genetics, lecture 3 (lecture notes)

8/8/2019 Genetics, Lecture 3 (Lecture Notes)

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Genetics - Lecture # 3

Wednesday 29-9-2010

Done by: Sara Al-Zu'bi

The Bubble Boy Syndrome and The Structure of DNA

Today, we will continue the last portion of the previouslecture, we already talked about: The different chemical reagents and drugs that are

used to interfere with nucleotide metabolism and howimportant they are in interfering with the pathways; as theyare used for treatment of some diseases such as; cancer andantiviral or antibacterial cells.

And we saw some structures of the drugs .

Today, I will talk about a disease caused by thedeficiency of an important enzyme which is: AdenosineDeaminase. Some people have this enzyme deficient. Theimportance of this enzyme is to convert adenosine ordeoxyadenosine to inosine or deoxyinosine.

When we have AMP or dAMP, we end with adenosine ordeoxyadenosine by nucleotidase, and we end up withinosine and deoxyinosine by adenosine deaminase(by deamination), then they will follow the scheme onthe next page to reach the uric acid, as weve seen in

GMP and UMP degradation.

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Some people have this enzyme deficient, and as a resultthere will be accumulation ofdATP.

It was found that dATP is toxic to the cell, because itinhibits the ribonucleotide diphosphate reductase

(RNDPR). The importance of this enzyme (RNDPR) is: The conversion

ofribonucleotides to deoxyribonucleoties, and thisconversion is important because it will give the substratesfor DNA synthesis, which are the fourdeoxyribonucleotides.

So, you block that enzyme because of theaccumulation of dATP, then the enzyme will be inhibited.

*** It was found that the immune systems cells, which inducethe different responses in immunity, are highly sensitive todATP, as a result, the whole immune system will be blocked

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in those people >> Because of this, it is called SEVERECOMBINED IMMUNODIFICIENCY DISEASE, or abbreviatedas SCID. As the doctor mentioned last time; it is called also

BUBBLE BOYSYNDROME. This deficiency was discovered in aboy with a complete immune deficiency, so he was put in anisolated environment in order to avoid infections >> thatdeficiency in the immune system is a result ofadenosinedeaminase deficiency.

What is interesting about this disease is that they triedthe first level of gene therapy for the treatment of thedisease -that was before 15 to 20 years from now-, BUT it

didnt succeed. They tried to introduce the adenosinedeaminase to the patient, but nowadays they are using thestem cells so they could introduce the healthy enzyme orthe gene of the enzyme, so the disease could be cured bythis technology.

** The doctor showed us a patient who is in an isolatedenvironment because of the immune system deficiency. BUTthat picture isn't found in the slides!!

SO, we are done with this part of the lecture and now we willtransfer to the next topic concerning this course We are going to talk about the DNA structure, genes,chromatin, and chromosomes. These are the topics that wewill cover in this lecture and in the next one.

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Lectures 3 & 4

DNA, Genes, & Chromatin

On slide 1, the objectives that will be covered today whichthe doctor wants us to know.>

The same thing here, you have to -after we finish these 2lectures (3 & 4) or during discussing them- ask yourselves

about these specific objectives if you understand them or not.So we are going to talk about:- The building blocks of the DNA in terms of; structure,

the bases, nucleosides, nucleotides and the doublestranded DNA.

- A little bit about the structure of the DNA, the basepairing, the complementarity, & the antiparallel.

- Some physical characteristics of DNA in terms of thekinetics of disassociation or regeneration of DNAbecause that will help you to identify the different typesof DNA.

- How this HUGE molecule of DNA is packaged in a verysmall size to be in the safe side to the nucleus.>> DNA is a huge molecule which has a big size that thespace where it is located is much much much muchsmaller than the size of this big molecule.

- Chromosomes and some structures of those

chromosomes.LET'S START

Genetic Dogma:You know that the genetic diseases happen because ofmutations in a gene or mutations that affect the repair

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system of the mutations (means that there will be a mutationin the gene because our DNA is always subjected to changesand mutations through time, because of the environment andthe chemicals that we are exposed to).

>> So our DNA is always subjected to mutations or changes,so there must be a mechanism in the body which is theREPAIR MECHANISM that will fix and repair any mistake orany mutation in the DNA.

Supposedly, those genes or enzymes -that are responsible forrepairing the mistakes- also have mutation >> then therepair system will also be blocked.

SO a genetic disease will result because of a mutation inthe gene that repairs it. Also the genetic flow (theinformation of the genetic constituents) will stop from onegeneration to another because of those mutations or whenthe repair is not replaced.

