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Haneen sba'ane & zainah matani .

<<Hyperchromicity >>
We will continue talking about physical properties of DNA, last time we talked about HYPERCHROMICTY >>
look at this figure:

A curve represents Hyperchromicity phenomenon of

: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" in DNA . solution notice that this nucleic acid absorbs maximally at 260 nm. * 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 of .nucleic acid of the double-stranded DNA single-stranded DNA absorbs light more than double-stranded * . << DNA << at 260 nm finally ..this phenomenon << The absorbance at 260 nm of * a DNA solution increases when the double helix is melted . into single strands >> is called hyperchromicity

Come on.. Give me the answer to these !!questions

Why are we so concerned about Hyperchromicity?! ? !?Whats the significance of this phenomenon

This is an important phenomenon BECAUSE it's used << ..for quantitative determination of nucleic acids ?!How could that happen ? let's assume that we want to quantitate an amount of<< nucleic acid preparation, you take blood sample from a patient, and then you isolate DNA for gene testing. Then you could quantitate the amount of DNA "that you cut" by .measuring the absorbance of that preparation at 260 nm Then we have an equation or law that correlates between absorbance at 260 nm "that you measured" and

.(the concentration in (microgram/ml SO.. From the reading you got (absorbance at 260 nm), you could know how many micrograms/ml or .milligrams/ml of DNA you have We said that the absorbance of single-stranded DNA is ? higher than that of the double-stranded DNA <at 260 !nm>. Explain the double-stranded DNA is a coiled structure , double<< helical , SO the surface area of exposed nitrogen bases to light in double-stranded DNA is less than the surface area of exposed nitrogen bases .in single-stranded Why does DNA absorb maximally at 260 ? !nm?? Not at any other wavelength you know that the basic units of DNA<< constituents are purines and pyrimidines, the chemistry of these constituents (the conjugated double bonds) has chemical .characteristics of absorbing at 260 nm So, single-stranded DNA absorbance is more than that of the double stranded at 260 nm; what is called Hyperchromicity property of nucleic .acids

DNA melting curve, Hyperchromicity here is used to follow the effect of increasing temperature on : denaturation pattern of DNA
melting DNA means : denaturation of double-stranded into * . single-stranded DNA . Y axis : percent of hyperchromicity ,, X axis : temperature * First we have a sample "or preparation" of DNA, we * started to measure the absorbance at 260 nm as we know. But here during measuring absorbance at constant wavelength "260, we are increasing temperature >> notice that increasing temperature will increase absorbance, until .we have 100% hyperchromicity at 90 C .hyperchromicity means that DNA is fully denatured 100% * 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 the * composition of that DNA molecule, if rich in G+C ,Tm will be .high, if rich in A+T then it will be less .Tm is very important for diagnostic purposes *

We said that Tm is very important for diagnostic purposes. All gene testing depend on !! what's called PCR remylo P noitcaeR > niah C <

eg yna ,WON ni deifilpma na rof detset itsongaid rof sid citeneg fo .

solution gradually rises above 50 >>> OS degrees C, the A-T regions will melt first giving rise to<< increase in the an riuqer si RCP .UV light absorbance gnitset eneg As the temperature increases gniwonk seriuqer >> further, more of the DNA will become single-stranded, further increasing the UV absorbance, until the DNA is fully denatured above 90 .degrees C The temperature at the mid-point of the melting curve is termed "melting temperature" and is abbreviated Tm. The Tm for a DNA depends on its average G+C content: the higher the G+C .content, the higher the Tm Note: G+C content, G-C content, and GC content are equivalent .terms

seriuqer RCP dneped taht Hyperchromicity can be used to ht fo mT eht follow the denaturation of DNA as a oc taht AND . retni fo eneg function of increasing temperature As up citsongaid the temperature of a DNA .

