Title: PowerPoint Presentation - Foundations of Biology
1Isaiah 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.
2Getting MeaningFromMolecular Data
- Timothy G. Standish, Ph. D.
3What 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
4Determination 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
5Indirect Estimates
6Denaturation 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
7Denaturation 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
8Determination 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
9Determination of GC Content
Tm is the temperature at which half the DNA is
melted
10GC 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
11Hybridization
- 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
12Hybridization
13Hybridization
- 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
14Reassociation 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
15Reassociation 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
16Reassociation Kinetics
Higher Cot1/2 values indicate greater genome
complexity
17Reassociation Kinetics
18Repetitive 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
19The 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
20Model 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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