The transformer gene in Bactrocera oleae: the genetic switch that determines its sex fate.

Transformer (tra) is the second gene of a regulatory cascade based on RNA splicing that determines sex in Drosophila melanogaster. Splicing of tra transcripts is regulated by the master gene Sex lethal and tra itself regulates splicing of the transcriptional regulator doublesex (dsx). We present the isolation and characterization of Botra, the olive fruit fly Bactrocera oleae orthologue to the Drosophila gene transformer. As in Drosophila, Botra transcripts are spliced in a sex-specific manner so that only females encode a functional polypeptide of 422 amino acids, whereas males encode presumably nonfunctional peptide(s). The identification of multiple TRA/TRA-2 binding sites within the Botra male-specific exons, suggests an autoregulation mechanism of tra, through TRA/TRA2 activities. The fundamental role of the TRA protein in sex determination of Bactrocera was investigated by RNA interference, where the introduction of Botra dsRNA into embryos resulted in complete transformation of XX flies into fertile males.

Expression and function of the Drosophila melanogaster ADH in male Ceratitis capitata adults: a potential strategy for medfly genetic sexing based on gene-transfer technology.

The aim of development of a Mediterranean fruit fly Ceratitis capitata genetic sexing strain derives from the large scale SIT programmes being carried out to control this pest. Toward this direction, we present here the male-specific expression of the Drosophila melanogaster alcohol dehydrogenase (ADH) in medfly transgenic adults generated by Minos-mediated germ line transformation. This expression pattern is obtained by using a promoter fragment of the male-specific gene MSSP-alpha2 of the medfly. We show that the heterologous enzyme is functional in the medfly oxidizing both ethanol and 2-propanol. Although leading to an approximately twofold increase of total ADH activity in male compared to female transgenic adults, these expression levels are not enough for performing genetic sexing when high doses of environmental alcohol are applied. This could be achieved either by further enhancement of the transgene expression or by generating an Adh- line to host the Minos insertions.

The chorion genes of the medfly. II. DNA sequence evolution of the autosomal chorion genes s18, s15, s19 and s16 in Diptera.

We present a total of approximately 15 kb of DNA sequences, encompassing four chorion genes Ccs18, Ccs15, Ccs19, Cc16 and their flanking DNA in the medfly C. capitata. Comparison of coding regions, introns and intergenic sequences in five Dipteran species, D. melanogaster, D. subobscura, D. virilis, D. grimshawi and C. capitata documented an extensive divergence in introns and coding regions, but few well conserved elements in the proximal 5' flanking regions in all species. These elements are related to conserved regulatory features of three of the genes, including tissue- and temporal regulation. In the fourth, gene s15, significant alterations in the 5' flanking region may be responsible for its changed temporal regulation in C. capitata. One long intergenic sequence, located in the distal 5' flanking region of gene s18, is homologous to ACE3, a major amplification control element and contains an 80-bp A/T-rich sequence, known to stimulate strong binding of the origin recognition complex (ORC) in D. melanogaster. Analysis of the nucleotide composition of all chorion genes in C. capitata and D. melanogaster showed that C. capitata exhibit less biased representation of synonymous codons than does D. melanogaster.

Two medfly promoters that have originated by recent gene duplication drive distinct sex, tissue and temporal expression patterns.

Genes encoding predominantly male-specific serum polypeptides (MSSPs) in the medfly Ceratitis capitata are members of a multigene family that are structurally similar to the genes encoding odorant binding proteins of insects. To study the transcriptional regulation of the genes MSSP-alpha2 and MSSP-beta2, overlapping fragments of their promoters, containing the 5' UTRs and 5' flanking regions, were fused to the lacZ reporter gene and introduced into the medfly genome via Minos-mediated germline transformation. Transgenic flies were functionally assayed for beta-galactosidase activity. Despite their extensive sequence similarity, the two gene promoters show distinct expression patterns of the reporter gene, consistent with previously reported evidence for analogous transcriptional activity of the corresponding endogenous genes. The MSSP-alpha2 promoter drives gene expression specifically in the fat body of the adult males, whereas the MSSP-beta2 promoter directs gene expression in the midgut of both sexes. In contrast, similar transformation experiments in Drosophila melanogaster showed that both promoters drive the expression of the reporter gene in the midgut of adult flies of both sexes. Thus, the very same MSSP-alpha2 promoter fragment directs expression in the adult male fat body in Ceratitis, but in the midgut of both sexes in Drosophila. Our data suggest that through the evolution of the MSSP gene family a limited number of mutations that occurred within certain cis-acting elements, in combination with new medfly-specific trans-acting factors, endowed these recently duplicated genes with distinct sex-, tissue-, and temporal-specific expression patterns.

