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DNA-KRAMATİN VE KROMOZOM Doç.Dr.Öztürk ÖZDEMİR Doç.Dr.Öztürk ÖZDEMİR Aralık 05 Sivas Aralık 05 Sivas.

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1 DNA-KRAMATİN VE KROMOZOM Doç.Dr.Öztürk ÖZDEMİR Doç.Dr.Öztürk ÖZDEMİR Aralık 05 Sivas Aralık 05 Sivas

2 What is a chromosome? Made up of CHROMATIN:- 1/3 DNA 1/3 HISTONE PROTEINS 1/3 OTHER PROTEINS

3 Proliferation signals

4

5 ATP-Dependent Chromatin Remodeling Complexes

6 Horn and Peterson. Science 297:1824, 2002 Chromatin Structure and Gene Expression by Elgin and Workman, Oxford University Press.

7 KROMOZOM Genomik DNA’nın türe özgü sayı ve morfolojide paketlenme şekline denir. Genomik 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 Birimleri 1- Primer DNA 20 Aº = 2nm Çift heliks zincir Şeker+Fosfat+baz+Hidrojen Bağları 2- Sekonder Nükleozom 100 Aº = 10nm 146 bç DNA Histon oktomeri (2XH 2 A,H 2 B,H 3, H 4 ) Histon oktomeri (2XH 2 A,H 2 B,H 3, H 4 ) 3- Tersiyer Selenoid 300 Aº =30nm 6 adet nükleozom kb’lık Looplar 4- Quarterner Süpercolid 600 Aº =60nm kb’lık Looplar  KROMOZOM : Pre ve Postmetafaz evrelerinde kromozom çapı Pre ve Postmetafaz evrelerinde kromozom çapı nm arasında değişir nm arasında değişir.

9 KROMOZOM DNA+HİSTON PROTEİNLERİ (bazik)  DNP  NÜKLEOZOM  SELENOİDYAPI  SÜPERCOİLD YAPI  KROMATİN  DNA+HİSTON PROTEİNLERİ (bazik)  DNP  NÜKLEOZOM  SELENOİDYAPI  SÜPERCOİLD YAPI  KROMATİN  KROMOZOM KROMOZOM

10 KROMOZOM 21 Tandem repeats (ardıl tekrarlar) %1.3 Tandem repeats (ardıl tekrarlar) %1.3 Exon %2.8 Exon %2.8 Repeats (tekrarlar) %38.1 Repeats (tekrarlar) %38.1 Diğer (Junk,konstitütif heterokromatin,sentromer) %57.8 Diğer (Junk,konstitütif heterokromatin,sentromer) %57.8

11 How does a Chromosome replicate? or 1. PROKARYOTES (bacteria) Circular chromosome Just one origin Two replication forks move round circle till all is replicated No mitosis - just a pulling apart of the 2 circles into 2 daughter cells

12 How does a Chromosome replicate? 2. EUKARYOTES several long, linear chromosomes hundreds of origins per chromosome each 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 molecule One long DNA molecule Average about 4 cm long ! Average about 4 cm long ! >1.5m in 46 chromosomes in each nucleus which is only 0.006mm diam. ! >1.5m in 46 chromosomes in each nucleus which is only 0.006mm diam. ! You have cells, so DNA in your body could stretch to sun and back 100 times! You have cells, so DNA in your body could stretch to sun and back 100 times! Over 10 8 bases in each DNA molecule Over 10 8 bases in each DNA molecule

14 Chromosome - Condensation/ Elongation Cycle Chromosome - Condensation/ Elongation Cycle INTERPHASE. INTERPHASE. Chromosomes extremely long, thin and not visible (unstainable). Chromosomes extremely long, thin and not visible (unstainable). GENES can be actively expressed (transcribed). GENES can be actively expressed (transcribed). INTERPHASE. INTERPHASE. Chromosomes extremely long, thin and not visible (unstainable). Chromosomes extremely long, thin and not visible (unstainable). GENES can be actively expressed (transcribed). GENES can be actively expressed (transcribed). MITOSIS. MITOSIS. Chromosomes are much shorter (40,000 x) and so thicker. Visible (stainable) GENES not able to be transcribed MITOSIS. MITOSIS. Chromosomes are much shorter (40,000 x) and so thicker. Visible (stainable) GENES not able to be transcribed

