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Good morning class,
nice to see you again.

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I trust you had a relaxing
weekend and a Happy Halloween,

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whatever a Happy Halloween is.
You recall that last time we were

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talking about the process
of cell transformation,

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and recall, what I said was that
transformation represents the

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conversion of a normal
cell into a cancer cell.

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In fact, there are a variety of
traits of a cell which suggest it's

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a cancer cell, it
changes its shape,

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it can get along with [foralized?
growth factors. Normal cells

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require tethering to the bottom
of a Petri dish in order to grow.

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Cancer cells, you can put
them into a semi-solid medium,

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like agar, and cancer cells
will often grow like this,

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as colonies in suspension,
without direct tethering, without

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direct adhesion to a
solid, underlying substrate.

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And that, that's a trait of cancer
cells, the phenotype of cancer cells,

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is sometimes called,
anchorage independence.

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But ultimately, the best litmus
test, whether a cell is truly

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transformed, is tumoraganisity,
i.e. the ability of a cell, when

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plucked out of a Petri-dish like
this, and implanted into a host

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mouse, to actually
grow into a tumor.

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So there are various gradations
of becoming transformed,

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but tumoraganisity is the ultimate
arbiter of whether or not a cell is

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truly transformed. Now,
you'll recall from our

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conversation last time that
if you put a transformed cell,

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a mixed a mono-layer of normal
cells, that the transformed cell will

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overgrow the mono-layer, it will
have lost contact inhibition,

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and that when viewed from above,
such a Petri-dish yields a thick

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clump of cells, which
is called a focus,

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plural, foci. And I will
tell you that beginning

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in the late 1960's, one
began to use a variety of

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different techniques, with
which to transform cancer cells

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into normal cells. One of
the techniques one used was

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to apply chemical carcinogens to
cells, and keep in mind that we're

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reserving the word,
carcinogen, for a chemical or a

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physical agent that causes
cancer. Ultraviolet radiation is a

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carcinogen, as are x-rays,
and there are many chemical

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carcinogens, such as
those in tobacco smoke.

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And certain experiments in the
early 1970's began to reveal that one

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could get foci of transformed cells,
by applying chemical carcinogen to

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the cell in-vitro, and
when I say in-vitro,

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I mean growing here, in the
Petri dish. And, in fact,

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one could begin to use a whole
variety of different types of

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carcinogens, chemical carcinogens,
and what seemed to be shared in

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common between all these carcinogens
was that they were all mutagenic.

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By mutagenic, I clearly mean the
ability to inflict damage on the

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genome of the cells that were being
exposed to these various compounds.

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In fact, one could draw an
interesting correlation because many

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of these mutagenic compounds had,
also by chance, been tested in

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laboratory animals for
their carcinogenicity.

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And so plots were derived in the
mid-1970's, between the mutagenic

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potential of a compound, and
the carcinogenic potential of a

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compound. And when
I say a carcinogen,

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how carcinogenic is a compound, I
mean, how many milligrams of this

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compound does it take
to make a tumor? And so,

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what one could do, is
plot over a log-log scale,

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how many milligrams of a
given compound was required,

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or micrograms, to make a
tumor in a rat or a mouse.

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And, at the same time, how
mutagenic were these compounds,

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i.e. how potent were they in
their ability to inflict damage

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on the genome?
Because it turns out,

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that if this log-log scale
is by orders of magnitude,

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powers of ten, compounds range
over at least five or six orders of

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magnitude in their potency
in inflicting mutations,

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and similarly, in their potency in
inducing tumors in mice and rats.

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And what one found was the
following, that there was a log-log

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relationship that was roughly linear,
but there was violations to this.

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Some compounds were extraordinarily
mutagenic, they could create

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mutations in very low doses,
and at the same time could create

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tumors, they were
carcinogenic in very low doses.

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Other compounds required an enormous
amount of material in order to

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induce mutations on the abscissa,
and an enormous amount of material

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in order to induce tumors on
the ordinate. And this log-log

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relationship over five orders of
magnitude, suggested the following

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obvious idea, that carcinogens are
mutagenic, and to the extent that

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carcinogens are able to induce
cancer, they do so through their

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ability to inflict mutational
damage on the cells within certain

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target tissues.
And this, therefore,

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obviously suggested the notion
that within cancer cells,

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as we said last time,
there are mutant genes,

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and that these mutant genes are
moreover instrumental in conferring

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the transformed phenotype on the
cells that carry these mutant genes.

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In the, in the late 1960's and
early 1970's, a man named Howard

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Temin, began to use a virus
called Rous sarcoma virus,

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which had first been discovered
in 1909 by a man named Peyton Rous.

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Rous was then a professor
at the Rockefeller Institute,

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later the Rockefeller University,
in New York. A Long Island chicken

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farmer brought in a prized hen
of his who had been growing a big

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muscle tumor, or sarcoma, in her
breast muscle, and asked Rous,

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the famous chicken doctor,
if he could cure this chicken,

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and Rous said, thank you,
cut off the hen's head,

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extracted the tumor,
and ground up the tumor,

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and after having homogenized
the tumor, passed the homogenate

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through a filter. And
this filter would trap all

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cellular material, but it
would allow non-cellular

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material, or material that was
smaller than the size of a cell,

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to pass right through. And so,
therefore, Rous took the material

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that passed through the filter,
which you can call the filtrate,

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and he injected the filtrate,
which passed through the filter,

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into a young chick. And what he
observed thereafter was that chick

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soon came down with sarcoma
after a period of some months,

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and when he ground up the tumor in
that chick, and once again injected

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into another chick, he
once again got a tumor.

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The fact that the agent,
which was inducing the cancer,

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and could be transmitted
from one animal to another,

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from one chicken to the other,
was filterable, suggested it was

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extremely small, and at
the time one had already

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begun to appreciate the fact that
there were sub-cellular infectious

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agents, which we now call viruses.
And Peyton Rous made this very

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important discovery in 1909,
1910, and in 1965 he was awarded a

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Nobel Prize for this.
It's a rather long wait,

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wouldn't you say? So he
only had to wait 55 years,

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anyhow, he died a happy man,
we can only presume. Now, what's

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interesting about this is the
life cycle of Rous Sarcoma virus.

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As we will discuss in greater
detail later, viruses are

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sub-cellular particles, they
don't have their own energy

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metabolism, and they parasitize on
the macromolecular machinery of the

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cell that they infect. And
therefore, what we can imagine

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here is the following scenario,
which actually happens to be true.

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A virus particle, which is
vastly smaller than a cell,

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enters the cell, the virus particle
carries into the cell its own genome,

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and this genome, in this
case of Rous Sarcoma virus,

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is single-stranded RNA, and this
genome, which is carried into the

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cell, carries the information
for making more virus particles.

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And what happens is, in the
case of Rous Sarcoma virus,

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as Temin later speculated in a
speculation that caused him ridicule

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and ostracism for many years, that
the single-stranded RNA of Rous

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Sarcoma virus, once
it gets into the cell,

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is reverse-transcribe, i.e.
copied into DNA molecules.

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So now we have double-stranded DNA,
a double-stranded DNA copy of the

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viral genome, and this
double-stranded DNA molecule,

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which came to be called a pro-virus,
then became integrated into the host

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chromosomal DNA. So
here's the host-chromosome.

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And now, the pro-virus, which
I'll depict here in the middle,

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in white, became physically inserted
as a double-stranded DNA molecule,

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it was slipped right into the
genome. We now realize that any,

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any of tens, of hundreds, of
millions of different sites in

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the genome of the host cell.
And this pro-virus, once

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established or integrated into
the genome, could thereafter,

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function essentially like
a cellular gene, i.e. from a

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molecular-biological perspective,
it was indistinguishable from a

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cellular gene, it was
double-stranded DNA,

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it had a promoter it carried
in a promoter with it,

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and it had a polyadenylation
signal, and therefore, this pro-virus

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thereafter, could use, or
parasitize, the host-cell RNA

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polymerase 2, to make
viral messenger RNA,

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on the one hand, and
progeny-genomic RNA.

