PART I: MATERIALS AND METHODS
Microbiology and Plasmids
Electron Microscopy
X-Ray Diffraction
Radioisotopes
Antibodies
Electrophoresis
Transfers & Probing
Sequencing
PCR
Oligonucleotide Synthesis
One-Minute Essay Questions and Answers: Archives from the Past
Outline:
INTRO: COURSE, TESTS, TERM PAPER
PART I: MATERIALS AND METHODS
PART II: THE CENTRAL DOGMA
PART III: REGULATING GENE EXPRESSION
PART IV: EUKARYOTIC HYPERDRIVE
INTRO: COURSE, TESTS, TERM PAPER
Course Questions
Tests & Term Paper
Q: Can you please keep lights on whenever possible? I'm trying to stay awake, I really am.
ANS: Yes. If there were more room in the classroom, and I noticed you were
getting sleepy, I might wander over and stand near you while I was lecturing--
but that is hard in this room. You might try moving to a different seat each
day (it helps some), or bringing along something to drink, or exercising some
before class.
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Q: Is this class doable?
ANS: Definitely. See the notes on "How to succeed as a student" in
the Syllabus portion of the course book. Keep up with the course; don't put
off the work until the weekend, or next week, or just before the test.... Use
all the help available, including coming to see me. I will do whatever I can
to help you succeed. As you realize, the course is supposed to be challenging,
and you are supposed to succeed.
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Q: Am I going to survive this class? The information looks confusing and scary.
Did I lose my brain during the course of the school year?
ANS: This course has had no fatalities yet. But seriously: one of the beauties
is that _all of the information makes sense_! All of the pieces fit together.
Really. And: you have already gotten through all the "weed-out" courses
(you've already gotten through Cell Bio, Gen Chem, O-Chem, and maybe also Genetics
and/or Biochem) so I know you have what it takes for this course. The rest is
just effort, and you can do it.
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Q: The foremost question I have is a general one about class and lab. I am unsure
as to how i am going to balance all the time needed for this class (lecuter,
lab, termpaper, lab reports, exams, etc.) with all the time for other classes.
This class seems _very_ interesting which means a lot of time goes into it in
order to get a lot out of it.
A: Hope it remains interesting. I intend for it to be challenging, expect for
it to be a lot of work, and hope for it to be fun. Remember that, as a 5 credit
course, it is supposed to be even more work than other biology lab courses.
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Q: How detailed do I have to know all these techniques?
Q: How in depth do we have to know this stuff for tests? Do we need to know how all these things work backwards and forwards? Or si the general just of it enough?
Q: Are we going to have to know what everything is called, or know the process by which things are done? I understand the processes, but I cant always remember what exactly the process is called.
ANS (for all 3): (1) Be sure you have a good handle on the vocabulary-- and
don't wait to know the vocab until the night before the test, because I will
be using it. There is a lot of vocab, and one of the best ways to learn it is
to make your own glossary. (2) While we are not going to do all of the M&M
that I describe (e.g. we will not do electron microscopy, X-ray diffraction,
chemical DNA synthesis, or sequencing), we will use most of these techniques
in lab, and certainly will be referring to all of these techniques in the course.
Remember, as I indicated on the first day (and in the syllabus), you will find
that the empirical basis of biology-- HOW we know-- is emphasized more in this
course than in most others.
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Q: How are we going to be tested on this stuff?
ANS: Several ways. The most obvious is the exams-- more on that below. But that's not all. You get tested each day, as I use terms and refer to results discussed in previous classes and in the readings. If you don't follow what I am saying because (for example) you don'thave a good handle on the difference between transcription and translation, you have just failed the first test. You get tested when you come into lab: do you understand what you and your team members are doing, or are you just following one step after another without being able to visualize, in your mind's eye, what is going on? You get tested when it's time to write up a lab report: if you understand what you are doing in the lab, and how it reflected things we talked about in lecture, you will be able to write a more informative and coherent lab report. And you are going to get tested when you are working on your term paper, reading primary and secondary research articles and working to analyze, compare, and summarize their findings: if you have a good handle on "this stuff", what you read will be much more informative, and much easier to summarize, than if you don't really know what the authors are talking about.
The exams will test your knowledge in several ways. Parts of the test will
be mainly recall, asking you to define or illustrate some terms, or give some
examples. Some of the questions will be multiple choice; some will involve short
written answers; some will involve drawing (nothing fancy-- it's not an art
course, but remember that an underlying theme of the course is developing your
mind's eye, and a good way to do that is to draw things. Draw pUC. Draw a bactriophage.
Illustrate chromatography. etc.). Some of the questions will ask you to go beyond
recall, and use what you know to interpret results from some experiment, or
to predict the results of an experiment. You will get some problem sets that
will give you practice and examples.
.....................................................................................................................
Q: Are we going to have to remember and know all of these procedures for the
test?
ANS: Yes.
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Q: How detailed does a prospectus have to be?
ANS: That's up to you. The more detailed it is, the more useful it will be
to you. You may want to look at the examples that are on reserve in the library.
A cursory prospectus is graded accordingly; while one that is thoughtful, well
organized, and informative would receive a higher grade.
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Q: The main question I have at this point would be concerning the term paper. What am I going to do? Which topic, where to start searching for info, etc. I would like to start this weekend.
A: Library-- especially journals and Annual Reviews (e.g. Annual Review of
Genetics)-- would be useful, also their search tools such as First Search.
.....................................................................................................................
PART I: MATERIALS AND METHODS
Microbiology and Plasmids
Electron Microscopy
X-Ray Diffraction
Radioisotopes
Antibodies
Electrophoresis
Transfers & Probing
Sequencing
PCR
Oligonucleotide Synthesis
Q: Do the sexes in bacteria occur together in one bacterium or separately i.e. can you have both sexes in one bacterium?
ANS: As we'll see in some more detail in a few weeks, it really is misleading
to say that bacteria "have sex", even though they do have mechanisms
for exchanging genes. When we think of sex (in the strictly biological sense,
that is) we really are thinking from a diploid perspective-- and, furthermore,
from the perspective of a complex, differentiated, multicellular organism. So
there aren't separate sexes, or hermaphrodites, that are really comparable to
what we find in plants or animals-- though there are some bacteria that "donate"
DNA and some that receive the DNA. I'll give you a handout that explains this
more clearly.
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Q: Explanation of various plasmids again?
ANS: Okay, hard to do well without pictures, so you might want to look some
up (e.g in the course book section on Lab #1, and maybe look in Weaver). Plasmids
are circular (almost always) pieces of DNA that carry just a few genes; they
are usually found in prokaryotes; they have an origin for replication and segments
of DNA that allow for the expression of structural genes on the plasmid. While
there are naturally occurring plasmids, some have been highly modified and are
now useful for research or commercial purposes. These plasmids are modified
so that it is easy to insert another piece of DNA in a precise place on the
plasmid (by restriction digest and ligation), and easy to determine whether
that plasmid has been taken up by bacteria and whether or not the foreign DNA
was successfully installed.
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Q: I'm still a little foggy about how isotopes are labled and used in experiments.
ANS: The answer, regardless of which isotope, is that the radioactive element
is made into a compound that is introduced to some metabolic process, and the
radioisotope allows us to identify some product of that metabolism. Think of
the Hershey & Chase experiments: these allowed H & C to identify nucleic
acids as the material that determined heredity in their "simple model system",
and not proteins. Our use of radioisotopes in sequencing may seem quite different,
but that is because we have isolated the "metabolism" part (actually
just an enzyme reaction) into a test tube.
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Q: I still don't completely understand how the rabbit/goat antibody thing works. I understand what they are trying to do, but I get lost in the steps.
ANS: Of all the questions, I think this one most needs pictures to answer. Take it one step at a time. Innoculating a vertebrate with a foreign substance (especially a protein) causes it to make antibodies, which are Y-shaped proteins that that will bind specifically with that antigen. You can take a blood sample from that animal (such as a rabbit) nd purify the antibodies (e.g. by column chromatography) and, if you want, put a label on the antibodies that allows you to detect them, e.g. a "fluorochrome" that glows under UV light. Then you can spread the antibodies on some tissue, look under a microscope, and find where the antigen is located. Or, you could spread your labled antibodies on a western blot, and they would bind to the antigen protein on the blot.
Using the combination of antibodies from two species allows you to generate a stronger response-- i.e. to "amplify the signal". Suppose your first antibody is made by innoculating a rabbit with your antigen (protein X). The rabbit then makes antibodies that will bind to X, but you do not label these antibodies. Instead, you obtain antibodies that have been made by a goat who was innoculated with rabbit antibodies. In this case, the rabbit antibodies are acting as antigens to the goat, who produces "goat anti-rabbit antibodies", and you label these goat antibodies with a fluorochrome. Now you can spread the rabbit antibodies on the tissue, and then spread the goat antibodies on too. Several rabbit antibodies bind to each molecule of protein X, and several goat antibodies bind to each of the rabbit antibodies. That means that you now have a bunch of fluorochrome molecules binding to each molecule of protein X, so it will be easier to find when you look under the microscope.
I still think I need pictures for that.....
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Q: How did electrophoresis come to be? Trial and error? Accidental?
ANS: I don't know the answer to this. I don't think it was accidental or trial
& error. Oliver Smities introduced the use of a gel (starch) in about 1957,
but I believe others had used paper before that. If you want to pursue the question,
I can give you a reference to Smithies' paper, and you could work back from
there.
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Q: What do we do with information that we get from these tests-- electrophoresis--
how do we make that information useful?
ANS: This depends entirely on what our experiment is-- on what it is we are
running on the gel, on what makes one lane different than another. You will
see some examples in lab, some more in lecture, and many more in your text.
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Q: Pulse-field electrophoresis still seems abstract.
I'm foggy on how pulse field electrophroesis tells us anything, or what it
does exactly.
.....................................................................................................................
ANS: If you were molecular sized, and wandering around inside a gel, what do
you think it would look like? Imagine you are swimming around in some big 3-dimensional
network or spiderweb, the strands being the polymers of agarose, or starch,
or polyacrylamide. That's roughly what a gel is like. An electrical field will
pull DNA molecules (or protein, for that matter) through this gel network, and
small molecules can slip through much more rapidly than large molecules. If
you have a mess of really, really big DNA molecules, they won't slip through
at all-- they will just be a tangled-up mess, stuck on the ropes. Now imagine
that, as the electrical field changes polarity, the little molecules are more
rapidly accelerated with each change, so they are gently teased away from the
larger molecules-- and eventually the larger molecules are teased away from
the very largest ones. As this continues, the changing electrical fields pull
th esmaller DNA molecules farther and farther away. By playing not only with
_velocity_ of the DNA but also _acceleration_, the pulsed-field system is able
to separate DNA strands that are hundreds of thousands or even millions of bases
long, producing bands on the gel that are (in some cases) even separate chromosomes.
