Genetic Characterization of Cytological Region 77A–D Harboring the Presenilin Gene of Drosophila melanogaster

Corresponding author: Mark E. Fortini, Department of Genetics, University of Pennsylvania School of Medicine, 709C Stellar-Chance Bldg., 422 Curie Blvd., Philadelphia, PA 19104-6069., fortini{at}mail.med.upenn.edu (E-mail)

Communicating editor: K. ANDERSON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We performed a systematic lethal mutagenesis of the genomic region uncovered by Df(3L)rdgC-co2 (cytological interval 77A–D) to isolate mutations in the single known Presenilin (Psn) gene of Drosophila melanogaster. Because this segment of chromosome III has not been systematically characterized before, inter se complementation testing of newly recovered mutants was carried out. A total of 79 lethal mutations were isolated, representing at least 17 lethal complementation groups, including one corresponding to the Psn gene. Fine structure mapping of the genomic region surrounding the Psn transcription unit by transgenic rescue experiments allowed us to localize two of the essential loci together with Psn within an ~12-kb genomic DNA region. One of these loci, located 3' to Psn, encodes a Drosophila protein related to the yeast 60S ribosomal protein L10 precursor. We also determined which of the newly recovered lethal mutant groups correspond to previously isolated lethal P-element insertions, lethal inversion breakpoints, and lethal polo gene mutants. Point mutations were identified in all five recovered Psn alleles, one of which results in a single amino acid substitution G-E at a conserved residue in the C-terminal cytoplasmic tail of the protein, suggesting an important functional role for this C-terminal domain of Presenilin. In addition, some viable mutations were recovered in the screen, including new alleles of the clipped and inturned loci.

MUTATIONS in the human Presenilin 1 and 2 (PS1 and 2) genes are a major cause of early-onset autosomal dominant familial Alzheimer's disease (reviewed in HAASS 1997 ; MATTSON et al. 1998 ; SELKOE 1998 ). Presenilins belong to an evolutionarily conserved family of integral membrane proteins present from nematodes to humans. Numerous studies have implicated Presenilins in amyloid deposition during Alzheimer's disease pathogenesis, Notch signaling, and apoptosis (reviewed in MATTSON et al. 1998 ; CHAN and JAN 1999 ). To generate genetic lesions in the Drosophila Presenilin (Psn) gene, we undertook a large-scale systematic mutagenesis of cytological region 77A–D, the genomic region known to harbor the Psn gene from polytene chromosome in situ hybridization data (BOULIANNE et al. 1997 ; HONG and KOO 1997 ; YE and FORTINI 1998 ). This region of the Drosophila genome has never been subjected to a systematic genetic characterization before and is relatively poorly represented by existing mutations and transposable element insertions (FLYBASE 1999 ). Because extensive databases of correlated genetic and molecular information are an important feature of the Drosophila genome project, we performed complementation tests and other analyses with all of the newly isolated mutations to genetically characterize the 77A–D cytological interval. We anticipate that this type of information will become particularly useful for the functional analysis of Drosophila genome project data, considering that sequencing of the euchromatic portion of the fly genome is likely to be completed within the next few years (MIKLOS and RUBIN 1996 ; RUBIN 1998 ).

After screening ~5200 EMS-mutagenized third chromosomes, we recovered a total of 79 lethal mutants that define 17 complementation groups consisting of 2–10 alleles each. In addition, new mutant alleles of known genes such as polo, inturned, and clipped were recovered, allowing the clipped mutation to be molecularly mapped much more precisely than its previously known assignment to the relatively large interval 75D4–79B1 (FLYBASE 1999 ). Several of our new lethal and viable mutant groups were correlated with known genetic lesions caused by chromosomal rearrangements and P-element insertions. One lethal complementation group, consisting of 5 lethal alleles, corresponds to the Psn gene by genomic rescue criteria. Rescue experiments with other lethal groups indicate that essential loci are located within ~7 kb of the 5' end of the Psn gene and within ~0.8 kb of the 3' end of the gene. One of these loci encodes a Drosophila protein related to the yeast mitochondrial 60S ribosomal protein L10 precursor. Mutations in all 5 Psn alleles were identified by DNA sequencing: four mutations correspond to nucleotide substitutions converting normal tryptophan codons into termination codons, while the fifth allele bears a mutation predicted to lead to a G-E amino acid substitution in the C-terminus of the Presenilin molecule. The latter mutation leads us to propose that the C-terminal tail of Presenilin may be an important functional domain of the protein. The collection of new lethal and viable mutants, together with their genetic complementation patterns relative to chromosomal rearrangements and P-element insertions that can be used to generate molecular tags, should contribute to the identification and characterization of other loci in the 77A–D region.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fly stocks, crosses, and EMS mutagenesis screen:
The following stocks were obtained from the Bloomington Drosophila Stock Center: Df(3L)rdgC-co2 th¹ st¹ in¹ ri¹ p^P, Df(3L)ri-79c, T(2;3)rdgC^co6 th¹ st¹ in¹ ri¹ p^P, In(3LR)Scr⁹ red¹ ed¹, In(3LR)225, l(3)04521⁰⁴⁵²¹, l(3)j7C3^j7C3, l(3)j10B2^j10B2, l(3)s2253^s2253, l(3)neo28⁰⁰¹⁰³, l(3)01673⁰¹⁶⁷³, l(3)77Aa^16-1, cp¹ in¹ ri¹ p^P, and Dp(1Ybb^-)B^s; ru¹ st¹ polo¹ e^s ca¹. In(3L)78Cb¹ was provided by Adelaide Carpenter (RUSSELL et al. 1996 ), Df(3L)ri-XT1 and Df(3L)ri^XT106 were provided by Jörg Großhans, and the Notch modifier mutant eDX5 was obtained from Esther VERHEYEN (personal communication; from the genetic screen described in VERHEYEN et al. 1996 ).

