Investigation of cis-acting sequences regulating expression of the gene encoding yolk protein 3 in Drosophila melanogaster.

Summary The regulatory sequences leading to the ovarian and fat body expression of yolk proteins 1 and 2 (YP1 and 2) of Drosophila melanogaster have been characterised in some detail. These genes (yp1 and yp2) share many enhancer elements, and some important regulatory sequences lie within the coding regions. We have begun to investigate the cis-regulation of the gene encoding yolk protein 3 (yp3). We describe a system for P element transformation using the complete and unaltered yp3 gene rather than reporter genes and describe sequences conferring correct expression in the ovary and carcass.     

Conservation and divergence in the control of yolk protein genes in dipteran insects

 We have investigated the conservation of regulatory elements for sex- and tissue-specific gene expression in three dipteran species, Drosophila melanogaster, Musca domestica and Calliphora erythrocephala, using the yolk protein (yp) genes. Yolk proteins of the fruitfly, medfly, housefly and blowfly are very well conserved both in their sequence and their expression in ovarian follicle cells and in fat bodies of adult females. Furthermore, yp regulation by both hormonal and nutritional factors shows similar features in all four species. To study conservation of yp regulation in dipteran insects, we tested 5′ flanking regions from one Musca yp gene and one Calliphora yp gene for enhancer functions in D. melanogaster. Two fragments of 823 and 1046 bp isolated from Musca and Calliphora yp genes, respectively, are able to direct correct expression of a reporter gene in the ovarian follicle cells of transformed Drosophila at specific stages during oogenesis. Surprisingly, these enhancers do not confer sex-specific reporter gene expression in the fat body, as expression was found in both sexes of the transformed flies. None-the-less by in vitro DNA/protein interaction assays, a 284-bp DNA region from the Musca yp enhancer was able to bind the Drosophila DOUBLESEX (DSX) protein, which in D.melanogaster confers sex-specific expression of yp. We speculate that the sex-determining pathway is not directly involved in yp regulation in Musca or Calliphora adult females, but depends instead on hormonal controls to achieve sex-specific expression of yp genes in the adult. Received: 17 April 1997 / Accepted: 12 July 1997     

A tissue-specific transcription enhancer from the Drosophila yolk protein 1 gene

The yolk protein 1 gene (yp1) of Drosophila melanogaster is expressed only in the ovarian follicle cells and the fat bodies of adult females. We have previously shown that a different cis-acting DNA region is required for each of these tissue specificities. In this paper we use germ line transformation to localize and characterized one of these tissue-specific regulatory regions. We demonstrate that a 125 bp segment of DNA located 196 bp upstream of the yp1 cap site is sufficient to determine the sex-, stage-, and fat body-specific expression of the yp1 gene. We also find that this region can confer yp1-specific expression on a heterologous Drosophila promoter. This specificity is retained when the region is in different orientations and at different distances from the heterologous promoter. Thus a small regulatory region acts in vivo as a positive enhancer to determine the fat body expression pattern of yp1.     

Differential reactivity of filarial antigens with human sera from bancroftian filariasis endemic zone

The reacting pattern of circulating filarial antigen fraction-2 fromWuchereria bancrofti and soluble antigen from adultBrugia malayi with bancroftian filarial sera were analysed by immunoblotting technique and enzyme linked immunosorbent assay. Microfilaraemic sera reacted specifically with proteins of molecular weight 200, 120, 97, 56, 54, 43, 26 and 17 kDa of circulating Filarial antigen fraction-2 and 44, 40, 38, 31, 22 and 18 kDa ofBrugia malayi adult soluble antigen. Clinical filarial sera identified protein molecules of 56, 54 and 42 kDa of circulating filarial antigen fraction-2 and 19, 16 and 14 kDa ofBrugia malayi adult soluble antigen. Some components of both the antigen preparation were also identified by endemic normal serai.e.proteins 120, 97, 62, 43 and 33 kDa of circulating filarial antigen fraction-2 and 170, 120, 43, 31 and 12 kDa ofBrugia malayi adult soluble antigen. One of the sodium dodecyl sulphate-polyacrylamide gel electropherosis fractions of circulating filarial antigen fraction-2 (CFA₂-8) andBrugia malayi adult soluble antigen fraction-6 when used in enzyme linked immunosorbent assay could differentiate microfilaraemic sera from endemic normal and clinical filarial sera. The other antigen fractions (CFA₂-2, 6 and 7 andBmA-2) showed a high geometric mean titre of filarial immunoglobulin G antibodies in endemic normal sera when compared to microfilaraemia and clinical filarial sera. These proteins need to be further studied to assess their involvement in protecting from filarial infection in an endemic area.     

