GEN 272 Introductory Molecular Genetics

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GEN 272Introductory Molecular Genetics

• Lecture One

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REMINDER!

• First test 27 February 2008 (during lecture
  period)

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Physiology
Behaviour
Molecular Biology
Ecology
Systematics
Cell biology
Evolution
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Johann Gregor Mendel

• An Augustitian monk, who lived in the 19th
  century in Europe
• Conducted a decade-long series of experiments
  using pea plants. From the
  experiments conclusions were made that
• traits are passed from parents to offspring in
  predictable ways
• for example, he was able to show that traits such
  as height and
  floral colour are controlled by discrete units of
  inheritance genes
• these genes exist in pairs and members of a gene
  pair are
  separated from each other during gamete formation
• his work got published in 1866 but remained
  largely ignored until it was duplicated by others
  in the early 1900s
• subsequently, Mendels work was recognized as the
  basis for the transmission of traits in pea
  plants and other organisms ? the field of
  genetics emerged

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Chromosomal theory of inheritance

• A few years after Mendels work, microscopy
  enabled the identification of chromosomes (chrs)
• It emerged that eukaryotes have a diploid number
  of chrs (i.e. 2n). Note diploidy is not always
  the rule especially in plants
• In diploid cells, chrs exist in pairs, termed
  homologous chromosomes (also called homologs)
• Homologs pair during mitosis and meiosis, with
  the latter step occurring during gamete formation
  to produce daughter cells containing a haploid
  (i.e. n) set of chrs
• Walter Sutton and Theodore Boveri independently
  noted that genes and chrs have common
  properties, i.e. genes exist in pairs (so does
  chrs) and each pair separate during gamete
  formation
• Based on the above information, they each
  proposed that genes are carried on chrs chr
  theory of inheritance
• inherited traits are controlled by genes residing
  on chrs that are faithfully transmitted thru
  gametes, maintaining genetic continuity from
  generation to generation

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Genetic variation

• About the same time of the chrs theory of
  inheritance, experiments studying the inheritance
  of traits in Drosophila melanogaster revealed a
  white-eyed fly in a red-eyed fly population
  (Fig.1-6)
• This variation was produced by a mutation, an
  inherited
  change in the gene controlling a trait, i.e. eye
  colour
• Note mutations can occur at both gene and
  chromosome levels
• The variant gene discovered in Drosophila,
  represents an
  allele (alternative forms of a gene) of the eye
  colour gene
• Different combinations of alleles for a
  particular trait
  (genotype) may produce differences in the
  phenotype
  (observable features) of an organism

Fig. 1-6
Mutant
Wild-type
Klug et al (2006). Concepts of Genetics. 8th Ed.
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Genetic Material DNA or Proteins?

• Until the 1940s, it was not clear what chemical
  component of the chr makes up genes and what
  constitutes genetic material
• However, many researchers favoured proteins over
  DNA as the genetic material
• Three major factors contributed to the idea, thus
• Proteins are abundant in cells these molecules
  account for more than 50 of the dry weight of
  cells
• The accepted proposal for the chemical structure
  of nucleic acids (1900s) DNA structure was
  first studied in 1868 (Swiss chemist, Friedrick
  Miescher)
• separated nuclei from cytoplasm and subsequently
  isolated an acidic substance from nuclei termed
  nuclein
• nuclein contained large amounts of phosphorus but
  lacked sulfur, a characteristic that
  differentiated it from protein
• analytical techniques were used to reveal the
  structure of nucleic acids, composed of molecular
  building blocks called nucleotides (Levenes
  tetranucleotide hypothesis)
• The active areas of research transmission
  genetics and mutation studies

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Evidence of DNA as genetic material

• In 1927, Frederick Griffith performed
  experiments using several different strains of
  the bacterium Streptococcus pneumoniae
• he used virulent (cause pneumonia especially in
  humans and mice) and avirulent strains
  virulence is a property related to the
  polysaccharide capsule of the bacterium
• virulent strains contain the capsule whereas
  avirulent strains do not
• the non-encapsulated bacteria (i.e. avirulent,
  rough colonies) are readily engulfed and
  destroyed by phagocytes in the hosts circulatory
  system
• encapsulated bacteria (i.e. virulent, smooth
  colonies) are not easily engulfed? able to
  multiply and cause pneumonia
• heat-killed virulent bacteria did not kill mice,
  as was the case with living avirulent bacteria

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Griffiths transformation experiment

• Inject mice with living avirulent cells (IIR)
  heat-killed virulent cells (IIIS) ? expect the
  injection not to kill mice
• after 5 days, mice injected with both types of
  cells died, but control mice injected with
  avirulent IIR cells alone remained healthy and
  alive
• blood analysis (of dead mice) revealed a large
  number of living IIIS cells, similar to the
  original strain used for the preparation of
  heat-killed cells
• alive control mice ruled out the theory of
  spontaneous change (i.e. mutation) of avirulent
  IIR cells ? virulent IIIS cells in the absence of
  heat-killed IIIS cells
• ? heat-killed virulent IIIS cells were
  responsible for converting avirulent IIR cells
  into virulent IIIS cells (proposed that the might
  be some transforming principle part of the
  polysaccharide capsule or some compound required
  for capsule synthesis )
• other scientists (Henry Dawson Lionel J.
  Alloway) subsequently tested the transforming
  principle in vitro (in a test tube) injection
  into mice was not necessary