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Q: Probes seem unclear.
Q: What is a probe? Making a library and screening process is still a little confusing.
ANS: A probe is a rather short piece of DNA (a few hundred bases long, typically)
that is made to find its complement, and carries some kind of label (e.g. a
radioisotope) that lets us find the probe itself after it has bound to its complement.
By "complement" I mean a piece of DNA that has the complementary sequence
of bases: where the probe has an A the complement has a T, where the probe has
a C the complement has a G, etc.
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Q: How does probing work? Some parts are still a little fuzzy.
ANS: There are bound to be some parts of the story that are still fuzzy, and
won't completely become clear until you do probing yourself, later in the semester.
But the basics are quite simple. We have a template DNA that we copy with an
enzyme (DNA polymerase) using labled nucleotides (by label we mean that they
carry a radioactive atom, or some other chemical tag that we can find later).
The copy that we made-- i.e. the probe-- will bind to any DNA to which it is
complementary (assuming we separate the strands of probe and target DNA-- see
question below).
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Q: What happens if the probe contains complementary strands to plaques other
than what we are looking for and how do we know this won't happen?
ANS: Same thing: the probe binds and we detect it. Now the really interesting
questions revolve around what the "mistakes" mean: i.e. there is a
match between the probe and the other plaques (or other target DNA molecules),
and we start to get wondering about what could cause this... which leads to
more research. Suppose you took DNA from a fly, cloned it into a plasmid such
as pUC, made a probe from that, and used that probe against DNA from a frog
(after it had been digested, run on a gel, and blotted onto a filter) and found
spots. What would you conclude?
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Q: I'm trying to remember/ understand the details on the very first page in
the handouts about probe synthesis where the primer is used. I think it is to
make the target DNA?
ANS: "Target" DNA usually refers to the piece of DNA that the probe
matches. "Template" DNA usually refers to the piece of DNA that serves
as the pattern for the DNA polymerase to copy.
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Q: How are the probes made into single strands, so they don't anneal to their
complements, but htey do anneal to the desired gene?
ANS: Like most other DNA, the probe starts out double-stranded (template strand
plus newly synthesized, labled strand). The strands are held to each other by
H-bonds which are easily separated by heating to about 95 C.
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Q: How does the probe recognize its matching target DNA?
ANS: It's all because of the complemenary binding: Adenine:Thymine, Guanine:Cytosine.
So a probe that has the sequence CATCATCAT will bind to DNA that has the sequence
( in the same direction) of GTAGTAGTA. Etc.
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Q: How do you create a probe to find a particular gene of interest? How do you
isolate the right piece after you replicate the plasmid containing the gene
of interest?
ANS: This is not a simple question, and there is not a single answer, since
the answer depends entirely on what the research project is you are attempting,
what starting materials and information you have, etc. The _easiest_ way to
get a probe for a gene of interest is to ask someone else for the one they are
using. I'm going to duck from giving a more complete answer, at least for now.
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Q: How accurate is the probe at finding the gene it is supposed to be "looking"
for?
ANS: As accurate as you want to make it. Since a probe binds to its target
by H-bonds, and then we wash away all of the excess probe that is not bound
to the target, we can control what is called the "stringency" of the
wash by altering the temperature (e.g. only probe/target combinations that are
very closely matched will hold together if the temperature is high) and the
ionization of the wash (e.g. by altering the salt concentration).
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Q: How does process of probing affect the culture? Does this change results
in any way?
ANS: This one I'm stuck on-- I don't really understand the question. We use
a probe against DNA (or RNA) on a filter (Tuesday I'll show examples of using
a probe against chromosomes).
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Q: What is the last figure we looked at all about, just transfers and hybridization?
ANS: Exactly, yes.
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Q: How do yo immerse a filter paper in a probe if it is just DNA? It seems like
you need a lot and something you can't immerse things in.
ANS: We dissolve the probe in a liquid: mostly water, of course, but also some
buffer to control the pH, salt to control the H-bonding strength (see question
above), and perhaps some other useful compounds.
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Q: I don't really understand the probe part. On the "synthesizing a probe"
page in the handout, is the probe on there in final form at all?
ANS: Barely, and not illustrated very clearly. Dang-- if I had a copy of Weaver's
text handy, I would find a diagram that illustrates this better. Will have to
look for that tomorrow, but you might look at the table near the front of the
book that lists where different techniques are illustrated in the text. I think
you will find this handy.
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Q: How is the probe made?
ANS: (I should have put this question up near the beginning). I hope I have
answered it adequately. If not PLEASE let me know--though really I think I need
to draw pictures to do a decent job of explaining how probes are made.
.....................................................................................................................
Q: How do transfers work? Going from double stranded to single stranded?
ANS: Transfers are extremely simple: like putting a piece of dry filter paper
onto a damp sponge (i.e. on the gel). That's all it takes. We can make the process
a little more complicated and a little more efficient by setting up a stack
of paper towels on top of the filter paper (so they will continue to draw up
liquid through the filter paper, even after it gets saturated) and by putting
the gel on a wick that goes into a pool of more buffer (mainly salt water) that
will continue to flow up through the gel, through the filter, and into the stack
of paper towels. You will make one later this semester in lab, and be surprised
at how simple it is.
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Q: Are replica plating and the filter paper method in your probing example (making
a library) examples of transfers? I'm still a little hazy on the topic of probing
and how it all works.
ANS: Yes, these are transfers. If the other questions and answers about probing
haven't clarified your understanding, please let me know.
.....................................................................................................................
Q: How in depth do we have to know this stuff for tests? Do we need to know
how all these things work backwards and forwards? Or is the general jist of
it enough?
ANS: I should have put this question in the set on "Tests and Term Papers"
below. I'll move a copy there, with answer.
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Q: How does the DNA stay in the proper vertical "lines" of the PFE
gel if there are side currents present and the DNA moves at different rates?
ANS: The only ones that I have seen had the same DNA in all of the wells, so
that all of the lanes had bands at the same places. I am not familiar with other
applications.
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Q: Are probes (e.g. finding complementary DNA) more effective or more widely
used than radiolabled antibodies? Is it easier to search for the gene or the
expressed protein?
ANS: They are used for different purposes. Probes are used for picking one
piece of DNA out of a bunch of others on a gel (or, more precisely, on a filter),
while radiolabeled antibodies would be used for picking one protein out of a
bunch of others. Which one is easier depends a lot on what the context for your
work is: if you have a probe from some other work, then that is easy to use;
if you have isolated a protein, then it is not too much work to get antibodies
made and use those on a gel (or more likely on a western blot). In general I
would say that DNA probing is easier.
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Q: What kind of info can you get from your "screened library"? What
true purpose do probes have in real life?
ANS: All of this depends on the context of the work. Here is an analogous question:
"What kind of info can you get from measuring voltage drop?" See,
the answer depends on what you are measuring: it could be voltage in a pH meter,
or in a spectrophotometer, or in a balance, or in a chromatograph, or in an
electronic thermometer, etc.: all of these instruments are built to measure
some kind of voltage in a circuit. And then, the significance of that measurement
of pH or absorbance or weight, etc.? That depends on the question you have set
up. Screening a library is merely a technique; it's significance depends on
the genius (and I mean the exact definition of that term) of the experimenter.
.....................................................................................................................
Q: Making and using DNA probes is still a little fuzzy to me.
A: Hmm-- I wonder what is the fuzzy part? Here's the key points: (1) a probe
is a piece of DNA carrying a "label" (such as a radioisotope or a
chemical side-arm, such as biotin, that we can identify later), that will stick
to any complementary DNA once it has been denatured. [These are two important
terms: "complementary" means having the corresponding sequence: GGG
is complementary to CCC, for example. "Denatured", for DNA, means
that the two complementary strands have been separated from each other.] We
can use the probe to find its complement in DNA that has been run on a gel and
then transferred to a nylon membrane, or to find colonies or plaques containing
corresponding DNA (the example from "screening a library").
.....................................................................................................................
Q: I'm still a little fuzzy about "making a library" and screening.
A: "Making a library" means chopping up a lot of DNA (usually with restriction enzymes), ligating the pieces into a vector (such as a plasmid, or into phage DNA), inserting the vectors into bacteria, and growing up lots of colonies or lots of copies of the vectors. If we are talking about bacteria, then we mean that the different bacterial colonies have plasmids with different pieces of DNA in them. If we are talking about phage, then the various plaques have phage with different pieces of DNA ligated into their DNA.
"Screening" means using a probe to find which of these colonies or plaques have the DNA that matches the probe. That identifies the colony or plaque that has the DNA that you want.
Suppose, for example, that you have identified a gene for hemoglobin in mice,
and you have cloned that gene into a vector, so you can use it to make a probe:
labled copies of the mouse hemoglobin gene. Then suppose you want to study hemoglobin
in newts, chickens, cows, and humans. You could take DNA from those species,
chop it up, clone it into phage, screen the phage, and isolate the phage that
have hemoglobin DNA from the four species you want to study. You now have DNA
that you can study, specific for that gene; you could analyze its sequences
to find out how they differ from one species to another, figure out what the
protein sequence would be-- or even find out how many copies of the gene exist
in each species. That's a simple example of the beginning; you'll find more
in just about any issue of Science, or Molecular Biology & Evolution, or
Journal of Molecular Ecology, or American Journal of Botany, or Cell, or Nature,
or many other biology journals in the library....
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Q: How is a probe used in a reaction? What does the probe do?
A: The probe binds to complementary DNA (the DNA has to be denatured, so that
the bases of the probe can H-bond to the bases of the target DNA). We find the
probe by locating some marker on the probe, such as having a radioisotope that
exposes X-ray film. That locates where the target DNA is located.
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Q: How exactly do you get different colors to show up when sequencing? Is each ddNTP labled with a radioisotope that glows differently?
A: Each lane is labled with a different "fluorochrome", so that it
fluoresces a different color. This works without radioactivity. Remember that
there are 4 reactions, done in parallel, one for each ddNTP. The labels can
be attached to any one of the bases used in each reaction, or to the primer
used (same primer sequence for each of the four, but a different fluorochrome
for each one).
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Q: How close are they to actually having the entire human genome sequenced?