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Now when I say genomic, I'm
not talking about the host-cell

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genome, I'm talking about the
viral genome. How big is the virus?

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Well, it's about nine or
ten kilo-bases in length,

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so it's genome, obviously,
is vastly smaller than the 3.

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mega- bases that constitute
the haploid human genome.

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The viral mRNA, once transcribed
in the nucleus, and exported to the

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cytoplasm, could make viral proteins,
and the viral proteins could then be

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used to encapsidate, and when
I use the word encapsidate,

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keep in mind, I never use a
simple word when a polysyllabic

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one is possible. So when
I, when the viral proteins

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encapsidate the viral-RNA, you
get a virus particle like this,

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which has virus proteins on the
outside, almost on the outside,

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viral RNA in the middle,
single-stranded RNA,

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and this single-stranded RNA
comes from the transcription of the

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pro-virus that has now been
integrated into the genome.

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Integration is an important
concept here, i.e. it becomes

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covalently linked. And
this suggests to us that the

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virus actually encodes
several specialized proteins.

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One set of specialized
proteins is required for the

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reverse-transcription, and
in fact, the virus actually

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carries with it, into the
cell, not only its RNA,

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but also, reverse transcriptase. So
if you isolate the virus particle,

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it has, in addition to this coat,
it has within it already reversed

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transcriptase, molecules,
so that the moment that

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this virion, a virion
is a virus-particle.

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The moment that this virion, or
virus-particle, penetrates into

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the cytoplasm of the cell,
there is then, immediately,

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an abundant supply of the
deoxyribonucleoside triphosphates,

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the process of reverse-transcription
can begin, the double-stranded DNA

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can be produced,
exported to the cytoplasm,

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where a second viral protein is
responsible for integrating the

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resulting double-stranded
reverse transcript into the

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host-cell genome. Again,
that's a highly specialized

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function. The forward
transcription, we just talked about

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reverse-transcription, but
the forward-transcription to

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make progeny RNA, obviously
can rely on the host cell

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polymerase, the virus doesn't need
to make that. The viral mRNA can be

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translated by host cell
ribosomes in the cytoplasm,

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the virus doesn't need to make that.
Some of the viral aren't, the new

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viral RNA, is genomic RNA, which
as I say, becomes encapsidated

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to make progeny virus particles.
And a cell, which is infected in

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this way, can suffer two fates.
It could be, as is in the case with

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many viruses like this, that
the viruses, that the cell is

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not actually killed
by this infection, i.e.

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that the cell can tolerate an
infectious, an infection like this,

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and therefore, if you look at such
a cell, for days and weeks later,

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it will be producing virus particles,
which are being released from the

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cell, continuously, continually
released from the cell,

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and are able then, to pass
and infect yet other cells.

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The alternative to this is what
is called a cytopathic effect,

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and the truth of the matter is,
many kinds of viruses, when they go

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and infect a cell, and
they produce their progeny,

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they end up killing the
cell that they've infected.

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So, for example, when we
get infected by a cold virus,

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which has a different
metabolism than this one,

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by the way, then the cells that are
infected and produce progeny-virus

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particles are rather quickly killed,
as a consequence of the infection,

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which is why we have
damage to our nasal mucosa.

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But in this case, in the
case of Temin's virus,

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actually RSV, Rous Sarcoma Virus,
that isn't the case. And therefore,

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in fact, one has RSV particles that
are continually being released from

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the cell. If one looks under
the electron microscope,

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at the surface of a virus-infected
cell, one sees structures like this.

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Where here is in the green
is the lipid by-layer,

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the plasma membrane of the
cell. And here, in the middle,

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is a viral-protein capsid,
a protein capsid like this,

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encoded by the virus, and
carried in the capsid is actually,

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are actually, the
viral-RNA molecules, and the

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reverse-transcriptase molecule.
And in saying that, what I mean to

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suggest to you, is that
actually the viral particle,

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the virion, is slightly more
complicated than I represented

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it to be. As the virus,
as progeny particles

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are made, they are pushed out
through the plasma membrane of the

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infected cell,
on which occasion,

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00:15:23,000 --> 00:15:26,000
they become enveloped with
a layer of plasma membrane.

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00:15:26,000 --> 00:15:30,000
And this layer of plasma membrane
is actually stolen as a patch from

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00:15:30,000 --> 00:15:34,000
the plasma membrane of the
infected cell. So in truth,

224
00:15:34,000 --> 00:15:37,000
actually, a virus particle like RSV,
has some membrane around it, it has

225
00:15:37,000 --> 00:15:41,000
a protein-capsid encoded by the
viral mRNA, and in the middle it has

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00:15:41,000 --> 00:15:45,000
RNA and reverse
transcriptase molecules.

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00:15:45,000 --> 00:15:49,000
Note, by the way, that
most viruses actually,

228
00:15:49,000 --> 00:15:53,000
everything that the virus carries,
the virus has encoded in it's own

229
00:15:53,000 --> 00:15:57,000
genome, in this case, the
virus has stolen, has absconded,

230
00:15:57,000 --> 00:16:02,000
with a patch of plasma
membrane from the infected cell.

231
00:16:02,000 --> 00:16:06,000
Now what Temin observered,
several other people before him had

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00:16:06,000 --> 00:16:10,000
done so, was that when he infected
a mono-layer of chicken cells with

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00:16:10,000 --> 00:16:15,000
Rous Sarcoma virus,
he was able to in fact,

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00:16:15,000 --> 00:16:19,000
observe the appearance of
foci of transformed cells.

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00:16:19,000 --> 00:16:23,000
And he observed that these foci of
transformed cells actually released

236
00:16:23,000 --> 00:16:27,000
Rous Sarcoma Virus. So, the
infection by Rous Sarcoma

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00:16:27,000 --> 00:16:31,000
Virus, had led to the production
of progeny virus particles,

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00:16:31,000 --> 00:16:35,000
which we kind of expect of a virus.
Keep in mind that if a virus can't

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00:16:35,000 --> 00:16:39,000
produce progeny,
it's out of business.

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00:16:39,000 --> 00:16:42,000
Or, to put it another way, the
only thing a virus is really

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00:16:42,000 --> 00:16:46,000
interested in, is making
more copies of itself.

242
00:16:46,000 --> 00:16:50,000
So, the cells in these foci of
transformants were transformed,

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00:16:50,000 --> 00:16:54,000
but they were also releasing
progeny virus particles.

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00:16:54,000 --> 00:16:57,000
And if you took these virus
particles, and he could isolate them

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00:16:57,000 --> 00:17:01,000
away from any contaminating cell,
the way Peyton Rous did, just by

246
00:17:01,000 --> 00:17:04,000
filtering them, or the
filter will trap all of the

247
00:17:04,000 --> 00:17:08,000
cells, and allow the much tinier
virus particles to pass through,

248
00:17:08,000 --> 00:17:11,000
then he could take these virus
particles and infect another plate

249
00:17:11,000 --> 00:17:15,000
of cells, and once again, he
would get foci of transformants.

250
00:17:15,000 --> 00:17:19,000
And therefore, this virus
was actually bipotential, it

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00:17:19,000 --> 00:17:22,000
could do two things.
It could replicate,

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00:17:22,000 --> 00:17:26,000
here I've been talking about the
life cycle or the replication-cycle

253
00:17:26,000 --> 00:17:29,000
of the virus, on the one hand,
and on the other hand, it could

254
00:17:29,000 --> 00:17:32,000
transform cells. And
subsequent work demonstrated

255
00:17:32,000 --> 00:17:36,000
that actually, the
replicated functions of Rous

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00:17:36,000 --> 00:17:39,000
Sarcoma Virus,
on the one hand,

257
00:17:39,000 --> 00:17:43,000
and the transforming functions
of Rous Sarcoma Virus,

258
00:17:43,000 --> 00:17:46,000
on the other hand, were
encoded in separable genes.

