Affolter, Markus

Gohl, Daryl M.

Müller, Martin

Sickmann, Michèle

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Dros. Inf. Serv. 100 (2017)   171  Mutation Notes   The Drosophila melanogaster straw locus is allelic to laccase2.    Sickmann, Michèle1, Daryl M. Gohl2, Markus Affolter1, and Martin Müller1.  1Biozentrum der Universität Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland;  2University of Minnesota Genomics Center, 2231 6th Street SE, Minneapolis, MN 55455, USA.      In the course of our studies on the apterous locus (Gohl et al., 2008; Gohl and Müller, unpublished), we have isolated a PiggyBac insertion in the nearby laccase2 gene (CG42345).  The new insert PBac{WH}laccase25151-37A was obtained by mobilization of PBac{WH}apf00451 and it was molecularly mapped to near the 3’ end of the laccase2 gene (see Figure 1).  The enzyme Laccase2 is part of the catecholamine pathway leading to pigmentation and sclerotization of the adult fly cuticle (Riedel et al., 2011).  It oxidizes dopamine to dopamine quinone, which, in the presence of the Yellow protein, polymerizes to form black melanin.  Laccase2 enzyme also oxidizes N-β-alanyldopamine to a quinone, which mediates cuticle protein cross-linking (sclerotization) (Riedel et al., 2011).             In order to genetically characterize the new allele, complementation crosses among stocks PBac{WH}laccase25151-37A/SM6, PBac{WH}laccase2f04551/CyO, PBac{WH}laccase2f03626/CyO, Df(2R)M41A10/ CyO, Df(2R)BSC630/CyO, and Df(2R)BSC696/CyO were set up (insertion sites and deficiency breaks are depicted in Figure 1).  The results indicated that laccase2 alleles PBac{WH}laccase2f04551, and PBac{WH}laccase2f03626 are lethal.  The new allele PBac{WH}laccase25151-37A is viable over deficiency as well as over both of the two lethal PBac{WH} alleles (see Table 1).  Such trans-heterozygous flies show a consistent pigmentation defect reminiscent of yellow null alleles:  bristles and wings lose their characteristic dark color.  However, and in contrast to yellow, pigmentation on abdominal tergites remains largely unchanged (data not shown).  Similar phenotypes were previously obtained by RNAi-mediated knockdown of the laccase2 gene (Riedel et al., 2011).  Figure 1.  700-kb genomic interval surrounding the laccase2 locus at the base of chromosome arm 2R (adapted from FlyBase).  In the upper part of the panel, gene spans of all loci in the region are shown.  Genes Nipped-A, laccase2, and apterous are highlighted.  Below the gene spans, all PiggyBac insertion sites mentioned in the text are indicated.  At the bottom of the panel, the complete length of deficiencies Df(2R)BSC630 and Df(2R)BSC696 are depicted by a red bar.  For the much larger deletion Df(2R) M41A10, only the approximate position of its distal break point is shown.  Mutation Notes Dros. Inf. Serv. 100 (2017)  172               Figure 2.  Adult phenotypes of stw1 and stw3.  In the top 4 pictures, the change in thoracic bristle pigmentation is shown.  Bristle color in OregonR (+/+) and heterozygous stw1 (stw1/+) flies is clearly darker than in stw1 (stw1/BSC696) and stw3 (stw3/BSC696) hemizygotes.  In the middle of the panel, the difference in appearance of the wing blade between heterozygous stw1/+ and stw3/f03626 flies is documented.  Note the yellowish appearance of the blade in stw3/f03626 as compared to stw1/+.  Wing blade stw3/f03626 is slightly damaged at the tip.  Wings were dissected and embedded in Hoyer’s before images were taken.  At the bottom left, blow-ups of proximal-anterior wing margins are shown.  Note the loss of dark pigment in hemizygous (stw1/BSC696, stw3/BSC696) or trans-heterozygous (stw1/f03626, stw3/f03626) stw1 and stw3 conditions as compared to OregonR (+/+) or stw1/+.  At the bottom right, blow-ups of adult legs are depicted.  Bristles acquire a distinct yellowish color in hemizygous (stw1/BSC696, stw3/BSC696) or trans-heterozygous (stw1/f03626, stw3/f03626) stw1 and stw3 conditions as compared to OregonR (+/+) or stw1/+.  