Dynamic expression of broad-complex isoforms mediates temporal control of an ecdysteroid target gene at the onset of Drosophila metamorphosis.

Metamorphosis in Drosophila melanogaster is orchestrated by the steroid hormone ecdysone, which triggers a cascade of primary-response transcriptional regulators and secondary effector genes during the third larval instar and prepupal periods of development. The early ecdysone-response Broad-Complex (BR-C) gene, a key regulator of this cascade, is defined by three complementing functions (rbp, br, and 2Bc) and encodes several distinct zinc-finger-containing isoforms (Z1 to Z4). Using isoform-specific polyclonal antibodies we observe in the fat body a switch in BR-C isoform expression from the Z2 to the other three isoforms during the third instar. We show that the 2Bc(+) function that corresponds presumably to the Z3 isoform is required for the larval fat body-specific expression of a transgenic construct (AE) in which the lacZ gene is under the control of the ecdysone-regulated enhancer and minimal promoter of the fat body protein 1 (Fbp1) gene. Using hs(BR-C) transgenes, we demonstrate that overexpression of Z1, Z3, or Z4, but not Z2, is able to rescue AE activity with faithful tissue specificity in a BR-C null (npr1) genetic context, demonstrating a partial functional redundancy between Z1, Z3, and Z4 isoforms. We also show that continuous overexpression of Z2 during the third instar represses AE, while conversely, expression of Z3 earlier than its normal onset induces precocious expression of the construct. This finding establishes a tight correlation between the dynamic pattern of expression of the BR-C isoforms and their individual repressive or inductive roles in AE regulation. Altogether our results demonstrate that the balance between BR-C protein isoforms in the fat body mediates, in part, the precise timing of the ecdysone activation of the AE construct but does not modulate its tissue specificity.

Sequence, structure and evolution of the ecdysone-inducible Lsp-2 gene of Drosophila melanogaster.

The Lsp-2 gene encodes a major larval serum protein (hexamerin) of Drosophila melanogaster. Transcription of Lsp-2 is controlled by 20-hydroxyecdysone. Here we report the analysis of the structure of the Lsp-2 gene including the adjacent 5' and 3' sequences. In contrast to all other known hexamerin genes, Lsp-2 does not contain an intron. The Lsp-2 mRNA measures 2312 bases, as deduced from experimental determination of the transcription-start and stop sites and conceptual translation results in a 718 amino acid hexamerin subunit, including a 21-amino-acid signal peptide. While the calculated molecular mass of the native 697-amino-acid subunit is 83.5 kDa, mass spectrometry gave a value of 74.5 kDa. We detected in the Lsp-2 gene a 2052-bp antisense ORF that probably does not code for any protein. An unusual accumulation of rarely used codon triplets was found at the 5' and 3' ends of the Lsp-2 ORF. The calculated secondary structure matches well with that of arthropod hemocyanins. Electron micrographs show for LSP-2 hexamers a cubic shape, which can not be easily reconciled with its hexameric structure. Phylogenetic analysis revealed that LSP-2 diverged from the LSP-1 like hexamerins after separation of the Diptera from other insect orders.

The Drosophila Lsp-1 beta gene. A structural and phylogenetic analysis.

In Drosophila melanogaster, metamorphosis and reproduction are thought to be supported in large by two immunologically distinct hexameric storage proteins (hexamerins), larval serum protein 1 (LSP-1), a mixed hexamer of three closely related subunits, Lsp-1 (alpha, beta and gamma) and larval serum protein 2 (LSP-2), a homohexamer of Lsp-2 subunits. To understand the structural and functional differences between these two storage hexamers, the nucleotide sequence of the coding region of the Lsp-1 beta gene was determined for comparison with LSP-2 and a number of other arthropod hexamerins. The G + C content of the coding sequence is 55%, with 92.8% of the codons containing G or C in the third position. Conceptual translation of the Lsp-1 beta open reading frame revealed a 789-amino-acid polypeptide of 94465 Da. The amino acid sequence of Lsp-1 beta is 65.8% identical to that of calliphorin, the major hexamerin of the blowfly, Calliphora vicina, and only 35.2% identical to Drosophila Lsp-2. This greater similarity to calliphorin is also reflected in high aromatic amino acid and methionine contents, in contrast to LSP-2 which is enriched to a lesser extent only in aromatic amino acids. Lsp-1 beta is also more closely related to calliphorin with respect to the protein domain structure, the presence of a single intron in its gene, and the absence of glycosylation sites. However, phylogenetic analysis based on multiple alignments revealed that LSP-1 calliphorin and LSP-2 form a distinct dipteran clade whose members are more similar to each other than to any previously sequenced lepidopteran hexamerin or arthropod hemocyanin.

