Mapping Second Chromosome Mutations to Defined Genomic Regions in Drosophila melanogaster.

Hundreds of Drosophila melanogaster stocks are currently maintained at the Bloomington Drosophila Stock Center with mutations that have not been associated with sequence-defined genes. They have been preserved because they have interesting loss-of-function phenotypes. The experimental value of these mutations would be increased by tying them to specific genomic intervals so that geneticists can more easily associate them with annotated genes. Here, we report the mapping of 85 second chromosome complementation groups in the Bloomington collection to specific, small clusters of contiguous genes or individual genes in the sequenced genome. This information should prove valuable to Drosophila geneticists interested in processes associated with particular phenotypes and those searching for mutations affecting specific sequence-defined genes.

The phenotypes associated with mutations often provide insights into the functions of genes. Indeed, much of genetics research involves explaining how mutations give rise to phenotypes. Newer methods for inducing mutations such as transposon excision, homologous recombination, and CRISPR-based disruption are particularly good for deleting coding sequences. Such knockout mutations are undeniably important in understanding the cellular roles of genes, but other kinds of mutations—such as those that reduce gene expression or protein activity or affect only certain protein isoforms or domains—can be informative in ways that knockout mutations are not (Venken and Bellen 2014). While point mutations can now be engineered, they usually reflect the biases of the investigator. Older methods that induce mutations randomly by chemical or irradiation treatments have the advantage of probing gene function blindly. They can reveal novel protein structure–function relations and elicit unexpected phenotypes.

Knowing the importance of mutations, the Bloomington Drosophila Stock Center devotes considerable effort to maintaining stocks carrying mutations. Many of these mutations have been characterized phenotypically, but they have not yet been associated with sequence-defined genes. These stocks are potentially valuable, but they are requested infrequently. Geneticists interested in particular sequence-defined genes often do not consider them as a source of potential alleles, because the mutations are generally not tightly mapped. Likewise, geneticists interested in particular processes might be more likely to study mutations with relevant phenotypes if they knew they would be relatively easy to associate with sequence-defined genes. The usefulness and popularity of these stocks would be improved tremendously by anchoring these mutations to the genome sequence map so that their relationships to annotated genes could be recognized more readily.

Our ability to map mutations to specific genomic intervals in Drosophila improved enormously when simple techniques became available for generating chromosomal deletions with breakpoints known at single-nucleotide resolution. Three large-scale projects, including one conducted at the Bloomington Drosophila Stock Center, generated deletions with molecularly defined breakpoints (Parks ; Ryder ; Cook ). Altogether, these deletions provide >98% genomic coverage and subdivide the genome into intervals of a median of nine genes (Cook ). Using these deletions, mutations can now be mapped to very small chromosomal regions, or even single genes, with simple complementation tests. Mutations can often be mapped even more closely with follow-up complementation tests involving chromosomal duplications (Cook ; Venken ), or mutations affecting single genes.

We report here the localization of 77 complementation groups in the Bloomington Drosophila Stock Center collection to defined genomic intervals and the mapping of eight complementation groups to individual genes. This work ties these mutations to single genes or small groups of closely linked genes, and increases the value of an underutilized set of stocks.

Materials and Methods

The data in this report came from fly crosses made on standard medium, reared under routine conditions, and evaluated by customary standards (details provided upon request). Genomic coordinates are given in terms of the Release 6 assembly, and gene annotations are those shown in the June 20, 2017 FlyBase release (FB2017_3). Supplemental Material, Table S1 in File S1 provides a list of stocks used and our sources.

Data availability

The accompanying tables contain complete mapping data. Stocks may be obtained from the Bloomington Drosophila Stock Center or Drosophila Genomics and Genetics Resources at the Kyoto Institute of Technology as indicated in Table S1 in File S1.

