1. Mapping and Characterizing temperature-sensitive, embryonic-lethal
C. elegans mutants with cell division defects
Sina Rahimi
Bowerman Lab

2. Lab Focus Early embryonic cell divisions
Find Genes required for cell division
Meiotic and mitotic spindle assembly and positioning
Cytoskeletal function

3. Caenorhabditis elegans
Free-living soil nematode
Feeds on microbes (E. coli )

4. Why C. elegans? Small and easy to maintain Short Life Cycle (3 days)
Large Number of progeny
Transparent DIC microscopy in living preparations
Completely sequenced genome
Five pairs of autosomes and one pair of sex chromosomes (Hermaphrodite XX, male XO)

5. wormbook
Next

6. The Approach Classical Genetics Leads to study of gene function
Find mutants with cell division defects
DIC microscopy
If interesting(?), find out what gene is mutated
Linkage group and 3-factor mapping
Leads to study of gene function

7. Summer Project Temperature Sensitive Mutants
or617
or625
Phenotype (homozygous): Hermaphrodite lays
Live embryo at 15°C
Dead embryo At 26° C
DIC movies
Linkage Group (chromosome) mapping

8. Wild Type (N2)
Pronuclei Pronuclei Neuclear centrosomal Anaphase Telophase Cleavage
Migration Meeting Complex

9. WT
or617 WT
or625

10. or617
or625 WT

11. Linkage Group mapping Which L.G is mutation on
Mapping Strains MT3751 and MT464
MT3751: dpy-5(I); rol-6(II); unc-32(III)
MT464: unc-5(IV); dpy-11(V); lon-2(X)
Crosses:
MT3751 X
MT464 X
or617; him-5
or625; him-5
or617; him-5
or625; him-5

12. or617 L.G. Mapping Done at Room Temp. (I) (II) (III) (III) (V) P.O.
We will assume or617 mutation is located on L.G. (III)
(I) (II) (III) (V) F1
Pick 3 or 4 L4 hermaphrodites (per plate, x20)
Put at 15°C to self fertilize

13. or617 L.G. Mapping (I) (II) (III) F2 Pick 30 of each class
Put at 26°C on separate plates
After ~ 2 days score the plates

14. How to Analyze If or617 is located on Linkage group III, then:
L.G. III
Will yield all hatching embryo
L.G. II
Will yield 1/3 plates with dead embryo
Will yield 1/3 plates with dead embryo
L.G. I

15. Linkage Group Mapping Results
or617
dpy-5(I)
rol-6(II)
unc-32(III)
unc-5(IV)
dpy-11(V)
lon-2(X)
Hatching 9 28 8 2 4
Non-hatching 1
or625
dpy-5(I)
rol-6(II)
unc-32(III)
unc-5(IV)
dpy-11(V)
lon-2(X)
Hatching 5 10 16 8
Non-hatching 2 4 6

16. or617 Confirmation Test Out of 26 plates, all had live embryos P.O.
Pick WT and put at 15°C to self fertilize F1
Pick 30 unc/rol to self fertilize at 26°C
Score F2
Out of 26 plates, all had live embryos

17. Or625 Dominance Test

18. Conclusions
Location of or617 mutation is most likely on linkage group II, however more genetics must be done to show confirm that it is NOT on L.G. I
Location of Or625 mutation can not be determined by the results. L. G. mapping must be repeated
More more DIC movies must be made for both strains to decide if they are useful for the focus of the lab and also how penetrant and reproducible they are

19. Acknowledgements