P{U2af50-tra[F]} is the only constitutively feminizing tra[+] transgene I know of that will rescue tra null mutant females to fertility. In addition to being useful in situations where it is necessary to provide fully feminizing Tra[F] activity, it also allows one to keep recessive tra mutant alleles around without them accumulating deleterious extraneous mutations that interfere with tra behavioral studies--a serious problem in the past. In this connection, it is convenient that the transgene is on chromosome II, while tra is on chromosome III. Moreover, when tra mutant alleles are kept in this way, there is a selection to maintain full tra[+F] feminizing activity of the transgene.

Note that this stock is set up so that the Bar-eyed red-eyed phenotypic females are actually sterile, sexually transformed chromosomal males (pseudofemales), while the Bar-eyed white-eyed phenotypic males are fertile true males. Conversely, the non-Bar-eyed red-eyed phenotypic females are the rescued, fertile chromosomal tra mutant females, while the non-Bar-eyed white-eyed phenotypic males are the sexually transformed chromosomal females (pseudomales) lacking the transgene.

I've maintained the line without much difficulty for many years at 18C, the only caution being that the females tend to become infertile faster than normal females. Whether this is because of some innate failing of the tra[F] transgene, or because there was some mildly deleterious genetic garbage closely linked to the tra[5] mutant allele that was present prior to this mutant allele being more cleanly balanced with the transgene, I can't say. On the other hand, since we know that the Tra[F] level is clearly critical to this gene's functioning (too much Tra[F] will actually masculinize females--a fact not widely known!) and since this Tra[F] transgene is certainly not using the endogenous tra promoter, it wouldn't be surprising if the rescued tra female phenotype were not entirely wild type. Obviously with half the adults in the stock being infertile in any event, the stock's maximum fertility will be a bit down anyway. I have always maintained two independent lines of this just in case one develops serious fertility problems, but over these several years I haven't had to replace either line. I've had quite a few requests for this particular stock. The line can also be kept at 22C or 25C.

The origin of the constitutively feminizing P{U2af50-tra[F]} transgene is described in:

Evans, D.S. and T. W. Cline, 2007. D. melanogaster male somatic cells feminized solely by TRAF collaborate with female germ cells to make functional eggs. Genetics 175: 631-642.

and we used it in the following papers:

Siera, S.G. and T.W. Cline, 2008. Sexual back talk with evolutionary implications: stimulation of the Drosophila sex-determination gene Sex-lethal by its target transformer. Genetics 180: 1963-1981.

Evans, D.S., and T.W. Cline, 2013. Drosophila switch gene Sex-lethal can bypass its switch-gene target transformer to regulate aspects of female behavior. Proc. Nat. Acad. Sci. U.S. 110: E4474-4481.

P{UASp-Sxl.alt5-C8} is a somewhat unusual UAS-Sxl["+"] which has more SXL[F] feminizing activity, and that feminizing activity is more NORMAL in its action than the only other UAS-Sxl[+] previously reported: the "Daniel Bopp" transgene (P{UAS-Sxl.H}) used and referenced by Horabin et al (2003). That other conditional expression construct actually has deleterious effects on females that I believe are artifactual. Our transgene can even rescue Sxl null adult females somatically (although their viability is not 100% even with the two doses of the construct that the chromosome that this line carries). Since even the two isoforms generated by this expression construct lack the exon-10 and exon-9,10 C-terminal alternative isoforms that are required for female germline development, such rescued null mutant females are necessarily sterile. The trick to this transgene is that it is designed to generate BOTH of the two major exon-8 C-terminus isoforms, proteins that are generated by alternative splicing into exon 5. This alternative splice seems to be evolutionarily conserved in Sxl orthologs at least out to mosquitoes, so it is clearly very important to SXL functioning. Hence this is the GAL4 target of choice in any experiment exploring wild-type SXL protein function. Being a P{UASp} construct, it should be applicable to germline as well as soma. The two copies of the transgene present on this third chromosome are quite far apart so they are easily genetically separable.

P{UASp-Sxl.alt5-C8} isolation and use is described in:

Evans, D.S., and T.W. Cline, 2013. Drosophila switch gene Sex-lethal can bypass its switch-gene target transformer to regulate aspects of female behavior. Proc. Nat. Acad. Sci. U.S. 110: E4474-4481.

