Copyright 6 1995 hy the Genetics Society of America Developmental Genetical Analysis and Molecular Cloning of the abnormal oocyte Gene of Drosophila melamgaster John Tomkiel,” Laura Fanti: Maria Berloco: Luigi Spinelli,+John W. Tamk~m,~ Barbara T. Wakimoto*9sand Sergio Pimpinellit” *Department of Genetics, University of Washington, Seattle, Washington 98195, +Zstituto di Genetica, Universita di Ban‘, 70126 Ban’, Italy, IDepartment of Biology, University of California, Santa Cruz, California 95064 and $Department of Zoology, University of Washington, Seattle, Washington 981 95 Manuscript received September 6, 1994 Accepted for publication March 8, 1995 ABSTRACT Studies of the abnormal oocyte (abo) gene of Drosophila mlanogaster have previously been limitedto the analysis of a single mutant allele, abnmmal oocyte’ (abo’). The abo’ mutation causes a maternal-effect lethality that can be partially rescued zygotically by the abo+ allele and by increasing the dosage of specific regions of heterochromatin denoted ABO. This report describes the propertiesof abo’, a new Pelement-induced allelethat allowed us to reexamine the natureof maternaleffect defect. Comparisons of the phenotype of progeny of abo‘/abo’ and abo’/abo’ females show that the preblastoderm lethality previously described asa component of the abo mutant maternal effect results froma recessive fertilization defect associated with the abo’ chromosome. We demonstrate here that the abeinduced maternaleffect lethality occurs predominately late in embryogenesis after cuticle deposition but before hatching. The phenocritical period forzygotic rescue by heterochromatin coincideswith this periodof late embryogene- sis. We have used the abo’ mutation to map and molecularly clone the gene.We show that the abo gene is located in the 32C cytogenetic interval and identify the putative abo transcript from mFWA isolated from adult females. Using germline transformation, weshow that a 9-kb genomic fragment to which the transcript maps, partially fulfills requirement for maternal andzygotic abo’ function. HE heterochromatin of Drosophila melanogaster is matin interact with both abo and dal (PIMPINELLIet al. T the best genetically and molecularly characterized 1985). heterochromatin of all organisms studied to date. A The heterochromatic regions that interact with abo wide variety of genetic functions are known to exist have been best characterized.These regions, desig- within Drosophila heterochromatin,including those nated ABO have been mapped by comparing the effect encoded by repetitive DNA sequences (reviewed by of cytologically defined duplications or deficiencies of GATTIand PIMPINELLI1992). These include the 18 + heterochromatin on the survival rates of progeny of 28s rDNA, the Rsp locus, and the ABO and DAL ele- do’ females. These studies have identified ABO loci in ments. The ABO and DAL elements are unusual in that the distal heterochromatin of the X, on the long and they are dispersed heterochromatic loci defined by short arms of the Y, and the right arm of the second their ability to interact with specific maternal-effect mu- chromosome (SANDLER1970,1977; PARRYand SANDLER tations that map to the euchromatin on the left arm 1974; HAEMER1978; YEDBOVNICK et al. 1980; PIMPINELLI of chromosome 2. The euchromatic mutations were et al. 1985; L. FANTI and S. PIMPINELLI,unpublished identified by SANDLER(1977) as a set of fiveclosely results). linked recessive, maternal-effect mutationsthat in- Recently, TOMKIELet al. (1991) demonstrated that an cludes: abnormal oocytd, hold up, wavoid-like, daughterless increase in the dosage of ABO heterochromatin in the and daughterless-abnormal oocyte-like. Thesemutations mother can partially rescue the maternaleffectlethality form a unique group among maternaleffect mutations caused by abo’. The ABO elements appear to function because the maternal defect can be partially compen- veryearly in embryogenesis (PIMPINELLIet al. 1985; sated by a paternal copy of the wild-type gene in the TOMKIELet al. 1991), ata time when genetic, molecular, zygote or by increasing the dosage of heterochromatin and biochemical studies all suggest that the zygotic ge- that the zygotes receive. Specificregions of heterochro- nome is transcriptionally silent (LAMBand LAIRD1976; MCKNIGHT ANDERSON Corresponding authorrJohn Tomkiel, Center for Molecular Medicine and MILLER1976; ZALOKAR1976; and Genetics, 5117 Biological Sciences Building, 5047 Gullen Mall, and LENGYEL1979; EDGARand SCHUBIGER1987). Exam- Wayne State University, Detroit, MI 48202. ination of the effects of various doses of ABO in abo’ E-mail: [email protected] mothers led to the conclusion that maternal ABO may ’ Present address: Dipartimento di Genetica e Biologia Molecolare “Charles