Molecular Genetics
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Molecular Genetics






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Molecular Genetics Molecular Genetics Presentation Transcript

  • Molecular Genetics The Central Dogma of Biology
  • What is a Genetic “Factor”?
    • From Mendel:
      • we now accepted that there was genetic transmission of traits.
    • Traits are transmitted by “factors”
      • Organisms carry 2 copies of each “factor”
    • The question now was: what is the factor that carries the genetic information?
  • Requirements of Genetic Material
    • Must be able to replicate , so it is reproduced in each cell of a growing organism.
    • Must be able to control expression of traits
      • Traits are determined by the proteins that act within us
      • Proteins are determined by their sequences
    • Therefore, the genetic material must be able to encode the sequence of proteins
    • It must be able to change in a controlled way, to allow variation, adaptation, thus survival in a changing environment.
  • Chromosomes – The First Clue
    • First ability to visualize chromosomes in the nucleus came at the turn of the century
      • construction of increasingly powerful microscopes
      • the discovery of dyes that selectively colored various components of the cell
    • Scientists examined cellular nuclei and observed nuclear structures, which they called chromosomes
    • Observation of these structures suggested their role in genetic transmission
  • Chromosome Observations
    • Variety of chromosome types found in the nucleus
    • 2 copies of each type of chromosome in most cells.
    • All of the cells of an organism, except gametes and rbc’s, have the same number of chromosomes.
    • All organisms of the same species have the same number of chromosomes.
    • The number of chromosomes in a cell doubles immediately prior to cell division
    • Gametes have exactly half of the number of chromosomes as the somatic cells of any organism.
      • Gametes have just one copy of each chromosome type.
      • Fertilization produces a diploid cell (a zygote), with the same number of chromosomes as somatic cells of that organism.
  • Implications
    • Chromosomes behaved like Mendel’s “factors”
      • Mendel's hereditary factors were either located on the chromosomes or were the chromosomes themselves.
    • Proof chromosomes were hereditary factors – 1905:
    • The first physical trait was linked to the presence of specific chromosomal material
      • conversely, the absence of that chromosome meant the absence of the particular physical trait.
    • Discovery of the sex chromosomes
      • "X" and "Y."
      • distinguished from other chromosomes and from each other
  • Importance of Sex Chromosomes
    • Somatic cells taken from female donors always contained 2 copies of the X chromosome
    • Somatic cells taken from male donors always contained one copy of the X chromosome and one copy of the Y chromosome
    • All of the other chromosomes in the nucleated cells of both male and female donors appeared identical
    • Thus, gender was shown to be the direct result of a specific combination of chromosomal material
      • The first phenotype (physical characteristic) to be assigned a chromosomal location
      • Specifically the X and Y chromosomes.
  • What Carries the Genetic Information?
    • Chromosomes are about 40% DNA & 60% protein.
      • Protein is the larger component
    • Protein molecules are composed of 20 different subunits
    • DNA molecules are composed of only four
    • Therefore protein molecules could encode more information, and a greater variety of information
      • protein had the possibility for much more diversity than in DNA
    • Therefore, scientists believed that the protein in chromosomes must carry the genetic information
  • The Discovery of DNA
    • First identified in 1868 by Friedrich Miescher, a Swiss biologist, in the nuclei of pus cells obtained from discarded surgical bandages.
    • He called the substance nuclein
    • Noted the presence of phosphorous
  • The Transforming Principle
    • Fredrick Griffith - 1928
    • Discovered that different strains of the bacterium Strepotococcus pneumonae had different effects on mice
      • One strain could kill an injected mouse (virulent)
      • Another strain had no effect (avirulent)
      • When the virulent strain was heat-killed and injected into mice, there was no effect.
      • But when a heat-killed virulent strain was co-injected with the avirulent strain, the mice died.
    • Concluded that some factor in the heat killed bacteria was transforming the living avirulent to virulent?
    • What was the transforming principle and was this the genetic material?
