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The Behemoth Biochem BoardReview
Part II
May 18, 2000
DNA Structure
Remember your base pairs: A pairs with T and C pairs with G. In order for
complimentary base pairing to occur, if one looks at the strands, the two strands
must be antiparallel. The have to go in opposite directions. Base pairing is also
called annealing or hybridization. The bottom line is that there is a sequence of
nucleotides, which is really a sequence of bases. There is a 5’ end and a 3’ end, and
the nucleotide sequence is always read 5’  3’. In some of the questions, you may
see a sequence in which the ends are not labeled. In this case, you must assume that
the sequence is written 5’  3’, left to right. Sometimes the little p will be in there;
that just represents the phosphodiester bond.
The 2 strands must be antiparallel, and that there are 2 H bonds between T and A
and 3 H bonds between G and C. G and C is a stronger base pair, so any sequences
that have a lot of G’s and C’s will be harder to separate. To separate strands of DNA
in the laboratory (called denaturation), we can increase the temperature or increase
the pH (make the solution alkaline). If this were RNA, increasing the pH would not
work because alkaline conditions degrade RNA because RNA has ribose, not
deoxyribose, and also has U instead of T.
This would not work in the cell obviously. To get DNA strands apart in the cell, I
use an enzyme called a helicase. They take apart the double helix, which is held
together by noncovalent forces.
The percent purines in a double stranded DNA sample is always equal to the percent
pyrimidines (50/50). This might be tested by giving you the percentage of A’s and
asking for the percentage of G’s. If the percentage of A’s was 15%, then the
percentage of G’s would have to be 35% because the percentage of purines always
adds up to 50.
There are some modifications to this. If you have a kid that has viral gastroenteritis,
the virus is most likely gonna be rotavirus, which is a double stranded RNA virus.
Everything still adds up, but you will have U instead of T. The other thing that
could happen is a situation where you have 15% A and 40% G. These should add up
to 50, but they don’t so what are they trying to tell you? This is a single stranded
DNA virus. The calculation only works with a double stranded molecule.
Replication
You should know the basics of replication, DNA repair, and transcription and
translation. Replication occurs for cell division. When one cell becomes 2, the
number of chromosomes doubles. In a bacterial cell, there is one chromosome – it is
haploid. The chromosome is circular and double-stranded. The two ends are fused,
so there are no free ends.
All of our cells, with the exception of sperm and ova, are diploid (44 autosomes,
and XY if you’re male, XX if you’re female). We have to replicate these
chromosomes, we get 92 and then 46 go to either cell. This occurs in the eukaryotic
cell cycle. DNA replication occurs in the S phase, which stands for DNA Synthesis.
This is between the two gap phases, G1 and G2.
When this is going to be replicated, the DNA opens up at an origin of replication
called the ori site. So in order to replicate any DNA molecule, there must be an
origin of replication. For prokaryotic cells, there is one ori site per DNA molecule.
In our cells, there are multiple ori sites on each chromosome. So on chromosome 1,
which is the longest, there are at least 300 origins of replication. That’s so the DNA
polymerases can replicate all the chromosomes in a reasonable amount of time.
DNA replication in a typical eukaryotic cell is about 8 hours. Without multiple ori
sites, it would take days.
At any of these ori sites, there is bi-directional replication, which forms a
replication bubble. That means that for every origin of replication, 2 replication
forks will form, one moving right and one moving left. Helicase is opening up
these strands so they can be copied by DNA polymerase.
Strands being copied are referred to as template strands. The parent (template)
strands are on the outside and the daughter, or newly synthesized strands, are in the
middle. One parent and one daughter strand will get together and form the
chromosome, which will go to one cell. The other parent strand and the other
daughter strand will go to the other cell. Because the chromosome consists of one
parent strand and one newly synthesized strand, this is called semi-conservative
replication.
The DNA is made either as a continuous long piece or in a discontinuous fashion
with a bunch of little pieces. The continuous strand is leading strand synthesis, and
the discontinuous strand is called the lagging strand. The little pieces of DNA are
called Okazaki fragments. Note that the long pieces are always being made going
into a replication fork and the short pieces are made going out of a replication fork.
The synthesis of ALL DNA strands is 5’  3’.(Lay down 5’3’ but read 3’
5’.) The order of the addition of the nucleotides is determined by the template
strands, the parental strands. They are copied by complimentary base pairing, so
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you should note that the template strand must be copied 3’  5’ (so complimentary
base pairing can occur).
Sometimes they will give you a diagram and ask you what the heck is going on
here. In that case, you should note that the (small) arrows always point toward the
3’ end of the newly synthesized strand.
Summary of the steps and proteins in DNA replication: First, you need to unwind
the DNA double helix at the origin of replication. The enzyme is helicase. I now
need to stabilize the unwound template strands to keep them from coming back
together. This is done by single-strand binding proteins (SSB).
DNA polymerases must have a primer to begin synthesis. So the next step is the
synthesis of an RNA primer, done by primase. DNA can now be made, and in
prokaryotic cells this is all done by DNA pol III. In our cell nuclei, eukaryotes have
DNA polymerases , , , and .  does the leading strand and you should associate
 with Okazaki fragment (lagging strand) synthesis.  is the only one not actually in
the nucleus because it replicates mitochondrial DNA.
At the end of all this, we need to remove the RNA primers (which are always found
at the 5’ end of any DNA being made) and replace them with DNA. This is done by
DNA pol I in the in the prokaryotic cell. DNA pol I must first remove the RNA
primer at the 5’ end (5’-3’ exonuclease) by cutting of nucleotides one at a time and
then putting in DNA. We don’t know what does it in eukaryotic cells.
