Identification of a sequence motif upstream of the Drosophila Dopa decarboxylase gene that enhances heterologous gene expression

In this paper we have examined the role that element S, a DNA sequence motif found approximately 215 bp upstream of the Dopa decarboxylase (Ddc) gene, might play in regulating Ddc expression. Nearly identical versions of the element are present upstream of four other Drosophila genes. For two of these, the element appears to be an important component of the upstream regulatory region, since mutations in it reduce expression of the downstream gene. Because an element S polymorphism differentiates the Ddc+ allele of an inbred laboratory strain from the Ddc+4 allele present in a strain isolated from the wild, we decided to test the activity of both forms. Oligonucleotides containing Ddc+ or Ddc+4 versions of element S were synthesized and their ability to drive the expression of an heterologous (Adh) reporter gene at the second molt was examined. Transgenic larvae carrying the element S – Adh fusion constructs consistently exhibited Adh levels that were elevated 1.5-fold above those seen in control organisms. We have also determined the effects of element S in white prepupae and once again, ADH expression levels were significantly above controls in both groups of transformants carrying the element S construct. The results point to a functional role for element S. Since reporter gene expression in third instar larvae was restricted to tissues where ADH is normally found, we conclude that element S is not involved in directing the tissue specificity of Ddc expression. However, its ability to enhance heterologous gene expression suggests that it may be the fifth in the set of cis-acting sequences in the complex regulatory domain known to specify epidermal Ddc expression during development.Key words: Drosophila, Dopa decarboxylase, regulatory element, reporter gene, transgenic flies.

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Targeting gene expression to the head: the Drosophila orthodenticle gene is a direct target of the Bicoid morphogen

Development ◽

10.1242/dev.125.21.4185 ◽

1998 ◽

Vol 125 (21) ◽

pp. 4185-4193 ◽

Cited By ~ 4

Author(s):

Q. Gao ◽

R. Finkelstein

Keyword(s):

Gene Expression ◽

Target Genes ◽

Regulatory Element ◽

Regulatory Region ◽

Repeated Sequence ◽

Drosophila Embryo ◽

Sequence Motif ◽

Specific Expression ◽

Gap Gene

The Bicoid (Bcd) morphogen establishes the head and thorax of the Drosophila embryo. Bcd activates the transcription of identified target genes in the thoracic segments, but its mechanism of action in the head remains poorly understood. It has been proposed that Bcd directly activates the cephalic gap genes, which are the first zygotic genes to be expressed in the head primordium. It has also been suggested that the affinity of Bcd-binding sites in the promoters of Bcd target genes determines the posterior extent of their expression (the Gene X model). However, both these hypotheses remain untested. Here, we show that a small regulatory region upstream of the cephalic gap gene orthodenticle (otd) is sufficient to recapitulate early otd expression in the head primordium. This region contains two control elements, each capable of driving otd-like expression. The first element has consensus Bcd target sites that bind Bcd in vitro and are necessary for head-specific expression. As predicted by the Gene X model, this element has a relatively low affinity for Bcd. Surprisingly, the second regulatory element has no Bcd sites. Instead, it contains a repeated sequence motif similar to a regulatory element found in the promoters of otd-related genes in vertebrates. Our study is the first demonstration that a cephalic gap gene is directly regulated by Bcd. However, it also shows that zygotic gene expression can be targeted to the head primordium without direct Bcd regulation.

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Hematopoietic regulatory domain of gata1 gene is positively regulated by GATA1 protein in zebrafish embryos

Development ◽

10.1242/dev.128.12.2341 ◽

2001 ◽

Vol 128 (12) ◽

pp. 2341-2350

Author(s):

Makoto Kobayashi ◽

Keizo Nishikawa ◽

Masayuki Yamamoto

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Regulatory Region ◽

Ectopic Expression ◽

Transgenic Fish ◽

Functional Domain ◽

Cis Acting ◽

Gfp Reporter ◽

Gata Motif

Expression of gata1 is regulated through multiple cis-acting GATA motifs. To elucidate regulatory mechanisms of the gata1 gene, we have used zebrafish. To this end, we isolated and analyzed zebrafish gata1 genomic DNA, which resulted in the discovery of a novel intron that was unknown in previous analyses. This intron corresponds to the first intron of other vertebrate Gata1 genes. GFP reporter analyses revealed that this intron and a distal double GATA motif in the regulatory region are important for the regulation of zebrafish gata1 gene expression. To examine whether GATA1 regulates its own gene expression, we microinjected into embryos a GFP reporter gene linked successively to the gata1 gene regulatory region and to GATA1 mRNA. Surprisingly, ectopic expression of the reporter gene was induced at the site of GATA1 overexpression and was dependent on the distal double GATA motif. Functional domain analyses using transgenic fish lines that harbor the gata1-GFP reporter construct revealed that both the N- and C-terminal zinc-finger domains of GATA1, hence intact GATA1 function, are required for the ectopic GFP expression. These results provide the first in vivo evidence that gata1 gene expression undergoes positive autoregulation.