A: Well, the complete sequence for the first chromosome was "published"
(i.e. loaded into one of the global databases) last fall. It was one of the
smallest chromosomes (#22 I think). There are different groups working on the
problem in different ways. One private group says that it will have the complete
sequence-- all the chromosomes-- done by the end of this year! So, even if it
is not this year, it will probably be very soon.
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Q: How does knowing a DNA sequence apply to a larger view?
A: Good question; an equally good answer could not be short, and is bound to
be full of hypotheticals. Knowing sequences can speed up specific projects (e.g.
designing primers to search for particular genes & markers), where the sequence
_per se_ is not the end of the study but just one of the means. In other cases,
knowing the sequence is closer to the end: by analyzing sequences we learn about
evolution of a family or order of plants, or the reasons that a gene is defective,
produces no protein product (or an altered product), and thereby results in
a pathology such as cancer.
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Q: I would like another description of the PCR. I don't know if I understand how it works.
A: Probably wouldn't help a lot for me to just use words: pictures would be
a lot more useful. I will put a couple articles on reserve and hope they clarify
the process. You could also try pp 79-84 in the WGWZ book.
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Q: What role do ddNTP's play in the PCR?
A: ddNTP's are not used in regular PCR; they are used in sequencing (and although
I did not discuss it in class, the PCR process can be modified fairly simply
by using ddNTPs to do sequencing).
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Q: What is the purpose of PCR?
A: To "amplify DNA", i.e. to make (lots) more of it. The specificity--
i.e. which DNA is amplified-- comes mostly from the primers. If you have primers
specific for hemoglobin, for instance, you would only get more copies of that
gene (assuming everything works right-- and that there are not multiple copies
of that gene! In fact there are multiple copies, and we have learned more about
them by doing PCR on the different hemoglobin genes).
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Q: How can we apply PCR? What does amplifying DNA synthesis of an organism allow
us to do?
Q: Why would you want to make so many copies of DNA using PCR? What is it used
for?
Q: So if you have PCR and you run it a whole bunch of times and get all this
DNA, why would you want so much DNA replicated?
A: (for all 3): The list has become so enormous it is hard to summarize. For
starters (examples), see the examples in chapter 6 of WGWZ. It is little exaggeration
to say that it is now hard to pick up a biological journal and _not_ run into
at least one project that used PCR. I will put an article on reserve that will
give some examples you might not have expected, e.g. in systematics, ecology,
etc.
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Q: In forensics, if they find blood, hair, or saliva and use PCR to replicate
the DNA and sequence the DNA using Sanger's method, then what? They have a series
of bases, but what good does that do the police? How is it possible to match
a sequence?
A: Usually the goal is not to compare sequences, but researchers are looking for genetic markers that differ from one individual to another. Some genes ("genes" used very loosely to refer to sections of DNA, that do not have to produce protein products) are highly polymorphic, meaning that there are many different alleles in the population, and these alleles differ in size: i.e. you may have 21 bases for that gene on one of your chromosomes, and 35 on the homologous chromosome, while I might be heterozygous for alleles with 24 and 39 bases. Because the DNA that flanks this region on either side is the same for all of the alleles, one set of primers can be used, and this will produce different-sized PCR products that can be separated by electrophoresis and identified on the gel.
Especially when there are many alleles for such a gene, and when forensics
experts can amplify many such polymorphic genes, it is possible to identify
a unique, multilocus genotype for each individual in a population.
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Q: How does PCR work when you take the DNA off the chimp hair for example? Do
you use the same high temps as you do with Thermus aquaticus? Do you put the
chimp DNA into the T. aquaticus first? So pretty much I don't understand the
application part of PCR.
Q: Another question is: in synthesis, does it (the PCR rxn) stop when the next primer is reached?
A: No. Remember that before the elongation takes place, the template strands
are denatured, so there is nothing at the position where the "other primer"
would attach.
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Q: What kind of limitations does PCR have? How old can DNA be (dinosaurs?) and
who owns the PCR-created DNA? The originator (for example, me) or the person
who ran the machine to duplicate it?
A: No dinos yet; that's at least 65 mya. DNA is remarkably sturdy stuff, but the world is full of hungry fungi and microbes (etc.) that would eat it up given half a chance, and mere chemistry alone can eventually degrade it: random thermal flux leading to hydrolysis, etc. (all that stuff from chemistry). In short, it falls apart eventually, and only RARELY are conditions correct to preserve an organic molecule for a long time (cold, anoxic, etc.).
Once the DNA is hydrolyzed (i.e. the backbone broken), amplification cannot continue, so over time the DNA becomes degraded (broken to short pieces etc, or bases broken off the backbone) and will not serve as a template.
People have amplified DNA from human remains several thousand years old, and
extinct ground sloths even older. The oldest published example I know of is
a plant (Magnolia) sample from the Miocene, 17 -20 MYA; the researchers are
very highly respected, but I do not know if their results are still considered
credible.
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Q: How can you possibly predict the correct conditions for the PCR reaction
without trial and error? Wow.
A: Well, it's called doing your homework and studying some P-chem: finding out information about optimal reaction conditions for the enzyme, such as the composition & strength of the buffer, requirments for ions (especially Mg++), temperature optima, estimating the temperatures necessary for primers to "stick" depending on their base composition (number of H-bonds), etc.
You will do this later in the semester.
.....................................................................................................................
Q: How do you know what primers to use in PCR? (Since you don't know the DNA
sequence).
A: Three answers: (1) In most applications you do have to know the sequence,
at least the sequences flanking the region you want to amplify. (2) There is
a particular variation where short (10-base) primers are used; because they
are short, they bind to various places scattered throughout the genome; this
method is called "randomly amplified polymorphic DNA (RAPD)", and
as the name suggests, the PCR products often differ in length from one individual
to another-- a kind of genetic polymorphism easily detected by agarose electrophoresis.
.....................................................................................................................
Q: My only question is about the primers. How do you know what primer sequence
to put into a PCR rxn?
A: You have to get this information some other way-- and how you get it will
depend on the kind of project you are working on, etc.
.....................................................................................................................
Q: Where or how is Kary Mullis now, is he reaping the profits of his idea? I've
heard he is quite an eccentric character.
A: Don't know the answers to these. He's moved on from Cetus (the company was
bought out by Perkin-Elmer, and then sold, and the PCR patent was sold to Hoffman-Roche).
If you're interested, he wrote an article describing the origins of PCR in Scientific
American, April of 1990.
.....................................................................................................................
Q: Not really sure how the last one (PCR) would detect mutations, unless it
is only a technique for making sure there weren't mutations in the DNA you were
making when you're making a bunch of copies.
A: Depends on the kind of mutation. Sequencing the DNA produced from PCR is
the most straightforward answer, but not the easiest technically. Basic question:
what kind of mutation are you looking for? If it is for insertions or deletions,
you might take the amplified DNA and do electrophoresis, looking for different
sized bands (see answer to earlier question); this is basically the technique
behind "DNA fingerprinting". Other modifications can be used for detecting
single-base point mutations (via analyzing things called single strand conformation
polymorphisms, which refers to the fact that the DNA will move through a gel
differently if the two strands match exactly or if they do not).
.....................................................................................................................
Q: How does the DNA strand know when to stop synthesis [the question had a diagram
included]?
Q: You mentioned Mullis was interested in detecting mutations. How does PCR help detect mutations? Perhaps by increasing the DNA sample to sequence?
A: Yes- and see the answer to the questions above. Sequencing with PCR is done
by using only _one_ of the two primers: you need to start with a fair amount
of the DNA, rather than just a few cells, but it will still increase the amount
of DNA, even though the increase is arithmetic rather than exponential: you
can't double the number of strands each time if you only duplicate one of them.
.....................................................................................................................
Q: On a related topic, in PCR, we get [diagram included]. Why don't we lose [the region of DNA "upstream" of the primers]?
A: This does get lost. Suppoe we have two primers, L (left) and R (right).
A close examination of a diagram of PCR would be helpful: the upstream region
of primer R can't get copied ... however, the strand that grows using primer
L and the opposite strand as a template can continue to grow, even past the
region where primer R would be. This kind of strand is called "the long
product", and more is made with each PCR cycle, but it is not _doubled_
each time as the region between the two primers is ("the short product").
.....................................................................................................................
Q: I don't understand how PCR could be modified to sequence DNA. How can repliating
large amounts of DNA be changed to sequencing it?
Q: How can you modify PCR to do sequencing?
A: Brief answer to this is above (maybe I have to start numbering these questions
so I can refer to them by number): basically, (1) first you do PCR to make lots
of the target region you want to sequence. Then (2) you just amplify _one_ of
the strands, by using _one_ primer. Radiolabled nucleotides are used, and a
different ddNTP mixed in with each of 4 rxns. Products run on a gel look just
like the "ladders" we see on Sanger's method of cloning the DNA into
M13.
.....................................................................................................................
Q: We were talking about errors in chains produced by DNA polymerase. How often
do they occur? Can one test for errors in the product? How do scientists deal
with these errors, if at all> Does repeated heating allow for more errors?
A: Errors are a problem, and for thermocycle sequencing, different manufacturers
produce enzymes from different organisms and advertise their accuracy rates.
Error rates are on the order of 1 in 10 billion nucleotides-- but that is in
vivo, in E. coli. In-vitro rates of errors for Taq can be around 1 in 10,000,
which would be way too high to generate reliable data. They can be reduced by
controlling reaction conditions; or detected by sequencing the complementary
strand; or perhaps repeating the work... etc.
.....................................................................................................................
Q: Is there a way we can eliminate or reduce the errors in DNA replication?
A: This will be the focus of the next lecture.
.....................................................................................................................
Q: How does the oligo synthesis work? I don't see the backwardness. Do you make the sequence yourself or does it replicate from some place?
A: The "backwardness" is that chain elongation occurs by addition
to the 5' end. In vivo, the elongation is always at the 3' end. You determine
the sequence by choosing which phosphoramidite-modified nucleotide solution
you add in each step.
.....................................................................................................................
Q: When would I use oligo synthesis?
A: When you wanted to make a primer (e.g. to amplify some DNA via PCR) and
had access to a synthesizer.
.....................................................................................................................
Q: I'm wondering what the purpose of chemists' reversing the direction of DNA
synthesis is. What's the advantage of doing it backward instead of the way the
cell does it?
A: I don't know enough o-chem to answer this one: why it is possible to set
up the kinds of protecting groups you need one way but not the other. Maybe
when I was taking O-chem I could have done so. Maybe I should send this question
to Carpenter-- it would make a great exam question, I imagine....
.....................................................................................................................
Q: Cell differentiation? how the hell do certain genes "know" to be turned on or off?