259
00:17:46,000 --> 00:17:49,000
For example, Howard Temin
was able to demonstrate,

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00:17:49,000 --> 00:17:53,000
and others later, that one
could get mutants of RSV

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00:17:53,000 --> 00:17:56,000
that had lost the ability
to transform cells,

262
00:17:56,000 --> 00:18:00,000
but could still
replicate perfectly well.

263
00:18:00,000 --> 00:18:03,000
And there were yet other mutants
that had lost the ability to

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00:18:03,000 --> 00:18:07,000
replicate, but could
transform perfectly well.

265
00:18:07,000 --> 00:18:11,000
And so there were two
classes of specialized genes,

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00:18:11,000 --> 00:18:15,000
one involved in replication,
the other in transformation. In

267
00:18:15,000 --> 00:18:18,000
1975 and 1976, the
laboratories of Harold Varmus

268
00:18:18,000 --> 00:18:22,000
and Mike Bishop, at
University of California,

269
00:18:22,000 --> 00:18:26,000
San Francisco, or UCSF, as
it's called in the trade,

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00:18:26,000 --> 00:18:30,000
began to exam the origin of
the viral transforming gene.

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00:18:30,000 --> 00:18:34,000
Now the viral transforming gene,
because it was assumed there was

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00:18:34,000 --> 00:18:38,000
only one of them, the
genome is so small,

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00:18:38,000 --> 00:18:42,000
it only has about ten kilo-basis,
and only enough room for three, or

274
00:18:42,000 --> 00:18:46,000
four, or five, genes in
it, not a hundred or a

275
00:18:46,000 --> 00:18:50,000
thousand, the viral transforming
gene came to be called SRC,

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00:18:50,000 --> 00:18:54,000
S-R-C. And what they
observed was the following,

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00:18:54,000 --> 00:18:58,000
they made a radio-labeled probe,
which was specific for the SRC gene.

278
00:18:58,000 --> 00:19:02,000
And they could use
the radio-labeled

279
00:19:02,000 --> 00:19:08,000
probe to anneal to two kinds of
viruses, wild-type Rous Sarcoma

280
00:19:08,000 --> 00:19:14,000
Virus, and a deletion mutant, I'll
use the Greek -delta, a version

281
00:19:14,000 --> 00:19:20,000
of Rous Sarcoma Virus, which
was lacking, which apparently

282
00:19:20,000 --> 00:19:26,000
through process of genetic deletion,
was lacking the ability to transform

283
00:19:26,000 --> 00:19:32,000
cells. And that loss of ability to
transform cells was ostensibly due

284
00:19:32,000 --> 00:19:38,000
to the lesion of it's
genome of the SRC gene.

285
00:19:38,000 --> 00:19:41,000
And what they observed is that the
SRC, the radio-labeled SRC probe,

286
00:19:41,000 --> 00:19:45,000
as one would've hoped, was able
to anneal to this wild-type genome,

287
00:19:45,000 --> 00:19:49,000
but it couldn't anneal to
the deletion mutant of RSV,

288
00:19:49,000 --> 00:19:53,000
which had lost the SRC gene through
a process of genetic deletion.

289
00:19:53,000 --> 00:19:56,000
So, so far, so good. By the way,
the fact that the SRC gene could

290
00:19:56,000 --> 00:20:00,000
transform cells, led
to it's being called,

291
00:20:00,000 --> 00:20:04,000
an oncogene. The term "oncos" in
Greek means, a tumor or a lump,

292
00:20:04,000 --> 00:20:08,000
an oncogene therefore,
was a cancer-causing gene.

293
00:20:08,000 --> 00:20:11,000
And therefore, Rous
Sarcoma Virus possessed at

294
00:20:11,000 --> 00:20:15,000
least one cancer-causing gene,
or oncogene. Now the mind-blowing

295
00:20:15,000 --> 00:20:19,000
result that happened shortly
thereafter, was the following.

296
00:20:19,000 --> 00:20:22,000
People in the Varmus-Bishop lab
began to look for the origins of the

297
00:20:22,000 --> 00:20:26,000
SRC oncogene of Rous Sarcoma Virus.
It turns out that the vast majority

298
00:20:26,000 --> 00:20:30,000
of genes that have been in virus,
that are present in viral genomes,

299
00:20:30,000 --> 00:20:34,000
have been in viral genomes,
as far as we know, for the last

300
00:20:34,000 --> 00:20:38,000
billion years. i.e. we
have every reason to think

301
00:20:38,000 --> 00:20:42,000
that the evolutionary origins
of viruses can be traced into the

302
00:20:42,000 --> 00:20:46,000
distant past. It could even
be the case, some people think,

303
00:20:46,000 --> 00:20:50,000
some perfectly sane people think,
that this whole retro-virus life

304
00:20:50,000 --> 00:20:55,000
cycle that I just told you about,
recapitulates one of the earliest

305
00:20:55,000 --> 00:20:59,000
stages of cellular evolution on
the planet. People believe now,

306
00:20:59,000 --> 00:21:03,000
with ever-increasing conviction,
and keep in mind, class, people who

307
00:21:03,000 --> 00:21:08,000
are convinced of something are
usually wrong, in a loud voice.

308
00:21:08,000 --> 00:21:11,000
But people believe with
ever-increasing conviction,

309
00:21:11,000 --> 00:21:15,000
that the first cells on earth
actually had RNA genomes,

310
00:21:15,000 --> 00:21:19,000
rather than DNA genomes, and that
the invention of double-stranded DNA

311
00:21:19,000 --> 00:21:23,000
genomes in cellular life forms,
came later. And if it did, then the

312
00:21:23,000 --> 00:21:26,000
conversion from an RNA to a DNA
state is reflected in the modern

313
00:21:26,000 --> 00:21:30,000
life cycle of Rous Sarcoma Virus,
and similar viruses, which as you

314
00:21:30,000 --> 00:21:34,000
may know, have come to be called
retroviruses, simply because they

315
00:21:34,000 --> 00:21:38,000
transcribe their
nucleic acid backwards.

316
00:21:38,000 --> 00:21:41,000
So, Varmus and Bishop were
interested in the origins of the SRC

317
00:21:41,000 --> 00:21:45,000
oncogene that was carried
by Rous Sarcoma Virus.

318
00:21:45,000 --> 00:21:49,000
I say, well it probably,
the Rous Sarcoma Virus, had

319
00:21:49,000 --> 00:21:53,000
antecedents, which existed
thousands and millions of years ago,

320
00:21:53,000 --> 00:21:56,000
and carried the SRC oncogene.
But the fact of the matter is,

321
00:21:56,000 --> 00:22:00,000
the SRC, the Rous Sarcoma Virus,
had only been isolated once in the

322
00:22:00,000 --> 00:22:04,000
20th century, when this
very trusting, and caring,

323
00:22:04,000 --> 00:22:08,000
Long Island chicken farmer
came in to Peyton Rous,

324
00:22:08,000 --> 00:22:12,000
hoping that Rous would cure his
chicken, rather than cutting the

325
00:22:12,000 --> 00:22:16,000
chicken's head off.
So what happened,

326
00:22:16,000 --> 00:22:21,000
then, was the following. They
used this radiolabeled probe

327
00:22:21,000 --> 00:22:26,000
to look at the DNA of infected,
RSV-infected chicken cells, and

328
00:22:26,000 --> 00:22:30,000
uninfected chicken cells.
So they probed the DNA of the

329
00:22:30,000 --> 00:22:35,000
infected chicken cells,
with this radiolabeled probe,

330
00:22:35,000 --> 00:22:40,000
and they probed the DNA
with uninfected chicken cell,

331
00:22:40,000 --> 00:22:45,000
of uninfected chicken cells,
once again with this radiolabeled

332
00:22:45,000 --> 00:22:49,000
probe. And
what they,

333
00:22:49,000 --> 00:22:52,000
what they expected to find was
the following, it's obvious,

334
00:22:52,000 --> 00:22:55,000
in uninfected chicken cells
you don't find any SRC,

335
00:22:55,000 --> 00:22:59,000
and in infected chicken cells,
you do find SRC, because the SRC

336
00:22:59,000 --> 00:23:02,000
gene has been brought into the
infected cells by the infecting

