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Fig. 9. Localization of transgene loci and their transcripts during endosperm development. Endosperm nuclei counterstained with DAPI (blue) were hybridized with probes for the gene flanking regions and vector sequences of pHMW-1Ax1 and pHMW-1Dx5 to detect the locus (green) and with an antisense probe for the 1Ax1 coding region to detect the transcript (red). The insets are 2x enlargements of the loci marked by an arrowhead in the overlay. The sizes of the nuclei vary considerably owing to the cell-cycle phase they are in and because of endoreduplication at later stages. A and B are probably G1 nuclei, and C-E and G are probably G2 nuclei. F shows an endoreplicated nucleus. Each image is a projection of serial confocal sections. (A) Non-expressing nucleus in tissue starting to express. One locus is seen as one focus in the centre of the nucleus, whereas the two loci above and below are seen as two foci each. (B) Nucleus starting to transcribe the transgene in the same tissue as in A. All three loci are seen as two foci. Only the left-hand part of the locus denoted by the arrowhead is transcribing. (C) Nucleus in which all three loci are transcribing and condensed. The inset shows that some areas of the transgene locus are not transcriptionally active. (D) Transcriptionally active nucleus in the centre of the endosperm starting to decondense. One region in the locus in the inset shows no transcript. (E,F) Nuclei with fully decondensed loci, which are largely but not completely transcriptionally active. (G) Dying nucleus with dispersed transgene DNA and very weak RNA labelling. (H) Nucleus with degrading chromatin and no hybridization signal for the transgenes and their transcripts. Bar, 10 µm; section spacing is 0.4-0.6 µm.
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