Organization, evolution and expression of a multigene family encoding putative members of the odourant binding protein family in the medfly Ceratitis capitata.

A multigene family encoding male specific serum polypeptides (MSSPs) that show significant structural similarity to the family of insect odourant binding proteins, has been characterized in the medfly Ceratitis capitata. This family comprises seven members classified in three subgroups, MSSP-alpha, MSSP-beta and MSSP-gamma. The genes of subgroups alpha and beta are clustered in tandem in a 35-kb genomic region, and present an exceptionally high degree of similarity not only in their coding but also in the surrounding regions, while the genes of the gamma subgroup are drastically divergent. Although MSSPs are predominantly expressed in the male fat body, detailed expression studies suggest that individual members of this family are expressed in a distinct sex- and tissue-specific manner.

Cloning and characterization of a cDNA encoding a male-specific serum protein of the Mediterranean fruit fly, Ceratitis capitata, with sequence similarity to odourant-binding proteins.

Male-specific serum proteins (MSSPs) are low molecular weight proteins which accumulate in high amounts in the haemolymph of adult males of the medfly Ceratitis capitata. By screening an expression library with anti-MSSP antibodies, we have isolated and determined the nucleotide sequence of a cDNA clone coding for one of the male-specific polypeptides (MSSP-alpha). The MSSP-alpha mRNA encodes a polypeptide of 144 amino acids with a secretory signal sequence of sixteen amino acids. Southern analysis indicated that there are multiple copies of MSSP genes in the medfly genome. Northern analysis showed that the MSSP mRNAs are synthesized only in adult males. The accumulation pattern of these mRNAs during development suggests that the expression of the MSSP genes is developmentally regulated at both transcriptional and translational levels. The predicted peptide sequence of MSSP-alpha shows significant similarity to a group of pheromone- and general odourant-binding proteins of insects.

The autosomal chorion locus of the medfly Ceratitis capitata. I. Conserved synteny, amplification and tissue specificity but sequence divergence and altered temporal regulation.

We report the isolation, full sequence characterization, amplification and expression properties of medfly chorion genes corresponding to the autosomal chorion locus of Drosophila. These genes are found adjacent to the paramyosin gene and are organized in the same order and tandem orientation as their Drosophila homologues, although they are spaced further apart. They show substantial sequence divergence from their Drosophila homologues, including novel peptide repeats and a new spacing of the tyrosines, which are known to be cross-linked in Dipteran chorion. The genes are amplified and expressed during oogenesis, as in Drosophila. Three of them are expressed in the same relative temporal order as in Drosophila but the fourth gene, the homologue of s15, shows a clear shift to an earlier expression period. This is the first known instance of changed temporal regulation in dipteran chorion genes.

The genome of the Mediterranean fruitfly Ceratitis capitata: localization of molecular markers by in situ hybridization to salivary gland polytene chromosomes.

We hybridized cloned DNA segments to salivary gland polytene chromosomes of the medfly, Ceratitis capitata, and thus established molecular markers for 24 sites on 6 out of 10 autosomal arms. An additional marker identified a medfly repetitive element that hybridizes to approximately 100 autosomal sites as well as a granular network that is thought to represent the X chromosome. Some of the markers correspond to 9 characterized transcription units, while 17 remain anonymous; at least 3 of the latter are restriction fragment length polymorphism (RFLP) markers. The characterized transcription units document that chromosomal arm 5L of C. capitata is homologous to the Drosophila melanogaster X chromosome, in agreement with previous inferences based on the extensive conservation of linkage groups in Diptera.

