake little bit or a
lot, depending on the phage, of bacterial DNA. Of chromosomal DNA from a
bacterial host. Thats the key. By gene transfer, genes from bacteria are going to be
transferred from donor to recipient via bacteriophage. Lytic or temperate, two
different types, well talk about those. So these phage that by mistake, instead of
having, being 100% viral phage DNA, are a little bit or a lot of bacterial DNA as well,
are called defective phages. Defective because theyre not 100% viral DNA. That
doesnt mean they cant infect new cells, new bacteria, new host. They do. And
when they do, they carry along some of the bacterial DNA from the donor to the
recipient. Genes from donor to recipient via the phage. So the phage are kind of like
the vehicle in transduction, carry across genes from a donor to a recipient.
Transcribed by Kevin Lin Lecture Date: July 22, 2014

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Transduce these genes to new host. There are two types, generalized and
restricted transduction. So at least know the definition of these two. And these are
pseudovirions. Those are another name for defective phage. Psuedo- are like
viruses, but they have some bacterial DNA in them as well.

[20] [REVIEW: Bacteriophage Replicative Cycles.]
[Dr. Boylan] So here we have the process, remember just to review the process. On
the left here we have lytic phage. Heres an infection, inject the DNA. Right away,
the phage DNA is shown here, begins to--, well it goes to the ribosome as you know.
Protein synthesis, all these new viral particles, capsomeres, capsids, nucleases,
polymerases, etc. They make new phage particles. So phage DNA and that proteins
are synthesized and inserted into new phage. And then they eventually, lytic phase,
they lyse the cell. They can infect new bacteria and repeat this process over and
over again. And this is a normal replicative cycle of a phage.
The other is the temperate phage. Okay. Here we have here, they inject the
DNA of the phage. It becomes a part of the--. Here, here and here.--. The phage DNA
circularizes in the lysogenic, in this case. The lysogenic cycle, becomes the
prophage. The phage DNA becomes a part of the chromosome. And then stays there
for many generations, perhaps. Becoming a prophage. Replicating over and over
again. Eventually and if the cell replicates over and over again, doubles repeatedly,
daughters formed all the time. It stays a part of the chromosome until finally it says,
okay, Im going to break free. So lysogenic bacteria, has the capacity to be lysed
when the prophage finally says, okay, Im going to excise from the chromosome and
Im going to go on into the lytic cycle and form more of myself. Form more and more
and more of my own temperate phage. But for a while, there is recall as a part of the
bacterial cell. So those are two types of replication of bacteriophage. What happens
in transduction?

[21] [Generalized and Specialized Transduction]
[Dr. Boylan] What happens in transduction? Okay, here we have, lets go through
the process. Here we have a bacterial cell. Heres a virulent phage, lytic phage. And
this is a stretch of the chromosome, between A and B. Phage injects its DNA. And
then it begins to replicate. And you see here, when these phage is replicating
remember its chaos inside this bacterial cellwithin 20 minutes, a hundred new
phage are produced. So the capsomeres are being formed. Theyre trying to get
together to assemble into capsids, and then surrounds the DNA or have the DNA
somehow injected into them. Different ways that this could happen. Sometimes by
mistake, as you see here, you pick up some of the chromosomal DNA. The dark
sequence shown here. A to B, here they picked up the B gene and some bacterial
genes are picked up. By this now defective phage, pseudovirion. These are released.
Transcribed by Kevin Lin Lecture Date: July 22, 2014