Pictures of the same morphological structures were taken under identical illumination conditions. Dros. Inf. Serv. 100 (2017) Mutation Notes 173  Table 1.  Results of complementation crosses. Genotype Viability straw phenotype Df(2R)M41A10 / Df(2R)BSC630 lethal  Df(2R)M41A10 / Df(2R)BSC696 viable no Df(2R)M41A10 / PBac{WH}laccase2f04551 viable no Df(2R)M41A10 / PBac{WH}laccase2f03626 viable no Df(2R)BSC630 / Df(2R)BSC696 lethal  Df(2R)BSC630 / PBac{WH}Iaccase2f04551 lethal  Df(2R)BSC630 / PBac{WH}Iaccase2f03626 lethal  Df(2R)BSC696 / PBac{WH}Iaccase2f04551 lethal  Df(2R)BSC696 / PBac{WH}Iaccase2f03626 lethal  PBac{WH}laccase2f03626 / PBac{WH}laccase2f04551 lethal     Df(2R)M41A10 / PBac{WH}laccase25151-37A viable no Df(2R)BSC630 / PBac{WH}laccase25151-37A viable yes* Df(2R)BSC696 / PBac{WH}laccase25151-37A viable yes* PBac{WH}Iaccase2f04551 / PBac{WH}laccase25151-37A viable yes PBac{WH}Iaccase2f03626 / PBac{WH}laccase25151-37A viable yes    Df(2R)M41A10 / stw1 viable no Df(2R)BSC630 / stw1 viable yes Df(2R)BSC696 / stw1 viable yes PBac{WH}laccase2f04551 / stw1 viable yes PBac{WH}laccase2f03626 / stw1 viable yes    Df(2R)M41A10 / stw3 viable no Df(2R)BSC630 / stw3 viable yes Df(2R)BSC696 / stw3 viable yes PBac{WH}laccase2f04551 / stw3 viable yes PBac{WH}Iaccase2f03626 / stw3 viable yes * These flies also show an apterous wing phenotype because the PBac{WH}laccase25151-37A chromosome has retained insert PBac{WH}apf00451 and Df(2R)BSC630 and Df(2R)BSC696 also take out apterous (see Figure 1). PBac{WH}laccase2f04551 / CyO (B#18785), Df(2R)M41A10/SM1 (B#741), Df(2r)BSC630 / CyO (B#25705), Df(2R)BSC696 / CYO (B#26548), stw1(B#412) and lt1 rl1 stw3 (B#1056 were obtained from the Bloomington stock center.  PBac{WH}apf00451 and PBac{WH}laccase1f03626 were purchased from the Exelixis stock collection at Harvard Medical School.   The first straw allele (stw1) was discovered by Calvin Bridges exactly 100 years ago in 1917 (Morgan et al., 1925).  According to FlyBase, the straw locus has not been annotated yet.  But information available on FlyBase suggests that straw could be allelic to laccase2:  (1) straw has been mapped to the base of 2R but distal to Df(2R)M41A10;  (2) the described straw phenotype is very similar to what we have observed for our new allele PBac{WH}laccase25151-37A.  This assumption was borne out by our observations obtained by complementation crosses between straw alleles stw1 and stw3 and deficiencies and laccase2 alleles shown in Figure 1 (see Table 1):    −  Df(2R)M41A10 complements both straw alleles but neither Df(2R)BSC630 nor Df(2R)BSC696 do.  Hemizygous stw1 and stw3 flies are well viable and show the typical straw phenotype (see Figure 2).   − Importantly, the straw alleles are also not complemented by PBac{WH}laccase2f04551 and PBac{WH}laccase2f03626 (see Figure 2).  In trans-heterozygous flies, pigmentation is lost in wings and bristles.  Mutation Notes Dros. Inf. Serv. 100 (2017)  174 In conclusion, these complementation tests demonstrate that straw is allelic to laccase2.  Therefore, we propose that according to established Drosophila nomenclature practices, the gene name of CG42345 should be changed to straw, as it was first called in the Morgan lab in 1917.  Our observation "Bridges" the historical gap in understanding the molecular nature of the straw mutants discovered 100 years ago.  References:  Gohl, D., M. Mueller, V. Pirrotta, M. Affolter, and P. Schedl 2008, Genetics 178: 127-143;  Morgan, T.H., C.B. Bridges, and A.H. Sturtevant 1925, The genetics of Drosophila melanogaster. Biblphia Genet. 2: 262pp;  Riedel, F., D. Vorkel, and S. Eaton 2011, Development 138: 149-158.   History of the FM7 balancer chromosome.  Merriam, John1.  Revised by Scott Hawley and Danny Miller, 1-22-2014.  Figure courtesy of Angie Miller.  1Molecular Cell and Developmental Biology, University of California, Los Angeles 90095.      