Worm Breeder's Gazette 9(3): 73

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.

Nuclease Hypersensitive Sites of the 16 kd Heat Shock Genes of C. elegans

D. Dixon, D. Jones and P. Candido

Recently our laboratory has cloned and sequenced six hsp16 genes at 
two chromosomal loci.  Regulatory sequences responsible for hsp16 heat 
induction have also been investigated by cloning the hsp16 genes, and 
in vitro constructed mutants thereof, into bovine papilloma virus 
vectors and transfecting the various constructions into mouse C127 
cells.  By assaying hsp16 heat inducibility in selected C127 clones, 
it has been shown that C.  elegans hsp16 regulatory sequences are 
correctly recognized in mouse cells and features of these 5' 
regulatory sequences important for proper heat induction have been 
We are now determining if the hsp16 genes in C.  elegans share any 
of the chromatin structural features identified in the heat shock 
genes of other organisms by examining nuclease hypersensitive sites 
within the genes and flanking sequences.  To facilitate this 
investigation we have developed a simple and rapid method for 
preparing large amounts of nuclei from frozen eggs, larvae and adults. 
These nuclei are essentially free of endogenous nuclease and protease 
activity and appear to be an excellent substrate for investigating 
chromatin structure in C.  elegans.The starting material consists of 
viable eggs or worms pelleted and resuspended with one-half the pellet 
volume of buffer A (250 mM sucrose; 10 mM Tris, pH 8.0; 10 mM MgC12; 1 
mM EGTA) and frozen into balls by dripping the slurry slowly into 
liquid nitrogen.  At all stages of the procedure, buffers contain 0.2 
mM PMSF and 7 mM mercaptoethanol, added fresh.  The frozen balls are 
stored at -70 C until needed.  Worms and larvae should be freed of 
bacteria by flotation in 30% sucrose, followed by a couple of washes 
in buffer A prior to freezing.
For a typical experiment nuclei are prepared by grinding one to two 
grams of frozen balls into a fine dust (Espresso grind) using a Waring 
blender equipped with a small, covered stainless steel cup (30 ml) 
that has been cooled on dry ice.  The grinding usually takes a couple 
of 15-30 second bursts at the highest speed.  Small dry ice chips may 
be added as a grinding agent and to maintain temperature, but be 
careful of pressure build up.  A chilled mortar and pestle also works, 
but is less convenient.  The worm dust is allowed to warm until it 
just melts, then 10 ml of ice-cold buffer A is added and the mixture 
blended at the highest speed for another 15-30 seconds.  The 
homogenate is collected and the blender cup rinsed with two more 10 ml 
aliquots of buffer A that are pooled with the homogenate.  All 
subsequent steps are carried out at 0-4 C.  Nuclei and debris are then 
pelleted at 4,000 x g for 5 minutes using a swing-out rotor (HB-4).  
The pellet is resuspended in 10 ml of buffer A containing 0.25% NP-40, 
plus 0.1% Triton X-100 and homogenized using ten strokes of a Dounce 
homogenizer with a tight fitting pestle.  The debris is removed by a 
low speed spin (40 x g for 5 minutes) and the supernatant containing 
the nuclei is carefully aspirated and kept on ice.  The debris pellet 
is reextracted by vortexing at maximum speed for 15-30 seconds in 10 
ml of buffer A without detergent.  The debris is removed again by a 
low speed spin and the nuclei pooled.  The debris pellet is 
reextracted one to two more times or until the turbidity of the 
supernatant drops significantly.  The pooled nuclei are pelleted at 4,
000 x g for 5 minutes, resuspended in 10 ml of buffer A and pelleted 
once again.
We routinely obtain the highest yield of nuclei from eggs, with 
yields in the order of one to two mg of DNA per gram of worms (wet 
weight).  Phase-contrast and fluorescence microscopy (using DAPI and 
ethidium bromide staining) indicate that the nuclei are clean, with 
little or no cytoplasmic tags.  There is some contamination by shards 
of cuticle, but this is estimated to be less than 1%.  Pelleting the 
nuclei through 2.5 M sucrose doesn't appear to significantly improve 
the purity.  We have used 18% SDS-PAGE to verify the absence of 
proteolytic activity during incubations up to 60 minutes at 37 C.  
Furthermore, all the histones are represented with little or no 
apparent contamination from ribosomal proteins.  Similarly, high 
percentage agarose gels and Southern blots have confirmed the absence 
of endogenous nuclease activity and micrococcal nuclease digests 
reveal a typical nucleosomal banding pattern with no evidence of 
nucleosome sliding.
We are currently using nuclei prepared by this procedure to map 
nuclease hypersensitive sites within the hsp16 loci from both control 
and heat shocked eggs and worms.  We have been able to obtain data for 
a variety of nucleases, including DNase I, micrococcal nuclease, S1 
and the N. crassa nuclease.  These data are beginning to reveal 
interesting features about chromatin structure and gene regulation in 
C.  elegans and, hopefully, the relative ease with which nuclei may be 
prepared will encourage other laboratories to undertake similar