Fly stocks were maintained and crosses were performed on cornmeal/molasses media at 25° unless otherwise noted. The EMS mutagenesis was carried out as follows: w¹¹¹⁸ males were starved for 12 hr, then fed 25 mM ethylmethane sulfonate (Sigma) for 16 hr (LEWIS and BACHER 1968 ) and crossed to y w; pros^86E/TM3 virgin females in bottles. Mated females were transferred to a new bottle every third day for three passages without removing the males. Because each mutant's allele designation bears a single letter representing one of the 25 separately mutagenized populations (A–Y) from which it was recovered, mutants with different letters represent independent events. Individual y w, */TM3 male progeny (the asterisk denotes the mutagenized third chromosome) of these crosses were mated to Df(3L)rdgC-co2/TM6C virgin females and third chromosomes bearing lethal mutations uncovered by the deficiency were recovered in progeny of this mating as balanced TM6C stocks. All putative mutant chromosomes were tested in crosses with Df(3L)ri-79c/TM3. Inter se complementation tests of mutations falling within the two deficiency intervals (lethal over only Df(3L)rdgC-co2 or lethal over both deficiencies) were performed to place the mutations into separate complementation groups. Viable mutants with visible phenotypes in trans to Df(3L)rdgC-co2 were also balanced over TM6C. P-element lines were crossed to multiple representatives of each complementation group, and 60–90 progeny were scored for all complementation tests.

Genomic DNA rescue fragments:
Genomic DNA rescue constructs are described in YE et al. 1999 , except for pPsn.6, which was constructed by SphI digestion and reclosure of pPsn.3 to remove a 2.7-kb SphI fragment extending from +584 of Psn to a site located ~800 bp downstream of the poly(A) site. Transgenic flies bearing these rescue constructs were produced by P-element-mediated germline transformation (RUBIN and SPRADLING 1982 ) and standard genetic crosses were used to assay their ability to rescue the lethal phenotype of each newly recovered lethal mutation in trans to Df(3L)rdgC-co2.

Cloning and sequencing of Psn mutant alleles:
Three pairs of oligonucleotides for polymerase chain reaction (PCR) were designed based on the nucleotide sequence of the Presenilin gene: primer A1, 5'-CGGAGGCGAACGAACGC-3'; primer A2, 5'-GAGAATCAGCCAGCCGTG-3'; primer B1, 5'-CCCTGATCCTGATGAGCG-3'; primer B2, 5'-GCCGCTGCTGCCTCTGG-3'; primer C1, 5'-TCCAGCAACTCCACCAC-3'; primer C2, 5'-GACACTTGATGTGTCCTTG-3'.

PCR reactions were performed using mutant genomic DNA as a template with the following parameters: hot start at 94° for 4 min, followed by 30 cycles at 94°, 30 sec; 54°, 30 sec; 72°, 1 min. Mutant genomic DNA was obtained by homogenization of Psn^-/Df(3L)rdgC-co2 larvae of each mutation, followed by three phenol/chloroform extractions, ethanol precipitation, centrifugation for 5 min at 10,000 x g, and resuspension of the pellet in TE buffer. PCR products were subcloned into the pCR II vector (TA cloning kit; Invitrogen, Carlsbad, CA) and sequenced from both insert ends using an ABI Prism 377 automatic sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA) with T7 and M13(rev) primers. The sequence of each mutant Psn was assembled and aligned with wild-type Psn genomic sequence using MacVector 6.0 and AssemblyLIGN sequence analysis software.

