Transcript No Slide Title

LECTURE 1.
The Genome Age
Lecture 1
MG701
The AGE of DNA:
Three Notions Converged
in the Construction of the
Double Helix Model for DNA
by Watson and Crick (1953)
Nobel Laureates: 1962
Francis Crick
James Watson
Maurice Wilkins
1. X-ray diffraction data
showed that DNA has the
form of a regular helix,
making a complete turn every
34 Å (3.4 nM),
with a diameter of ~20 Å (2 nM).
Since previous studies indicated
that the distance between
adjacent nucleotides is 3.4 Å,
there must be 10 nucleotides
per turn of the helix.
Maurice Wilkins
Rosalind Franklin
1920-1958
2. The density of DNA suggests that the helix must contain
two polynucleotide chains. The constant diameter of the
helix can be explained if the bases in each chain face inward
and are restricted so that a purine is always opposite a pyrimidine
3. Irrespective of the
actual amounts of each
base, the proportion
of G is always
the same as the
proportion of C,
and the proportion
of A is always the
same as that of T.
Thus the composition
of any DNA can be
described by the
proportion of its
bases that is G + C,
which ranges
from 26% to 74%
for different species.
The Watson-Crick Model:
1. TWO ANTI-PARALLEL
DNA STRANDS
2. COMPLEMENTARY
A:T AND G:C BASE PAIRS
3. BACKBONE FORMS A RIGHT
HANDED DOUBLE HELIX
4. BASES LIE PERPENDICULAR
TO THE AXIS OF THE HELIX
AND ARE "STACKED"
5. PITCH OF THE HELIX
IS 10 BP/TURN
Double-stranded DNA can be MELTED and REANNEALED
T(M)=temperature at which 1/2 of a DNA sequence of known
composition will be denatured (single stranded)
The T(M) is Directly Proportional to G-C Content of the DNA
T(M): DNA Concentration and Time are Critical parameters
At equal DNA concentrations, a smaller genome will
reanneal more quickly than a larger genome
Genome Analysis by Hybridization:
C0t1/2 for different Genomes
Is Proportional to Genome Size
Mammalian Genomes Contain
Three Classes of DNA
Highly Repetitive
10K-50K copies/cell
Middle Repetitive
100-10,000 copies/cell
Unique
1. Unique Sequences
A. Genes
B. Regulatory Sequences
2. Middle Repetitive
A. Gene families, e.g. histones
B. Ribosomal, tRNAs, other small RNAs
C. Non-coding sequences
-variable number tandem repeats (minisatellite)
-short tandem repeats (microsatellite)
3. Highly Repetitive sequences
A. Sequences involved in chromosome structure/stability
-centromeric (satellite) and telomeric
B. Transposable elements
-Long interspersed elements (LINES)
-Short interspersed elements (SINES)
THE AGE OF CLONING: Restriction Enzymes Allow DNA
to be fragmented in a sequence specific fashion
Discovery of
Restriction in
Bacteria(1962):
-phage produced in E.
Coli K12 could infect
K12 but not E. Coli B:
K12 phage growth
was "restricted" in B
strain.
Nobel Prize
1978:
Arber,
Nathans,
Smith
Subsequently
demonstrated that
bacteria contained
enzymes that were
responsible for phage
restriction:
A) Each bacterial strain
produced an
endonuclease that
cleaved foreign DNA at
specific sites: Restriction
enzyme.
B) The bacteria also
produced a methyl
transferase that modified
it's own DNA, thus
protecting against it's
cognate restriction
enzyme.
Restriction Enzymes allow Molecular Cloning of DNA
Polymerase Chain Reaction: Molecular Cloning
without vectors
Nobel Prize
1993
Kerry Mullis,
Michael Smith
THE AGE OF GENOMICS
Genome Size and Gene Number in Model Organisms and Man
50 genes
4100 genes
6000 genes
18,000 genes
35-70,000 genes?
14,000 genes
Goals of Genome Projects:
1) Complete Genetic Maps of the entire genome.
2) Complete set of contiguous clones that span the entire
genome.
3) Complete Nucleotide sequence of the genome
Yeast genome project*
http://genome-www.stanford.edu/Saccharomyces/
C. elegans genome project*
http://www.sanger.ac.uk/Projects/C_elegans/
Drosophila genome project*
http://flybase.bio.indiana.edu/
Mouse Genome Project*
http://www.informatics.jax.org/
Human genome project*
http://www.ncbi.nlm.nih.gov/genome/guide/
*all three goals completed
Future Laureates?
Craig Venter(Celera)
Francis Collins (NIH)
Genomics:
1) Structural genomics: the original Goals
of Genome Projects; largely complete for
the Human Genome Project
What do we do with all this information?
2) Functional Genomics:
Development and Application of GenomeWide Experimental Approaches to Assess
Gene Function by making use of the
information and reagents provided by
STRUCTURAL GENOMICS
In the Age of Cloning:
Identify and Study One Gene at a Time
In the Age of Genomics:
Study ALL GENES AT THE SAME TIME
Genome Projects are the Periodic Table for
Biological Sciences: will allow the
organization of 30,000-70,000 genes
Goals of Functional
Genomics:
1)DNA
2)RNA
3) Protein
4) Whole organism
5) Society
Lander, E. 1996. The New Genomics: Global
Views of Biology. Science 274: 536-539.
1. DNA level:
a) Systematic identification of all common variants
in human genes, both the coding
and non-coding regions.
These are the "isotopes" to gene "elements"
b) resequencing of entire genomes
of individuals
c) comparison of fully sequenced genomes of
related (and unrelated) species
EG: man and chimp
This requires sequencing of many genomes.
2. RNA
Simultaneous monitoring of the expression of all
genes
EG: What do gene expression patterns look like in
tumor vs. normal cells? What about following
chemotherapy? Will reveal Regulatory Networks.
3. Protein
a) monitoring the expression and modification state
of all proteins in a cell
b) systematic catalogs of all protein interactions
(e.g., yeast two hybrid interactions). Already
underway in yeast.
c) application of structural biochemistry to
genomics: classifying proteins by their shapes.
4. Whole organism
Genetic tools for manipulating cell circuitry
Model Systems are especially important.
a) systematic knockout and mutation of genes (already
underway in yeast): both stable and transient
b) transgenic studies
c) redesigning of cellular circuits (e.g., drosophila gal4
enhancer traps)
5. Society
a) for Scientists: Increased attention to ethical, legal
and social issues (ELSI)
b) For non-scientists: public education