Genetics – Lecture 5

Wednesday 6/10/2010
Done By: Rasha Al-3ibeeni

We'll talk about the gene structure, DNA packaging, and the structure of the
chromosomes.

DNA reassociation curve (Cot curve)

Concerning this curve it represents the reassociation of the human genome or what’s
called cot curve (Cot is the product of Co (initial DNA concentration) and t (time)).

And thus the DNA concentration is constant, the x-axis is simply the time course of
the reaction, with the percentage of DNA reassociation on the Y-axis starting from
0% reassociation till 100% reassociation.

If the curve looks in one profile and ends, that means we have only one type of DNA.

One profile DNA

Since we have many profiles, then we have many types of
reassociation curve (one
DNA in our genome. The reassociation depends on the size
type
of theDNA)
DNA (the length of the base pairs in DNA) which is
termed DNA complexity.

Prof 1: reassociation fast – low complexity

(repeated too much)
Prof 2: reassociation in between – intermediate complexity (repeated, lesser extent
than 1)
Prof 3: reassociation slow – high complexity (single copy gene)

As profile 1 is highly repeated, it will reassociate quickly because the single stranded
DNA will find the corresponding genes on each other fast and easily. While in profile
3 with no repeated sequences, the single stranded DNA will find it difficult to match
the corresponding genes (human genome is very big) so will take a lot of time to
reassociate.

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DNA reassociation correlates with DNA complexity (gene repetition) as mentioned
before, also correlates in the curve with k2 (reassociation rate constant – second
order).

To determine the value of k2 we take every profile look at its beginning and end and
take 50% between them drop a vertical line and see the cot value this cot value =
Cot1/2 = 1 / k2 as in the figure above.

We notice that when the Cot1/2 is high the rate or k2 will be low and vice versa.

Prof1: Cot1/2 is small while k2 is big – fast

Prof3: Cot1/2 is big while k2 is small – slow

** The arrow between the profiles indicates 100% reassociation of the previous
profile and at the same time 0% reassociation of the next profile.

(?) Why have we taken cot value of 50% reassociation as an indicator of k2 not
the cot value of 100% reassociation?

Dr.: it’s a definition between the scientist, like km in the enzymes 50% of Vmax . if we
take at 100% well get another rate but not k2 .

(?) What’s the meaning of repeated?

Dr.: small sequences may be 20 -30 bp repeated randomly, interspersed within the
genome may be million times.

(?) Why will it affect the speed of reassociation?

Dr.: Because it's present everywhere in many areas, at 0% reassociation we have 2

single strands when they strike each other they bind directly because their
concentration is high.

Gene structure
Human genome consist of 46 chromosomes, each chromosome has one complete
molecule of double stranded linear DNA. The following figure shows part of this DNA
(a gene).

Eukaryotic genes are called split genes because they are not continuous; they are
interrupted: there are introns and exons in the gene.

Typical gene parts:

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 • The promoter region or what’s called 5` flanking region: specific sequence
of DNA nucleotides that specialized enzymes and proteins bind to, to activate
or to promote the gene expression (important to regulate & promote the
transcription process).

• +1: the first nucleotide to be transcribed, any nucleotide before it in the

promoter is labeled as minus -1, -4, -20.

• Exons: the coding regions of the gene, will be transcribed to mRNA then
translated to polypeptide chains (protein), their number varies from one gene
to one another.

• Introns: non coding region of the gene, will be transcribed to mRNA but
won’t be translated. (Small hint: introns are important in: 1. variability between
individuals 2. decreases the risk of having harmful mutations as they are non
coding). Some genes don’t have introns.

• 3` flanking region: last region, not transcribed nor translated.

Transcription of the gene will result in a primary mRNA that includes the previous
regions but not the flanking regions, then further processing, modification and
splicing of the primary mRNA takes place in order to have a mature mRNA that
consist of exons only (we’ve split the introns). The mature mRNA consists of 3 parts:

 5` region: first region of the mature mRNA (part of the first exon)

 Exons: continuous, not interrupted.

 3` region: last region of the mature mRNA (part of the last exon)

Now the last process is translation to AA to form polypeptide chains, the region that’s
translated is the exon part of the mature mRNA but also not 5`or 3` regions. So, not
all parts of the mRNA are translated.

(?) Is there any possibility that an intron will be converted to exons or vice
versa?

Dr.: Yes, by the alternative splicing of the mRNA. Last time I said that every gene
may give 3 proteins (depending on the cell type, tissue type, and the protein that’s
required) but not coming from the same mRNA.
Transcription of gene  primary mRNA  by alternative splicing some introns will be
changed to exons and so  different mature mRNA  translation  different
proteins.

The figure in slide 49 - Some examples of Genes:

 Histone: 1 exons, no introns (one continuous coding piece), 400bp.