We used the attached-X in stock #58746 in combination with this X-chromosome balancer (58485) to easily generate very large populations of flies in which all adult viable progeny carry only one specific XX genotype and no XYs (the scheme also requires shi[1] on one of the chromosomes that are balanced).

Although I've heard of other balancer chromosomes carrying the "heat-shock hid" transgene P{hs-hid} that confers heat-shock inducible dominant embryonic lethality in this case, I don't know any for which just a 1-hr heat shock of embryos (we use a water bath) has been reported to reliably kill 100% of the animals carrying the chromosome--and probably kill as embryos, so that useless larvae aren't sharing the food with animals to be screened. Moreover, despite the fact that heat sensitivity at 37C is so tight, we found that both balancer chromosomes seem to be perfectly happy at 29C (an important consideration in our screen using shi[1] whose nonpermissive temperature is 29C). It really is only heat-shock per se--37C--that generates the lethal HID protein.

The cross strategy we used for producing large numbers of virgins automatically, as well as large numbers of males automatically, such that THEIR mating would then produce only one class of daughters is described in Fig. 2 of

Evans, D.S., and T.W. Cline, 2013, Drosophila switch gene Sex-lethal can bypass its switch-gene target transformer to regulate aspects of female behavior. Proc. Nat. Acad. Sci. U.S. 110: E4474-4481.

This strategy allowed us, in a matter of weeks, to carry out a genetic screen so powerful that using EMS for blind mutagenesis, we were able to generate a constitutive allele of the endogenous tra locus--one easily identified survivor out of 300,000 adults generated, all with the same initial genotype. Unfortunately the point of our isolation of a constitutive tra mutation prevented us from actually recovering it (the sterility of the particular mutant female carrying it answered an important question that we were asking in that experiment), but a minor modification of the screen (for example, using the Sxl[f2593] allele in a stock described below) would allow recovery of a constitutively feminizing tra mutant  allele--something that would be even more valuable for studying tra than even our tra[F] transgene described in above. (Note that tra is the "Sxl" in most insects, the only other cell-fate-determining gene known to operate by a pre-mRNA splicing positive feedback loop).  On the other hand, there are now methods for effective site-directed modification of fly genes that might be as effective or more for generating a constitutive endogenous tra mutant (but of course these kinds of modification lack the power of a true mutagenesis to deliver something not planned for).

Both lines also have the following other genetic items of interest, novelty, and utility in connection with Sxl studies:

Stock #58746 carries on its third chromosome three copies of a snf[+] transgene, P{snf[+],dhd[+]}, that allow one to greatly increase the dose of this fascinating splicing regulator. I thought that Bloomington already carried a third chromosome with one copy of this transgene (the transgene designated SL1) but all I could find recently looking through the stock list was a transgene designated SL2 on chromosome I (#4557) which is significantly less useful than one on III, particularly since it is cis to a deficiency nearby (it wasn't used to study snf). These multiple transgenes boost Sxl positive feedback greatly. We used the triple transgene in an extremely powerful genetic selection for P-element insertions into Sxl that demonstrates unequivocally the preference for P-elements inserting near ACTIVE promoters (unpublished). Others could certainly have other uses for this transgene.

Unfortunately I no longer keep a snf[1] mutant line rescued by these snf[+] transgenes or I would have sent that to replace the snf[1] line you are presently keeping (#4316) which is not kept in a way that would keep additional spontaneous mutations from arising that are deleterious to female viability or fertility (and I'm not in a position to construct such a stock at the moment). I can, however, provide a stock in which the snf[1] germline defect is specifically suppressed by an old germline-specifically-expressed Sxl transgene, P{otu-Sxl.cF1}. Kept in this fashion, snf[1] won't pick up any unrelated genetic garbage. See stock 58491 below.

The X-chromosome of the male in this line carries the null Sxl[f1] allele, both to act against loss of the attached-X by nondisjunction and to ensure that males are not harmed by the extra copies of snf[+].  Although Bloomington does already carry Sxl[f1], you do not have a version flanked by the closely linked benign markers cm[1] and ct[6]--marking that is very handy in stock construction.  By the way, although we haven't published the fact, we know that Sxl[f1] is a roo transposon insertion at 13258-62 in exon 8, consistent with its behavior as a genetic null.