337
00:23:02,000 --> 00:23:05,000
viral genome. Stands to reason,
right? Shouldn't be any in the

338
00:23:05,000 --> 00:23:08,000
uninfected cells, after
the cell's infected,

339
00:23:08,000 --> 00:23:11,000
now they have a SRC, at least one
copy, they may have multiple copies

340
00:23:11,000 --> 00:23:15,000
of the SRC oncogene, because
I haven't really dictated

341
00:23:15,000 --> 00:23:18,000
how many pro-viruses should
be integrated into the genome

342
00:23:18,000 --> 00:23:22,000
of an infected cell. And
what they found was puzzling,

343
00:23:22,000 --> 00:23:27,000
and eventually mind-blowing.
Because they found that in the DNA

344
00:23:27,000 --> 00:23:33,000
of uninfected chicken cells,
they could find a SRC gene, and

345
00:23:33,000 --> 00:23:38,000
these uninfected chicken cells had
never experienced Rous Sarcoma Virus

346
00:23:38,000 --> 00:23:43,000
in any form, whatsoever. And
as a consequence, they began to

347
00:23:43,000 --> 00:23:48,000
develop a theory, a
model, which turned out,

348
00:23:48,000 --> 00:23:54,000
actually, to be right on,
and the model was as follows,

349
00:23:54,000 --> 00:23:59,000
that there was a retrovirus,
like Rous Sarcoma Virus, that was

350
00:23:59,000 --> 00:24:04,000
the precursor of RSV, and
this retrovirus had replication

351
00:24:04,000 --> 00:24:10,000
genes, but it lacked a
transforming oncogene.

352
00:24:10,000 --> 00:24:14,000
This retrovirus went into a chicken
cell, and when it emerged from the

353
00:24:14,000 --> 00:24:18,000
chicken cell, it carried not
only the replication genes,

354
00:24:18,000 --> 00:24:22,000
but now the SRC oncogene.
It had acquired a new gene,

355
00:24:22,000 --> 00:24:26,000
which it could then use to
subsequently transform other cells

356
00:24:26,000 --> 00:24:30,000
that it had infected. And
this, itself, turned out to be

357
00:24:30,000 --> 00:24:34,000
absolutely right. This
SRC gene was of cellular

358
00:24:34,000 --> 00:24:38,000
origin, and in fact,
homologs of the SRC gene,

359
00:24:38,000 --> 00:24:42,000
were present in all vertebrates,
in all chordates, in all metazoan,

360
00:24:42,000 --> 00:24:46,000
there's even a distant homolog
of the SRC gene that's present in

361
00:24:46,000 --> 00:24:50,000
sponge cells, which are
obviously rather primitive.

362
00:24:50,000 --> 00:24:54,000
So this SRC gene is not a recent
invention, it's been sitting around

363
00:24:54,000 --> 00:24:58,000
in the eukaryotic genome, at
least in the genome of metazoan

364
00:24:58,000 --> 00:25:02,000
and its precursors,
for a very long time.

365
00:25:02,000 --> 00:25:07,000
It was kidnapped, picked up
by the Rous Sarcoma Virus,

366
00:25:07,000 --> 00:25:11,000
and subsequently exploited by the
virus to transform cells that it

367
00:25:11,000 --> 00:25:15,000
happened to infect. And this
acquisition and activation

368
00:25:15,000 --> 00:25:19,000
of a gene was obviously a rare event,
because RSV, as I've just told you,

369
00:25:19,000 --> 00:25:24,000
was only picked up once,
was only generated once.

370
00:25:24,000 --> 00:25:28,000
It didn't exist in nature, and
Rous Sarcoma Virus was never

371
00:25:28,000 --> 00:25:32,000
seen to, go from one chicken flock
to the other, like most infectious

372
00:25:32,000 --> 00:25:36,000
agents. The ecology of Rous Sarcoma
Virus is not so much of interest to

373
00:25:36,000 --> 00:25:41,000
us, what is of greatest interest
to us in our discussion today,

374
00:25:41,000 --> 00:25:45,000
is the following notion that,
within the normal genome of a

375
00:25:45,000 --> 00:25:49,000
chicken cell, there exists a normal
gene which came to be called a

376
00:25:49,000 --> 00:25:54,000
proto-oncogene, a
precursor of the,

377
00:25:54,000 --> 00:25:58,000
A proto-oncogene which
resides in normal chicken DNA,

378
00:25:58,000 --> 00:26:02,000
and the fact that the
proto-oncogene is highly conserved,

379
00:26:02,000 --> 00:26:06,000
evolutionary, dictates to
us that this proto-oncogene,

380
00:26:06,000 --> 00:26:10,000
this SRC proto-oncogene, must
mediate essential functions,

381
00:26:10,000 --> 00:26:14,000
otherwise it wouldn't long
ago been lost. In fact,

382
00:26:14,000 --> 00:26:18,000
just to repeat myself,
virtually identical copies of the

383
00:26:18,000 --> 00:26:22,000
SRC oncogene, proto-oncogene,
excuse me, lie, can be found in the

384
00:26:22,000 --> 00:26:26,000
genomes of all vertebrates.
So, a proto-oncogene is a normal

385
00:26:26,000 --> 00:26:30,000
cellular, growth-regulating gene,
which, on this occasion, became

386
00:26:30,000 --> 00:26:34,000
activated, and subverted,
and converted into an active

387
00:26:34,000 --> 00:26:39,000
transforming gene, i.e.
an oncogene. So the term

388
00:26:39,000 --> 00:26:43,000
-proto in this case,
implies a normal gene,

389
00:26:43,000 --> 00:26:47,000
which has the potential,
under certain circumstances,

390
00:26:47,000 --> 00:26:51,000
to become an active oncogene.
In the years that followed,

391
00:26:51,000 --> 00:26:55,000
it's been almost 30 years now,
more than 30 proto-oncogenes have

392
00:26:55,000 --> 00:26:58,000
been discovered, by looking
at retroviruses like RSV.

393
00:26:58,000 --> 00:27:02,000
SRC is not the only proto-oncogene
that lies in our genome,

394
00:27:02,000 --> 00:27:05,000
and therefore, we begin to
appreciate on the basis of this,

395
00:27:05,000 --> 00:27:09,000
that our genome carries a
whole repertoire of these

396
00:27:09,000 --> 00:27:13,000
growth-regulating genes that,
when a retrovirus happens to swoop

397
00:27:13,000 --> 00:27:16,000
in, can be activated into active
oncogenes, they can be converted

398
00:27:16,000 --> 00:27:20,000
into active oncogenes, and
thereafter, they can induce

399
00:27:20,000 --> 00:27:24,000
cancer. And this
obviously leads to the

400
00:27:24,000 --> 00:27:28,000
suggestion that the seeds
of human cancer don't lie,

401
00:27:28,000 --> 00:27:33,000
necessarily, on the outside of cells
because most kinds of human cancers

402
00:27:33,000 --> 00:27:38,000
are not caused by infecting viruses.
That was a puzzle that was already

403
00:27:38,000 --> 00:27:42,000
apparent in the late-1970's. If
Rous Sarcoma Virus, or similar

404
00:27:42,000 --> 00:27:47,000
viruses, could not be invoked
to explain many kinds of viruses,

405
00:27:47,000 --> 00:27:52,000
how could one get cancer? And this
work suggested an obvious solution.

406
00:27:52,000 --> 00:27:55,000
Let's imagine that there
are a repertoire of a dozen,

407
00:27:55,000 --> 00:27:58,000
or two-dozen, or three-dozen,
proto-oncogenes that reside in our

408
00:27:58,000 --> 00:28:01,000
normal genome, their purpose
there is not to create

409
00:28:01,000 --> 00:28:05,000
cancer, their purpose is to
regulate normal cell proliferation.

