Microbiology 7-9

• Microbial Genetics is the study of the mechanisms of heritable information in microorganisms (bacteria, archaea, viruses and some protozoa and fungi)
• This also involves the study of the genotype of microbial species and also the expression system in the form of phenotypes
• Nucleotide


  Structural unit of nucleic acids, consisting of a nitrogenous base, a pentose sugar, and a phosphate group

  
• Nitrogenous bases


  • Purines: Adenine (A), Guanine (G)
  • Pyrimidines: Cytosine (C), Thymine (T), Uracil (U)

  
• DNA


  • Consists of two strands that form a double helix structure
  • Each DNA strand is composed of nucleotides
  • The sequences of nitrogenous bases on the two strands are complementary
  • The nitrogenous base pairs are joined by hydrogen bonds
  • The two strands of DNA are antiparallel
• RNA


  • Uses the sugar ribose instead of deoxyribose
  • Generally single-stranded
  • Contains uracil in place of thymine

  
• Types of RNA


  • mRNA (messenger RNA)
  • rRNA (ribosomal RNA)
  • tRNA (transfer RNA)

  
• Central Dogma of Molecular Biology

  • DNA contains the complete genetic information that defines the structure and function of an organism
  • Proteins are formed using the genetic code of the DNA
  • Conversion of DNA encoded information to RNA is essential to form proteins

  
• Genotype


  The organism's genetic makeup - all its DNA - the information that codes for all the particular characteristics of the organism
• Phenotype


  Refers to actual, expressed properties (proteins)
• DNA replication


  1. Semi-conservative mode

  2. Resulting daughter molecules each have one parental (old) strand and one newly synthesized strand

  3. Watson and Crick base pairing maintained

  4. DNA is synthesized in the 5' to 3' method

  5. A primer is needed for initiation

  
• Origin of Replication


  Sequence of DNA at which replication is initiated on a chromosome, plasmid or virus

  
• Stages of Replication: Initiation

  1. DNA gyrase and topoisomerases relaxes supercoiling ahead of the replication fork

  2. Replication fork - the point at which replication actively occurs

  3. The two strands of parental DNA are unwound by helicase

  4. Primers - signal the starting point of DNA replication, synthesized by primase

  
• Stages of Replication: Elongation

  1. Leading strand - continuous, one primer, DNA polymerase, 5' to 3'

  2. Lagging strand - synthesized opposite to the fork movement, discontinuous, several primers, DNA polymerase, 5' to 3', Okazaki fragments

  
• Stages of Replication: Termination

  1. Forks converge until all intervening DNA is unwound

  2. Any remaining gaps are filled and ligated (DNA ligase)

  3. Replication proteins are unloaded

  
• Transcription


  1. RNA polymerase binds to the DNA at a site called the promoter

  2. RNA polymerase synthesize mRNA in the 5' - 3' direction

  3. RNA synthesis continues until RNA polymerase reaches a site on the DNA called the terminator

  
• Codon


  Groups of 3 nucleotides on mRNA that determine the sequence of amino acids in the protein being synthesized

  
• There are 61 possible codons but only 20 amino acids, so most amino acids are signaled by several alternative codons
• Third Base Degeneracy or Wobble Hypothesis


  Codons for the same amino acid usually differ in the 3rd position only
• Translation


  1. The ribosome binds to mRNA at a specific area

  2. The ribosome starts matching tRNA anticodon sequences to the mRNA codon sequence

  3. Each time a new tRNA comes into the ribosome, the amino acid it was carrying gets added to the elongating polypeptide chain

  4. The ribosome continues until it hits a stop sequence, then it releases the polypeptide and the mRNA

  5. The polypeptide forms into its native shape and starts acting as a functional protein in the cell

  
• Mutation


  Any heritable alteration in the base sequence of the genetic material

  
• Types of Mutation


  • Spontaneous mutation
  • Induced mutation

  
• Base Substitution


  • A single base at one point in the DNA sequence is replaced with a different base during replication
  • Can be a transition (purine to purine or pyrimidine to pyrimidine) or a transversion (purine to pyrimidine)

  
• Mutation


  The initial and final states of the organisms
• When established, mutation may be permanently present whether or not the conditions of development of the mutated organism allow their detection

  
• Mutation


  Can either be spontaneous or induced
• Spontaneous mutation


  Occurs without external intervention, and most result from occasional errors in the pairing of bases by DNA polymerase during DNA replication

  
• Induced mutation


  • Caused by agents in the environment and include mutations made deliberately by humans
  • Results from exposure to natural radiation that alters the structure of bases in the DNA, or from a variety of chemicals that chemically modify DNA

  
• Base Substitution


  • Also called as point mutation or base pair changes
  • A single base at one point in the DNA sequence is replaced with a different base during replication
  • Can either be (1) transition – purine to purine (A→G) or pyrimidine to pyrimidine (C→T) – or (2) transversion – purine to pyrimidine or vice versa

  
• Consequences of Base Substitutions

  • Missense mutation - Changes a codon for one amino acid to a codon for another amino acid, resulting in an amino acid substitution in the protein product
  • Nonsense mutation - Changes a codon for an amino acid with a codon for chain termination (UAG, UAA, UGA)
  • Silent mutation - A change in codon composition that has no effect on the resulting polypeptide

  
• Frameshift mutation


  • Adds or deletes one or two bases (or any non-multiple of 3) from a coding sequence in a DNA, so that the genetic code is read out-of-phase
  • Consequences: (a) incorrect amino acid or premature termination and/or (b) severe phenotypic effects

  
• Deletion


  A mutation in which a region of the DNA has been eliminated
• Insertion


  Occurs when new bases are added to the DNA
• Mutagens


  Physical or chemical agents that changes the genetic material
• Physical mutagens


  • High energy radiations that penetrate living cells
  • Electromagnetic radiations (gamma rays, X-rays and ultraviolet rays)
  • Particulate radiations (alpha particles, beta particles and neutrons)
• Ionizing radiations (Gamma rays and X-rays)


  • Direct effect: Single or double-stranded breaks in the DNA molecules
  • Indirect effect: Free radicals created; form compounds, HO2 – initiate harmful chemical reactions within the cells; can lead to cell death
• Non-ionizing radiations (UV rays)

  Formation of pyrimidine dimers – most are immediately repaired, but some escape repair and inhibit replication and transcription
• Particulate radiations


  • Are in the form of sub-atomic particles emitted from the atoms with high energy
  • Penetrating power: Beta particles > alpha particles because of its smaller size
  • Neutrons: extremely penetrant, and can cause severe damage to the living tissues as well as genetic material
  • Result: Single strand or double strands break in the DNA
• Chemical mutagens


  • Deaminating agents (nitric oxide, nitrous acid and N-nitrosoindoles)
  • Base Analogs
  • Alkylating Agents (methylmethane sulphonate (MMS), ethyl methane sulfonate (EMS), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG))
  • Intercalating Agents (acridine derivatives, nitrogen mustards, and ethidium bromide)
• Recombination


  • The physical exchange of DNA between genetic elements
  • Horizontally-transferred DNA from a donor should undergo recombination with the recipient's DNA in order to be retained in the recipient