Scanning electron microscopy:
Control and V5/Df(3L)rdgC-co2 flies were dehydrated through a 25, 50, 75, and 100% (twice) ethanol series for 15–20 hr for each step. Samples were prepared by critical point drying using hexamethyldisilazane (Sigma, St. Louis) and mounted onto scanning electron microscopy (SEM) stubs using T.V. tube coat (Ted Pella, Redding, CA). SEM images were collected on a Phillips scanning electron microscope and imported into Adobe Photoshop 4.0.1.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation and complementation testing of new lethal mutants in the 77A–D region:
A large-scale systematic EMS mutagenesis screen (Figure 1) was performed to recover lethal mutations uncovered by Df(3L)rdgC-co2 (77A–D region), a deficiency known to uncover the Psn gene by molecular criteria (BOULIANNE et al. 1997 ; HONG and KOO 1997 ; YE and FORTINI 1998 ). 5160 mutagenized third chromosomes were screened for lethality over Df(3L)rdgC-co2, resulting in the isolation of 79 new lethal mutants as well as 15 viable mutations with visible phenotypes (see below), giving a frequency of about one new 77A–D mutation for every 55 chromosomes screened. Mating of all lethal mutations to the overlapping, more proximal deficiency Df(3L)ri-79c/TM3 (77C–F) allowed us to assign 38 mutants to the 77A–B region [uncovered by Df(3L)rdgC-co2 but not by Df(3L)ri-79c] and 41 mutant lines to the 77C–D region (uncovered by both deficiencies as well as a third deficiency Df(3L)ri^XT106; see Table 1).

View larger version (20K): In this window In a new window Download PPT slide   Figure 1. Scheme of the systematic lethal mutagenesis of the 77A–D region of the D. melanogaster third chromosome uncovered by Df(3L)rdgC-co2 (*, mutated third chromosome). In the first generation, mutagenized males were crossed to females bearing the third chromosome balancer TM3. In the second cross, individual males with a mutagenized and balanced third chromosome were mated to virgin females of genotype Df(3L)rdgC-co2/TM6C, resulting in four progeny classes. Not <60 progeny were scored to test for lethality of the */Df(3L)rdgC-co2 genotype. Lethal and viable mutations were recovered as third chromosomes balanced over TM6C from these test crosses, and balanced stocks of each mutant allele were used in complementation tests as described in Table 1.

All lethal mutations were assigned to complementation groups by inter se crosses within each of the two sets defined by mapping using the two deficiencies described above. The results of this analysis are summarized in Table 1. Lethal mutations mapping to the distal 77A–B region of Df(3L)rdgC-co2 fall into 7 complementation groups containing multiple alleles and four "single-hit" mutations that do not fall into complementation groups. Similarly, 10 lethal complementation groups and three single hit mutants map to the proximal 77C–D region of Df(3L)rdgC-co2. Because mutations were recovered over the TM6C balancer chromosome marked with the dominant Tubby (Tb) marker, which can be reliably scored in late larvae and pupae, we were able to perform a preliminary survey of the lethal phases of the various mutant complementation groups by examining the phenotypes of any non-Tb pupae in crosses of each mutant to Df(3L)rdgC-co2/TM6C. If non-Tb pupae never appeared on the vial wall, and viable non-Tb larvae and/or abundant larval corpses were absent from the fly food, the mutation was scored as an "early" lethal. If the test cross produced some viable non-Tb larvae and numerous dead larvae but no pupariating non-Tb larvae on the vial wall, it was scored as a "larval" lethal mutant. Those with non-Tb flies that died as late larvae after pupariation but prior to pupation were scored as "late prepupal" lethal mutants, those dying after pupation but prior to the formation of adult structures were scored as "pupal" lethal mutants, and those dying with well-developed adult structures were scored as "pharate adult" lethal mutants (Table 1). Most groups contain mutants that all display similar lethal phases in trans to Df(3L)rdgC-co2, except for group l(3)77ABb, which contains mutants with lethal phases ranging from early to late. This group also displays weak partial complementation for viability involving certain heteroallelic combinations in a pattern consistent with the notion that the recovered alleles represent an allelic series of weak to strong loss-of-function alleles (data not shown).