P{snf[+],dhd[+]} is described in:

Larochelle et al. 1998, Cdk7 is essential for mitosis and for in vivo Cdk-activating kinase activity. Genes Dev. 12: 370–381

and we used it in:

Cline et al. 1999, Functioning of the Drosophila integral U1/U2 protein independent of U1 and U2 small nuclear ribonucleoprotein particles is revealed by snf[+] gene dose effects. Proc. Nat. Acad. Sci. U.S. 96: 14451-14458.

Stock 58485 is balancing a chromosome with two important new tools for sex-determination studies. Both are described in:

Evans, D.S., and T.W. Cline, 2013, Drosophila switch gene Sex-lethal can bypass its switch-gene target transformer to regulate aspects of female behavior. Proc. Nat. Acad. Sci. U.S. 110: E4474-4481.

Sxl[f18,f32]  This is a new, female-viable sex-transforming (masculinizing) loss-of-function double-mutant Sxl allele. The allele from which it was derived, Sxl[f18], is specifically defective with respect to all germline-autonomous feminizing functions of Sxl[F] almost certainly because it cannot generate the exon 10 and exon 9-10 alternative C-terminal isoforms. It is a G>A transition at bp #13472 (relative to the transcription start site of the maintenance promoter) that destroys the exon 8 3'ss used to generate the alternative exon nine and ten isoforms. It also substitutes aspartic acid for glycine in exon-8 C-terminal isoforms. We know that loss of the wild-type alternative 3'ss activates a cryptic 3'ss further downstream in exon 8, but use of this cryptic site causes a frameshift that would render the protein products nonfunctional. Since the allele has wild-type somatic functions (including full female viability), we can infer that the exon-8 C-terminal isoforms it generates are wild type. The Sxl[f32] lesion in trans to Sxl[f18] was generated as an intragenic suppressor of the germline defects of Sxl[f18] in an F0 genetic screen so powerful that it also yielded four true revertants of the Sxl[f18] lesion.

Remarkably, although the Sxl[f32] point change in the double mutant allows female germlines to develop normally without the exon10 and exon9-10 alternative C-terminal isoforms that would normally be required, this suppression comes at the expense of the double-mutant allele's ability to provide any somatic feminizing function, and at the expense of its ability to provide full somatic dosage compensation (female viability) function. It is the ability to regulate tra[+] that is lost, and as a consequence homozygous double-mutant chromosomal females are sexually transformed into pseudomales. These pseudomales are viable because dosage compensation function is not nearly so adversely affected as tra regulation. Nevertheless, these homozygous Sxl[f18,f32] mutant chromosomal females are less than fully viable (15-75%, with viability depending on genetic background). This reduced viability is likely due to modestly reduced positive autoregulatory activity of the Sxl[F] proteins generated, since if wild-type autoregulatory activity is provided just at the blastoderm stage (given a "kickstart"--see below), adult viability of these pseudomales reliably increases to near 100%. Importantly, Sxl[f18,f32] diplo-X animals are defective not only with respect to transformer-dependent feminization, but also with respect to transformer-insufficient feminization (an important point of the 2013 Evans and Cline paper).

Dp(1;1)SxlDeltaPm is a partial chromosomal duplication we refer to as "Sxl kickstarter." It is a 5'-truncated duplication of the entire Sxl locus that can provide a positive autoregulatory "kickstart" at the blastoderm stage--and only at that early stage--to raise the viability of Sxl[f18,f32] mutant females (phenotypically pseudomales) without affecting the specific activity of the mutant Sxl[F] proteins produced from Sxl[f18,f32]. This "kickstarter" can do the same for many other partial loss-of-function mutant Sxl alleles. Indeed as expected it can rescue females homozygous mutant for the classic "initiation defective" allele Sxl[f9] described below. It has no obvious deleterious effects on males, since the kickstarter relies on the Sxl[Pe] "establishment" promoter (its ONLY promoter), which is still properly responsive to X-chromosome dose (hence silent in haplo-X animals). For reasons that are unclear (perhaps a subtle deleterious effect of generally non-essential genes at the specific site of insertion of this chromosomal duplication), the kickstarter seems somewhat less effective when homozygous than when heterozygous. Hence the highest viability of homozygous Sxl[f18,f32] females (see above) is when they are heterozygous for the kickstarter allele, not homozygous. The use/study of Sxl[f18,f32] females should not be hindered in any way by the presence of the kickstarter that raises their viability to essentially 100%, since its products are only generated for a few hours soon after fertilization.