Results and Discussion

We identified a large set of second chromosome mutations in the Bloomington Drosophila Stock Center collection that had not been associated with annotated genes and used mapping information archived in FlyBase (http://flybase.org/), or recorded in publications to estimate the chromosomal positions of the mutations (Table S2 in File S1). We then made complementation crosses between stocks carrying the mutations and molecularly defined chromosomal deletions to place the mutations in defined genomic intervals that refine previous mapping (Table S3 in File S1). Subsequent crosses tested the mutations for allelism with mutations in sequence-defined genes. Table 1 summarizes our results. The number of candidate genes in each interval was initially determined by the overlap of deletions with transcribed gene regions. (We recognize this criterion is potentially misleading as it is possible for a deletion to remove gene regulatory regions and disrupt gene function even if transcribed gene regions are not deleted. Nevertheless, it is a reasonable and commonly employed practice for deletion studies.) From this total, we subtracted the number of genes with complementing mutations. (This criterion could also be misleading, because partial loss-of-function alleles can show intragenic complementation. Nevertheless, it is also a reasonable simplification for a preliminary mapping study.) We have provided a list of candidate sequence-defined genes for each complementation group in Table S4 in File S1. Table S5 in File S1 provides a full list of the informative mapping crosses. In every cross, we had experimental evidence indicating that both stocks were valid as follows. Every mutation being mapped failed to complement at least one deletion. Most stocks used to map the mutations were validated with independent control crosses to stocks carrying relevant, previously characterized, loss-of-function mutations or chromosomal deletions (Table S6 in File S1). For a dozen deletion stocks, noncomplementation of the deletion with one of the mutations we were mapping was taken as evidence the stock was intact, and no independent control cross was undertaken.

