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.
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 delineated. 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 investigations.