Meiotic pairing and disjunction of mini-X chromosomes in drosophila is mediated by 240-bp rDNA repeats and the homolog conjunction proteins SNM and MNM

Abstract

In most eukaryotes, segregation of homologous chromosomes during meiosis is dependent on crossovers that occur while the homologs are intimately paired during early prophase. Crossovers generate homolog connectors known as chiasmata that are stabilized by cohesion between sister-chromatid arms. In Drosophila males, homologs pair and segregate without recombining or forming chiasmata. Stable pairing of homologs is dependent on two proteins, SNM and MNM, that associate with chromosomes throughout meiosis I until their removal at anaphase I. SNM and MNM localize to the rDNA region of the X-Y pair, which contains 240-bp repeats that have previously been shown to function as cis-acting chromosome pairing/segregation sites. Here we show that heterochromatic mini-X chromosomes lacking native rDNA but carrying transgenic 240-bp repeat arrays segregate preferentially from full-length sex chromosomes and from each other. Mini-X pairs do not form autonomous bivalents but do associate at high frequency with the X-Y bivalent to form trivalents and quadrivalents. Both disjunction of mini-X pairs and multivalent formation are dependent on the presence of SNM and MNM. These results imply that 240-bp repeats function to mediate association of sex chromosomes with SNM and MNM.

Figure 1.—

Organization of sex chromosome pairing region and composition of transgenes and chromosomes used in the experiments. (A) Organization of X and Y heterochromatin (rectangular regions of X and Y diagrams). Circles, centromeres. Shaded rectangles, 359-bp repeats (1.688 g/cm³ satellite). Crosshatched rectangles, rDNA. Arrows indicate pre-rRNA transcription units; arrowheads indicate 240-bp repeats. (B) Composition of [rib7] (Karpen et al. 1988) and [240⁺x8] transformation vectors. [240⁺x8] was generated by self-ligation of a single subcloned 240-bp repeat from [rib7]. (C) Two-step scheme for remobilization of [rib7] and [240⁺x8] (P[rib-i, ry⁺]) from X to Dp1187. [Δ2,3] is a stably inserted P transposase source on a third chromosome carrying the dominant marker Sb (Robertson et al. 1988). Selection for y⁺ ry⁺ males in the first generation ensures that the transposon has hopped off the X (which is transmitted only to daughters) and that Dp1187 is recovered. Selection for cosegregation of y⁺ and ry⁺ in the second generation selects for hops to Dp1187. (D) Mini-X chromosomes carrying rDNA insertions generated by remobilization and used in this study. Diagonally cross-hatched triangles represent [rib7] trangenes; horizontally cross-hatched triangles represent [240⁺x8] transgenes.

Figure 2.—

Analysis of mini-X pairing by FISH. (A) Examples of spermatocytes from Dp62-bearing males hybridized with a 359 Rpt FISH probe with zero, one, or two Dps (arrowheads) visible. DNA stained with DAPI. See Table 5 for frequencies. Bar, 5 μm. (B) Dp62 prometaphase I spermatocyte showing two 359 FISH signals [X and Dp62 (arrow)] within the X–Y bivalent. Bar, 5 μm.

Figure 3.—

Altered morphology of X–Y bivalent in Dp62 spermatocytes. Prometaphase I spermatocytes from Dp62-bearing males and wild-type males stained with an antibody against MNM. Note the broadened edge at the upper (MNM-stained) edge of the X–Y bivalent and localized finger of strong DAPI staining in the Dp62 spermatocytes compared to the more regular appearance of the X–Y bivalent in the wild-type spermatocytes. Bar, 5 μm.

Figure 4.—

(A) Representative FISH results from y/Y/Dp62; mnm^Z3-5578/Df(3R)T16 and y/Y/Dp62; snm^Z3-0317/snm^Z3-2138 males hybridized with a 359 Rpt FISH probe. Arrowheads indicate mini-X chromosomes. Bar, 5 μm. (B) Frequencies of prometaphase I and metaphase I spermatocytes with or without a visible mini-X chromosome in snm, mnm, and heterozygous snm/+ or mnm/+ control males carrying one copy of either Dp1187 or Dp62.

Figure 5.—

Illustration of chain-quadrivalent model for Dp-Dp segregation. Dp1 and Dp2 pair with opposite large sex chromosomes and each orients to the pole opposite that of its pairing partner. rDNA loci and transgenes are cross-hatched.