Like all modern balancer chromosomes in D. melanogaster, FM7 was constructed from a series of progenitor balancer chromosomes.  Inversion heterozygotes, but not homozygotes, suppress crossing over at the inverted portion of the chromosome.  Sturtevant (1913) discovered the first example of an inversion, which he named In(3R)C, for crossover suppressor in the right arm of chromosome 3.  This inversion reverses much of the distal third of the chromosome (from section 92D1 to 100F2) so that the chromosome sections in the right arm distal to the break at 92D1 are in the order centromere–100F2–92D1–telomere, and it suppresses crossing over for this region (from the marker Dl (Delta) to the telomere).  Muller (1918) used In(3R)C to make the first permanent heterozygous stock, or “balanced stock”, with the marker Bd, an allele of the Serrate (Ser) gene, that has both a dominant wing notching and recessive homozygous lethality.  In the language of its time, this stock was “pure breeding”—all progeny had the same phenotype and genotype of their parents—because Bd/Bd and In(3R)C/In(3R)C homozygotes did not survive, leaving only Bd/In(3R)C heterozygotes each generation.  In order for this stock to remain heterozygous each generation there must be suppression of crossing over, as it keeps the wild-type Bd+ allele from In(3R)C from being placed onto the Bd chromosome, which would allow for recovery of Bd+/Bd non-In(3R)C progeny.   The importance of this example was instantly recognized, leading to the identification of dominant crossover suppressor lines for all the linkage groups, and it was applied to maintaining mutant alleles with poor viability and/or multiply-marked chromosomes.  Because Drosophila stocks cannot be maintained through frozen lines, essentially all of the thousands of mutant alleles in different genes now available must be maintained in balanced stocks without selection.  The balancer chromosomes responsible have improved to contain multiple inversions for more complete crossover suppression, as well as a dominant marker for identification and recessive lethal or sterile mutants to prevent the stock from becoming homozygous for its balancer and losing the mutant allele.  Along with balancing mutant alleles, the inverted chromosomes also became essential in screens for new mutants.  Muller (1928) recovered a balancer on the X (or 1st) chromosome, In(1)Cl, also carrying the visible markers sc v f (all recessive) and B (dominant), as well as a lethal allele in an unknown gene.  The middle two-thirds of this chromosome was inverted from 4A5 to 17A6.  Maintained as the “ClB” stock, it formed the basis for Muller’s assay to determine the fraction of sperm that carried a new X-linked lethal mutation after exposure to X-rays, work for which he received the Nobel Prize in 1946.  Balancers were also used to identify lines with segregating recessive lethal mutations following mutagenesis, which could be identified as stocks which only gave heterozygous mutagenized-chromosome/balancer progeny.  One highlight example is a 1980 paper by Nusslein-Volhard and Wieschaus describing their identification of the embryonic patterning genes through lethal alleles, for which they received the Nobel Prize in 1992.  Both the stock-maintenance and selective-screening uses of balancers depend on their effectiveness in suppressing crossing over.  The goal of balancer construction has, therefore, been to add multiple inversions in order to cover as much of the chromosome as possible.  In(1)Cl suppressed crossing over for most X regions except most proximally, but was less useful because of its own recessive lethality.  In(1)dl-49, the second X 

The Drosophila melanogaster straw locus is allelic to laccase2.

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The Drosophila melanogaster straw locus is allelic to laccase2.

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