Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion

Zhang, Hui; Liu, Changjun; Jiang, Hui; Zhou, Lu; Li, Wenying; Zhu, Lingyun; Wu, Lei; Meng, Er; Zhang, Dongyi

doi:10.1042/bsr20160608

“…The construct h1prom0-pGL3 contained the whole 600 bp genomic sequence (Chr5:133340586-133341185); h1prom1-pGL3 contained a 200 bp genomic sequence (chr5:133340586-133340786) and, similarly, h1prom2-pGL3 (chr5:133340786-133340986), and h1prom3-pGL3 (chr5:133340986-133341185). pcDNA3.0-NRF-1 and pcDNA3.0-HIF-1 constructs were obtained by PCR cloning strategy (22)(23)(24). In brief, coding sequences of TFs amplified from HeLa cDNA were modified at terminal ends by adding sequences complementary to pcDNA 3.0 vector and recombinants generated by PCR.…”

Section: Plasmid Constructs and Mutagenesismentioning

confidence: 99%

NRF-1 and HIF-1α contribute to modulation of human VDAC1 gene promoter during starvation and hypoxia in HeLa cells

Guarino¹,

Zinghirino²,

Mela³

et al. 2020

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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VDAC (Voltage Dependent Anion Channel) is a family of pore forming protein located in the outer mitochondrial membrane. Its channel property ensures metabolites exchange between mitochondria and the rest of the cell resulting in metabolism and bioenergetics regulation, and in cell death and life switch. VDAC1 is the best characterized and most abundant isoform, and is involved in many pathologies, as cancer or neurodegenerative diseases. However, little information is available about its gene expression regulation in normal and/or pathological conditions. In this work, we explored VDAC1 gene expression regulation in normal conditions and in the contest of metabolic and energetic mitochondrial dysfunction and cell stress. The most active area of the putative promoter region was characterized in terms of transcription factors responsive elements both by bioinformatic studies and promoter activity experiments. In particular, we found a predominant presence of NRF-1 together with other transcription factors binding sites, involved in cell growth, proliferation, development and we studied their prevalence in gene activity. Furthermore, upon depletion of nutrients or controlled hypoxia, as reported in various pathologies, we found that VDAC1 transcripts levels were significantly increased in a time related manner. VDAC1 promoter activity was also validated by gene reporter assays. According to PCR real-time data, it was confirmed that VDAC1 promoter activity is further stimulated when are exposed to stress. A bioinformatic survey suggested NRF-1 e HIF-1 as the most active TFBS. Their validation was obtained by mutagenesis and overexpression experiments. In conclusion, we demonstrated experimentally the involvement of both NRF-1 and HIF-1 in the regulation of VDAC1 promoter activation at basal level and in cell stress conditions..

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“…Based on our electrophysiology analysis of the wild-type GluK1-1a/GluK1-2a receptors, structural analysis of GluK1-1a EM , and recent reports that suggest that the presence of the positive patches in GluK2 ATD affects the interaction with Neto proteins (He et al, 2021b; Li et al, 2019; Vinnakota et al, 2021), we performed SDM to understand the role of splice residues in the receptor kinetics. All splice mutations were introduced in the wild-type (species) GRIK1-1a pRK7 construct for electrophysiology, as well as the GRIK1-1a EM -EGFP-His 8 -pEGBacMam construct for surface expression and pull-downs using the (overlap PCR) ligation-free cloning approach (Zhang et al, 2017). In brief, we performed the 2 sets of PCR using standard cloning primers (∼300 bp upstream and downstream of the mutation) and the mutant primers as listed in Supplemental Table 1 to obtain the fragment containing our mutation of interest and flanking regions corresponding to the wild-type GluK1-1a.…”