Table 1

Mapping complementation groups to specific genomic intervals

Complementation group	Genomic interval from deletion mapping	Complementing mutations	Noncomplementing mutations	Candidate genesa	Comments
abb	2R:23666959;23713811			13
bhe		Gs11, Sam-S1	dbrEP9, l(2)gl4		Our complementation tests with deletions (Table S4 in File S1) and these mutations indicate bhe1 is a multigene, terminal deletion (Df(2L)bhe) with a breakpoint between dbr and Sam-S. Polytene analysis showed a breakpoint at 21A5-B1. This is consistent with J. Kennison’s observation of at least one bhe allele failing to complement l(2)gl (cited in Lindsley and Zimm 1992). The mutant embryonic phenotype likely results from the disruption of several genes.
blo	2R:8733630;8898753	RyRQ3878X, snsMI12892, snsMI03001		11
bub	2L:7702880;7718010			6
c	2R:15918423;15950652			10	Our data are consistent with unpublished identifications of c as Strn-Mlck by Rodriguez (2004) and E. Spana and E. Green (personal communication).
cass	2L:17473293;17482011		Aac11k06710	1	These results show cass is the same gene as Aac11.
dw-24E	2L:4361214;4403405	Tps1k08903		9	Szidonya and Reuter (1988) mapped dw-24E left of Tps1, reducing candidates to five. Curry (1939) showed l(2)cg1 was originally present in the nearby dpy to cl region, but our crosses to spanning deletions showed no lethality, suggesting it was removed before the current stock was established.
eay	2R:18173570;18230554			11
flz	2R:8976399;9031045	NpMI00240, NpMI10279	CG8213MI04680	1	Our identification of flz as CG8213 is consistent with the independent, unpublished results of Anne Uv (cited in Geberemedhin 2011).
Frd	2R:23001651;23068684	twi1		6	Our mapping is based on the recessive lethality of Frd1. Frd1 mutants carry an intragenic deletion in PPO3 (Sugumaran and Chase 2004).
fs(2)abc	2R:15375176;15386324	dupPA77		1
fs(2)lto3	2L:19464056;19517610			14
fs(2)ltoQE45	2L:3656901;3713827			24
fs(2)ltoRM7	2R:23385467;23395914			2
hum	2R:7395885;7447410	soD		18	Heitzler et al. (1993) mapped hum left of so, reducing candidates to nine.
l(2)21Ba	2L:67365;159063	Sam-SR23, Sam-S1, Gs11		23	Caggese et al. (1988) showed l(2)21Ba is not the same gene as Gs1. Larsson et al. (1996) mapped l(2)21Ba right of Sam-S and left of Gs1, reducing candidates to five.
l(2)23Ab	2L:2677694;2753125			9	Littleton and Bellen (1994) mapped l(2)23Ab left of Pgk, reducing candidates to seven.
l(2)24Dc	2L:4162968;4197800			5
l(2)24Dd	2L:4031318;4162968	ed1		5	Szidonya and Reuter (1988) showed l(2)24Dc is not the same gene as ed.
l(2)24De	2L:4162968;4197800			5
l(2)25Cg	2L:5073453;5145500			5	Szidonya and Reuter (1988) showed l(2)25Cg is not the same gene as Msp300, reducing candidates to four.
l(2)34Db	2L:13721648;13800829			16	John Roote reported l(2)34Db maps right of P{lacW}TM9SF4k07245 (http://flybase.org/reports/FBrf0129261.html) and left of Df(2L)Exel7059 (personal communication), reducing candidates to nine. This is consistent with the tentative conclusion that l(2)34Db is the same gene as Sec71 (Ashburner et al. 1999).
l(2)35De	2L:15821840;15912343			15	Our data maps l(2)35De to one of two intervals, but this interval is consistent with the mapping of Ashburner et al. (1999).
l(2)36Ba	2L:16824908;16886557			14
l(2)36Fe	2L:18606977;18617225			3
l(2)37Ab	2L:18673286;18689053	msl-1kmB		4
l(2)37Ac	2L:18753432..18753444;18795820			2
l(2)37Dc	2L:19438065;19452918			4
l(2)37De	2L:19438065;19452918			4
l(2)37Di	2L:19464056;19517610	swm37Dh-1		13	Brittnacher and Ganetzky (1983) showed l(2)37Di is not the same gene as swm.
l(2)37Ea	2L:19517610;19528383			5
l(2)37Fb	2L:19576108..19576133;19586375			6	Rutledge et al. (1992) and Gay and Contamine (1993) showed l(2)37Fb is not the same gene as spi, reducing candidates to five.
l(2)37Fc	2L:19576108..19576133;19586375			6	Rutledge et al. (1992) and Gay and Contamine (1993) showed l(2)37Fc is not the same gene as spi, reducing candidates to five.
l(2)37Fe	2L:19586375;19753324	Lar13.2		7	Butler et al. (2001) showed l(2)37Fe is not the same gene as scw, reducing candidates to six.
l(2)38Ab	2L:20085397;20120504	nebk05702		7
l(2)38Db	2L:20449190..20458307;20638580			24–26
l(2)38Eb	2L:20680624;20770538	Hr3802306, Fs(2)KetRX3		11	Butler et al. (2001) and Kozlova et al. (2009) showed l(2)38Eb is not the same gene as dia, reducing candidates to 10.
l(2)38EFb	2L:20831386;20851900			6
l(2)43Ba	2R:7187225;7326951			9	Heitzler et al. (1993) showed l(2)43Ba is not the same gene as pwn, reducing candidates to eight.
l(2)43Cc	2R:7493197;7533553	dpaEY04015, dpa1		11	Heitzler et al. (1993) mapped l(2)43Cc right of dpa, reducing candidates to nine. MacIver et al. (1998) tentatively identified l(2)43Cc as didum.
