Genetics 4 - Corrected

7/31/2019 Genetics 4 - Corrected

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Welcome to genetics world ..

the music of life.. in the key of DNA!

Haneen sba'ane &zainah matani .


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We will continue talking about physical properties

of DNA, last time we talked aboutHYPERCHROMICTY >>look at this figure:

A curve represents Hyperchromicity phenomenon of


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nucleic acids under different physical conditions:*the Y axis represents the absorbance of light at 260 nm

wavelength. The X axis represents the wave length (nm.(*these are measurements of the absorbance of nucleic acids

under different wavelengths "as you see in the figure" inDNAsolution.

*notice that this nucleic acid absorbs maximally at260nm.And this is a constant physical property of nucleic acids. " you

can conclude this number (260 nm) from the experiment"*if you study this curve carefully you will see that it represents a

spectra of nucleic acid which is single-stranded -the one above-"look again at the curve" , while the other one is spectrum ofnucleic acid of the double-stranded DNA.

*single-stranded DNA absorbs light more than double-strandedDNA


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5/23

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DNA melting curve, Hyperchromicity here is usedto follow the effect of increasing temperature on

denaturation pattern of DNA: *melting DNA means : denaturation of double-stranded intosingle-stranded DNA.

*Y axis : percent of hyperchromicity ,, X axis : temperature.

*First we have a sample "or preparation" of DNA, westarted to measure the absorbance at 260 nm as we know.But here during measuring absorbance at constant

wavelength "260, we are increasing temperature >> noticethat increasing temperature will increase absorbance, untilwe have 100% hyperchromicity at 90 C.

*100%hyperchromicity means that DNA is fully denatured.*when hyperchromicity is 50% , the temperature then is

called "melting temperature" or Tm and at this point(midpoint), 50% of DNA is denatured, here we have 50%transition from "hypo to hyper" chromicity.

*every DNA molecule has its own Tm >> it depends on thecomposition of that DNA molecule, if rich in G+C ,Tm will behigh, if rich in A+T then it will be less.

*Tm is very important for diagnostic purposes.


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Hyperchromicity can be used tofollow the denaturation of DNA as a

function of increasing temperature.As the temperature of a DNAsolution gradually rises above 50degrees C, the A-T regions will meltfirst giving rise to an increase in the

UV light absorbance.As the temperature increasesfurther, more of the DNA willbecome single-stranded, furtherincreasing the UV absorbance, untilthe DNA is fully denatured above 90degrees C.

The temperature at the mid-point ofthe melting curve is termed"melting temperature" and isabbreviated Tm. The Tm for a DNAdepends on its average G+Ccontent: the higher the G+C

content, the higher the Tm.Note: G+C content, G-C content,

and GC content are equivalentterms.

We said that Tm is very

important for diagnostic

purposes. All gene

testing depend on

what's called PCR!!

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When a solution of double-stranded DNA is placed in a spectrophotometer

cuvette "an instrument to measure light intensity " and the absorbance of the

DNA is determined across the electromagnetic spectrum, it characteristically

shows an absorbance maximum at 260 nm (in the UV region of the spectrum).

If the same DNA solution is melted (denatured), the absorbance at 260 nm

increases to approximately 40%. This property is termed "hyperchromicity."The hyperchromic shift is due to the fact that unstacked bases absorb more

si tahW?


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This curve is thesame like theprevious one,but here wehave differenttypes of DNA

molecules:*We have

different Tmvalues for thesedifferentmolecules, all at

the same hyperchromicity percent "50.%*We said that Tm depends on its average of G+C content.

ALSO, it depends on the ionic strength of the solution.*At fixed ionic strength there is a linear relation between Tm

and G+C content.*the curve at the left is for the DNA with the lowest amount

of G+C content ...(lowest Tm.(So .. we have proportional relation between Tm and G+Ccontent of DNA.

*Under the conditions used in this experiment, E. coli DNAwhich has an average G+C content of about 50%, meltedwith a Tm of 69 degrees C.

The previous pictures were to explain

denaturation of nucleic acid.

*Denaturation is important in:

-Genes expression.

-DNA replication.

-For diagnostic purposes in vitro.

*Factors that cause denaturation:

-Extremities of PH.

-Temperature.

What's the PCR?


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-Organic solvents.

if it's so big then reassociation "or rewinding" will takelong time.