410
00:28:05,000 --> 00:28:08,000
These genes, being genes,
are subject to damage,

411
00:28:08,000 --> 00:28:11,000
to mutation, and therefore we
can imagine that in cases of human

412
00:28:11,000 --> 00:28:15,000
cancer, where there are no viruses
involved, there can be genetic

413
00:28:15,000 --> 00:28:18,000
alteration of the DNA sequences
of a proto-oncogene that converted

414
00:28:18,000 --> 00:28:22,000
into an oncogene. i.e
chemical changes to the DNA,

415
00:28:22,000 --> 00:28:27,000
mutagenic changes to the DNA,
can mimic the conversion of a

416
00:28:27,000 --> 00:28:32,000
proto-oncogene to an oncogene,
without any virus. There are other

417
00:28:32,000 --> 00:28:37,000
ways by which you can skin this cat.
And one experiment to demonstrate

418
00:28:37,000 --> 00:28:42,000
that is the following. Did we
talk at the end of last time

419
00:28:42,000 --> 00:28:47,000
about the guy who got a bladder
carcinoma after 40 years of smoking?

420
00:28:47,000 --> 00:28:51,000
I'm glad we did. Good. So,
let's say this person gets a,

421
00:28:51,000 --> 00:28:55,000
has a bladder carcinoma, and
he got the bladder carcinoma,

422
00:28:55,000 --> 00:28:59,000
and it was called an EG bladder
carcinoma, and he got it for reasons

423
00:28:59,000 --> 00:29:03,000
we described last time, and
I can't imagine you have any

424
00:29:03,000 --> 00:29:07,000
illusions about whether
smoking is good or bad for you,

425
00:29:07,000 --> 00:29:10,000
after last time. But anyhow,
so, EG bladder carcinoma, pig DNA

426
00:29:10,000 --> 00:29:14,000
from the tumor, so
we make tumor DNA.

427
00:29:14,000 --> 00:29:18,000
And now, and by the way, we
presume correctly that viruses

428
00:29:18,000 --> 00:29:22,000
have nothing to do with
this particular cancer,

429
00:29:22,000 --> 00:29:26,000
with the development of this cancer,
and by the way, the development of a

430
00:29:26,000 --> 00:29:30,000
disease is another wonderful
polysyllabic Greek word,

431
00:29:30,000 --> 00:29:34,000
pathogenesis. Pathogenesis
means the study of how

432
00:29:34,000 --> 00:29:38,000
a disease is caused, what
generates the disease.

433
00:29:38,000 --> 00:29:42,000
So the pathogenesis of bladder
carcinoma has nothing whatsoever to

434
00:29:42,000 --> 00:29:46,000
do with any viruses. Maybe
it had to do with the fact

435
00:29:46,000 --> 00:29:50,000
that cigarette smoke mutated genes,
mutated, proto-oncogenes in the DNA

436
00:29:50,000 --> 00:29:54,000
of Mr. Jones, that happens
to have been his name,

437
00:29:54,000 --> 00:29:58,000
or his pseudonym, who knows,
Mr. Jones' bladder cells.

438
00:29:58,000 --> 00:30:02,000
So one takes
tumor DNA here,

439
00:30:02,000 --> 00:30:06,000
and one uses the procedure of
transfection, where you take the DNA,

440
00:30:06,000 --> 00:30:10,000
make a DNA, and you put it into
normal cells, by a gene transfer,

441
00:30:10,000 --> 00:30:14,000
or a transfection procedure.
So, this is transfection,

442
00:30:14,000 --> 00:30:18,000
or gene transfer, these are
equally applicable names,

443
00:30:18,000 --> 00:30:23,000
and what was found on this
occasion was, that one got foci of

444
00:30:23,000 --> 00:30:27,000
transformed cells. And
these foci, for all practical

445
00:30:27,000 --> 00:30:31,000
purposes, looked just like the foci
that Howard Temin had gotten years

446
00:30:31,000 --> 00:30:35,000
earlier, by infecting monolayers
of chicken cells, with Rous

447
00:30:35,000 --> 00:30:39,000
Sarcoma Virus.
And therefore,

448
00:30:39,000 --> 00:30:43,000
that suggested very strongly,
that the DNA of the bladder

449
00:30:43,000 --> 00:30:47,000
carcinoma, carried within it,
an oncogene that was capable of

450
00:30:47,000 --> 00:30:50,000
transforming these recipient
cells, into which the DNA had been

451
00:30:50,000 --> 00:30:54,000
introduced by the transfection
procedure. It remained,

452
00:30:54,000 --> 00:30:58,000
of course, to actually find that.
I'll mention to you in passing,

453
00:30:58,000 --> 00:31:02,000
that one does a
control experiment here.

454
00:31:02,000 --> 00:31:05,000
If you take normal DNA, you do
the exact same experiment you

455
00:31:05,000 --> 00:31:09,000
never get foci. So that
means, it's not as if all

456
00:31:09,000 --> 00:31:12,000
human DNA carries oncogenes in
it. The normal DNA doesn't give you

457
00:31:12,000 --> 00:31:16,000
foci, the DNA from the bladder
carcinoma does give you foci.

458
00:31:16,000 --> 00:31:19,000
And so, about 20 years ago, one
actually looked at the normal DNA

459
00:31:19,000 --> 00:31:23,000
and the tumor DNA, and one
came up with the following,

460
00:31:23,000 --> 00:31:26,000
that the bladder carcinoma oncogene,
was about, let's say, 6KB long,

461
00:31:26,000 --> 00:31:30,000
there was a corresponding,
normal proto-oncogene.

462
00:31:30,000 --> 00:31:36,000
It was also 6 kilobasis long.
This was a normal DNA, this was

463
00:31:36,000 --> 00:31:43,000
extracted by cloning, gene
cloning, from the genome of the

464
00:31:43,000 --> 00:31:50,000
bladder carcinoma. And
having extracted it,

465
00:31:50,000 --> 00:31:57,000
one then began to look at how
different these two genes were from

466
00:31:57,000 --> 00:32:03,000
one another. This transformed
cells, this did not transform cells.

467
00:32:03,000 --> 00:32:10,000
If one did restriction enzyme
mapping, one found the identical

468
00:32:10,000 --> 00:32:15,000
array of restriction enzyme sites.
So it was clear that even though

469
00:32:15,000 --> 00:32:19,000
these two genes, we can
call the normal one a

470
00:32:19,000 --> 00:32:22,000
proto-oncogene, these two
genes were structurally

471
00:32:22,000 --> 00:32:26,000
identical, they couldn't
be absolutely identical,

472
00:32:26,000 --> 00:32:29,000
because biologically, they
were behaving very differently.

473
00:32:29,000 --> 00:32:33,000
And so, when the sequence analyses
were done, it was discovered that

474
00:32:33,000 --> 00:32:36,000
the difference between the normal
proto-oncogene and the oncogene,

475
00:32:36,000 --> 00:32:40,000
was a single point mutation,
a single-base pair change.

476
00:32:40,000 --> 00:32:44,000
And that single-base pair
change created a potent oncogene.

477
00:32:44,000 --> 00:32:49,000
And that single-based pair change,
one could show in comparable tumors,

478
00:32:49,000 --> 00:32:53,000
was a somatic mutation. Remember,
we said somatic mutations are

479
00:32:53,000 --> 00:32:58,000
mutations that strike the
genomes of cells in our soma,

480
00:32:58,000 --> 00:33:03,000
rather than the germ line.
That somatic mutation had almost

481
00:33:03,000 --> 00:33:07,000
surely had struck one of the
epithelial cells lining the bladder

482
00:33:07,000 --> 00:33:12,000
of Mr. Jones' urinary
bladder. How did this work?

483
00:33:12,000 --> 00:33:16,000
Well, let's go back to our
discussion of growth factor

484
00:33:16,000 --> 00:33:20,000
receptors, you recall we
talked about them last time.