A: That, my friend, is the number one question of molecular and developmental
biology for the next few decades coming, at least. We will spend a few weeks
beginning to understand how.
.....................................................................................................................
Q: How do they know where a gene starts and where it ends?
A: Brings up the question of "what is a gene?" If you mean a section
that codes for a protein.... well, we get into that when we start studying transcription.
Soon.
.....................................................................................................................
Q: In cell biology I learned that with aging DNA becomes "damaged"
every time DNA is replicated, some of the end chain is lost. With current technology,
why is it not possible to re-attach those lost chains?
A: Yes-- and if you think about what is needed to replicate DNA, the question
does come up of how the _ends_ of a linear DNA molecule can both get replicated.
Short answer is that they don't-- not directly, so as cells divide, each generation
the DNA gets a bit shorter (crude generalization). More crudely, that is part
of how a cell knows that it has differentiated, and is not supposed to keep
dividing. The ends are called "telomeres", and there is an enzyme
called "telomerase" whose sole job in life is to extend the telomers
to their full length, which has to happen for cells that keep dividing. However,
most cells aren't supposed to keep dividing-- which is why excess telomerase
activity is found in some cancers.
.....................................................................................................................
Q: WHERE ARE MY KEYS!?
A: That happens to me, too--- usually I get to my car and realize they're back
in the office.
.....................................................................................................................
Q: Could you make a basic timeline which has when what breakthroughs were made?
It gets confusing trying to remember which tools were available when so and
so was discovered/developed.
A: I have not made one up. If I were going to, I would start with the material
on reserve by Alberts et al., Molecular Biology of the Cell (the photocopied
material about materials and methods from their chapter 4)-- so that is where
I would recommend you start. You may want to add some things to that (e.g. I
don't know if Jacob & Monod's work would be listed there; it was in the
early 1960's; Mullis's invention of PCR (published 1985), etc.
.....................................................................................................................
Q: What kind of job is going to allow me to use the micro techniques and lab experience that I know and love but also take advantage of my theatre background and my love for public speaking and creative oral presentation?
A: Hmmm...... Well, as my mom said to me, "Write when you get work". ;)
More seriously, you might not find one job that encompasses all of that, but
might find a life with those opportunities in different parts-- I say that because
I used to live in a small town known widely for its outstanding community theatre
(classic & original works, guest directors, etc.). Some amazing performances
there, and had the advantage of bringing together people from all sorts of different
walks of life: lawyers and nurses and teachers and carpenters and .....
.....................................................................................................................
Q: How and where do people find ideas like these?
A: As Pasteur put it: "Chance favors the prepared mind". And Edison:
"Genius is 1 % inspiration and 99% perspiration".
.....................................................................................................................
Q: I really want to learn how all this is controlled? What decides if recombinant
DNA is/should be replicated?
ANS: I don't know whether you mean "controlled" in a physical or
legal sense--?? If you mean legal, the first line of regulations are institutional:
the U. requires reporting of work that involves recombinant DNA with higher
organisms (animals, plants) or hazardous microbes. We won't be doing either
(and have previously reported the kind of recombinant work we are doing). But
on a broader note, there really are few if any regulations on recombinant DNA
per se-- but there are restrictions on _releasing_ organisms carrying genes
transferred by recombinant DNA. This includes, for example, engineered corn,
soybeans, cotton, and fish.
.....................................................................................................................
When will this all tie together into less "background" or materials
we use and get into the nitty gritty?
ANS: by next week I expect we will be through the M&M, and you will see
that our discussion of the structure and replcation of DNA will begin to draw
on this M&M info.
.....................................................................................................................
Can the techniques we learned today be used in a quantitative way (electrophoresis,
probing, combinations), or can they only tell us "yes, we have _this_"
or "no we don't have _this"?
ANS: Yes, we can use these quantitatively. In one sense, a quantitative application
would be to estimate the size of a piece of DNA that moved through a gel. In
another, we could estimate the amount of DNA in that piece. We could do this
by staining the gel, or by probing. In either case, we would then compare the
resulting band with bands of different known amounts of DNA.
.....................................................................................................................
Q: How DNA is an acid if it is made of bases?
Q: How can DNA be an acid if it is composed of bases?
ANS: I won't tell you the whole answer yet, just one more hint: dig up a figure
illustrating a DNA molecule, that shows all of the atoms in the bases as well
as the sugar-phosphate backbone.
.....................................................................................................................
Q: Why the 3 questions?
ANS: Three main reasons: 1) The questions let me know how well the information
is coming across. 2) They give me a chance to clarify points that remain fuzzy
from lecture, or address concerns that you might have, etc. 3) They make you
think about the material, and you learn it better when you are trying to recall
the info and come up with questions, rather than just record the information
being presented.
.....................................................................................................................
Q: I'm curious about the legality and laws surrounding patenting of enzymes and organisms. I find it interesting that this can be done.
A: Yes, though I'm afraid the legal issues are an area I have not at all tried
to keep up on (though the first mouse patented was back in Science just this
week, and the PCR patent keeps getting argued & new decisions even after
all these years).
.....................................................................................................................
Q: Who will regulate the ethics of what this technology (molecular biology)
is used for some day? This can no doubt improve life for some, but it can also
no doubt threaten lives and mankind (and also other species) also. Who will
police this someday?
A: Well, who regulates the ethics of any technology? Universities establish
bioethics committees and offices, NIH (National Institutes of Health) has their
own review boards and sets of standards & procedures that are supposed to
be followed... but that's only part of it. Did anyone "regulate" the
Soviet Union when it's scientists cloned together genes from different viruses
to make more lethal viruses? Or, at the other end, what about the conflicts
over the use of genetically engineered crops?
.....................................................................................................................
Q: I read in the book other uses for PCR such as sex determination in embryos,
and using this to deal with X-linked diseases (only implanting female embryos
into mom) -- can you talk more about this? Very interesting! Ethics??
A: Well.... the technical issues, the biological issues, are not really in
the realm of molecular biology as much as in physiology (e.g. controlling reproduction)
and cell biology ( e.g. handling such small embryos). As for the ethical issues...
why don't you ask me sometime when we're waiting for reactions or something
in lab? I'm not interested in lecturing about ethics; I think they're better
approached as discussions/conversations.
.....................................................................................................................
MISCELLANEOUS
Q: How and where do people find ideas like these?
A: As Pasteur put it: "Chance favors the prepared mind". And Edison:
"Genius is 1 % inspiration and 99% perspiration".
.....................................................................................................................
Q: I really want to learn how all this is controlled? What decides if recombinant
DNA is/should be replicated?
ANS: I don't know whether you mean "controlled" in a physical or
legal sense--?? If you mean legal, the first line of regulations are institutional:
the U. requires reporting of work that involves recombinant DNA with higher
organisms (animals, plants) or hazardous microbes. We won't be doing either
(and have previously reported the kind of recombinant work we are doing). But
on a broader note, there really are few if any regulations on recombinant DNA
per se-- but there are restrictions on _releasing_ organisms carrying genes
transferred by recombinant DNA. This includes, for example, engineered corn,
soybeans, cotton, and fish.
.....................................................................................................................
When will this all tie together into less "background" or materials
we use and get into the nitty gritty?
ANS: by next week I expect we will be through the M&M, and you will see
that our discussion of the structure and replcation of DNA will begin to draw
on this M&M info.
.....................................................................................................................
Can the techniques we learned today be used in a quantitative way (electrophoresis,
probing, combinations), or can they only tell us "yes, we have _this_"
or "no we don't have _this"?
ANS: Yes, we can use these quantitatively. In one sense, a quantitative application
would be to estimate the size of a piece of DNA that moved through a gel. In
another, we could estimate the amount of DNA in that piece. We could do this
by staining the gel, or by probing. In either case, we would then compare the
resulting band with bands of different known amounts of DNA.
.....................................................................................................................
Q: How DNA is an acid if it is made of bases?
Q: How can DNA be an acid if it is composed of bases?
ANS: I won't tell you the whole answer yet, just one more hint: dig up a figure
illustrating a DNA molecule, that shows all of the atoms in the bases as well
as the sugar-phosphate backbone.
.....................................................................................................................
Q: Why the 3 questions?
ANS: Three main reasons: 1) The questions let me know how well the information
is coming across. 2) They give me a chance to clarify points that remain fuzzy
from lecture, or address concerns that you might have, etc. 3) They make you
think about the material, and you learn it better when you are trying to recall
the info and come up with questions, rather than just record the information
being presented.
.....................................................................................................................
DNA Replication
Okazaki's Work
Translation
Misc
Q: What is a Klenow fragment? How can I do really well on this exam?
ANS: Remember that pol I has three functions: 5'-->3' polymerase, 3'-->5'
exonuclease, and 5'-->3 exonuclease. Each function is located in a different
part of the protein. The part that does the 5'-->3 exonuclease function is
at one end of the protein, and can separated by relatively mild proteolytic
digestion, leaving the polymerase/proofreading portions. This portion is referred
to as the "Klenow fragment" after the person who first isolated it.
.....................................................................................................................
Q: Still a little confused on primers and how they are formed and added to lagging
strand. Is this lecture covered on the test? Still a little confused on pulse
labeling.
Q: I'm confused still on all of the CH3 bonds. What does it bond to that causes something bad?
Q: What does pol II do?
Q: Are we supposed to just get the general message from the two Okazaki graphs and if so why did we have to spend so much time on it when you could have said if you want to know more about the graphs come talk to me otherwise you just have to know the general idea of the graphs and Okazaki's experiments?
ANS: I like this question because it gives me a chance to emphasize the real
reason for studying this stuff-- and this goes back to the main themes of the
course: the emphasis on empirical evidence, and what we see with our "mind's
eye". The goals are to learn how to go back and forth between the "model"
or theory that we have, how an experiment is designed, what the results are,
how they are interpreted, and how that modifies our model or theory. Okazaki's
work, then, is a case study for all of these. You see, then, that getting the
general message has very little to do with why we have been studying this.
.....................................................................................................................
Q: What are Okazaki fragments-- why don't they get repaired and why don't they
form on the leading strand?
ANS: At each origin of replication, where the two DNA strands separate, one
will be a leading strand and the other will be a lagging strand. Replication
begins with the primosome laying down a RNA primer, which is then extended by
pol III. On the leading strand, this just keeps on going towards the fork where
the strands are separating, making one long, continuous piece.
.....................................................................................................................
Q. I'm still a little shaky on how the wobbling of the third bas fits into the grand scheme of things.
A: Perhaps I should not have even mentioned this without having some decent
illustrations. See page 573-574 in Weaver-- though even these are not particularly
good illustrations. How it fits into the grand scheme of things is beautiful.