Darwin”, Universita di Roma“La Sapienza”, pie. Aldo Moro function either in oogenesis or in the egg beforecom- 5, 00185 Rome, Italy. pletion of meiosis I (TOMKIELet al. 1991). Analysis of Genetics 140 615-627 (June, 1995) 616 J. Tomkiel et a1 survival of embryos mosaic for various amounts of ABO 1988) into males that were also heterozygous for two of suggests that the developmental timing of zygotic res- the P elements andheterozygous for the wild-type and domi- nant mutant alleles of the flanking markers Jammed and cue by ABO is before completion of the first zygotic (J) Tufted ( Tft). division. Thus, bothmaternally and zygotically supplied The four different P elements, each marked with the ry+ AB0 appear to exert theirrescuing effect between fertil- gene, were as follows: CHBA-89 P[ry+] and Cp7OAB P[ly+] ization and the first zygotic division. isolated by SIMONrt al. (1985), fs(2)ly5PI?’] provided by D. Several models have been proposed to explain the MCKEARINand g65 P[ry+] isolated by Bou~ou~sand RI(:HARI)S (1985). These P[ry+] elements are located at SIB, 32C/D, rescue of the abo maternal-effect defect by hetero- ABO 32E and 32F, respectively. The f(2)95 chromosome that we chromatin. One model postulates that abo and AB0 received also contained a second P-element insertion that we carry out similar functions, perhapsby encoding related could detect by Southern blotting. With the exception of the products. The location of loci in heterochromatin may fs(2)ly5insertion that results in a recessive female sterility that allow expression of a necessary product during times fully complements abo’, these P-element insertions do not cause mutant phenotypes. The cross used to obtain males when the euchromatin is obligately silent (PIMPINELLI carrying P elements on both chromosome 2 homologues is et al. 1985). A second class of models proposes that abo described in Figure 1. It was necessary to repress P-element and ABO loci have different developmental functions transposition in the preceding generation. This was accom- with some interaction required, either at the level of plished by introducing the X-linked I35 element kindly pro- their gene products or via regulatory interactions. For vided by R.W. PHYLLIS.I35 is a mutated Pelement thateffec- tively represses transposition because it encodes a truncated instance, the product of one gene may be required to transposase protein. activate the expression of the other. Alternatively, the From among the progeny of males that were J Cp7OAB heterochromatin may exert its effect directly or indi- P[ry+] TJ/J’ P[$] Tjf,we recovered chromosomes with rectly on the expression of the euchromatic abo locus nonparental combinations of the flanking markers (J Tf+ or by titration of regulatory factors or by influencing chro- ,I’ Tj).A total of 52 recombinant chromosomes were tested for complementation of the abo’ maternal effect lethality. matin structure (TOMKIEL1990). Finally, a third model Twelve were from J Cp7OAB Tj/Cp7OAB fathers, six were proposes that the observed interaction of ABO with abo from J Cp7OAB 73/g65 fathers, eight were from J Cp7OAH reflects the specific nature of the abo’ allele, rather than Tj/ CHBA89 Fathers and 26 were from J Cp7OAB 73/&(2) 7y5 a requirementnecessary for normalembryogenesis. LA- fathers. One of these was a noncomplementing J T+ 73+ VORGNA et al. (1989) have suggested that the abo’ allele chromosome recovered from a J Cp7OAB Tj/ji(2) tyi;A2- 3(YYB) father. This new mutation will be referred to as ab$. is associated with a copia-like transposable element in- (This same mutation was described as nbo3by TOMKIEI.(1990) serted in or near the gene. They attribute the mutant but is renamed here). phenotype to the disruptive effect of the insertion on Recombination mapping of do2: The abo’ mutation was gene expression and postulate that the effect of ABO mapped by recombination relative to Jammed (2-41.0), thery’ is due to the suppressive influence of heterochromatin on the abo’ chromosome, andScutoid (2-51.0). The distance between J and Sco in this experiment was 12.2 map units on the transposable element. (131 recombination events/1070), compared to the reported The above models are not easily distinguished by ge- distance of 10.0 map units (LINDSLEY andZIMM 1992). Forty- netic means. A clearer understandingof the mechanism threeJSca recombinants were tested for the ry and abo pheno- of interaction between the euchromatic mutation and types. These data map abo’ -2.1 map units proximal toJand theheterochromatic elements requires a molecular 9.3 map units distal to Sco. This places abo’ between 41.7 and analysis of’ both abo and ABO. Toward this end, we have 43.1 map units, in close proximity to the previously reported map position of abo’ (44.0). In