Download DNA Replication and recombination

Announcements
1. Exam 2 next week: 10/17, 18, and 20. Same format as
exam 1, a bit shorter. You will need a bluebook again.
2. Answers from exam 1 will be removed from lab when
exam 2 answers go up.
3. Homework/ problem set 4 (15 pts) due next week in lab.
4. Handout for transformation lab available Monday.
Homework (15 pts) - BIO 326 Genetics
Due 10/15,16 in lab
1. Are sex chromosome aneuploids or autosomal aneuploids more common in humans?
Give one reason to explain your answer.
2. Red-green color blindness is an X-linked recessive disorder. A young man with a 47,
XXY karyotype (Klinefelter syndrome) is color blind. His 46, XY brother is also color
blind. Both parents have normal color vision. Where did the nondisjunction occur that
gave rise to the young man with Klinefelter syndrome?
3. Would a human with 2 X chromosomes and 2 Y chromosomes be male or female?
4. Predict the sex of Drosophila with the following chromosome compositions (A =
haploid set of autosomes):
a.
4X 4A
b.
3X 4A
c.
1X 2A
5. Mark true or false for the following relations regarding the percentages of bases in a
double-stranded DNA molecule:
a.
A+T=G+C
b.
A+G=T+C
c.
A+C=G+T
d.
A + T = 1.0
C+G
e.
A=T
G C
Review of Last Lecture
1. Polyploidy
2. Variation in structure/arrangement of chromosomes
- deletion
- duplication
- inversion
-translocation
3. What are criteria for genetic material?
Outline of Lecture 19
I. Evidence that DNA is genetic material
II. DNA and RNA: Composition and Function
III. History of solving the structure of DNA
I. DNA Structure and analysis
What is the genetic material?
Chromosomes contain protein and DNA - which is it?
What must genetic material do?
1. Replication
2. Storage of information
3. Expression of information
4. Variation by mutation - evolution
The Flow of Genetic Information
(The Central Dogma)
Trait
Is the Genetic Material Protein or DNA?
• Many favored proteins until the mid-1940’s.
• DNA is simple chemically (4 nucleotides
known); how could it then hold complex
genetic information?
• Proteins are much more complicated
chemically (20 amino acids) and more
abundant; perhaps they hold genetic
information.
Evidence for DNA as Hereditary Molecule
• Transformation studies
– Griffith (1927)
– Avery, MacLeod and McCarty (1944)
• Hershey-Chase experiment (1952)
• Chargaff’s Rules
• Molecular Studies
Griffith’s Transformation Expt.
Bacteria Used
Living smooth
(virulent)
Living rough
(avirulent)
Killed smooth
Living rough +
killed smooth
Expected mouse to live
Conclusion:
Killed smooth
converted living
rough to virulent cells
A Transforming
Principle (some
smooth component)
is responsible.
Avery, MacLeod, and McCarty Expt:
DNA is the “Transforming Principle”
IIIS
IIIS filtrate
IIR + IIIS filtrate
Active factor
Is DNA!
Hershey-Chase Experiment
• Study of infection of E. coli by T2 phage
• Radioactively labeled DNA and protein:
– 32P atom is in phosphate molecules in DNA
and RNA, only at low levels in protein
(phosphorylated proteins).
– 35S atom is in sulfur-containing amino acids
(cysteine and methionine); not in DNA or
RNA.
Phage Made Radioactive
Non-radioactive medium
+ bacteria
Phage Infect Cells
32P
Phage
Label
(DNA)
is inside
35S
Phage
Label
(protein)
is outside
Is DNA the genetic material in eukaryotes?
Indirect evidence -DNA and RNA absorb UV Light
• Action spectrum of UVinduced mutations in
bacteria correlates with
absorption spectrum of UV
light for nucleic acids, not
protein.
• Can use to quantify amounts
of nucleic acid and protein.
Direct evidence?
RNA is the Hereditary Material in RNA
Viruses, e.g. TMV
Tobacco Mosaic Virus
Reconstitution of Hybrid TMV
(Fraenkel-Conrat & Singer)
Strain 1
Strain 2
Lesions corresponded
to RNA
II. Structure of DNA/RNA
Bases and Sugars
pyrimidines
C
T
U
purines
G
Ribose
sugars
A
Bases and Sugars in DNA and RNA
• In DNA: deoxyribose + A, T, G or C
– dA
deoxyadenosine
– dT
deoxythymidine
– dG
deoxyguanosine
– dC
deoxycytidine
• In RNA: ribose + A, U, G, or C
–A
adenosine
–U
uridine
–G
guanosine
–C
cytidine
Nucleoside = Base + Sugar
Nucleotide = Nucleoside + Phosphate
U
dAMP
dNDP’s and dNTP’s:
Note Errors in the Text
deoxy
deoxy
deoxy
dNDP (dTDP)
deoxy
dNTP (dATP)
3’ to 5’ Phosphodiester Bonds Make the
Sugar-Phosphate Backbone
New monomers
add here
Strand has 5’-PO4
end and 3’-OH end
Chargaff’s Rules - also evidence for
DNA as genetic material
• 1949-1953, quantified amounts of each base in DNA
from different species.
• In every species, amount of A = amount of T, and
amount of G = amount of C
• If that’s true, then A + G = C + T
• The % GC and % AT varied from species to species,
but always adds up to 100%.
III. History of structure of DNA:
Rosalind Franklin and Maurice Wilkins found
X-ray Diffraction Evidence for DNA Double Helix
Linus Pauling and
Robert Corey at
Cal Tech were in
the Race to
Determine DNA
structure
James Watson and Francis Crick Used
Molecular Modeling to Deduce DNA
structure
The DNA Double Helix
DNA structure
• Double helical
–
–
–
–
major, minor grooves
right-handed
bases are 3.4 Å apart (10 Å = 1 nm)
10 bases/turn
• Complementary Base Pairing
– through H bonds: A=T, GC
• Antiparallel Strands
– 5’ to 3’
– 3’ to 5’
Right- and Left-handed DNA
Base-Pairing in DNA
A=T
GC
Reading DNA Strands
Single strand of DNA:
5’-AGCATTCG-3’
3’-TCGTAAGC-5’
Complementary strand of above, usually written 5’ to 3’:
5’-CGAATGCT-3’
Double-stranded fragment is written:
5’-AGCATTCG-3’
3’-TCGTAAGC-5’
Denaturation/Renaturation
Which DNA has higher GC content and why?
Nucleic Acid Hybridization
Molecular probes can “fish out”
a specific DNA from a mixture
Nucleic Acid Gel Electrophoresis