l(2)43Da	2R:7493197;7533553	dpa1, dpaEY04015		12	Heitzler et al. (1993) mapped l(2)43Da right of dpa, reducing candidates to 10.
l(2)43Db	2R:7493197;7533553	dpaEY04015		12	Heitzler et al. (1993) mapped l(2)43Db right of dpa, reducing candidates to 10.
l(2)43Ef	2R:7665795;7708707	tor4, U2A1		9	Heitzler et al. (1993) showed l(2)43Ef is not the same gene as U2A or tor and maps right of U2A. Nagengast and Salz (2001) showed a U2A transgene did not rescue l(2)43Ef mutations. This reduces candidates to four.
l(2)43Eg	2R:7665795;7708707	U2A1		10	Heitzler et al. (1993) showed l(2)43Eg maps right of U2A and is not the same gene as tor, reducing candidates to four.
l(2)46Ca	2R:9875312;9922003..9927457	tea1755	Etf-QOf05640	1	These results show l(2)46Ca is the same gene as Etf-QO.
l(2)46Cb	2R:9875312;9922003..9927457	Etf-QOf05640, tea1755		8	O’Brien et al. (1994) showed l(2)46Cb is not the same gene as FMRFa, reducing candidates to seven.
l(2)46Cd	2R:9958120;10025288	eve3, eve5, Pal1ta-1, eIF3jk13906		9
l(2)46Db	2R:9959818;10025288	eve3, eve5, Pal1ta-1, eIF3jk13906, TER94k15502, TER94EY03486	TER9403775, TER9426-8, TER9422-30	1	These results show l(2)46Db is the same gene as TER94, even though l(2)46Db26 shows a complex complementation pattern with other TER94 alleles.
l(2)46Dc	2R:10025288;10030539			3
l(2)46Dd	2R:10030539;10078293			16
l(2)46De	2R:9959818;10025288..10025310	eve5, eIF3jk13906		8	O’Brien et al. (1994) mapped l(2)43De right of eve, reducing candidates to four.
l(2)46Df	2R:10030539;10078293			16
l(2)49Dc	2R:12894105..12894116;12940453..12949055			12–13
l(2)49Fa	2R:13197974..13198492;13219347..13219349			10	Lasko and Pardue (1988) showed l(2)49Fa is not the same gene as Orc3, reducing candidates to nine.
l(2)49Fg	2R:13219130;13249241			14	Lasko and Pardue (1988) mapped l(2)49Fg left of Dp, reducing candidates to three.
l(2)51Ea	2R:15218008;15262942		scb2	1	These results show l(2)51Ea is the same gene as scb.
l(2)57Ba	2R:21000163;21056798			8
l(2)57Bd	2R:21056798;21088247			12
l(2)57Cb	2R:21056798;21088247			12
l(2)57Cc	2R:21143577;21177310			12	J. M. O’Donnell et al. (1989) suggested l(2)57Cc is adjacent to Pu or overlaps it. Reynaud et al. (1999) suggested l(2)57Cc is not the same gene as Xpd.
l(2)57Cd	2R:21143577;21177310			12	J. M. O’Donnell et al. (1989) suggested l(2)57Cd is adjacent to Pu or overlaps it. Reynaud et al. (1999) suggested l(2)57Cd is not the same gene as Xpd.
l(2)57Ce	2R:21180990;21215223			9	J. O’Donnell et al. (1989) showed l(2)57Ce is not the same as tud. This reduces candidates to eight.
l(2)57Db	2R:21301798;21341647			23
l(2)57Eb	2R:21497209;21607081			16	J. O’Donnell et al. (1989), Price et al. (1989), and Schejter and Shilo (1989) showed l(2)57Eb is not the same gene as Egfr, reducing candidates to 15.
l(2)57Ec	2R:21497209;21607081			16	J. O’Donnell et al. (1989) showed l(2)57Ec is not the same gene as Egfr, reducing candidates to 15.
l(2)DA2	2L:9522946;9560489			7
l(2)DB2	2L:9897536;9908459			2
l(2)DB4	2L:9205076;9388129			20	Lane and Kalderon (1993) showed l(2)DB4 is not the same gene as Cks30A, reducing candidates to 19.
l(2)FE3			hoipk07104	1	These results show l(2)FE3 is the same gene as hoip. It was mapped with cytologically defined deletions (Table S4 in File S1), but not molecularly defined deletions, so no genomic interval is given.
l(2)N7-6	2L:9622987;9699225			5
l(2)N7-8	2L:9205076;9388129	Cks30ARA74		19	Lane and Kalderon (1993) showed l(2)N7-8 is not the same gene as Cks30A.
l(2)PC4-A	2R:18051197;18118348	stau1		11
l(2)PC4-D	2R:17782032;17792649			4	Mohr and Gelbart (2002) mapped l(2)PC4-D to Ubc10, CG5033 or Dhit, reducing candidates to two.
l(2)PC4-M	2R:17716263..17716471;17739901..17739916			11
l(2)PC4-P	2R:18621522;18639268			6
l(2)PC4-Q	2R:18621522;18639268			6
mat(2)syn-E	2L:10349604;10381214			11	Our data place mat(2)syn-E in the same general region as Clegg et al. (1993), but they placed it right of da and RpS27A.
moa	2R:22729367;22764935			3
ms(2)35Eb	2L:15912343;16025369			16
nrd	2L:2517598..2551864;2621016			16–19	Littleton and Bellen (1994) mapped nrd right of Drp1, reducing candidates to five. Our results are compatible with nrdD20 being associated with a small deletion as proposed by Littleton and Bellen.
P{f+13}30B	2L:9205076;9388129			20	These crosses show a lethal mutation (hereafter l(2)30ABa1) is caused by the P{f+13}30B insertion or is closely linked to it.
pd	2R:23811400;23844351	shu2		7
qui	2R:23659139;23713811			12
sat	2R:7493197;7533553		Orc1KO	1	These results show sat is the same gene as Orc1.
sie	2R:13034847;13159579			10

