Lampbrush loop-specific protein of Drosophila hydei.

Abstract

By immunofluorescence techniques and protein blotting experiments we have shown that an antiserum specifically reacts with a Mr 80,000 protein (the "Ps protein") in the lampbrush loop "pseudonucleolus" in spermatocyte nuclei of Drosophila hydei. Comparative studies of X/Y and X/0 testes indicate that the gene encoding the Ps protein is not located on the Y chromosome but on an autosome or the X chromosome. The Ps protein is tissue specific. It is likely to be a rather conserved protein since the antigenic determinant recognized by the antiserum could be detected in the spermatocyte nuclei of a number of other Drosophila species. For those species with prominent Y chromosomal lampbrush loops, it could be shown that the cross-reaction is, as in D. hydei, associated with a specific Y chromosomal loop.During the primary spermatocyte stage the Y chromosome of Drosophila hydei (and many other Drosophila species) develops characteristic lampbrush loops of distinct morphology (1). The loops (2) result from transcription (3) MATERIALS AND METHODS Drosophila Stocks. D. hydei, wild type, Drosophila neohydei, wild-type, and Drosophila melanogaster, wild-type, were from our laboratory collection. D. eohydei was obtained from H. Beck, Geneva. All other Drosophila species came from the Drosophila Stock Center (Austin, TX).The 74/0 containing a Y chromosomal fragment with the pseudonucleolus (complementation groups B and C); and -X/ms (Y)C4 male sterile mutation in complementation group C, which "lacks" the pseudonucleolus in phase contrast. Immunological Procedures. Preparation of the antiserum against sph155 and the immunoblotting procedure have been described (14).For immunofluorescence staining, spermatocyte regions were collected in a drop of testis preparation buffer (10 mM Tris'HCl, pH 6.8/47 mM NaCl/183 mM KCl) on a microscopic slide. Coverslips were applied, and the preparations were gently squashed and then frozen in liquid nitrogen for 15 s. The coverslips were removed with a surgical knife and the preparations were passed through a series of ethanol washes (90%, 60%, 30%, 10%, 2 min each step). They were washed twice with phosphate-buffered saline (P1/NaCl), fixed in 3.7% formaldehyde in P1/NaCl for 10 min, and then incubated for 10 min with 3.7% formaldehyde/45% acetic acid in Pi/NaCl and extensively washed with Pi/NaCl. For immunofluorescence staining of spermatids and spermatozoa, the preparations were permeabilized for 5 min with 1% (wt/vol) Triton X-100 in Pi/NaCl and then washed. This treatment was omitted in cases in which spermatocyte nuclei were examined. The preparations were incubated for 30 min with 50 ,ul of antiserum or with pre-immune serum (both diAbbreviation: Pi/NaCl, phosphate-buffered saline. 3404The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Autosomal control of the Y-chromosome kl-3 loop of Drosophila melanogaster

“…The peculiar molecular structure of the loop-forming regions, consisting of large portions of satellite DNA interspersed with transposable elements, is maintained in different Drosophila species, although the DNA sequences involved are very different (Hennig et al 1974;Vogt and Hennig 1983;Hujiser et al 1988;Lankenau et al 1990;Hochstenbach et al 1994). Another conserved feature of the lampbrush-like loops is their association with a number of non-Y-encoded antigens, which include DNA-interacting proteins (Marhold et al 2002), RNA-interacting proteins (Risau et al 1983;Melzer and Glätzer 1985;Bonaccorsi et al 1988;Eberhart et al 1996;Cheng et al 1998;Heatwole and Haynes 1996), and testis-specific antigens that are incorporated either in nuclei (Svensson et al 2003) or in sperm tails (Hulsebos et al 1984;Bonaccorsi et al 1988;Pisano et al 1993;Lu and Beckingham 2000) during late stages of spermiogenesis.…”

The localization of histone H3.3 in germ line chromatin of Drosophila males as established with a histone H3.3-specific antiserum

“…On the other hand, one cannot exclude the possibility that the histones are at least partially bound to transcripts. Finally, it should be noted that, in immunological studies, a variety of proteins have been reported to be present in the Ychromosomal lampbrush loops (Hulsebos et al 1984 …”

The sphere organelle contains small nuclear ribonucleoproteins.

“…They were then rinsed in PBS and processed immediately for immunofluorescence. RNase A (100 ,ug/ml) and RNase T1 (50 units/ml) were used together in 0.3 M NaCl/0.03 M sodium citrate, pH 7.0.Immature oocytes from Acheta and Achaearanea were isolated in OR2 saline (11) and squashed between a microscope slide and coverslip as described (12). The slide was frozen in liquid N2, the coverslip was pried off with a razor blade, and the preparation was postfixed in 2% paraformaldehyde in 95% (vol/vol) ethanol for 30 min.…”