Generation of a useful roX1 allele by targeted gene conversion

Abstract

Methods for altering the sequence of endogenous Drosophila melanogaster genes remain labor-intensive. We have tested a relatively simple strategy that enables the introduction of engineered mutations in the vicinity of existing P-elements. This method was used to generate useful alleles of the roX1 gene, which produces a noncoding RNA involved in dosage compensation. The desired change was first introduced into a genomic clone of roX1 and transgenic flies were generated that carry this sequence in a P-element. Targeted transposition was then used to move the P-element into roX1. Remobilization of the targeted insertion produced large numbers of offspring carrying chromosomes that had precisely introduced the engineered sequences into roX1. We postulate that this occurred by gap repair, using the P-element on the sister chromatid as template. This strategy was used to introduce six MS2 loops into the roX1 gene (roX1(MS2-6)), enabling detection of roX1 RNA by a MCP-GFP fusion protein in embryos. The roX1(MS2-6) remains under the control of the authentic promoter and within the correct genomic context, features expected to contribute to normal roX1 function. The ability to replace relatively large blocks of sequence suggests that this method will be of general use.

Keywords: dosage compensation; gene conversion; mutagenesis; roX.

Figure 1

The roX1^MS2-6 restores X-chromosome MSL localization. (A) MS2 loops in RNA enable transcript visualization with MS2 coat protein (MCP) fused to GFP. (B) Structure of the p[w^+mC GM roX1^MS2-6] transgene. Six tandem MS2 loops (322 bp) are inserted in a 4.9-kb genomic roX1 clone. (C) Polytene chromosomes from a male roX1^ex6roX2Δ /Y; p[w^+mC GM roX1^MS2-6] /+ larva were immunostained with MSL1 antibody detected by Texas Red. DNA is counterstained with DAPI. Restoration of X localization and spreading of MSL1 into the autosome flanking the p[w^+mC GM roX1^MS2-6] insertion site (arrow) is observed.

Figure 2

Strategy for targeted transposition into roX1. (Top) A p[w^+mC GM roX1^MS2-6] insertion on the third chromosome was mobilized in roX1^mb710 males with plArB (ry⁺) in roX1. (Bottom) Tandem insertions (roX1^[MS2-6]T2A, roX1^[MS2-6]T4B) retain plArB. The roX1^[MS2-6]R36A is a precise replacement of plArB by p[w^+mC GM roX1^MS2-6].

Figure 3

All predicted products of homology-dependent gene conversion are recovered. (A) The roX1^[MS2-6]T2A is a tandem insertion of p[w^+mC GM roX1^MS2-6] at the 3′ end of plArB. Alignment of the engineered roX1^MS2-6 (gray line) is shown collinear to and below the corresponding genomic sequence. The MS2 loops are 430 bp from the plArB insertion site. (B and C) Predicted products of homology-dependent gap repair and gene conversion. Left panels depict short repair tracts that do not incorporate MS2 loops; right panels depict longer tracts incorporating MS2 loops into the repaired chromosome. (B) Homology in roX1 precisely substitutes a portion of roX1^[MS2-6]T2A (thick gray line) at the plArB insertion site. (C) Homology in roX1 and at P-ends leads to retention of the 3′ P-end and duplication of 5′ roX1 sequence. (D) An imprecise excision removing w^+mC from roX1^[MS2-6]T2A. (E) MS2 loop incorporation was detected by PCR using primers (arrows) flanking the MS2 loop insertion site (top). The roX1^MS2-6 produces an 869-bp amplicon and roX1⁺ produces a 547-bp amplicon. Three representative excisions in each category are shown. Contraction of the MS2 loop array in excision 36A.1 was detected by a reduction of the amplicon to 800 bp (right). (F) Blot of EcoR1-digested DNA probed with the roX1 promoter (black bar, E). Hybridization to a single 4.9-kb roX1 fragment is seen in wild-type (WT) flies and in a gene conversion that did not incorporate MS2 loops or retain a P-end (roX1⁺). A single 5.2-kb fragment is detected in two precise conversions incorporating MS2 loops (lines 2A.1 and 4B.1). Hybridization to a single 5.1-kb band is observed in excision 36A.1, consistent with the reduced MS2 loop array observed by PCR. Line 2.5 is the imprecise excision depicted in (D). A 5.2-kb band from p[w^+mC GM roX1^MS2-6] and a 2.5-kb band produced by disruption of genomic roX1 by insertion of plArB are present.

Figure 4

The roX1^MS2-6 supports focal recruitment of MCP-GFP in male embryonic nuclei. Embryos were generated by mating roX1^MS2-6 roX2Δ; [w^+mC MCP-GFP] females to males carrying an X-linked [w^+mC Sqh-mCherry] transgene. Sons (roX1^MS2-6 roX2Δ/Y; [w^+mC MCP-GFP]/+) lack w^+mC Sqh-mCherry (A–E). Females express mCherry (F–J). A wild-type embryo reveals autofluorescence limited to the vitelline membrane (K–N). Details in (E) reveal MCP-GFP recruitment to a single domain within the male nucleus, consistent with X-chromosome painting. MCP-GFP recruitment is absent in the female nucleus (I, J). Each set of panels is derived from a single Z-plane image. The brightness of mCherry signals was uniformly enhanced for reproduction (C, H, and M). See Materials and Methods for details of photography and image processing.