Excludes genes with complementing mutations from the set of contiguous genes defined by deletion breakpoints (Table S3 in File S1). Ranges reflect deletion breakpoint uncertainty. Candidate genes are listed in Table S4 in File S1.

We were able to map 77 complementation groups to the smallest chromosomal intervals possible using existing molecularly defined deletions (Table S3 in File S1). With follow-up complementation tests using existing point mutations and transposon insertions in annotated genes, we were able to map eight complementation groups to single annotated genes, but we did not exhaust all possible tests of this sort. In the final tally, we were able to map 84 of the 85 complementation groups to 26 genes or fewer. (We found the remaining complementation group, , to be a multigene deletion.)

Table 2 summarizes information on the mutations mapped to single annotated genes, and shows the diversity of interesting genes affected. This work has identified the first nontransposon alleles of two genes ( and ), and has added potentially important EMS- or irradiation-induced alleles to the other genes. While we have not attempted to assess the allelic strength of most of the mutations, we know the female-sterile mutation sat mapped to must be a partial loss-of-function allele because knockout alleles are recessive lethal (Park and Asano 2008). Mutations affecting a particular motif in the protein have been shown to cause the same defective eggshell phenotypes as sat (Park and Asano 2012), which suggests it too is domain specific. This result illustrates the importance of point mutations maintained in stock for their loss-of-function phenotypes: they can reveal aspects of gene function that would not be apparent from the phenotypes of gene knockouts.

Table 2

Complementation groups mapped to single genes

Complementation group	Summary
cassowary (cass)	cass mutations were isolated as recessive lethal mutations that result in lack of adhesion between wing surfaces in homozygous mitotic clones (Prout et al. 1997). cass is allelic to Aac11, which encodes an inhibitor of apoptosis homologous to human Apoptosis Inhibitor 5 (API5) (Morris et al. 2006).
filzig (flz)	flz mutations were isolated as recessive lethal mutations affecting the patterning of the embryonic cuticle (Nüsslein-Volhard et al. 1984). We found flz to be allelic to CG8213, which encodes a serine protease (Ross et al. 2003). Subsequently, we learned flz was also identified as CG8213 by Anne Uv (unpublished results cited in Geberemedhin 2011).
fs(2)abc	fs(2)abc (abnormal chromatin) mutations were isolated as recessive maternal-effect lethals causing abnormal embryonic nuclear divisions and defective chorions (Schüpbach and Wieschaus 1989; Vessey et al. 1991). fs(2)abc is allelic to SRPK, which encodes a Serine–Arginine Protein Kinase necessary for dorsoventral egg patterning, karyosome formation, and meiotic divisions (Barbosa et al. 2007; Loh et al. 2012).
l(2)46Ca	The recessive lethal l(2)46Ca is allelic to Electron transfer flavoprotein-ubiquinone oxidoreductase (Etf-QO), which encodes a component of the electron-transport chain that generates ATP from the breakdown of fatty acids (Watmough and Frerman 2010).
l(2)46Db	The recessive lethal l(2)46Db is allelic to TER94, which encodes a chaperone that targets ubiquitin-tagged proteins to the proteasome (Meyer et al. 2012).
l(2)FE3	The recessive lethal l(2)FE3 is allelic to hoi-polloi (hoip), which encodes a small nuclear ribonucleoprotein component of spliceosomes (Mount and Salz 2000).
satin (sat)	Schüpbach and Wieschaus (1991) showed homozygous satSC46 females lay eggs with thin eggshells. sat is allelic to Origin recognition complex 1 (Orc1), which is needed for chorion gene amplification (Park and Asano 2008).
l(2)51Ea	The recessive lethal l(2)51Ea is allelic to scab (scb), which encodes an α integrin involved in cell adhesion (Stark et al. 1997).

In conclusion, we have refined the mapping of a large number of second chromosome mutations that have been preserved at the Bloomington Drosophila Stock Center for their mutant phenotypes. This information will provide Drosophila workers opportunities to make connections between these mutations and genes they might be studying in defined chromosomal regions.

Supplementary Material

Supplemental material is available online at www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300289/-/DC1.

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