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What happens here, this phage here, injects bacterial DNA into the recipient cell.
Not viral, but bacterial. So, and then, transduced cell has bacterial genes from donor
cell. So, this is the way that lytic phage do it.
Another point here is that any gene, not just A or B orany part of this
chromosome up here can be packed into the capsid of the lytic phage shown here.
Not just certain genes repeatedly and often, but any gene can theoretically can be
picked up. Any bacterial gene can be picked up in a lytic phage replication cycle by
these phage that are produced. And here we have the other type of transduction
called specialized transduction. So that was kind of the generalized transduction,
any gene of the bacterial cell, not specific ones. But with the temperate phage, only
specific genes are gonna be transferred from a donor to a recipient, called
specialized transduction. See only theheres the red. Injected the DNA. DNA
finally incorporates itself as a prophage. And then when its released from the
chromosome of the cell, look what happens. It picks up some of the chromosomal
DNA adjacent to it. So only those bacterial genes adjacent to the prophage will be
transduced. The specialized transduction, only adjacent genes, whereas in
generalized transduction, any gene in the chromosome could theoretically be
transduced. DNA is chopped up into fragments and its guess which segment will
wind up inside the capsid in lytic. But in specific [specialized], only adjacent genes
to integration of the prophage are picked up. So here we see this D gene here. This
integrates into the chromosome of the recipient cell. Takes the bacterial gene along
with it. So those are two types of transduction, generalized lytic phage, specialized
temperate phage.

[22] [Conjugation -1]
[Dr. Boylan] Once again, gene transfer in bacteria. Third type is conjugation.
Conjugation. Gene transfer in bacteria. Cell-to-cell contact is needed. Mating, sex in
bacteria. Mating, we called them male and female. There are different properties.
Here you can see the difference in this slide right here between the ugly little male
right here and this beautiful female with a nice, clean sheen. But there are all pili on
the male. But this is a special pilus produced by the male called the sex pilus. And
this is the one thats produced that sort of reaches out and grabs ahold of the female
and brings her close, so they can come in contact with each other and form a bridge
between them as we will see. And between that bridge, the male can donate some of
his DNA to the female. Maybe a small amount, maybe a large amount, maybe his
whole chromosome. So male and female. Heres the male. The plasmid. And what
makes this male a male and able to produce a conjugation pilus is the fact that it has
a plasmid called the fertility factor. The F factor. Fertility. Plasmid has genesone
of the genes is for the production of the special conjugation pilus. These are not
found in female. May self transfer. These plasmids, these F factors, are called
Transcribed by Kevin Lin Lecture Date: July 22, 2014

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conjugative because they enable the male to conjugate or mate with the female cell.
And many of them carry also genes for antibiotic resistance. And then are also
resistance transfer, genes for resistance as well.

[23] [CONJUGATION 1 -Transfer of plasmid alone]
[Dr. Boylan] Heres what happens, conjugation. Big point, first of all, is that in
conjugation, cells mate. Sometimes only the plasmid, the fertility factor is
transmitted from male to female. Thats one type of conjugation only the
plasmid. In the second type of conjugation, bridge is formed, but as a result of this
plasmid becoming a part of the chromosome of the bacterial cell, some
chromosomal bacterial genes will also be transformed exchanged during
conjugation. So sometimes, only the plasmid, other times, plasmid plus bacterial
DNA. So here we have F factor itself. Only the plasmid, this plasmid, is gonna be
transferred from the donor to the recipient as a result of conjugation occurring.
Conjugation of pilus. The bridge forms. The plasmid replicates. Its DNA. And one
of the two strandsits replicating its DNA, the plasmid isone of the two strands
goes across. While its going across, it replicates. And so we now wind up with this
kind of conjugation, both cells have a plasmid [bottom row]. So what happened
here, this female cell, recipient, F

cell. The donor is called the F

+
for possession of
the F factor. No F factor here [most top right cell]. This cell [most bottom right cell]
we called the female or F

, is now a male. Now you have two male cells, why?

Because this cell here, this recipient has picked up the fertility factor. It has
undergone a sex change operation in a way. By definition, and now it has the F
factor in it. Both of these are now male, this cell could go on and produce a sex pilus
as well. So the point here, sometimes only the plasmid can be transferred during
conjugation. But were more concerned about

[24] [CONJUGATION 2 -Transfer of bacterial genes with the help of the F factor]
[Dr. Boylan] and thats important because genes are transferred. Genes and drug
resistant genes are transferred via plasmids among bacteria all over the world. This
is the main reason we have so much of a problem with drug resistance. More and
more bacteria picking up resistance to antibiotics because plasmids carry genes that
they transfer among bacteria in types of experiments thats shownjust there, the
conjugation. Yeah, they can transfer from cells, but they also have various genes
that enable the bacteria to become resistant to antibiotics. The second type of
conjugation, transfer of bacterial genes with the help of this same F factor. F factor
integrates. So these F factors that can integrate into the chromosome are called
episomes. So in the previous slide, they werent episomes. They went and
transferred by themselves. But now the F factor, well see, is gonna integrate into
the chromosome of the bacterial cell. So here it does here. Heres the F factor
Transcribed by Kevin Lin Lecture Date: July 22, 2014

