Discovery of supernumerary B chromosomes in Drosophila melanogaster

Abstract

B chromosomes are small, heterochromatic chromosomes that are transmitted in a non-Mendelian manner. We have identified a stock of Drosophila melanogaster that recently (within the last decade) acquired an average of 10 B chromosomes per fly. These B chromosomes are transmitted by both males and females and can be maintained for multiple generations in a wild-type genetic background despite the fact that they cause high levels of 4(th) chromosome meiotic nondisjunction in females. Most curiously, these B chromosomes are mitotically unstable, suggesting either the absence of critical chromosomal sites or the inability of the meiotic or mitotic systems to cope with many additional chromosomes. These B chromosomes also contain centromeres and are primarily composed of the heterochromatic AATAT satellite sequence. Although the AATAT sequence comprises the majority of the 4(th) chromosome heterochromatin, the B chromosomes lack most, if not all, 4(th) chromosome euchromatin. Presumably as a consequence of their heterochromatic content, these B chromosomes significantly modify position-effect variegation in two separate reporter systems, acting as enhancers of variegation in one case and suppressors in the other. The identification of B chromosomes in a genetically tractable organism like D. melanogaster will facilitate studies of chromosome evolution and the analysis of the mechanisms by which meiotic and mitotic processes cope with additional chromosomes.

Keywords: meiosis; minichromosomes; mitotic instability; supernumerary chromosomes.

Figure 1

B chromosomes contain centromeres. Fixed prometaphase I oocytes were prepared with antibodies against CID (yellow) and the DNA dye DAPI (blue). (A) Wild-type oocytes display the expected 8 CID foci. Arrows indicate the location of the 4^th chromosomes. (B) A mtrm¹²⁶/TM3 oocyte displaying 37 CID foci (mean of 29.3, SD 7.5, range 10–43, n = 49). (C) A WT+B oocyte with 19 CID foci.

Figure 2

B chromosomes are composed of 4^th chromosome heterochromatic sequences. Localization of FISH probes in fixed prometaphase I oocytes with DAPI (blue) and probes to the 359-bp heterochromatic repeat on the X (green) and to the AATAT heterochromatic repeats on a large region of the 4^th chromosomes and a small region of the X chromosomes (red). (A) Wild-type oocyte showing the expected localization of both probes with arrows indicating the location of the 4^th chromosomes. (B) A mtrm¹²⁶/TM3 oocyte showing the AATAT probe displaying additional localization to B chromosomes.

Figure 3

B chromosomes do not contain 4^th chromosome genes. (A) Schematic of the 4^th chromosome showing the distribution of the seven genes that were assayed by genetic approaches (Rsp3A, ci, and ey) or qPCR (ci, Crk, Asator, mav, and Caps). (B) qPCR analysis showing approximately equal relative quantities of the gene products tested in the wild-type and WT+B stocks. The 2^nd chromosome gene ord and the X chromosome gene y were used as controls.

Figure 4

B chromosomes in Drosophila melanogaster are smaller in size than the 4^th chromosomes. Larval neuroblasts from 3^rd instar larvae of (A) a normal wild-type female karyotype, (B) a male from the mtrm¹²⁶ stock displaying 12 B chromosomes, and (C) a WT+B female displaying 5 B chromosomes. As measured during cytological examination, the B chromosomes are ∼0.36 µm in size, while the 4^th chromosomes averaged 0.56 µm. Arrows indicate the location of 4^th chromosomes.

Figure 5

B chromosomes are maintained in a wild-type background. (A) Plot of the number of B chromosomes in individual metaphase preparations of WT+B larval neuroblasts from generations 2–6 in set 1 and set 2. Set 1 was initiated from crosses using mtrm¹²⁶/TM3 males, and set 2 was initiated with mtrm¹²⁶/TM3 females. For each set, the experiment was performed twice and the data were pooled. Larval neuroblast data from the mtrm¹²⁶ stock are shown for comparison (green). (B) Plot of the number of B chromosomes in individual metaphase preparations of larval neuroblasts from mtrm¹²⁶/TM3 flies outcrossed to wild type six consecutive times. Of the 52 larvae scored (482 cells total) in generation 6, only 12 still contained at least one cell with at least one B chromosome. Of these, only two larvae contained at least one cell with more than one B chromosome. Outcross A was started with mtrm¹²⁶/TM3 males, and outcross B was started with mtrm¹²⁶/TM3 females. The mtrm¹²⁶ stock data shown in A are again included for comparison. For each outcross, the experiment was started four times and the data were pooled. (A and B) In each experiment, generation 1 was not scored. For each plot, the centerline indicates the mean, with the shaded area containing the middle 50% of the observations. Notches represent the 95% confidence interval for each observation, and points represent outliers.

Figure 6

B chromosomes are mitotically unstable. Number of CID foci for 28 (brain A) and 30 (brain B) cells from two neuroblast preparations illustrating the mitotic instability of the B chromosomes. Dashes represent cells that we imaged but were unable to score. Bars, 2 µm.

Figure 7

B chromosomes significantly modify position-effect variegation. For each line, females carrying the reporter gene were crossed to males of the genotypes listed. (A) In(1)w^m4 flies were examined for their degree of PEV from crosses with a wild-type control, WT+B, or mtrm¹²⁶/TM3. (B) Four insertion reporter lines with the w⁺ gene inserted in the pericentric region of chromosomes 2 (39C-3 and Hs-5), 4 (118E-10), and the X (118E-32), were examined when crossed to a wild-type control or WT+B.