The joining of the Okazaki fragments is done by DNA ligase. It just makes a
phosphodiester bond. That is called sealing, joining, ligating.
A couple of other things down here: as helicase moves ahead of each replication
fork, the DNA becomes positively supercoiled ahead of these replication forks and
the DNA gets jumble up. Under these conditions, DNA synthesis will stop, so to
prevent this there is an enzyme called topoisomerase II (DNA gyrase in
prokaryotes) that removes positive supercoils. Topoisomerase I cuts one strand of
the DNA helix, topo II cuts them both. These are targets for drugs, and you should
remember from pharmacology: the analogues of nalidixic acid, such as norfloxacin
and ciprifloxacin. These are your fluoroquinolones and they work against the
bacterial DNA gyrase. The anticancer drugs that work in our cells are etoposide and
teniposide., which both target topoisomerase II.
Finally, the ends of chromosomes that are linear are called telomeres. They are
specific sequences that make sure that our chromosomes don’t get too short. They
are not in prokaryotes because they have circular chromosomes. In our cells,
telomeres are made by telomerase. The activity of this enzyme is thought to
decrease with age. In cancer cells, telomerase is more active.
DNA repair
These repair mechanisms put the cell cycle on hold until the DNA can be repaired.
One of the major things that has been tested is the formation of thymine
(pyrimidine) dimers. Thymine dimers form in our DNA because of sunlight. The
two tissues you should be concerned with are the skin and the cornea of the eye.
Thymine dimers form with adjacent thymines on the same strand.
We have an enzyme that runs around scanning for thymine dimers, and that is
referred to as an excision endonuclease or excinuclease. It cuts and removes a
sequence of nucleotides in one strand that contains the thymine dimer. This leaves a
gap that will be filled in by a DNA polymerase and then ligated by DNA ligase. The
main thing to know is the excision endonuclease, and this is actually the main
enzyme that is deficient in xeroderma pigmentosum. So a clinical scenario might
be a young kid that comes in with corneal ulcerations, excessive freckling,
photosensitivity and skin tumors of all types.
Another example of a DNA repair defect is ataxia telangiectasia.it is characterized
by uncoordinated movements, spider veins, and lymphomas. Another example is
nonhereditary polyposis coli. This leads to colon cancer and is a defect in base
mismatch repair
The main thing to note is that C can be converted to U. That is a deamination, the
loss of ammonia, and has been implicated in colon cancers caused by nitrates and
nitrosoureas. U should not be in DNA, so the enzyme that will get rid of this U is
called uracil DNA glycosylase. Identify the term glycosylase with an enzyme
whose job it is to look for unusual bases. The glycosylase removes the base, but the
DNA backbone remains in tact, so this is called base excision repair.
At this point, we have a site missing a base, which is referred to as the AP site. The
enzyme that looks for AP sites is an AP endonuclease. This cuts the DNA
backbone (making a larger hole) and then DNA polymerase and ligase come in and
fix it up and the C is restored. The other thing to note is that with the temperature of
our bodies, we are probably losing 10,000 or so of these everyday. So this is a
normal occurrence.
Methylation: C is the base most often methylated which turns the genes off.
DNA polymerase  replicates mitochondrial DNA, which is circular. The other thing is
reverse transcriptase is pretty much guaranteed to be there, probably in association with
AIDS. The thing to note is that HIV-1 is a single stranded RNA virus. In order to put that
genome into the host chromosome, the RNA is made into cDNA and is then put into a host
chromosome. Reverse transcriptase is the enzyme that makes the DNA from the RNA and
then integrase puts it in the chromosome. Both of these enzymes are required to establish an
infection. When the DNA copy of the virus is put into a chromosome, it is known as a
provirus. Also note that reverse transcriptase is an RNA-dependent DNA polymerase. It’s
copying an RNA template and making DNA. This does require a primer (all DNA
polymerases do)!
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DNA and RNA Synthesis
Protein Synthesis (Translation)
DNA polymerase is copying the template and it requires an RNA primer (made by
primase, an RNA polymerase) to get started. Note the U in the primer because it is
RNA. Synthesis proceeds in a 5’  3’ direction, so nucleotides are added to the 3’
end and the nucleotide depends on base pairing with the template DNA. If the DNA
pol puts in the wrong nucleotide, it can back up and remove it so it has 3’
exonuclease activity. This is called proofreading activity and it insures that DNA
replication occurs with high fidelity.
Protein synthesis always occurs in the 5’  3’ direction along the message, so the
protein is made from the amino to the carboxyl end.
RNA polymerases don’t need a primer. RNA synthesis also occurs 5’  3’. RNA
polymerases do not have proofreading activity. If they mess up, they just keep on
going. Remember that there is U in RNA instead of T.
Note that the difference in the codons for proline comes in the 3 rd position. That is
referred to as the wobble position. The advantage to this is that if a mutation occurs
in the 3rd position, it will generally not result in a change in the amino acid. A
mutation that does not result in a change in the amino acid is called a silent
mutation. Know that AUG codes for methionine and it is the initiation codon for
protein synthesis. Methionine always starts out at the amino end of a protein, even
though it may not always stay there.
The substrates for DNA synthesis are deoxynucleotide triphosphates (dATP,
dCTP, dTTP, dGTP). The substrates for RNA synthesis are nucleotide phosphates,
but not deoxy (ATP, UTP, GTP, CTP).
Transcription (RNA synthesis) There are 2 main differences from replication. First
of all, the strands of DNA are separated, but only one strand is copied. That is called
the template strand and the other strand that does not participate physically is
called the coding strand. The 2nd thing is that the entire template strand is not being
copied. It is copied from a start site to a stop site, and that is referred to as a
transcription unit. Under these conditions, the start site is always labeled in the
coding strand as the +1 base.