The flow of the genetic material:The flow of the genetic material, as you can see on the nextpage, the genetic material flows from DNA to DNA by DNAreplication (this means that the genetic material is producedand transferred from one generation to another via DNAreplication or DNA synthesis).

From DNA to RNA when a gene is expressed by aprocess called: transcription process. From RNA to protein in order to have the proper

phenotype of the genetic character.So, it starts from the DNA (or from the gene) in order toconserve this genetic information. DNA must replicate,so the genetic flow went from DNA to a new DNA viareplication. And DNA to RNA via transcription, in order

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for the genetic traits (or information) to be transferred tothe protein. mRNA must be translated into protein, andthe protein will be processed in order to give the properfunction for the cell or for the organism, so this is how

the genetic material flows.>> This flow of genetic information is called THE CENTRALDOGMA of MOLECULAR BIOLOGY, and the components of thecentral dogma of molecular genetics are: DNA to DNA, DNA to RNA, RNA to proteinQuestion: Is there any genetic flow from RNA to DNA?

The answer: There is, sometimes, a reverse genetic flow.

Some viruses, in order to keep transferring the flow of thegenetic material, RNA must be converted into DNA by aprocess called reverse transcription. And there areenzymes responsible for this reverse transcription.

Question: Is there any genetic flow from protein to DNA orRNA?

The answer: Till now, it is not clear if there is any flow of

genetic information from protein to DNA or RNA, but in futurethere might be something discovered. So far, some enzymes(which are proteins) work back on other enzymes to modifyRNA, so this could open an area to start looking if there is anygenetic flow from protein to DNA or RNA.

NOW, we will talk about the DNA structure and its chemistry,and in this aspect we will talk about some evidences that theDNA is the genetic material, not protein or any other

compound.>> SO, we will talk about: DNA transformation and someexperiments to do that, transgenic experiments (that alsowill give us evidence that the genetic material is transferredvia DNA), some tools of mutations on some genes and look

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if the phenotype will be changed after we change thegenotype.

* NOTE: The phenotype is the appearance.

The genotype is the genetic constituents.

Question: What are the evidences that the genetic materialis the DNA (The molecule that transfers the geneticinformation from one generation to another is the DNA)?

The answer: There are 2 famous experiments done invivo and in vitro:

In vivo: They took specific bacterial cells (streptococcus),and then some of the virulent bacteria were heat-killed (thismeans they are not infective agents anymore), the othergroup of cells are viable but not virulent (not infectiveagents). And then they injected a mouse with these twobacterial populations.So we have 2 types of bacterial cells: heat-killed virulentbacteria, and viable non-virulent bacteria.

>>> When you kill the bacteria, you kill all the enzymes andproteins and every other infective agent.

What do you expect as a result? When the mouse wasinjected with these two types of bacteria what happened tothat mouse, was it infected or not?The result: The mouse was infected. Why?

Few students answered, but the answers werent heard,unfortunately, but I have written the doctors comment onthem:

1- Yeah, but you killed the virulent strain by heat, and thestrain which is not virulent is not an infective agent.

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Although we have these assumptions, the mouse wasinfected!

2- Yeah, but it needs some components from the virulentin order to replicate, so it will replicate but the result of

this replication is non-virulent, and why do we needsome of those components although it could replicate byitself?! Its a good explanation or conclusion, which is:the other components in the heat-killed virulent bacteriadon't help the non-virulent to do any new thing.

3- (The correct answer) yeah this is exactly what happens,although the effective strain was heat-killed but still theDNA is there, and the DNA was mixed with the DNA ofthe non-infective, and thus produced microorganisms of

the virulent that caused death of the mouse.What happened is a transfer or mixing of the DNAwhich carried the infectious character.

>> So, this is an evidence that the DNA is responsible for thetransfer of genetic material (this is in vivo).In vitro: They looked at another two strains of bacteria withspecific phenotypes in vitro in a test tube. Suppose we havestrain A, which has a smooth colony -for example- andanother strain which has a rough colony. The smooth-colony-producing-bacteria were killed and mixed with the rough-colony-producing-bacteria and the result was: The roughcolonies have grown, although the rough colony was mixedwith the viable smooth colony bacteria.

So the genetic information was transferred from a specificagent that was damaged but DNA wasn't damaged, so it

transferred the genetic information to the next generation.Concerning transgenic experiments, you could introduce agene to an egg and look if that gene was expressed. Youcould insert that gene in the ovum and the animal that isproduced after inserting a full gene in the egg is called atransgenic animal.