  • The Transforming Principle is DNA
    • Avery, Macleod, & McCarty – 1943
    • Attempted to identify Griffith’s “transforming principle”
    • Separated the dead virulent cells into fractions
      • The protein fraction
      • DNA fraction
    • Co-injected them with the avirulent strain.
      • When co-injected with protein fraction, the mice lived
      • with the DNA fraction, the mice died
    • Result was IGNORED
      • Most scientists believed protein was the genetic material.
      • They discounted this result and said that there must have been some protein in the fraction that conferred virulence.
  • The Hershey-Chase Experiment
    • Hershey & Chase – 1952
    • Performed the definitive experiment that showed that DNA was the genetic material.
    • Bacteriphages = viruses that infect bacteria
    • Bacteriphage is composed only of protein & DNA
    • Inject their genetic material into the host
  • The Experiment
    • Prepared 2 cultures of bacteriophages
    • Radiolabeled sulphur in one culture
      • there is sulphur in proteins, in the amino acids methionine and cysteine
      • there is no sulphur in DNA
    • Radiolabeled phosphorous in the second culture
      • there is phosphorous in the phosphate backbone of DNA
      • none in any of the amino acids.
    • So this one culture in which only the phage protein was labeled, and one culture in which only the phage DNA was labeled.
  • Experiment Summary
    • Performed side by side experiments with separate phage cultures in which either the protein capsule was labeled with radioactive sulfur or the DNA core was labeled with radioactive phosphorus.
    • The radioactively labeled phages were allowed to infect bacteria.
    • Agitation in a blender dislodged phage particles from bacterial cells.
    • Centrifugation pelleted cells, separating them from the phage particles left in the supernatant.
  • Results Summary
    • Radioactive sulfur was found predominantly in the supernatant.
    • Radioactive phosphorus was found predominantly in the cell fraction, from which a new generation of infective phage was generated.
    • Thus, it was shown that the genetic material that encoded the growth of a new generation of phage was in the phosphorous-containing DNA.
  • Chargaff’s Rule
    • Once DNA was recognized as the genetic material, scientists began investigating its mechanism and structure.
    • Erwin Chargaff – 1950
      • discovered the % content of the 4 nucleotides was the same in all tissues of the same species
      • percentages could vary from species to species.
    • He also found that in all animals (Chargaff’s rule):
    • %G = %C %A = %T
    • This suggested that the structure of the DNA was specific and conserved in each organism.
    • The significance of these results was initially overlooked
  • Base Pairing in DNA
    • Not understood ‘til Watson & Crick described double helix
    • Adenine & guanine are purines
      • 2 organic rings
    • Cytosine & guanine are pyrimidines
      • 1 organic ring
    • Pairing a purine & a pyrimidine creates the correct 2 nm distance in the double helix
    • A – T joined by 2 hydrogen bonds
    • G – C joined by 3 hydrogen bonds
  • The Double Helix
    • James Watson and Francis Crick – 1953
    • Presented a model of the structure of DNA.
    • It was already known from chemical studies that DNA was a polymer of nucleotide (sugar, base and phosphate) units.
    • X-ray crystallographic data obtained by Rosalind Franklin, combined with the previous results from Chargaff and others, were fitted together by Watson and Crick into the double helix model.
  • Watson and Crick shared the 1962 Nobel Prize for Physiology and Medicine with Maurice Wilkins. Rosalind Franklin died before this date.
  • DNA Structure
    • The double helix is formed from two strands of DNA
    • DNA strands run in opposite directions
    • They are complementary
      • attached by hydrogen bonds between complimentary base pairs:
      • A - T and G - C
    • This complementary pairing of the bases suggests that, when DNA replicates, an exact duplicate of the parental genetic information is made.
      • The polymerization of a new complementary strand takes place using each of the old strands as a template.
  • Messelson and Stahl
    • How does DNA replicate?
