LECTURE 1. The Genome Age

Lecture 1MG701

The AGE of DNA: Three Notions Converged in the Construction of the Double Helix Model for DNA by Watson and Crick (1953)

Francis Crick James Watson Maurice Wilkins

Nobel Laureates: 1962

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 Franklin1920-1958

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 theactual amounts of eachbase, the proportion of G is alwaysthe 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 rangesfrom 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

Middle Repetitive

Unique

10K-50K copies/cell

100-10,000 copies/cell

1. Unique SequencesA. GenesB. Regulatory Sequences

2. Middle RepetitiveA. Gene families, e.g. histonesB. Ribosomal, tRNAs, other small RNAsC. Non-coding sequences -variable number tandem repeats (minisatellite) -short tandem repeats (microsatellite)

3. Highly Repetitive sequencesA. 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 DNAto 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                                                                

4100 genes 6000 genes 18,000 genes 14,000 genes

35-70,000 genes?

50 genes

THE AGE OF GENOMICSGenome Size and Gene Number in Model Organisms and Man

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 Genome-Wide 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)DNA2)RNA3) Protein4) Whole organism5) 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 chimpThis requires sequencing of many genomes.

2. RNASimultaneous 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. Proteina) monitoring the expression and modification state of all proteins in a cellb) 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 circuitryModel 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. Societya) for Scientists: Increased attention to ethical, legal and social issues (ELSI)b) For non-scientists: public education