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Transgenic animal: is an engineered animal in which aforeign gene is introduced in the ovum and then the fertilizedegg grows, and the produced organism is called a transgenicanimal.

NOW, what they did with these transgenic animals is thatthey started to look at some . (the doctor was interrupted),when they put the foreign gene, they changed the genotypeand they noticed that the phenotype has changed, forexample: if they insert a gene of a growth hormone in atransgenic animal, they noticed that the transgenic animalnow is growing in size and volume, it is bigger than thenormal one which is not transgenic or the foreign gene wasntinserted in.So, this is another evidence that proves that the DNA isresponsible for changing the phenotype when the geneticconstituent is changed.

Another thing that was built in with transgenic animals, ifthey want to study the effect of specific gene on a specificcharacter, they destroy that gene and start looking at thephenotype of that new organism, that type of experimental iscalled knockout transgenic animals, (Knocking out agene will produce a knockout transgenic animal).

This is also an evidence that DNA or part of the DNA willchange the phenotype because the genetic constituent was

changed.

Now, also another evidence that DNA is responsible forchanging the phenotype or transmitting genetic informationfrom one generation to another is the mutation that we havein our DNA.

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If we look at each other we will not see any two individualslooking the same except in identical twins, and they will alsohave some little differences.

>> this is an evidence that the genetic information istransmitted from one generation to another or the phenotypeis different because of something in the genetic constituenthas changed in the individuals due to mutations (andmutation happens only to genes or genetic constituents orthe DNA).

So because of the variation in the DNA sequence (i.e. our

DNA is polymorphic, if you look at the DNA sequence of anytwo individuals, you will see some differences in some regionsof the DNA) we are not the same in tolerating a disease - forexample -, or we are different in tolerating a drug - forexample - because our genetic constituents are different fromone person to another, so a patient taking a specific drug,that patient could improve using that drug, but anotherpatient having the same disease using the same drug couldnot improve because of the variation of individuals in thegenetic constituent due to polymorphic sequences of our DNAdue to mutations.

these are the three principal concepts concerned asevidences that DNA is the agent responsible for the transferand conservation of genetic material from one generation toanother, and transmitting that information from onegeneration to another (it's not protein, it's not carbohydrates,

it's not amino acids, its a DNA molecule that is responsiblefor those things).. Slide #8

And those evidences are:

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DNA transformationTransgenic experimentsMutation alters phenotype

NOW, you are familiar with the structures of bases; purines(adenine and guanine), pyrimidines(thymine and cytosine).

In the previous two lectures, you sawhow they are biosynthesized and hownucleosides are also biosynthesized.BUT, what concerns us is: Themodification of some nitrogen bases.

One of the important modifications thathappened to our DNA is themethylation (specifically to cytosine).

So you will see (in our DNA): 5-methylcytosine which is amodified N. base ofcytosine.

Methylation is a very important mechanism to regulategene expression and control it (regulation is an importantmechanism to control a specific gene expression)I.e. supposedly, if you want to stop expression of gene, therate of methylation of that gene will be increased; thosemethyl groups will cover that gene and prevent otherenzymes to come and initiate transcription of that gene.But, it was found that 5-methylcytosine is rich in thepromoter regions, (they are very important to control geneexpression & to control transcription). SO there are a collection of cytosines (C & G) >> they arecalled hot spots CG islands. They are located or clustered nearthe promoter regions (you know the promoter regions are theprincipal region to control gene expression).

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When those are methylated, the gene expression will bedecreased and when they are demethylated, the geneexpression will be increased.

The danger comes if: 5-methylcytosine wasdeaminated.I.e. Our DNA is always exposed to modifications, likemethylation, amination, and deamination. All these chemicalreactions could happen to our DNA and among those is: thedeamination of 5-methylcytosine.

** If 5-methlcytosine was deaminated, it will be converted to

THYMINE.Question: If 5-methlcytosine is converted to thymine, whatare the consequences for that?

The answer: Mutations. If mutations happen to our DNA, wehave the repair system to discover that mistake andautomatically change it and put the right base in the gene orin the DNA. BUT in this case, our repair system will break

down; it will not discover that this thymine was 5-methylcytosine, because thymine is not a foreign base to ourDNA, so the repair won't take place.

If the repair doesn't take place, then we will havemutations. Mutations caused by the deamination of 5-methylcytosine cause lots of cancer types in our bodies.

Modification by methylation is important, specifically forthe CG islands which are located near the promoter.Amination will cause cancers and other disorders.

