ABS1hACT Continuous cell lines that secrete both insulin and somatostatin were established by two cooprating groups of investigators from a serially transplantable, radiation-induced, rat islet cell tumor. The cell lines, named RIN-r and RIN-m, were initiated from tumors maintained in inbred rats or in athymic nude mice, respectively. The cultured cells are ppithelioid, free of fibroblast.contamination, and can be cloned.They have a hypodiploid chromosome number, are tumorigenic, and possess amine-handling properties, including high evels of L-dopa decarboxylase and formaldehyde-induced fluorescence. Preliminary analysis of clones revealed a spectrum of insulin secretion from undetectable to relatively high. These clonal cell lines provide important systems to study the biology of insulin and somatostatin. MATERIALS AND METHODS Origin of the cultures. Cultures were initiated from a transplantable islet cell tumor (6) induced by high-dose x-irradiation in an inbred NEDH (New England Deaconess Hospital) rat. The tumor was maintained by serial transplantation in NEDH rats. After nine transplants, it was successfully heterotransplanted into athymic nude mice, BALB/c background (ARS/Sprague-Dawley, Madison, WI). Continuous cell lines were derived either from rat transplants (Joslin group) or from nude mouse heterotransplants (NCI-VA group). These cell lines were named RIN-r and RIN-m, respectively.Establishment of RIN-r Cell Line. Tumors were removed aseptically from recipient rats and cut into small fragments The tumor cells were separated from the connective tissue stroma by washing the fragments with tissue culture medium 199 containing 0.1% fetal bovine serum, 16.5 mM glucose, and 400 units of penicillin per ml. The culture medium used throughout the remainder of the procedure was similar, but contained 10% fetal bovine serum. Erythrocytes present in the original cell suspension were removed by centrifugation (750 X g, 10 min) on a discontinuous gradient consisting of a solution of 25% di-

Gel mobility-shift assays have been used to identify proteins that bind specifically to the promoter region of the Drosophila s15 chorion gene. These proteins are present in nuclear extracts of ovarian follicles, the tissue where s15 is expressed during development, and bind to specific elements of the promoter that have been shown by transformation analysis to be important for in vivo expression. The DNA binding specificity has been used for molecular cloning of two components from expression cDNA libraries and for their tentative identification with specific DNA-binding proteins of the nuclear extracts. The mRNAs for both of these components, CF1 and CF2, are differentially enriched in the follicles. DNA sequence analysis suggests that both CF1 and CF2 are novel Drosophila transcription factors. CF2 is a member of the C2H2 family of zinc finger proteins, whereas CF1 is a member of the family of steroid hormone receptors. The putative DNA-binding domain of CF1 is highly similar to the corresponding domains of certain vertebrate hormone receptors and recognizes a region of DNA with similar, hyphenated palindromic sequences. The nature of CF1 raises the possibility of hormonal control of choriogenesis in Drosophila.

The Drosophila chorion factor 1/ultraspiracle (CF1/USP) transcription factor, a homologue of the retinoid X receptor, is a developmentally important member of the family of nuclear (steroid) hormone receptors. Using newly developed monoclonal antibodies and a full-length bacterially produced protein, we have studied in detail the in vitro DNAbinding properties of this factor and aspects of its distribution in vivo. During oogenesis, CF1/USP is present both in germline cells and in the somatic follicular epithelium. We have determined the optimal binding site of partially purifiled bacterially produced CF1/USP by an in vitro selection procedure and also have characterized its binding to the follicular-specific chorion siS promoter. In vito this bacterially produced factor is unusual in binding to a single element ("half-site"); simultaneous but noncoordinate binding to a second half-site is possible if these repeated elements are organized in direct orientation and spaced adequately. However, the factor interacts synergistically with several other nuclear hormone receptors: notably, it can form in vito heteromers with mammalian thyroid and retinoic acid receptors, binding to two half-sites that are organized in either direct or inverted orientation. In vivo the factor most probably functions as a heterodimer, but its partner(s) remains to be determined.Steroid and other nuclear hormone receptors are a family of ligand-modulated transcription factors that regulate cell differentiation and development as well as homeostasis and reproduction (1, 2). These receptors bind to DNA promoter or enhancer motifs ("hormone response elements" or HRE), which typically contain two copies of short, receptor-specific sequences called "half-sites," oriented either as direct (e.g., refs. 3-5) or as inverted (e.g., refs. 6 and 7) repeats. Although some members of the family bind to HREs as homodimers, others can heterodimerize, potentially enhancing the range of regulatory processes that can be served by a single HRE (8).In Drosophila melanogaster, the paradigm for metazoan genetics, nine members of this family have been identified so far, with strategic roles in embryogenesis, postembryonic development, and morphogenesis (2). We first cloned one of these factors by its binding to a potential HRE in the chorion siS promoter and named it chorion factor 1 (CF1) (9); this receptor was also cloned independently by homology to the DNA-binding domain of vertebrate hormone receptors (10,11). This orphan receptor, CF1/USP, is encoded by the ultraspiracle locus (usp) (10), a maternally and zygotically required gene with multiple functions in development (12). CF1/USP is most closely related to the subfamily of vertebrate retinoid X receptors (RXRs), suggesting that its ligand might be a retinoid metabolite (13)(14)(15).

No abstract

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.