DNA-KRAMATİN VE KROMOZOM

... konulu sunumlar: "DNA-KRAMATİN VE KROMOZOM"— Sunum transkripti:

1 DNA-KRAMATİN VE KROMOZOM
Doç.Dr.Öztürk ÖZDEMİR 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 Chromatin Structure and Gene Expression by Elgin and Workman,
Oxford University Press. Horn and Peterson. Science 297:1824, 2002

7 KROMOZOM 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 Aº = 2nm Çift heliks zincir Şeker+Fosfat+baz+Hidrojen Bağları 2- Sekonder Nükleozom 100 Aº = 10nm bç DNA Histon oktomeri (2XH2A,H2B,H3, H4) 3- Tersiyer Selenoid Aº =30nm 6 adet nükleozom kb’lık Looplar 4- Quarterner Süpercolid Aº =60nm kb’lık Looplar  KROMOZOM : Pre ve Postmetafaz evrelerinde kromozom çapı nm arasında değişir.

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

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

11 How does a Chromosome replicate?
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 or

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 Average 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 : Seconder Further folding and shortening of length Selenoid : Tercier Very tightly packed METAPHASE chromosome : Quarterner How to squeeze 100 cm of DNA into one tiny cell !

16 Chromosomal Structure of the Genetic Material

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

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 2 M Hücre Nukleus 5 x 10-8 M

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

21 Superdönümler Çoğu 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 Nucleozomdur Histonlar H1, H2A, H2B, H3, H4

24 DNA daha da paketlenir

25                                                                                            Slide2 of 23                                   Genetics home page

26                                                                                            Slide3 of 36                                   Genetics home page

27                                                                                            Slide5 of 36                                   Genetics home page

28                                                                                            Slide13 of 36                                   Genetics home page

29                                                                                            Slide6 of 14                                   Genetics home page

30                                                                                            Slide8 of 14                                   Genetics home page

31                                                                                            Slide10 of 14                                   Genetics home page

32                                                                                            Slide11 of 14                                   Genetics home page

33                                                                                            Slide12 of 14                                   Genetics home page

34                                                                                            Slide13 of 14                                   Genetics home page

35                                                                                            Slide8 of 28                                   Genetics home page

36                                                                                            Slide9 of 28                                   Genetics home page

37                                                                                            Slide14 of 28                                   Genetics home page

38                                                                                            Slide14 of 14                     Genetics home page

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 direction 3. 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 alleles a g q Non-sister chromatid

41 What does a eukaryotic gene look like?
q Promoter -transcription starts here Protein-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 of sugar and phosphate Nitrogenous bases (purines and pyrimidines) inside

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

45 Memorize the bases Purines Pyrimidines Adenine Thymine
Guanine Cytosine Large Small weak bonding, lower M.W. strong bonding, higher M.W.

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

47 Building a DNA molecule
base 5 O 4 1 Deoxyribose sugar 3 2 phosphate The carbon atoms are numbered The bases attach at # 1 phosphate attaches at # 3 on one sugar and and # 5 on the next one

48 Building a DNA molecule
The gold structure represents the phosphate The bronze structure represents the bases 5 1 3 The silver structure represents the deoxyribose sugar O Deoxyribose sugar This 3-part structure is called a nucleotide

49 Building a DNA molecule
5 1 3 The silver structure represents the deoxyribose sugar The bronze structure represents the bases Without 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

51 Building a DNA molecule
Etc etc !

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

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’ direction 3’ 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 RNA Thymine Uracil 5 5 4 1 4 1 3 2 3 2 2-Deoxyribose Ribose

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
Isolation of a particular gene or sequence Production of large quantities of a gene product Protein or RNA Increased production efficiency for commercially made enzymes and drugs Modification/improvement of existing organisms 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:- 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 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 1. Obtain a DNA vector which can replicate inside a bacterial cell (plasmid or virus) which 2. Insert DNA into vector - use restriction enzyme 3. 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 cell A famous plasmid. The circular molecule in this electron micrograph is pSC101, the first plasmid used successfully to clone a vertebrate gene. Its name comes from the fact that it was the one-hundred-and-first plasmid isolated by Stanley Cohen. The DNA of interest must be inserted into the vector.

63 Restriction Enzymes Target or recognition sequence
Cuts here Restriction 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
AATT TTAA Plasmids are usually chosen to have only one target site. DNA of interest can then insert into this site Recombinant 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

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

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 2. 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 ? Enhancers Introns ? Poly-T (from poly-A tail)?

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