Genetic Mapping of prod ^E.3.3 , a New Lethal Allele of prod

Abstract

The E.3.3 mutation was generated in a Flp/FRT EMS screen for conditional mutations that cause growth and developmental defects in a genetic background that blocks apoptosis. The mutations were conditional, based on the Dark ⁸² allele being present on the starting chromosome, and blocking canonical apoptosis in a homozygous state. The E.3.3 mosaic eyes exhibit defects in eye development including patches of rough eye and irregular surface structure. Whole Genome Sequencing and complementation mapping revealed E.3.3 as an allele of prod . Prod is a DNA-binding protein that binds satellite repeats and is involved in chromocenter formation during mitosis. Here we present a novel allele of prod , prod ^E.3.3 , that disrupts the functional region of the Prod protein resulting in disruption of typical eye structure, likely due to disruption of chromatid separation during development.

Figure 1. Characterization of the prod ^E.3.3 allele in mosaic eyes and genetic sequencing.

FRT42D, Dark ⁸² control mosaic eyes ( A ) have similar red (mutant cells): white (wild-type cells) ratio to FRT42D, prod ^E.3.3 , Dark ⁸² mosaic eyes ( B ). The FRT42D, prod ^E.3.3 , Dark ⁸² mosaic eyes are mis patterned (blue arrow), have potentially necrotic areas (white arrow), and have concave regions on the surface of the eye (red arrow). Sequencing of prod PCR products amplified from FRT42D, Dark ⁸² /CyO and FRT42D, prod ^E.3.3 , Dark ⁸² /CyO heterozygous flies identified a heterozygous peak ( D , G→A, red arrow) present in the mutant flies that was not present in the control ( C ). This nucleotide change at position 2R:18970530 creates a premature stop codon in the protein at amino acid 138 ( E , Q→STOP, red box).

Description

Undergraduate students in the Fly-CURE project (Merkle et al., 2023) characterize and map mutations identified in a screen for mutations that alter cell growth within the context of an undergraduate course. The mutant lines were generated in an EMS screen designed to identify genes that regulate cellular growth and development (Kagey et al., 2012) . The right arm of Chromosome 2 was screened using the Flp/FRT system to generate mitotic clones in a Dark ⁸² background that suppresses apoptosis (Kagey et al., 2012) . The Dark ⁸² loss-of-function mutation is the result of a transposon insertion that disrupts the Dark coding sequence, but also inserts the mini-white ⁺ gene allowing the identification of homozygous mutant tissue in the eye based on color. The growth defects that result from the E.3.3 mutation were examined by generating mitotic clones as a result of crossing w ^- , ey>FLP; FRT42D flies that express the FLP recombinase in the eye with flies that carry both the E.3.3 allele and Dark ⁸² allele on the FRT42D chromosome ( w ^- ; FRT42D, Dark ⁸² , E.3.3/CyO ). Tissue that was homozygous mutant for E.3.3 and Dark ⁸² was identified based on red color. These mosaic eyes were compared to the mosaic eyes from a control cross that did not include the E.3.3 mutation ( w ^- , ey>FLP; FRT42D x w ^- ; FRT42D, Dark ⁸² /CyO ). The E.3.3 mosaic eyes have a rough eye phenotype (blue arrow) with concave regions on the eye surface (red arrow) and some regions that appear glossy compared to the control mosaic eye (see Fig. 1A, B ). While the overall ratio of red to white tissue does not have a dramatic difference between the control and E.3.3 , the overall patterning and organization of the eye has been disrupted and the overall eye size does appear a bit smaller, suggesting E.3.3 has a role in eye development. Additionally, some mosaic E.3.3 eyes also have black spots suggesting potential necrotic tissue in the mutant eye (white arrow).

In parallel, female w ^- ; FRT42D, Dark ⁸² , E.3.3/CyO flies were crossed with males from the Bloomington Drosophila Stock Center 2R Deficiency Kit in order to map the E.3.3 mutation (Cook et al., 2012) . The Fly-CURE utilizes independent mapping experiments at each institution, so each cross was set up independently four times. For a cross to be designated a failure to complement, at least 50 curly winged flies have to be observed without any straight winged F1 progeny. These results were confirmed by independent replicates at each institution. Two of the 2R Deficiency Kit stocks failed to complement the FRT42D, Dark ⁸² , E.3.3 chromosome: Df(2R)ED2747 and Df(2R)BSC331 . Both of these deficiencies include the Dark locus (Gramates et al., 2022) , so it is likely that the failure to complement is due to the lethal Dark ⁸² allele. All of the remaining 2R Deficiency Kit stocks complemented the E.3.3 mutation, indicating that either the E.3.3 mutation lies in the same region of the chromosome as Dark , the E.3.3 mutation is not lethal (and, thus, could not be identified through complementation testing) or the 2R Deficiency Kit used did not cover the region of the chromosome that contained the E.3.3 . mutation. At this stage we took a similar approach to that of mutant B.2.16 (Vrailas-Mortimer et al. 2021) . Whole Genome Sequence analysis of the w ^- ; FRT42D, Dark ⁸² , E.3.3/CyO genome identified a point mutation (G→A) in the proliferation disrupter ( prod ) gene consistent with a premature stop codon (Vrailas-Mortimer et al. 2021) . The prod gene maps to nucleotides 18,969,404 to 18,971,133 on chromosome 2R ( D. melanogaster r6.56), which is located between two non-overlapping deficiencies in the Deficiency Kit, Df(2R)ED3610 and Df(2R)Exel6069 (Gramates et al., 2022) . The presence of this mutation in the prod gene and the location of prod in a region that is not covered by the Deficiency Kit used suggests that E.3.3 was an allele of prod , prod ^E.3.3 .