So in transcription, RNA is made 5’  3’ and the template is copied from 3’ to 5’
by a DNA-dependent RNA polymerase. This is done by complimentary base pairing
with the template strand. So the RNA strand that is synthesized is going to look like
the coding strand.
RNA finds a gene or a set of genes by binding to a promoter. This is the RNA pol
binding site and this is where you’re gonna see signals to start transcription. To the
left, from the +1 start site, the bases in the coding strand are given negative numbers
(that’s upstream) and the bases to the right are given positive numbers
(downstream). The +1 base in the coding strand will be the base at the 5’ end of the
RNA.
The RNA that is being made doesn’t contain the promoter. Transcription starts at
+1, which is downstream from the promoter, so the promoter is not transcribed.
So what is a transcription unit? In eukaryotic cells, it’s simple: it’s a gene. In
prokaryotic cells, a transcription unit may be a gene but most often they are sets of
genes, and sets of genes under the control of one promoter are called operons.
In the genetic code, we have 64 codons, and 61 of them code for an amino acid. We
only need 20 amino acids to make proteins, so this tells me that the genetic code is
degenerate. There are multiple codons for each amino acid. But also note that it is
unambiguous – a particular codon only codes for one amino acid.
Also note that there are 3 stop codons: UAA, UAG, and UGA. Know what they are
in the DNA too (TAA, TAG, TGA).
Point mutations are single base substitutions. The gene and the mRNA will be of
normal length, but the protein may be different. A missense mutation is a type of
point mutation, where here the T in leucine has been converted to C. That converts
the codon for leucine into one for serine. This may or may not cause genetic
disease, depending on where it occurs (this happens in sickle cell disease). A
nonsense mutation is also a single base mutation, but that new codon codes for one
of the stop codons, not another amino acid. This results in premature termination of
protein synthesis, leading to a truncated protein.
A frameshift mutation is a 1 or 2 bp insertion or deletion, not a substitution. If I
insert (or delete) one or 2 bps, I will throw the reading frame out of kilter to the
right or downstream or toward the carboxy end. If I delete 3 bps, then an amino
acid will be deleted, and this is what happens in cystic fibrosis – phenylalanine is
missing. Note that if I take out a base (as in this example), the amino acid sequence
on the carboxyl end of the mutation is going to be different and generally, it will
generate a premature stop codon, so these proteins will also be truncated.
You should also be aware of triplet repeat expansions. This is when I put in
several extra sets of 3 bases; basically, this is just adding a bunch of codons. The
protein will be too long. This generally causes neurological disorders and the 2 you
should be aware of are Huntington’s Chorea, which is a CAG (glutamine)
expansion, and Fragile X syndrome.
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Transcription and Translation
Mammalian cells do not have operons. The promoter sequence at –10 binds RNA
pol and positions it at the +1 transcription site. RNA pol will then transcribe the
template strand of the gene from the +1 start site to the stop site. At each end of the
operon is a region that will not be translated into protein, the 5’ untranslated region
(UTR) and the 3’ UTR. I have 3 genes, and in between them is just junk DNA.
There are no introns and exons in prokaryotic cells.
To bind to the promoter, RNA pol needs a sequence at about –10 called the TATA
box (also called the Pribnow box, but only in bugs). This is the start site for
transcription. They may give you a mutation where TATA is TCTA. This would
result in a problem with transcription initiation obviously, not translation.
There is a protein that helps RNA pol bind to the TATA box and it is called sigma.
This binds to the TATA box and helps RNA pol find the start site. In essence, sigma
is a subunit of RNA pol; it binds and then recruits the rest of the RNA pol to the
promoter. RNA pol will then make a transcript that is an exact replica of the coding
strand from 5’  3’.
There are 3 genes in the operon, so there will be 3 in the message, and that is
referred to as a polycistronic mRNA. At this point, transcription is stopped by a
stem-loop structure in the mRNA. The stem is a bunch of C-G bases that are base
paired and the loop is a bunch of bases that aren’t base paired to anything. When
that structure forms, the RNA pol falls off the DNA and transcription is over. This
is rho-independent termination. In other cases, a protein called rho is required to
stop termination, and that is called rho-dependent termination.
In prokaryotes, all of this occurs in the cytosol. As the RNA is being made, it is also
being translated. Ribosomes assemble at the beginning of each gene and then
translate to the end. AUG is the initiation codon for translation, so it must be the
first codon at the beginning of every gene. There is also a stop codon at the end of
every gene.
Shine-Delgarno sequence: This is where the ribosome binds. The S-D is just
upstream from the start codon, so it’s how the ribosome finds the start codon.
In mammalian cells, the transcription unit corresponds to a single gene, thus there
are no polycistronic genes in us. Again, the gene has a +1 start site and a stop site,
something called the poly-A site. There is a promoter to the left. The RNA will bind
at the TATA box, which is situated at –25 in us. RNA pol II is the enzyme that
transcribes all of our protein coding genes. The protein coding genes are referred to
as class II gene because they are transcribed by RNA pol II. We don’t have sigma,
but every gene that is going to be transcribed requires a set of transcription factors
(TF). If they are helping RNA pol II, they are called TF-II’s. Of these, only TF-IID
is important. This binds the TATA box. TBP stands for TATA binding protein, and
that’s just the part of TF-IID that binds the TATA box.
