Molecular analysis ofcis-regulatory sequences at the ?-amylase locus inDrosophila melanogaster

Hawley, Sylvia A.; Doane, Winifred W.; Norman, R. A.

doi:10.1007/bf00553754

Cited by 7 publications

(4 citation statements)

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“…Amylases and amylase genes in insects have been best described in species of Drosophila where there is evidence for conservation of genome organization and generegulation (Payant et a/., 1988). Here they are present in general as multigene families producing proteins with similar molecular weights in the range of 45-55 kD (Boer & Hickey, 1986;Gemmill et a/., 1986;Hawley et a/., 1990Hawley et a/., , 1992DaLage et a/., 1992). Some multigene families include silent or pseudogene members Doane et a/., 1990).…”

Section: Discussionmentioning

confidence: 99%

The salivary glands of the vector mosquito, Aedes aegypti, express a novel member of the amylase gene family

Grossman¹,

James

1993

Insect Molecular Biology

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Several cDNA clones with similarity to alpha-amylases have been characterized from a library made from adult female salivary gland RNA isolated from the vector mosquito, Aedes aegypti. The corresponding gene, designated Amylase I (Amy I), is expressed specifically in the proximal-lateral lobes of the adult female salivary gland, a pattern overlapping that of another gene, Mal I, involved in carbohydrate metabolism. The deduced amino acid sequence of Amy I indicates that this gene encodes a protein, approximate M(r) = 81,500, that appears to be a novel member of the amylase gene family. The mosquito protein contains a putative signal peptide for secretion and several consensus sites for asparagine-linked glycosylation. The Amy I protein shows significant similarity to invertebrate and vertebrate amylases including the conservation of four reactive and substrate binding sites. However, the amino-terminal region of the Amy-I protein is unique to the mosquito. Similarity with the Drosophila melanogaster protein is evident only after the first 260 amino acids in the mosquito sequence. The identification of this gene and its expression pattern adds to the observed relationship between spatial-specific gene expression in the female salivary glands and the specific feeding mode of the adult mosquito.

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Section: Discussionmentioning

confidence: 99%

The salivary glands of the vector mosquito, Aedes aegypti, express a novel member of the amylase gene family

Grossman¹,

James

1993

Insect Molecular Biology

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“…In the 5'-flanking sequence of the distal genes, sequences from 0 to the position corresponding to the EcoRI site in the 5'-flanking sequence of the distal gene of D. sechellia were determined. We next examined whether the putative regulatory elements identified from the nucleotide sequence of D. melanogaster (Boer and Hickey 1986;Hawley, Doane, and Norman 1992;Choi and Yamazaki 1994) are conserved among the species or not. In figure 2, they are indicated by boxes.…”

Section: Multiple Alignment and Putative Regulatory Elementsmentioning

confidence: 99%

Molecular evolution of the 5'-flanking regions of the duplicated Amy genes in Drosophila melanogaster species subgroup

Okuyama

Shibata

Tachida³

et al. 1996

Molecular Biology and Evolution

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The nucleotide sequences of the 5'-flanking regions of the duplicated Amy genes in eight sibling species belonging to the melanogaster species subgroup are analyzed. In Drosophila melanogaster, a region of about 450 bp immediately upstream of the translation initiation site of the two paralogous genes (the proximal and distal genes) has sequence similarities. However, we could not detect any significant sequence similarity in the region more upstream than -450. This result indicates that the coding regions of the ancestral Amy gene were duplicated together with 450 bp of the 5'-flanking region as one unit. Multiple alignment of these 450-bp sequences in the proximal and distal genes of all eight species revealed a mosaic pattern of highly conserved and divergent regions. The conserved regions included almost all the putative regulatory elements identified in previous analyses of the sequences. A phylogenetic analysis of the aligned sequences shows that these 450-bp sequences are clustered into the proximal and the distal groups. As a whole, the divergence between groups in this region is very large in contrast to that in the coding regions. Based on the divergence between groups, the 450-bp region is divided into two subregions. We found that the ratios of the divergence between groups to that within groups differ in the two subregions. From these observations, we discuss a possibility of positive selection acting on the subregion immediately upstream of the Amy coding region to cause divergence of regulatory elements of the paralogous genes.

