A binary gene expression system using the yeast GAL4 DNA-binding protein and the upstream activating sequence (UAS) of galactose-driven yeast genes is an established and powerful tool for the analysis of gene function. However, in the domesticated silkworm, Bombyx mori, this system has been limited in its utility by the relatively low transcriptional activation activity of GAL4 and by its toxicity. In this study, we investigated the potential of several established GAL4 variants (GAL4Δ, GAL4VP16, GAL4VPmad2, GAL4VPmad3, and GAL4NFκB) and of two new GAL4 variants, GAL4Rel and GAL4Relish, which contain the transcription-activating regions of the BmRel and BmRelish genes, respectively, to improve the utility of the GAL4/UAS system in B. mori. We generated constructs containing these GAL4 variants under the control of constitutive or inducible promoters and investigated their transcription-activating activity in cultured B. mori cells and embryos and in transgenic silkworms. GAL4VP16 and GAL4NFκB exhibited high transactivation activity but appeared to be toxic when used as transgenes under the control of a constitutive promoter. Similarly, GAL4VPmad2 and GAL4VPmad3 exhibited higher transactivation activity than GAL4, combined with strong toxicity. The transcription-activating activity of GAL4Δ was about twice that of GAL4. The two new GAL4 variants, GAL4Rel and GAL4Relish, were less active than GAL4. Using GAL4VP16 and GAL4NFκB constructs, we have developed a very efficient GAL4/UAS binary gene expression system for use in cultured B. mori cells and embryos and in transgenic silkworms.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1002/arch.20402 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Distinguishing gene variant severity for mild, severe, and atypical peroxisome biogenesis disorders in .
bioRxiv
November 2024
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston.
Peroxisomal biogenesis disorders (PBD) are autosomal recessive disorders caused by loss-of-function mutations of one of the genes responsible for peroxisomal formation. Impaired peroxisome assembly causes severe multisystemic failure with patient phenotypes ranging from epilepsy, liver disease, feeding issues, biochemical abnormalities, and neurodegeneration. Variants in the same gene can produce wide differences in severity, ranging from individuals with death in the first year of life to adults with milder complications.
View Article and Find Full Text PDFDe novo variants in UPF1 associated with intellectual disabilities: Human genetic and functional evidences using Drosophila model.
Eur J Med Genet
December 2024
Center for Medical Genetics, Keio University School of Medicine, Tokyo, Japan. Electronic address:
Nonsense-mediated mRNA decay represents a biologic clearing system against aberrant mRNAs harboring nonsense and frameshift mutations and depends on three factors, UPF1, UPF2, and UPF3 (UPF3A, UPF3B). While germline pathogenic variants of UPF3B and UPF2 are known to be associated with neurodevelopmental disorders, germline variants in UPF1 have not been reported, until date, as being associated with any human disorders. Herein, we report two unrelated patients with de novo UPF1 variants.
View Article and Find Full Text PDFTransgenic Expressing Active Human LH Receptor in the Gonads Exhibit a Decreased Fecundity: Towards a Platform to Identify New Orally Active Modulators of Gonadotropin Receptor Activity.
Pharmaceuticals (Basel)
September 2024
Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheba 8410501, Israel.
Background/objectives: The gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and their receptors are major regulators of reproduction in mammals and are absent in insects. We previously established transgenic lines expressing a constitutively active human LH receptor variant (LHR) and the wild-type receptor (LHR; inactive in the absence of an agonist). That study showed that ubiquitously expression of LHR-but not of LHR-resulted in pupal lethality, and targeted expression in midline cells resulted in thorax/bristles defects.
View Article and Find Full Text PDFA yeast two-hybrid system to obtain triple-helical ligands from combinatorial random peptide libraries.
J Biol Chem
November 2024
Waseda Research Institute for Science and Engineering, Waseda University, Shinjuku, Tokyo, Japan; Department of Chemistry and Biochemistry, School of Advanced Science and Engineering, Waseda University, Shinjuku, Tokyo, Japan. Electronic address:
Article Synopsis
- Researchers developed a strategy to create and select triple-helical peptides that can interact with specific bioactive proteins, specifically using a yeast-based library.
- The selection process utilized a two-hybrid system to identify peptides that bind to the pigment epithelium-derived factor (PEDF), which is known for its anti-angiogenic and neurotrophic properties.
- The study revealed new binding sequences with strong affinities for PEDF, including a variant that differed from previously known collagen motifs, showcasing the potential of the library approach to discover more effective peptides.
Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants.
Mol Syst Biol
July 2024
MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, EH4 2XU, UK.
Nonsense and missense mutations in the transcription factor PAX6 cause a wide range of eye development defects, including aniridia, microphthalmia and coloboma. To understand how changes of PAX6:DNA binding cause these phenotypes, we combined saturation mutagenesis of the paired domain of PAX6 with a yeast one-hybrid (Y1H) assay in which expression of a PAX6-GAL4 fusion gene drives antibiotic resistance. We quantified binding of more than 2700 single amino-acid variants to two DNA sequence elements.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!