Genetics - Lec.

4
Monday 4-10-2010

 Some words and sentences are Bold, these are either important
expressions we should memorize, or additional notes I made
myself. 
 Before starting, the doctor made an announcement about the first
and second exams. The first exam will be on Monday 18-10 from
9-11 am, and the second exam on 12-5.

 The Doctor said:

Last time I talked about the forces that contribute to the

stability of the DNA. And now I want to continue talking about
them, and how they stabilize or destabilize the DNA structure.
Then I will talk about the human Genome and general
characteristics about the genes, and then about chromosomes
and how DNA is packaged to fit in a single cell in a very small
space in the nucleus. And finally about telomeres and important
structures in chromosomes. These are the general topics that will
be covered in this lecture.

The internal forces in DNA are important for replication and

transcription, because when the cell wants to replicates its genetic
material, the two strands of the DNA must open, and for the
strands to open, these forces must be removed. That's why we
should study about these forces and how to remove them.

We talked about the hydrophobic interactions. Hydrogen

bonding between the nitrogen bases will prevent the water from
entering the double helix, thus making hydrophobic interactions
at the inner side of the helix. Additional forces are the stacking
forces, which result from interactions between nitrogen bases.
They are very close to each other in the double helical structure
that, so having these bases over each other will provide additional
forces. This can be easily understood by imagining putting coins
over each other in a graduate cylinder. We should also remember
that these forces are very weak, but because they are so
numerous and additive, we'll have a resultant of a strong force.

Now we all remember that the backbone of the DNA is made of

sugars and phosphates, and phosphates will be negatively charged
due to its low pKa (2.15) in comparison to the cellular pH. These
charges will exhibit a repulsion force among each other, leading to
destabilizing of the structure. However, there are other
neutralizing agents that will neglect those repulsion forces and
stabilize the structure. The best example is Histones, which are
rich in basic Amino Acids, especially Arginine and Lysine.
Additional stabilizers of these charges are the positively charged
ions such as Na, K, and others.

Now we'll study kinetics of how the two strands will open for
translation and transcription of DNA. This process is called
'Denaturation', and the reverse process is 'Renaturation'.

Specific conditions, especially extreme pH( either high or low)

and high temperatures, will remove the hydrogen bonds and
stabilize the single strands of the DNA. However, this happens in
abnormal situations outside the cell, but what are the reasons
that cause that inside the cells and in normal situations? How can
we separate the 2 strands? Is it pH? Or high temperature? Could
we boil the cell so we separate the strands?

It is a process of enzymatic reactions of specific cellular enzymes

called Replication Enzymes that will separate the 2 strands, and
then some proteins will stabilize the single ones, and DNA
replication will starts. The DNA will have some Replication
Bubbles that will be formed at separation points so DNA starts
replication, and these bubbles are in fact the place where
hydrogen bonds originally existed. a Replication Fork is also
formed and grows with the continuous unwinding of the double
strands. Remember that not all the DNA is replicated at the same
time; we'll have a maximum of 70% of the base pairs replicated at
one time.

- The bubble is the empty space formed, while the fork is the
two single strands with the neighboring double-strands still
not unwinded yet.

It was noticed that the regions with more A-T pairs will open
first, because regions of high G-C presence will resist the
separation more, so what is the reason for that? Why does it take
more time for the DNA to be separated at G-C regions? Easily we
can know conclude that it is the 3 hydrogen bonds between this
pair, compared to 2 in A-T pair.

This separation, or what we'll call from now on Unwinding, is

cooperative, which means that will have a gradual increase in the
unwinding and the conversion of double-stranded DNA into
Single-Stranded, and this will make a sigmoidal curve not
hyperbolic. All this mean that as the first pair is unwinded, this will
lead to many other pairs separation.

Now we'll talk about a phenomenon called Hyperchromicity of

DNA. If we do an experiment of measuring the light absorbance of
2 samples of DNA, one is single-stranded and the other is double-
stranded, we will have relatively different results. In both cases
we'll have the maximum absorbance at 260 nm, but the
absorbance of the single stranded sample will be higher than the
double-stranded one, and this is the phenomena of
hyperchromicity: the increase of absorbance of light in single
stranded DNA. The question now is WHY? This is simply because
when the DNA is denatured, the nitrogen bases will be more
exposed to light than before, and thus absorbing more light than
double-stranded sample. Another question that you may ask is
why does DNA absorbs maximally at 260 nm? The answer is that it
depends highly on the chemical structure of the nitrogen bases.
They have aromatic ring, with conjugated double bonds, and
chemically speaking these compounds absorb maximally at 260
nm, in comparison to proteins which absorb maximally at 280 nm.
And this range of 260-280 nm is very important in knowing the
purity of DNA samples and if they have proteins in them, which is
a major test made by molecular genetics researchers.

*A guy at that point asked: We studied in biochemistry that

denaturation by heat is mostly irreversible, but you said that
renaturation always happens.
A: This is NOT true. If you raise the temperature of DNA to 150 °C
for example, and you return it back to normal temperature slowly,
it will rewind again. But if you put it directly in ice after exposing
to very high temperature, rewinding will not happen.

