DNA Structure & Chromosomal Organisation PDF

12/23/2024 Week 7 DNA discovery – humble beginnings Nuclei were first purified from pus cells in the 1860’s DNA Structure & Chromosomal...

12/23/2024 Week 7 DNA discovery – humble beginnings Nuclei were first purified from pus cells in the 1860’s DNA Structure & Chromosomal Friedrich Miescher Organisation First organelle ever purified based on eBook chapter 7 DNA discovery – humble beginnings Chromosomes carry Genetic Information Purified the nuclei Early 1900’s – chromosomes carry the genetic information Chemical analysis revealed Hydrogen, Carbon, Oxygen, Chromosomes contain an acidic portion (DNA) and a basic Nitrogen and Phosphorous portion (protein) This same substance was found in all cells he examined - DNA – 4 different nucleotides Nuclein Proteins – 20 different amino acids Which was the important bit? DNA Protein 1  12/23/2024 Pneumonia answered the question Pneumonia answered the question Two strains of Streptococcus pneumoniae Frederick Griffith tried different experiments Strain S – capsule allowed infection Mice were injected with heat-killed S-strain Strain R – no capsule, no infection Mice injected with heat-killed S-strain Frederick Griffith injected mice proving infection Mice injected with live R strain Mice injected with live S strain Mice live. No live R-strain cells in Mice die. Live S-strain cells in their Mice live. No live S-strain cells in their blood their blood blood Pneumonia answered the question Transformation Mice injected with heat-killed S strain, plus live R-strain R-strain cells could now make capsules and infect Genetic information had passed from the dead S-strain Mice injected with heat-killed S-strain, plus live R-strain to the live R strain - Transformation Mice die. Live S-strain cells in their blood 2  12/23/2024 Nucleic Acid carries the Genetic Material Nucleic Acid carries the Genetic Material 1944 Avery, MacLeod & McCarty proved nucleic acids, Purified the components of S-strain cells not proteins were the genetic material Carbohydrates, fats, proteins and nucleic acids Again experimented with Pneumonia Each was mixed with live R-strain cells and injected into mice Live R-strain plus Carbohydrates Fats Proteins Nucleic Acid Live Live Live Died DNA not RNA carries the Genetic Material DNA carries the Genetic Information Purified nucleic acid was treated with either Rnase (destroys RNA) or Dnase (destroys DNA) DNA and RNA were added separately to the R-strain of S. pneumoniae Only the bacteria with the DNA added developed into S- strain DNA carries the genetic information DNA controls the synthesis of specific products 3  12/23/2024 Viruses to the rescue Bacteriophage Bacteriophages finally proved DNA’s importance – 1950’s They are viruses that infect bacteria Contractile Tail They are made up of only protein and DNA fibres Perfect for determining which carries the genetic information Hershey and Chase Experiments Hershey and Chase Experiments Used two radioactive compounds Bacterial Cell Radioactive protein Most 35S found in 35S capsule (35S) supernatant used to label protein coats 32P used to label DNA Infected two different cultures of bacteria and figured DNA out where the radioactivity was Labelled Phages infect Blender separates Phages Cells and Phages are bacteria outside the bacteria from separated by cells and their components centrifugation Bacterial Cell Protein capsule Most 32P found in pellet Radioactiv e DNA 32P 4  12/23/2024 Hershey and Chase Experiments Summary Nucleic Acid Structures DNA gets injected into bacteria, protein remains outside Two different types DNA directs synthesis of new viruses DNA (deoxyribonucleic acid) DNA carries the genetic information RNA (ribonucleic acid) Both are made up of nucleotide subunits Phosphate group Sugar (deoxyribose or ribose) Nitrogen-containing base Purines (A & G) or Pyrimidines (C & T/U) DNA RNA Nucleotide Components - Bases Nucleotide Components – Sugars and Phosphate Purine short name, big structure Double ring Sugars Adenine & Guanine Adenine (A) Thymine (T) Uracil (U) Purine (in DNA) (in RNA) Pyrimidine Pyrimidine Pyrimidine Deoxyribose Ribose long name, (in DNA) (in RNA) small structure Single ring Phosphate Cytosine, Thymine & Uracil Guanine (G) Cytosine (C) Purine Pyrimidine 5  12/23/2024 DNA vs RNA DNA Nucleotides Adenine (A) Thymine (T) Deoxyribose Phosphate Ribose sugar group sugar Thymine base Uracil base Sugar (deoxyribose) Guanine (G) Cytosine (C) Double- Single- stranded stranded Nucleotides join to form Polynucleotides Chargaff’s rules of DNA composition Edwin Chargaff examined the DNA of numerous species A Nucleotides are joined to form a in the 1940’s and discovered: chain 1. The number of Adenine residues always equals the Polynucleotides have a direction G number of Thymine residues A=T 5’ end - phosphate 2. The number of Cytosine residues always equals 3’ end – sugar C the number of Guanine residues This sequence is C=G T 3. The number of Purines always equals the number 5’-AGCT-3’ of Pyrimidines (A+G) = (C+T) 6  12/23/2024 Rosalind Franklin Watson and Crick Used X-ray crystallography to study the physical In 1953 described the model for DNA structure of DNA Used information from research available at the time Concluded: Chagraff’s chemical nature of DNA DNA is a helix Franklin’s physical structure of DNA Phosphates are on the outside