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integration. So the F factor that was separate by itself in the previous slide, now this
F factor integrates into the chromosome you see here. The green is the plasmid, the
F factor DNA. The red is the chromosomal DNA. And this bacterial cellone more
termis called the Hfr cell, the donor. This is a donor. For High frequency of
recombination. Why? It just means that this donor cell is very likely to give up its
DNA, to transfer its DNA as a result of the integrated F factor. High frequency of
recombination cell. Bacterial cell. Once it has the F factor integrated into it, its
much more likely to give up its chromosomal material than a bacterial cell without
this integrated F factor. So once again, okay. Heres this donor cell. Its male. It
formed a conjugation pilus. Heres the female cell. And it seems to be what happens
often with the integrated condition herenow replication occurs. You have the
integrated plasmid. The strands kind of separate a little bit because replication of
the strands is going to occur. So this mixture, chromosome with plasmid DNA, is
going to replicate this entire thing. Its got to be separated a little bit. And as with
bacterial chromosomal replication, it begins at where we call the origin. Note here
that the originso the two strands separate. And in the origin off replication, the
integrated plasmid is right in the middle somewhere of the plasmid. Not in the
chromosome, but once its integrated and the whole mixture begins to replicate,
somewhere along in the middle of the plasmid is where the origin of replication
begin. oriT. And as it does, one strand, the beginning strand of the plasmid is
pushed across the conjugation bridge to the recipient. And as its doing this, this one
strand is being replicated. So that it gets into the recipient cell, its double stranded,
and replication occurs. So you see, once this happens, two cells separate, you get
back to another donor Hfr cell, just like before. But in the process of this type of
conjugation, part of the plasmid DNA goes across and takes along with it some
chromosomal bacterial DNA. So the third type of gene transfer conjugation, where
bacterial DNA is transferred from donor toe recipient via a plasmid, the fertility
factor. Gets inside the recipient cell, and then sometimes it sort of breaks up and
becomesyou can see here it integrates into the recipient, the females
chromosome. Does this female cell become a male cell? Only if this process goes on
for about a 100 minutes. Its gonna take a 100 minutes for this whole complex to
replicate. To go from origin to here, to go all the way around, come back to the
beginning. And if these two cells are in contact for 100 minutes, the whole bacterial
chromosome can go across. And finally if this tail end, kind of like the--, heres the
plasmid and heres the head car on the train. Its the head car on the train. And the
other half of it, the smaller piece, is the caboose of the train. The head kind of goes
across first, leading the way. The caboose kind of pushing the rest of the bacterial
DNA across until finally a 100 minutes of contact, the whole chromosome could be--,
but usually in conjugation, this bridge formed doesnt last for more than say 15 to 20
minutes, so you only get maybe part of the chromosome of the donor into the
Transcribed by Kevin Lin Lecture Date: July 22, 2014

