Worm Breeder's Gazette 8(3): 93

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.

Expression of a Chimeric Gene Transformed into the Worm

R. Jefferson, D. Hirsh

We have constructed fusions of the col-1 gene from the worm with the 
E.  coli  -glucuronidase gene, introduced them into the germline of 
the worm, and studied their expression.  The fusions consist of the 
promoter region and the first several nucleotides of the col-1 coding 
sequence, the entire coding sequence of the bacterial  -glucuronidase 
gene, col-1 sequences encoding its 3' intron and the terminator region.
The fusions were either in frame or out of frame with the col-1 
initiator codon.  These constructions were injected into the gonad of 
gus-1(DH408) worms and detected in the progeny by dot blot 
hybridization.  DH408 is a strain with no detectable  -glucuronidase 
activity, and is used to increase the sensitivity with which we can 
measure the chimeric activity (Bazzicallupo, Jefferson and Hirsh, 
-glucuronidase activity was measured in extracts of the transformed 
worms.  DH408 transformed with the in-frame fusion showed 
glucuronidase activity, while two independent transformed lines 
carrying the out-of-frame fusion showed no detectable activity.  As 
the DNA was in the long extrachromosomal tandem array, we could use 
its characteristic instability to perform a cosegregation analysis.  
There was always a strict correlation between the presence of the 
transforming DNA and the glucuronidase activity.  We verified that the 
glucuronidase activity in extracts from the transformed line was due 
to the bacterial enzyme by using affinity purified antibodies directed 
against the purified bacterial glucuronidase.
col-1 has been shown to be transcribed primarily in the embryo (
presumably during the synthesis of the L1 cuticle) but also at lower 
levels in later stages (Kramer, Cox and Hirsh, in press).  To see if 
the col-1: glucuronidase fusion was being regulated temporally, we 
prepared extracts from staged populations of transformed worms and 
measured glucuronidase specific activity.  Specific activity is 
highest in eggs, and decreases markedly in later stages.  These 
experiments are being repeated, but although they are consistent with 
the wild-type col-1 transcription profiles, they are not sufficient to 
indicate temporal regulation.  To analyze spatial distribution of the 
glucuronidase activity, we have used enzyme histochemistry and immuno-
cytochemical techniques.  Although we can visualize activity in eggs 
using the histochemical stains, we have not yet achieved the 
resolution to say anything about localization.  These experiments are 
in progress.