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“…MIG1 is the component that is specific to genes subject to catabolite repression, and its binding site has been shown to be important in the glucose repression of yeast GAL genes (29). In addition to the basic transcription signals, such as TATA, and the glucose repression elements inferred here, the Drosophila amylase promoter also contains signals for tissue-specific expression in flies (30)(31)(32). These signals must have been added to the promoter relatively recently in the course of evolution-i.e., after the divergence of metazoans from other primitive eukaryotic lineages.…”

Section: Discussionmentioning

confidence: 99%

A Drosophila gene promoter is subject to glucose repression in yeast cells.

Hickey

Benkel

Fong

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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Previous work has shown that the a-amylase gene of Drosophila Ml Glucose repression is a common feature in the expression of microbial genes, and this phenomenon has been the subject of extensive genetic and molecular studies (1-3). The Drosophila amylase gene provides a rare example of glucose repression in multicellular animals (4, 5). As in the microbial systems, this Drosophila gene is required for the utilization of nonpreferred carbohydrate resources (6), and the repression is mediated by promoter sequences close to the transcriptional start site (7,8 Fig. 1A).The recombinant gene Amy/Luc (Fig. 1B) Aphid (15). This approach facilitated the introduction of the restriction enzyme sites that were used for assembly of the gene components, as well as the transfer of hybrid genes between vectors.The Act/Luc plasmid ( Fig. 1 C) was constructed by deleting the amylase promoter of the Amy/Luc plasmid and replacing it with a 427-bp firgment containing the yeast actin promoter (13). First, a fragment of the yeast actin gene was amplified from plasmid pTZAct34 (obtained from A. Wildeman, University of Guelph) by using oligomers that converted the sequence beyond -427 into a Not I site and the ATG environment into an Nco I site. Then, the fly amylase promoter was removed by digestion ofthe Amy/Luc plasmid with Not I and Nco I followed by agarose gel fractionation.Finally, the recovered, promoterless fragment was ligated with the actin promoter fragment to give the Act/Luc plasmid.Yeast Transformation and Expression Assays. Yeast cells from the AH22 cir+ strain of Saccharomyces cerivisiae (16) were transformed with recombinant shuttle vectors by using standard techniques (17,18). Cells were plated on selective medium (leu-dropout, Bio 101), and leu prototrophs were grown at 30TC in rich liquid medium (yeast extract/peptone) with the carbon source [5% (vol/vol) glycerol or 5% (wt/vol) glucose] as indicated.Amylase activity is secreted from transformed yeast cells. For the enzyme assays, cells were removed by centrifugation, and aliquots ofthe extracellular medium were separated on polyacrylamide gels under nondenatring conditions. Gels were processed and stained for amylase activity as described (5).Luciferase is not secreted from the transformed cells. To assay for this enzyme, transformed cells were cultured for 24 hr. Cell densities of the cultures were estimated by ODIN measurements. Cells were harvested by centrifugation, resuspended in cell lysis buffer (Promega), and frozen for at least 24 hr at -70°C. Cells grown on glycerol-containing medium were resuspended in a volume of cell lysis buffer 11109The pubion csts ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Molecular analysis ofcis-regulatory sequences at the ?-amylase locus inDrosophila melanogaster

Cited by 7 publications

References 51 publications

The salivary glands of the vector mosquito, Aedes aegypti, express a novel member of the amylase gene family

The salivary glands of the vector mosquito, Aedes aegypti, express a novel member of the amylase gene family

Molecular evolution of the 5'-flanking regions of the duplicated Amy genes in Drosophila melanogaster species subgroup

A Drosophila gene promoter is subject to glucose repression in yeast cells.

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