    • Matthew Meselson and Franklin Stahl - 1957
    • Did an experiment to determine which model best represented DNA replication:
    • semiconservative replication
      • the two strands unwind and each acts as a template for new strands
      • each new strand is half comprised of molecules from the old strand
    • conservative replication
      • the strands do not unwind, but somehow generate a new double stranded DNA copy of entirely new molecules
  • The Experiment
    • The original DNA strand was labeled with the heavy isotope of nitrogen, N-15.
    • This DNA was allowed to go through one round of replication with N-14 (non-labeled)
    • the mixture was centrifuged so that the heavier DNA would form a band lower in the tube
    • the intermediate (one N-15 strand and one N-14 strand) and light DNA (all N-14) would appear as a band higher in the tube
  • The expected results for each model were:
  • The actual results were as expected for the semiconservative model and thus Watson and Crick's suspicion was borne out.
  • Enzymes & Replication
    • DNA replication is not a passive, spontaneous process.
    • More than a dozen enzymes & other proteins are required to unwind the double helix & to synthesize & finalize a new strand of DNA.
    • The molecular mechanism of DNA replication can best be understood from the point of view of the machinery required to accomplish it.
    • The unwound helix, with each strand being synthesized into a new double helix, is called the replication fork .
  • The Enzymes of DNA Replication
    • Topoisomerase
      • Responsible for initiation of unwinding of DNA.
      • The tension holding the helix in its coiled and supercoiled structure is broken by nicking a single strand of DNA.
    • Helicase
      • Accomplishes unwinding of the original double strand, once supercoiling is eliminated by topoisomerase.
      • The two strands want to bind together because of hydrogen bonding affinity for each other, so helicase requires energy (ATP) to break the strands apart.
  • Single-stranded Binding Proteins
    • Important to maintain the stability of the replication fork.
    • Line up along unpaired DNA strans, holding them apart
    • Single-stranded DNA is very unstable, so these proteins bind bind to it while it remains single stranded and keep it from being degraded.
  • Beginning: Origins of Replication
    • Replication begins at specific sites called origins of replication
    • In prokaryotes, the bacterial chromosome has a specific origin
    • In eukaryotes, replication begins at many sites on the large molecule
      • 100’s of origins
    • Proteins that begin replication recognize the origin sequence
    • These enzymes attach to DNA, separating the strands, and opening a replication bubble
    • The end of the replication bubble is the “Y” shaped replication fork , where new strands of DNA elongate
  • Elongation
    • Elongation of the new DNA strands is catalyzed by DNA polymerase
    • Nucleotides align with complimentary bases on the template strand, and are added by the polymerase, one by one, to the growing chain
      • DNA polymerase proceeds along a single-stranded DNA molecule, recruiting free nucleotides to H-bond with the complementary nucleotides on the single strand
    • Forms a covalent phosphodiester bond with the previous nucleotide of the same strand
      • The energy stored in the triphosphate is used to covalently bind each new nucleotide to the growing second strand
    • Replication proceeds in both directions
  • The Replication Fork
  • DNA Polymerase
    • There are different forms of DNA polymerase
      • DNA polymerase III is responsible for the synthesis of new DNA strands
    • DNA polymerase is actually an aggregate of several different protein subunits, so it is often called a holoenzyme .
    • Primary job is adding nucleotides to the growing chain
    • Also has proofreading activities
  • Proofreading & Repair
    • DNA polymerase proofreads each nucleotide added against its’ template as it is added
    • Removes incorrectly paired nucleotides & corrects
  • DNA Strands are Anti-parallel
    • The sugar phosphate backbones of the 2 parent strands run in opposite directions
      • “ upside down” to each other
    • DNA is polar
      • There is a 3’ end and a 5’ end
      • At the 3’ end, a OH is attached to the 3’ C of the last deoxyribose
      • At the other end a phosphate group is attached to the 5’ C of the last nucleotide
    • DNA polymerase adds nucleotides only to the 3’ end of the growing chain
      • So new DNA strand elongates only in the 5’  3” direction
  • Why 5’  3’?