Your mitochondria can synthesize proteins-- they have their own DNA, their own
ribosomes (which are prokaryotic-type ribosomes, by the way, more similar to
those of the bacteria in your gut than to the ribosomes in the cytoplasm of
your cells!). Your mitochondria can read the complete set of codons despite
the fact that you only have genes for about 20 different tRNA molecules.
.....................................................................................................................
Q. I am unclear on initiating factors, eukaryotic elongation factors, etc.
A: Weaver discusses this in quite a bit more detail than we will cover in the
course. The important parts to know, for our purposes, are that (1) ribosomes
and mRNA are not all there is to translation: specific proteins carry out roles
at different parts of the procedure; (2) these proteins are similar but not
identical between prokaryotes and eukaryotes; and (3) the energy for several
steps of protein synthesis comes from GTP.
.....................................................................................................................
MISC
Q: I have a question about the CIP addition in lab. CIP cleaves off the 5'phosphate so that pUC cannot re-bind with itself, I get that much (but correct me if I'm wrong). How then can the lambda DNA bind to it? Does it get re-phophorylated? Perhaps I missed something, but as I see it no 5' phosphate, no phophodiester bonds.
A: No, this is a good question. The answer is that, with the P gone from the pUC, the only ligation that can take place is on the other strand, i.e. the piece of lambda that carries a P. The two pieces of DNA are then held together by the H-bonds between the sticky ends, and only one of the two DNA strands. However, as we see from the results, this is enough for the recombinant plasmid to be replicated, and the progeny plasmids are all intact. If that isn't clear in words, maybe I can try to draw it.
This still leaves us with the question, though, of how the dephosphorylated pUC molecules can re-ligate-- and have this occur with such high frequency. We know that this happens (rather than simply being a failure to be cut apart in the first place) not only because we let the digestion go a very long time (overnight-- though four hours would probably be enough) but also because we have examined the results by our first electrophoresis, and we see that the pUC are digested completely, and do not show the bands of the uncut pUC.
My guess is that the low temperatures at which we conduct the ligation (16
C), which represents a compromise between activation energy for the enzyme and
stabilty of the H bonding between the sticky ends, is low enough that pUC molecules
held together only by the H-bonds can be replicated. It might be interesting
to conduct a series of ligation experiments at different temps, from (say) 15
to 35 C, and see if there is a decline in the percent of blue colonies as the
temp gets higher. On the other hand, though, if the pieces we were attempting
to ligate in are very large (as is the case for the lambda/Eco fragments), we
might not see much change because higher temps might make the DNA just to squggly
for the ligase to tie them together.
.....................................................................................................................
PART III: REGULATING GENE EXPRESSION
Bacterial Genetics
Mapping
Enzymes
Regulation
LAC
ARA
TRP
Autogenous Regulation
Binding Proteins
Q. What exactly is a partial diploid and how do they get made?
Q. Partial diploids? Huh?
Q. What are partial diploids?
Q. I don't understand cis/trans test of partial diploids
A: We know that bacteria have large, circular chromosomes, and that they can
also carry plasmids that have a few genes. Bacterial geneticists have been able
to move some genes from the main chromosome onto plasmids. When these plasmids
are inserted into other bacteria, it effectively makes the bacteria "diploid"
for the loci that were transferred. A diagram would really help. Look at figure
1-18 on p. 48 of the handout from Friefelder's Molecular Biology text, on bacterial
matings. The diagram shows a partial diploid, after DNA has been transferred
from one bacterium to another by means of a plasmid (the right hand part of
the diagram shows what would happen after more steps, when the new DNA becomes
recombined with the chromosome, but that is a later step we don't have to worry
about here).
.....................................................................................................................
Q. The mechanics and experimental methods of operon mapping are pretty fuzzy to me. That's not a question but the topic is a question mark in my head.
A; The key to this is summarized in the Friefelder extract, on p. 48, in the
bottom paragraph. Perhaps the key additional information not obvious is that
bacterial geneticists generated a variety of mutant strains, e.g unable to synthesize
B-galactosidase _or_ unable to turn off synthesis of the enzyme. Because they
also worked with the F' plasmid, which would instigate the transfer of a linear
array of genes from one bacterium to another, they could map the locations of
the different genes (the different mutants) based on how long it took for the
genes to transfer to the recipient bacteria.
.....................................................................................................................
Q. Is it possible to determine exact (not relative) locations of genes by the
frequency method? How? It seems like an imprecise science.
A: All measures of position are relative. The accuracy is remarkably precise.
Consider that jacob and Monod were able to map locations of promoter and operator,
between the repressor and B-galactosidase genes-- and recall the diagram that
I showed in class on Thursday: there really are only a few hundred bases in
that entire stretch.
.....................................................................................................................
Q. I'm still confused about the mapping distances in the problem, etc.
A: The key here is to use the data from the table on the handout to make a
graph such as that in figure 1-19 of the Friefelder handout. The graph leads
directly to the map, as Friefelder explains on pages 48-49.
.....................................................................................................................
Q. Is there any test to do to know if our maps made from graphs are right
A: Experimentally, one would conduct similar experiments with several different
sets of mutants, and compare the results.
.....................................................................................................................
Q. How sure are they on the validity of these genome maps?
A: Very sure. Very, very sure. That's how pUC got built, etc. You might want
to look at Muller-Hill's book.
.....................................................................................................................
(Back to Top)
Q. How do enzymes change their shapes?
A: Well, they don't-- at least not in the way proposed by the "enzyme
instruction" hypothesis. They make slight changes in shape as they bind
substrate (or an allosteric control molecule), but not the major changes envisioned
when people thought that, for instance, the change of bacteria from metabolizing
glucose to metabolizing lactose came from the changing shape of the enzymes.
.....................................................................................................................
Q. What is enzyme induction?
A: Turning on production of an enzyme.
.....................................................................................................................
Q. I don't get the "tool kit".
(see below)
Q. From the metabolic tool kit, what was the purpose of studying the decoy molecules? What was their function, and how do they they help?
(see below)
Q. Decoy substances turning on or not turning on, substrates and inducers.
A: I hope I clarified this on Thursday, during the review at the beginning
of class. The "tool kit" provided some substances that would change
color when the enzyme acted on them, giving researchers a way to measure enzyme
activity. There are also substances that allowed the researchers to turn on
the production of the enzymes, or to turn it off. They could not use lactose
for doing all of this because it is invisible, its breakdown products are invisible,
and it is both the inducer and substrate for the enzyme.
.....................................................................................................................
Q. I don't really understand the last question on the handout we worked on today,
so my quesition would be on what is going on there.
A: Do you mean "Diagram a flow chart for this experiment"? Start with the two bacterial strains you defined in problem 3. Then prepare media that would be able to would differentiate between the first strain and a strain (e.g. unable to metabolize lactose, galactose, or biotin, and sensitive to the T1 phage) and a strain that had contrasting alleles for all of the genes (e.g. can metabolize lactose, galactose, biotin, and resistant to T1)-- e.g. media that contain different combinations of these substances. Mix the two strains together and then remove aliquots at various times, agitate to separate, and plate on your various media. Record which traits appear together at different times after the two strains are put together, which gives you the measure of how far apart the genes are.
This is only a short summary, and pictures help. I opened the Klug & Cummings
text used in the Genetics course right to the pages where you will find a better
description: Chapter 7, especially pages 182-183, and the problems1-3 on the
bottom of page 201 and on p. 202.. You might also find this chapter useful in
a few weeks, when we discuss regulation in lambda.
.....................................................................................................................
Q: Big picture of operon/repressor/promoter.
ANS: Expression of genes must be _regulated_, not having all genes on all the
time: it must respond to the cellular milieu and to the environment of the cell.
.............................................................................................
Q: Could you briefly review the rule of thumb for operons?
ANS: The first 4 are on p. 33 of the course book (you may have found them by now). The fifth is: "Proteins bring information abouit the status of cellular metabolism AND about conditions outised the cell."
.............................................................................................
Q: What is positive and negative control again?
ANS: Positive: protein binding turns the gene ON.
Negative: Protein binding turns the gene OFF.
.............................................................................................
Q: Could you give me a heads up on what the differences are between negative
control and negative feedback as well as between positive control and positive
feedback?
ANS:
Negative control: it takes a specific event to shut something off; in the case
of gene regulation, it takes a protein binding to a regulatory region to shut
the gene off.
Positive control: it takes a specific event to turn something on; in the case of gene regulation, it takes a protein binding to a regulatory region to turn the gene on.
Negative feedback means that a system operates to maintain a given state variable. Best example is a thermostat set to 20 C: if colder, the heat turns on; if warmer, it shuts off; thus the system converges on a specified value.
Positive feedback means that a system operates to increase (or decrease) from a given state variable. Rarely do engineers have reason to build positive feedback mechanisms, so we don't have much for examples. "Autocatalysis" would be one, where the product of a reaction catalyzes the reaction that produces more of the product--- but I can't think of a specific example. A nuclear chain reaction would be an example of positive feedback.
Thus, for example, in the ARA operon we see examples of positive regulation,
negative regulation, and negative feedback. Be sure you can specify how each
of these works in ARA.
.............................................................................................
Q: Define operon. If we were asked to define lac operon or ara operon, what
answer would you like us to give?
.............................................................................................
Q. More of a statement: I think gene regulation is going to be confusing.
A: It is easy to be confused by this. Remember being confused by mitosis? By meiosis? By calculating concentrations and dilutions? The number one best way I know for you to learn this is to EXPLAIN IT TO SOMEONE ELSE. Fortunately, there is much sense and pattern in gene regulation (because of the "argument for simplicity", we find the rules of thumb often hold true).
I know that the lac operon, for example, is awfully theoretical: you can't
actually look at one and watch the parts move. But remember that you have seen
it work, when you plated bacteria that turned blue, and you saw what happened
when you "broke" one of the parts, so the bacteria turned white. You
might have seen what happens when you did not provide the bacteria with the
inducer and substrate (IPTG and X-Gal), perhaps by not spreading it across the
entire plate, and you ended up with colonies near the edges that grew but did
not turn blue, just because they lacked these two items from the "tool
kit". So you can tie the theoretical models discussed in class and in your
book with the results you have seen in lab, as well as with the problem set
kinds of questions.
.....................................................................................................................
Q. Can genes be turned on by other genes?? Not just their substrates?
A: Sure. "Repressor" is an example of one. We will see more.
.....................................................................................................................
Q. What are the processes that activate and deactivate genes?
A: That's what this part of the course is all about, and will lead us into
the next part. In other words, I'll be answering that question for a few weeks.
