6 Chromatin Structure and Gene Expression by Elgin and Workman, Oxford University Press.Horn and Peterson. Science 297:1824, 2002
7 KROMOZOMGenomik DNA’nın türe özgü sayı ve morfolojide paketlenme şekline denir.- Ökaryotik hücrelere özgüdür- Kompleks DNA’nın bölünme esnasında yavru hücrelere eşit ve mutasyonsuz pay edilmesi esasına dayanır.- En ideal formasyon hücrenin metafaz evresinde olur- DNA-gen paketlenmesi dışında “gen aktivitesi” bakımından negatif aksiyona sahip. Bu formda DNA replike -transkribe olamaz ve gen ekspressiyonu yoktur.
8 KROMOZOM -DNA PAKETLENMESİ Kademe Birim Adı Büyüklüğü Alt Birimleri1- Primer DNA Aº = 2nm Çift heliks zincir Şeker+Fosfat+baz+Hidrojen Bağları2- Sekonder Nükleozom 100 Aº = 10nm bç DNAHiston oktomeri (2XH2A,H2B,H3, H4)3- Tersiyer Selenoid Aº =30nm 6 adet nükleozomkb’lık Looplar4- Quarterner Süpercolid Aº =60nm kb’lık LooplarKROMOZOM :Pre ve Postmetafaz evrelerinde kromozom çapınm arasında değişir.
11 How does a Chromosome replicate? 1. PROKARYOTES (bacteria)Circular chromosomeJust one originTwo replication forks move round circle till all is replicatedNo mitosis - just a pulling apart of the 2 circles into 2 daughter cellsor
12 How does a Chromosome replicate? 2. EUKARYOTESseveral long, linear chromosomeshundreds of origins per chromosomeeach origin replicated bidirectionally - forming a series of replication ‘bubbles’takes place in S period of mitotic cell cycle
13 Chromosome facts (Humans) One long DNA moleculeAverage about 4 cm long !>1.5m in 46 chromosomes in each nucleus which is only 0.006mm diam. !You have 1013 cells, so DNA in your body could stretch to sun and back 100 times!Over 108 bases in each DNA molecule
14 Chromosome - Condensation/ Elongation Cycle INTERPHASE.Chromosomes extremely long, thin and not visible (unstainable).GENES can be actively expressed (transcribed).MITOSIS.Chromosomes are much shorter (40,000 x) and so thicker. Visible (stainable)GENES not able to be transcribed
15 DNA PACKING DNA :Primer Wrapped round beads of histone protein =nucleosomes: SeconderFurther folding and shortening of lengthSelenoid : TercierVery tightly packed METAPHASE chromosome : QuarternerHow to squeeze 100 cm of DNA into one tiny cell !
17 Doğal DNA B-DNAdır. Major Minör 20Å İki iplik sağ dönümlüdür. ~20Å çap Bazların bir dönümü: 10 (~34Å)major ve minör girintiler oluşurMajorMinör20Å
18 DNA yapısının çözülmesi: dört ana bilim insanı DNA yapısının çözülmesi moleküler biyoloji ve genomiks bilimlerinin gelişmesini sağladı.Watson, Crick and Wilkins “Nobel Prize in Physiology and Medicine, 1962” kazandılar.Rosalind Franklin 1958 kanserden öldüğünden ödül alamadı.
19 DNA hücre içinde paketlenmiş halde bulunur DNA tek bir insan hücresinde açılsa ~2 uzunluktadırDNA2 MHücre Nukleus5 x 10-8 M
20 Paketlenme mekanizması DNA Replikasyonu ve Transkripsiyonu için önemlidirSuperdönümlerKromatini oluşturmak üzere proteinler etrafındaki dönümlerDNA paketlenmesinde görev alan enzimler Topoisomerazlardır
21 SuperdönümlerÇoğu DNA negatif süper dönümlüdür.Daha superdönüm
22 Topoisomerazlar Topoisomerazlar Moleküler makastırlar DNA’da bir kesim yaparak ikinci ipliğin aradan geçişini sağlarlar
23 DNA Histon proteinleri etrafında döner Bu yapı NucleozomdurHistonlar H1, H2A, H2B, H3, H4
39 Chromosomes and Genes A g Q Non-coding DNA gene 1. Coded material (Genes) only accounts for a small amount of the DNA in a chromosome - in fact < 5% of DNA ( HUMAN GENOME PROJECT -only 31,000 genes in human genome)2. Genes aren’t all read in same direction3. Many genes interrupted by non-coding sequences
40 Chromosomes and Genes A g Q Sister chromatids A g Q 3. Sister chromatids MUST be identical - made by COPYING.Non-sisters can have different allelesagqNon-sister chromatid
41 What does a eukaryotic gene look like? qPromoter -transcriptionstarts hereProtein-coding regions(Exons)Termination of transcription
42 What does a eukaryotic gene look like? Introns (non-coding regions)Spacer
43 Features of Watson and Crick’s DNA model Outer backbone made ofsugar and phosphateNitrogenous bases(purines and pyrimidines) inside
44 Features of Watson and Crick’s DNA model Constant width10 bases per turnPurine facespyrimidineandvice-versa
46 Building a DNA molecule OHHOOxygen atomCarbon atomDNA contains:-1. the sugar deoxyribose (above)2. phosphates3. Purine and pyrimidine basesHow do these fit together to make a DNA molecule ?