9
recipient. But in actuality, long stretches of DNA can possibly go from donor to
recipient. But in this case, only a portion of the donor DNA enters the recipient cell
before the conjugation bridge breaks. The new strand replaces the complementary
portion of the recipients DNA. Recombination is now complete and the new genes
can be expressed by the recipient. So now this recombined cell can express the
genes that it got from the donor over here. Okay. Itll take about 100 minutes. and
yes, if indeed, it is possible for them to be in contact for a 100 minutes or more, so
that the whole plasmid can get across, and in that case, these recipient cells will
become male cells. But ordinarily, the process is interrupted after 20 minutes, 40
minutes, and only a partsometimes very large parts of the bacterial chromosome,
all those genes can be transferred. And of the three processes, transformation,
transduction, and conjugation, this is the best process weve followed for mapping of
the chromosome. For mapping the chromosome. That meaning to tell the sequence
of genes in the chromosome of bacterial cell. How do they map? A, B, C, D, E. How
do we know that? And so we do the experiments in the lab. Done many of them.
And we let them go on for 20 minutes, 40 minutes. And we have a way to see how
much of the chromosomal DNA got across. And then we match them up piece by
piece to determine the sequence of genes in a bacterial chromosome because big
segments get across. And you interrupt the mating. Mating interrupted, they call it.
And see which genes have gotten across and which ones have not. Those are the
three types of gene transfer. Transformation, transduction, conjugation. We saw
that in conjugation, we can also get the fertility factor being transferred. But in this
case here, we see bacterial chromosomal DNA also can be transferred by
conjugation.
Oh, and one more point. Okay. This is a very unusual type of replication of
DNA. Its not bidirectional. We didnt have any idea untilwell, now its been quite
a few years. How this could possibly occur? We knew about bidirectional
replication of the bacterial chromosome. But how could that go on when this
occurred when the replicating chromosome had half of it go to the recipient cell?
And now we call thisgoing from all the way around, one direction onlythe
rolling circle. So its rolling circle of replication of the chromosome when someone
has the integrated F factor in it. Rolls around, one of the two strands goes across,
and is replicated. You have the strands staying inside the cells and its replicated.
Question?
[student] The recipient cell, does it turn to F positive after it receives the--
[Dr. Boylan] this one? Well, only if the whole plasmid got across. Looks here like it
might have. If all this green material was only due to prophage, sorry, only due to
plasmid, gets across, then it would be an F
+
cell. But here it says the recombined cell
has some bacterial DNA. I dont see the red much. It says here that its still an F

cell
because it does not have apparently all the plasmid DNA. Theres another term I
Transcribed by Kevin Lin Lecture Date: July 22, 2014

10
dont wanna get into, F-prime. But thats the key. The important key is the third
way of gene transfer. And this is going on all the time in all of us. Youll see when
Dr. Tierno talks about resistance to antibiotics and how its occurring and how they
use animal feed. And they give animals in their feed, they give them antibiotics.
Cattle, sheep. Why? Because they have an infection? No. Just to fatten them up.
Give these animals antibiotics. Maybe it makes them partly resistant to infection,
but it fattens them up. But these drugs we give animals can, well see, eventually
wind up in our gut. And bacteria that resistant to these antibiotics can wind up in
our gut. And well talk about the danger of that. The fact that its already happening.
But its the result of these processes of gene transfer among bacteria. Genes for drug
resistance, genes for toxins, genes for enzymes that are dangerous for us that
transfer among all of them all the time. And leading to more and more infections,
too, that are very becoming very difficult to treat sometimes.

[25] [Conjugation (showing the rolling circle type of replication]
[Dr. Boylan] So lets see here. The Rolling circle.

[26] [The E. coli chromosome]
[Dr. Boylan] Heres the map of E. coli chromosome. Once again, here it starts.
Plasmid begins--. And if they replicate the whole thing, itll be about a 100 minutes,
they say. Let me see if I have enough time for a story. One more thing. Okay, I like
this story. Some of you will be offended. I wont. Okay here, 100 minutes to
replicate the whole chromosome, right? Imagine, heres an E. coli cell. 20 minutes
later under the appropriate conditions (nutrition, temperature) it can divide into
two cells. So this one cell, 20 minutes later, there are two daughter cells. This one
cell, will only live for 20 minutes. the two daughter cells that are formed, they
divide in 20 minutes, you get 4 cells. Those two daughter cells, they lived, have a
lifespan of just 20 minutes. Thats their whole total existence. Theyre gone.
Daughter cells. However, when bacteria mate, have sex, they can live for 100
minutes. that proves that sex is good for you because these bacteria have a 5 times
longer lifespan when they mate. So guys, if you want to use that line, its okay. It
never worked for me. Hahaha. But its true! Sex is good for you. Well, maybe not
tonight. I dont know.

[27] [A summary of Genetic Recombination through Horizontal Gene Transfer]
[Dr. Boylan] Anyhow this ishahahaha A review of the uhh. We just went
through all this, this synopsis. Three types of gene transfer.

