intersex, a gene required for female sexual development in Drosophila, is expressed in both sexes and functions together with doublesex to regulate terminal differentiation
Carrie M. Garrett-Engele1,2,*,
,
Mark L. Siegal1,,
,
Devanand S. Manoli1,
Byron C. Williams3,
Hao Li1,
and
Bruce S. Baker1
1 Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA
2 Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA
3 Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853-2703, USA
* Present address: Howard Hughes Medical Institute and Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
Present address: Department of Functional Genomics, Novartis Pharmaceuticals, 556 Morris Avenue, Summit, NJ 07901, USA
These authors contributed equally to this work
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Fig. 1. The Drosophila somatic sex-determination hierarchy. The ratio of X chromosomes to sets of autosomes determines the on/off state of the Sex-lethal (Sxl) gene. In females, where the X:A ratio is 1, active SXL protein is made and its production is maintained via autoregulation. The presence of SXL causes splicing of the transformer (tra) pre-mRNA such that active TRA protein is made. When TRA is present with the protein product of the transformer-2 (tra-2) gene, the pre-mRNA of the doublesex (dsx) gene is spliced into its female-specific form, which encodes the DSXF protein. Similarly, the pre-mRNAs from the 5'-most promoter of the fruitless (fru) gene are spliced in a female-specific manner, and do not produce any detectable protein (three other promoters of fru produce transcripts that do not differ between the sexes). DSXF interacts with the products of the hermaphrodite (her) and intersex (ix) genes to activate female terminal differentiation and to repress male terminal differentiation. In males, where the X:A ratio is 0.5, no active SXL is made, so the tra pre-mRNA is spliced into its default, male-specific form, which does not produce active TRA protein. Although it is present in males, TRA-2 cannot act without active TRA, so the dsx and fru pre-mRNAs are spliced into default, male-specific forms. The male-specific DSXM protein activates male terminal differentiation and represses female terminal differentiation, interacting to some extent with HER. Although ix is expressed in males, like tra-2 it has no detectable function. The male-specific FRUM protein activates male courtship behavior. Arrows indicate positive regulation, bars indicate negative regulation and gray shading of gene names indicates that active proteins are not produced in the given sex.
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Fig. 2. The cytological and physical localization of ix. (A) Deficiency mapping of ix. The boxes indicate the region of the chromosome deleted for each deficiency chromosome (reported breakpoints in parentheses after deficiency names). The black boxes represent the deficiencies that fail to complement ix, and the white boxes represent the deficiencies that complement the loss-of-function alleles of ix. (B) Chromosomal walk spanning ix. Cosmid and phage clones spanning the cytological region 47F are indicated by lines. The relevant deficiency breakpoints are indicated above the DNA walk, and the probes used for RFLP mapping are indicated below the DNA clones. Map in A, reproduced (with permission) from Bridges and Bridges (Bridges and Bridges, 1939 ).
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Fig. 3. The ix region defined by RFLP mapping and P-element-mediated germline transformation. (A) 65 kb ix region. cDNAs and known genes are indicated below the phage and cosmid clones in the ix region, and the genomic rescue constructs 2GB and 1GS are shown below the cDNAs. (B) Restriction-site map of the ix region defined by the 2GB genomic rescue construct. Transcripts included in the 2GB construct are indicated as arrows. The extents of germline transformation constructs are shown below the map, with rescue results indicated. Triangles indicate the positions of inserted stop codons. B, BamHI; P, PstI; R, EcoRI; S, SalI.
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Fig. 4. Cuticle preps of wild-type and ix-mutant females. (A) Abdomen of wild-type female. (B) Abdomen of ix2/ix2 female. (C) Abdomen of ix2/ix2 female carrying one copy of the 3GBG* transgene. (D) Abdomen of ix2/ix2 mutant female carrying two copies of the heat-shock-inducible G cDNA transgene.
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Fig. 5. ix DNA sequence and predicted protein product. (A) DNA and protein sequences. The ix DNA sequence (GenBank Accession Number, AF491289) is shown with the predicted protein sequence in single-letter code below the corresponding nucleotides. 5' RACE experiments defined the start of the 5' UTR (underlined), and 3' RACE experiments determined the 3' end of the mRNA (position at which polyadenylation begins underlined). The putative upstream exon suggested by results of RT-PCR experiments is indicated in bold. (B) Schematic of the predicted ix protein. The asterisk indicates the stop codon inserted in the 3GBG* knockout construct. The gray and black boxes represent regions of the ix protein with sequence similarity to known proteins and ESTs. (C) Sequence alignment of the N-terminal region of the ix protein (gray in B) with the mammalian SYT and C. elegans sur-2 proteins. The consensus sequence is shown below. (D) Sequence alignment of a region of the ix protein (black in B) with the predicted proteins of human and mouse EST sequences. The consensus sequence is shown below.