485
00:33:20,000 --> 00:33:24,000
Let's say, here's a growth factor
receptor at the cell surface,

486
00:33:24,000 --> 00:33:29,000
the growth factor receptor
sends signals into the cell,

487
00:33:29,000 --> 00:33:33,000
and such a sequence of signals,
where you go from a to b, to c to d,

488
00:33:33,000 --> 00:33:37,000
is sometimes called a signaling
pathway, sometimes it's called a

489
00:33:37,000 --> 00:33:42,000
signaling cascade, It's
a molecular bucket brigade

490
00:33:42,000 --> 00:33:46,000
where a passes signals to b,
passes signals to c, to do, and so

491
00:33:46,000 --> 00:33:51,000
fourth, and they cross-communicate
with one another to process this

492
00:33:51,000 --> 00:33:55,000
incoming signal from the growth
factor activated receptor.

493
00:33:55,000 --> 00:34:00,000
Now it turned out that the
protein product of this,

494
00:34:00,000 --> 00:34:04,000
of a normal proto-oncogene, and
the bladder carcinoma oncogene,

495
00:34:04,000 --> 00:34:09,000
sat right down here, in
the signaling cascade,

496
00:34:09,000 --> 00:34:14,000
downstream in the signaling cascade
of the growth factor receptor.

497
00:34:14,000 --> 00:34:18,000
And the protein product was
a very interesting protein,

498
00:34:18,000 --> 00:34:22,000
it was a protein, which came
to be called RAS, in fact,

499
00:34:22,000 --> 00:34:26,000
the original proto-oncogene had
previously been associated with a

500
00:34:26,000 --> 00:34:30,000
retrovirus. But in this case, it
was clear that the activation of

501
00:34:30,000 --> 00:34:34,000
the proto-oncogene to the oncogene
had nothing to do whatever,

502
00:34:34,000 --> 00:34:38,000
with the intervention, by a
retrovirus, or by the acquisition of

503
00:34:38,000 --> 00:34:42,000
a retrovirus, by a
normal proto-oncogene.

504
00:34:42,000 --> 00:34:47,000
So RAS has the normal, the
following normal lifestyle.

505
00:34:47,000 --> 00:34:53,000
RAS normally exists in a
quiet state, an inactive state,

506
00:34:53,000 --> 00:34:58,000
so here's RAS, and while it's
in the quiet state it binds GDP,

507
00:34:58,000 --> 00:35:04,000
guanosine diphosphate, GDP. What
happens, then, is that RAS on

508
00:35:04,000 --> 00:35:10,000
occasion, gets an incoming signal
from some upstream activator,

509
00:35:10,000 --> 00:35:16,000
and you can imagine what the
upstream activator is from here.

510
00:35:16,000 --> 00:35:21,000
Keep in mind, I'm only
focusing now on, let's say,

511
00:35:21,000 --> 00:35:26,000
component c of this signaling
cascade, so an upstream activator

512
00:35:26,000 --> 00:35:31,000
comes in, and impinges on RAS.
And in so doing, it wants to switch

513
00:35:31,000 --> 00:35:36,000
RAS from a quiet to an
activated state. So what happens,

514
00:35:36,000 --> 00:35:42,000
when RAS gets a signal from,
an upstream signal from here,

515
00:35:42,000 --> 00:35:47,000
RAS will shed its GDP, and
will instead, allow a GTP

516
00:35:47,000 --> 00:35:52,000
to jump aboard. So now
a GTP can jump aboard,

517
00:35:52,000 --> 00:35:56,000
and now RAS is in its active state
up here. And while it's in its

518
00:35:56,000 --> 00:36:01,000
active state, it can emit growth
stimulatory signals into the cell,

519
00:36:01,000 --> 00:36:05,000
like that. I'm not showing
you exactly what it looks like,

520
00:36:05,000 --> 00:36:10,000
but it can emit signals.
And this is, by the way,

521
00:36:10,000 --> 00:36:14,000
called a signal transducing protein,
a protein that tranduces signals

522
00:36:14,000 --> 00:36:19,000
receives signals from higher up
in the cascade, and passes them on

523
00:36:19,000 --> 00:36:23,000
lower into the cascade. So
it's transducing these signals,

524
00:36:23,000 --> 00:36:28,000
and so RAS is put, here I
should've capitalized RAS here,

525
00:36:28,000 --> 00:36:33,000
so RAS is now in
its active state.

526
00:36:33,000 --> 00:36:36,000
It's received upstream signals,
it's shed its GDP, its bound GDP,

527
00:36:36,000 --> 00:36:40,000
and while it's there, RAS passes
signals on further downstream in the

528
00:36:40,000 --> 00:36:44,000
pathway. So, if want to relate
it to the signaling cascade,

529
00:36:44,000 --> 00:36:48,000
and we imagine that RAS is
component c of this cascade,

530
00:36:48,000 --> 00:36:52,000
it receives signals from b, and
then RAS passes signals onto d,

531
00:36:52,000 --> 00:36:56,000
it's sitting in-between
them, it's an intermediary,

532
00:36:56,000 --> 00:37:00,000
it's a member of this
molecular bucket-brigade.

533
00:37:00,000 --> 00:37:04,000
Now what happens is that RAS is in
this active state for only a period

534
00:37:04,000 --> 00:37:09,000
of usually milliseconds, and
after it's in a period of this

535
00:37:09,000 --> 00:37:14,000
active stage, and it's emitting
growth stimulatory signals,

536
00:37:14,000 --> 00:37:18,000
downstream, for a period of
milliseconds, RAS does something

537
00:37:18,000 --> 00:37:23,000
very interesting. It
hydrolyzes the GTP,

538
00:37:23,000 --> 00:37:28,000
and when it hydrolyzes the GTP
into GDP, obviously an inorganic

539
00:37:28,000 --> 00:37:32,000
phosphate comes out,
RAS switches itself off,

540
00:37:32,000 --> 00:37:37,000
i.e. RAS has an intrinsic
GTPase activity, GTPase means

541
00:37:37,000 --> 00:37:41,000
it can cleave GTP. So it's
as if there was a switch

542
00:37:41,000 --> 00:37:45,000
here, and I could turn it on,
well I'm not going to mess with it

543
00:37:45,000 --> 00:37:48,000
since I still haven't figured
out how the switches work,

544
00:37:48,000 --> 00:37:52,000
but it's as if there was a light
switch here that I could switch on,

545
00:37:52,000 --> 00:37:55,000
and the lights would be on
for a short period of time,

546
00:37:55,000 --> 00:37:59,000
and then the switch would
automatically shut itself off.

547
00:37:59,000 --> 00:38:02,000
A negative-feedback control to
ensure that the period of activation

548
00:38:02,000 --> 00:38:06,000
of the RAS protein is only
a period of milliseconds,

549
00:38:06,000 --> 00:38:09,000
and that the pulse of downstream
signals that are released is finite,

550
00:38:09,000 --> 00:38:13,000
circumscribed, limited, it's
only a quantum of signals that

551
00:38:13,000 --> 00:38:16,000
are released. After
which occasion,

552
00:38:16,000 --> 00:38:20,000
RAS hydrolyzes it's GTP and shuts
itself off. It's a nice system,

553
00:38:20,000 --> 00:38:24,000
and it actually works because,
as I've told you before,

554
00:38:24,000 --> 00:38:27,000
we go through ten of the
sixteenth cell divisions,

555
00:38:27,000 --> 00:38:31,000
with RAS proteins in our cells,
and rarely, if we lead virtuous

556
00:38:31,000 --> 00:38:35,000
lives and listen to everything
I've said in the last lecture,

557
00:38:35,000 --> 00:38:38,000
do we ever get cancer. So, what
happens when the RAS encoding

558
00:38:38,000 --> 00:38:42,000
gene becomes mutated in cancer?
That's another one of those

559
00:38:42,000 --> 00:38:46,000
questions that I'm really glad I
asked, because what happens is that

560
00:38:46,000 --> 00:38:50,000
the ability of the GTPase activity
to function, is knocked out.

561
00:38:50,000 --> 00:38:55,000
The GTPase can no longer operate,
and therefore, the ability of RAS to

562
00:38:55,000 --> 00:39:00,000
shut itself off, by
cleaving GTP down to GDP,

563
00:39:00,000 --> 00:39:05,000
is now compromised, and as a
consequence, RAS is trapped for

564
00:39:05,000 --> 00:39:10,000
extended periods of time, for
minutes and hours, and maybe

565
00:39:10,000 --> 00:39:15,000
even days, in this excited
signal-emitting configuration,

566
00:39:15,000 --> 00:39:20,000
on which occasion, it sends out
a continuous flood of signals.

