Download 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
or617
dpy  5  


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