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Fig. 6. Regulation of ix transcription. (A) Northern hybridization of female and male polyA+ RNA with probes from ix and ninaE. The arrow points to the position to which a 750 nucleotide molecule would migrate, as determined by a size marker run on the gel that was blotted (not shown). The relative abundance of female and male ix transcripts is given at the bottom, normalized to the amounts of ninaE transcript in each lane. (B) Scheme for RNase protection assay. A restriction map of the genomic region surrounding the putative ix translation start site (indicated by arrow labeled ‘Met...’) is shown, with the locations indicated of the putative transcription start site (as identified by 5' RACE, labeled ‘RACE start’), of the polyadenylation signal sequence (as identified by 3' RACE, labeled ‘polyA’), and of the potential intron from a transcript originating 5' to the RACE start (as identified by RT-PCR analysis, labeled ‘RT-PCR intron’). Aligned below the map is the full-length, 527 nucleotide probe used for the assay, which stretches from the MscI site to the 5'-most PstI site, and includes sequences from the T7-promoter-containing vector used to produce it (dashed region of arrow). Below the probe are the predicted protected fragments corresponding to the different potential ix transcripts. An unspliced transcript originating 5' to the MscI site would protect a probe fragment of 457 nucleotides, whereas an unspliced transcript originating at the site identified by 5' RACE would protect a probe fragment of 203 nucleotides. If a transcript originating 5' to the MscI site were spliced at the donor and acceptor sites identified by RT-PCR, this processed transcript would protect two probe fragments, 46 nucleotides and 132 nucleotides in length. (C) RNase protection assay. Female and male polyA+ RNA samples were each hybridized in solution with the probe shown in B, then digested with RNase and electrophoresed. Yeast RNA controls were also performed, either with (‘Y+’) or without (‘Y–’) RNase. Size markers are in lanes marked ‘M’ and the sizes of marker bands are indicated at right. The arrow points to the position to which a 203 nucleotide molecule would migrate.
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Fig. 7. ix and dsx act interdependently to activate Yp reporter expression in females. (A) Progeny from pML-58/pML-58; ix3/SM; ry/ry mothers crossed to w/Y; ix2/CyO; P[ix+ 9.5]/MKRS fathers. (B) Progeny from pCR1/pCR1; ix3/CyO; dsx pp/MKRS mothers crossed to w/Y; Df(2R)enB/CyO; dsx127/MKRS fathers. Mean lacZ activity is plotted for each progeny genotype, in units of OD574/minute/mg fly, based on the CPRG assay. Error bars are +1 s.e.m. Each genotype was assayed at least in triplicate.
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Fig. 8. Interaction of IX and DSXF to form a DNA-binding complex. (A) Results from yeast two-hybrid analysis. Fusion constructs between IX, DSXF or DSXM protein-coding sequences and either the Gal4 activation domain coding sequence (column 1) or the Gal4 DNA-binding domain coding sequence (column 2) were co-transformed into yeast containing Ade, His and lacZ reporters. A minus sign in column 1 or 2 indicates that no construct bearing the given domain sequence was transformed. The presence of an interaction between the fusion proteins (plus sign in column 3) was inferred by the ability of a transformed strain to grow on restrictive medium and to express lacZ, using a colony lift assay. (B) Co-immunoprecipitation of DSXF and IX. Nuclear extracts containing equivalent concentrations of protein from Drosophila S2 cells co-transfected with AU1-epitope-tagged IX and V5-epitope-tagged DSXF (lane 2) or DSXM (lane 3) were immunoprecipitated with monoclonal anti-AU1 antibody and analyzed via western blot with rabbit polyclonal anti-V5. The band at approximately 57 kDa in lane 2 corresponds to DsxF-V5. Lane 4 contains the supernatant from extracts precipitated in lane 3, indicating that DsxM-V5 protein was expressed but not precipitated with Ix-AU1. (C) DSXF and IX form a DNA-binding complex. EMSA was performed by incubating nuclear extracts from S2 cells expressing DSXF-V5 and/or IX-AU1 with a 32P-labeled DNA probe containing the 185 bp FBE region of the Yp enhancer, and resolving by native PAGE. Lane 1, free probe (no extract); lanes 2-5, probe plus tagged protein (indicated above lanes). Extracts from cells co-transfected with DSXF-V5 and IX-AU1 were probed in the presence of anti-AU1 or anti-V5 monoclonal antibodies or mouse IgG (lanes 6-8, respectively) to assay super-shifting of the DNA-binding complex.
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© The Company of Biologists Ltd 2002