Again, in eukaryotic genes, there is a 5’ UTR and a 3’ UTR. The stop signal is in
the 3’ UTR, and inside the gene there are exons and introns. The exons contain the
actual code; the introns are just sequences that are in between the exons. There is
always one less intron that there are exons. The introns are generally longer.
RNA pol binds to the promoter, transcribes the gene, and comes to the so-called
poly-A site. This is not poly-A; it’s just called the poly-A site. When that signal
appears in the mRNA, an endonuclease cuts on the 3’ side of it. That stops
transcription. RNA pol may not have fallen off the DNA, but the transcript has been
cut so it’s a moot point. This is done in the nucleus. This RNA is not functional. It
cannot be translated until 3 things have been done to it. So this is an immature
precursor of mRNA called pre-mRNA, also called hnRNA (heterogeneous nuclear
RNA).
Processing of pre-mRNA: capping, tailing, and splicing. These are posttranscriptional modifications. All of this occurs in the nucleus, and none of this
occurs in bugs. The capping occurs at the 5’ end. The cap is a methyl-G (guanine,
guanylate) cap which comes from SAM the methyl man. The cap serves as the
ribosome-binding site because we don’t have S-D sequences.
Tailing occurs at the 3’ end and it is a poly-A tail. It does have a bunch of A’s.
Once you cut at the poly-A site, an enzyme called poly-A polymerase comes along
and puts on the poly-A tail. The substrate for this enzyme is ATP.
Splicing is the removal of the introns as lariats or lassos and the joining together of
the exons. There is a big complex called a spliceosome that forms around these
exon-intron junctions. Spliceosomes contain proteins, but they also contain certain
RNA’s (snRNA), and it’s these RNA’s that do the catalytic work.
This then produces a mature mRNA. It has the cap, the tail, and the introns are
gone. The exons have been fused together and I am ready to translate. This is
sometimes referred to as an open reading frame. The ribosome will bind at the
beginning of the first exon and then stop and the end of the last exon. This must
mean that AUG is always the first codon in exon 1. The stop codon is always going
to be at the end of the last exon. Exon 1 codes for the amino end of the polypeptide
and the last exon codes for the carboxyl end.
Remember that the mature mRNA is made in the nucleus and then exported through
nuclear pores and out into the cytoplasm where the ribosomes are waiting for it.
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Regulation of Transcription
Ribosomes and tRNA
If I turn on a gene and the rate of transcription inc. the amount of mRNA will inc.
The mRNA will be translated by ribosomes and the amt of protein in the cell will
inc. That’s enzyme induction. By the same token, if I dec transcription I dec the
amt of protein in the cell  repression.
In a prokaryotic cell, to turn on genes I need an activator protein, and to turn genes
off I need a repressor protein. Operons can be turned on by one activator protein
and turned off by one repressor protein. Activator proteins bind to activator sites
and repressor proteins bind to operator sites. You need to know this!
lac operon: The idea is an activator protein to turn it on, a repressor protein to turn it off, and
a set of genes called an operon. In order to turn the operon on and make mRNA, I have to add
an activator protein (CAP) and remove a repressor protein. I remove the repressor protein by
the addition of lactose and I put on the activator protein by the addition of cAMP. So I need
lactose and cAMP to turn this on. Glucose must be low, because high glucose inhibits cAMP
production and the activator cannot bind. So as long as there is any glucose present, the lac
operon is turned off. If they’re looking for a way to measure whether the operon is on or off,
you’re mostly likely gonna look at the activity of -galactosidase. If the activity of this
enzyme is high, the operon is on, if its activity is low, then the operon is off.
In our genes, we need a set of activator proteins to activate a gene. Our activator
proteins can bind far away, close to or w/in intron. If they enhance the rate of
transcription, the binding sites are called enhancers. For the binding of a repressor
protein, we don’t have operators, we have silencers. Additionally, for our DNA to
be transcribed, it must be decondensed to heterochromatin from euchromatin. Also,
methylation turns genes off and demethylation makes them active.
Enhancers/ silencers can be located far away from the promoter region but it still
interacts with RNA pol, so wherever it binds it has to get close to the RNA pol. The
DNA has to loop out in order for the activator protein to interact with the RNA pol.
There are enhancer-binding proteins (enhancers) during development. These are
called homeodomain proteins, and they are encoded by genes called homeotic or
homeobox genes. These genes therefore make TFs that appear at certain times
during fetal development that set down the body plan. So if there are multiple birth
defects that can be traced to a single gene mutation it is probably a homeobox gene.
These binding proteins/enhancers, gene activator proteins, are also intracellular
receptors for hormones. All steroid hormone receptors are enhancer-binding
proteins. Also remember the fat-soluble vitamins A and D, as well as thyroid
hormone (these are all zinc finger proteins). These activator proteins all have certain
structural domains required to bind the DNA and turn on genes. So if you hear
something like a leucine zipper or a zinc finger, they’re talking about a
transcription factor.
For protein synthesis to occur, mRNA, ribosomes and tRNA are needed. You want
to know the difference between eukaryotic ribosomes and prokaryotic ribosomes.
The eukaryotic ribosome has even numbers: 80s, 60s, 40s. In prokaryotes, they are
smaller and they are odd numbers: 70s, 50s, and 30s. Don’t memorize the RNA’s
inside, but just keep in mind that the number 8 seems to stick out in the eukaryotic
ribosome.
Note that ribosomal RNA is the most abundant RNA in any cell.
