1. Candidate Gene Approach - 1
Lecture 5
Candidate Gene Approach - 1
BSE652

2. To understand genetic basis of development
“How genes might specify the complex structures found in higher organisms is a major unsolved problem in biology.” Sydney Brenner, 1974 – “THE GENETICS OF CAENORHABDITIS ELEGANS”
“To discern the genetic contribution clearly, the thing to do is to keep the environment constant and change the genes.” From the Gene to Behavior, Seymour Benzer, 1971
“This is not easy to do with human beings; they are notoriously uncooperative and unwieldy experimental subjects, particularly if one must wait generations for the results.” Seymour Benzer, 1971

3. Which genes’ role should one study?
To understand genetic basis of development
Question
There are 15-25,000 genes in a given multi-cellular organism.
Which genes’ role should one study?

4. Saturation mutagenesis screen
Candidate gene approach
Mutate all genes, one gene at a time, to see which developmental process is being affected
Saturation mutagenesis screen

5. Drosophila as the model organism
Morgan, Sturtevant ………………
A culture was started where the gene is named after the mutant phenotype, white
C. elegans as the model organism
Sidney Brenner, Sulston and Horvitz ………………
Mutants were named in series based on gross phenotype – unc, lin, lon etc.
Zebrafish as the model organism
Chuck Kimmel, Christiane Nüsslein-Volhard
First vertebrate saturated mutagenesis screen, transparent embryo, very helpful for hematopoiesis research
Mouse as the model organism
Kathryn Anderson, Monica Justice
Very difficult to conduct. However, some key genes were discovered using this approach.

6. Nature, 1980

7. Outline of the crossing schemes used in the Heidelberg screen
Please read about balancer chromosomes
These are specific for specific chromosomes and prevent homologous recombination
Balancer/balancer animals are dead
Please remember that there is no meiotic recombination in drosophila males
And drosophila stocks cannot be frozen for later use, it has to be continuously maintained
The art and design of genetic screens: drosophila melanogaster
Daniel St. Johnston
VOLUME 3 | MARCH 2002
Compulsory reading

8. Cuticular patterning defect – the assay for the screen
Wingless
Staufen

9. Wild type
Dumpy
Small
Long
In C. elegans the tradition was not to name individual phenotype rather to name series. This screen also identified some mutants with movement defects – Uncoordinated or Unc mutants.
THE GENETICS OF CAENORHABDITIS ELEGANS
S. BRENNER

10. The lin mutants

11. Vulval defect mutants identified Notch pathway members – lin-12

12. Large scale genetic screen in a small vertebrate: zebrafish

13. Fig. 4. Examples of mutations with specific defects in the development of zebrafish embryos. AI! embryos shown are 24 hours-old. (A)Wildtype. (B) cyclops mutant with partial!y fused eyes (Hatta et a/ ). (C) Wildtype. (D) cyclops mutant showing the absence of a floor plate (Hatta et al., 1993). (E) Wildtype, (F) no tail mutant which lacks a differentiated notochord, has no tai! and abnormally shaped somites (Halpern et al.,1993). (GI Wildtype, (H) spade tail mutant accumulates trunk somitic mesoderm precursor cells in the tail (Ho et al., 1990).

17. Which genes will escape the scan?
Maternally supplied genes i.e. the reason why maternal effect screen had to be conducted separately.
Involved in patterning/differentiation of internal structures
Only first instance of essential function may be scored
Genes having redundancy