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NOW, look at the following picture these are thenomenclature of a nucleoside and nucleotide.

You can see the base andthe glycosidic bond (always the numbering of the nitrogenbase takes the priority; the numbering for the sugar will takethe primes).

REMMEMBER THAT:The glycosidic bond will be between 1' and N9 of thenitrogen base.If we talk about DNA then the sugar is deoxy sugar(deoxyribose).When we have the nitrogen base + the sugarthecompound is called nucleoside >> it's not guanine it'sguanosine, it's not adenine its adenosine.

The Phosphate group is going to be esterefied at the 5' and3' regions, and if the phosphate attaches then that nucleosidebecomes a nucleotide (or nucleoside mono phosphate ornucleoside diphosphate or nucleoside triphosphate).

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The picture below (or in slide 14) shows how thepolynucleotide is formed; in the picture is what we call apolynucleotide.

The DNA has twotermini. Theconventionalstarting head of thepolynucleotide iscalled the 5' endand the lastnucleotide (the

other terminal ofthe polynucleotide)is called the 3' end.

They (the 5 end and the 3 end) are connected with eachother via 3,5 diester bond (or phosphodiester bond), so 3'from the first one and 5' from the second nucleotide in thephosphate, and the same thing repeated; anotherphosphodiester bond, so they run from 5' to 3'.

5' end in most cases has a free phosphate group and the 3'end always has free hydroxyl group (you will see theimportance of having a free hydroxyl group in this region forthe expression of any gene or the synthesis of the DNA or forthe transcription).

You have to memorize: the phosphodiester bonds from the5' and 3. The 5end is the beginning and the 3' end is theend of the polynucleotide, glycosidic bonds, and if you wantto read a polynucleotide you have to read it from the 5' endto 3' end not vice versa.

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The DNA was discovered in 1951-1953 by Watson & Crick.They published a paper in nature saying: the DNA is a doublehelical structure, and all those conclusions came from a lot ofexperiments for many years in order to reach thoseconclusions.

We are going to review some of those conclusions aboutDNA double helical structure:

- They found that the DNA is found in a double helicalstructure and they discovered that later on (Watson and

Crick found by another evidences that it's a doublehelical structure).

- and after 10 years another scientist did an experimentcalled x-ray diffraction, and found the solid evidence thatthe DNA is found in a double helical structure, anddiscussed it with Watson and Crick and improved whatthey have estimated or concluded.

They found that in the DNA strands there must bebase pairing; they found that the cytosine paired withguanine and adenine paired with thymine.

According to the accurate measures and figures thatthey concluded in their experiments in x-ray diffraction,they found that the adenine couldn't paired with anyother nitrogen base except the thymine, the same thingfor guanine and cytosine

The base pairing takes place by hydrogen bonds (Hbonds form when we have two highly electronegativeatoms).

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These side chains of oxy carbonyl and amino grouparen't found for NO reason!! Those side chains arevery important for the stabilization of the doublehelical structure via the hydrogen bonding.

For the A-T base pairing, TWO hydrogen bonds couldbe formed, and for the G-C base pairing - according tothe chemistry of the structure of these nitrogen bases- THREE hydrogen bonds could be formed.

As a conclusion: DNA is a double helical structure with twostrands; each strand is paired to the opposite strand and they

are complementary to each other (in terms of A VS T & C VSG). They are anti-parallel (one is running from 5' to 3', & theother is running the opposite direction (from 3 to 5).

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There are different forms of DNA, depending on how muchborder is found in that molecule, but the famous form iscalled B DNA form.

DNA forms are: B form, A form, Z form and other forms.

These forms are different from each other in: terms ofdimensions, for example in the B form: we have 10 basepairs every turn, while in another form of DNA may bethe number of base pairs is less or more.

the rise between the N base and another in the B formhas a specific dimension, but in another form that distanceis different, so it gives us different shapes of these differentforms of DNA.

Now, an important thing is the two structures: the majorgroove (which is deep and narrow), & the minor groove(which is shallow and wide).

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The importance of these grooves is forproteins or the enzymes that willinteract with the DNA, control the DNAexpression, then their binding to the

DNA must be specific according tospecific sequence, that enzyme (whichcomes and bind to affect the controlof any gene expression) must be ableto read that specific sequences tobind there or not to bind there.

The Major and minor grooves willprovide a place for those enzymeswhen they come to bind to DNA toread the sequence in that region.

SO, the function of these grooves: isto build for specific binding for thoseenzymes or protein to read whatsequence they are looking for to bind

Major groove

Minor groove


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Now, talking about the composition of nucleotides of the DNA,always the A=T and C=G.

