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

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

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

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)

wormbook Next

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

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

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

WT or617 WT or625

or617 or625 WT

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

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

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

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

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

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

Or625 Dominance Test

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

Acknowledgements