15 DNA :Primer Wrapped round beads of histone protein =nucleosomes : Seconder Very tightly packed METAPHASE chromosome : Quarterner DNA PACKING How to squeeze 100 cm of DNA into one tiny cell ! Further folding and shortening of length Selenoid : Tercier

16 Chromosomal Structure of the Genetic Material

17 Doğal DNA B-DNAdır. İki iplik sağ dönümlüdür. İki iplik sağ dönümlüdür. ~20Å çap ~20Å çap Bazların bir dönümü: 10 (~34Å) Bazların bir dönümü: 10 (~34Å) major ve minör girintiler oluşur major ve minör girintiler oluşur 20Å Major Minör

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 uzunluktadır DNA tek bir insan hücresinde açılsa ~2 uzunluktadır Hücre Nukleus 5 x M DNA 2 M

20 Paketlenme mekanizması DNA Replikasyonu ve Transkripsiyonu için önemlidir DNA Replikasyonu ve Transkripsiyonu için önemlidir Superdönümler Superdönümler Kromatini oluşturmak üzere proteinler etrafındaki dönümler Kromatini oluşturmak üzere proteinler etrafındaki dönümler DNA paketlenmesinde görev alan enzimler Topoisomerazlardır DNA paketlenmesinde görev alan enzimler Topoisomerazlardır

21 Superdönümler Çoğu DNA negatif süper dönümlüdür. Çoğu DNA negatif süper dönümlüdür. Daha superdönüm

22 Topoisomerazlar Topoisomerazlar Moleküler makastırlar Topoisomerazlar Moleküler makastırlar DNA’da bir kesim yaparak ikinci ipliğin aradan geçişini sağlarlar DNA’da bir kesim yaparak ikinci ipliğin aradan geçişini sağlarlar

23 DNA Histon proteinleri etrafında döner Bu yapı Nucleozomdur Bu yapı Nucleozomdur Histonlar H1, H2A, H2B, H3, H4 Histonlar H1, H2A, H2B, H3, H4

24 DNA daha da paketlenir

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39 Chromosomes and Genes 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) AgQNon-coding DNA gene 3. Many genes interrupted by non-coding sequences 2. Genes aren’t all read in same direction

40 Chromosomes and Genes AgQ 3. Sister chromatids MUST be identical - made by COPYING. Non-sisters can have different alleles AgQ Sister chromatids agq Non-sister chromatid

41 What does a eukaryotic gene look like? Protein-coding regions (Exons) Termination of transcription Promoter - transcription starts here agq

42 Introns (non-coding regions) Spacer What does a eukaryotic gene look like?

43 Features of Watson and Crick’s DNA model Outer backbone made of sugar and phosphate sugar and phosphate Nitrogenous bases (purines and pyrimidines) inside

44 Features of Watson and Crick’s DNA model 10 bases per turn Constant width Purine faces pyrimidineandvice-versa

45 PurinesPyrimidines AdenineThymine GuanineCytosine LargeSmall Memorize the bases weak bonding, lower M.W. strong bonding, higher M.W.

46 DNA contains:- DNA contains:- 1. the sugar deoxyribose (above) 1. the sugar deoxyribose (above) 2. phosphates 2. phosphates 3. Purine and pyrimidine bases 3. Purine and pyrimidine bases OH H H H H H O Oxygen atom Carbon atom How do these fit together to make a DNA molecule ? Building a DNA molecule

47 The carbon atoms are numbered The carbon atoms are numbered O base The bases attach at # 1 phosphate attaches at # 3 on one sugar and and # 5 on the next one phosphate Deoxyribose sugar Building a DNA molecule

48 O Deoxyribose sugar The gold structure represents the phosphate The silver structure represents the deoxyribose sugar The bronze structure represents the bases This 3-part structure is called a nucleotide Building a DNA molecule

49 5 1 3 The silver structure represents the deoxyribose sugar The bronze structure represents the bases Without the phosphate it would be a nucleoside Building a DNA molecule

50 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 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 5’ 3’ 5’ Building a DNA molecule

