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MOLECULAR BIOLOGY 2003-4Michal Linial

Topic A1 - Hybridization, COT (1st wk)

Topic A2 - Genes & Genomes (2nd wk)

Topic B1 - Analyzing N.A. & ProteinsTopic B2 - Recombinant techniques, lambdaTopic B3 - Large scale analyses

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Nucleic Acids (principles in brief)

CoT, hybridization ..Genome size & moreChapter 4 (MBC, edition 4)Chapter 2 & 3 (Genes VII)

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4.1 Nucleic acids (in brief)

� DNA contains the information prescribing the amino acidsequence of proteins

� This information is arranged in units termed genes (Mendel, Morgan)

� RNA serves in the cellular machinery that chooses and linksamino acids in the correct sequence

� The (basic) central dogma: DNA � RNA � Protein

� DNA and RNA are linear polymers of nucleotide subunits

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In the cell

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4.1 Native DNA is a double helix ofcomplementary antiparallel chains

Figure 4-4

Hydrogen bondingbetween complementarybase pairs (A-T or G-C)holds the twostrands together

Right-handedB-form

From the polymer to3D conformation

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4.1 DNA and complementary base pairs Rules

A-T (2 h-h) G-C (3 h-h)

But other combinations are possible(G-T or C-T or C-U)

Why not ?

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4.1 Nucleic acids General view

� Deoxyribonucleic acid (DNA) is very long (>106 -10 8 nt)Plant 30,000 genes 1011

Fly 12,000 108

Bact. 4,000 107

dsDNA virus 300 105 Vaccinia

ssRNA virus 10 104 SARS

Ribonucleic acid (RNA) is short-medium (70 - 4000) ? most RNA are defined in size (t-RNA, rRNA)

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4.1 DNA can undergo reversible strandseparation

Figure 4-8

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4.1 DNA strand separation (denaturation)when ? how?

Figure 4-9

� Natural

Making RNA from DNAUnwinding by proteinsUnwinding by DNA topology

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�Experimental HeatingLow ionic strength Disruption hydrogen bonds Urea, formamide, Alkaline PH

NH2-C -NH2

0||

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4.1 Analysis of DNA denaturation

Figure 4-9

Change in light absorption UV - 260nm

Tm

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4.1 N.A. denaturation - renaturationSome more

Denaturation -

DNA circular ?

Circular DNA & denaturation - a problem, why ?

Renaturation - only by complementary, time dependent

no competition if non related seq.

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DNA contentDNA sizeDNA complexity

DNA dynamics and characteristics

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DNA content

C-valueTotal amount of DNAin (haploid) genome

C-paradox

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Genome sizeMinimal genome

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Genome size

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Genome sizeGene number

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Genome size, Gene number

Larger genome - more genes.

Same gene appear many times?? Different?

What is the complexity of a genome???Kinetics studies

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4.1 DNA can undergo reversible strandseparation

Figure 4-8

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Cot value

Co -initial concentration single strand at t=0C -Concentration of single strand at time=t

50% of the reaction completed Cot 1/2

Cot 1/2 = 1/k (k= reassociation rate constant)

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Rate ofassociation& DNAlength

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Rate ofassociation& DNAlength

Cot 1/2 is directly related to the amountof DNA in the genome

A fixed amount of DNA, 12pg (Co) = 3000 E. colibut only 4 eukaryotic genomes

X 750 fold in DNA concentration for specific sequence

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Rate ofassociation& DNAlength

Cot 1/2 indicates the total length (bp) of different sequences = complexity

Cot 1/2 of a genome complexity of a genomeCot 1/2 of E. coli 4.2 x106 bp

=

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Rate of association& DNA complexity

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Rate of association& DNA complexity

The slow componentis a non repetitive DNA

The fast componentis a repetitive DNA

moderate

high

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9.2 Simple sequence DNA(fast association)

Figure 9-6

Short tandem repeats: 5-10 nt X n

Long tandem repeats: 20-200 nt X n

Satellite - in specific sites in chromosomesnear centromers, telomers

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9.2 Simple-sequence DNAs areconcentrated in specific chromosomal

locations

Figure 9-6

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9.2 Tandemly repeated genes of rRNA,tRNA, Histones

Figure 9-6

Almost identical - head to tail, many repeats

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Embryo -24 hr doubling time , with 5-10 million ribosomes !!Need >100 copies, fully active

Frog 20,000 of 5S RNA !!

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9.2 Reassociation experiments revealthree major fractions of eukaryotic DNA

• Single copy DNA (50-60%)• Moderately-repeated/intermediate-repeat DNA (25-40%)

Includes mobile DNA elements• Simple-sequence DNA (10-15%)

• Single protein product - Solitary (i.e., lysosome)

• Duplicated protein-coding genes

• Gene family: immunoglobulin, kinases (hundreds copies)

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Unique vs repeated

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Structural genes arein the non-repetitive DNA

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To Genomes , Genes ,

From chapters 2 & 3 GENES VII

What is a gene?

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Chapter 9

Molecular Structure of GenesNon coding regionsGene families

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9.1 Molecular definition of a gene

� A gene is the entire nucleic acid sequence that isnecessary for the synthesis of a functional polypeptide

� DNA regions that code for RNA molecules such as tRNAand rRNA may also be considered genes

� In eukaryotes, genes lie amidst a large expanse ofnonfunctional, noncoding DNA and genes may alsocontain regions of noncoding DNA

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Gene organization, transcription, andtranslation in prokaryotes

Figure 4-17a

Functional groupOPERON

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4.3 Gene organization, transcription, andtranslation in eukaryotes

Figure 4-17b

Another level, mostgenes have exon-intron organization

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The

feat

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of a

gen

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d a

mat

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tran

scri

pt

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9.1 Simple and complex transcription units arefound in eukaryotic genomes

Figure 9-2

Simple transcription unitComplex transcription unit

poly A different sites exon skipping

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9.1 Most eukaryotic mRNAs are monocistronic andcontain introns

Figure 9-1

Extreme examples:

DMD (Duche Muscular Dystrophy) gene >2,000,000 bp (bigger than H. influenza)

Fibronectin (an ECM protein) many repeat of 3 different exons

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Gene variations in higher Eukaryotes

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Intron size

Figure 9-1

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Exon size

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Some DNA code for more than one gene

startframe

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Protein family

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Pseudogenes

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Globinfamily

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Globin gene evolution

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Globin cluster‘ tree ’

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Globin &disease

βα

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ADDITIONAL MATERIAL

FOR YOUR OWN FUN…

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4.1 There are five principal bases innucleic acids

Figure 4-2

fused ring

A, G, T, C are present in DNAA, G, U, C are present in RNA

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DNA - few characteristic shapes

RNA - many characteristic shapes RNA folds:Hairpin Stem-loop Pseudo-knots

This difference is critical for the function..

4.1 From a linear polymer to 3Dconformation in nucleic acids

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mRNA Cap

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Introns and ORFs

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Exon size conservation

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Open questions

The minimal set for ‘real life’ ?

Fly - only 2.5 fold the bacterial genes

Human - How many ???

Multi-cellular organism only x2 of single cell

Plant -Arabidopsis (2001) >27,000 genes

The role of gene duplication for specialization

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Gene families

Duplication

Specialization

Divergence

Paralogs - within the same organism, optimized function

Ortologs - conservation in different species, taxa

Examples: tubulin, actin, intermediat filaments…