567
00:39:20,000 --> 00:39:24,000
In fact, we now know
that the point-mutation,

568
00:39:24,000 --> 00:39:28,000
which happens in the gene here,
and is seen, by the way, in about

569
00:39:28,000 --> 00:39:32,000
20% of human cancers that have
virtually identical point-mutations,

570
00:39:32,000 --> 00:39:36,000
those, that point mutation is in
the GTPase domain of the RAS protein,

571
00:39:36,000 --> 00:39:41,000
that is normally responsible
for cleaving GTP into GDP.

572
00:39:41,000 --> 00:39:45,000
So now we can begin to get a very
concrete and specific understanding,

573
00:39:45,000 --> 00:39:49,000
an insight, into the mechanism
by which a point mutation,

574
00:39:49,000 --> 00:39:53,000
a somatic mutation, can have a
dramatic effect on the ability of a

575
00:39:53,000 --> 00:39:58,000
cell to grow.
Note, by the way,

576
00:39:58,000 --> 00:40:03,000
that if RAS is sending signals
constitutively down here,

577
00:40:03,000 --> 00:40:09,000
and you may recall that
the word constitutive means,

578
00:40:09,000 --> 00:40:14,000
at a constant and unrelenting
fashion. So the ability of RAS,

579
00:40:14,000 --> 00:40:19,000
which I depicted as residing right
in here in the signaling cascade,

580
00:40:19,000 --> 00:40:24,000
to send out signals
unrelentingly down like this,

581
00:40:24,000 --> 00:40:30,000
means that the signals up here
are no longer so important.

582
00:40:30,000 --> 00:40:34,000
RAS might require a brief stimulus
to get into this activated state,

583
00:40:34,000 --> 00:40:38,000
but then it can sit around for
hours and days, pushing the cell to

584
00:40:38,000 --> 00:40:42,000
proliferate. And the subsequent
exposure of a cell carrying a RAS

585
00:40:42,000 --> 00:40:47,000
oncogene is now gratuitous,
it's unnecessary, because this

586
00:40:47,000 --> 00:40:51,000
downstream signal emitter has gone
wild, and is firing and pushing the

587
00:40:51,000 --> 00:40:55,000
cell to grow unrelentingly.
Interestingly, that signal is very

588
00:40:55,000 --> 00:40:59,000
critically important in pushing
cells to move through the G1 phase

589
00:40:59,000 --> 00:41:04,000
of their growth
and division cycle.

590
00:41:04,000 --> 00:41:08,000
In other words, it's
during that phase of the

591
00:41:08,000 --> 00:41:12,000
division cycle, that RAS
is actually able to send

592
00:41:12,000 --> 00:41:16,000
it's signals that perturb cell
cycling, and push the cell to move

593
00:41:16,000 --> 00:41:20,000
from G1 up to the restriction point,
and then into the remaining part of

594
00:41:20,000 --> 00:41:24,000
the cell cycle. So now we
see how somatic mutations,

595
00:41:24,000 --> 00:41:28,000
and we now know about dozens of such
genes, can convert proto-oncogenes

596
00:41:28,000 --> 00:41:33,000
to oncogenes, without the
intervention of any retrovirus.

597
00:41:33,000 --> 00:41:37,000
There are yet another class
of genes, which are called,

598
00:41:37,000 --> 00:41:42,000
tumor suppressor genes, and
these tumor suppressor genes

599
00:41:42,000 --> 00:41:46,000
operate in exactly the opposite way
as proto-oncogenes and oncogenes.

600
00:41:46,000 --> 00:41:51,000
The proto-oncogenes and oncogenes,
from what I've told you, you can

601
00:41:51,000 --> 00:41:56,000
imagine are functioning analogously
to accelerator pedals on a car,

602
00:41:56,000 --> 00:42:00,000
they push the cell to proliferate
and, when you have a cancer,

603
00:42:00,000 --> 00:42:05,000
the accelerator pedal gets stuck
to the floor. In other words,

604
00:42:05,000 --> 00:42:09,000
it's no longer well regulated.
The tumor suppressor genes work in

605
00:42:09,000 --> 00:42:13,000
an opposite fashion, as a
breaking system to slow down

606
00:42:13,000 --> 00:42:17,000
cell proliferation. And,
as such, tumor suppressor

607
00:42:17,000 --> 00:42:21,000
genes operate to counteract,
and counterbalance, and limit,

608
00:42:21,000 --> 00:42:25,000
the growth stimulatory signals
that are coming from the RAS gene,

609
00:42:25,000 --> 00:42:28,000
and other growth promoting genes.
So there's two sides to the coin,

610
00:42:28,000 --> 00:42:32,000
indeed as you can imagine
from circuit theory,

611
00:42:32,000 --> 00:42:36,000
any positive signal must be counter
balanced by a negative signal,

612
00:42:36,000 --> 00:42:40,000
so that you end up having some
kind of physical balance that is

613
00:42:40,000 --> 00:42:44,000
compatible with normal,
biological function.

614
00:42:44,000 --> 00:42:48,000
So these tumor suppressor genes
are normally functioning as break,

615
00:42:48,000 --> 00:42:52,000
break linings of a cell, if you
will, and that also describes how they

616
00:42:52,000 --> 00:42:56,000
become involved in cancer. The
mutation that struck the RAS

617
00:42:56,000 --> 00:43:00,000
gene caused a hyper-activation
of the proto-oncogene.

618
00:43:00,000 --> 00:43:04,000
The proto-oncogene
was hyper-activated,

619
00:43:04,000 --> 00:43:08,000
and now it began sending out an
unrelenting stream of growth-stimulatory

620
00:43:08,000 --> 00:43:12,000
signals. As
you can imagine,

621
00:43:12,000 --> 00:43:16,000
as it is intuitively obvious,
in the case of tumor suppressor

622
00:43:16,000 --> 00:43:20,000
genes, when they became involved
in cancer, what kind of mutations

623
00:43:20,000 --> 00:43:24,000
affect them? Inactivating mutations.
So there's a very interesting gene,

624
00:43:24,000 --> 00:43:28,000
it's called the retinoblastoma
gene, and the retinoblastoma gene,

625
00:43:28,000 --> 00:43:33,000
the retinoblastoma is a
rare eye-tumor of children,

626
00:43:33,000 --> 00:43:37,000
it happens about 1/20,000 kids,
and the retinoblastoma gene, if you

627
00:43:37,000 --> 00:43:41,000
look at the retinoblastoma gene
in the genomes of tumor cells,

628
00:43:41,000 --> 00:43:45,000
these are the eye cells
that form the precursors,

629
00:43:45,000 --> 00:43:49,000
the rods and the cones, and
other neuronal cells lining the

630
00:43:49,000 --> 00:43:54,000
retina. Here's the
normal retinoblastoma

631
00:43:54,000 --> 00:43:58,000
gene, and it's 190 kilo basis long.
So it's a pretty nice size gene,

632
00:43:58,000 --> 00:44:02,000
it's not the biggest,
it's not the smallest.

633
00:44:02,000 --> 00:44:06,000
If you look at many retinoblastoma
tumors, what you find is that there

634
00:44:06,000 --> 00:44:10,000
are major portions of the gene
that have been just deleted.

635
00:44:10,000 --> 00:44:14,000
Here I'll show you one deletion,
here's another deletion, here's a

636
00:44:14,000 --> 00:44:18,000
third deletion, sometimes
the whole gene is deleted,

637
00:44:18,000 --> 00:44:21,000
and cut out. Obviously
such deletions are not

638
00:44:21,000 --> 00:44:25,000
enhancing the function of
the retinoblastoma gene,

639
00:44:25,000 --> 00:44:29,000
obviously they're wiping it out.
And therefore, we can begin to

640
00:44:29,000 --> 00:44:32,000
imagine that the way that the tumor
suppressor genes are recruited into

641
00:44:32,000 --> 00:44:36,000
the process of forming cancer
is through their elimination,

642
00:44:36,000 --> 00:44:39,000
rather than through their
hyper activation. In fact,

643
00:44:39,000 --> 00:44:43,000
as you might also imagine, a
cell, which has the following

644
00:44:43,000 --> 00:44:47,000
genotype, RB+, RB-,
actually grows normally.