47 Building a DNA molecule base5O41Deoxyribose sugar32phosphateThe carbon atoms are numberedThe bases attach at # 1phosphate attaches at # 3 on one sugar and and # 5 on the next one
48 Building a DNA molecule The gold structure represents the phosphateThe bronze structure represents the bases513The silver structure represents the deoxyribose sugarODeoxyribose sugarThis 3-part structure is called a nucleotide
49 Building a DNA molecule 513The silver structure represents the deoxyribose sugarThe bronze structure represents the basesWithout the phosphate it would be a nucleoside
50 Building a DNA molecule 5’3’3’5’Nucleotides are joined together into long chains by bonds connecting the 3’ atom of one sugar via a phosphate to the 5’ sugar of the next
52 Building a DNA molecule 5’3’As a result they have different structures at each end of the chainTermed 3’ ends and 5’ ends
53 Building a DNA molecule 5’Any linear DNA molecule, no matter how long will always have 3’ ends and 5’ ends3’
54 Building a DNA molecule Growth of a chain is always at the 3’ end - never the 5’ end.DNA polymerases are the enzymes which add nucleotides one at a time to the 3’ end.We say that growth is in the 5’ to 3’ direction3’3’3’
55 Building a DNA molecule 3’5’5’3’DNA is normally in double helical form. It then consists of two chains of nucleotides paired together in OPPOSITE orientations (i.e one is ‘upside-down’ with respect to the other)Note the 3’ ends and 5’ ends of each chain are at opposite ends.
56 DNA vs RNAThymineUracil55414132322-DeoxyriboseRibose
58 Outline/ReadingsOutline: DNA cloning, PCR, DNA Sequencing, Applications of DNA TechnologyBackground Readings: Chap 20Assigned Readings: Key Concepts, Self quiz p 400
59 Goals of DNA Technology Isolation of a particular gene or sequenceProduction of large quantities of a gene productProtein or RNAIncreased production efficiency for commercially made enzymes and drugsModification/improvement of existing organismsCorrection of genetic defects
60 Amplifying DNAOften we need large quantities of a particular DNA molecule or fragment for analysis. Two ways to do this:-1. Insert DNA mol. in a plasmid and let it replicate in host >>> many identical copies (= ‘DNA cloning’)2. Use PCR technique - automated multiple rounds of replication >>> many identical copies.
61 DNA CloningPurpose:- to amplify (bulk up) a small amount of DNA by inserting it into in a fast growing cell e.g. bacterium, so as bacterium divides we will have many copies of our DNA1. Obtain a DNA vector which can replicate inside a bacterial cell (plasmid or virus) which2. Insert DNA into vector - use restriction enzyme3. Transform host cells i.e. insert vector into host cell (e.g. bacterium)4. Clone host cells (along with desired DNA)5. Identify clones carrying DNA of interest
62 The DNA of interest must be inserted into the vector. Vectors are convenient carriers of DNA. They are often viruses or plasmids.Usually are small circular DNA molecules and must be capable of replicating in the host cellA famous plasmid. The circular moleculein this electron micrograph is pSC101,the first plasmid used successfully to clonea vertebrate gene. Its name comes fromthe fact that it was theone-hundred-and-first plasmid isolated byStanley Cohen.The DNA of interest must be inserted into the vector.
63 Restriction Enzymes Target or recognition sequence Cuts hereRestriction enzymes (R.E.) recognise target sequences and cut DNA in a specific manner.This R.E. leaves TTAA single stranded ends (‘sticky ends’)If you cut DNA of interest and plasmid with same restriction enzyme then you will have fragments with identical sticky ends.
64 Sticky ends will readily rejoin - so its possible to join 2 DNA’s from different sources AATTTTAAPlasmids are usually chosen to have only one target site. DNA of interest can then insert into this siteRecombinant plasmid
65 Transformation of host and selection of desired clones Bacteria are made to take up the recombinant plasmid & grown (cloned) in large numbers (TRANSFORMATION)Bacteria carrying desired sequence can be selected.Large amounts of DNA or proteins can be extracted
67 Making a Genomic Library Genomic library = a complete collection of DNA fragments representing an organism’s entire genome.1. Cut up genome into thousands of fragments with an R.E.3. Result - a collection of bacterial colonies (clones) carrying all the foreign DNA fragments i.e. a genomic library2. Insert each of these into separate plasmids and then into separate host cells.
68 Making a cDNA LibrarycDNA library = a collection of DNA fragments representing the active genes in a tissue.1. Extract all mRNA molecules from a tissue2. Use enzyme ‘reverse transcriptase’ to make a DNA copy of these mRNA’s ( = cDNA)3. Result - a collection of bacterial colonies (clones) carrying cDNA fragments representing a cell’s active genes = a cDNA library (= copy DNA)3. Insert each of these DNA mols. into individual plasmids and then into individual host cells
69 A question for you - how will a cDNA library differ from a genomic library ? Which would have more genes ?What would be present in the clones in each case?Promoters ?EnhancersIntrons ?Poly-T (from poly-A tail)?
70 How do we identify DNA mols. of different sizes ? long DNAshort DNAGel ElectrophoresisStandards of known M.W.Run DNA fragments through a gel under influence of an electric current. Each of the DNA fragments travels through the gel at a constant speed appropriate for its size.Longer molecules move more slowly so don’t travel as far.See Fig 20.8
71 Polymerase Chain Reaction (PCR) Small amount of DNA can be amplified greatly - automated process involves:-A DNA polymerase which is stable at high temperaturesspecific primers to start off replication at known position.Three step cycle:Heat to separate DNA strands = DenaturationCool and allow primers to bind (Annealing)Polymerize new DNA strands (Extension)Repeat steps 25 – 35 times >>> millions of copies of original DNA
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