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The Drosophila eggshell consists of three major proteinaceous layers: the vitelline membrane, the inner chorionic layer, and the outer endochorion. During the latter stages of oogenesis, the proteins that comprise these layers are synthesized and secreted by epithelial follicle cells which surround the maturing oocyte. While there is considerable knowledge of the structural units which comprise the eggshell layers, there is little knowledge of how individual proteins function or interact with one another to form the structure. Immunoelectron microscopy was used to follow the distribution of four different eggshell proteins in the assembling and mature eggshell. sV23 and sV17, follicle cell proteins synthesized during the early stages of eggshell formation (stages 8-10), were distributed within the vitelline membrane layer at all stages. Despite marked temporal differences in their accumulation profiles, s36 and s18, putative chorion proteins, were similarly distributed throughout the floor, pillars, and roof of the endochorion. Although the vitelline membrane appears to be morphologically complete by stage 11, developmental Western blots and immunolocalization data indicate that molecular dynamism persists within the layer throughout the subsequent choriogenic stages. During early chorion formation the vitelline membrane appears to act as a reservoir for chorion proteins since s36 was found predominantly in the vitelline membrane layer of stage 12 egg chambers. During the late choriogenic stages (13-14), both sV17 and sV23 are processed to smaller derivatives. Interactions between the eggshell layers were suggested by ultrastructural analysis of a sV23 protein null mutant which showed that the structural integrity of the outer chorion is dependent upon the presence of a vitelline membrane component.

We have isolated and characterized a dispersed middle repetitive DNA sequence from Drosophila melanogaster that is concentrated on the euchromatic portion of the X chromosome. In situ hybridization of the repeat unit to salivary gland chromosomes shows the sequence is distributed among approximately 10 major and 20 minor X chromosomal sites. Based on DNA sequence analysis of homologous sequences from three different cytogenetic regions, the 372-base-pair repeat unit appears to be (A+T)-rich and noncoding and shows strong sequence conservation among units from different chromosomal regions. The nature and distribution of this sequence are suggestive of the hypothetical X chromosome DNA sequences thought to be involved in the primary establishment of sex determination and dosage compensation in Drosophila.The Drosophila genome consists of three kinetic components, highly repetitive, middle repetitive, and unique DNA (1). The middle repetitive fraction constitutes -417% of the total genomic DNA and includes the tandemly repeated rRNA, 5S RNA, and histone genes. Most of the middle repetitive DNA in Drosophila melanogaster, however, is not tandemly repeated but rather is dispersed over the entire genome (2). It is composed of at least 50-100 different sequence families, each of which generally consists of 10-100 closely related sequences (2). The number of sequences within a family seems to be conserved in different strains of the species, but their chromosomal positions are highly variable. In marked contrast, in this paper we describe a dispersed middle repetitive family with repeats at chromosomal positions that are conserved between strains. More importantly, the repeat units are concentrated in the euchromatic portion of the X chromosome. A functional link between family members is suggested by the extensive sequence homology found between repeats from different chromosomal regions. The intriguing possibility that these sequences may play a role in the primary establishment of sex determination and dosage compensation in Drosophila is considered. MATERIALS AND METHODSPreparation of Nucleic Acids. The Maniatis X bacteriophage library of D. melanogaster genomic DNA (3) was screened by in situ plaque hybridization (4) under low stringency hybridization [4x SET (0.6 M NaCl/8 mM EDTA/0.12 M Tris HCl, pH 8), 43% formamide, 37°C] and wash conditions (0.3 M NaCl/30 mM sodium citrate, pH 7, 37°C) with the 32P-labeled single-stranded RNA probe described in the text.Phage from single plaques were purified from liquid cultures by centrifugation in CsCl block gradients (5); phage DNA was purified with NaDodSO4, proteinase K, and phenol. For smaller quantities, DNA was prepared from phage lysates with RNase, NaDodSO4, and phenol.Subclones were generated by inserting gel-purified restriction fragments into the appropriate sites of the pGem-1 and pGem-2 transcription vectors (Promega Biotec, Madison, WI). Recombinant plasmids were used to transfect Escherichia coli HB101. To confirm their identity, alkaline minipreparat...

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