Expression of an amylase - alcohol dehydrogenase chimeric gene in transgenic strains of Drosophila melanogaster

Genome ◽  
1993 ◽  
Vol 36 (5) ◽  
pp. 954-961 ◽  
Author(s):  
Allan A. Grunder ◽  
Ada Loverre-Chyurlia ◽  
Donal A. Hickey
Keyword(s):  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Dna Analysis ◽  
Glucose Repression ◽  
Chimeric Gene ◽  
P Element ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Promoter Sequences ◽  
Adh Gene

A chimeric gene, consisting of 428 bp of the promoter sequences of the α-amylase gene of Drosophila melanogaster, fused to the transcribed region of the alcohol dehydrogenase (Adh) gene, was introduced into the genome of an Adhnull stock of Drosophila via P element mediated transformation. DNA analysis (Southern blotting) of three transformant strains confirmed the insertion of either one or two copies of the chimeric gene per strain. A histochemical study of ADH enzyme activity in dissected tissues of the transgenic larvae revealed that the chimeric Amy–Adh gene was expressed only in the posterior larval midgut and that this expression was repressed by dietary glucose, thus representing an expression pattern characteristic of the Amy gene. This indicates that the Amy upstream promoter sequences contain signals mediating both tissue specificity and glucose repression of the Adh structural gene in the transgenic larvae. The level of ADH activity expressed in transgenic flies was relatively low. This was paralleled by a low level of Adh mRNA, indicating a reduction in the transcriptional rate of the chimeric gene.Key words: Drosophila, germline transformation, chimeric gene, cis-regulatory sequences, α-amylase, alcohol dehydrogenase, tissue-specific expression, glucose repression, mRNA levels.

Rapid spread of a P element/Adh gene construct through experimental populations of Drosophila melanogaster

Genome ◽  
1993 ◽  
Vol 36 (6) ◽  
pp. 1169-1175 ◽  
Author(s):  
G. A. Meister ◽  
T. A. Grigliatti
Keyword(s):  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Transposable Elements ◽  
Transposable Element ◽  
Dehydrogenase Activity ◽  
P Element ◽  
P Elements ◽  
Alcohol Dehydrogenase Activity ◽  
Adh Gene ◽  
Experimental Populations

Transposable elements may be potential tools for the dispersal of engineered DNA through target insect populations. The utility of this hypothesis is predicated on the ability of transposable elements carrying a large DNA insert to rapidly disperse through a population. In addition, the inserted DNA must be replicated with a high degree of fidelity during this dispersal. We have monitored the ability of a transposable element with an inserted gene to spread through experimental populations and tested whether the passenger gene retains its ability to encode an active protein. Several Drosophila melanogaster laboratory populations were initiated with female flies that were null for alcohol dehydrogenase activity and contained no P elements. Most of the females were mated to males of the same strain; however, 1 or 10% of the females were mated to males from a strain that had previously been transformed with a helper P element and a P element/Adh gene construct. The dispersal of P elements to new genomes was monitored at each generation by randomly selecting females and performing DNA hybridization assays on dissected ovarian tissue. In addition, each female was tested for alcohol dehydrogenase activity using a simple histochemical assay. We find that, despite an approximate threefold increase in size, the P element constructs containing a functioning gene are still capable of rapid dispersal through the experimental populations. We also show that many of the inserted Adh genes still encode an active product.Key words: P element, transformation, Adh, transposable element.

scholarly journals Molecular control of the induction of alcohol dehydrogenase by ethanol in Drosophila melanogaster larvae.

Genetics ◽  
1990 ◽  
Vol 124 (4) ◽  
pp. 881-888 ◽  
Author(s):  
A M Kapoun ◽  
B W Geer ◽  
P W Heinstra ◽  
V Corbin ◽  
S W McKechnie
Keyword(s):  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Dna Sequence ◽  
P Element ◽  
Start Site ◽  
Major Pathway ◽  
Molecular Control ◽  
Adh Gene ◽  
Regulatory Dna ◽  
Transcript Start Site

Abstract The activity of alcohol dehydrogenase (ADH:EC 1.1.1.1), the initial enzyme in the major pathway for ethanol degradation, is induced in Drosophila melanogaster larvae by low concentrations of dietary ethanol. Two lines of evidence indicate that the metabolic products of the ADH pathway for ethanol degradation are not directly involved in the induction of Adh. First, the accumulation of the proximal transcript in Adhn2 larvae was increased when the intracellular level of ethanol was elevated. In addition, the ADH activity, the proximal Adh mRNA, and the intracellular concentration of ethanol were elevated coordinately in wild-type larvae fed hexadeuterated-ethanol, which is metabolized more slowly than normal ethanol. An examination of P element transformant lines with specific deletions in the 5' regulatory DNA of the Adh gene showed that a DNA sequence between +527 and +604 of the distal transcript start site is essential for the induction of the Adh gene [corrected]. The DNA sequence between -660 and about -5000 of the distal transcript start site was important for the down-regulation of the induction response.

scholarly journals Effects of a transposable element insertion on alcohol dehydrogenase expression in Drosophila melanogaster.

