UNIT 1:GENETICS:NUCLEIC ACIDDNA(Campbell & Reece:Chapters, 5, 16)
LOCATION OF DNA INA CELL• Chromatin is a complex of DNA andprotein, and is found in the nucleus ofeukaryotic cells.• Histones are proteins that areresponsible for the first level of DNApacking in chromatin• The chromatin network in the nucleus ofa cell will coil up tightly during celldivision and form individualchromosomes.
• Chromosomes are always duplicatedduring this process (2 sets of identicalgenetic information to ensure each cellreceives identical genetic info to theparent cell during cell division).• A duplicated chromosome consists of 2chromatids attached to each other by acentromere.• Each chromatid consists of several genes.• Genes consists of a long DNA strand.• A string of DNA coiled around a fewhistones is called a nucleosome.
LOCATION OF DNA INA CELLLocus: Position of gene on chromosome
2. DNA STRUCTURE• DNA molecules are polymers calledpolynucleotides.• Each polynucleotide is made ofmonomers called nucleotides.• Each nucleotide consists of :• a nitrogenous base (Adenine,Thymine, Cytosine or Guanine)• a pentose sugar (DNA =Deoxyribose sugar),• and a phosphate group.
• Nucleotide monomers are linkedtogether to build a polynucleotide.• Adjacent nucleotides are joined bycovalent bonds that form between the –OH group on the 3’ carbon of onenucleotide and the phosphate on the 5’carbon on the next nucleotide.• These links create a backbone of sugar-phosphate units with nitrogenous basesas appendages.• The sequence of bases along a DNApolymer is unique for each gene.
The different nitrogenous bases in DNASingle ring structureDouble ring structure
• A DNA molecule has two polynucleo-tides spiralling around an imaginary axis,forming a double helix.• In the DNA double helix, the twobackbones run in opposite 5’ → 3’directions from each other, anarrangement referred to as antiparallel.• One DNA molecule includes many genes.• The nitrogenous bases in DNA pair upand form hydrogen bonds: adenine (A)always with thymine (T), and guanine(G) always with cytosine (C).
3. DISCOVERY OF THEDNA STRUCTURE• Early in the 20th century, the identifi-cation of the molecules of inheritanceloomed as a major challenge to biologists.
The Search for the GeneticMaterial: Scientific Inquiry• T. H. Morgan’s group showed that genesare located on chromosomes, the 2components of chromosomes are DNA &protein which became the candidates forthe genetic material.• Key factor in determining the geneticmaterial was choosing appropriateexperimental organisms - bacteria & theviruses that infect them were chosen.
• Discovery of the genetic role of DNA beganwith research by Frederick Griffith in 1928.• Griffith worked with 2 strains of abacterium, 1 pathogenic (S cells) & 1harmless (R cells)• Heat-killed pathogenic strain were mixedwith living cells of harmless strain and theresult = some living cells becamepathogenic.• This phenomenon was calledtransformation, now defined as a changein genotype & phenotype due toassimilation of foreign DNA.
• More evidence for DNA as the geneticmaterial came from studies of viruses thatinfect bacteria.• Such viruses, called bacteriophages (orphages), are widely used in moleculargenetics research.Bacterial cellPhage headTail sheathTail fiberDNA
• 1952: A. Hershey & M. Chaseexperiments showing that DNA isthe genetic material of T2 phage.• To determine the source of geneticmaterial in the phage, they designedan experiment showing that only 1 /2 components of T2 (DNA orprotein) enters an E. coli cell duringinfection• They concluded that the injectedDNA of the phage provides thegenetic information.
• After most biologists becameconvinced that DNA was the geneticmaterial, the challenge was todetermine how its structure accountsfor its role…• M Wilkins & R Franklin used X-raycrystallography to study molecularstructure.• Franklin produced a picture of the DNAmolecule using this technique.
A picture of the DNA molecule usingcrystallography by Franklin.
