PowerPoint Presentation - Foundations of Biology

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Isaiah 404, 5 4 Every valley shall be exalted,
and every mountain and hill shall be made low
and the crooked shall be made straight, and the
rough places plain 5 And the glory of the LORD
shall be revealed, and all flesh shall see it
together for the mouth of the LORD hath spoken
it.
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Getting MeaningFromMolecular Data

• Timothy G. Standish, Ph. D.

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What are Genes?

• The one gene one enzyme hypothesis has been
  refined to mean each gene codes for a polypeptide
• Things get fuzzy when a specific locus codes for
  more than one polypeptide
• For the purposes of this class, we will define
  genes as segments of DNA that are transcribed and
  associated regions that control their
  transcription
• Genes may code for both polypeptides or RNAs

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Determination of Gene Numbers

• DNA sequences are considered to be the gold
  standard for determining the number of genes in
  an organisms genome
• The problem is that most organisms have
  un-sequenced genomes and, even when genomes are
  sequenced, deciding if a segment of DNA
  represents a region that is transcribed can
  frequently be difficult
• Searching DNA for open reading frames seems to be
  the most logical way of finding genes, but just
  because an open reading frame exists does not
  definitively answer whether it is transcribed

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Indirect Estimates

• DNA hybridization etc.

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Denaturation and Renaturation

• Heating double stranded DNA can overcome the
  hydrogen bonds holding it together and cause the
  strands to separate resulting in denaturation of
  the DNA
• When cooled relatively weak hydrogen bonds
  between bases can reform and the DNA renatures

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Denaturation and Renaturation

• DNA with a high guanine and cytosine content has
  relatively more hydrogen bonds between strands
• This is because for every GC base pair 3 hydrogen
  bonds are made while for AT base pairs only 2
  bonds are made
• Thus higher GC content is reflected in higher
  melting or denaturation temperature

Low melting temperature
High melting temperature
Intermediate melting temperature
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Determination of GC Content

• Comparison of melting temperatures can be used to
  determine the GC content of an organisms genome
• To do this it is necessary to be able to detect
  whether DNA is melted or not
• Absorbance at 260 nm of DNA in solution provides
  a means of determining how much is single
  stranded
• Single stranded DNA absorbs 260 nm ultraviolet
  light more strongly than double stranded DNA does
  although both absorb at this wavelength
• Thus, increasing absorbance at 260 nm during
  heating indicates increasing concentration of
  single stranded DNA

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Determination of GC Content
Tm is the temperature at which half the DNA is
melted
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GC Content Of Some Genomes
Organism GC
Homo sapiens 39.7
Sheep 42.4
Hen 42.0
Turtle 43.3
Salmon 41.2
Sea urchin 35.0
E. coli 51.7
Staphylococcus aureus 50.0
Phage l 55.8
Phage T7 48.0
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Hybridization

• The bases in DNA will only pair in very specific
  ways, G with C and A with T
• In short DNA sequences, imprecise base pairing
  will not be tolerated
• Long sequences can tolerate some mispairing only
  if -?G of the majority of bases in a sequence
  exceeds the energy required to keep mispaired
  bases together
• Because the source of any single strand of DNA is
  irrelevant, merely the sequence is important, DNA
  from different sources can form double helix as
  long as their sequences are compatible
• Thus, this phenomenon of base pairing of single
  stranded DNA strands to form a double helix is
  called hybridization as it may be used to make
  hybrid DNA composed of strands which came from
  different sources

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Hybridization
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Hybridization

• Because DNA sequences will seek out and hybridize
  with other sequences with which they base pair in
  a specific way much information can be gained
  about unknown DNA using single stranded DNA of
  known sequence
• Short sequences of single stranded DNA can be
  used as probes to detect the presence of their
  complimentary sequence in any number of
  applications including
• Southern blots
• Northern blots (in which RNA is probed)
• In situ hybridization
• Dot blots . . .
• In addition, the renaturation or hybridization of
  DNA in solution can tell much about the nature of
  organisms genomes

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Reassociation Kinetics

• An organisms DNA can be heated in solution until
  it melts, then cooled to allow DNA strands to
  reassociate forming double stranded DNA
• This is typically done after shearing the DNA to
  form many fragments a few hundred bases in length
• The larger and more complex an organisms genome
  is, the longer it will take for complimentary
  strands to bum into one another and hybridize
• Reassociation follows second order kinetics

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Reassociation Kinetics

• The following equation describes the second order
  rate kinetics of DNA reassociation

Cot1/2 is the point at which half the initial
concentration of single stranded DNA has annealed
to form double-stranded DNA
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Reassociation Kinetics
Higher Cot1/2 values indicate greater genome
complexity
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Reassociation Kinetics
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Repetitive DNA
Organism Repetitive DNA
Homo sapiens 21
Mouse 35
Calf 42
Drosophila 70
Wheat 42
Pea 52
Maize 60
Saccharomycetes cerevisiae 5
E. coli 0.3
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The Globin Gene Family

• Globin genes code for the protein portion of
  hemoglobin
• In adults, hemoglobin is made up of an iron
  containing heme molecule surrounded by 4 globin
  proteins 2 a globins and 2 b globins

• During development, different globin genes are
  expressed which alter the oxygen affinity of
  embryonic and fetal hemoglobin

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Model For Evolution Of The Globin Gene Family
Pseudo genes (y) resemble genes, but may lack
introns and, along with other differences
typically have stop codons that come soon after
the start codons.
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