At the bottom are transfer RNA molecules. These bind amino acids and proceed to
insert them at the right position in the polypeptide chain by reading codons. This is
the so-called cloverleaf structure, which is the secondary structure and the tertiary
structure is the overall 3 dimensional structure. Pictures of these have been shown
before. The 3d structure of the tRNA looks like an L with the CCA at the end.
tRNA’s are small; they’re short guys. They are capable of base pairing among
themselves and that’s how they form this cloverleaf structure. Some of these base
pairs are unusual. tRNA is the most modified RNA; it contains the most modified
bases and the most unusual base pairs. It contains bases like ribothymidine,
pseudouridine, dihydrouridine, etc. These are found in the loops on the sides.
Note that at the 3’ end of the tRNA, every tRNA has the sequence CCA. That is a
triplet but it is not a codon. It’s just CCA. It is this end that binds the amino acid.
The amino acids are attached to tRNA by enzymes called amino acyl tRNA
synthases. These are in the cytoplasm, where translation occurs. The amino acid
must be attached to a tRNA before it can participate in protein synthesis. This
overall process is called amino acid activation.
Each amino acid has its own tRNA. They read the codon in mRNA with something
called an anticodon. This is at the bottom of the anticodon loop, and it is 3 bases
that read codons by complimentary base pairing. Note that this is RNA, so you will
see A-U base pairs. Also recall that for base pairing to occur, the strands must be
oriented antiparallel to one another. Where is the wobble position in the anticodon?
Base 1!
At the bottom are the enzymes that make RNA. Recall that RNA pol II makes all
our mRNA and it also turns out that it makes most of the snRNA. This is also the
enzyme that is most sensitive to -amanitin, a poisonous mushroom. This causes
GI distress and eventually hepatic encephalopathy. The very young and the elderly
are particularly susceptible to this. rRNA is made by RNA pol I. Ribosomal
subunits and rRNA are made in the nucleolus. RNA pol III makes tRNA. In a
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bacterial cell, one RNA pol does it all! This bacterial enzyme is inhibited by
rifampicin (rifamycin), useful in treating TB and bacterial meningitis.
Steps in Translation (page 10)
Here is an mRNA in the cytoplasm. The ribosome will translate from the 5’  3’
end. Its looking for AUG. The binding site in the prokaryote is Shine-Delgarno and
in our cells it is the methyl Cap. It is always the small ribosomal subunit that binds
first (40 in us and 30 in them).
When the full ribosome assembles around the start codon AUG, there is a P and an
A site. The AUG is in the P site. To assemble the ribosome, I need energy in the
form of GTP. The tRNA that carries methionine will go into the P site and one thing
they can ask you here is that in bugs, its not just a Met that is the first amino acid,
it’s a formyl-methionine (f-met).
In terms of elongation, we’re doing 3 things. Codon #2 is in the A site, in comes a
tRNA and recognizes that codon. Secondly, the 2 amino acids get together and form
a peptide bond. The A site is full, but I want to keep putting stuff in the A site. So
the ribosome then moves one codon to the right. This moves the whole thing to the
P site, freeing up the A site for the next amino acid to be added. If they show a
diagram, as they have in the past, its gonna come from elongation.
Initiation and elongation require factors. If you hear IF, that’s a prokaryotic
initiation factor (eIF in eukaryotes). EF is an elongation factor in prokaryotes (eEF
in eukaryotes). They may be nasty enough to ask you something like this: for
elongation in our cells, I need eEF 1 and 2. What do they do? They both bind GTP;
they need GTP to work, so they’re G-proteins. eEF-1 brings stuff into the A site
and eEF-2 moves the ribosome (translocation). Peptidyl transferase forms the
peptide bond.
2 toxins inhibit eukaryotic protein synthesis. The 2 they will test are diphtheria
toxin and pseudomonas toxin. They inhibit at the end, so they inhibit eEF-2. These
toxins are actually enzymes that put an ADP-ribose on this factor and knock it out
(ADP-ribosylation).
The other thing to worry about are the bugs. So I have to remember EF-Tu and EFG in prokaryotes. These are the equivalent of eEF-1 and eEF-2 in us. They do the
same thing.
As far as antibiotics are concerned, you have to remember the aminoglycosides.
These inhibit initiation by preventing the ribosome from assembling. If the question
is from a biochemist, it will be streptomycin. If it’s from a pharmacologist, it will
be gentamycin or tobramycin.
Antibiotics that inhibit elongation are tetracyclines (they prevent things from
entering the A site), chloramphenicol (inhibits peptidyl transferase), and
macrolides (erythromycin) and clindamycin [these prevent translocation]. The
pharmacologists have been nasty enough to ask where these antibiotics bind, and if
you keep this diagram in mind, the answer is top, top, bottom, bottom from the
aminoglycosides to the macrolides. The small ribosomal subunit is on top and the
large ribosomal subunit is on bottom.
Termination occurs when the stop codon enters the A site.
Synthesis of secretory, membrane and lysosomal proteins (page 11)
This is a secretory pathway. Proteins are made in the cytoplasm and some proteins
will be sent out into the plasma membrane or to lysosomes. The ribosome makes the
protein amino end to carboxyl end. If a protein is going to go outside of a cell, into
the plasma membrane, or to lysosomes, all of them must have an N-terminal
hydrophobic sequence. This is encoded for by exon 1. How can this be tested?
Concentrate on things that are going out into the blood – from the liver, from the
pancreas, from B cells. So watch out for a secretion question.
A particle in the cytoplasm called SRP (signal recognition peptide) binds to it and
takes the whole thing, ribosome and all, to the ER, making some rough ER. At this
point the SRP leaves, protein synthesis resumes, and the protein is threaded, amino
end first, into the lumen of the ER. At that point, an enzyme called signal peptidase
removes the signal peptide.
The protein is then glycosylated in the ER, so with few exceptions (e.g. insulin),
proteins that are secreted, in the membrane, or in lysosomes, are glycoproteins.