NOW , if we have a repetitive sequence in the DNA molecule thenreassociation will take place faster than having non-repetitivesequence "also non-repetitive sequence is called one-single copy of

gene."we mean by one-single copy of gene thatthe gene is found only

once in that big DNA molecule . "presented in one copy per haploidgenome"


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>>Renaturation steps


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A curve showing the kinetics of reassociation of a DNAmolecule, we are not concerned with the details here,this curve is called "Cot curve for human genomic DNA

" shows a DNA Renaturation (reassociation) reaction:*The reaction follows ideal second-order kinetics. "Cot" is

the product of Co (initial DNA concentration) and t (time(*This curve is composed of three regions "three profiles


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Human genome consist of these types of DNA:-Slow fraction "single copy: it makes 75% of our

genome


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*

E

X

A

M

P

L

E

S

o

f Interspersed Repetitive sequences:

-ALU sequence: they are sequences of

deoxynucleotides , about 300 base pairs

(BP) in length , they have specific sequence and they are

repeated 300000 times in the genome , they could be

everywhere "in front of the gene, at the end of the gene,within the gene INTERSPERSED". As we said they are

found every where, not only in the genes!! We should

know that our genes represent 1.5% of our genome and

the rest represents DNA that does not code for any

protein "is not considered as gene. SO Alu sequence could

be found every where in DNA .Their presence can

sometimes leads to the occasional disruption of genes.

-VNTR "variable number of tandem repeats" : short repeated

sequences of only a few base pairs , variable in length ,

they interspersed throughout the genome.

They are highly polymorphic "differ in length or number of

repeats from individual to individual" SO they are useful

for mapping genes.


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*The importance of these repeats:

>This is important to test paternity

problems. NOW concerning Identical

twins they have the SAME DNA

fingerprint, but non-identical twins have

different DNA fingerprints


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You may read in your BOOK about what is called "satellite..."

*First "as you know", the satellite repetitive of

DNA sequence has the same meaning ofTandem

repetitive of DNA sequence.

*We have the term minisatellite: number of

nucleotide in that repeat is between 10_60

nucleotides.

*Microsatellite: number of deoxynucleotides

below 5.

>>Also you could see dinucleotides repetitive

sequences

"e.g. GCGCGC" 100000 of times within your

genome.

>>Also you could see trinucleotide repetitive

sequence in all over your genome.

ALL these repetitive sequences have significance in

diagnosis identification and linkage to disease as

you would see through the coarse.

Knowing the complete sequence of the human

genome will allow medical researchers to more

easily find disease-causing genes. In addition, it

should become possible to understand how

differences in our DNA sequences from individual

to individual may affect our predisposition to

diseases and our ability to metabolize drugs.

Because the human genome has ~3 billion bp of

DNA and there are 23 pairs of chromosomes in


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>

*The Gene contains:

- Promoter region.

- Exons.

- Introns.

- 3' & 5' regions.

chromosome has ~130 million bp DNA.

*A picture to show you the

complexity and size of

genomes in different

organisms.

*the human genome projectwas finished at 2000.

*it was thought that the

number of human genome

was between

(80000_140000) genes, but

after they did all the

sequences of human

genome they discovered

that the total number of our

genes ranges between(30000_40000) genes or

Let's take a piece of

chromosome, eachchromosome has a single

double stranded DNA

molecule , specifically

this is a picture shows a

sample of a gene within

the double stranded DNA

molecule on the


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NOW, let's talk about gene structure in

details:

1) Promoter region: it's a specific

DNA sequence that contains

regulatory sequences, they will

not code for any protein>> JUST

regulatory sequences.

These regulatory sequences are important to be

recognized by specific proteins, to initiate the DNAreplication or transcription or any other activity of the

gene.

USUALLY, promoters are found in the 5' region of any

gene.

"May be found within the gene OR in 3' region too"

>> the first nucleotide after promoter region will take

the number +1, it's found at the 5' end of the first exon.

The nucleotides which are before it are going to take

the minus numbers.

In order to assign for those regulatory sequences in the

promoter region, one will identify to say for example:the sequence at -25 regions are important in this

promoter for this gene etc.

2) Exons: they are sequences that code for proteins

when they are transcribed and translated into amino

acids.


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3) Introns: between Exons, they are DNA sequences in

any gene that don't code for any protein.

>> Most genes in the human genome are called "split

genes" because they are composed of "exons" separated

by "introns."

*SOME NOTES :>> The transcribed region of a gene (double-endedarrow :The arrows here are stop signals meaning , just to

show you the boundaries and it is the ends) starts at the +1

nucleotide at the 5' end of the first exon and

includes all of the exons and introns. (Initiation of

transcription is regulated by the promoter region of

a gene, which is upstream of the +1 site). To givewhat's called immature mRNA or immature RNA.

>> Not only mRNA will be transcribed, there are

genes for tRNA (transfer RNA) and rRNA (ribosomal

RNA) that also will be transcribed.