* Human DNA nucleotide composition is different frombacterial or viral DNA.

What stabilizes the DNA double helical structure? The presence of the double stranded is very important

for the DNA replication or transcription. In a moment, you saw how N bases are arranged and

how the backbone of the DNA is arranged: The backbonewhich is outside (the backbone from outside is:phosphate sugar) and the N bases are inside the doublehelical structure.

The existence of N bases in this arrangement is a stackedform (stacked y3ne fo8 b39hom l b39) that will produce a

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The phosphatesugar backboneoutside the doublehelical structure of

The N basesinside the doublehelical structure ofthe DNA


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force that will stabilize the double helical structure and thatforce is the hydrophobic force.

The 1st important force that stabilizes the double helical

structure of the DNA is the hydrophobic force that comesfrom the stacking of the N bases.Another important force that stabilizes the double helicalstructure of the DNA is the H bonding between the basepairs, although the strength of the H bond is NOT sufficientbut because we have tremendous, numerous numbersof Hbonds so this collection of H bonds between N bases in thedouble helical structure will stabilize the double helicalstructure. We have two forces that stabilize the double helicalstructure of the DNA are: Hydrophobic force that comes from the stacking of the Nbases H bonds between the N bases

NOW, in the phosphate sugar backbone, Phosphate isnegatively charged and near it sugar then Phosphate (which

is negatively charged) >>> so, thatwill produce a destabilizing force for the double helicalstructure that is: the repulsion of the negative chargesbetween the Phosphates, but in vivo, those negative chargesare covered or neutralized by positive charges from somemetals, such as: Na, K, or by some basic proteins which arepositively charged and rich with basic amino acids. SO thiscovering with positively charged molecules will prevent thedestabilization process of the DNA.

FINISHED -------------------------------------------------

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STUDENTS QUESTIONS

These are the questions of the students, some of them Iheard them clearly, but most of them unfortunately Icouldnt :D

If you want to go over them, here they are and I advise you toread them because they contain some information that maycome in the exam, and its still up to you.

The proteins or enzymes that will bind to the major or minorgroove will stabilize or destabilize the DNA?

The answer: They will bind to the negative charge, either inthe minor or in the major, 6b3an when there is atranscription, there will be changes when the gene will be

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expressed, instead of being packaged to each other it will beunwrapped and all the enzymes will be exposed.

** The positive ion makes stabilization and of course the

negative ion destabilization.Is there a reverse transcription in animal cells??

The answer: They are mainly in viruses, but there areprocesses in human being where we will see the reversetranscription.

The prions microorganisms have ribose, so it has protein

not genetic material, so how it does it replicate?The answer: there is no microorganisms that have onlyprotein, prions are proteins produced by sensitivemicroorganisms, and they are not themselvesmicroorganisms, so these are proteins produced byspecific microorganisms not microorganism itself. It makescow madness disease.

What controls the regulation process? (A good question)

the answer: there are enzymes that will methylate DNAand those enzymes and the gene responsible on thoseenzymes, when we talk about transcription you will see alot of DNA-protein interaction, that will stimulate, forexample, the gene 4-methylase in order to stop thetranscription of a gene or it will stimulate the expressionof demethylase gene in order to stimulate the expressionof the gene.

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So, its a DNA-protein interaction that will control thismethylation.

If we have 20 nucleotides, how many phosphates we

have?The answer: 20 phosphates, 1 free OH group, 19

glycosidic bonds.

I couldnt hear :D

The answer: The stability doesnt allow or the distancebetween the double helical DNA structure dont permit

because the H bond for it are linked so it doesnt fit orstabilize the chemistry of the double helical.

>> And another students asked about mutations I guess andthe doctor said:

- Its a dangerous mutation to have the transformation ofcytosine to thymine, especially because they are locatedon promoter regions.

I couldnt hear :D

The answer: Histones: are basic proteins that are richin basic amino acids that will bind to the negative chargeand neutralize it.

They will bind to the negative charges either in theminor or in the major groove, of course when there istranscription there will be changes, when the gene isgoing to be expressed the region will be changed, soinstead of being packaged on each other it will beunwrapped and it will be exposed to the enzymes.

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I would like to thank one of my best freinds who helped me as possibleas she could,,, i cant thank her enought with words just THANKS.....

And i cany forget my elder sister who stood me alot,

THANKS .......

.

.

Done by: Sara Alzubi

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genetics, lecture 3 (lecture notes)

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