Lecture 2: The code of life Review of the Essentials

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Lecture 2The code of lifeReview of the
Essentials
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Reading material

• Three textbooks
• Mentioned last time
• Your introductory biology textbook
• Reading material
• Will be put on homepage
• Selected bibliography
• Will be used in exams
• Tuesdays lecture
• Carvalho GR (1998) Molecular ecology origins and
  approach. In Advances in Molecular Ecology,
  IOSPress, Amsterdam. Pp. 1-23
• Required sections 1-4 (pp. 1-10)

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Milestones in DNA history

• 1859 - Charles Darwin
• On The Origin of Species by Means of Natural
  Selection or The Preservation of Favoured Races
  in the Struggle for Life (1859)
• No clear idea of how variability was created
• Our ignorance of the laws of variation is
  profound
• 1866 Gregor Mendel (Austrian monk)
• Hybridization experiment with peas
• parents provide particles that are passed onto
  offspring.

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Milestones in DNA history

• 1900
• Rediscovery of Mendels work
• 1949 Erwin Chargraff
• Composition of DNA
• GC AT
• 1952 - Rosalind Franklin
• X ray crystallography
• Crystallized DNA
• Reflects X rays
• Positions of atoms in 3D

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Milestones in DNA history

• 1953 - James Watson (biologist) Francis Crick
  (physicist)
• Calculated 3D structure of DNA
• Based on Franklins X-rays
• Published 50 years ago (almost to the day)
• Watson, J. D. and F.H.C. Crick. 1953 A Structure
  for Deoxyribose Nucleic Acid. Nature 171 page
  737
• 1962 Nobel Prize
• Rosalind Franklin had died

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The structure of DNA

• Sugar-phosphate backbone
• 5 C-atoms in the sugar
• Chain is directional
• 3 on one side
• 5 on the other
• Two different sugars
• DNA
• RNA
• Nitrogenous base
• Purines A, G
• Pyrimidines C, T

Pyrimidines
Purines
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The structure of DNA

• Complimentary binding
• Hydrogen bonds
• Purine with Pyrimidine
• A T
• G C
• Chain is antiparallel

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The structure of DNA

• Each base pair causes a twist
• 360o turn every 10 base pairs
• The double helix
• Human Genome
• 3billion bases
• 3,000,000,000
• About 2.3 m long
• How does that fit into each small cell?

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How does lots of DNA fit into small cells?

• DNA double helix
• Coiled
• Wrapped around protein pellets
• Nucleosomes
• Pearls on a chain
• Coiled again
• Chromatin
• Wrapped into chromosome
• Genome consists of many chromosomes
• 3 several hundred
• Needs to unwrap for
• Replication
• Cell division
• Transcription
• Gene expression

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The Karyotype

• Chromosome number
• Often species-specific
• Even populations
• Ploidy
• Number of sets of chromosome
• 2N diploid
• N haploid
• XN polyploid
• Ploidy change
• Reproductive isolation
• Speciation mechanism
• Kinds of chromosomes
• Sex chromosomes
• Heterogametic sex
• Males XY (mammals)
• Females WZ (birds)
• autosomes

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DNA Replication
It has not escaped our notice that the specific
pairing we have postulated immediately suggests
a possible copying mechanism for the genetic
material. Watson Crick, 1953
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DNA Replication

• DNA polymerase
• Catalyzes the synthesis of new DNA strand from
  free nucleotides
• Only works 5 ? 3
• Bits connected by DNA ligase
• Very fast and very accurate
• Proofreading enzymes
• 1 in 1 billion error
• mutation

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DNA replication and the cell cycle

• DNA replication
• Chromosomes duplicated
• Chromatids
• Split up during mitosis
• Cell cycle
• DNA replication happens in interphase between
  cell divisions
• Before and after periods of metabolic activity
• G1 single chromosomes
• Cell grows
• S DNA replication
• G2 chromosomes with 2 chromatids
• Cell produces proteins essential to cell division

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MitosisCell division
centrosome
Mitotic spindle
Interphase
Prophase
Metaphase
Metaphase
Anaphase
Telophase Cytokinesis
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DNA Replication in the TubePCR

• Polymerase Chain Reaction
• Most important recent discovery (1985)
• Patented all PCR reactions pay royalty
• Repeated replication of specific DNA sections
• Small quantities
• Feathers, hair etc.
• Specific regions of DNA
• Target specific sequences
• Need PCR primers
• Polymerase can only start synthesizing from
  double stranded DNA
• Start where primer anneal
• 5 ? 3
• Short artificial DNA sequences
• 15-20 bp
• Match template DNA
• Can pick where we want to start PCR