[28] [Figure 10: Construction of a Recombinant DNA Molecule.]
Transcribed by Kevin Lin Lecture Date: July 22, 2014

11
[Dr. Boylan] And what I want to wind up with. Oh no, I didnt have this. Sorry I
didnt show those slides. Do you have a different slide than this? A quote from TIME
magazine? About recombinant DNA. Okay. Well. What happened to that.
The last part here, just briefly. Youve already gone through this, I know, but
I would think, we wanna focus on the importance of plasmids in the world for many
different reasons, not just for transmissions of drug resistance and transmission of
pathogenic properties among bacteria, but also in recombinant DNA work.
Recombinant DNA work. Some TIME quote said something like, this is the most
important thing weve learned since the splitting of the atom. Importance of genetic
engineering. The effect it has on human kind. Its been amazing over the past 40
years what has happened. Transfer of genes. Recombining genes. What does it
mean to recombine DNA? Well essentially, in recombinant DNA workand I had
much better slides, Ill try to give them to youwe have plasmids--. So recombined
DNA means getting DNA from two different sources and combining them with each
other. One of the two sources of DNA is going to be plasmid DNA. Thats one source
always. The other source is some foreign DNA. It can be a DNA or it can be a gene
from anything. Plant, animal, human. Get a gene. So you combine this gene, this
DNA from some source, any type of cell, and combine it with plasmid DNA. Get
these two types of DNA together. Thats recombinant DNA. And that gene that you
wanna work with. Youre gonna deal with from this other source. Maybe its a gene
for insulin production. Maybe its a gene for human growth hormone. The gene for
interferon. You get that gene and you add it to a plasmid. The goal being, you want
that gene for insulin to be--, put it into a bacterial cell thats going to mass produce
insulin. So the plasmid in this case is going to be the vector, the carrier, the vector of
some important gene, recombinant DNA work, that we want to have multiplied
thousands, if not, millions of times, so that its gene product will be mass produced.
And the way we do it is get the plasmid. Heres the gene from some other source.
The key is to use the same enzyme to cleave the plasmid DNA and the foreign DNA.
And as you know, those enzymes are called restriction endonucleases. These
enzymes cleave the DNA from the plasmid and the gene at the same sites in each. As
a result of this, same bonds being broken in the DNAs, they form what are called
sticky ends. Sticky ends is shown here. And the foreign DNA and the plasmid
DNA, you combine the two together and the sticky ends stick to each other, they
round up. And they form recombinant DNA molecule. So you have the plasmid, the
foreign DNA. use restriction enzymes, like endonuclease I. Can be many different
that are used. They form these sticky ends that match up here with each other and
they recombined. Recombinant DNA molecules. So, here I have the plasmid vector.
The plasmid with this new gene in it. What happens next?

[28] [Figure 12: Developing New Products Using Genetic Engineering]
Transcribed by Kevin Lin Lecture Date: July 22, 2014