• Franklin’s X-ray crystallographicimages of DNA enabled Watson todeduce:• that DNA was helical• the width of the helix• the spacing of the nitrogenousbases• Width suggested that the DNAmolecule was made up of 2 strands,forming a double helix
• Watson and Crick: built models of adouble helix to conform to the X-rays &chemistry of DNA.• Franklin concluded there were 2antiparallel sugar-P backbones, withthe N bases paired in the molecule’sinterior.• But: How did bases pair? A-A?/A-T?/A-C?/A-G?......
• But: How did bases pair?• They worked it out by using thefollowing image:Purine + purine: too widePyrimidine + pyrimidine:too narrowPurine + pyrimidine: widthconsistent with X-ray data
• W & C noted that the pairing of theNitrogen bases was specific, dictatedby the base structures.• Adenine (A) paired only with thymine(T), & guanine (G) paired only withcytosine (C)• The W-C model explains Chargaff’srules which states that; in anyorganism the amount of A = T, & theamount of G = C
4. THE ROLE OF DNA• DNA is vital for all living beings – evenplants.• It is important for:• inheritance,• coding for proteins and• the genetic instruction guide forlife and its processes. DNA holds the instructions for anorganisms or each cell’s developmentand reproduction and ultimately death. DNA can replicate itself.
NON-CODING DNA Multicellular eukaryotes have manyintrons(non-coding DNA) within genes andnoncoding DNA between genes. The bulk of most eukaryotic genomesconsists of noncoding DNA sequences,often described in the past as “junk DNA” Much evidence indicates that noncodingDNA plays important roles in the cell. Sequencing of the human genome revealsthat 98.5% does not code for proteins,rRNAs, or tRNAs.
About 24% of the human genomecodes for introns and gene-relatedregulatory sequences. Intergenic DNA is noncoding DNAfound between genes: Pseudogenes are former genes thathave accumulated mutations and arenonfunctional Repetitive DNA is present inmultiple copies in the genome
5. DNA REPLICATION• Replication begins at special sitescalled origins of replication, wherethe 2 DNA strands separate, openingup a replication “bubble” (eukaryoticchromosome may have many originsof replication.• The enzyme helicase unwinds theparental double helix.• Single stranded binding proteinstabilizes the unwound templatestrands.
• A replication fork forms.• The enzyme: Topoisomerase breaks,swivels and re-joins the parental DNAahead of the replication for, toprevent over winding.• The unwind complimentary strandsnow act as individual template for 2new strands.• RNA nucleotides are added to eachDNA template by the enzyme RNAprimase to form RNA primers on bothtemplates
• The enzyme DNA polymerase III addfree DNA nucleotides to the RNAprimer 3’ carbon.• The free nucleotides bond with H-bonds to their complimentary baseson the DNA templates.• Along one template strand of DNA,the DNA polymerase synthesizes aleading strand continuously, movingtoward the replication fork.
• To elongate the other newstrand, called the lagging strand, DNApolymerase must work in thedirection away from the replicationfork.• The lagging strand is synthesized as aseries of segments called Okazakifragments.• Each fragment has an RNA primer andadded DNA strand.• All the fragments are then joined withthe help of the enzyme DNA ligase.
• DNA polymerase I then removes RNAprimers and replaces it with DNAnucleotides.
6. PROOFREADING ANDREPAIRING DNA• DNA polymerases proofread newly madeDNA, replacing any incorrect nucleotides• In mismatch repair of DNA, repairenzymes correct errors in base pairing.• DNA damaged by chemicals, radioactiveemissions, X-rays, UV light, & certainmolecules (in cigarette smoke for example)• In nucleotide excision repair, a nucleasecuts out & replaces damaged stretches ofDNA.
DNA CLONING–Cloning is the reproduction of geneticallyidentical copies of DNA, cells ororganisms through some asexual means.–DNA cloning can be done to producemany identical copies of the same gene –for the purpose of gene cloning.–When cloned genes are used to modify ahuman, the process is called genetherapy.
