The I.3.2 developmental mutant has a single nucleotide deletion in the gene centromere identifier

Abstract

The mutation I.3.2 was previously identified in a FLP/FRT screen of chromosome 2R for conditional growth regulators. Here we report the phenotypic characterization and genetic mapping of I.3.2 by undergraduate students participating in Fly-CURE, a pedagogical program that teaches the science of genetics through a classroom research experience. We find that creation of I.3.2 cell clones in the developing eye-antennal imaginal disc causes a headless adult phenotype, suggestive of both autonomous and non-autonomous effects on cell growth or viability. We also identify the I.3.2 mutation as a loss-of-function allele of the gene centromere identifier ( cid ), which encodes centromere-specific histone H3 variant critical for chromosomal segregation.

Figure 1. Phenotypic characterization, genetic mapping, and molecular analysis of mutation I.3.2 .

A) Adult fly eye phenotype (control) showing mosaic pigmentation caused by Dark ^82[w+] (red) and wild-type (white) mitotic cell clones; B) Adult “headless” phenotype (dissected pharate) in flies creating I.3.2, Dark ^82[w+] and wild-type mitotic cell clones. Residual structures resemble adult mouthparts and arise from tissues not creating mutant clones. Headless I.3.2 clone phenotype from ventral (C) and dorsal (D) views. All images are 55X magnification. E) Genomic view of the cid locus (FlyBase) showing chromosomal deficiencies (red bars, bottom) that delete cid . The highlighted region (yellow) indicates the interval of overlap between Df(2R)BSC273 and Df(2R)BSC274 , both of which fail to complement I.3.2 . This region includes thirteen protein-coding genes (blue bars), one of which is cid (red arrow). F) Alignment of the wild-type and I.3.2 cid protein coding nucleotide sequences (first 150 nucleotides only), highlighting the predicted single nucleotide deletion (136delA) in I.3.2 (red). G) Alignment of the wild-type and I.3.2 amino acid sequences, showing the truncation of CID in the I.3.2 mutant well before the CID DNA binding domain (aa 119-223, gray region; SMART/EMBL). H) DNA sequencing chromatograms demonstrating the cid gene single nucleotide deletion in I.3.2 . Coding DNA control sequence (upper panel) from CyO homozygous embryos, which matches both the FlyBase reference sequence and the DNA sequence from Dark ^82[w+] / CyO control flies (as described in the text). In comparison, coding DNA sequences from I.3.2 / CyO heterozygous adults (middle panel) and I.3.2 homozygous embryos (lower panel) indicate a deletion present in the I.3.2 chromosome. Note that the identities of homozygous embryos ( I.3.2 / I.3.2 and CyO / CyO ) were inferred from the resultant DNA sequences. The relevant CAG nucleotide triplet is indicated in the control panel (C, gray highlight; A, red asterisk; G, blue highlight), with the A (136) predicted to be deleted in I.3.2 (Poly Peak Parser; Hill et al. 2014). This A peak becomes an A/C doublet in the I.3.2 / CyO heterozygote DNA sequence (middle, though the doublet is called an A because of slightly higher fluorescence), and many double fluorescence peaks continue to the left (arrow) but not to the right (from G, blue, and onward). This is pattern is consistent with deletion of the A prior to the G on the coding strand (when sequencing the template strand/reverse reaction). This is confirmed in I.3.2 homozygotes (lower panel) where sequencing shows the A nucleotide peak is missing and the flanking C (gray) and G (blue) nucleotides are directly adjacent, and that flanking nucleotides are all in register