Adult expression of the Drosophila Lsp-2 gene.

Previous work has demonstrated expression of the larval serum protein-2 gene, Lsp-2, uniquely during the late larval and pupal stages of development in Drosophila melanogaster. Here we report that the LSP-2 polypeptide accumulates in the hemolymph throughout adult life as well. Western blot analysis using an LSP-2 antiserum reveals notable differences in the molecular weight of the larval and adult polypeptides. Lsp-2 transcription results in a unique mRNA of 2.3 kb, exhibiting the same 5' end in both larvae and adults. However, adult Lsp-2 mRNa is only expressed at 1% of the high level detectable in late third-instar larvae. Whereas Lsp-2 mRNA accumulates uniformly in all fat body cells of third-instar larvae, over 80% of the adult Lsp-2 transcript is expressed in the adipose tissue of the head. These results suggest a differential regulation for expression of the Drosophila Lsp-2 gene in adults and larvae.

sry h-1, a new Drosophila melanogaster multifingered protein gene showing maternal and zygotic expression.

Low-stringency hybridization of the Drosophila serendipity (sry) finger-coding sequences revealed copies of homologous DNA sequences in the genomes of members of the family Drosophilidae and higher vertebrates. sry h-1, a new Drosophila finger protein-coding gene isolated on the basis of this homology, encodes a 3.2-kilobase (kb) mRNA accumulating in eggs and abundant in early embryos. The predicted sry h-1 protein product, starting at an internal initiation site of translation, is a 868-amino-acid basic polypeptide containing eight TFIIIA-like fingers encoded by three separate exons. Links separating individual fingers in the sry h-1 protein are variable in length and sequence, in contrast with the invariant H/C link found in most multi-fingered proteins. The similarity of the developmental pattern of transcription of sry h-1 with that of several other Drosophila finger protein genes suggests the existence of a complex set of such genes encoding an information which is, at least partly, maternally provided to the embryo and required for activation of gene transcription in early embryos or maintenance of gene activity during subsequent development.

Identification and molecular analysis of a multigene family encoding calliphorin, the major larval serum protein of Calliphora vicina.

A library of Calliphora vicina genomic DNA was constructed in the lambdaEMBL3 vector and screened for recombinant phages containing chromosomal segments encoding calliphorin, the major larval serum protein (LSP) of Calliphora. A large series of recombinants hybridizing with in vitro labelled poly(A) RNA from Calliphora larval fat bodies and with specific probes derived from the LSP-1 genes of Drosophila melanogaster was isolated. Five of these phages, chosen at random, were shown by hybrid selection to retain calliphorin mRNA specifically. Eleven calliphorin mRNA-homologous regions were located on restriction maps of these phages by hybridization with 5' end-labelled poly(A) RNA from Calliphora larval fat bodies. Each phage contains at least two calliphorin genes arranged in direct repeat orientation and seperated by 3.5-5 kb intergenic regions. The genes display similar but not identical restriction patterns. Filter hybridization and heteroduplex analysis indicate that they share a detectable homology with the LSP-1beta gene of D. melanogaster. Whole genome Southern analysis showed that these genes belong to a large family of closely related calliphorin genes which were found by in situ hybridization to polytene chromosomes of trichogen cells to be clustered in region 4a of chromosome 2 of Calliphora vicina.

In situ hybridization: a routine method for parallel localization of DNA sequences and of their transcripts in consecutive paraffin sections with the use of 3H-labelled nick translated cloned DNA probes.

A routine in situ hybridization method is described and discussed, which allows parallel detection of repeated DNA sequences and of their abundant transcripts in consecutive tissue sections of a same biological sample with a unique probe. The protocol, based on the use of classical 3H-labelled nick translated cloned DNA probes and of conventional paraffin sections of ethanol-acetic acid-fixed tissues, consists of a simple combination of procedures in current use for separate detection of either RNA or DNA. Different treatments recommended in other methods are omitted or simplified, making the protocol suitable for routine use. The method is successfully applied here to a test-system where ribosomal sequences are sought in the ovarian follicles of the Lepidopteran Ephestia kühniella, by using a recombinant plasmid containing a Drosophila melanogaster rDNA repeating unit as a probe. Specific and reproducible results are obtained. Sensitivity is sufficient though moderate specific activities are used. Background level is very low. The regionalized distribution of sequences of both types in the chosen model allows to demonstrate that specific detection of RNA requires the systematic removal of DNA from the tissue sections prior to hybridization.

AT-rich linkers in long range organisation of Drosophila DNA.

