Expression In Fat Body Research Articles

Cloning and sequencing of a cDNA for the hemolymph juvenile hormone binding protein of larval Manduca sexta.

A cDNA for the hemolymph juvenile hormone binding protein (JHBP) of larval Manduca sexta has been cloned and sequenced. The JHBP was purified to homogeneity from fifth instar larval hemolymph using gel filtration chromatography, ion exchange chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal rabbit antibodies, generated in response to this protein, were used to identify and isolate JHBP cDNAs from a fat body expression library in bacteriophage lambda ZAPII. Eleven putative JHBP cDNA clones were isolated and subcloned into Bluescript plasmid; cDNA inserts were approximately 750 base pairs in length. A 36-kDa immunoreactive protein was expressed from these plasmids; this beta-galactosidase fusion protein, like the authentic 32-kDa JHBP, was specifically photoaffinity labeled with [3H] epoxyhomofarnesyl diazoacetate (EHDA). Single-stranded DNA from one clone was sequenced by the Sanger dideoxynucleotide method, using deletion and custom primer techniques. A mature translation product was identified which had 226 amino acid residues, a molecular mass of 25,111 daltons, and a predicted isoelectric point (pI) of 5.40. The cDNA correctly predicts the N-terminal amino acid sequence and the amino acid composition of an authentic M. sexta hemolymph JHBP. A computer search of protein and nucleic acid data bases failed to reveal any related sequences. Thus, M. sexta hemolymph JHBP appears to be the first member of a new superfamily of insect hormone binding proteins.

Temporal and spatial utilization of the alcohol dehydrogenase gene promoters during the development of Drosophila melanogaster

The enzyme alcohol dehydrogenase of Drosophila melanogaster is encoded by a single structural gene ( Adh) with two promoters, distal and proximal ( P D and P P ). During development these two promoters are used differently: the major Adh transcript of larvae is from P P , the major transcript of adult flies is from P D . At a few discrete times in development transcription occurs simultaneously from both promoters. In situ hybridization has been used to investigate the spatial and temporal aspects of promoter activity at these stages of development. Maternally inherited Adh transcripts are not localized in the embryo; they decay very rapidly after fertilization. Zygotic expression of Adh RNA begins after germ-band retraction, 10.5 hr after fertilization. Expression is confined to the fat body, but occurs from both distal and proximal promoters. By 15 hr expression is first seen in the gut, from P P . By the same time fat body expression from P D has ceased, and transcription in this tissue is exclusively from P P for the next 4 days. The steady-state level of Adh transcript begins to decline at the end of larval development. There is then the transient accumulation of transcripts from P D , but predominantly in the larval fat body, rather than in the gut. These data illustrate a surprising complexity in the tissue and temporal regulation of Adh expression in D. melanogaster. Moreover, they show that transcripts from two different promoters of the same gene can, at certain well-defined stages of development, accumulate in the same cells.

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.

The effect of 20-hydroxyecdysone and juvenile hormone on transcription and specific gene expression in larval fat body in Galleria

The effects of 20-hydroxyecdysone and of juvenile hormone on transcription in fat body were studied in ligated abdomens of day-5 last-instar Galleria mellonella larvae. 20-Hydroxyecdysone stimulated synthesis of both total and poly(A) RNAs while juvenile hormone had no effect on transcription. The stimulatory effect of 20-hydroxyecdysone was observed within 3 h after hormone application and peaked at 6–8 h, at which time the specific activity of total and poly(A) RNAs was double that of controls. Fifteen hours after hormone application the specific activity of poly(A) RNA in treated and control larvae was identical. The specific activity of total RNA, however, remained higher than those of controls even 26 h after hormone treatment. The effect of juvenile hormone and 20-hydroxyecdysone on specific gene expression in these fat body cells was also determined by electrophoretic analysis of the polypeptides synthesized in vitro from the fat body poly(A) RNAs from experimental and control larvae. 20-Hydroxyecdysone induced synthesis of mRNAs that code for 22K and 26K Dalton proteins, which were not detectable in preparations from untreated, Ringers- or juvenile hormone-treated larvae. These mRNAs appeared between 1 and 6 h after hormone treatment and persisted up to 24h. Application of juvenile hormone 4 h before ecdysteroid treatment inhibited ecdysteroid induction of one of the inducible mRNAs.

Developmental control of Drosophila yolk protein 1 gene by cis-acting DNA elements.

In this paper we have demonstrated that at least two tissue-specific cis-acting DNA elements are necessary for the normal developmental pattern of Drosophila yp1 gene expression. One of these elements is necessary for expression in the adult female follicle cells and the other is necessary for expression in adult female fat bodies. We have localized the fat body expression element to a 125-bp fragment that lies between nucleotides -196 and -321 of the yp1 gene. This small fragment has at least one of the characteristics of enhancer sequences because it can direct a heterologous Drosophila promoter to be transcribed with the yp1 fat body expression pattern.