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Griffiths transformation experiment
Fig. 10-3
Klug et al (2006). Concepts of Genetics. 8th Ed.
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Avery, MacLeod McCarty experiment

• Following Griffiths experiment, the critical
  question remained as to what molecule serves as
  the transforming principle?

Fig. 10-4
Klug et al (2006). Concepts of Genetics. 8th Ed.
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Hershey-Chase experiment
Lytic Cycle

• Hershey and Chase already knew that,
• T2 phages consist 50 protein and 50 DNA
• Infection is initiated by adsorption of the
  phage by its tail fibers to the bacterial cell
• The production of new viruses occurs within the
  bacterial cell
• Key point of experiment strategy
• DNA contains phosphorus (P) and not sulphur (S),
  whereas proteins contain S and not P
• ? used radioisotopes 32P and 35S to follow the
  molecular components of phages during infection

Fig. 10-5
Klug et al (2006). Concepts of Genetics. 8th Ed.
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Hershey-Chase experiment
Fig. 10-6

• Results indicate that the protein of the phage
  coat remains outside the host cell and is not
  directly involved in directing the production of
  new phages
• On the other hand, phage DNA enters the host
  cell and directs phage reproduction

unlabeled bacteria
Klug et al (2006). Concepts of Genetics. 8th Ed.
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Transfection Experiments

• Following the Hershey-Chase experiment,
• studies demonstrated that the outer (cell) wall
  of E. coli can be removed without destroying the
  cell
• this can be achieved by treating the bacterial
  cells with enzymes called lysozymes
• resultant naked cells are known as protoplasts
  (or spheroplasts)
• John Spizizen and Dean Fraser independently
  reported using spheroplasts to initiate phage
  reproduction with disrupted T2 particles (i.e.
  outer protein coat)
• virus does not have to be intact for infection
  to occur
• outer protein coat structure may only be
  essential for DNA movement through the intact
  cell wall, but not essential for infection
• Similar observations were also made by Guthrie
  and Sinsheimer using bacteriophage øX174
• infection by only the (viral) nucleic acid,
  transfection, proved conclusively that øX174 DNA
  contains the necessary information for production
  of mature viruses

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Genetic Material in Eukaryotes

• Indirect evidence
• distribution of DNA
• genetic material is found only where primary
  genetic function occurs, e.g. DNA exists in
  mitochondria, chloroplasts whereas protein is
  found everywhere in the cell
• also, there is a positive correlation between
  ploidy of a cell and the quantity of molecule
  that functions as genetic material, e.g. DNA and
  protein content in gametic and somatic cells (see
  Table 10.2)
• mutagenesis
• the molecule serving as the genetic material
  is expected to absorb UV light at the
  wavelength(s) found to be mutagenic UV light is
  most mutagenic at ? 260 nm
• DNA and RNA absorb UV light most strongly at
  this wavelength
• for proteins, absorption occurs at ? 280
  nm, but no significant mutagenic effect is
  evident at the same wavelength nucleic acid
  (i.e DNA) is genetic material
• Direct evidence recombinant DNA technology,
  e.g. eukaryotic gene cloned into bacteria. New
  bacterial cells following replication also
  contain the inserted gene. Similar observations
  were made in mice by cloning the human betaglobin
  gene.

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RNA as genetic material

• In some cases, e.g. viruses, RNA is the genetic
  material (and not DNA)
• Tobacco Mosaic Virus (TMV)
• RNA isolated and spread on tobacco leaves
  characteristic lesions caused by viral infection
  subsequently appeared on leaves
• RNA is genetic material (see also Fig. 10-8)
• Other studies revealed that RNA is capable of
  replication
• RNA isolated from phage Q? and replicated in
  vitro
• replication was dependent on RNA replicase,
  which was isolated from E.coli
• RNA replicated in vitro added to E. coli
  protoplasts ? infection and viral multiplication
  occurred RNA is the genetic material
• One other group of RNA-containing viruses is
  retroviruses (e.g. HIV)
• replication occurs under the direction of an
  RNA-dependent DNA polymerase called reverse
  transcriptase
• the process, reverse transcription, involves
  RNA ? DNA ? RNA

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RNA as Genetic Material Example
Fig. 10-8
Klug et al (2006). Concepts of Genetics. 8th Ed.
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Take Home Message

• Genetics is central to the field of biology
• Genes are carried on chrs (chrs theory of
  inheritance)
• DNA (and in some cases, RNA) is the genetic
  material, but not protein