They contain sugars. In the ER, the type of glycosylation is N-glycosylation. There
is a lipid in the membrane of the ER that has a bunch of sugars attached to it. It
transfers the oligosaccharide onto the protein. The membrane lipid is called dolichol
or dolichol-P. The amino acid to which the oligosaccharide is attached is
asparagine. The main sugar in this oligosaccharide is mannose.
At this point, the glycoproteins are bundled up in vesicles and sent to the Golgi for
sorting. They can then either be secreted outside or sent to the cell membrane. There
is a targeting sequence that sends them to the lysosome. The signal for this is
phosphorylation of mannose. Some mannose residues become mannose 6phosphate. The enzyme that does this is in the Golgi, and its called
phosphotransferase.
This can be tested with a disease called I-cell disease. This is a defect in the gene
encoding phosphotransferase. Phosphorylation cannot occur and these will not go to
lysosomes. They will follow the normal path and be secreted into the blood. The
clinical signs of this are diffuse. You will find craniofacial abnormalities, swollen
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gums, macroglossia, skeletal problems, cardiac problems; the kids are in bad shape.
How do I differentiate this? High concentrations of multiple lysosomal enzymes in
the serum. Cellular garbage will accumulate in the lysosomes, and these are called
inclusion bodies.
Genetic Tests (page 12)
In terms of testing for different molecules, we are concerned with DNA, RNA and
protein. These are nucleotide or amino acid chains of varying lengths. A DNA test
is called a Southern blot, an RNA test is a Northern blot, and a protein test is a
Western blot.
In the case of Western blot, we are extracting proteins from a cell and they will have
different lengths, so we can separate them by length. The same thing is true for
RNA: we will find mRNA, tRNA of different lengths in the cytosol. But generally,
for DNA something must be done first because that is in a nucleus. So we have to
get some WBC’s or a buccal swab. We take out the chromosomes, but they’re too
damn long. So we have to cut these up with a restriction endonuclease to produce
smaller fragments.
The electrophoresis separates by size (regardless of its DNA, RNA, or proteins);
long chains are at the top, smaller chains at the bottom. Then I have to transfer to
paper because I can’t do anything with the original gel. Then I have to add a probe
to detect what I’m interested in. that means I have to know something about these
molecules that I’m interested in before I even start the experiment.
If I want to probe a DNA or RNA sequence, I use a cDNA* probe. * indicates that
its labeled – radioactive, whatever – so when it binds I can find it. This is nothing
more than a single strand of DNA that I can make in the lab. To find a sequence, it
will hybridize, or form complimentary base pairs, with it. So I have to know
something about the thing that I want to probe so I can go into the laboratory and
make something that will complimentary base pair with it.
For a protein, the probe is an antibody. It should be specific for the protein under
investigation, which means that I must know what the protein is, isolate it, and then
immunize the animal to get the antibody before I use it as a probe. The detection
here is from the formation of an antigen-antibody complex.
How can these thing be tested? If I want to know if a gene is present in a particular
cell type, I will have a probe that will bind to that particular gene (gene-specific
probe) and they will digest DNA from a bunch of different species, or cells, or
whatever, electrophorese it, put it on paper and then hybridize with the probe. In
this case (on the far left) I see three lanes and three reactions. That tells me that the
gene is present in whatever DNA I put in every lane. It doesn’t matter that these are
different lengths and a different number of bands. This just tells you that the gene is
there. You might get multiple bands if your restriction enzyme cuts up your gene
and the probe recognizes both pieces. The Southern blot is most used for genetic
testing.
The Northern blot will be used for gene expression. If a gene is active, its being
transcribed, the mRNA will be made, and a Northern blot can detect this. An
example of this is glucokinase, the gene for which is present in all cells but is
expressed only in liver cells and not in other cells, such as muscle cells. The
Western blot can test the induction of a protein in a cell – is it being translated. It is
also a confirmatory test for an ELISA for HIV/AIDS testing. They will want you to
know that you first do and ELISA and if it is positive, it may be true or it may be a
false positive. You then confirm it with a Western blot. For an infant less than 6
months of age, you must do a DNA test because the infant could still have the
mother’s antibodies.
The polymerase chain reaction (PCR) is shown on the right. This is basically
cloning in a test tube. What I’m trying to do is identify a region of DNA between A
and B in this particular example to make multiple copies. I can make several million
copies from a single one.
What they’ll want to know are the general ways that one does this. 3 things are
required. I first separate the 2 strands of the target sequence that I’m interested in.
This is done in a thermocycler by increasing the temperature. I then have to bind
primers to each end of the sequence that I want to amplify. At this point, I add a
thermostable DNA polymerase (Taq polymerase). This is the only enzyme used in
PCR. As the reaction proceeds through the various cycles, the region between the
primers becomes preferentially amplified until, at the end, most of what you have is
exactly the region between the 2 primers. So I must know the sequence of the DNA
flanking my gene of interest.
The DNA you are amplifying, as well as the DNA polymerase, is present in small
amounts. Present in excess are the primers and the substrates for DNA polymerase
(dATP, dTTP, dCTP, dGTP). Substrates are in the most excess.
As far as applications are concerned, I can amplify genes, use it for forensic testing
and paternity issues, gene sequencing, and amplification of foreign sequences such
as the HIV provirus. If a mother is HIV positive, I can’t do a Western blot on the 3month-old kid because he might just have her antibodies. I can use PCR and
southern blot to find out if he is infected.