Much of it will boil down to proteins messing around with DNA.
.....................................................................................................................
Q. I still really don't understand constituitive synthesis. Also, the "tool
kit" is fuzzy-- what doe sit do when it initiates synthesis?
A: There were some comments about the "tool kit" above. "Constituitive
synthesis" should make more sense after Thursday's class, when we worked
with the problem set. At least I hope so.
.....................................................................................................................
Q. What is positive and negative control?
A: Another one I hope is more clear after Thursday; Positive control: protein
binding to DNA turns the gene ON. Negative control: Protein binging to DNA turns
a gene OFF.
.....................................................................................................................
Q. How exactly do positive and negative control work? How are regulators controlled
A: See above. For "How are regulators controlled", stay tuned over
the next couple weeks, as we look at other operons and the regulation of genes
in lambda.
.....................................................................................................................
Q. I don't know what the lac operon is and what it does an dhow it fits into
everything we talked about today. How does it work?
A: I didn't expect that this would be clear on Tuesday-- at that point we were
just beginning to look at lac, and had really only looked at some background
info. Hopefully Thursday's discussion of lac clarified how the pieces are put
together and how they work.
.....................................................................................................................
Q. What does all of this mean?
A: It means life works, mostly.
.....................................................................................................................
Q: How do all of these small things (operons, etc.) fit into the entire big picture of life?
A: We live on a planet that is just the right distance from its star, and has an atmosphere composed of just the right gasses, so that the temperature is precisely warm enough so that water is in the liquid state but H-bonds can still form. Incredible! This is an extremely narrow window of temperatures. Also, we have a crust containing the right elements so that carbon, oxygen, nitrogen, and phosphorus are fairly abundant. You should be able to insert a few more sentences or paragraphs here about how this planet happens to be suited to the chemical reactions of life.
But life can't exist unless those reactions can be controlled! Losing the control
of these reactions is precisely what we mean by the term "death".
Our understanding of how control works-- indeed, our understanding of how genes
work, and even what a gene is, has been built up over the last few decades largely
from studying these small things. Using "model systems" such as E.
coli and lambda gives us tools (both techniques and questions) to use with more
complicated systems, such as bacteria or yeast engineered to produce useful
proteins, or genetically engineered crops, or understanding how things like
HIV work.
.........................................................
Q: I was wondering if you have a Oc Z+ / O+ Z+ will the lac products produced
by the Oc Z+ bind to the repressor protein and restrict the repressor from binding
to the Oc, thus allowing O+ Z+ to also produce lac products?
.............................................................................................
Q: Why did we think it was a tetramer not just a dimer? [I think this refers
to the configuration of lac repressor binding to the operator].
ANS: I am not familiar with the evidence, but have tracked it down to these
original reports, cited in the article by Lewis et al. reporting the 3-D structure
of the lac operon:
Bourgois, S. and M. Pfahl. 1976. Adv. Prot. Chem 30:1
Farabaugh, P.J. 1978. Nature 274:765
Beyreuther, K., N. Alder, A. Geiseler, and A. Klemin. 1973. Proc. Nat. Acad.
Sci. 70:3576.
.............................................................................................
Q: My main question deals with O1 and O3 : how can there be two operators?
.............................................................................................
Q: What's the difference between O1 & O2, O2 & O3, etc.? Is it just
the structures or functions or what?
.............................................................................................
Q: How does lactose become allolactose in the presence of B-galactosidase when
B-galactosidase splits the lactose into galactose and glucose?
A: I don't know: you are asking a question about a specific reaction mechanism;
there is a diagram of lactose and allolactose in Weaver (fig 7.3) but that doesn't
answer your question. It is fascinating that this enzyme produces two different
products from the same substrate, and that one has a regulatory function while
the other is the "main" product, that gets fed into glycolysis.
..........................................................................
Q: When does the cell use the ara operon?
ANS: When arabinose is present but glucose is not (i.e. low energy state: hungry).
.............................................................................................
Q: If C binds like [diagram of two C molecules attached one on top the other]
in loop form w/o arabinose, how does it change to [diagram of the two C molecules
attached next to each other] when arabinose binds?
ANS: Arabinose binds to an allosteric site on C, changing the shape of the
protein so that it no longer binds to the O2site, but will bind to I2. This
can be tested by DNA binding tests with and without arabinose present, or by
looking at the results of electrophoresis: see W2 figs. 7.27 and 7.28 for an
example.
.............................................................................................
Q: In your diagram of the ara function: in step 2 is it because the DNA is folded
that represses the BAD genes, or something else?
Q: Do we know _how_ the BAD transcription is repressed in the ara operon? Does
the loop interfere with binding?
ANS: Good question. While the lac operon has been studied in great detail with
regard to this question (e.g. "Just what does RNA pol produce when the
lac gene is repressed? How many bases are strung together?"), I haven't
read any comparable work for the ara operon.
.............................................................................................
Q: What's the purpose of the ara operon? What does an operon do (I must of forget).
ANS: Think "coordinated gene expression": a group of genes whose
products (enzymes) work together, all controlled by one regulatory region. The
purpose of the ara operon is to produce the enzymes necessary for metabolizing
arabinose, when it is present and the cell is "hungry".
.............................................................................................
Q: How do BAD come into play with the arabinose operon?
ANS: These are the abbreviations for the genes that produce enzymes that metabolize
arabinose-- see the handout for their names and the reactions they run.
.............................................................................................
Q: Do BAD all have the same promoter and basically if you have one being made are they all being made?
ANS: Yes.
.............................................................................................
Q: How were they able to conclude what they did about the looping through the experiments discussed?
ANS: Remember that they had some other clues: first, the two operators (O1
and O2) were mapped far apart, about 250 bp from each other, so it seemed likely
that the DNA must be looping to bring the regulatory sites close enough for
proteins to reach them. Second, Lobell and Schleif did some other experiments,
wherein they controlled the number of bases in the minicircle of DNA containing
the operators, so that they could control which direction the binding portion
of the DNA would face-- i.e. pointing into the circle or out of the circle,
and in the latter case the protein-induced looping did not occur (Weaver describes
some of this work on p. 194).
.............................................................................................
Q: Rules of thumb for arabinose-- I know I can figure this one out.
ANS: Yup, I know you can too, and that it would be better review for you to
do this than for me to hand you the answer.
.............................................................................................
Q: How is so much known about the ara operon if it (arabinose) is kind of a
last resort as far as an energy source?
ANS: There really isn't much reason to expect that how fast a metabolic pathway
produces ATP would be tightly correlated with how much we know about it. (However,
I have never looked into the history of this research, as I have for the lac
operon.). The finding that the regulatory regions looped in space was a real
breakthrough, leading to further research and energizing our ideas about how
genetic regulation might work, so people (e.g. Lobel et al) pursued it.
.............................................................................................
Q: Why does supercoiled DNA travel faster in the gel than uncoiled DNA? Why
does it supercoil at all when the C protein is bound?
Q: Why does when it is bound in loop does it travel farther down electrophoresis
gel? Didn't quite understand reasoning.
Q: I'm curious, why does the supercoiling cause the DNA to go faster in the
gel, is it because it's more compact and doesn't collide with other particles
often?
ANS: Because when the C protein can bind the two sites of the minicircle together,
the rest of the DNA can twist around itself, forming an even tighter, smaller
construct. Think of a small rubber band that you have twisted together: it moves
through the gel faster because it has less surface area and fits through the
pore space of the gel more easily.
.............................................................................................
Q: What is the difference between the two mutants used in the ara experiment
to show the differences between the binding of I?
ANS: One has a mutation in the ara O2 gene, the other has a mutation in the
ara I gene. Remember that these are the two sections of DNA held together by
the C protein, so that when either of them is mutated, the C protein cannot
hold the DNA as strongly.
.............................................................................................
Q: Why does the labeled DNA, but not the unlabled DNA, show up on the gel in
a competitive binding assay?
ANS: What we are looking at is not the gel itself, but an autoradiograph of
the gel, so we only see the labled DNA.
.............................................................................................
Q: On the competitive binding assay our handouts show unlabled ara I and the overhead showed unlabled O2, Why is this different and would it change the result?
ANS: Actually, what Lobel and Schleif used for the competition was unlabled
minicircles, so they had both the O2 and the I sites on them, both of which
competed for binding with the C protein. My overhead and handout should probably
show this.
.....................................................................................................................
TRP
Q: How does it work? I missed the day we covered it and the double hairpin stuff
is over my head right now.
A: Weaver gives a good, thorough discussion of how this works. Remember that
there are two separate mechanisms, and that both bring information about the
concentration of tryptophan (the end product) to the transcription apparatus.
The first mechanism is the aporepressor/co-repressor binding that prevents transcription,
in a manner similar to what we saw with the lac operon. The second mechanism,
attenuation, is quite different. This involves the synthesis of a "leader
peptide" before the structural (enzyme) genes. Synthesizing the leader
requires tryptophan: if there is lots of trp around, then attenuation shuts
down transcription of the enzyme genes. If trp levels are low, attenuation does
not occur and the enzyme genes are transcribed so the cell can synthesize more
tryptophan. The mechanism uses a terminator, just like the rho-independent terminator
we looked at a few weeks ago.
.....................................................................................................................
Q: I am unclear about the hairpin formation in the trp operon.
A: Again, perhaps Weaver's illustrations might help, or the figures on the
handout in class. The key is that transcription and translation are happening
at the same time, on the same piece of RNA-- since this is a prokaryote. If
translation stalls, transcription can continue. If translation goes quickly,
then transcription is terminated. Drawing the steps will be an important part
of learning how this works.
.....................................................................................................................
Q: With the attenuator/trp thing, there is a picture in the book (p. 201). When trp is low, it goes through the attenuator and is still read, but how does the RNA polymerase go through the UGA stop signal to continue transcription?
A: Transcription and translation are fundamentally different processes, and
what stops translation has no effect on transcription: a stop codon such as
UGA makes no difference to the transcription apparatus-- and you will recall
that mRNA is synthesized with a 3' noncoding region that comes after the stop
codon.
.....................................................................................................................
Q: I don't understand the example on one of the handouts about autogenous regulation:
16S ribosomal RNA and mRNA both having a binding site for protein S7. What is
the regulation part of this? How is this autogenous?
A: Regulation comes at the level of translation: if there is lots of S7 protein
around, it will bind not only to the rRNA but also to the mRNA that codes for
S7, which will reduce the level of translation that takes place, decreasing
the level of S7 in the cell. "Autogenous" means "its own origin",
and this applies to S7's regulation of its own translation.
