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Facultative heterochromatization in parahaploid male mealybugs: involvement of a heterochromatin-associated protein

Dipartimento di Agrobiologia e Agrochimica, Università della Tuscia, 01100 Viterbo, Italy

View larger version (46K): [in a new window]   Fig. 1. (A) Western blot of protein extracts from Drosophila melanogaster and Planococcus citri probed with the D. melanogaster anti-HP-1 antibody as the primary antibody, and with goat anti-mouse IgG-HRP as the secondary antibody. Note the intense single band detected in D. melanogaster (lane 2), migrating, as expected (James and Elgin, 1986), at an apparent molecular weight of 34 kDa. P. citri protein extracts (lanes 3, 4) show two bands with apparent molecular weights of 29 and 46 kDa, respectively. The high molecular weight band is also present in control immunoblot (lane 6) probed with only the secondary antibody. M, molecular weight marker. (B) In situ immunofluorescence negative control, obtained by incubating cytological preparations with the secondary antibody only. Note the absence of any immunostaining on nuclei. Shown here are nuclei from a late blastoderm embryo. Scale bar: 10 µm.

View larger version (43K): [in a new window]   Fig. 2. HP-1 immunostaining pattern in interphase nuclei from blastoderm embryos. (A) Female nuclei. Immunostaining for HP-1 antibody (left) and staining for DNA (middle). The merged image (right) shows the punctate distribution of the HP-1 immunolabeling (green) over the DAPI-stained nuclei (red). (B,C) Male nuclei. Immunostaining for HP-1 antibody (left) and staining for DNA (middle). The merged image (right) shows the co-localization of the HP-1 immunolabeling (green) with the chromocenter in DAPI-stained nuclei (red). Note in B the two nuclei in the middle with the chromocenter split into two masses. In C, a prometaphase cell (top right) is also shown. Scale bar: 10 µm.

View larger version (29K): [in a new window]   Fig. 3. HP-1 antibody staining pattern in mitotic nuclei from blastoderm male and female embryos. Left: DAPI staining. Right: merged image, with DAPI fluorescence in red and HP-1 immunolabeling in green. The 10 euchromatic chromosomes of the female complement are shown in A. Note the scattered chromosomal distribution of HP-1 immunostaining. (B,C) Chromosomes from male nuclei in early (B), and late (C) metaphase. HP-1 immunostaining is concentrated over heterochromatic chromosomes (arrowheads), with the euchromatic homologs lacking any labeling (small arrow in B indicates the secondary constriction corresponding to the nucleolus organizing region of a chromosome). Note that, in C, the five euchromatic chromosomes reached a high degree of condensation and the differentiation between euchromatic and heterochromatic chromosomes by DAPI staining is less apparent. Scale bar: 10 µm.

View larger version (29K): [in a new window]   Fig. 4. Karyotype of female P. citri chromosomes based on the HP-1 antibody banding pattern. Note that the pattern, while remarkably similar between homologs, is not always completely coincident between the homologs and between different cells.

View larger version (18K): [in a new window]   Fig. 5. Comparison of the HP-1 antibody pattern (A) with C-bands (B). C-bands that coincide with HP-1 signals are indicated by arrows. Shown here are nuclei from a late blastoderm embryo. Scale bar: 10 µm.

View larger version (99K): [in a new window]   Fig. 6. A field of DAPI stained nuclei from an embryo at the seventh nuclear division (128 nuclei). Only one nucleus (insert) exhibits some DAPI brilliant spots over a dull mass of chromatin. Note that DAPI-positive spots are labeled by HP-1 antibody signal in this nucleus. No other cells show an appreciable HP-1 immunostaining. Insert shows DAPI staining (left) and merged images (right). In merged images, DAPI fluorescence is in red and HP-1 immunolabeling in green. Scale bar: 10 µm.

View larger version (52K): [in a new window]   Fig. 7. Male and female embryos during blastoderm formation (128-256 nuclei stage). (Left) Male embryo with different embryo sectors at different stages of heterochomatization shown. (A) Not all the nuclei show the typical chromocenter (DAPI staining alone on the top, and merged image on the bottom, with DAPI in red and HP-1 immunolabeling in green). Note that HP-1 immunolabeling is apparent even in nuclei that do not yet exhibit the chromocenter. (B) All nuclei have already formed a chromocenter (DAPI staining alone on the left, and merged image on the right, with DAPI in red and HP-1 immunolabeling in green). (Right) In the female embryo, nuclei do not show formation of heterochromatin. In the insert, some nuclei are shown at higher magnification, evidencing the dispersed appearance of HP-1 signal (DAPI staining alone on the left, and merged image on the right. In merged images, DAPI is in red and HP-1 immunolabeling in green). Scale bars: 10 µm.

View larger version (35K): [in a new window]   Fig. 8. Male embryo during blastoderm formation (256 nuclei stage). DAPI staining (left), HP-1 immunolabeling fluorescence (middle) and merged image (right), with DAPI in red and HP-1 immunolabeling in green. Note the large size serosa nuclei with no apparent chromocenter or HP-1 immunolabeling. By contrast, nuclei of the embryo proper, show a prominent chromocenter with the HP-1 immunolabeling signal on it. Scale bar: 10 µm (bottom right).

View larger version (82K): [in a new window]   Fig. 9. An advanced stage of blastoderm formation in a male embryo (512-1024 nuclei stage). Serosa cells forming a cap over the anterior pole. In all panels, DAPI staining alone is on the left and merged image on the right, with DAPI in red and HP-1 immunolabeling in green. (A) Higher magnification of embryo nuclei displaying a bright DAPI-stained chromocenter labeled by the HP-1 antibody. (B) Most of the serosa nuclei show immunolabeling over a differentiated mass of chromatin (arrows) not yet showing the characteristic bright DAPI staining of the chromocenter. Scale bars: 10 µm.

View larger version (50K): [in a new window]   Fig. 10. Male embryo at gastrulation with serosa cells covering the whole embryo length. Serosa cells show a well-formed chromocenter labeled by the HP-1 antibody. DAPI staining (left), merged images (right), with DAPI fluorescence in red and HP-1 immunolabeling in green. HP-1 immunolabeling is not apparent over the chromocenters of all embryo nuclei, because the immunofluorescent signals are scattered over many focal planes. Scale bar: 10 µm.