A Distal Heterochromatic Block Displays Centromeric Activity When Detached from a Natural Centromere  J.Suso Platero, Kami Ahmad, Steven Henikoff  Molecular Cell  Volume 4, Issue 6, Pages 995-1004 (December 1999) DOI: 10.1016/S1097-2765(00)80228-2

Figure 1 FLP-Induced Release of a Chromosomal Fragment (A) The second chromosome centromere is represented as a circle, FRT sites as grey arrows, and bwD as a closed triangle. Upon FLP induction, an unequal exchange can occur between sister chromatids. When this event becomes irreversible (because products drift away from one another), a dicentric chromosome and a released fragment result. The fragment is monitored for its ability to move during the next anaphase. (B) Cartoons depict the behavior expected for an acentric fragment (left) and for a fragment that moves to the pole (right), where white dots represent labeling of the bw gene region on distal 2R and the grey ovals represent labeling of the AACAC satellite marking 2R heterochromatin. (C) An example of released chromosome arm 2R in a homozygous larval salivary gland 1 week after FLP induction. The presence of band 55C4 on the proximal tip and band 55C5 on the distal tip indicates that the inverted FRT sites are close to one another. Ectopic fibers are often seen to connect bwD with 2R heterochromatin (Talbert et al. 1994). Molecular Cell 1999 4, 995-1004DOI: (10.1016/S1097-2765(00)80228-2)

Figure 2 Anaphase Movement of a Chromosomal Fragment Released by FLP Typical result of releasing a chromosomal fragment that either (A) lacks or (B) contains bwD. The acentric fragment (arrow) is identified by the 59E hybridization probe (green). In (B), two 59E hybridization spots are now present in the DAPI (blue) region of the lower nucleus. Occasional green spots that did not coincide with DAPI staining were not scored. Another probe, (AACAC)n (red), marks the base of the 2R chromosome arm. Molecular Cell 1999 4, 995-1004DOI: (10.1016/S1097-2765(00)80228-2)

Figure 3 Shapes of Released Chromosomal Fragments Carrying bwD (A) Anaphase figure illustrating the typical appearance of a released bwD-containing fragment that has not migrated to the pole. (B and C) Metaphase examples of a folded (B) and a straight (C) fragment. Chromosomes are stained with DAPI (blue). Anti-GAGA (red), which stains (AAGAG)n, identifies bwD on the fragment. Molecular Cell 1999 4, 995-1004DOI: (10.1016/S1097-2765(00)80228-2)

Figure 4 The Number of Minute Bristles in Flies Subjected to FLP-Induced Fragment Release Larvae that were 5 days old were heat shocked, and 12 major thoracic bristles were scored on all adults that showed scalloped wings, an indicator of dicentric formation just prior to determination of adult cell fate. Open bars, bw+-bearing flies; closed bars, bwD-bearing flies. Molecular Cell 1999 4, 995-1004DOI: (10.1016/S1097-2765(00)80228-2)

Figure 5 Mitotic Transmission of a bwD-Containing Chromosome Third instar larval neuroblast metaphase (A) and anaphase (B) after induction of FLP during embryogenesis. Note the presence of two chromosome fragments with bwD insertions (arrows) in each case. One arm of the parent second chromosome is truncated, direct evidence that the original dicentric broke and was transmitted. Chromosomes are stained with DAPI (blue). Anti-GAGA (red) identifies bwD on the fragments. Molecular Cell 1999 4, 995-1004DOI: (10.1016/S1097-2765(00)80228-2)

Figure 6 Mitotic Defects in Syncytial Embryos Surface (A) and interior (B) views of a wild-type (Amherst) embryo stained with DAPI. Surface (C) and interior (D) views of a bwD/bwD embryo illustrating the high frequency of fallen nuclei. Interior plane is 10 μm below the top surface plane. (E) Typical example (of nine observed) of an anaphase bridge from bwD embryos stained with DAPI (blue) and anti-GAGA (red) showing that bwD is not present between the nuclei. (A)–(D) were photographed with a 20× objective and (E) with a 63× objective. Molecular Cell 1999 4, 995-1004DOI: (10.1016/S1097-2765(00)80228-2)

Figure 7 Anti-3F3/2 Staining Colocalizes with bwD Third instar bwD/+ larval neuroblast metaphase figure stained with anti-3F3/2 (green) and anti-GAGA (red) and DAPI (blue). Arrow points to the location of bwD. Molecular Cell 1999 4, 995-1004DOI: (10.1016/S1097-2765(00)80228-2)