Worm Breeder's Gazette 9(2): 11

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

A Drosophila Heat Shock Promotor Functions in C. elegans

A. Fire

Figure 1

In the last gazette, I reported that coinjection of one plasmid 
carrying the amber suppressor sup-7 (used as a selectable marker) with 
a second, unselected plasmid could be used to obtain stably 
transformed lines which contain sequences from the second plasmid in 
addition to sup-7 DNA.  I have used this to introduce a plasmid pShZ2 (
from Sean Munro) carrying a Drosophila heat shock promoter (HSP) fused 
to sequences coding for E.  Coli  -galactosidase (see diagram).  Of 
three independent stably transformed lines derived from these 
injections, two (CB4027 and CB4028) were analyzed on southern blots 
and found to contain the HSP- -gal fusion segment in apparently 
unrearranged form.   -galactosidase activities from these transformed 
lines have been compared with control lines both by soluble enzyme 
assays and by histochemical staining.
1.  SOLUBLE ENZYME ACTIVITIES: Worms were grown on a bacterial 
strain deleted for the  -galactosidase gene , harvested as L4s and 
homogenized by sonication (painful on the ears) or by a new 
'sandblasting' method (see below).  The resulting lysates were 
incubated with ONP  -Galactoside (yellow product) or 4-
Methylumbelliferyl  -Galactoside (fluorescent product) and enzyme 
activities determined spectrophotometrically.  A very low basal level 
of enzyme activity was detected both in the parental line, and in sup-
7 transformed lines not carrying the HSP- -gal fusion.  This basal 
level was not affected by heat shock (3 hours at 34 C).  Under normal 
growth conditions (20 C) the two lines carrying the HSP- -gal fusion 
produce levels of  -galactosidase similar to the parent line (within 2-
fold).  After heat shock, a striking increase of 10-20 fold in 
galactosidase level is seen with both CB4027 and CB4028.
2.  HISTOCHEMICAL STAINING: Whole worms were fixed and stained for 
gal activity as described below.  No staining was observed in the 
parental line (with or without heat shock) or in CB4027 or CB4028 
growing at 20 C.  After heat shock, strong and reproducible staining 
of both CB4027 and CB4028 was observed (>95% of the animals stain).  
The staining appears almost exclusively in the pharynx (all stages) 
and in embryos.  The localization of the stain is unlikely to reflect 
a simple permeability difference, since squashed and/or cut animals 
show similar patterns of staining.  Additionally, injection of pure 
enzyme into the gonad, gut or pharynx of wild type animals followed by 
fixation and staining as above resulted in specific staining in each 
case of only the injected tissue.
Histochemical staining is being used to examine the segregation of 
the inducible galactosidase activity: Preliminary evidence suggests 
that multiple loci (2-3) in CB4027 carry functional HSP- -gal segments.
In particular at least one locus unlinked to the amber suppression 
activity (which maps on chromosome IV(L) near unc-17) can confer heat 
inducible gal activity.  This is consistent with estimates of copy 
number for the gene fusion, between 5 and 20 for the two strains.  The 
possibility of extrachromosomal inheritance for the transgenic HSP- -
gal loci cannot yet be ruled out but seems unlikely due to the DNA 
copy number and the fact that inducible gal activities have been 
maintained in the two strains in the absence of selection for over 30 
A rough estimate of the induced  -Galactosidase as a fraction of 
total protein can be obtained either from soluble enzyme assays or 
from histochemistry (by comparison with injections of pure enzyme).  
These estimates on the order of .001% and .01% respectively (the 
difference may reflect losses in solubilizing the enzyme).
Why is staining in induced animals limited to the pharynx? Obvious 
suspects include the nature of the worm heat shock response, the 
synthetic rates and stabilities of  -gal protein and message in 
different tissues, and possible fortuitous expression signals in the 
HSP- -gal fusion plasmid that was used.  In any case the strains may 
prove useful in finding nematodes on warm summer nights.
SAND EXTRACT: To worms (10-100 l) in a 1.5ml microfuge tube are 
added about equal volumes of buffer and acid washed fine grain sand.  
The tube is then agitated vigourously-- I do this by striking the 
bottom of the tube along the pegs of a plastic (Gilson) test tube rack 
10-20 times (like bad, loud guitar playing).  Sand can be removed by 
centrifugation or filtration.  All the worms are lysed and completely 
broken up by this procedure and recovery of total protein and enzyme 
activity appear comparable to sonication.
-GAL STAIN: Wash worms in water and place in drop on a slide (I use 
4-well multitest slides).  Dry slides 5-10 min in a dessicator jar 
under vacuum (<2mbar).  Dip the dried slides in acetone and dry on 
benchtop.  Add 100 l of staining solution [0.2M NaPhosphate pH 7.5, 1.
0mMDTT; saturate with substrate (6Bromo2NapthylBDgalactoside from 
Sigma) and add 0.004% SDS] for 1 hr at room temperature.  Then add 20 
l of 0.125% 'fast blue B diazonium salt' [BDH], mix well, and staining 
should appear within 10-20 min.  The substrate is cleaved by  -
galactosidase to yield a local insoluble precipitate of 6Bromo2Napthol;
this reacts with the diazotized Fast Blue to form insoluble turquoise 
dye (Rutenberg et al J. Histochem. and Cytochem.  6, 122).  The fast 
blue must be dissolved (H20) just prior to use or a nasty orange stain 
develops which can obscure the reaction product.  Some staining near 
the mouth in induced live worms can be obtained using this protocol 
without drying and acetone steps.
[See Figure 1]

Figure 1