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Fig. 1 (A-E) Tpl94D is transiently expressed during the switch between histones and protamines. (A,D) Anti-core histone antibody staining of transgenic flies (A) expressing protamine-eGFP and (D) bearing tpl94D-eGFP to compare histone and protamine expression in the same fly tissue. Core histones are detectable in young elongating nuclei and in early canoe stage spermatids but not in later stages. (B) Protamine-eGFP expression starts at the late canoe stage when histones have already gone and stays in the individualising spermatids. (C) Anti-Flag antibody staining of flies expressing histone H3.3-Flag. Histone H3.3 is detectable in young elongating nuclei and in early canoe stage spermatids and shows the same pattern as the core histones (A,D). (E) Expression of Tpl94D-eGFP starts in the early canoe stage and becomes highest in late canoe stage spermatids. In individualising sperm, it is not detectable (see D for histone staining of the identical slide). Bars, 5 µm. (F) Tpl94D is a HMG protein. The deduced 164 amino acid protein sequence of the tpl94D transcript is shown. The N-terminal HMG domain is underlined in red and lasts from amino acid 4 to 84. The HMG box was determined using the Ensembl genome browser (www.ensembl.org). (G) tpl94D expression is testes specific. RT-PCR of tpl94D from wild-type testes (t), carcass males (m), embryos (e), adult females (f) and larvae (l). The tpl94D-specific primers amplified a 361 bp cDNA fragment from the open reading frame of tpl94D in testes and in larvae. In carcass males and in adult females a 429 bp DNA fragment was amplified, whereas in embryos no fragment was detectable. A 397 bp cDNA fragment of the 
3-tubulin gene was amplified as control. The cDNA 3-tubulin fragment was visible in all samples, whereas DNA contamination shown by the 492 bp fragment was visible only in adult females and larvae.
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