AT-rich segments were mapped in 6 different domains of the Drosophila melanogaster genome by partial denaturation of cloned genomic DNA segments and observation in the electron microscope (EM). As found in a previous investigation (Moreau et al., 1982), three types of AT-rich segments could be distinguished: 1) AT-rich linkers with a very high AT content, 600 +/- 200 bp long; 2) clusters of such AT-linkers extending over up to 10 Kbp and 3) AT-rich stretches which are shorter and of lower AT content compared to AT-linkers. Six genes previously localized in these domains were found to lie in relatively GC-rich segments framed by AT-rich elements of the 3 types: the Larval Serum Protein LSP-1 alpha gene is framed by 2 AT-linkers, the LSP-1 beta and LSP-2 genes by two AT-rich stretches, an the LSP-1 gamma gene by a cluster and a stretch. The 55 Kbp genomic segment encompassing the P6 gene contains and AT-cluster of about 15 Kbp and several AT-linkers and AT-rich stretches. The 85 Kbp domain containing the P1 gene includes 3 AT-rich clusters of about 10 Kbp each framing GC-rich domains punctuated by AT-linkers and stretches. This study shows that AT-mapping allows a rapid diagnosis of large genomic DNA domains in relation to the AT-rich segments which, possibly, are of significance with regard to genome organisation and function.

A complete and a truncated U1 snRNA gene of Drosophila melanogaster are found as inverted repeats at region 82E of the polytene chromosomes.

A phage containing two sequences homologous to U1 snRNA was isolated from a Drosophila melanogaster genomic library, and identified with a previously cloned D. melanogaster U1 snRNA gene. DNA sequence analysis showed that complete and truncated U1 snRNA genes are present, both of which have base substitutions relative to U1 snRNA. These genes show conservation of 5' and 3' flanking regions relative to other U1 and U2 snRNA genes of Drosophila. Intramolecular renaturation experiments and electron microscope mapping demonstrates that the two U1 snRNA sequences are present as inverted repeats about 2.7kb apart, separated by a smaller pair of inverted repeats of an unrelated sequence. These U1 snRNA sequences were located by in situ hybridization at 82E, and related sequences were found at 21D and 95C on the polytene chromosome map. The results are discussed with reference to the origin and function of snRNAs.

Ecdysone-inducible functions of larval fat bodies in Drosophila.

Late in the third instar larval stage of Drosophila melanogaster, the titer of the steroid hormone ecdysone increases sharply. This increase is blocked in the temperature-sensitive mutant ecd(1) after a temperature shift from 20 degrees C to 29 degrees C. The mutant was used to prepare three samples of late third instar larvae with different titers of ecdysone; the titer was low in one sample because of an earlier temperature shift, high in a second sample because the larvae were subsequently transferred to ecdysone-supplemented food, and also high in a third sample that was kept at 20 degrees C, providing a control for normal development. The effect of the high titer of ecdysone on proteins of the larval fat bodies was examined by comparing two-dimensional gel electrophoresis patterns of total proteins in stained gels. There were proteins at five positions in the gels for the high-ecdysone samples that were not detected at the corresponding positions in the gel for the low-ecdysone sample. The effect of ecdysone on these proteins was further studied by injecting [(35)S]methionine into the larvae at both early and late third instar stages, in order to label proteins synthesized before and after the increase in ecdysone titer. The results indicate that ecdysone induces two major responses in the fat bodies; certain proteins that were synthesized earlier in the fat bodies and secreted into the hemolymph are incorporated back into the fat bodies, and other proteins are newly synthesized. Attempts to induce prematurely the synthesis of the new proteins by exposing early third instar larvae to exogenous ecdysone were unsuccessful, suggesting that development must proceed further before the fat bodies can respond to ecdysone. By in vitro translation of RNA isolated from fat bodies of low-and high-ecdysone samples of larvae, it was shown that ecdysone greatly increases the amount of translatable messenger RNA for one of the newly synthesized proteins. A clone of DNA complementary to the induced messenger RNA has been isolated from a population of lambda bacteriophage carrying segments of the Drosophila genome. Using the cloned DNA to measure amounts of complementary poly(A)-RNA in the fat bodies by DNA.RNA hybridization, we detected about 50 times more complementary poly(A)-RNA in the high-ecdysone sample of larvae than in the low-ecdysone sample. This finding provides direct evidence that ecdysone induces an increase in the amount of the messenger RNA. The ecdysone-induced appearance of a major messenger RNA in late third instar larval fat bodies represents a developmental response to ecdysone that appears to be gene-specific, tissue-specific, and stage-specific, and it has exceptionally favorable features for further molecular studies of the control of gene expression by a steroid hormone.