645
00:44:47,000 --> 00:44:50,000
Why? Because one of the two
alleles has been inactivated,

646
00:44:50,000 --> 00:44:54,000
the minus, but the other allele
is still, is still active,

647
00:44:54,000 --> 00:44:58,000
and still functional, and still able
to create an adequately functional

648
00:44:58,000 --> 00:45:03,000
break lining. Only when
a cell becomes RB- like

649
00:45:03,000 --> 00:45:09,000
this, homozygous minus, does
it begin to grow uncontrollably,

650
00:45:09,000 --> 00:45:15,000
because now it lacks all ability
to manufacture the normally required

651
00:45:15,000 --> 00:45:21,000
break lining. And this begins, as
well, to explain certain kinds of

652
00:45:21,000 --> 00:45:27,000
hereditary cancers, because
in certain individuals,

653
00:45:27,000 --> 00:45:33,000
they inherit a defective allele
of the RGB, and therefore,

654
00:45:33,000 --> 00:45:40,000
at the moment of conception,
they have the following genotype.

655
00:45:40,000 --> 00:45:44,000
Well, you'll say that's all,
that's fine, because each of their

656
00:45:44,000 --> 00:45:49,000
cells has a wild-type copy of
their RB gene, and a mutant,

657
00:45:49,000 --> 00:45:53,000
defective copy. And the wild-type
copy, as you will correctly say,

658
00:45:53,000 --> 00:45:58,000
suffices to template, to orchestrate,
to program, normal cell behavior.

659
00:45:58,000 --> 00:46:03,000
Why do they then
get retinoblastomas?

660
00:46:03,000 --> 00:46:07,000
Because the surviving wild-type
gene, or the gene copy,

661
00:46:07,000 --> 00:46:12,000
the wild-type allele, can
be lost with a certain,

662
00:46:12,000 --> 00:46:17,000
finite probability per cell
generation, through chromosomal

663
00:46:17,000 --> 00:46:22,000
mistakes, accidents, or
through crossing-over.

664
00:46:22,000 --> 00:46:26,000
And so roughly, one in
ten to the sixth cells,

665
00:46:26,000 --> 00:46:31,000
one has an event, a genetic accident,
which causes the accidental loss of

666
00:46:31,000 --> 00:46:35,000
the surviving RB allele,
leaving, RB wild-type allele,

667
00:46:35,000 --> 00:46:40,000
leading now to a genotype that
looks like this, homozygous mutant,

668
00:46:40,000 --> 00:46:44,000
or homozygous inactive, and now a
rare cell in which that has happened,

669
00:46:44,000 --> 00:46:49,000
now begins to proliferate
uncontrollably because of the

670
00:46:49,000 --> 00:46:53,000
absence of the RB break lining,
which is normally required to

671
00:46:53,000 --> 00:46:58,000
control cell proliferation. By
the way, I'll tell you in passing,

672
00:46:58,000 --> 00:47:02,000
without describing the gory details,
that the RB protein works at the end,

673
00:47:02,000 --> 00:47:06,000
at the restriction point. We
talked about the restriction

674
00:47:06,000 --> 00:47:10,000
point last time, you
remember? The RB protein

675
00:47:10,000 --> 00:47:14,000
actually prevents the cell from
advancing through the restriction

676
00:47:14,000 --> 00:47:18,000
point gate, unless it becomes
inactivated, and then the cell can

677
00:47:18,000 --> 00:47:22,000
sail through into the
rest of the cell cycle.

678
00:47:22,000 --> 00:47:28,000
In cells that lack the RB protein,
the guardian of the restriction

679
00:47:28,000 --> 00:47:35,000
point gate is no longer there, and
therefore the gate is held open,

680
00:47:35,000 --> 00:47:42,000
and cells can sail all the way
through G1, in an unimpeded fashion,

681
00:47:42,000 --> 00:47:48,000
without the RB protein standing
at the restriction point gate and

682
00:47:48,000 --> 00:47:55,000
saying, advance no further, unless
and until, certain conditions

683
00:47:55,000 --> 00:48:00,000
have been satisfied. And so
we can begin to understand

684
00:48:00,000 --> 00:48:03,000
that the tumor suppressor gene,
the RB gene, is able to act as a

685
00:48:03,000 --> 00:48:07,000
quality control, to
ensure that cells don't

686
00:48:07,000 --> 00:48:10,000
inadvertently, inappropriately,

687
00:48:10,000 --> 00:48:13,000
pass through the restriction point
gate into the rest of the cell cycle.

688
00:48:13,000 --> 00:48:16,000
And by now, there are 40 or 50
different kinds of tumor suppressor

689
00:48:16,000 --> 00:48:20,000
genes that are found to be
inactivated through various

690
00:48:20,000 --> 00:48:23,000
mechanisms, in human cancers.
This was only the progenitor,

691
00:48:23,000 --> 00:48:26,000
the harbinger, of those. How many
tumors does a child who's born like,

692
00:48:26,000 --> 00:48:30,000
this get in his
eyes, or her eyes?

693
00:48:30,000 --> 00:48:34,000
Well, it might get two or
three or four, in both eyes,

694
00:48:34,000 --> 00:48:39,000
but that reflects the fact that
there maybe a million or two million

695
00:48:39,000 --> 00:48:44,000
cells in each of the retina,
which are susceptible to this loss

696
00:48:44,000 --> 00:48:49,000
of the surviving wild-type gene
copy. Having heard all that,

697
00:48:49,000 --> 00:48:54,000
you will ask me, well why don't
they get tumors all over the body,

698
00:48:54,000 --> 00:48:59,000
since it is the fact that the RB
protein regulates the restriction

699
00:48:59,000 --> 00:49:04,000
point gate in all cells
throughout the body?

700
00:49:04,000 --> 00:49:07,000
So therefore, why isn't the
child who's born constitutionally,

701
00:49:07,000 --> 00:49:10,000
whose genetic makeup is this,
why isn't a child like this

702
00:49:10,000 --> 00:49:13,000
sensitive to developing tumors
all over his or her body?

703
00:49:13,000 --> 00:49:17,000
In fact, children who are born
genetically with this genotype,

704
00:49:17,000 --> 00:49:20,000
get retinoblastoma's with
high probability early in life,

705
00:49:20,000 --> 00:49:23,000
and as teenagers, they often
come down with osteosarcomas,

706
00:49:23,000 --> 00:49:27,000
which are tumors of the bones.
But otherwise, they don't get many

707
00:49:27,000 --> 00:49:30,000
kinds of cancer, and the
answer to your question is,

708
00:49:30,000 --> 00:49:34,000
I haven't the vaguest idea.
No one knows why loss of this

709
00:49:34,000 --> 00:49:38,000
critical gene that plays
a key role in the biology,

710
00:49:38,000 --> 00:49:43,000
in the metabolism, of all
cells throughout the body,

711
00:49:43,000 --> 00:49:47,000
can be lost to yield cancer
in the eye and in the bone,

712
00:49:47,000 --> 00:49:52,000
whereas when the same gene,
which must be lost elsewhere in

713
00:49:52,000 --> 00:49:56,000
other tissues in the body are lost,
tumors do not arise. And on that

714
00:49:56,000 --> 00:50:01,000
puzzling note, I wish
you a pleasant day,

715
00:50:01,000 --> 00:50:05,000
another happy Halloween, and see you
on, oh yes, tomorrow is election day.

716
00:50:05,000 --> 00:50:10,000
Don't forget to vote. And
remember what the mayor of

717
00:50:10,000 --> 00:50:15,000
Boston once said,
vote early and often.