Correlation of recovered mutant loci with known genetic lesions:
Although the chromosomal interval uncovered by Df(3L)rdgC-co2 has not previously been subjected to a systematic mutagenesis, some lethal mutations caused by P-element insertions (BERKELEY DROSOPHILA GENOME PROJECT, personal communication) and chromosomal rearrangements with putative breakpoints in the region (FLYBASE 1999 ) have accumulated over the years. We attempted to correlate these lesions, some of which are being used as sequence-tagged sites to assemble genetic and molecular contig data for the Drosophila genome project, with our newly isolated mutations in the hopes of producing a preliminary genetic characterization of this chromosomal interval that can be integrated with emerging genomic database information for the region. We tested six nonallelic lethal P-element insertions for complementation against our new lethal groups and were successful in assigning the four insert lines l(3)neo28⁰⁰¹⁰³, l(3)j10B2^j10B2, l(3)01673⁰¹⁶⁷³, and l(3)04521⁰⁴⁵²¹ to individual complementation groups recovered in our screen (Table 1). The remaining two lines, l(3)s2253^s2253 and l(3)j7C3^j7C3, do not appear to be lethal in trans to either Df(3L)rdgC-co2 or Df(3L)ri-79c, although l(3)j7C3^j7C3 exhibits a viable, small bristle phenotype in trans to either deficiency (see below). We also tested several inversions and determined that lethal lesions associated with In(3LR)Scr⁹ and In(3L)78Cb¹ correspond to one or more of the lethal complementation groups for each inversion (Table 1). The lethal lesions associated with the inversion In(3LR)225 map outside of the Df(3L)rdgC-co2 interval. We found that other deficiencies previously mapped to this region either do not overlap with Df(3L)rdgC-co2 (deficiency Df(3L)ri-XT1) or have a distal breakpoint very close to the presumed distal breakpoint of Df(3L)ri-79c [deficiency Df(3L)ri^XT106, which exhibits an identical complementation pattern with all lethal groups and single hit mutants as Df(3L)ri-79c]. Additional complementation testing of representatives of all lethal groups and single hit mutants revealed that a single lethal group consisting of three mutants is allelic to both polo¹ and the previously isolated mutant l(3)77Aa^16-1 (ROSEMAN et al. 1995 ), as well as to the lethal P-element insertion l(3)01673⁰¹⁶⁷³ (Table 1). Escapers doubly heterozygous for various combinations of these new polo alleles and either polo¹ or Df(3L)rdgC-co2 displayed highly penetrant abdominal tergite patterning defects as reported previously for polo¹/polo² heterozygous survivors (LINDSLEY and ZIMM 1992 ). Finally, we screened through all uncharacterized, chromosome III single-hit mutants recovered in a genetic screen for dominant modifiers of an activated Notch eye phenotype (the screen is described in VERHEYEN et al. 1996 ), because Presenilin is known to facilitate Notch/Lin-12 signaling in flies, worms, and mice (reviewed in CHAN and JAN 1999 ). We identified a single recessive lethal Notch modifier mutant, termed eDX5, that is allelic to mutations in the l(3)77CDg group (Table 1).

Identification and description of new viable mutations:
A relatively small number (15) of viable mutations with obvious morphological phenotypes were also recovered from the screen (Table 1). Three mutants (G5, R3, and X2) exhibit a typical inturned phenotype of whorled bristles in trans to Df(3L)rdgC-co2 and fail to complement inturned¹. The inturned locus maps to the 77B region and encodes a novel putative transmembrane protein (PARK et al. 1996 ). A group of 11 mutants display a shortened, thinned macrochaetae phenotype and may be allelic to the P-element line l(3)j7C3^j7C3, although our complementation data were inconclusive due to the partial penetrance of these viable alleles, possibly explaining the anomalous lethal complementation data reported for the l(3)j7C3 gene vs. Df(3L)rdgC-co2 and Df(3L)ri-79c (BERKELEY DROSOPHILA GENOME PROJECT, personal communication). Aside from these viable mutations, a semilethal allele of the otherwise lethal complementation group l(3)77CDj, termed V5 (Table 1), displays a distinctive rough eye phenotype and loss of thoracic microchaetae in rare escapers of genotype l(3)77CDj^V5/Df(3L)rdgC-co2 (Figure 2). Most flies of this genotype (>90%) die as pharate adults, and preliminary analysis of the mutant eye phenotype of the escapers indicates that they resemble "furrow-stop" mutants, affecting progression of the morphogenetic furrow during Drosophila eye development (data not shown; HEBERLEIN et al. 1993 ).

View larger version (145K): In this window In a new window Download PPT slide   Figure 2. Scanning electron micrographs of morphological defects during eye and thorax development of flies bearing a semilethal allele (V5) of l(3)77CDj. (A) w1118/Df(3L)rdgC-co2 control eye phenotype; (B) l(3)77CDjV5/Df(3L)rdgC-co2 mutant eye phenotype; (C) w1118/Df(3L)rdgC-co2 control thorax phenotype; and (D) l(3)77CDjV5/Df(3L)rdgC-co2 mutant thorax phenotype, arrows indicate missing bristles. (A and B) anterior at right, dorsal at top; (C and D) anterior at top, dorsal view.

One viable mutation that deserves comment is the cp^I5 mutant, which maps to the distalmost region of Df(3L)rdgC-co2 and is not uncovered by Df(3L)ri-79c. This mutant displays missing wing margin structures in trans to Df(3L)rdgC-co2 and also fails to complement the mutation clipped¹ (MAINX 1936 ). Furthermore, in the course of our genetic studies, we observed that T(2;3)rdgC^co6, a viable reciprocal translocation that breaks within the rdgC gene (STEELE et al. 1992 ), fails to complement both cp^I5 and clipped¹, and that the translocation itself shows a very subtle clipped phenotype affecting only a small region of anterior wing margin adjacent to the wing hinge (Figure 3). The clipped gene has previously been localized only to a rather large segment of chromosome III extending from 75D4–79B1 and has not been molecularly characterized (FLYBASE 1999 ). However, because the molecular breakpoint of T(2;3)rdgC^co6 has been precisely mapped within a 7.0-kb SalI restriction fragment of the rdgC gene, which encodes a novel serine/threonine protein phosphatase, our genetic data allow the clipped gene to be much more precisely mapped to the 77A–B region, probably in very close proximity to the rdgC gene. Intriguingly, the clipped wing phenotype of cp^I5 lethal pharate adult homozygotes is considerably more severe than that of cp^I5/Df(3L)rdgC-co2 viable adult flies, suggesting that the cp^I5 mutation may represent an unusual type of recessive gain-of-function mutation in the clipped gene (data not shown). This mutation may result in a gene product that interferes with other factors needed for wing margin differentiation or that has acquired a neomorphic activity. Further genetic and molecular analysis of clipped, perhaps aided by these mapping data, may help resolve this issue.