    • Why can DNA polymerase only add nucleotides to the 3’ end ?
    • Needs a triphosphate to provide energy for the bond between an added nucleotide & the growing DNA strand.
    • This triphosphate is very unstable
      • can easily break into a monophosphate and an inorganic pyrophosphate
    • At the 5' end, this triphosphate can easily break
      • It is not be able to attach new nucleotides to the 5' end once the phosphate has broken off
    • The 3' end only has a hydroxyl group
      • As long as new nucleotide triphosphates are brought by DNA polymerase, synthesis of a new strand continues, no matter how long the 3' end has remained free.
  • Leading & Lagging Strands
    • The new strand made by adding to the 3’ end = leading strand
      • Parent strand is 3’  5’
      • New strand is 5’  3’
    • How can DNA polymerase synthesize new copies of the 5'  3' strand, if it can only travel in one direction?
    • To elongate in the other direction, the process must work away from the replication fork
    • The new strand formed on the 5’  3’ parent strand is called the lagging strand
  • Building the Lagging Strand
    • DNA polymerase makes a second copy in an overall 3’  5’ direction
    • First, it produces short segments, called Okazaki fragments
      • These are built in a 5’ –3’ direction
    • Okazaki fragments are joined together by ligase to produce the new 3’  5’ lagging strand
  • Ligase
    • Catalyzes the formation of a phosphodiester bond given an unattached adjacent 3‘ OH and 5‘ phosphate.
    • This can join Okazaki fragments
    • This can also fill in the unattached gap left when an RNA primer is removed
    • DNA polymerase can organize the bond on the 5' end of the primer, but ligase is needed to make the bond on the 3' end.
  • Primers
    • DNA polymerase cannot start synthesizing on a bare single strand.
      • It only adds to an existing chain
    • It needs a primer with a 3'OH group on which to attach a nucleotide.
    • The start of a new chain is not DNA, but a short RNA primer
      • Only one primer is needed for the leading strand
      • One primer for each Okazaki fragment on the lagging strand
  • Primase
    • Part of an aggregate of proteins called the primeosome.
    • Attaches the small RNA primer to the single-stranded DNA which acts as a substitute 3'OH so DNA polymerase can begin synthesis
    • This RNA primer is eventually removed by RNase H
      • the gap is filled in by DNA polymerase I.
  • Ending the Strand
    • DNA polymerase only adds to the 3’ end
    • There is no way to complete the 5’ end of the new strand
    • A small gap would be left at the 5’ end of each new strand
    • Repeated replication would then make the strand shorter and shorter, eventually losing genes
    • Not a problem in prokaryotes, because the DNA is circular
      • There are no “ends”
  • Telomeres
    • Eukaryotes have a special sequence of repeated nucleotides at the end, called telomeres
    • Multiple repititions of a short nucleitide sequence
      • Can be repeated hundreds, or thousands of times
      • In humans, TTAGGG
    • Do not contain genes
    • Protects genes from erosion thru repeated replication
    • Precvents unfinished ends from activating DNA monitoring & repair mechanisms
  • Telomerase
    • Catalyzes lengthening of telomeres
    • Includes a short RNA template with the enzyme
    • Present in immortal cell lines and in the cells that give rise to gametes
    • Not found in most somatic cells
    • May account for finite life span of tissues
  • Further Proofreading & Repair
    • Some errors evade initial proofreading or occur after synthesis
    • DNA can be damaged by reactive chemicals, x-rays, UV, etc
    • Cells continually monitor DNA for mutations & repair
    • Contain 100’s of repair enzymes
  • Nucleotide Excision & Repair
    • A segment of DNA containing damage is cut out by a nuclease (a DNA cutting enzyme)
    • The gap is filled & closed by DNA polymerase and ligase
    • Thymine dimers
    • Covalent linking of thymine bases
    • Causes DNA to “buckle”
    • One common problem corrected this way
  • Proteins of DNA Replication
  • Summary View of Replication