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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 .light than stacked bases
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This curve is the same like the previous one, but here we have different types of DNA :molecules We have * different Tm values for these different molecules, 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+C .content of DNA Under the conditions used in this experiment, E. coli DNA * which has an average G+C content of about 50%, melted .with a Tm of 69 degrees C
he previous pictures were to explain .enaturation of nucleic acid :Denaturation ?What's the PCRis important in .Genes expression .DNA replication .For diagnostic purposes in vitro :Factors that cause denaturation .Extremities of PH .Temperature T d * * -

.Organic solvents Renaturation is also important to go back to the << original molecule, so that original molecule is functional .as in DNA replication or transcription etc NOW, when you remove the factors that cause denaturation, these two strands will start looking for . each other and REWIND again :The main factors that determine the rate of reassociation are . Complexity of DNA molecule (1 .Repetitive or non-repetitive sequences in DNA (2 Complexity of DNA molecule is related to the SIZE of DNA molecule >> if it's so big then reassociation "or rewinding" will take .long time NOW , if we have a repetitive sequence in the DNA molecule then reassociation will take place faster than having non-repetitive sequence "also non-repetitive sequence is called one-single copy of ."gene we mean by one-single copy of gene that the gene is found only once in that big DNA molecule . "presented in one copy per haploid "genome

<< Renaturation steps >>

A curve showing the kinetics of reassociation of a DNA molecule, 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 * <<<<three profiles in the big curve these profiles are different from each other because we * have different types of DNA in this chromosome ... DIFFERENT types means we may have sequences in high repetition in that big molecule of that DNA , we may have sequences in intermediate repetition ..And we may have .sequences with no repletion "one-single copy of the gene We know that high repetition means very fast reassociation * .and low complexity at time-zero the DNA is denatured into single strands and * the conditions are adjusted to promote DNA base pairing to allow the renaturation of the DNA; during the reaction the various kinetic fractions find partners (complements) and reassociate until the reaction is complete (100% . (reassociation

This illustrates the concept of how sequence complexity affects the rate of DNA reassociation. Imagine two different DNA sequences in a genome, one present one time per haploid genome (right) and the other present 1,000,000 times per haploid genome (left). They would be present at a 1:1,000,000 ratio with respect to each other. If these sequences were mixed together (which is what would happen if total genomic DNA was isolated for analysis), then fragmented, denatured and allowed to reassociate, the repeated sequences would reassociate much more rapidly because it would be much easier for them to find complementary strands to base pair with. The repeated sequences would reassociate with a very low Cot1/2 and therefore with a very high k2, .consistent with a rapid rate of reassociation

:Human genome consist of these types of DNA Slow fraction "single copy: it makes 75% of our <<genome <<includes most genes :repetitive "fast and intermediate fraction Tandemely repeated: satellite DNAs, "fast fraction" , highly (1 ,repeated sequences of our genome >> they are sequences that are repeated many 10% ~ time adjacent to each other and have low complexity. Usually located in .(centromers and telomeres. (E.g. the sequence TTAGGG Interspersed: means randomly, "intermediate fraction", (2 intermediate repeated, ~15% of our genome >> EXAMPLES: ( ALU .(sequences, VNTR

* E X A M P L E S o :f Interspersed Repetitive sequences they are sequences ofdeoxynucleotides , 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
ALU sequence:

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
VNTR "variable number of tandem repeats" :

: The importance of these repeats*

although these repetitions don't have any physiological << function in our DNA or in our genome, they are important because they are polymorphic, "number of repeats is different .from individual to another They are important to identify individuals according to what's called DNA fingerprint! Every one of us has a specific pattern of DNA called: DNA fingerprint..These fingerprints are different .because the number of repetition differs

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

:To summarize what we were talking about

First "as you know", the satellite repetitive of* DNA sequence has the same meaning of Tandem .repetitive of DNA sequence We have the term minisatellite: number of* nucleotide in thatyour BOOK about what is10_60 ..."You may read in repeat is between called "satellite . 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 genome3 has ~3 billion bp of DNA and there are 23 pairs of chromosomes in

diploid human cells, the average metaphase .chromosome has ~130 million bp DNA

A picture to show you the* complexity and size of genomes in different .organisms the human genome project* .was 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 .even less

<< GENE structure >>

* The Gene contains:

Promoter region. Exons. Introns. 3' & 5' regions.