.....................................................................................................................
Q: I'm going to ask it today after class-- about S7.
Q: Could you go over autogenous regulation again, or tell me where to find it in the book? I am confused about this concept.
A: Weaver does not talk about this, so you'll have to examine the handout I distributed. For a bit more discussion, you could go to the source I took the figure from: Molecular Biology of the Gene, by Watson et al., 1987 (there is a copy in the library).
Q: How does autogenous regulation work? Diagram?
A: Sorry, a weak point of this communication is the inability to share diagrams.
I'll have to refer you to the handout, and point out the similarity of the the
2-D structures of the mRNA and rRNA that the S7 protein binds to.
...................................................................................................................................................
Q: Is it just the repressor that does this h-t-h binding or do the other proteins (cro) do this too?
A: Lots of others do this; the Kinemage program showed several examples. The first images we looked at overlaid the h-t-h section of cro, lambda repressor, and lac repressor, emphasizing their similarity.
.....................................................................................................................
Q: I'm not really sure what helix-turn-helix is. I am still really confused about pretty much everything that has to do with lambda!
A: Hopefully the Kinemage session clarified what the h-t-h section is, along
with some of the figures on the handout. Lambda certainly can be confusing!
There are a lot of players in that story: different proteins, different genes,
etc. Mastering the cast of characters is the first step to understanding what's
going on. The second is being able to DRAW various parts-- e.g. how repressor
binds to the operator and how that affects expression of the CI and cro genes,
etc. If I knew a simple/easy way to convey the story, I would do it!
.....................................................................................................................
Q: I'm still confused on helix-turn-helix. Could you explain it one more time please? How does it associate with the lambda repressor?
A: Tough to do well with words: this leans heavily on our mind's eye, and the
Kinemage pictures are the best way I know of coveying what a h-t-h is and how
it interacts with DNA. The best is the figure that shows the H-bonding of the
h-t-h with the DNA bases in the double helix. See Kinemage figures for Chapter
7 (c7dnabnd), especially Kinemage #3. Lambda repressor and cro both bind to
DNA by this kind of structure, though both their amino acid sequences and the
DNA sequences they bind to have slight differences.
.....................................................................................................................
Q: Since the sugar phosphate part of nucleotides is the same throughout, and the basees are stuck in the middle, how do proteins recognize the difference? Is that where the major groove comes into play?
A: Again, this is best answered with pictures, and the Kinemage pictures do
the best job. See c7dnabnd especially Kinemage #3.
.....................................................................................................................
PART IV: EUKARYOTIC HYPERDRIVE
Note: Links not yet installed in this section
INTRO TO EUKARYOTES
Q: How much do we really know about genetic regulation in eukaryotes since it is so complex? How much more is there to learn? And how much of what has been experimentally described is actually correct?
ANS: This is incredibly complex, and grows more so by leaps and bounds every
month. This is a scientific revolution happening. I see my task as helping you
to prepare to understand what is unfolding.
.....................................................................................................................
EUKARYOTIC mRNA
Q: What is the purpose of the cap structure?
ANS: Four functions: (1) They protect the mRNA from degradation by RNAses.
92) They enhanse the translation of mRNAs. In doing so, they may "tell"
the ribosome where to start. Euk. mRNA lacks the Shine-Dalgarno sequence found
in prokaryotes, that distinguishes a start AUG from an internal AUG. The 5'
cap acts to indicate that "this is a messenger RNA" to the ribosome,
which binds to the 5' end and slides downstream until it gets to an AUG, and
there starts translation. (3) They enhance the transport of the mRNAs into the
cytoplasm (note that not all RNA goes there). (4) They enhance the efficiency
of splicing of RNA (a topic we will get to later).
.....................................................................................................................
Q: What is the role of having a bass-ackward change in the mRNA of eukaryotes?
ANS: see above
Q: Explain again the process of cap blocks on the 5' end of mRNA. Didn't understand the caps or the reversal of the one base.
ANS: see above. Distinguishes mRNA from the other RNAs
.....................................................................................................................
Q: Do the internal promoters get cut out before translation begins?
ANS: Wait-- what kinds of RNA are transcribed from these promoters? Why are
you wondering about translation.......??
.....................................................................................................................
Q: If the promoters are downstream, how does this affect transcription? Will the areas that the mc [?] are bound to release after trans. starts so the are will be trans.?
Q: The role of RNA pol III and the function of different promoters.
Q: Were you overall just trying to show (with the last concept you were talking about today) how RNA pol III, when binding to either rRNA 5s or tRNA requires different transcription factors and has different internal promoters?
ANS: Yes: (1) The promoters can be internal (different than what we would expect from prokaryotes etc.), (2) they work in conjunction with TF proteins.
.....................................................................................................................
POLYADENYLATION
Q: I would like to know why the polyadenine region is present... however, I don't think the scientific community has figured this one out totally.
Q: What's the role of the poly A region? I don't get it.
Q: I am unclear on the role of polyadenylation.
Q: What is the purpose of polyadenylation? Is it required for translation?
ANS: Polyadenylation has at least two functions. (1) it protects the mRNA from---
makes it more stable. (2) It enhances the "translatability" of mRNA--
there is a protein that binds to the poly(A) tail and seems to increase the
efficiency of translation, somehow.
.....................................................................................................................
CAT ASSAYS
Q: The CAT assay thing-- I'm fuzzy on how we can tell if the promoter is on by chromatography.
Q: For the method of studying eukaryote genes where you use chloramphenicol, can you use it only to study the CAT gene?
Q: I would like to hear more about that CAT thing. I'm a little confused.
ANS: See the figure on the handout (from Watson et al., Recombinant DNA, 2nd
ed.). I think that figure and its caption do a better job than I can here, without
graphics.
.....................................................................................................................
MISCELLANEOUS
Q: Can we deduce anything predictable about gene regulation if there's all these other factors that may or may not be present, e.g. RNA editing, transposons, alternative splicing.
A: Definitely. Initiation of transcription remains the principle method of
genetic regulation, in eukaryotes as in prokaryotes. It's just that eukaryotes
have all these additional mechanisms for regulating expression. Another pattern
we recognize is that proteins play important roles in regulation-- not only
as repressors but as other kinds of transcription factors, and members of information
pathway cascades, and thus lend exquisite specificity to the expression of genes.
.....................................................................................................................
Q: How do you define "alive" What characteristics must something posess to be considered alive?
A: Many biologists would say the two principle characteristics of life are
metabolism and heredity. Viruses show heredity but not metabolism, so some people
don't think of them as alive. My own perspective places some more emphasis on
heritabiltiy (including mutability and adaptability), but I am quite comfortable
with having a fuzzy boundary between "living" and "non-living"--
and not only in terms of the scale of life from molecules to megafauna and megaflora
(whales and redwoods), but other ways as well: if a person is killed in a car
crash, but organs (heart, kidneys, bones, corneas) are taken from that person,
are the parts alive?
.....................................................................................................................
Q: What is the significance of the differences between the locations of TATA, GC, CAT boxes between eukaryotes and prokaryotes?
ANS: The size of the RNA pol complexes and the roles of various proteins interacting
with them to initiate transcription.
.....................................................................................................................
Q: How do I keep all this straight in my head for the exam?
ANS: Study early, study often. Do the problem set questions, and make up your
own.
.............................................................................................
Q: How can we prepare our next lab report better?
ANS: I hope that the comments I made will help with this. Starting early, and
getting feedback from others, will help. So will consulting Pechenik (though
his Short Guide to Writing in Biology is no longer short, but it does have good
advice for writing lab reports). And be sure to look at examples of published
papers.
.............................................................................................
Q: How am I going to finish all my papers on time?
ANS: One of the best pieces of advice that I got (when I worked in construction)
was "Plan the work, work the plan". Some people make it look easy,
but that is not my experience.
.............................................................................................
Q: What readings will be on the test? Will a copy of the old test be available
again?
ANS: For Spring 2003 readings up through Part III (except NOT on Lambda), plus the first part of Part IV, week 9 and 10: Reporter gene transcription (121-122), sec.5.6 through DNAse footprinting (p. 126), RNA pols 261-266; and just barely beginning promoters, 279-283.
By now I've already handed out most of what's shown up on several tests from
the last few years.
.............................................................................................
Q: How are we going to keep all these genes and gene products straight and not
confuse them with one another?
ANS: Draw pictures. What are pieces? How do they interact?
.............................................................................................
Q: What am I going to do with my biology degree after I graduate?
ANS: Beyond me, but send me a postcard when you get there.
TRANSPOSONS
Q: How do transposons move in DNA? And what is the difference between transposons
and transposases?
A: A transposon is DNA, and can move from one location on a chromosome to another.
A transposase is the enzyme that does the work of moving the transposon into
another location of a cell's DNA, and is coded for by a gene on the transposon.
There is a little information on pages 730-742 in Weaver's text.
.....................................................................................................................
Q: Do humans have transposons and if so how do they affect us?
A: Yes, we do-- lots. The last page of the handout on transposons included
an abstract from a paper describing the discovery of a new transposon, and lists
the names of several other families of transposons found in humans. I don't
know anything about their effects on humans.
........................................................
IMMUNE SYSTEM/ANTIBODY GENETICS
Q: How do T-cells tell B-cells what to do?
A: I really don't know enough immunology to answer this. I know a little bit but (1) what I know off the top of my head may be more inaccurate than useful, and (2) it is complex and takes a while to explain, and is better done with pictures. I have some books in the office here that you could look at. It quickly gets into the arcane and fascinating realm of helper T cells, cytokines, antigen presenting cells-- well out of my field. Sorry.
Q: If we have a mechanism in our body that gets rid of anything foreign to our our body, then how does organ transplantation work?
A: Have to turn off that rejection. There are drugs that can suppress the immune
system and prevent rejection. The more similar a tissue is to the host, the
less of those drugs is needed, and the less the immune system is suppressed.
.....................................................................................................................
Q: You said macrophages could eat up E. coli in our body-- so what about the E. coli naturally sitting in our body? Do they attack that too?
A: Well, most of the E. coli "in" us are in our large intestine--
"outside" of our tissues. That helps! They are therefore not subject
to attack by the B cells and their antibodies that I mentioned. But they can
get inside, through any opening into our blood system, for instance, and then
move to a nice comfy spot and set up home. Can be lethal.
.....................................................................................................................
Q: How is it (again) that so many antibodies are produced? Do they all just randomly create variable regions for proteins to bind?
