Isolation of Nuclei from Crayfish Tissues and Demonstration of Nuclear Ecdysteroid Receptors

Margarethe Spindler-Barth 1 , Ursula Bassemir 2 , Paul Kuppert 3  and Klaus-Dieter Spindler 3
  • 1 Diabetes-Forschungsinstitut an der Universität, A ufm Hennekamp 65, D-4000 Düsseldorf 1
  • 2 EMBL, Meyerhofstr. 1, D-6900 Heidelberg
  • 3 Institut für Zoologie III, Universität Düsseldorf, Universitätsstr. 1, D-4000 Düsseldorf 1

Nuclei from hypodermis and midgut gland of the crayfish, Orconectes limosus, have been isolated with a slightly modified Chauveau-method. The isolated nuclei are well preserved and free from cytoplasmic contamination. They are able to incorporate [3H]UTP into acid-insoluble material. This labelling is α-amanitin sensitive. The isolated nuclei are devoid of the mitochondrial/microsomal ecdysone-C-20-hydroxylase. The DNA: RNA: protein ratio is 1:0.8: 3.2 (using Triton X-100 during the isolation procedure the ratio is 1:0.4: 4.6) for nuclei from the hypodermis and 1:1: 3.8 for nuclei from the midgut gland. The absolute amount of DNA per cell is 1.6, resp. 3.5 pg. The final DNA-recovery is about 20%. The yield increases to about 60% if the final ultrazentrifugation step is omitted. The isolated nuclei bind 3H-ecdysone and 3H-20-OH-ecdysone specifically. As revealed by Scatchard analysis the affinity of the nuclear 20-OH-ecdysone receptor is 1-2 × 10-9

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A Journal of Biosciences: Zeitschrift für Naturforschung C (ZNC) is an international scientific journal for the emerging field of natural and natural-like products. ZNC publishes original research on the isolation, bio-chemical synthesis and bioactivities of natural products, their biochemistry, pharmacology, biotechnology, and biological activity and innovative developed computational methods for predicting their structure and/or function.