Let's take a piece of chromosome, each chromosome 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 chromosome, and its
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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 DNA replication 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.

<it will be transcribed into mRNA.. then translated into proteins>

3)

Introns: between Exons, they are DNA sequences in any gene that don't code for any protein. <they will be transcribed but not translated >

>> 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-ended arrow : 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 give what'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 called 5' 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)

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 some sequences 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 from transcription, 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

?!!the function of introns

Some of them are for regulation of gene expression; some (1 .of those sequences will participate in the regulation Others are for protection of our genes, as you know our (2 genome 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. 13

Those 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 empty oxes ) could undergo transcription but b an't undergo translation because it c oesn't have to this point the d . ethionine codon m

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

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 gene structures 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 in
stabilizing the double helical structure of DNA. Beta-globin gene: is the gene that is responsible for * synthesizing the beta globin polypeptide chain which is the .subunit 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 in . our cells (dystrophin ) that has hundreds of exons

So the message here is that variable genes have << different number of exons ,and different genes have This figure shows examples of the wide variety of gene structures seen in the human genome. Some . variable sizes

(very few) genes do not have introns. One example is the histone genes, which encode the small DNAbinding proteins, histones H1, H2A, H2B, H3, and H4. Shown here is a histone gene that is only 400 base pairs (bp) inany gene composed close one exon. The beta-globinare plus orexons and Exons in length and is are of of only size , there sizes gene has three two introns. The hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT) gene has nine exons and is over 100-times larger than the histone gene, yet has an mRNA that is only about 3-times larger than the histone mRNA (total exon length is 1,263 bp). This is due to the fact that introns can be very long, while exons are usually relatively short. An extreme example of this is the factor VIII gene .which has numerous exons 3

minus.. the same .sizes in genes

,while introns in genes are different in

SO .. VARIATION IN GENE SIZES IS DUE TO VARIATION IN INTRON SIZES AND NOT EXON SIZES>> THE INTRON .DETERMINES THE SIZE OF THE GENE

Properties of the human << :genome

We have two types ofgenomes; here we describe the :properties of each type

Mitochondrial genome:*
circular genome of ~17000 pb ( the fact that the mitochondrial genome is circular like bacteria has a connection to evolution) , contains <40 genes ((the rest of the genes are nuclear there are some proteins found in mitochondria , some of those proteins are transcribed there by mitochondrial genes , however not all mitochondrial proteins are transcribed in the mitochondria, .some are transcribed in the nucleus

* :Nuclear genome
.The haploid human genome has ~3 X 109 bp of DNA .Single-copy DNA comprises ~75% of the human genome .The human genome contains ~30,000 to 40,000 genes .Most genes are single-copy in the haploid genome :Genes are composed of from 1 to >75 exons you saw it earlier in one of the pictures ,that histone ) genes has one exon , and the dystrophin has 200 exons (on an average of 1 to 75 exons Genes vary in length from <100 to >2,300,000 bp.- -Alu .sequences are present throughout the genome

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 who are 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 _____________________________________________________________^ ^____

- The happy end ^_^ , I'm happy of this end aktar wa7de feekom :P Bidayatan b3rf enu el mo7adara 6weele $wai ! w ma b3rf $o el fikra elli 3ind el 6olab lamma tkon el mo7adara 6weele bi9eero yid3o 3la elli fara3'ha :P but really it's not my fault :P >>mo3zam el slides elli en$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 <3 for her help thanx zaina ;) W 6ab3an l2inu hay 9af7it el ehda2 w mn 7a22i 23mal elli bidi eyah :P I will say some words >> it's not being normal that's important , 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 ..