Built models out of cardboard and wire Constant diameter Eventually made one to satisfy all the rules Watson and Crick DNA is a double-stranded helix DNA has two chains Sugars and Phosphates on the outside Chains are antiparallel Chains coil to form a helix Bases are paired across the chains via hydrogen bonds Pairing is specific A-T, C-G Watson and Crick and Strands are complementary their wire model 7  12/23/2024 DNA strands are antiparallel Watson & Crick’s Model Genetic information is stored in the sequence of bases in the DNA The model offers a molecular explanation for mutation Reads The complementary sequence of bases explains how DNA is copied before each cell division Reads DNA allows complexity An explanation for mutation At any point in the sequence there can be any one of 4 Because DNA is a linear string of bases, any change in bases - A, C, G or T the order or number of bases can lead to an altered For a string of 3 nucleotides there are 64 (43) possible phenotype combinations (AAA, AAC, AAG, AAT, ACA, ACC…) For a string of n nucleotides there are 4n 10 nucleotides = 410 = 1,048,576 possibilities Human genome ~3.2 billion bases! 8  12/23/2024 But what is RNA? DNA vs RNA A nucleic acid Single stranded A, C, G and U (Uracil) Ribose instead of deoxyribose Transfers genetic information from the nucleus to the cytoplasm The message for protein synthesis Also a component of ribosomes Three main types of RNA Three main types of RNA Messenger RNA (mRNA) Carries information from the DNA to the cytoplasm for protein synthesis Transfer RNA (tRNA) Transports amino acids to the ribosome Ribosomal RNA (rRNA) Combines with proteins to make the ribosome 9  12/23/2024 DNA’s Semi-conservative Replication DNA Replication – Semi-conservative One old strand is conserved in each new DNA molecule One old strand and one Semi-conservative and one new one synthesized new strand in each daughter DNA molecule Each complementary strand of the DNA molecule is used as a template Conservative Two DNA molecules Proceeds in a 5’  3’ direction generated, but one entirely Occurs during the S phase of the cell cycle new and one entirely old Dispersive Two DNA molecules generated, bits of old and bits of new in each DNA Replication DNA Replication – 4 Major Enzymes Begins at origins of replication Hydrogen bonds are broken by helicase Primase forms a starting point DNA Polymerase III “reads” the sequence and joins on complementary bases DNA Polymerase III also does checks New DNA is made continuously on one strand, but in Okazaki fragments on the other DNA ligase fills in the gaps Proteins wind the strands into a helix 10  12/23/2024 Helicase Primase Finds the origin of replication Creates a short RNA primer to act as starting point for Unzips the DNA by breaking hydrogen bonds, creating two DNA replication single strands that can be used as templates Provides a free OH group for DNA Polymerase III to add bases to Primase DNA Polymerase III DNA replication – Leading and Lagging DNA polymerase III synthesizes the new strand 5’-3’ Two new strands are formed It binds to the 3’ end of the RNA primer, reads the base on One is continuous – leading strand the single strand and pulls a complementary base towards One is formed from short fragments known as Okazaki it, joining it with a hydrogen bond fragments – lagging strand Uses excess phosphates to provide the energy required 11  12/23/2024 Okazaki Fragments DNA Ligase DNA Polymerase I converts RNA primers to DNA DNA Ligase joins the Okazaki fragments together to produce one long strand DNA Polymerase III Quality Control Bi-directional DNA replication DNA Polymerase III also checks and corrects errors as it Two replication forks are created from each origin of goes replication DNA synthesis occurs in both directions There is a leading strand and a lagging strand in both directions Replication Replication fork fork 12  12/23/2024 The Organisation of DNA into Chromosomes The Organisation of DNA into Chromosomes Over 2m of DNA in the nucleus Nucleosomes condense DNA 6-7 times Each chromosome consists of highly coiled double- stranded DNA DNA winds around histones to form nucleosomes 8 histone molecules join to form the histone core But DNA is compacted 5,000-10,000 times during replication! Compaction of DNA into Chromosomes Centromeres and Telomeres Centromere Region where sister chromatids attach Several models have “nucleosome” Telomere been proposed Capping on the ends of chromosome arms Chromatin (DNA + proteins) folds into loops that attach to a protein scaffold 13  12/23/2024 Role of Centromeres Role of Telomeres Different location on each chromosome TTAGGG repeated hundreds of times Kinetochore forms at the centromere during cell division - Provide a structure to maintain DNA stability important for attachment of spindle fibres Not every repeat gets copied during replication Contain long stretches of repetitive DNA Chromosomes get progressively shorter Rate limiting step in determining the number of times a cell can divide 50-60 divisions Role of Telomeres Nuclear Organisation Telomerase protects telomeres during meiosis Each chromosomes has a designated region in the Binds to the end of the DNA and provides a short stretch nucleus – chromosome territory of dsDNA Organisation is linked with function Acts as a starting point for DNA Polymerase III to add Flexible more DNA 14