View larger version (128K): In this window In a new window Download PPT slide   Figure 3. Adult wing phenotypes of flies of the following genotypes: (A) cp1/Df(3L)rdgC-co2; (B) cpI5/Df(3L)rdgC-co2; (C) cp1/cpI5; (D) cp1/T(2;3)rdgCco6; (E) cpI5/T(2;3)rdgCco6; (F) T(2;3)rdgCco6/Df(3L)rdgC-co2; and (G)T(2;3)rdgCco6 homozygote, arrow indicates missing anterior margin bristles just distal to the wing hinge. (H) Higher magnification view of the missing anterior margin bristles of the wing shown in G. Interrupted LII wing veins in A, D, F, G, and H are due to the ri1 marker present on some of these chromosomes (see MATERIALS AND METHODS). Anterior at top.

Characterization of the Psn genomic region by rescue experiments:
To determine the genetic structure of the Psn transcription unit and surrounding DNA region, transgenic Drosophila were produced bearing different segments of wild-type genomic DNA from the Psn region in 77C (YE et al. 1999 ). An ~12-kb segment of genomic DNA containing the functional Psn gene was used as the starting point of this analysis, and this fragment was found to rescue only two other lethal complementation groups in addition to Psn, namely l(3)77CDa and l(3)77CDc (Figure 4). Further 5', 3', and internal deletions of this ~12-kb segment were assayed for their rescue ability, mapping essential sequences of the l(3)77CDa locus to the ~3-kb region located ~4 kb 5' to the Psn transcriptional start site and mapping essential sequences of the l(3)77CDc locus to within ~0.8 kb of the 3' end of the Psn polyadenylation signal. These results confirm our previous finding that the Drosophila Psn gene is a compact functional unit that maximally occupies only ~3.8 kb of genomic DNA, including ~1.2 kb of 5' flanking sequences, ~2.5 kb of transcribed DNA, and ~100 bp of 3' flanking sequences (YE et al. 1999 ).

View larger version (19K): In this window In a new window Download PPT slide   Figure 4. Genetic mapping of the genomic DNA region surrounding the Presenilin gene. Rescue constructs pPsn.3–8 were obtained by appropriate restriction enzyme digestions of an ~12-kb genomic DNA fragment containing the Psn transcription unit (YE et al. 1999 ; see MATERIALS AND METHODS). Restriction enzyme sites are indicated below the line denoting the Psn genomic region. Transgenic flies bearing a given rescue fragment were mated to at least two representatives of each complementation group to assay their ability to rescue the lethal phenotype of each group in trans to Df(3L)rdgC-co2.

All alleles of Psn display indistinguishable lethal phenotypes just prior to pupation and arrested larval imaginal disc phenotypes when assayed in all possible heteroallelic Psn genotypes or in trans to Df(3L)rdgC-co2, suggesting that they are likely to represent strong or complete loss-of-function alleles (YE et al. 1999 ; our unpublished data). The lethal phases of all five Psn mutations are remarkably uniform and appear to correspond to the larval-pupal transition stage referred to as the P4(i) prepupa, which occurs ~12 hr after pupariation (BAINBRIDGE and BOWNES 1981 ). Mutant pupating larvae collapse into a homogeneous oily mass devoid of pigment or coalescing adult structures within the pupal case, and almost all of the resulting dead pupae contain a trapped air bubble at their posterior cavity, diagnostic of the P4(i) prepupal stage.

The Psn gene is flanked by two essential loci, with all three loci together occupying only ~12 kb of genomic DNA. Because l(3)77CDc is located very close to the 3' end of Psn, we obtained partial sequence of the XhoI/BamHI fragment corresponding to l(3)77CDc and determined that this gene is likely to encode a Drosophila protein showing 35–40% amino acid sequence identity to the yeast mitochondrial 60S ribosomal protein L10 precursor (GROHMANN et al. 1991 ), which belongs to the L15P family of ribosomal proteins. This mutant group has a developmental arrest phenotype in which larvae cease to grow beyond the second instar phase, although they survive at this stage for a few weeks before dying (data not shown).

