Transcript unc-32(III)
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
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
Migration
Pronuclei
Meeting
Neuclear centrosomal
Complex
Anaphase
Telophase
Cleavage
W
T
or617
W
T
or625
or617
WT
or625
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:
or617; him-5
MT3751 X
or625; him-5
MT464 X
or617; him-5
or625; him-5
or617 L.G. Mapping
Done at Room Temp.
(I)
P.O.
(II)
(III)
(III)
(V)
dpy 5 rol 6 unc 32 or617 him 5
;
;
;
dpy 5 rol 6 unc 32 or617 him 5
We will assume or617 mutation is located on L.G. (III)
(I)
F1
(II)
(III)
dpy 5 rol 6 unc 32 him 5
;
;
;
or617
Pick 3 or 4 L4 hermaphrodites (per plate, x20)
Put at 15°C to self fertilize
(V)
or617 L.G. Mapping
(I)
F2
(II)
(III)
dpy 5 rol 6 unc 32
1/4
1/4 dpy 5 1/4
rol
6
unc
32
dpy 5 rol 6 unc 32
1/2 ;1/2 ;1/2 or617
or617
1/4
1/4
1/4
or617
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:
dpy 5 rol 6
2 /3 unc 32
;
;
L.G. III
Will yield all hatching embryo
1/3
unc 32
dpy 5
unc 32
2
/3
dpy 5 rol 6
or617
;
L.G. II
;
Will yield 1/3 plates with dead embryo
or617
rol
6
1/3
or617
rol 6 unc 32
dpy 5 2 /3 or617
;
Will yield 1/3 plates with dead embryo
;
L.G. I
dpy 5
or617
1/3
or617
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
2
4
Nonhatching
0
0
1
1
4
2
or625
dpy-5(I)
rol-6(II)
unc-32(III)
unc-5(IV)
dpy-11(V)
lon-2(X)
Hatching
5
10
16
8
8
10
Nonhatching
2
4
5
5
8
6
or617 Confirmation Test
unc 4()rol 1 ()or617() him 5
;
unc 4()rol 1 ()or617() him 5
P.O
.
F1
F2
()or()
unc()rol
()or()
1/ 4
()or()
()or()
1/2
unc()rol
1/ 4 unc()rol
unc()rol
•Pick WT and put at 15°C to self fertilize
•Pick 30 unc/rol to self fertilize at 26°C
•Score
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