(?) What’s the function of histones?

Answer: bind DNA for stabilization and expression regulation.

 β globin: 3 exons (990 bp), 2 introns, total = 1,660 bp

(?) What’s the function of β globin?

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Answer: hemoglobin consist of 4 polypeptide chains: 2 α globin and 2 β globin.

If we compared the total size of the β globin gene with the total size of the histone,
we'll find that they are only 4 times. And the exon size of them is nearly doubled.
We’ll see what that means in a moment.

 HGPRT: 9 exons (1263 bp), 8 introns, total = 42,830 bp.

As we saw this enzyme is important in the salvage pathway of Purines.

If we compared the total size of the HGPRT gene with the total size of β globin, we'll
find that they are more than 20 times. Whereas the exon size of them are nearly the
same.

We conclude that the exons size is nearly constant, but the introns size is widely
different, so what determine the size of genes big or small are the introns.

 Factor VIII: many many exons and introns, total = ~186,000 bp,
exons = ~9,000 bp

Another comparison between factor VIII and the other genes but this time the ratio
between the (exon size / total size) we find no comparable difference.

(?) As histones don’t have introns, what produces the variability between the
different types of histones?

Dr.: the different types are due to the difference in the 5`region in the mature mRNA.
Different sequences of nucleotides in this region give me different histones.

One student asked about the 5`flanking region how it causes variability without being
transcribed but the doctor ensured that he meant the 5` region of the transcribed
mRNA.

Alu sequences can be “mutagenic”

To remind us the doctor asked couple of questions:

(?) What are Alu sequences?

Answer: Alu sequences are about 300 bp in length and are repeated about 300,000
times in the genome. They can be found adjacent to or within genes in introns or
nontranslated regions.

(?) What’s the general characteristic of Alu sequences?

Answer: They are polymorphic in terms of number of repeated sequences among
individuals.

Alu sequences can cause mutations that result in serious diseases an example is
familial hypercholesterolemia (FH) is a lethal disease where cholesterol levels rises
away above normal levels.

We know that cholesterol is very important and has a specific physiological

concentration in our blood, in average ranges 150-200 mg/dl. In FH patients,

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cholesterol levels could rise to 800-1000 mg/dl or more, that leads to cardiovascular
diseases, cardiac infarction and atherosclerosis and may lead to death.

The figure in slide 52 represents the plasma cholesterol levels in 3 cases:

• Normal people = 150 - 250 ----- average 200.

• Patient 1: cholesterol accumulates in ranges of 300-500 (above normal).

• Patient 2: ranging 600-1000 (way above normal ranges).

We synthesize cholesterol in the metabolism and we take it from diet as well. It's
carried in the blood via a carrier LDL molecules (low density lipoprotein) which are
mostly composed of cholesterol. Excess cholesterol must be taken to the liver to get
rid of it, LDL will bind to LDL receptors on the surface of hepatocytes, then
engulfment of these (LDL+LDL receptors) and other processes takes place in order
to metabolize cholesterol, this is the normal mechanism.

In FH patients the LDL receptors are defected and (their gene is mutated) so they are
non functional, so LDL will accumulate in the blood resulting in high concentration of
cholesterol.

The molecular mechanism of FH and Alu sequences

Above is the gene of LDL receptors, which consists of many exons and introns.The
arrows indicate the sites of Alu sequences. Alu sequences are present all over in
introns and in exons as well. We have interspersed Alu sequences found
everywhere.

During cell division (meiosis), crossing over must take place. Crossing over occurs
between homologous chromosomes (identical genes). Crossing over requires
homologous sequences at the site of the synapse to exchange their genetic
information, so the non-sister chromatin must be aligned and well positioned. For this
gene (e.g) (exon 4 is under exon 4), (Alu under Alu), (exon 5 under 5), and so on.
Then, one chromosome`s half will go to complete the other one`s half.

In this case, the Alu sequences were used as the homologous sequences required in
crossing over. So, the 2 chromosomes were mispositioned (out-of-register

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misalignment) leading to unequal crossing over of 2 products one of which has a
deleted 5 exon and consequently will not be translated. This mutation will result in
defected LDL receptors and FH.

If one of the parents has FH (the gene is defected) while the other parent is normal
that will give heterozygous patient offspring (patient 1 above) with cholesterol levels
300-500 mg/dl. Patient 2 is called homozygous, and results when both parents have
a mutation in this gene, total defect the receptor doesn’t function at all so they have
even higher cholesterol concentration in their blood than patient 1.

(?) What about the other product of this unequal crossing over?

Dr.: it will result having all the exons but repeated exon 5, and that doesn’t matter too
much; maybe a little, but normal LDL receptors. Maybe exon 5 was defected during
crossing over. (We're not concerned about it).