This 5'-truncated duplication of Sxl[M1] is located very near Sxl's close neighbor, cm. Its close proximity (a fraction of a map unit) to Sxl makes it a pain to construct different combinations of kickstarter and Sxl mutant alleles, but by the same token it makes it easy to keep intact any cis combination that has been constructed, even when not balanced. Complementation tests showed that the truncated duplication provides full Sxl[Pe] function but no Sxl[Pm] function, consistent with the molecular characterization of it as a copy of Sxl that carries everything in Sxl downstream of exon 1 (the exon generated by expression of the "maintenance" promoter--now absent from the truncated duplication), including a fully functional "establishment" promoter. The truncated duplication was generated by mobilization of a P{lacW} inserted in Sxl at +820-823 (relative to the transcription start site of the maintenance promoter--just downstream of exon 1). Mobilization was in the germline of an otherwise sterile Sxl[M1], P{lacW}B/Sxl[f4] female, the screen being an F0 screen for the recovery of fertility. My guess is that the P{lacW} first duplicated itself by hopping centromere distal to Sxl, and in a second mobilization step the two P{lacW}'s conspired to duplicate all of Sxl[M1] running from the original site of P{lacW} insertion to well beyond the most 3' end of Sxl. Finally, this duplication mobilized and inserted centromere distal to the original Sxl[M1] (leaving an intact Sxl[M1] allele behind in cis to complement Sxl[f4]--which we then replaced by recombination later). By the way, we determined that the duplicate is wild type for Sxl[f4], the allele in trans to the original P{lacW} insertion. Functionality of the 5' truncated Sxl[M1] duplication was assessed by complementation after replacing the intact Sxl[M1] with the deletion, Df(1)Sxl[f7B0] (a stock I no longer have).

A later note in response to a question about the breakpoints of Dp(1;1)SxlDeltaPm: With regards to Dp(1;1)SxlDeltaPm, the P{lacW} insertion which we mobilized to generate this peculiar duplication was one of our own. The original P{lacW} insertion site--using the release 6 coordinates (with the Sxl most centromere-proximal transcription start site as 7,098,053)--would be 7,097,233 (or 3 bp less; there was a 3 bp ambiguity due to duplication of sequence at the point of insertion). We know that the duplicated Sxl sequence lacks everything 5' of this point (centromere proximal), and that the duplication has full Sxl function, which means it undoubtedly goes beyond the most 3' end of Sxl (listed as 7,074,550). It does carry the roo transposon insertion at 7,087,891 (or 7,087,887 due to 5 bp ambiguity as a consequence of insertion site duplication) of Sxl[M1], the starting gain-of-function mutant allele, but that fact is likely irrelevant, since the "establishment" transcripts, which are all this allele appears to make (no "maintenance" transcripts), would splice around that transposon to make the female splice constitutively even in the absence of any SXL-F protein activity. It's P-element complement is a bit more ambiguous. It certainly carries miniwhite from the original P{lacW}, and since the duplication (or at least that miniwhite) can still be mobilized by P-element transposase (although I didn't manage to get it to reinsert on an autosome), it probably has P sequence of some kind at both ends of the duplicated, transposed region (perhaps all of the original P{lacW}). We made some effort to characterize it by Southern Blots once we got a deletion of the endogenous Sxl locus in cis but the results were never really clear cut and we didn't think it was worth pushing the study. How far downstream (3') of Sxl the duplication extends we don't know, only that it carries all of Sxl itself (listed as 7,074,550 in release 6). I doubt that there is very much duplicated or the construct would be more deleterious than it seems to be and would likely more drastically alter the cm-Sxl recombination distance interval into which it falls.

Bloomington comment: We have shown the breakpoints of Dp(1;1)SxlDeltaPm as ?-6F3;6F5;6E4, X:?..7074550;7097230..7097233;7004151..7009006 (R6). The distal end of the duplicated segment is ?-6F3, X:?..7074550, because the minimal extent (6F3, X:7074550) corresponds to the distal end of Sxl and the maximal extent is unknown. The proximal end of the duplicated segment corresponds to the position of the progenitor P{lacW} insertion (6F5-6F5, X:7097230..7097233). The insertion site of the duplicated segment is 6E4-6E4, X:7004151..7009006, because 6E4, X:7004151 is the proximal end of Inx2 and 6E4, X:7009006 is the distal end of cm.

