Worm Breeder's Gazette 6(1): 32

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.

Hybridization of DNA Probes to RNA in situ

M.K. Edwards

In situ hybridization should prove useful for analyzing changes in 
RNA populations during C.  elegans development without isolating 
biochemical quantities of similarly staged embryos or specific tissues.
RNA can be detected in various fixed preparations with well-
preserved morphology.  Embryos, squashed according to the procedure of 
Gossettand Hecht (1979 C.  elegans meeting), maintain their cell 
boundaries so that the number of cells, and thus the developmental age,
is easily determined.  Larvae and adults, fixed in ethanol/acetic 
acid (3:1), frozen in O.C.T. compound from Tissue-Tek, and sectioned 
at -20 C, retain good tissue morphology.  With Nomarski optics, well-
preserved muscle, gonad and intestinal tissues can be recognized, and 
with polarization optics, muscle birefringence and gut granules are 
visible.  Alternatively, adults can be cut open in M9 such that the 
gonad and intestine extrude as separate tissues from the body of the 
worm.  When squashed in 45% acetic acid, frozen,and then fixed in 
ethanol/acetic acid, these tissues retain good Nomarski morphology.  
After hybridization of a radioactive DNA probe to these preparations, 
followed by autoradiography, grain densities can be determined for 
particular embryonic cells or adult tissues.
I can currently detect abundant RNAs using recombinant DNA probes.  
In situ hybridizations with cloned nick-translated DNAs containing 
ribosomal, histone, actin (from Jim Files), and myosin (from John Karn)
genes produce strong signals (10 to 200 fold over background after 10-
day exposure) under conditions in which non-homologous DNA (Charon 3) 
gives no signal over background.  In addition, three cloned DNAs from 
our Charon 3 library that plaque-hybridize with C.  elegans cDNA also 
hybridize to RNAs in situ.  These three probes show differential 
hybridization between young embryos (1-50 cells) and old embryos (
comma to hatching) and between two adult tissues (gonad and intestine).
Present work involves more precisely characterizing the amount of 
hybridization as functions of developmental time and tissue 
specificity for these and other clones.