Sequencing of Psn mutant alleles:
To confirm that the putative Psn complementation group defined by our genomic rescue experiments represents Psn mutations and to determine the nature of the mutant lesions, we sequenced the Psn coding region of the alleles. We obtained genomic DNA from larvae bearing different Psn mutations in trans to Df(3L)rdgC-co2 and used it as template for PCR reactions with three different pairs of primers designed to cover the exonic sequences of Psn. Genomic sequence data were obtained for overlapping PCR fragments representing all coding region sequences of the five mutants, and potential mutations were confirmed by resequencing using independent PCR reactions to exclude potential PCR errors. This analysis resulted in the identification of the following nucleic acid and predicted amino acid alterations for all five Psn mutants: GGA GAA (G516E amino acid substitution) in Psn^B3, TGG TGA (W478Stop) in Psn^K2, TGG TGA (W278Stop) in Psn^I2, TGG TGA (W237Stop) in Psn^S3, and TGG TAG (W152Stop) in Psn^C4. Amino acid position numbers are as in YE and FORTINI 1998 . We also found a polymorphism (R38W) in the N-terminal tail of Drosophila Presenilin; in Psn^S3, Psn^K2, and Psn^I2, the arginine-38 residue is replaced by tryptophan due to a CGG TGG base pair substitution.

Four alleles, Psn^C4, Psn^S3, Psn^I2, and Psn^K2, bear nucleotide substitutions predicted to replace tryptophan codons with termination codons, and they would therefore be predicted to encode prematurely truncated Psn proteins ending at the beginning of the second transmembrane domain, in the middle of the small hydrophilic loop between the fourth and fifth transmembrane domains, in the middle of the sixth transmembrane domain, and at the beginning of the eighth transmembrane domain, respectively (Figure 5). These molecular lesions are consistent with our assessment of these Psn mutations as strong or complete loss-of-function alleles by genetic criteria (see above; YE et al. 1999 ). The Psn^B3 mutation is predicted to result in an amino acid substitution (G516E) in the C-terminal tail of Presenilin at a residue conserved in almost all known Presenilin family members, including even an Arabidopsis thaliana Presenilin, but excluding the nematode Sel-12 and Hop-1 proteins. Because the Psn^B3 mutation is genetically equivalent to Psn^C4, Psn^S3, Psn^I2, and Psn^K2, this result suggests that the G-to-E amino acid substitution at residue 516 severely or completely abrogates Presenilin activity and that the C-terminal cytoplasmic domain of Presenilin may thus be an important functional domain of the protein.

View larger version (23K): In this window In a new window Download PPT slide   Figure 5. Predicted amino acid substitutions caused by Psn mutations recovered in the F2 mutagenesis screen shown relative to the proposed structure and topology of the Psn protein within the membrane (modified from MATTSON et al. 1998 ), where transmembrane domains are depicted as shaded barrels 1–8 connected by hydrophilic loop regions. The positions of the mutations present in PsnB3, PsnK2, PsnI2, PsnS3, and PsnC4 are indicated by asterisks.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

As has been shown previously, D. melanogaster possesses one known Presenilin gene mapping to the proximal left arm of the third chromosome in cytological location 77B/C (BOULIANNE et al. 1997 ; HONG and KOO 1997 ; YE and FORTINI 1998 ). This region of the Drosophila genome has not been subjected to systematic mutational analysis. Only a few genes in the area have been extensively studied, most notably polo, which encodes a protein kinase needed for mitosis and meiosis (LLAMAZARES et al. 1991 ), inturned, encoding a novel transmembrane protein required for tissue polarity (PARK et al. 1996 ), and retinal degeneration C (rdgC), encoding a serine/threonine protein phosphatase functioning in the visual system (STEELE et al. 1992 ). Because our attempts to find Psn mutants among the few extant mutants mapping to the 77B/C region were unsuccessful, we elected to perform a systematic lethal mutagenesis of the 77A–D region uncovered by Df(3L)rdgC-co2 to isolate lethal Psn mutants. Our mutant alleles of Psn have been useful in studies of the role of Psn in proteolytic processing of the Notch receptor (YE et al. 1999 ), but we also wished to genetically characterize the 77A–D region further, given the paucity of information about this region and the potential value of such data for the Drosophila genome project (MIKLOS and RUBIN 1996 ; RUBIN 1998 ). Using a standard F₂ recessive lethal screening protocol, we recovered 79 new lethal mutants and 15 new viable mutants that map within the region uncovered by Df(3L)rdgC-co2. The lethal mutants define 17 complementation groups, approximately half of which also fail to complement the overlapping, more proximal deficiencies Df(3L)ri-79c and Df(3L)ri^XT106. Seven single-hit lethal mutants could not be assigned to the complementation groups. Although some essential genes defined by our screen are only represented by a single mutant allele, the fact that our 17 lethal complementation groups are represented by 2–10 alleles each suggests that our screen has detected all or most of the essential genes in the 77A–D cytological interval uncovered by Df(3L)rdgC-co2. The recovery of new alleles of all known mutants with lethal or obvious morphological phenotypes in this interval, such as polo, clipped, and inturned, is additional evidence of the reasonably exhaustive scale of our screen. As expected, we did not recover any alleles of retinal degeneration C, because this phenotype is not apparent unless the internal structure of the adult compound eye is closely examined.

