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First published online 22 April 2008
doi: 10.1242/jcs.025320

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Drosophila melanogaster kl-3 and kl-5 Y-loops harbor triple-stranded nucleic acids

¹ Department of Genetics and Molecular Biology – "Sapienza" Università di Roma, 00185 Rome, Italy
² Department of Evolutionary Biology, University of Siena, I-53100 Siena, Italy

View larger version (23K): [in this window] [in a new window]   Fig. 1. Schematic representation of the Y chromosome of Drosophila melanogaster. From top to bottom: cytological localization of satellite DNAs (Bonaccorsi and Lohe, 1991; Lohe et al., 1993), Y chromosome diagram showing N-banding and Hoechst 33258 banding (Pimpinelli et al., 1976; Gatti and Pimpinelli, 1992), cytological localization of transposable elements (Pimpinelli et al., 1995; Berloco et al., 2005), Y chromosome diagram showing the cytological localization and organization of the fertility factors (Gatti and Pimpinelli, 1992), other genetic elements mapping on the Y chromosome (Gatti and Pimpinelli, 1992; Russell and Kaiser, 1993; Gepner and Hays, 1993; Carvalho et al., 2000; Carvalho et al., 2001).

View larger version (163K): [in this window] [in a new window]   Fig. 2. Cytological characterization of primary spermatocyte nuclei of D. melanogaster using Jel318 and Jel466 antibodies. (A,B) Phase contrast images. (A',B') Immunostaining images. (A,A') Jel318 immunostaining of slides pre-treated with trypsin; the antibody specifically recognizes the kl-3 loop but no other intranuclear structure. (B-B') Jel466 immunostaining; the antibody specifically recognizes the kl-5 loop although in some cases also a faint fluorescence on kl-3 loop is present. Note that Jel318 decorates a continuous kl-3 filament whereas Jel466 recognizes a granular matrix inside the kl-5 loop.

View larger version (67K): [in this window] [in a new window]   Fig. 3. Analysis of DNA fibers prepared from primary spermatocyte nuclei of D. melanogaster. (A-C) Staining with the DNA dye Hoechst 33258. (A'-C') Immunostaining produced by either Jel318 (yellow) or Jel466 (red) merged with the DNA staining (blue). (A') Jel318 specifically recognizes a long, continuous filament barely visible after Hoechst 33258 staining; (B') an almost integer loop occasionally visible in these preparations; image B is slightly overexposed compared with B', to show the DNA nature of the structure decorated by the antibody. (C') Jel466 recognizes a granular, dot-like matrix inside the kl-5 loop (see also Fig. 2); sometimes it is possible to see aligned dots along a DNA axis. Note that none of the antibodies shows cross reactions with the chromatin clumps which are present in these cells.

View larger version (75K): [in this window] [in a new window]   Fig. 4. Jel466-immunostaining of young primary spermatocytes of D. melanogaster. Blue, Hoechst 33258 staining; red, Jel466 immunostaining. Young primary spermatocytes were identified according to Cenci et al. (Cenci et al., 1994). All nuclei, prepared according to standard protocols (Piergentili, 2006) show a maximum of four to five small dots that sometimes congregate into large clumps.

View larger version (178K): [in this window] [in a new window]   Fig. 5. Cytological characterization of primary spermatocyte nuclei of Drosophila hydei using Jel318 and Jel466 antibodies. Both antibodies specifically recognize the pseudonucleolus and clubs loops formed by the Y chromosome of D. hydei. Note that Jel318 and Jel466 immunostainings are comparable. No trypsin pre-treatment has been performed on slides incubated with Jel318.

View larger version (34K): [in this window] [in a new window]   Fig. 6. Electrophoretic analysis of high molecular weight (HMW) ploypeptides expressed in D. melanogaster testes. (A) Testis HMW dynein complement from wild-type males reared at either 24±1°C (lane 1), or at the non-permissive temperature of 31±0.5°C (lane 2). The HMW dynein bands named as band 3 and band 5 (Goldstein et al., 1982) are clearly visible only in lane 1. (B) HMW electrophoretic pattern of testis extracts from males reared at 31±0.5°C (lane 1), and extracts of testis dissected from males reared at 24±1°C and immediately transferred at 32±0.5°C for either 2 hours (lane 2) or 1 hour (lane 3); note that lanes 2 and 3 exhibit the same electrophoretic pattern as controls (panel A, lane 1), indicating that bands 3 and 5 are stable under these experimental conditions.

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