>>we have a region in the Promoter region called5' flanking region, and the region at the end of the

gene that doesn't contain any exons or introns is

called 3' flanking region, they could have some

regulatory sequences also.. (it will not be transcribed

nor translated)


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SO >> 3' flanking region, and 5' flanking region will not

be transcribed nor translated!!!

>> The 5' region of exon number one will not be

translated, it will just be transcribed. Also somesequences of them are in order to identify the

beginning of translation. Look at the figure again ...

look at the 3' & 5' regions in the transcribed RNA, these

light in color regions are not translated to proteins"

>>Primary RNA (could be mRNA, tRNA, rRNA ... all

called primary transcript) that resulted fromtranscription, will be processed by removing of

introns and connecting of exons by specific

proteins before being translated. This process

takes place in

the nucleus.

?What's the

function of

introns!?

You know that

the whole

genome size is

3 billion base

pair, now...

Only 1.5 % of

these base

pairs are

GENES.

What is the

function of the rest 98.5%? In another meaning what's


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the function of introns?!!1(Some of them are for regulation of gene expression; some

of those sequences will participate in the regulation.

2(Others are for protection of our genes, as you know ourgenome is always exposed to external factors and mutations,

and the presence of these extra pairs will protect our genes

from destruction from those external factors, so the presence

of this huge extra portion in DNA will protect it from mutations.

?What are those empty boxes? In mRNA.. Figure p. 13Those empty boxes are part of the exons that are not

translated ,you know that at the beginning of translation, the

transfer RNA recognize for the methionine codon to start the

translation process .We have many codons before the transfer

RNA sees methionine codon ,so all these codons will not be

used for translation ,there could be 20 or 30 or 40 codons ,but

the transfer RNA sees the methionine codon and begins the

translation process from there , so this region (the emptyboxes ) could undergo transcription but

can't undergo translation because it

doesn't have to this point the

methionine codon.

?Why arent there exons in the 3' flanking

region?

Because the gene ended,

every gene is composed

of a specific number of

exons ,there is no fixed


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number of exons for all the genes ,so the genes are

variable in size and in the number of exons .The 3'

flanking region is a noncoding region ,they will not code

for any proteins or any amino acids ,here we have a signal

of DNA sequence in the gene that will tell the protein that

this is the end of this gene and another gene will start , so

these introns or flanking regions will make boundaries

between genes within the genome.

look at this figure :

* this figure shows example of the wide variety of genestructures seen in the human genome .

*some genes do not have introns, example is histone

genes. They have one exon , they function in synthesizing

histones. Histones are: Basic proteins that are important instabilizing the double helical structure of DNA.

*Beta-globin gene: is the gene that is responsible for

synthesizing the beta globin polypeptide chain which is thesubunit of hemoglobin.

*HGPRT: hypoxanthine guanine phosphoribosyl transferase,

has exons.

*Factor XIII: which is a clotting factor, has exons.

And here's also a gene for dystrophin which is very important

in muscle contraction and function, this is the biggest gene inour cells (dystrophin ) that has hundreds of exons.


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minus.. the same ,while introns in genes are different in

sizes in genes.

SO .. VARIATION IN GENE SIZES IS DUE TO VARIATION IN

INTRON SIZES AND NOT EXON SIZES>> THE INTRON

DETERMINES THE SIZE OF THE GENE.


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This curve represents 3 columns, first column represents

cholesterol concentration in blood in normal people ranging

from ( 100 to 220 mg cholesterol /100 ml blood) , second one

represents the concentration of cholesterol in people who are

heterozygous for familial hypercholesterolemia the

accumulation ranging between ( 300 to 500 mg cholesterol /

100 ml blood) which is higher than normal ,while people whoare homozygous for the disease have the highest

accumulation of cholesterol with a concentration ranging from

(700 to 1000 mg cholesterol /100 ml blood) and next lecture

I'll explain it on the molecular level.

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^____


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- The happy end _^ , I'm happy of this end aktar

wa7de feekom :P

Bidayatan b3rf enu el mo7adara 6weele $wai ! w ma b3rf

$o el fikra elli 3ind el 6olab lamma tkon el mo7adara6weele bi9eero yid3o 3la elli fara3'ha :P but really it's not

my fault :P

>>mo3zam el slides elli en$ara7 3leehom are included

here ,every thing mentioned also is included here , I did

also include some notes of al 8o9oor lectures "la tawdee7

el 2$ya2 elli kan bidha tawdee7 "

Special thanx from me to zainah matani > it's not being normal that'simportant , but learning to accept our being different : to

live and love as fully as we can ;)

w el ehda2 la kol 9a7bati , w kol el $okor wl ta8deer lal

nas elli kanat wa2fe m3i and giving support thanx !

DONE BY :

HANEEN SABA3NE

ZAINAH MATANI ..

Genetics 4 - Corrected

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