Sxl[f7,M1]/Sxl[M1,fDelta33] This heteroallelic combination survives well as essentially completely masculinized (somatically) chromosomal females. Those animals are defective not only with respect to tra-dependent somatic feminization, but also with respect to tra-independent somatic feminization (the subject of Evans and Cline, 2013). It is similar to the classic masculizing combination Sxl[f7,M1]/Sxl[M1,f3] but more viable.

The Sxl[f7,M1] mutant allele has played a key roll in just about every paper I've published on Sxl since 1984. For that reason alone I think it is worth keeping at Bloomington. Since it is a Sxl[M1] derivative, it would not be trivial to reconstruct the allele even with the powerful new site-directed mutation strategies. Incidentally, having characterized an allele (independently isolated, remarkably enough) with the same point change (Sxl[f7]) but without the Sxl[M1] roo insertion, I know that the Sxl[M1] insertion is important for the functioning of Sxl[f7,M1]. Sxl[f7,M1] is the allele that in combination with Sxl[f9] generates fully viable females whose ovaries specifically disintegrate during metamorphosis--providing an assay (the only one known) for the surprising effect of TRA protein on Sxl autoregulation (see Siera and Cline, 2008, Genetics 180:1963-1981).

Sxl[M1,fDelta33] is an intragenic partial internal deletion (missing bp +3,410 through 7947) that is interesting in part because it generates partially functional SXL proteins missing the recently evolved N terminus. These proteins seem to correspond to more ancestral forms of SXL, proteins made when Sxl was not a sex switch (and probably still made in Drosophila via the newly discovered "exon Z"--see Cline et al. 2010, Genetics 186:1321-1336). Lacking the "establishment" promoter while still possessing a functional "maintenance" promoter, Sxl[M1,fDelta33] lacks all Sxl activity during the early X-chromosome counting period when sex-specific expression is set up, but constitutively provides its N-terminally truncated forms of SXL isoforms soon thereafter (and throughout development) when the "maintenance" promoter becomes active. It is described in Fig. 1 and  Materials and Methods of:

Evans, D.S., and T.W. Cline, 2013. Drosophila switch gene Sex-lethal can bypass its switch-gene target transformer to regulate aspects of female behavior. Proc. Nat. Acad. Sci. U.S. 110: E4474-4481.

In line 58486, Sxl[f7,M1] is effectively balanced by a precise duplication of the entire Sxl[+] gene: P{Sxl.+tCa}9A. This duplication contains all of Sxl[+] with nothing else duplicated. This duplication of the normally X-linked Sxl[+] is inserted on chromosome III. Such autosomal duplications of Sxl[+] are ideal for maintaining Sxl loss-of-function alleles free of spontaneously accumulating genetic garbage, something particularly important for female-sterile alleles like Sxl[f18] (see 58490 below) whose value is in the phenotype of homozygous mutant adult females. Moreover, this type of Sxl[+] duplication is especially important in the proper design of controls for Sxl mutant studies. When such duplications are allowed to freely recombine with a wild-type autosome in trans for several generations, they allow one to provide a Sxl[+] control for comparison with Sxl mutant situations in which the controls and experimentals are properly matched for genetic background--a critical point in many such studies, given the sensitivity of many Sxl mutant phenotypes to genetic background differences.

The derivation of this Sxl[+] duplication, and its use, are described in:

Cline et al., 2010. Evolution of the Drosophila feminizing switch gene Sex-lethal. Genetics 186: 1321-1336.

While we are on the subject of Sxl[+] precise autosomally located duplications, this stock might be useful for anyone wanting to increase Sxl[+] dose without increasing the dose of any other genes. This third chromosome carries two copies of Sxl[+] about 34 cM apart.

I was surprised to discover that Bloomington doesn't already carry Sxl[f9], the Sxl allele that is specifically defective for the "establishment" step (reading of X-chromosome dose) of the sex-determination process. This allele mimics zygotically the maternal-effect of daughterless on Sxl. As mentioned above, it is the Sxl[f9]/Sxl[f7,M1] heteroallelic females whose ovaries literally disappear during metamorphosis, the phenotype (or more appropriately, the sensitized genotype) that we used to demonstrate the surprising effect of maternal and zygotic Tra[+] protein on Sxl autoregulation. Sxl[f9] has been used in several of my papers, going back to 1994, but most recently in 2008 where its true molecular nature as a nonsense mutation in exon E1 was described (an A>T transition at position +5362 relative to the transcription start site of the maintenance promoter):

Siera, S.G. and T.W. Cline, 2008. Sexual back talk with evolutionary implications: stimulation of the Drosophila sex-determination gene Sex-lethal by its target transformer. Genetics 180: 1963-1981.