–Otherwise, the organisms are calledtransgenic organisms – theseorganisms today are used to produceproducts desired by humans.–A. Recombinant DNA (rDNA) and B.polymerase chain reaction (PCR) aretwo procedures that scientists can useto clone DNA
A. CLONING A HUMAN GENEHOW IS INSULIN MADE BY DNA CLONING?(A. RECOMBINANT DNA)• A large quantity of insulin are being producedby recombinant DNA technology.• This process is as follows:1. DNA that codes for the production ofinsulin is removed from the chromosome of ahuman pancreatic cell.2. Restriction enzymes cut the gene from thechromosome (isolating the gene for insulin)I
Insulin gene – cutout with restrictionenzymeIsolated insulin gene
3. A plasmid (acting as a vector/carrier ofnew gene) is removed from the bacteriumand cut open with a restriction enzyme toform sticky endsPlasmid removed frombacteriumCut by restrictionenzymeplasmidNucleusBACTERIUM CELLSticky ends
• 4. Ligase (enzyme) is added to join theinsulin gene to the plasmid of thebacterium cell - forming recombinantDNA.• 5. The recombinant DNA can then bereinserted into the bacterium, thebacterium will then produce moreinsulin, therefore cloning the gene.Insulin gene placed in plasmidby enzyme Ligase ( attachedto sticky ends)
• 6. When the bacterium reproduces it makesthe insulin inserted into the plasmid.• 7. The bacteria are kept in huge tenks withoptimum pH, temperature and nutrientvalues, where they multiply rapidly, producingenormous amounts of insulin, this is thenpurified and sold.Recombinant DNA placed into bacterium cell
B. POLYMERASE CHAIN REACTION• PCR – Used in genetic profiling.• To solve crimes – criminals usually leave DNAevidence at the scene of the crime in the formof saliva, blood, skin, semen and hair. These allcontain DNA. If only a little bit of DNA is foundor the DNA is old, we can make copies of theavailable DNA by means of PCR.• From the DNA produced through PCR, DNAfingerprint can be generated.
PCR method• 1. Sample containing DNA is heated in atest tube to separate DNA into singlestrands.• 2. Free nucleotides are added to the testtube with DNA polymerase (enzyme), toallow DNA replication.• 3. DNA is cooled to allow free nucleotidesto form a complementary strand along sideeach single strand.• 4. In this way the DNA is doubled givingsufficient amount of DNA to work with.
DNA SCREENING AND FINGERPRINTTECHNIQUE• 1. Sample of DNA is cut into fragments by means ofrestriction enzymes.• 2. Negative charged electrode at one end of arectangular flat piece of gel and a positiveelectrode is placed at the other end.• 3. The DNA is placed at the negative end of the geland starts to move to the positive end. Smallerfragments move faster than the larger ones.Separation occurs on the basis of size. This processis called gel electrophoresis.
• 4. DNA is then pressed flat against the gel andtransferred to filter paper.• 5. Radioactive probes bind to special DNAfragments.• 6. X-rays are taken of the filter paper. The DNAprobes show up as dark bands on the film. Thepattern of these bands is the DNA fingerprint.
BIOTECHNOLOGY PRODUCTS• Today transgenic bacteria, plants and animalsare called genetically modified organisms(GMO’s).• The products that GMO’s produce are calledbiotechnology products.
GENETICALLY MODIFIED BACTERIA• Recombinant DNA is used to make transgenicbacteria.• They are used to make insulin, clotting factorVIII, human growth hormone and hepatitis Bvaccine.• Transgenic bacteria is used to protect theroots of plants from insect attack, byproducing insect toxins.
GENETICALLY MODIFIED PLANTS• Example = pomato• Genetically modified to produce potatosbelow the ground and tomatos above theground.• Foreign genes transferred to cotton, corn, andpotato strains have made these plantsresistant to pests because their cells nowproduce an insect toxin.• Read p. 253 for more examples