A. DNA PROPERTIES
1. DNA structure
1.1 The four bases
1.2 Polymer structure (covalent bonds)
1.3 Deductions from X-ray diffractions
1.4 Hydrogen donors & acceptors
1.5 Hydrophobic base interactions: stacking
1.6 Summary of major forces
2. DNA strand separation
2.1 Cellular replication by strand separation
2.2 Denaturation: strand separation in vitro
3. Genome size & potential gene number
4. Formation of duplexes from single strands
4.1 DNA renaturation--reformation of duplexes
4.2 How fast do duplexes reform?
4.3 Duplex formation between different DNAs--Hybridization
5. Estimate of gene number from RNA:DNA hybridization
B. SIZING & MAPPING DNA
MOLECULES
6. Cutting & sizing DNA molecules
6.1 Restriction enzymes
6.2 Measuring DNA fragment sizes
7. Mapping DNA molecules with restriction enzymes
7.1 Complete digestion
7.2 Partial digestion
C. CLONING BASICS
8. Putting a genome into a cloning library
8.1 Overview
8.2 Vectors, hosts and transformation
8.3 Joining DNA fragments to a vector
D. COMPLEMENTATION
9. Genetic Heterogeneity & Complementation
E. GENE CLONING & ANALYSIS
10. Cloning & analysis of cloned genes
10.1 Cloning by selection
10.2 Cloning by homology
10.3 Analysis of a cloned insert
A. Deletion Analysis
B. Sequencing
C. Site-specific mutagenesis
D. Polymerase chain reaction
F. MUTATION & THE GENETIC CODE
11. Reversion of mutations
11.1 Exact reversion
11.2 Equivalent reversion
11.3 Pseudo-reversion: suppression
12. "General nature" of the code
13. Mutations creating STOP
13.1 Discovery of translation termination mutations
13.2 Hypothesis to explain rIIsus
13.3 Discovery of extragenic suppression of STOP
13.4 How does E. coli KB3 suppress the rIIsus mutation?
13.5 Example of a STOP! mutation in the human b-globin gene
G. GENE STRUCTURE & RNA SPLICING
14. Split genes & splicing
14.1 Continuous genes in bacteria
14.2 Split genes in eukaryotes
14.3 The mechanism of joining exon information
14.4 Splice signals in the nuclear RNA
14.5 Genome size & gene number revisited
15. Alternative splicing
15.1 Overview
15.2 Multiple products from one tissue
15.3 Different tissues/different splice products
15.4 Alternate products determining different developmental fates
H. RECOMBINATION MAPPING
16. Mapping genes by meiotic linkage
16.1 Assortment & "crossingover" during meiosis
16.2 Recombination mapping
16.3 Traits at many "levels" can be mapped
I. MAPPING & CLONING DISEASE GENES
17. DNA polymorphisms & linkage.
17.1 Restriction site polymorphisms (RSPs) as alleles
17.2 Meiotic linkage between gene and DNA polymorphism
17.3 How many DNA polymorphisms are needed?
17.4 Multiple allele DNA polymorphisms
18. Chromosome-scale DNA polymorphism Maps
18.1 Mapping by fluorescence in situ hybridization
18.2 Mapping with somatic cell hybrids
19. Linkage of disease trait & DNA polymorphisms
19.1 Meiotic linkage in pedigree analysis
19.2 Cytogenetic "shortcuts" to linkage: chromosome
abnormalities
19.3 Cloning the sequences between DNA polymorphisms
19.4 Finding genes within the contig
19.5 How determine if a "candidate" gene is actually
the disease gene?
19.6 Genome size & gene number revisited
20. Analyzing "disease" genes
20.1 Probable protein domain functions from database searches
20.2 Functional information from "model" organisms
20.3 "Interplay" of work on humans & "model" organisms
J. USING CLONED GENES
21. Gene therapy
2.1 Kinds of defects & the prospects for gene therapy
2.2 Some approaches to gene therapy
22. Screening for disease alleles.
23. Making mouse mutants: "knockouts" & "models"
23.1 Engineering a "cystic fibrosis" mouse
23.2 Toward understanding cleidocranial dysplasia (CCD)
24. DNA fingerprinting
K. MITOCHONDRIAL GENES
25. The mitochondrial genome
25.1 Mitochondrial genetics of yeast
25.2 Human mitochondrial DNA
26. Mitochondrial DNA diseases.
26.1 (revisited) Mutation rates: nuclear DNA vs. mtDNA
26.2 Some mtDNA Diseases
26.3 "Transmission" of mutant mtDNAs can be variable
L. RNA POLYMERASE
27. What RNA polymerase sees in a gene
27.1 How many different RNA polymerases?
27.2 Where (exactly) does transcription begin?
27.3 What sequence does RNA polymerase recognize?
27.4 Where on the DNA does RNA polymerase bind?
28. RNA polymerase mechanics
28.1 Opening the DNA duplex
28.2 Does complementary base pairing really guide polymerization?
28.3 How does RNA polymerase bind tightly and specifically to
a promoter
28.4 How does RNA polymerase loosen up enough to move along the
DNA?
28.5 How does Sigma give specificity to RNA polymerase binding?
28.6 Why doesn't Sigma bind to the promoter by itself?
M. REGULATION OF TRANSCRIPTION
29. Transcriptional activation.
29.1 Alternate sigmas to create alternate holoenzymes
29.2 Transcriptional activators--holoenzyme "clamps"
30. Transcriptional repression
30.1 The "inducible" E. coli lac system
30.2 Mutations affecting lac regulation
30.3 Where does the lac i repressor act?
30.4 Inactivation of the inducer-binding site of the lac repressor
30.5 "Repressible" gene systems
31. Summary of positive & negative control
N. DEVELOPMENT
32. Determination of the body axes
32.1 Drosophila development
32.2 Screening for developmental mutants
32.3 "Maternal" contributions to early development
32.4 "Maternal effect" mutations
32.5 1950's Observations on axis development
32.6 Maternal anterior determinants
32.7 Further development
33. Selector genes & master genes
33.1 Segment identity
33.2 Selector genes
33.3 Related homeobox genes in other organisms?
33.4 Master control genes
O. MUTATION
34. DNA damage & repair
34.1 Exogenous damage
34.2 Endogenous ("Spontaneous") damage
34.3 The special problem with 5-methylcytosine
35. Errors in DNA Replication
35.1 DNA polymerization.
35.2 Post-replication mismatch scanning
Q. CANCER
36. Cancer.
36.1 Suggestions of multiple steps in tumorigenesis
36.2 The cell cycle & proliferation
36.3 Proliferation "activator" genes--proto-oncogenes
36.4 Proliferation "inhibitor" genes--tumor suppressors
36.5 Different genes can be mutated in different cancer cells
36.6 Mutations that increase the frequency of other mutations
36.7 Programmed cell death--apoptosis genes
36.8 A lifetime of cell division--many oppportunities for mutation