To confirm that prod ^E.3.3 is a lethal allele of prod , w ^- ; FRT42D, Dark ⁸² , E.3.3/CyO virgins were crossed with prod ^E (hypomorphic) and prod ^k08810 (null)males that carry lethal alleles of the prod gene (Török et al., 1997; Gramates et al., 2022) . The prod alleles did not complement prod ^E.3.3 , supporting the hypothesis that the lethal hit in E.3.3 is a null allele, prod ^E.3.3 . Based on newly available data on Flybase, there are only three of the ten prod alleles that have been characterized based on the nature of the allele. prod ^E.3.3 is the fourth characterized prod allele (Öztürk-Çolak et al. (2024)).

To confirm the molecular nature of the prod ^E.3.3 mutation, we sequenced PCR products from prod in w ^- ; FRT42D, Dark ⁸² , E.3.3/CyO flies and w ^- ; FRT42D, Dark ⁸² /CyO flies. Three different sets of primers were designed to ensure that the products overlapped and covered the entire coding sequence of the prod gene. PCR products of the expected size were visualized by gel electrophoresis and purified products were sequenced (Eurofins Genomics). The sequence chromatograms for both the mutant and control strains were examined for overlapping peaks that indicated heterozygosity. Only one heterozygous site was present in the E.3.3 sequences that was not present in the control sequences (see Fig. 1C, D ), and this site aligned with the mutation identified in the Whole Genome Sequence analysis. The sequenced mutation introduces a premature stop codon in the prod transcript ( Fig. 1E ) that interrupts the predicted coiled-coil domain of the protein, as well as terminates the protein sequence in a region that has been determined to be essential for DNA binding (Török et al., 2000). Thus, it is likely that the prod ^E.3.3 allele does not produce a functional Prod protein.

Based on both the genetic and molecular results, we conclude that prod ^E.3.3 is a newly identified allele of prod . The prod gene was initially identified as l(2)88/10 in a P-element screen that identified lethal P insertions with overgrowth phenotypes (Török et al., 1993) . It was later demonstrated to play a role in chromatid separation during mitosis, with homozygous mutants exhibiting a decrease in mitotic index and cell death in highly proliferative cell types (Török et al., 1997) . The previously identified functions of prod are consistent with the identification of an allele in a mosaic screen for growth defects.

Reagents

Bloomington Stock Center 2R Deficiency Kit
Deficiency Stocks	BDSC Stock #	Chromosomal Deletions	Complementation Results
Df(2R)ED2747	9278	2R:16829073..17097303	Failed to complement (overlaps Dark 82 )
Df(2R)BSC331	24356	2R:16869330..17139923	Failed to complement (overlaps Dark 82 )
Mutant alleles of individual genes
Gene	BDSC Stock #	Allele	Complementation Results
p rod	42688	prod E	Failed to complement
prod	10814	prod k08810	Failed to complement

w ^- ; FRT42D, Dark ⁸² /CyO (Akdemir et al. 2006)

w ^- ; FRT42D, Dark ⁸² , E.3.3/CyO (this study)

w ^- , ey>FLP; FRT42D (BDSC 5616)

Bloomington Drosophila Stock Center 2R Deficiency Kit (Cook et al. 2012)

y ¹ w ^67c23 ; P{w[+mC]=lacW} prod ^k08810 /CyO (BDSC 10814)

y ¹ w ^67c23 ; prod ^E /CyO, y ⁺ (BDSC 42688)

prod forward primer 1: 5' CAT CGA GCA CGC AGG C 3'

prod reverse primer 1: 5' CTC CAT CTC GTC GTC GTG C 3'

prod forward primer 2: 5' GAT GCT CAA TCT GAA GTC CG 3'

prod reverse primer 2: 5' AGC TTA TTG CCG GAG GAG G 3'

prod forward primer 3: 5' ACG CCG TCG AGT ACG TCA C 3'

prod reverse primer 3: 5' CGA CTG CTT AGA CCC ACT GAT C 3'