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PCR

• How does it work
• Separate the two strands (94oC)
• Anneal primers (55oC)
• Replication start
• Extension (72oC)
• replication
• Repeat 20 30 times
• Produces billions of copies of template DNA

94
94
72
55
5
3
5
3
5
3
3
5
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PCR
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PCR in practice

• Reaction ingredients
• Template DNA
• Primers
• As a starting point
• Forward and reverse
• Nucleotides
• To synthesize DNA
• Polymerase
• Taq polymerase
• thermostable
• PCR machines
• Automatic cycling of temperature
• Very accurate

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PCR primers

• PCR needs sequence information
• Usually species specific
• Where do we get primer sequences from?
• Somebody may have isolated them
• Check databases
• Freely available on internet (GenBank)
• Results not publishable without primer
  information
• Heterologous primers
• Isolated from related species
• Very useful for many applications
• Problem
• may not exactly match
• PCR does not always work
• Primer isolation
• Very lengthy and expensive procedure
• 6 months work

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PCR primers

• Annealing temperature
• Optimal temperature for primers to attach to the
  template DNA
• Too high
• Bonds dont work
• Primer doesnt anneal
• Too low
• Primer may attach anywhere
• Non-specific amplification
• Depends on strength of bonds
• Remember
• G-C three hydrogen bonds
• A-T two hydrogen bonds
• Annealing temperature dependson GC content

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Summary of Key Concepts

• Milestones of DNA history
• Darwin, Mendel, Franklin, Watson Crick
• The structure of DNA
• Nucleotides
• Sugar - Phosphate backbone, nitrogenous base
• Complimentary binding
• Directionality of DNA
• 5 end, 3 end, antiparallel strands
• Coiling of DNA
• Double helix, histones, nucleosomes, chromatin,
  chromosomes
• Karyotype
• Chromosome number, ploidy
• Autosomes, sex chromosomes

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Summary of Key Concepts

• DNA replication
• Complimentary binding
• DNA polymerase, ligase
• Cell cycle phases, mitosis
• PCR
• Purpose, principles
• Produce millions of copies of specified DNA
  region
• PCR in practice
• Primers
• Where can we get them from?
• Annealing temperatures

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Lab Project

• Rockfish (Sebastes sp.)
• 65 species along the US west coast
• Long-lived (up to 100 years)
• Ovoviviparous
• Slow reproduction
• Commercially very important

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Rockfish Fisheries

• Most are overexploited
• Some below 10 of the initial population
  abundance
• Very slow reproduction
• Maturation 20-30 y
• Conservation status
• Almost all overfished
• Not yet endangered
• Many considered vulnerable
• E.g. copper, quillback and brown in Puget Sound

Changes in biomass of yelloweye rockfish (CA)
Decrease in abundance
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Multi-species Fisheries and Bycatch

• 65 species categorized as
• Near-shore, Shelf, Slope
• Detailed management plans only for 10 species
• Many caught as by-catch
• E.g. Halibut fishery (BC)
• 77,000 t (2003)
• 21 species
• 15 30 discarded (AFS)
• Usually die anyway
• Species identification
• Difficult
• Color, spines, morphology
• Not possible in
• Fillets
• larvae

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Fillet Identification

• Some species may become threatened
• Currently vulnerable
• Important to identify illegal catches
• Some rockfish are more equal than others
• Difference in price
• 8 c - gt 1
• Labeling of fillets can be an issue
• Fair trading
• Consumer protection

?
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Larval identification

• Many species not identifiable morphologically
• Abundant
• Most abundant fish larva
• Important
• Larval distribution
• Ecology
• Stock assessment
• Recruitment

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Whos who?
Copper (S. caurinus)
Pacific Ocean Perch (S. alutus)
Shortspine Thornyhead (Sebastolobus alascanus)
Rougheye (S. aleutianus)
Shortraker (S. borealis)
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How to identify rockfish larvae and fillets

• Molecular markers
• Can separate species
• DNA
• Proteins (enzymes)
• We will use
• Two mystery fillets
• Bought in Pike Place Market
• Two molecular methods
• Mitochondrial DNA RFLP
• Allozymes (protein marker)
• More later