12
[Dr. Boylan] Here we go through it again. So heres the plasmid. Heres the foreign
DNA. You use an enzyme, endonuclease. That combines, forms recombinant DNA.
So you have that gene, the gene for insulin production. You wanna get a lot of
insulin produced by bacteria. What you do is you infect these cells. This is actually
called a type of transformation. You inject the recombinant DNA into E. coli cells.
You want that bacterial cell to have that plasmid that has the gene for insulin
production. This bacterial cell now reproduce over and over and over again.
Thousands and millions of copies of itself. And that gene in the plasmid also will be
turned over thousands of times as well. And that gene will produce its product. The
insulin gene, as shown in the red here, will be used over and over again to mass
produce insulin. And as a result of that, we can know very cheaply get things like
insulin and many other hormones, products thats shown on the bottom right here.
Blood clotting factors, anti-clotting factors, insulin, growth hormone, some antiviral
proteins, animal vaccines, interferon. 30 years ago, interferonyoull learn more
about what interferon doeshelps us ward off viral infections. We produce
interferons in our white blood cells. We used to isolate interferon from our human
white blood cells. It was a laborious process, took forever, and you only get
nanogram quantities of interferons. This wonder antiviral agent, we called it. Now,
as results of recombinant DNA work, we have the gene for interferon. Put it in a
plasmid, put it in a bacterial cell. We can mass produce interferon and get it very
cheaply, saving billions of dollars over the years. Same thing, its been remarkable
to discover recombinant DNA. other things as well.
And the last point I think Ill to make is that youve all gotten the hepatitis B
vaccine, right? That vaccine is result of recombinant DNA work. There used to be a
vaccine to prevent hepatitis B, but it was derived from the virus itself in the blood of
patients who had hepatitis B. So hepatitis B. Theyre sick with hepatitis B, theyve
got a lot of that virus in them. And there are these important viral particles, from
the virus itself, in their blood as well. So they would isolate the virus and these
important particles of the hepatitis B virus from the blood of these patients. Purify
them, and use that as a source of the vaccine for hepatitis B. It was great it worked.
But the people were a little hesitant about getting that vaccine. Why? Well you
know, many times, people who have hepatitis B also have AIDS. So many then the
HIV virus. What if the purification scheme was used to purify the hepatitis B antigen
was not controlled adequately, with a little bit of the HIV viruses? Well that never
happened, but it was always a fear. Stopped a lot people from getting the hepatitis B
vaccine. Now as a result of being able to get the gene for the hepatitis B, what we
call the surface antigen. The important part of the hepatitis B virus is the surface
antigen. The surface antigen. That virus has a gene for the surface antigen. Thats
the important part. Thats the important component in a vaccine. That one protein,
that one antigen. The surface antigen of hepatitis B virus. We got the gene for that
Transcribed by Kevin Lin Lecture Date: July 22, 2014

13
antigen. Put the gene in the plasmid. Put the plasmid, in this case, not in the
bacterial cell, but in the yeast cell. So I wanna point out here, not just E. coli, but
other bacteria. And in this caseI think I remember thisthe recombinant vaccine
we got the hepatitis B vaccine, result of recombinant DNA work, where the gene for
the important constituent of the virus was picked up by a plasmid vector, and then
injected into yeast cells. And the same thing happened. The yeast cells multiplied,
the plasmid multiplied, the gene for the surface antigen multiplied. And now we can
purify that and not have to worry about where the antigen came from. It didnt
come from patients who had infections. It came from chemical experiments carried
out in the lab. You get this pure vaccine. And now everybody gets it, even children
get it, we recommend. We re gonna wipe out hepatitis B, and we thought we
wouldve by now because the vaccine is so effective. Were gonna wipe out hepatitis
B if its the next thing on our list right after polio. We hope we wipe out.


[29] [Pathogenicity islands]
[Dr. Boylan] And the last thing I want to mention about genetics of bacteria is that
some bacteria, the bacteria that are pathogenic. Today were studying bacteria that
are good for us and their neighbors that are pathogens. And trying to see the
difference between the non-pathogens of members of a species, and the pathogenic
members of a species. Well, there are genes, like. What genes do, they have to make
them pathogenic, so now we can sequence genes, we can see what genes are there.
And which genes pathogens have, and non-pathogens of the same species dont
have. And the process of doing this, we find out that many of the genes for the
virulence of bacteria. The toxins they produces, the pili they produce, the flagella.
Any other properties they have that make them toxigenic or pathogenic. The genes
for those components, those factors they produce, are located on the same stretches
of bacterial chromosome. They are near each other. Theyre not just a 12 oclock, 3
oclock,7 oclock. Most of the time, theyre linked together. Theyre called
pathogenicity islands. These are genes involved with the production of virulence
factors of bacteria that make the pathogenic. And they sort of coordinate their
expression and that theyre all close to each other. Maybe adjacent operons, try
them on and off because of the fact that theyre neighboring genes, not just scattered
willy-nilly throughout the chromosome. So pathogenic. To the islands, theyre
gonna be transfer of other bacteria. Coordinated expressed or repressed. Okay, any
questions? Go over that overview. And I guess about bacterial genetics and email
me if you do. And Ill be here on Thursday to introduce Dr. Tierno.