(?) What are the causes of this error in crossing over?

Dr.: no causes, all the conditions are present, but what increased the chance are the
highly repeated Alu sequences.

DNA packaging
The figure in slide 56 shows TEM picture of the chromosomes, like beads on a string
they found that those beads are DNA and proteins in structures called
nucleosomes.

After the scientists saw that picture, they took chromatin and subjected it to DNAase
(enzymes that digests DNA) DNAase started to chop off DNA, then they took the
product and ran electrophoresis and found DNA bands in a ladder form of 200 bp.
What does it mean?

Some students answered:

- that DNAase cuts DNA at specific sites (Dr.: could be)
- the same structure is repeated which is the nucleosomes and there is a
space between them.
- Dr.: DNAase cuts the DNA between those beads, DNA size of nucleosomes
is 200 bp.

Nucleosomes structure:

The figure in slide 58 shows the nucleosome`s core and the nucleosome proper. The
nucleosomes parts are:

• Octomer of histones: 4 dimers of histones: 2 subunits of each of these

histones (H2A , H2B ,H3 , H4), found in the center of the nucleosome.

• Histone’s tail: a tail like structure oriented outside the histone, consists of
amino acids mainly Arg and Lyc (K), has a very important regulatory role as
we'll see in a minute.

• DNA: the DNA wrap around the octomer of histones nearly 1 ¾ turns. It's
nearly 150 bp in length.

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 • The spacer: about 50 bp of DNA that connects one nucleosome with the
other.
DNA + the spacer = 200 bp (as they found in electrophoresis)

• H1 histone: a monomer of histone that will bind the spacer with the 2
nucleosomes.

Histone`s tail role:

As we saw the tail consist of basic AA that carry positive charges, and these AAs can
be extensively modified by addition of certain groups to them like:
1) Acetylation (Ac): addition of acetyl group
2) Methylation (met): addition of methyl group
3) Phosphorylation
4) Upiquination
5) Adipophosphorylation
6) Poly (ADP) ribosylation.

Now these modifications affect the nucleosomes structure and so the gene
expression. To know how, we'll take 2 examples of modification:

The phosphorus groups in the DNA are negatively charged, so to be stabilized it

reacts with the positive charges of the basic histones. Any factor that changes this
binding of charges will affect the transcription process.

• Hyperacetylation: by histone acetylase (facilitated by TFs): when acetylase

is active it will acetylate the tail (add to the basic AA), these acetyl groups will
cover the positive charges of the histones causing them to loosen their grip on
the DNA to allow transcription factors to bind.

(Hyperacetylation  “loose” nucleosomes  genes are exposed  polymerase

protein will bind  transcriptional activation)

• Hypoacetylation: by histone deacetylase (facilitated by the retinoblastoma

Rb enzyme). Deacetylase will remove the already existing acetyl groups
exposing more positive charges to bind with phosphorus of DNA so more tight
nucleosomes are formed. The enzyme polymerase is unable to bind to the
DNA anymore (transcriptional repression).

Chromosomes packaging:
Nucleosomes discovery opened the area for the scientist to think of the mechanism
of packaging 6-feet-DNA inside a restricted space which’s the nucleus. If we put the
DNA in a solution every 500 to 800 will turn around the other. While in the
nucleosome, every 150 – 200 nucleotides will wrap around the histones.

A multistage-process:
• Naked DNA will wrap around histones to give nucleosomes (beads on
string) 2 nm to 11 nm that’s 5 folds packaging of DNA.

• Looping of nucleosomes to give selinoid.

• Selinoid by further wrapping, coiling, and folding forms the fibers (30nm).

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 • Fibers will bind to a scaffold protein to give a more packaged structure.

• Wrapping, coiling, recoiling, & binding to specific proteins to recoil again and
again to form the last shape of a chromosome.
2 nm – 11 nm – 30 nm – 300 nm – 700 nm – 1400 nm

‫ و صلى‬،‫الحمد لله الذي بنعمته تتم الصالحات‬

‫الله وسلم وبارك على حبيبنا محمد و على اله و‬
‫صحبه أجمعين‬..

‫تحية إليك أردن المعالي شامخة‬

‫راياتك في افرست أعلى قمة في‬
‫العالم الى البحر الميت أخفض نقطة‬
‫ نم قرير العين فطلب‬.... ‫في العالم‬
‫ دفعتنا سيسهرون على صحة أبنائك‬.
Ya 25wan,, sorry for any mistake. W 3thrn 3la shrbi llmr6bat 2thna2 tfree`3 l
mo7a`9arh lkn y78 li ma la y78 l`3airi .w m3ko 10 seconds to switch off your
mobiles.

(Sorry doctor Nabeel but its great fun when you say these things during the
lecture)

Done by: Rasha Al-Ebbini