- What we studied about in biochemistry was the

denaturation of proteins, not DNA. And denaturation of
proteins by heat is mostly irreversible.

* Refer to slide 28 to perfectly understand the following

paragraph. 

Now if you look at the curve of chromicity, you'll start with low
percentage as we have double-stranded DNA, and we'll end with
100% hyperchromicity when the whole DNA is single-stranded.
Notice also the sigmoidal curve due to the cooperativity in the
unwinding. An important expression that you must know is the
'Melting Temperature', Tm, which is the temperature at which
50% of the DNA is denatured. This temperature varies from one
type of DNA to another, depending on the A-T and G-C
percentages in it, and there are equations that can calculate the
Tm for any sample of DNA after you know the percentages of the
nitrogen bases.

If you look at the chart in Slide 37, you'll see the different
curves of different types of DNA, depending on the G-C content.

Now we'll go on to our next topics: Complexity of DNA, and the

structure of the gene and the chromosome.

Renaturation of DNA from single-stranded to double-stranded is

a multi-step process. The first step is a slow one, rate-limiting, and
second order, because it has 2 substrates. So the nitrogen bases
will look for their complementary regions so they attach to each
other, they will nucleate in an intermediate step called
Nucleation. Then a faster step will proceed to form the double
helical structure.

Renaturation speed depends on many factors, and the most

important one is the complexity of the DNA. Complexity means
how much size is the DNA; the larger the size means the more
complex it is. And for calculating the speed of the renaturation we
should also know something called the log Cot1/2 value, the
logarithm of the initial concentration of DNA, multiplied by half of
the time the step takes. The log Cot1/2 value is inversely
proportional to the rate constant of the step; this means if you
have very small value of the rate constant, the DNA will
reassociate very fast. So we have the possibility of fast,
intermediate, or slow reassociation depending on the rate
constant.
 We can also know the rate constant and speed of the reaction
by knowing the existence of genes on the DNA. For example, the
slow one is called 'Single-Copy' which means that the DNA has
very high number of genes, and they are present only once in the
whole genome, so the reassociation will be very slow because it
will take time for the gene on one strand to find its
complementary one on the other strand. Intermediate repetition
of genes gives intermediate speed, and high repetition makes it
very fast. Most of our human genes –About 75%- are found as
single-copy, and 15% are intermediately repeated and the rest are
highly repeated.

The repetition of genes has two types: some genes are

interspersed or repeated randomly, so you have a copy, then
other genes, then another copy, then maybe an intron, then
maybe an exon, then again a copy, and so on...and the best
example of them are the Alu sequences which are 300 base-pair
long and repeated 300,000 times in the genome. Alu are
restriction enzymes, an endonuclease enzyme that will recognize
specific sequence and cut the DNA at that region as a scissor. They
are also polymorphic in human genome, which means that the
number of it differs from one individual to another, and because
of this, they could be used to map some diseases, they could be
linked to genes that could be defected and cause specific diseases,
and there are many other sequences that could be used for the
mapping of diseases other than the Alu sequences, and we will
talk later about these techniques when we talk about diagnosing
genetic diseases.

The other type is the tandem-genes or satellite-genes which are

repeated regularly, have low complexity, and are usually present
in telomeres or centromeres of the genome. These genes are
usually short compared to the dispersed ones, with an average of
13 base-pairs per gene.
 Now we'll move to talk about human genome. The project of
determining the human genome started about 15 years ago. On
June 26, 2000, the Human Genome Project and Celera Genomics
Corporation jointly announced that the sequencing of the human
genome was 90% completed, and within the last 10 years, the
percentage raised to 99.9% of the 3 billion base pairs in our
human genome. This is very important for medical research, and
to understand the molecular basis of diseases. It was believed that
we have 100,000 genes, but when they found out the whole
genome they knew we have a range of 25,000-30,000 genes.
However, 50% of these genes produce proteins of unknown
function. So as you see, there are many things that were made to
advance science, but we still have a long way to finish everything.

It was also found that 99.9% of the genome sequence is similar

among individuals, and only 0.1% of the base pairs are different.
But this 0.1% is however a large number: 3 million base pairs are
different among us. These differences opened an area in medicine
called personalized medicine, how much the person is responding
to a drug, because the genomes of different individuals will give
different responses for the same drug for treating the same
disease, tolerance to diseases is different because of this
variations. Another thing to mention is that we use only 2% of our
genes, and the other 98% are not used.

The genomes of different creatures are different in size, human

genome is the longest and plasmids have the shortest one.

Before the genome project was done, scientists thought that

one gene will produce only one protein, but they then discovered
that we have only 30,000 genes and 120,000 proteins. How could
one gene produce 3-4 different protein in average? The answer is
in two words only, Alternative Splicing, which we will talk about
later when we study transcription.
 Forgive for any mistake I did ^_^ nobody is
perfect 

"What we have done for ourselves alone dies with us;

What we have done for others and the world remains and
is immortal". - Albert Pike

Sin Cere 
Omar Shunnar