Genetics ◽  
1995 ◽  
Vol 140 (2) ◽  
pp. 667-677 ◽  
Author(s):  
R C Dunn ◽  
C C Laurie
Keyword(s):  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Transposable Element ◽  
Dna Sequence ◽  
Natural Populations ◽  
P Element ◽  
Dna Polymorphisms ◽  
Threefold Increase ◽  
Adh Gene ◽  
High Level

Abstract Variation in the DNA sequence and level of alcohol dehydrogenase (Adh) gene expression in Drosophila melanogaster have been studied to determine what types of DNA polymorphisms contribute to phenotypic variation in natural populations. The Adh gene, like many others, shows a high level of variability in both DNA sequence and quantitative level of expression. A number of transposable element insertions occur in the Adh region and one of these, a copia insertion in the 5' flanking region, is associated with unusually low Adh expression. To determine whether this insertion (called R142) causes the low expression level, the insertion was excised from the cloned R142 Adh gene and the effect was assessed by P-element transformation. Removal of this insertion causes a threefold increase in the level of ADH, clearly showing that it contributes to the naturally occurring variation in expression at this locus. Removal of all but one LTR also causes a threefold increase, indicating that the mechanism is not a simple sequence disruption. Furthermore, this copia insertion, which is located between the two Adh promoters and their upstream enhancer sequences, has differential effects on the levels of proximal and distal transcripts. Finally, a test for the possible modifying effects of two suppressor loci, su(wa) and su(f), on this insertional mutation was negative, in contrast to a previous report in the literature.

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

Regulated expression of a chimeric histone gene introduced into mouse fibroblasts

1985 ◽  
Vol 5 (9) ◽  
pp. 2316-2324
Author(s):  
R B Alterman ◽  
C Sprecher ◽  
R Graves ◽  
W F Marzluff ◽  
A I Skoultchi
Keyword(s):  
Histone Gene ◽  
Chimeric Gene ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Histone Genes ◽  
Mouse Fibroblasts ◽  
Cis Acting ◽  
Regulated Expression ◽  
Chimeric Rna ◽  
The Stability

The regulated expression of a mouse histone gene was studied by DNA-mediated gene transfer. A chimeric H3 histone gene was constructed by fusing the 5' and 3' portions of two different mouse H3 histone genes. Transfection of the chimeric gene into mouse fibroblasts resulted in the production of chimeric mRNA at levels nearly equal to that of the total endogenous H3 histone mRNAs. Most chimeric RNA transcripts had correct 5' and 3' termini, and the chimeric mRNA was translated into an H3.1 protein that accumulated in the nucleus of the transfected cells. Expression of the chimeric gene was studied under several conditions in which the rate of transcription and the stability of endogenous H3 transcripts change. Chimeric mRNA levels were regulated in parallel with endogenous H3 mRNAs, suggesting that cis-acting regulatory sequences lie within or near individual histone genes. In addition to correctly initiated and terminated chimeric mRNA, we also detected a novel H3 transcript containing an additional 250 bases at the 3' end. Surprisingly, the longer transcript is polyadenylated and accumulates in the cytoplasm.

scholarly journals Identification of cis-regulatory elements required for larval expression of the Drosophila melanogaster alcohol dehydrogenase gene.

Genetics ◽  
1990 ◽  
Vol 124 (3) ◽  
pp. 637-646 ◽  
Author(s):  
V Corbin ◽  
T Maniatis
Keyword(s):  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Dna Sequences ◽  
Regulatory Elements ◽  
Malpighian Tubules ◽  
Proximal Promoter ◽  
Start Site ◽  
Wild Type ◽  
Tissue Specific ◽  
Adh Gene

Abstract The Alcohol dehydrogenase (Adh) genes of two distantly related species, Drosophila melanogaster and Drosophila mulleri, display similar, but not identical, patterns of tissue-specific expression in larvae and adults. The regulatory DNA sequences necessary for wild-type Adh expression in D. mulleri larvae were previously reported. In this paper we present an analysis of the DNA sequences necessary for wild-type Adh expression in D. melanogaster larvae. We show that transcription from the proximal promoter of the melanogaster Adh gene is regulated by a far upstream enhancer and two or more elements near the transcription start site. The enhancer is tissue specific and stimulates transcription to high levels in fat body and to lower levels in midgut and malpighian tubules whether linked to the proximal promoter or to a heterologous promoter. The enhancer activity localized to at least two discrete regions dispersed over more than 1.7 kb of DNA. Deletion of any one of these subregions reduces Adh transcription in all three larval tissues. Similarly, two regions immediately upstream of the proximal promoter start site are necessary for wild-type transcription levels in all three tissues. Thus, each of the identified regulatory elements is sufficient for low levels of Adh gene expression in all three larval tissues, but maximal levels of expression requires the entire set.

scholarly journals Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene.