Don’t forget about something called reverse transcriptase PCR. Nowadays, they’re looking for the
extent of viremia, so they’re trying to find the genome of the virus in the blood. That’s RNA and PCR
only works for DNA. So I have to make DNA from RNA using reverse transcriptase and then amplify
the DNA by PCR.
Gene Cloning (page 13)
Page 8 of 10
How do I clone if I don’t have a test tube? I clone in bugs! Here is genomic DNA; it
is a chromosome from a cell. I cut it up into smaller pieces with a restriction
endonuclease. This restriction enzyme is EcoRI. They won’t expect you to know
them, but at least you know it is from E. coli, strain R, 1st enzyme discovered. The
fragments produced by a restriction endonuclease are restriction fragments.
All of these have “sticky ends”. Restriction enzymes only work on double stranded
DNA, and cutting with most of them produces overhangs on each end – little single
stranded sections that are not base paired with anything. These are sticky ends.
We put these into a bacterium and let it replicate them. You can’t directly do this,
but you can put them in a vector that will be taken up by E. coli. This is a plasmid
vector, and all vectors must contain 3 things to be useful. First, you must cut it and
then put these fragments in it. In order to cut it, it must have at least 1 restriction
enzyme site. The 2nd thing is need is an ori site so the fragment can be copied. The
last thing I need is something so I can tell which bacteria got the plasmid. That’s
where I need an ampicillin resistance gene. Think microbiology; some bacteria
become resistant to antibiotics because they take up plasmids with an antibiotic
resistance gene.
You need to cut both the DNA you want to clone and the plasmid with the same
restriction enzyme. They will have the same sticky ends so they can come together
and base pair. Each vector will get a different fragment (DNA inserts) and now
plasmids with new DNA inserted in them are called recombinant plasmids. You
need DNA ligase to join the fragment and the plasmid.
So now I have a bunch of plasmids that all contain different inserts. I now put them
into E. coli cells in a process called transformation. Each E. coli cell will take up a
different plasmid, and if you think of these as genes, you have a bunch of E. coli
cells all with different genes. Now I grow these up in the presence of ampicillin and
those that do grow must have a plasmid, otherwise the ampicillin would have killed
them. In this case, each of these represents a colony of bacteria, they’re called
clones, so I have cloned different genes because each different colony contains a
different fragment of DNA.
To find out which colony has the gene you’re interested in, you plate these out and
then use a probe (ssDNA that will bind to your gene) to find it. So you have to
know something about the sequence of your gene. When your probe hybridizes, you
can grow up millions of copies of the cell that has your gene, so you will have
millions of copies of your gene. Now you have to cut your gene back out with the
same restriction enzyme. The main thing here is that this is just a way to amplify the
gene of interest.
There are different types of vectors: plasmids, bacteria phages ( phage),
cosmids, and YAC (yeast artificial chromosome). As you go plasmid  phage 
cosmid  YAC, you can put increasingly larger pieces into each of these vectors.
Plasmids, phages, and cosmids go into bugs, YAC into yeast, which is a eukaryotic
cell.
Restriction Enzymes (page 14)
Restriction enzymes cut at palindromes. Restriction enzymes are not in your cells,
they are in bugs, but if you put them your cells, they will cut up your DNA. What
they look for are cutting sites – palindromes.
Palindromes only make sense on a double stranded DNA molecule. If you read the
sequence of one strand in one direction, the sequence of the other strand in the same
direction will be the same. We always read 5’  3’, so if both of these strands are
read in this direction, they will be the same. This is a palindrome.
Restriction sites are generally not that long (about 4-6 base pairs). A is the sequence
for EcoRI. You should be able to recognize it. Sometimes they will give you a
single stranded sequence and ask you if it is a palindrome. If it is, you should be
able to fold it in the middle and the two ends should base pair with one another.
This is fold-back DNA.
They may ask you something like “if this is a palindrome, do I know where the
enzyme will cut?” T he answer is no. I know it will cut here, don’t know where.
Restriction enzymes can cut in different ways. In these examples, the first cuts on
the left, the 2nd in the middle, and the 3rd on the right. Notice that the one that cuts
right in the middle doesn’t give you sticky ends. It gives you blunt ends. In that
case, I have to put sticky ends on in the lab, and I can do that.
On the right at the bottom, I’m making cDNA. A genomic library is a series of clones that contain all
the pieces of DNA in my genome. So if take my chromosomes and cut them up with restriction enzymes,
I’m going to get pieces of everything. I will get promoters, enhancers, junk DNA, etc. Sometimes, I will
cut my gene up into pieces, which means it will be in different clones. There will also be introns because
I am cutting up entire chromosomes.
So what I would really like is a continuos piece of piece of DNA that I can translate. So a better approach
is the cDNA approach, making complimentary DNA. Here I start with mature mRNA from the
cytoplasm. That means that these genes aren’t just in the cell, they are also being expressed. So I start
with the single stranded mRNA and make a double stranded DNA, called complimentary DNA. The key
enzyme is reverse transcriptase.
The advantage here is that all the introns have been cut out; this will be a continuos coding sequence. So
if I want to give a patient a gene that can be translated into protein, this is what I want to give them.
Remember that you have to use cells that express the gene you want to give the patient. For example, if
you want to give a diabetic the gene for insulin, you can’t use buccal cells or liver cells because these cell
types don’t express the gene for insulin and therefore won’t have any mRNA for insulin in the
cytoplasm. You need to use beta cells of the pancreas.