Tandemly repeating peptide motifs and their secondary structure in Ceratitis capitata eggshell proteins Ccs36 and Ccs38.

Evidence from amino acid composition, Fourier transform analysis of primary structure and secondary structure prediction suggests a tripartite structure for Ceratitis capitata eggshell proteins Ccs36 and Ccs38, which consists of a central domain and two flanking 'arms'. The proteins, apparently, contain tandemly repeating peptide motifs specific for each domain of the tripartite structure. The central domain of both proteins, which exhibits extensive sequence homology with the corresponding domains of Drosophila melanogaster proteins s36 and s38, is formed by tandem repeats of an octapeptide-X-X-X-Z-Z-Z-Z-Z- (where X = large hydrophobic residue and Z = beta-turn former residue) and its variants. It is predicted to adopt a compact, most probably twisted, antiparallel beta-pleated sheet structure of beta-sheet strands regularly alternating with beta-turns or loops. The central domains of Ccs36 and Ccs38 share structural similarities, but they are recognizably different. The 'arms' of the proteins presumably serving for protein and species-specific functions differ substantially from those of Drosophila melanogaster. In Ccs36, the C-terminal 'arm' is formed by, almost precise, tandem repeats of an octapeptide-Y-X-A-A-P-A-A-S- (X = G or S), whereas the N-terminal 'arm' contains repeats of the octapeptide -Z-Z-Z-A-X-A-A-Z- (X = Q, N or E and Z a beta-turn former). In both 'arms' alpha-helices are predicted, alternating with beta-turns.(ABSTRACT TRUNCATED AT 250 WORDS)

The chorion genes of the medfly, Ceratitis capitata. II. Characterization of three novel cDNA clones obtained by differential screening of an ovarian library.

We have isolated three new chorion cDNA clones from a Ceratitis capitata ovarian library. Their isolation was accomplished by differential screening of the library using as probes 32P-labeled poly(A)+ mRNAs obtained from hand-staged medfly choriogenic versus prechoriogenic follicles. RNA blot hybridization analysis revealed that the genes corresponding to these clones have unique temporal profiles of mRNA accumulation, restricted to specific choriogenic stages. In addition, in vitro translation products encoded by these cDNAs approximately comigrated with polypeptides synthesized de novo in culture by choriogenic follicles. All three genes are located in regions of the medfly genome that are specifically amplified in female ovaries. DNA sequence analysis has revealed that one of these clones is derived from a homolog of the Drosophila melanogaster s38 chorion gene. It appears that, although D. melanogaster and C. capitata are separated by at least 120 million years of evolution, the mechanisms by which chorion genes are expressed and regulated during development have been well maintained. We suggest that the regulatory elements controlling the expression of sex-specific (e.g., chorion) genes may be isolated and used to construct transgenic medfly strains from which females could be eliminated by negative selection; such strains could be used as part of an effort to control this agricultural pest.

The chorion genes of the medfly, Ceratitis capitata, I: Structural and regulatory conservation of the s36 gene relative to two Drosophila species.

We have used low stringency screening with the Drosophila melanogaster s36 chorion gene to recover its homologue from genomic and cDNA libraries of the medfly, Ceratitis capitata. The same gene has also been recovered from a genomic library of D. virilis. The medfly s36 gene shows similar developmental specificity as in Drosophila (early choriogenesis). It is also specifically amplified in ovarian follicles; this is the first report of chorion gene amplification outside the genus Drosophila. Alignments of s36 sequences from three species show that, in addition to its regulatory conservation, the s36 gene is extensively conserved in sequence, in a region corresponding to a central protein domain, and in short regions of 5' flanking DNA that might correspond to cis-regulatory elements.

Structural and biochemical studies on four sex-linked chorion mutants of Drosophila melanogaster.