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557 ◽  
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

Improved gene expression in Aspergillus nidulans

1995 ◽  
Vol 73 (S1) ◽  
pp. 876-884 ◽  
Author(s):  
William E. Hintz ◽  
Inge Kalsner ◽  
Ewa Plawinski ◽  
Zimin Guo ◽  
Peter A. Lagosky
Keyword(s):  
Gene Expression ◽  
Alcohol Dehydrogenase ◽  
Aspergillus Nidulans ◽  
Glucose Repression ◽  
Expression System ◽  
Therapeutic Proteins ◽  
Regulatory Sequences ◽  
Transcriptional Regulatory ◽  
And Function

A variety of gene expression systems have been developed that utilize the promoter and transcriptional regulatory sequences derived from carbon-catabolite repressed genes for the expression of heterologous genes. The alcA expression system of Aspergillus nidulans utilizes the promoter and regulatory sequences derived from the alcohol dehydrogenase I (alcA) gene. Expression of the alcA gene is repressed by a DNA-binding protein (CreA) in the presence of glucose and induced by ethanol under glucose-depleted conditions. One problem encountered during the expression of therapeutic proteins in A. nidulans is the coexpression of secreted proteases at the time of maximal secretion of heterologous product. To avoid the proteases we created an alcA promoter variant that is no longer sensitive to glucose repression hence could drive expression at earlier time points during the fermentation. The use of this promoter variant in the expression of recombinant interleukin-6 is discussed. A second problem encountered during the expression of high-quality human therapeutic proteins in Aspergillus is aberrant glycosylation. Lower eukaryotic systems, such as Aspergillus, tend to add highly branched mannosidic chains to heterologous secreted protein products. N-Glycans can be important for both the structure and function of specific glycoproteins, hence efforts are being made to in vivo alter the type and complexity of N-glycans substituted by A. nidulans. Key words: Aspergillus, gene expression, alcohol dehydrogenase, glycosylation.

Regulatory sequences of the Sgs-4 gene of Drosophila melanogaster analysed by P element-mediated transformation

Chromosoma ◽  
1987 ◽  
Vol 96 (1) ◽  
pp. 8-17 ◽  
Author(s):  
Annemarie Hofmann ◽  
Arne Keinhorst ◽  
Anton Krumm ◽  
G�nter Korge
Keyword(s):  
Drosophila Melanogaster ◽  
P Element ◽  
Regulatory Sequences

scholarly journals Tissue-specific expression phenotypes of Hawaiian Drosophila Adh genes in Drosophila melanogaster transformants.

Genetics ◽  
1990 ◽  
Vol 125 (3) ◽  
pp. 599-610
Author(s):  
C Y Wu ◽  
J Mote ◽  
M D Brennan
Keyword(s):  
Drosophila Melanogaster ◽  
Tissue Distribution ◽  
Germ Line ◽  
P Element ◽  
Specific Expression ◽  
Position Effects ◽  
Tissue Specific ◽  
Hawaiian Drosophila ◽  
Adh Genes ◽  
Adh Gene

Abstract Interspecific differences in the tissue-specific patterns of expression displayed by the alcohol dehydrogenase (Adh) genes within the Hawaiian picture-winged Drosophila represent a rich source of evolutionary variation in gene regulation. Study of the cis-acting elements responsible for regulatory differences between Adh genes from various species is greatly facilitated by analyzing the behavior of the different Adh genes in a homogeneous background. Accordingly, the Adh gene from Drosophila grimshawi was introduced into the germ line of Drosophila melanogaster by means of P element-mediated transformation, and transformants carrying this gene were compared to transformants carrying the Adh genes from Drosophila affinidisjuncta and Drosophila hawaiiensis. The results indicate that the D. affinidisjuncta and D. grimshawi genes have relatively higher levels of expression and broader tissue distribution of expression than the D. hawaiiensis gene in larvae. All three genes are expressed at similar overall levels in adults, with differences in tissue distribution of enzyme activity corresponding to the pattern in the donor species. However, certain systematic differences between Adh gene expression in transformants and in the Hawaiian Drosophila are noted along with tissue-specific position effects in some cases. The implications of these findings for the understanding of evolved regulatory variation are discussed.

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