Page 9 of 10
What can I do with this cDNA? This is shown on page 15. On the left, toward the
bottom, I can make an expression vector. I can use a plasmid that will copy the
gene (cDNA) and then once it goes into the bug, the bug will transcribe this gene
and then translate it to make protein. So the bug will make gobs of protein for me
each time it replicates. Note what I must have: ori site, restriction site, and an
antibiotic resistance gene. In addition, to make sure the gene is transcribed, I need a
promoter sequence to bind RNA pol and a Shine-Delgarno sequence to bind the
ribosome for translation. This can be used to make insulin, clotting factor VIII,
vaccines, EPO, etc.
The 2nd thing I can do is gene therapy. This is at the top, its somatic cell gene
therapy. The first thing to note here is that you NEVER do this with germ cells. It
is illegal. But what you’re trying to do here is put a gene in a cell type that is
causing major clinical problems. The vector I will use is a retrovirus or
adenovirus. I can put in the gene that I have cloned, it goes inside a chromosome, it
gets transcribed, translated, I will get good protein in the cell and that’s how I can
help the patient. These viruses are made replication defective, because you don’t
want these to replicate in your patient. Under no circumstances would you use HIV,
because you just don’t want to take that chance.
A couple of types of people are really helped by this: SCID patients (adenosine
deaminase deficiency – causes problems in both T and B cells) and cystic fibrosis
patients (put this in adenovirus because it likes the respiratory system – its
aerosolized and goes to the respiratory epithelium). All gene therapy has been
halted because a young man undergoing therapy for cystic fibrosis died of liver
disease secondary to gene therapy using an adenovirus vector.
The last part over here is making transgenic animals. In this case, the cloned
cDNA for a gene is put into mouse ova and the mother has the babies, some of
which are transgenic because they have the gene, called a transgene. Remember that
ALL the tissues in the transgenic animal will have the gene, because the animal
started out as a single cell. You can use Southern blot to find out which animals
have the gene (A and C) and then you can use the Northern blot to see which
animals, if any, are expressing the gene (only C). This is not possible in humans.
This is used for model systems, not gene therapy
Patterns of Inheritance (page16)
There is a good chance of seeing a pedigree. These are the 4 most common, which
is everything except X-linked dominant. So there’s autosomal recessive,
autosomal dominant, X-linked recessive, and mitochondrial. Mitochondrial
inheritance has become popular lately. What one would look for in these pedigrees,
what one would like to see if it’s not recessive is a sick person in every generation
(solid circle/square). If you see that, try to rule out mitochondrial. If it’s not
mitochondrial, it’s autosomal dominant.
In mitochondrial, bear in mind that we get all our mitochondria from our mother. So
if the mother is sick, every kid is sick; it the father is sick, none of the kids are sick.
The pattern itself might tell you something. In #1, 1 out of 4 is sick. What does that
tell you? It tells you that that’s a homozygous individual and this is autosomal
recessive. 50% are carriers. They may ask you for certain diseases here, and that’s
basically pathology. In autosomal recessive, I’d want to remember things like sickle
cell anemia, cystic fibrosis, and a number of enzyme deficiencies (PKU). For
mitochondrial, it will usually involve things that code for oxidative phosphorylation
enzymes. This will present with nervous system and muscle problems.
For autosomal dominant, remember familial hypercholesterolemia, Huntington’s
Chorea, and connective tissue diseases (Marfan’s, Ehlers-Danlos).
For X-linked recessive, remember glucose 6-phosphate dehydrogenase deficiency,
Lesch-Nyhan syndrome, the muscular dystrophies and the hemophilias.
The last thing on this page involves the use of a Southern blot of an RFLP analysis.
On the top is a normal beta-globin gene. Here are restriction sites on that gene. The
normal beta-globin gene will produce a fragment of a specific length. I have a probe
that will bind to the beta-globin gene, good or bad. In B, you see the Southern blot
test, and the 7.6 kb fragment is the marker for the normal allele.
There is a mutation in the gene that causes sickle cell anemia (glu  val), and when
that mutation occurs, a mutation also occurs which changes the cutting site of the
enzyme so that the enzyme can no longer cut (at the site in the middle). When I cut
this mutated gene up, I get a much larger fragment (14.0 kb). That is a marker for
the mutant allele. These are restriction fragment length polymorphisms.
The other type of probe you might remember is the allele-specific oligonucleotide
probe (ASO). This is also called dot blotting.In this case, these are short
deoxyribonucleotides that are allele-specific. I can get one, synthesize it, and it will
base pair only with the normal gene. I can make another one that will bind only to
the sickle gene. Now I can test directly, and I can see if someone contains that
allele. In order to make these probes, I have to know what the mutation is. RFLP
can be done without knowing the mutation.
The last page has some test examples. In the first one, you are using RFLP and
Southern blot. Looking at the Southern blot, you know that the 11.0 band is useless
because everyone has it. The disease is autosomal recessive, so the child must
inherit 2 mutant alleles to have the disease. II-1 has the disease, so the 9.7 band is
the marker for the mutant allele. Mom and Dad are carriers, II-2 is a carrier, II-3 is
Page 10 of 10
normal and the fetus is normal. So the correct answer is D. B is true, but it’s not the
best answer. You’re doing the test so you can tell conclusively.
The last one is a dot blot. This is used with ASO. You take buccal cells from the
mother and father and the cells from the fetus via amniocentesis. You amplify the
beta-globin gene and take 2 samples. You react each sample from each individual
with either a normal probe or a mutant probe. This is pretty simple: if the circle is
dark, I have a reaction. So if the reaction is with a normal probe, the circle will be
dark and the gene is normal, and the reverse is true for the sickle allele.
The man has the disease, but the mother is phenotypically normal. This is autosomal
recessive, so the man should only show a reaction with ONLY the sickle probe. Its
C, so the fetus will be a carrier and the mom is genotypically normal.
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