A: Here are the main parts: (1) there are multiple germ-line genes for the
variable regions of the antibodies, (2) random combinations of each segment
(i.e. one V and one D and one J), (3) slippage of the joining, which alters
the reading frame, (4) nucleotide insertion at the joining, which also alters
the reading frame, (5) somatic hypermutation, meaning that in these somatic
cells the DNA replication does not occur with the same fidelity and repair that
takes place in the germ line, and finally (6) combinatorial joining of any heavy
chain with any light chain. Most of what I discussed focused on (1) and (2),
since the broader topic is "how the central dogma gets altered in euk.
molecular genetics", and this illustrates a novel mechanism we have not
previously encountered, but which bears some similarity to the movement of DNA
we see in transposons.
.....................................................................................................................
Q: I have seen discovery channel programs on immunology and the allergies that can develop in response to "improper mechanics" in the immune system. Can you address allergies and maybe anaphylactic shock in class?
A: No, I can't. I wish I knew more about this, too.
.....................................................................................................................
VIRUSES
Q: Not sure yet how the TMV thing works-- you went over it pretty quickly.
A: Tough to do without a diagram, so you might want to look at the picture
on the handout while you read this. The RNA genome is 6395 bases long, and codes
for four proteins. It does this by using two different reading frames. One reading
frame starts a protein that can stop at two different places, giving different
sized products; these work together as the polymerase theat replicates the RNA
genome. That same reading frame then has another portion that is translated
and makes another protein (the one that wraps around the RNA as a coat protein).
The fourth protein is also translated from this RNA, but using a different reading
frame, and translating the stretch between the polymerase and coat proteins
(though it overlaps a bit with the polymerase). The function of this protein
is not known (well, my info on TMV is dated-- not known as of 1987).
.....................................................................................................................
Q: In the poliovirus figure in the handout I understand how it cuts itself before it's even fully translated, but I missed it when you explained how it makes more of itself.
A: The poliovirus genome is + RNA, meaning it is translated directly to make
a long polypeptide; the COOH end of this is cleaved off to make a replicase:
an RNA-dependent RNA polymerase enzyme that will copy the RNA genome into RNA
(- RNA) that serves as a template for more + RNA. This replication is somewhat
unusual and I did not talk more about it in class: the RNA molecule folds back
on itself at one end, where a poly-A stretch is H-bonded to a poly-U stretch,
and this serves as a primer for this unusual RNA polymerase (remember that RNA
pol molecules usually don't need primers). This - strand serves as template
for synthesis of several + RNA strands simultaneously, and these + strands are
translated to produce the capsid proteins.
.....................................................................................................................
Q: What is the difference between + and - RNA? Why is + RNA translated directly, while - RNA needs to be copied first?
A: + RNA is translated to produce protein; - strand RNA is replicated to produce + strand RNA. My guess as to "why" is that because the - strand can produce a bunch of + strands (even simultaneously), all of which can be translated (even simultaneously), this gives the cell a way of replicating the genome rapidly, and rapidly packaging it up for dispersal. Perhaps there also might be some advantage to being able to go through this whole life cycle in the cytoplasm, without having to get into or out of the nucleus.
HIV
Q: Why does HIV carry RNA and reverse transcriptase? It would seem more efficient
to just carry the DNA.
A: Agreed, and the broader question is "why don't we find DNA viruses that integrate into the genome, the way lambda does?" I can't answer that.
Q: Are all viruses as mutatious as HIV? If antibodies can attack foreign organs from a sibling, how do they make transplants work?
A: RNA viruses have very high mutation rates. This is not only because the RNA is unstable, but also because the reverse transcriptase enzymes tend to be error-prone, lacking the kinds of proofreading mechanisms that we saw in DNA polymerases. Furthermore, because the genome is single-stranded, there is not the kind of "backup copy" that could be used to check the newly replicated genome, as we saw happens with mismatch repair in DNA.
Q: Why doesn't the cell recognize virus overproduction and stop? How is the cell's own protein production altered in lysis, and how is lysis and controlled growth regulated?
A: Sorry, I don't know. How would a cell recognize that the proteins and RNA it is producing are "wrong"? In fact, this is one of the areas of HIV/AIDS research, attempting to develop a vaccine that enables killer T cells (oops-- I only told you about the "Helper T Cells", not about this other kind of T cell) recognize the viral proteins on the cell and destroy them.
Q: Why is it so hard to find something that won't allow HIV to bind to T-cell surface proteins?
A: Several reasons, but this has been suggested: quite some time ago it was
suggested that the gene for GP 120 should be cloned so lots of the protein could
be produced, and then given to people; that GP 120 would bind to the T-cells
so the virus couldn't bind. Problem came up, though, that GP 120 is harmful,
all by itself: causes neurological problems. Another approach would be to make
something that would bind to the GP 120, so it would be "full" and
not able to bind to the T-cells, I don't know of any work on this path-- or
even know enough to guess what pitfalls might be there.
.....................................................................................................................
Q: Has anyone ever been cured of the HIV virus? Is there any sort of "predators" to virus as they are pathogens to us or bacterial cells?
A: I have heard of a few cases. In some of these, the people are infected with
the HIV virus but they do not develop AIDS, even over 8 years later, and have
healthy populations of T cells (specifically the CD 4 and CD 8 cells targeted
by the HIV; these drop to virtually nothing when the viral load overwhelms the
person). I have also heard of one person, in this country, who apparently eliminated
the HIV virus from his body. Sorry, I can't recall anything more about that
case-- who, where, when-- and nobody knew why.
.....................................................................................................................
Q: More about HIV in general? It's interesting how it works... I don't really have a specific question right now.
Q: Progress or process of finding a HIV vaccine-- what are the researchers trying?
A: Sorry, I haven['t been keeping up on that literature. The last I heard was
that some DNA vaccines were being tried. That is an extremely unusual kind of
approach for vaccine development. The two major routes for vaccine development
are (1) develop a live, "attenuated" virus (i.e. people get the virus
but it doesn't make them sick), or (2) develop a killed virus (typically denatured
by heat or chemicals that denature the proteins). Problems with these are that
the live virus can mutate or otherwise acquire virulence, and not all people
react the same (i.e. it can be safe for some people but lethal for others),
and killed viruses may not be as effective because they are denatured and thus
not a good model for the antibody-mediated clonal selection I described. With
AIDS the problems compound: the RNA itself can move in and cause problems, so
attenuation seems extremely risky; the high mutation rate holds similar hazard;
and the high mutation rate also means that the vaccine might not be very effective--
antibodies might develop but a new strain differing in GP 120 might come along
and not be recognized.
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Q: Why poison someone with AZT? Will the body learn not to use AZT dTTP's before the virus, so DNA can be replicated by the cell but not by the virus? How can we stop HIV? What angle have we not considered?
A: No, our DNA polymerase will use AZT all too effectively. Remember, "learning" in this case is a shorthand for speaking of the process that really is natural selection.
I don't know how we can stop HIV. In class I emphasized how it seems diabolically
well designed to focus on the key weak point of the human immune system: it
is exquisitely tuned to our biology. But furthermore, it is exquisitely well
tuned to our biology in a much larger sense, as well: not just the details of
the immune system, but the fact that we are characteristically a highly mobile
and promiscuous species. Thus, it is all too readily passed from one host to
another. Moreover, the extremely long latent period between infection and development
of the disease (years in some cases) means that it can be spread many, many
times. Diabolical indeed.
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Q: Why isn't there a cure for AIDS yet? Why can't its binding be hindered or why don't our antibodies take care of it?
A: Most of what I know (and then some) is in the above answers.
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Q: How did the HIV virus become so specific in its binding to T-cells?
A: Great question that I can't answer. When it was first discovered, it was
dubbed HTLV-III, meaning it was the third type of human T-cell lymphotrophic
virus: it followed the discovery of two other retro viruses that specialize
on the T-cells.
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Q: Where and how did HIV develop so that it could infect humans? Wasn't it the Rhesus monkey?
Q: Where did this HIV virus come from?
Q: Where did HIV come from? How come it is so specific to the immune system? Did it start out like that or just develop into the virus it is today?
A: To the best of my knowledge this has not been answered yet. I think I recall reading that blood dating from the late 1930's had been found to contain either the viral genes or antibodies against the virus, indicating the person had acquired the disease, but it may be that the oldest such samples came from the 1950's. A semi-popular hypothesis favored by those who like conspiracies (and popularized by a former writer for the Rolling Stone) fingers polio vaccine testing in Africa in the 1950's, but I think that was pretty well debunked by scientists with good data-- I don't recall what. At one time it was suggested that humans picked it up from Green Monkeys, another time suggested from Chimpanzees; routes of transfer included eating these animals ("bushmeat" has become more popular and accessible in Africa) and also via some sexual practices that involved rubbing monkey/ape blood into cuts or scratches in female genitals, to cause itching etc. It is a strange world. These and other simians carry one or more kinds of simian immunodeficiency viruses (SIV), that seems to be an ancestor to the HIV virus, based on analyses of the RNA sequences. The answer isn't in yet. However, there is another question, since there are a variety of these retroviruses that all have similarities but infect very different animals: birds, cats, humans: how did the first retroviruses arise?
Q: What is the other mechanism for fighting HIV infection besides AZT?
A: A protease inhibitor. I will mention more about this next week when we look more at the functions of the HIV genome.
Q: The main question I have is this: If viruses like HIV (& others I'm sure) have evolved so much, why haven't our bodies come up with ways to combat them? You'll probably say something about generation time being short for viruses vs. plants and animals.
A: Yes, that is the heart of what I would say, along with comments about mutability of the RNA and DNA genomes and fidelity of their respective polymerases.
But I would not stop there-- after all, there are other retroviruses, and they do not all cause the damage that HIV does. In fact, there is more than one kind of HIV, and HIV-2 is less damaging to people than is HIV-1. Chimps can be infected with HIV and will harbor the virus, but do not develop AIDS: their immune system does not collapse. The SIV virus does not always cause an AIDS-like disease. So perhaps in other cases the host species has evolved some mechanism of resistance.
CANCER
Q: What are eumetazoa studied for?
A: "Eumetazoa" simply refers to multicellular animals, excluding
colonial animals like corals. Thus it includes worms, flies, humans, etc.
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Q: Can plants get cancer? Can the Rous Sarcoma Virus affect humans and other animals, or only chickens?
A: Plants don't really get cancer. There is a bacterium (Agrobacterium tumefaciens) that carries a plasmid which causes a proliferation of tissue, in either woody or non-woody tissue. The plasmid can leave the bacteria, be translocated through the plant's vascular system, and induce growth elsewhere. You may have seen large masses growing on tree trunks o