Description

X-linked female sterile screens in Drosophila have led to a tremendous increase in our understanding of the genetic control of oogenesis (Gans et al. 1975; Mohler 1977; Komitopoulou et al. 1983). However, many of the loci in these screens have not been mapped to a single gene and therefore remain a rich resource for further elucidating the genetic control of female fertility. fs(1)A1304¹ is one such allele that is germline dependent and results in a degenerative ovary phenotype (Gans et al. 1975; Khipple and King 1976; Mulligan 1981; Wieschaus et al. 1981; Mulligan and Rasch 1985; Lamnissou and Gelti-Douka 1985). We were interested in determining the mutation that leads to sterility in fs(1)A1304¹ females. Previous recombination mapping had placed fs(1)A1304¹ at 19±2 cM on the X chromosome (Gans, Audit, and Masson 1975; Khipple and King 1976). We confirmed the previous mapping interval by meiotically mapping fs(1)A1304¹ to the right of crossveinless (12 cM) and to the left of singed (22 cM). We began complementation tests for female sterility with known deficiencies tiling the crossveinless and singed region and placed the lesion within a roughly 235 kb region (Figure 1A, non-complementing Df(1)BSC276, BSC285, BSC286, BSC297, BSC351, BSC535, and sov) (Parks et al. 2004; Cook et al. 2012). Two duplications within this narrow region rescued fs(1)A1304¹ sterility and thus further narrowed down the possible location of the causal mutation (Figure 1A, Dp(1;3)DC486 and Dp(1;3)DC026) (Venken et al. 2010). The mapping results were somewhat ambiguous within this narrow region (discussed below). However, the smallest non-complementing deficiency, Df(1)sov, contains only the protein coding gene small ovary (sov) and non-coding RNA gene CR43496. We therefore decided to complementation test fs(1)A1304¹ with known alleles of sov. Flies homozygous for hypomorphic alleles of sov show a similar female sterility phenotype to flies bearing fs(1)A1304¹ while amorphic sov alleles are embryonic lethal (Wayne et al. 1995; Jankovics et al. 2018; Benner et al. 2019). We found that amorphic alleles sov^EA42 and sov^ML150 failed to complement fs(1)A1304¹ female sterility while the hypomorphic sov² complemented fs(1)A1304¹ sterility. Collectively this indicates that fs(1)A1304¹ is a sov allele (sov^A1304-1).

To determine the molecular lesion, we performed paired-end DNA sequencing on sov^A1304-1 females. The sov locus contains three annotated transcripts; sov-RA has an annotated upstream transcriptional start site while sov-RB/RC are annotated to use a downstream transcriptional start site (Thurmond et al. 2019). Our sequencing data suggested that sov^A1304-1 flies contained a deletion within the sov gene region that would delete a majority of the sov-RA 5′ UTR. Genomic PCR of this potential deletion confirmed the presence of a deletion in sov^A1304-1 flies (Figure 1B). Sanger sequencing of the sov^A1304-1 genomic PCR product showed that there was a 324 nucleotide deletion (chrX:6,756,385-6,756,709) and a 10 nucleotide insertion (TCAACCTTCG) in the sov-RA 5′ UTR and would therefore remove most of the annotated 5′ UTR and donor splice site (Figure 1C).

We are unsure why a duplication (Dp(1;3)DC026) and a deficiency (Df(1)BSC535) to the left of the sov region rescued and failed to complement sov^A1304-1, respectively. We also found that the small duplication of just sov and CR43496 (Dp(1;3)sov^{tCH322-191E24}) failed to rescue. We were not able to find any deleterious mutations or structural variants in our sequencing data to the left of sov that might indicate the presence of a second-site suppressor or long-range genomic interactions with the sov locus that are necessary for its proper expression. It is interesting that sov^A1304-1 had not been previously mapped to sov since the Mohler and Gans X-linked female sterile collections had been previously complementation tested inter se (Perrimon et al. 1986). We found that one of the original Mohler alleles, sov², complemented sov^A1304-1sterility and is thus possible that the other two Mohler alleles, sov¹ and sov³, behaved similarly, providing an explanation as to why sov^A1304-1 was not previously recognized as belonging to the sov locus. It would be interesting to determine if the 5′ UTR deletion of the sov-RA transcript found in sov^A1304-1 flies affects sov activity in other tissues of the body other than the ovary. There is no indication that sov-RA, or sov-RB/RC, is differentially expressed in the ovary or other adult tissues (Benner et al. 2019). Pole cell transplantation studies of sov^A1304-1 indicated that defects are germline dependent (Wieschaus et al. 1981; Lamnissou and Gelti-Douka 1985), however, sov is an essential gene that has been shown to dominantly suppress position-effect variegation in tissues such as the eye (Jankovics et al. 2018; Benner et al. 2019). It is possible that the deletion solely affects sov-RA and that the Drosophila ovary is more sensitive to loss of sov-RA, or sov transcripts in general, in comparison to other tissues since sov^A1304-1 females are viable but sterile. However, we have not directly measured the deletions effects on sov-RB/RC transcript levels, which might also be perturbed. The nature of the sov^A1304-1 deletion therefore provides a unique mechanism to further elucidate the function of Sov at potentially both the transcript and regulatory level in Drosophila.