Tools of the Trade

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Tools of the Trade

• Introduction to Plasmids and Cosmids
• Discovery of Restriction Endonucleases and Other
  DNA Modifying Enzymes

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Plasmids

• Plasmids are like Mini-chromosomes
• Covalently closed circular
• Autonomous replication

• All plasmids have an origin of replication (ori)
  and genes.

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Plasmids

• Differs from chromosomes
• One or more copies (700)
• Not all bacteria have plasmids
• Range in size from 1200 kbp (kilobase pairs)
• Functions
• Cryptic no known function
• Phenotypic antibiotic resistance, conjugative,
  virulence, etc.

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The 1950s and 1960s

• Saw great breakthroughs in DNA enzymology
• 1955 - Arthur Kornberg and Severo Ochoa isolated
  DNA polymerase I (1959, Nobel)
• B. Weiss and C.C. Richardson isolated DNA ligase
  (1966)
• H.O. Smith, K.W. Wilcox, and T.J. Kelley isolated
  and characterized the first sequence specific
  restriction nuclease (1968)

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DNA Transformation More Discoveries

• In the 1950s people observed that DNA introduced
  into different strains of Escherichia coli (E.
  coli) would not always replicate well in all
  strains.
• 1966, chemical analysis of small viral DNA that
  survive in bacteria were shown to be methylated.
• Methylation was achieved enzymatically.

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Late 1960s

• Stewart Linn and Werner Arber isolated
• Methylase
• A random Restriction nuclease
• Additional discovery of methylases in two
  additional strains of E. coli along with
  companion restriction nucleases got scientists
  excited.
• Scientists thought that there may be restriction
  endonucleases that were specific.

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Why?

• Opened the possibility that site-specific
  nucleases might exist.
• Bursting their bubble
• These early restriction nucleases only cleaved
  DNA at random locations far removed from the
  recognition sites.
• Nobody else got excited because television was
  here and we could now watch the race for space.

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Restriction Nucleases

• Today, these are known as Type I restriction
  endonucleases.
• You can find a listing of Type I enzymes at
• http//www.internalmed.wustl.edu/divisions/enzymes
  /index.htm
• CfrA I GCA(N)8GTGG

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Type II

• 1970 Hamilton Smith, K.W. Wilcox, and T.J.
  Kelley, Johns Hopkins University.
• Accidentally found that Haemophilus influenzae
  rapidly broke down foreign DNA.
• Purified enzyme
• Broke down (digested) E. coli DNA
• Did not digest H. influenzae DNA
• Enzyme called HindII

5-GTPy PuAC-3 3-CAPu PyTG-5
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Restriction Nucleases

• Today, over 900 restriction nucleases are known
• Isolated from over 230 strains of bacteria
• Names reflect origin
• Ex. EcoRI E. coli strain RY13
• Ex. HindII H. influenzae strain Rd
• Ex. HindIII - ????????

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Types of Restriction Endonucleases

• Type I - Cuts nonspecifically a distance greater
  than 1000 bp from its recognition sequence and
  contains both restriction and methylation
  activities
• Type II - Cuts at or near a short, and often
  symmetrical, recognition sequence. A separate
  enzyme methylates the same recognition sequence
• Type III - Cuts 24-26 bp downstream from a short,
  asymmetrical recognition sequence. Requires ATP
  and contains both restriction and methylation
  activities

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Restriction Endonucleases

• Scans the DNA molecule
• Recognizes a specific sequence of nucleotides
• Cuts the sugar-phosphate bond specifically
  between two nucleotides

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Restriction Endonuclease

• Base recognition
• 4 bases higher probability of finding
• 6 bases more common amount for most REs less
  sites on DNA and 4 base REs
• Ex. T7 DNA (40,000 bp) has no EcoRI sites
• 8 bases rare cutters
• 10 bases or more

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Sticky Ends, Blunt Ends
Sau3AI
5-G AATTC-33-CTTAA G-5
5-N GATC-3 3-CTAG N-5
EcoRI
5-G GATCC-3 3-CCTAG G-5
5-GTPy PuAC-33-CAPu PyTG-5
HindII
BamHI
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Restriction Endonucleases

• Some REs are unique, ex. BamHI
• Others have Isoschizomers
• Cla I - ATCGAT
• These enzymes have the same recognition site.
• Aag I,Ban III,Bsa29 I,Bsc I,BseC I,BsiXI,Bsp106
  I,BspD I,BspX I,Bsu15 I,

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EcoRIand recombination
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Restriction Digests Exercise

• In the early 1970's, there were reports of
  bacterial enzymes that would attack foreign DNA,
  cutting them into little pieces
• RE TheI recognizes the and cuts between h and
  e
• Count the letters between fragments of the
  sentence
• Line them up biggest to smallest

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Your Gel Top (Big Stuff, minus side)
There were reports of bacterial enzymes that
would attack foreign DNA, cutting them into
little pieces.
ere were reports of bacterial enzymes that would
attack foreign DNA, cutting th (64 letters) em
into little pieces (18 letters) e early 1970's
th (13 letters) In th (4 letters)
Your Gel Bottom (Small Stuff, plus side)