The regulation of <Emphasis Type="Italic">yp3</Emphasis> expression in the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> fat body

The regulation of the Drosophila melanogaster yolk protein genes 1 and 2 have been well characterised. Cis-acting DNA elements and trans-acting factors regulating ovarian fat body and sex-specific expression have been identified. In this paper we have analysed the regulation of yolk protein 3, which is separated from the other two genes on the X-chromosome. We have separated sex-specific control from fat body control in some constructs in transgenic flies. We propose that the organisation of the regulatory elements in yp3 differs from yp1 and yp2 for control of fat body expression and that it closely resembles the regulation of a reporter gene using Musca and Calliphora yp promoter enhancer sequences in transgenic Drosophila.     

Yolk synthesis in ovarian follicles ofDrosophila

Summary The autonomous synthesis of yolk proteins in ovarian follicles ofDrosophila melanogaster was analyzed. Vitellogenic follicles were labelled with³⁵S-methionine in vitro and the newly synthesized yolk proteins were separated by SDS-polyacrylamide gel electrophoresis. Possible contamination of the follicle preparations caused by adhering fat body cells could be excluded by culturing follicles in males prior to labelling in vitro. When labelled follicles were cut at the nurse cell/oocyte border the three yolk proteins (YP1, YP2, YP3) were found only in posterior fragments containing ooplasm and follicle cells, whereas two radioactive protein bands (A and B) were detected in nurse cells (anterior fragments). The yolk proteins of these five bands were characterized by peptide mapping. Band A protein, migrating a little more slowly than YP2, is closely related to both YP1 and YP2 while band B contains a yolk protein which is very similar to YP3. Hence, the nurse cells have been identified as a site of vitellogenin synthesis within the ovary ofDrosophila.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

Cloning and characterization of mouse mSox13 cDN

A novel SRY-related cDNA, mSox13, was isolated from a λ phage library derived from mouse embryo. The cDNA encodes a protein of 595 amino acids containing the SRY-type high mobility group (HMG) box and a putative leucine zipper motif. A sequence comparison of mSox13 and other type-D SOX proteins shows that the leucine zipper and a neighboring glutamine-rich sequence stretch, which was named Q box, are well conserved among known type-D SOX proteins. The expression of mSox13 is restricted to the kidney and ovary. The electrophoretic mobility shift assay indicates that the recombinant mSox13 protein is capable of binding to the AACAAT sequence.     

Binding ofNicotiana nuclear proteins to the subterminal regions of theAc transposable element

Specific binding ofNicotiana nuclear protein(s) to subterminal regions of theAc transposable element was detected using gel mobility shift assays. A sequence motif (GGTAAA) repeated in both terminal regions ofAc, was identified as the protein binding site. Mutation of two nucleotides in this motif was sufficient to abolish binding. Based on a series of competition assays, it is deduced that there is cooperative binding between two repeats, each similar to the GGTAAA motif. The binding protein is probably similar to a previously characterized maize protein which binds to a GGTAAA-containing motif located in the ends ofMutator. Moreover, we show that DNA fromDs1 competes for protein binding toAc termini, and we show, by sequence analysis, that GGTAAA binding sites are present in the terminal region ofTgm1, Tpn1, En/Spm, Tam3 andDs1-like elements. This suggests that the binding protein(s) might be involved in the transposition process.     

Incorporation of palmitic acid-1-14C into lipids of pharate adult tissues of Bombyx mori

Incorporation of palmitic acid-1-¹⁴C into pharate adult tissues and their lipid components of Bombyx mori was investigated. Rapid incorporation of radioactivity took place predominantly in fat body and haemolymph lipids, and partially in ovarian lipids immediately after the injection at the middle stage of pharate adult development. The major parts of the radioactivities in fat body, haemolymph and ovary were distributed in triglycerides and phospholipids, diglycerides, and triglycerides, respectively. The patterns of time course of incorporation of radioactivity into lipid components of pharate adult tissues suggest that the major form of lipid released from fat body may be diglycerides and the diglycerides in haemolymph are probably the main source of ovarian triglycerides.     

Cloning and DNA-binding properties of ethylene response factor,LeERF1 and LeERF2, in tomato

Two new genes, LeERF1 andLeERF2, were isolated from a tomato (Lycopersicon esculentum cv. Lichun) cDNA library. Phylogenetic analysis indicated that they encoded Ethylene Responsive Element Binding Proteins (EREBPs), characterized by a conserved ERF (ethylene response factor) domain of specific binding plant cis-acting elements GCC box. Both LeERF1 and LeERF2 proteins were obtained via prokaryotic expression and purification. Electrophoretic mobility shift assay showed that LeERF1 and LeERF2 protein could bind to the promoter of the NP24 gene coding for pathogenesis-related protein osmotin precursor but not the mutant promoter where its GCC box was deleted. Polyclonal antibodies of LeERF1 and LeERF2 blocked their binding in vitro.Revisions requested 4 January 2005; Revisions received 28 January 2005