Sxl has an important role in germ cells both with respect to the control of cystocyte proliferation and with respect to meiotic recombination. Sxl has been shown to control the sex of germ cells, but it is unclear how that sex-determination function, a function required even before the gonad is formed, is related to these two more familiar germline functions. Sxl[f24,M1], in compound with Sxl[f4] or Sxl[f5], two more germline-specifically-defective alleles that Bloomington currently maintains, allows one to separate the cystocyte proliferation function (which is normal in that heteroallelic genotype) from the meiotic recombination function (which is not only grossly deficient, but more deficient on the autosomes than on the X chromosome). This heteroallelic genotype has the highest fertility by far of any strong meiotic-recombination-defective mutant known, whether involving Sxl or not. Sxl[f24,M1] is balanced by the classic (large) chromosomal duplication that was the only Sxl[+] duplication available prior to our generating the P{Sxl.+tCa} variety describe above (in connection with stocks 58486 and 58747). Sxl[f24,M1] is described in:

Sun, S. and T.W. Cline, 2009. Effects of Wolbachia infection and ovarian tumor mutations on Sex-lethal germline functioning in Drosophila. Genetics, 181: 1291-1301.

Although Bloomington currently carries Sxl[M1], the original "constitutively feminizing" gain-of-function allele, a dominant male-specific lethal (as stock #3719), we reported in 1995 that Sxl[M1] is not really a fully constitutive allele; instead, it has to ramp up through positive autoregulation over time to a level of full female transcript splicing, probably not reaching the fully constitutive level until late in embryogenesis. Moreover, mutations reducing Sxl positive autoregulation (such as snf[1]) can suppress completely its gain-of-function character. Hence I think it would be appropriate and useful for Bloomington to maintain a Sxl allele that is truly fully constitutive and whose constitutive character cannot be modified by mutations in other genes that affect pre-mRNA splicing. Sxl[M8] is just such an allele. It is a 110bp deletion that eliminates bases +9212 through +9321 (relative to the transcription start site of the maintenance promoter), taking out both of the 3' splice sites of the male-specific Sxl exon, as well as the polypyrimidine tract that those splice sites depend on. Consequently, the fully female pre-mRNA Sxl splicing pattern occurs by default in this allele, regardless of X-chromosome dose, and the gene is fully constitutive from the moment that the "establishment" promoter is active (recall that it is active in both sexes). The allele was recovered in the sisA suppressor screen described in:

Barbash, D.A. and T.W. Cline, 1995, Genetic and molecular analysis of the autosomal component of the primary sex determination signal of Drosophila melanogaster. Genetics 141: 1451-1471

However, that paper doesn't actually describe and characterize Sxl[M8]. For that information, the only reference available is the Ph.D. thesis of Dan Barbash:

Barbash, D. A., 1995, Genetic and molecular analysis of suppressors of the Drosophila melanogaster female-specific lethal mutation sisterlessA. University of California at Berkeley.

In line 58489, Sxl[M8] is conveniently and stably maintained in a balanced condition in trans with a male-viable deletion of the entire Sxl locus, Df(1)Sxl[fP7B0] (also known simply, though somewhat incorrectly, as Sxl[fP7B0]). This deletion cleanly (no residual P element) eliminates the entire Sxl locus; however, it does extend somewhat beyond Sxl, although not enough to reduce the viability of hemizygous mutant males. While in my lab as a postdoctoral fellow, Dr. Sha Sun used microarray analysis of mRNA in male heads that happened to characterize this deletion further with respect to its extent. Her unpublished data indicate that the centromere proximal breakpoint likely extends at least 10 kb past Sxl to eliminate fz4. The centromere distal breakpoint must NOT extend as far as nullo (ca 25 kb away), since nullo is an essential zygotic gene.  Although we know that Sxl[f1] is functionally a null allele (as I mentioned in connection with line (stock number coming soon) above), in some future studies of Sxl or of genes with which it interacts, it might be useful for the investigator to have a line in which all Sxl DNA is absent, but relatively little beyond that DNA is perturbed.