51 Etc etc ! Building a DNA molecule

52 5’ 3’ As a result they have different structures at each end of the chain As a result they have different structures at each end of the chain Termed 3’ ends and 5’ ends Termed 3’ ends and 5’ ends Building a DNA molecule

53 Any linear DNA molecule, no matter how long will always have 3’ ends and 5’ ends Any linear DNA molecule, no matter how long will always have 3’ ends and 5’ ends 5’ 3’ Building a DNA molecule

54 Growth of a chain is always at the 3’ end - never the 5’ end. 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. 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’ direction We say that growth is in the 5’ to 3’ direction 3’ Building a DNA molecule 3’

55 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) 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. Note the 3’ ends and 5’ ends of each chain are at opposite ends. 5’ 3’ 5’ Building a DNA molecule

56 DNA vs RNA DNA vs RNA 2-Deoxyribose Ribose Thymine Uracil

57 The Tools of Molecular Biology

58 Outline/Readings Outline: DNA cloning, PCR, DNA Sequencing, Applications of DNA Technology Background Readings: Chap 20 Assigned Readings: Key Concepts, Self quiz p 400

59 Goals of DNA Technology 1.Isolation of a particular gene or sequence 2.Production of large quantities of a gene product 1.Protein or RNA 3.Increased production efficiency for commercially made enzymes and drugs 4.Modification/improvement of existing organisms 5.Correction of genetic defects

60 Amplifying DNA Often we need large quantities of a particular DNA molecule or fragment for analysis. Two ways to do this:- Often 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 Cloning 2.1. Obtain a DNA vector which can replicate inside a bacterial cell (plasmid or virus) which 3.2. Insert DNA into vector - use restriction enzyme 3.3. Transform host cells i.e. insert vector into host cell (e.g. bacterium) 4.4. Clone host cells (along with desired DNA ) 5.5. Identify clones carrying DNA of interest 1.Purpose:- 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 DNA

62 Vectors are convenient carriers of DNA. They are often viruses or plasmids 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 cell The DNA of interest must be inserted into the vector.

63 Target or recognition sequence 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. Cuts here Restriction enzymes (R.E.) recognise target sequences and cut DNA in a specific manner. Restriction Enzymes

64 Sticky ends will readily rejoin - so its possible to join 2 DNA’s from different sources Plasmids are usually chosen to have only one target site. DNA of interest can then insert into this site Recombinant plasmid AATT TTAA

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 are made to take up the recombinant plasmid & grown (cloned) in large numbers (TRANSFORMATION) Bacteria carrying desired sequence can be selected. Bacteria carrying desired sequence can be selected. Large amounts of DNA or proteins can be extracted Large amounts of DNA or proteins can be extracted

66 work with genework with protein

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. 2. Insert each of these into separate plasmids and then into separate host cells. 3. Result - a collection of bacterial colonies (clones) carrying all the foreign DNA fragments i.e. a genomic library

68 Making a cDNA Library cDNA library = a collection of DNA fragments representing the active genes in a tissue. 1. Extract all mRNA molecules from a tissue 3. Insert each of these DNA mols. into individual plasmids and then into individual host cells 3. Result - a collection of bacterial colonies (clones) carrying cDNA fragments representing a cell’s active genes = a cDNA library (= copy DNA) 2. Use enzyme ‘reverse transcriptase’ to make a DNA copy of these mRNA’s ( = cDNA)

69 A question for you - how will a cDNA library differ from a genomic library ? Which would have more genes ? Which would have more genes ? What would be present in the clones in each case? What would be present in the clones in each case? Promoters ? Promoters ? Enhancers Enhancers Introns ? Introns ? Poly-T (from poly-A tail)? Poly-T (from poly-A tail)?

70 How do we identify DNA mols. of different sizes ? 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. Gel Electrophoresis Standards of known M.W. long DNA short DNA 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 temperatures A DNA polymerase which is stable at high temperatures specific primers to start off replication at known position. specific primers to start off replication at known position. Three step cycle: Three step cycle: 1. Heat to separate DNA strands = Denaturation 2. Cool and allow primers to bind (Annealing) 3. Polymerize new DNA strands (Extension) Repeat steps 25 – 35 times >>> millions of copies of original DNA


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