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4855231 Regulatory region for heterologous gene expression in yeast

Biotechnology Advances ◽

10.1016/0734-9750(90)91080-z ◽

1990 ◽

Vol 8 (2) ◽

pp. 361

Keyword(s):

Gene Expression ◽

Heterologous Gene Expression ◽

Regulatory Region ◽

Heterologous Gene

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Human cellular homeostasis buffers trans-acting translational effects of heterologous gene expression with very different codon usage bias

10.1101/2021.12.09.471957 ◽

2021 ◽

Author(s):

Arthur J JALLET ◽

Antonin Demange ◽

Fiona Leblay ◽

Mathilde Decourcelle ◽

Khadija El Koulali ◽

...

Keyword(s):

Gene Expression ◽

Codon Usage ◽

Codon Usage Bias ◽

Heterologous Gene Expression ◽

Human Cells ◽

Heterologous Gene ◽

Translation Efficiency ◽

Cellular Homeostasis ◽

Cis Acting ◽

The Impact

The frequency of synonymous codons in protein coding genes is non-random and varies both between species and between genes within species. Whether this codon usage bias (CUBias) reflects underlying neutral mutational processes or is instead shaped by selection remains an open debate, especially regarding the role of selection for enhanced protein production. Variation in CUBias of a gene (be it natural synonymous mutations or biotechnological synonymous recoding) can have an enormous impact on its expression by diverse cis- acting mechanisms. But expression of genes with extreme CUBias can also lead to strong phenotypic effects by altering the overall intracellular translation homeostasis via competition for ribosomal machinery or tRNA depletion. In this study, we expressed at high levels in human cells six different synonymous versions of a gene and used matched transcriptomic and proteomic data to evaluate the impact of CUBias of the heterologous gene on the translation of cellular transcripts. Our experimental design focused specifically on differences during translation elongation. Response to expression of the different synonymous sequences was assessed by various approaches, ranging from analyses performed on a per-gene basis to more integrated approaches of the cell as a whole. We observe that the transcriptome displayed substantial changes as a result of heterologous gene expression by triggering an intense antiviral and inflammatory response, but that changes in the proteomes were very modest. Most importantly we notice that changes in translation efficiency of cellular transcripts were not associated with the direction of the CUBias of the heterologous sequences, thereby providing only limited support for trans- acting effects of synonymous changes. We interpret that, in human cells in culture, changes in CUBias can lead to important cis- acting effects in gene expression, but that cellular homeostasis can buffer the phenotypic impact of overexpression of heterologous genes with extreme CUBias.

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Optimization of Heterologous Gene Expression for In Vitro Evolution

BioTechniques ◽

10.2144/01303bm01 ◽

2001 ◽

Vol 30 (3) ◽

pp. 474-476 ◽

Cited By ~ 5

Author(s):

Ichiro Matsumura ◽

Mark J. Olsen ◽

Andrew D. Ellington

Keyword(s):

Gene Expression ◽

Heterologous Gene Expression ◽

Heterologous Gene ◽

In Vitro Evolution

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Production of foreign proteins in the yeast Pichia pastoris

Canadian Journal of Botany ◽

10.1139/b95-336 ◽

1995 ◽

Vol 73 (S1) ◽

pp. 891-897 ◽

Cited By ~ 46

Author(s):

James M. Cregg ◽

David R. Higgins

Keyword(s):

Gene Expression ◽

Pichia Pastoris ◽

Heterologous Gene Expression ◽

Expression System ◽

Methylotrophic Yeast ◽

Culture Broth ◽

Heterologous Gene ◽

Foreign Gene ◽

Heterologous Proteins ◽

Yeast Pichia Pastoris

The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest. An industrial yeast selected for efficient growth on methanol for biomass generation, P. pastoris is readily grown on defined medium in continuous culture at high volume and density. A unique feature of the expression system is the promoter employed to drive heterologous gene expression, which is derived from the methanol-regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters known. The strength of the AOX1 promoter results in high expression levels in strains harboring only a single integrated copy of a foreign-gene expression cassette. Levels may often be further enhanced through the integration of multiple cassette copies into the P. pastoris genome and strategies to construct and select multicopy cassette strains have been devised. The system is particularly attractive for the secretion of foreign-gene products. Because P. pastoris endogenous protein secretion levels are low, foreign secreted proteins often appear to be virtually the only proteins in the culture broth, a major advantage in processing and purification. Key words: heterologous gene expression, methylotrophic yeast, Pichia pastoris, secretion, glycosylation.

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