Four female-sterile mutants, fs(1)K451, fs(1)K1214, fs(1)K575TS, and fs(1)384, were studied in terms of chorion structure and chorion protein composition. The first three of these mutants cause morphological defects, ie, substantial underproduction and disruption of the endochorion, correlated with underproduction of the six major chorion proteins, s15-s38; the phenotypes are consistent with the observation that these mutants interfere with amplification of the major chorion genes that encode the s15-s38 proteins [Orr et al.: Proc Natl Acad Sci USA 81:3773-3777, 1984; Komitopoulou et al.: Dev Genet 7:75-80, 1986]. The fourth mutant, fs(1)384, and its alleles do not interfere with production of the major chorion proteins and the morphologically detectable bulk of the endochorion but lead to failure of endochorionic organization. Apparently this complementation group is responsible for a minor chorion product, which is evidently important morphogenetically and which is processed posttranslationally in a complex manner [Bauer and Waring: Dev Biol 121:349-358, 1987].

A temperature-sensitive mutant affecting the process of chorion gene amplification in Drosophila melanogaster.

K575 is a temperature-sensitive female sterile mutant which shows abnormal chorion structure and subnormal amounts of the major chorion proteins at the restrictive temperature. These phenotypes apparently result from a temperature-sensitive defect in amplification. Both clusters of chorion genes are affected, indicating that the gene operates in trans.

Mutants suppressing in trans chorion gene amplification in Drosophila.

Two recessive female-sterile mutants, K451 and K1214 , disrupt chorion formation by causing underproduction of all major chorion proteins. We present evidence that this effect is due to underaccumulation of the chorion mRNAs and that, in turn, this is caused by a substantial reduction in the level of chorion gene amplification. The mutants are X-linked but located at two sites far from the chorion gene cluster at 7F1 -2; their effect is even more pronounced on the third chromosome chorion gene cluster, and thus the wild type gene must act in trans. The time course of amplification in mutant and wild-type follicles is documented.

Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER with Special Attention to Eggshell Mutants.

To study genes that function mainly or exclusively during oogenesis, we have isolated and analyzed female-sterile mutations, with special emphasis on those that affect eggshell formation. Following treatment that induced 61 to 66% lethals, 8.1% of the 1071 X chromosomes tested carried recessive female sterility mutations (87 isolates), and 8.0% carried partial female-sterile mutations (86 isolates), respectively. In addition, three dominant female steriles were recovered. Some of the mutants had very low fecundity, and others laid morphologically normal eggs that failed to develop. A third category included 29 mutants that laid eggs with morphological abnormalities: 26 were female steriles, two were partial female steriles and one was fertile. Mutants of this third category were characterized in some detail and compared with 40 previously isolated mutants that laid similarly abnormal eggs. Approximately 28-31 complementation groups with morphological abnormalities were detected, some of which were large allelic series (11, 9, 7, 6 and 5 alleles). Twenty-four groups were mapped genetically or cytogenetically, and 21 were partially characterized by ultrastructural and biochemical procedures. Of the latter, one group showed clear deficiency of yolk proteins, and nine showed prominent ultrastructural defects in the chorion (at least eight accompanied by deficiencies in characterized chorion proteins). At least six groups with clear-cut effects were found at loci not previously identified with known chorion structural genes.

Genetic Analysis of Three Dominant Female-Sterile Mutations Located on the X Chromosome of DROSOPHILA MELANOGASTER.

Three dominant female-sterile mutations were isolated following ethyl methanesulfonate (EMS) mutagenesis. Females heterozygous for two of these mutations show atrophy of the ovaries and produce no eggs (ovo( D1)) or few eggs (ovo(D2)); females heterozygous for the third mutation, ovo(D3), lay flaccid eggs. All three mutations are germ line-dependent and map to the cytological region 4D-E on the X chromosome; they represent a single allelic series. Two doses of the wild-type allele restore fertility to females carrying ovo(D3) and ovo(D2), but females carrying ovo(D1) and three doses of the wild-type allele remain sterile. The three mutations are stable in males but are capable of reversion in females; reversion of the dominant mutations is accompanied by the appearance, in the same region, of a recessive mutation causing female sterility. We discuss the utility of these mutations as markers of clones induced in the female germ line by mitotic recombination as well as the nature of the mutations.