Section: Methodsmentioning

confidence: 99%

Functional Implications of the Exon 9 Splice Insert in GluK1 Kainate Receptors

Dhingra,

Chopade,

Vinnakota

et al. 2023

Preprint

0

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Kainate receptors are key modulators of synaptic transmission and plasticity in the central nervous system. Different kainate receptor isoforms with distinct spatiotemporal expression have been identified in the brain. The GluK1-1 splice variant receptors, which are abundant in the adult brain, have extra fifteen amino acids inserted in the amino-terminal domain (ATD) of the receptor resulting from alternative splicing of exon 9. However, the functional implications of this post-transcriptional modification are not yet clear. We employed a multi-pronged approach using cryogenic electron microscopy, electrophysiology, and other biophysical and biochemical tools to understand the structural and functional impact of this splice insert in the extracellular domain of GluK1 receptors. Our study reveals that the splice insert alters the key gating properties of GluK1 receptors and their modulation by the cognate auxiliary Neuropilin and tolloid-like (Neto) proteins 1 and 2. Mutational analysis identified the role of key splice residues that influence receptor properties and their modulation. Furthermore, cryoEM structure of the variant shows that the presence of exon 9 in GluK1 does not affect the receptor architecture or domain arrangement in the desensitized state. Our study thus provides the first detailed structural and functional characterization of GluK1-1a receptors, highlighting the role of the splice insert in modulating receptor properties and their modulation.

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“…Based on our electrophysiology analysis of the wild-type GluK1-1a/GluK1-2a receptors, structural analysis of GluK1-1a EM , and recent reports that suggest that the presence of the positive patches in GluK2 ATD affects the interaction with Neto proteins (He et al, 2021b ;Li et al, 2019 ;Vinnakota et al, 2021 ), we performed SDM to understand the role of splice residues in the receptor kinetics. All splice mutations were introduced in the wild-type GRIK1-1a pRK7 construct for electrophysiology, as well as the GRIK1-1a EM -EGFP-His 8 -pEGBacMam construct for surface expression and pull-downs using the ligation-free cloning approach (Zhang et al, 2017 ). Clones were confirmed by sequencing.…”

Section: Site-directed Mutagenesis (Sdm)mentioning

confidence: 99%

Functional Implications of the Exon 9 Splice Insert in GluK1 Kainate Receptors

Dhingra,

Chopade,

Vinnakota

et al. 2024

Preprint

0

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Kainate receptors are key modulators of synaptic transmission and plasticity in the central nervous system. Different kainate receptor isoforms with distinct spatiotemporal expression have been identified in the brain. The GluK1-1 splice variant receptors, which are abundant in the adult brain, have extra fifteen amino acids inserted in the amino-terminal domain (ATD) of the receptor resulting from alternative splicing of exon 9. However, the functional implications of this post-transcriptional modification are not yet clear. We employed a multi-pronged approach using cryogenic electron microscopy, electrophysiology, and other biophysical and biochemical tools to understand the structural and functional impact of this splice insert in the extracellular domain of GluK1 receptors. Our study reveals that the splice insert alters the key gating properties of GluK1 receptors and their modulation by the cognate auxiliary Neuropilin and tolloid-like (Neto) proteins 1 and 2. Mutational analysis identified the role of key splice residues that influence receptor properties and their modulation. Furthermore, cryoEM structure of the variant shows that the presence of exon 9 in GluK1 does not affect the receptor architecture or domain arrangement in the desensitized state. Our study thus provides the first detailed structural and functional characterization of GluK1-1a receptors, highlighting the role of the splice insert in modulating receptor properties and their modulation.

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Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion

Cited by 9 publications

References 17 publications

NRF-1 and HIF-1α contribute to modulation of human VDAC1 gene promoter during starvation and hypoxia in HeLa cells

NRF-1 and HIF-1α contribute to modulation of human VDAC1 gene promoter during starvation and hypoxia in HeLa cells

Functional Implications of the Exon 9 Splice Insert in GluK1 Kainate Receptors

Functional Implications of the Exon 9 Splice Insert in GluK1 Kainate Receptors

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