Some of the viable mutations recovered in our screen are also of potential interest. An unusual, possible gain-of-function allele of the clipped gene was isolated, and complementation testing of this allele and the original allele clipped¹ permitted us to map the clipped locus to the distal region of Df(3L)rdgC-co2 that does not overlap with Df(3L)ri-79c. Weak clipped-like phenotypes and failure to complement known clipped alleles revealed that a lesion associated with the reciprocal translocation T(2;3)rdgC^co6 is likely to partially impair clipped gene activity, suggesting that the clipped transcription unit may be located near the molecularly mapped translocation breakpoint of T(2;3)rdgC^co6 (STEELE et al. 1992 ). Finally, a semilethal mutation of the l(3)77CDj locus displays loss of thoracic microchaetae and a rough eye phenotype reminiscent of the furrow-stop class of eye development mutants, such as hedgehog, Bar, and Drop (HEBERLEIN et al. 1993 ). Our recovery of these mutations and the preliminary mapping data we present here should facilitate the further molecular characterization of these loci.

Genetic analysis of the genomic region immediately surrounding the Psn transcription unit was accomplished by rescue experiments using several partially overlapping genomic DNA fragments. These studies led to our previous identification of one lethal complementation group as Psn gene mutations (YE et al. 1999 ), and in this article we identify two additional essential genes located in the same ~12-kb genomic DNA region as the Psn gene. Psn is a compact gene residing in a 3.8-kb genomic DNA fragment that is capable of providing full Psn gene function. This DNA segment contains ~1.2 kb of 5' flanking DNA, the ~2.5-kb Psn transcribed region, and ~100 bp of 3' flanking DNA (YE et al. 1999 ). An essential gene corresponding to l(3)77CDa is located ~4 kb upstream of Psn, and an essential gene corresponding to l(3)77CDc, encoding a fly protein with some similarity to the yeast mitochondrial 60S ribosomal protein L10 precursor (GROHMANN et al. 1991 ), is located <800 bp downstream of Psn.

Sequence analysis confirmed that all five Psn mutants bear lesions in the Psn gene as deduced from the genomic DNA rescue experiments. Psn^C4, Psn^S3, Psn^I2, and Psn^K2 are predicted to encode prematurely truncated Presenilins, in agreement with our genetic studies suggesting that these mutant Psn alleles are likely to represent strong or complete loss-of-function mutants (YE et al. 1999 ). Psn^B3 is predicted to cause an amino acid substitution at a conserved residue of the C-terminal region of Presenilin, directing our attention to this portion of the molecule. Most Alzheimer's disease-associated mutations in the human Presenilins occur in either transmembrane domain 2 (TM2) or an N-terminal stretch of the large hydrophilic loop between TM6 and TM7 (reviewed in HAASS 1997 ; MATTSON et al. 1998 ; SELKOE 1998 ), and only one mutation has been recently discovered that maps to the C-terminal tail of PS1 (PALMER et al. 1999 ). In the case of Psn^B3, a single amino acid substitution (G516E) at a conserved position 26 residues before the C-terminus results in a complete or nearly complete loss of Presenilin function. The affected glycine residue presumably plays an important role in normal Presenilin function, perhaps as part of a protein-protein interaction site or in contributing to the conformation of a functionally important C-terminal Presenilin domain. The significance of the C-terminal tail of Presenilin family members for the normal biological functions of these proteins will require further genetic and molecular analysis.

	ACKNOWLEDGMENTS

We thank Yihong Ye for making the pPsn.8 transgenic flies, Yuri Veklich for help with scanning electron microscopy, Esther Verheyen, Todd Laverty, Adelaide Carpenter, Doujia Pan, Jörg Großhans, Taras Bulba, and the Bloomington Drosophila Stock Center for fly stocks, the Berkeley Drosophila Genome Project for P1 phage stocks, and Yihong Ye, Simon Petliura, Goran Periz, and Rachel Drysdale for advice and comments on the manuscript. This work was supported by National Institutes of Health RO1 grant AG14583, the Alzheimer's Association, and the Life and Health Insurance Medical Research Fund.

Manuscript received May 1, 1999; Accepted for publication August 23, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

BAINBRIDGE, S. P. and M. BOWNES, 1981  Staging the metamorphosis of Drosophila melanogaster.. J. Embryol. Exp. Morphol. 66:57-80[Medline].

BOULIANNE, G. L., I. LIVNE-BAR, J. M. HUMPHREYS, Y. LIANG, and C. LIN et al., 1997  Cloning and characterization of the Drosophila presenilin homologue. Neuroreport 8:1025-1029[Medline].