Since this Sxl[-] deletion is necessarily recessive female-specific lethal, and since the fully constitutive Sxl[M8] allele is dominant male-specific lethal, balancing the two mutations in this way maintains both chromosomes free of extraneous deleterious mutations. Because the stock as originally constructed was heterozygous for carmine (with the mutant cm[1] allele cis to Df(1)Sxl[fP7B0]) but homozygous for white, we can't tell simply by inspection whether the stock has remained heterozygous for cm[1] over the nearly two decades that we have maintained it this way. Hence the ambiguity indicated with respect to the cm genotype in the stock description.

The isolation of Df(1)Sxl[fP7B0] is described in:

Salz et al. 1987, Functional changes associated with structural alterations induced by mobilization of a P element inserted in the Sex-lethal gene of Drosophila. Genetics 117: 221-231.

We determined that this deletion eliminates the entire Sxl transcription unit and first refer to it as a "Sxl deletion" in:

Bell et al. 1991, Positive autoregulation of Sex-lethal by alternative splicing maintains the female determined state in Drosophila. Cell 65: 229-239.

Bloomington comment: We estimated the breakpoints of Df(1)Sxl[fP7B0] as follows. The distal breakpoint is somewhere between nullo and Sxl, so it is shown as 6F1-6F3, X:7050321..7074550 (Release 6), where 6F1; X:7050321 is the proximal end of nullo and 6F3, X:7074550 is the distal end of Sxl. The proximal breakpoint is shown as 6F5-7B1, X:7110321..7307939, where 6F5, X:7110321 is the proximal end of fz4 and 7B1, X:7307939 is the distal end of brk. We chose brk because it is the closest gene on the proximal side of fz4 known to be mutable to lethality.

I would recommend maintaining this old partial-loss-of-function Sxl allele because it is an excellent heat-sensitive allele. Its female-specific activities can be manipulated to a considerable degree by varying temperature. Indeed at low temperature, homozygotes survive, but they are intersexual ("true" intersexes, that is intersexual at the level of individual cells, unlike mosaic intersexes). Remarkably the original paper on this allele by Marshal and Whittle, 1978, mistook these viable intersexual animals for males, rather than the diplo-X mutant flies that they are, and as a consequence incorrectly concluded that the master regulator of fly sex determination, Sxl (then called Fl), was unrelated to sex determination! Ironically the allele was generated in the lab that I did my postdoc in, but before I arrived. Its isolation was never mentioned to me.  Importantly, Sxl[f2593] in combination with the Sxl[f18,f32] allele (see 58485 above) at the appropriate temperature generates highly viable diplo-X females who are almost completely masculinized somatically, but in contrast to the other masculinized adult viable genotypes described above, are NOT defective with respect to transformer-insufficient feminization (a fact we have not yet published).

P{Sxl.alt5-C8} is a transgene we briefly mentioned in the Materials and Methods section of Evans and Cline, 2013. It generates the same pair of exon-8-C-terminal Sxl protein isoforms as the conditional expression construct, P{UASp-Sxl.alt5-C8} described above in connection with the conditional expression construct of line 58484. However, unlike P{UASp-Sxl.alt5-C8}, P{Sxl.alt5-C8} does so constitutively, using 2.36 kb of Sxl DNA upstream of the maintenance promoter. The transgene can be mobilized by P transposase, so one could select for increased expression. 

Although Bloomington already maintains this allele (currently listed as Sxl[fs3]), it would be far better to have this female-sterile but adult viable allele maintained over a duplication only of Sxl[+] (see discussion in connection with stock 58486 above) rather than being balanced the way the Bloomington stock currently is, with no selection against the accumulation of extraneous deleterious mutations unrelated to Sxl. For those wanting to study Sxl germline functions, it is important that the X chromosome not be allowed to pick up extraneous mutations outside of Sxl that reduce female fertility or viability for reasons unrelated to Sxl. Moreover, the Sxl[f18] allele in this stock is marked with the very closely linked cm[1] eye-color marker mutation that can be a convenience in genetic stock construction.

The P{otu-Sxl.cF1} transgene above expresses one SXL-F isoform specifically in the germline (driven by the otu promoter) which is sufficient to counteract the deleterious effect of the snf[1] mutation on Sxl autoregulation in the germline--the basis for snf[1] female sterility. The P{otu-Sxl.cF1}A insertion is on chromosome II. Its isolation and characterization is described in:

Hager, J.H. and T.W.Cline, 1997, Induction of female Sex-lethal RNA splicing in male germ cells: implications for Drosophila germline sex determination. Development 124: 5033-5048.