CHAN, Y.-M. and Y. N. JAN, 1999  Presenilins, processing of ß-amyloid precursor protein, and Notch signaling. Neuron 23:201-204[Medline].

FLYBASE,, 1999  The FlyBase database of the Drosophila genome projects and community literature. Nucleic Acids Res. 27:85-88. http://flybase.bio.indiana.edu/([Abstract/Free Full Text].

GROHMANN, L., H. R. GRAACK, V. KRUFT, T. CHOLI, and S. GOLDSCHMIDT-REISIN et al., 1991  Extended N-terminal sequencing of proteins of the large ribosomal subunit from yeast mitochondria. FEBS Lett. 284:51-56[Medline].

HAASS, C., 1997  Presenilins: genes for life and death. Neuron 18:687-690[Medline].

HEBERLEIN, U., T. WOLFF, and G. M. RUBIN, 1993  The TGFß homolog dpp and the segment polarity gene hedgehog are required for propagation of a morphogenetic wave in the Drosophila retina. Cell 75:913-926[Medline].

HONG, C. S. and E. H. KOO, 1997  Isolation and characterization of Drosophila presenilin homolog. Neuroreport 8:665-668[Medline].

LEWIS, E. B. and F. BACHER, 1968  Method of feeding ethyl methanesulfonate (EMS) to Drosophila males. Dros. Inf. Serv. 43:193-194.

LINDSLEY, D. L., and G. G. ZIMM, 1992 The Genome of Drosophila melanogaster. Academic Press, San Diego.

LLAMAZARES, S., A. MOREIRA, A. TAVARES, C. GIRDHAM, and B. A. SPRUCE et al., 1991  polo encodes a protein kinase homolog required for mitosis in Drosophila.. Genes Dev. 5:2153-2165[Abstract/Free Full Text].

MAINX, F., 1936  Eine neue Faktormutation von Drosophila melanogaster. Z. indukt. Abstamm.-u. VerebLehre 71:303-304.

MATTSON, M. P., Q. GUO, K. FURUKAWA, and W. A. PEDERSEN, 1998  Presenilins, the endoplasmic reticulum, and neuronal apoptosis in Alzheimer's disease. J. Neurochem. 70:1-14[Medline].

MIKLOS, G. L. G. and G. M. RUBIN, 1996  The role of the genome project in determining gene function: insights from model organisms. Cell 86:521-529[Medline].

PALMER, M. S., J. A. BECK, T. A. CAMPBELL, C. B. HUMPHRIES, and P. K. ROQUES et al., 1999  Pathogenic presenilin 1 mutations (P436S & I143F) in early-onset Alzheimer's disease in the UK. Hum. Mutat. 13:256[Medline].

PARK, W. J., J. LIU, E. J. SHARP, and P. N. ADLER, 1996  The Drosophila tissue polarity gene inturned acts cell autonomously and encodes a novel protein. Development 122:961-969[Abstract].

ROSEMAN, R. R., E. A. JOHNSON, C. K. RODESCH, M. BJERKE, and R. N. NAGOSHI et al., 1995  A P element containing suppressor of hairy-wing binding regions has novel properties for mutagenesis in Drosophila melanogaster.. Genetics 141:1061-1074[Abstract].

RUBIN, G. M., 1998  The Drosophila genome project: a progress report. Trends Genet. 14:340-343[Medline].

RUBIN, G. M. and A. C. SPRADLING, 1982  Genetic transformation of Drosophila with transposable element vectors. Science 218:348-353[Abstract/Free Full Text].

RUSSELL, S. R. H., G. HEIMBECK, C. M. GODDARD, A. T. C. CARPENTER, and M. ASHBURNER, 1996  The Drosophila Eip78C gene is not vital but has a role in regulating chromosome puffs. Genetics 144:159-170[Abstract].

SELKOE, D. J., 1998  The cell biology of ß-amyloid precursor protein and presenilin in Alzheimer's disease. Trends Cell Biol. 8:447-453[Medline].

STEELE, F. R., T. WASHBURN, R. RIEGER, and J. E. O'TOUSA, 1992  Drosophila retinal degeneration C (rdgC) encodes a novel serine/threonine protein phosphatase. Cell 69:669-676[Medline].

VERHEYEN, E. M., K. J. PURCELL, M. E. FORTINI, and S. ARTAVANIS-TSAKONAS, 1996  Analysis of dominant enhancers and suppressors of activated Notch in Drosophila. Genetics 144:1127-1141[Abstract].

YE, Y. and M. E. FORTINI, 1998  Characterization of Drosophila Presenilin and its colocalization with Notch during development. Mech. Dev. 79:199-211[Medline].

YE, Y., N. LUKINOVA, and M. E. FORTINI, 1999  Neurogenic phenotypes and altered Notch processing in Drosophila Presenilin mutants. Nature 238:525-529.

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