scholarly journals Characterization of a novel Fgf10CreERT2 knock-in mouse line targeting postnatal lung Fgf10 lineages

2021 ◽  
Author(s):  
Xuran Chu ◽  
Sara Taghizadeh ◽  
Ana Ivonne Vasquez-Armendariz ◽  
Susanne Herold ◽  
Lei Chong ◽  
...  
Keyword(s):  
Binding Sites ◽  
Stop Codon ◽  
Start Codon ◽  
Wild Type ◽  
Functional Allele ◽  
Wild Type Counterpart ◽  
Putative Transcription Factor ◽  
Exon 1 ◽  
Fgf10 Expression

AbstractFgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a Fgf10CreERT2 line (with the CreERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10Ki-v1, to target Fgf10Pos cells. While this line allowed fairly efficient and specific labeling of Fgf10Pos cells during the embryonic stage, it failed to target these cells after birth, particularly in the postnatal lung, which has been the focus on our research. We report here the generation and validation of a new Fgf10CreERT2 (called thereafter Fgf10Ki-v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. This new Fgf10Ki-v2 line exhibited comparable Fgf10 expression level to their wild type counterpart. However, a disconnection between the Fgf10 and the Cre expression was observed in Fgf10Ki-v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf10Ki-v2 mice. Bio-informatics analysis shows that the 3’UTR, where the CreERT2 cassette is inserted, contains numerous putative transcription factor binding sites. By crossing this line with tdTomato reporter line, we demonstrated that tdTomato expression faithfully recapitulated Fgf10 expression during development. Significantly, Fgf10Ki-v2 mouse is capable of significantly targeting Fgf10Pos cells in the adult lung. Therefore, despite the aforementioned limitations, this new Fgf10Ki-v2 line opens the way for future mechanistic experiments involving the postnatal lung.

scholarly journals Validation of a Novel Fgf10Cre–ERT2 Knock-in Mouse Line Targeting FGF10Pos Cells Postnatally

2021 ◽  
Vol 9 ◽  
Author(s):  
Xuran Chu ◽  
Sara Taghizadeh ◽  
Ana Ivonne Vazquez-Armendariz ◽  
Susanne Herold ◽  
Lei Chong ◽  
...  
Keyword(s):  
Binding Sites ◽  
Stop Codon ◽  
Bioinformatic Analysis ◽  
Start Codon ◽  
Mouse Line ◽  
Wild Type ◽  
Functional Allele ◽  
Expression Levels ◽  
Putative Transcription Factor ◽  
Exon 1

Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a knock-in Fgf10Cre–ERT2 line (with the Cre-ERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10Ki–v1, to target FGF10Pos cells. While this line allowed fairly efficient and specific labeling of FGF10Pos cells during the embryonic stage, it failed to target these cells after birth, particularly in the postnatal lung, which has been the focus of our research. We report here the generation and validation of a new knock-in Fgf10Cre–ERT2 line (called thereafter Fgf10Ki–v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. Fgf10Ki−v2/+ heterozygous mice exhibited comparable Fgf10 expression levels to wild type animals. However, a mismatch between Fgf10 and Cre expression levels was observed in Fgf10Ki–v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf10Ki–v2 mice. Bioinformatic analysis shows that the 3′UTR, where the Cre-ERT2 cassette is inserted, contains numerous putative transcription factor binding sites. By crossing this line with tdTomato reporter line, we demonstrated that tdTomato expression faithfully recapitulated Fgf10 expression during development. Importantly, Fgf10Ki–v2 mouse is capable of significantly targeting FGF10Pos cells in the adult lung. Therefore, despite the aforementioned limitations, this new Fgf10Ki–v2 line opens the way for future mechanistic experiments involving the postnatal lung.

scholarly journals Functional Cooperation between Multiple Regulatory Elements in the Untranslated Exon 1 Stimulates the Basal Transcription of the Human GnRH-II Gene

2003 ◽  
Vol 17 (7) ◽  
pp. 1175-1191 ◽  
Author(s):  
Chi Keung Cheng ◽  
Ruby L. C. Hoo ◽  
Billy K. C. Chow ◽  
Peter C. K. Leung
Keyword(s):  
Gene Transcription ◽  
Binding Sites ◽  
Molecular Mechanisms ◽  
Mutational Analysis ◽  
Regulatory Elements ◽  
Start Codon ◽  
Basal Transcription ◽  
Untranslated Exon ◽  
E Box ◽  
Exon 1

Abstract The wide distribution of GnRH-II and conservation of its structure over all vertebrate classes suggest that the neuropeptide possesses vital biological functions. Although recent studies have shown that the expression of the human GnRH-II gene is regulated by cAMP and estrogen, the molecular mechanisms governing its basal transcription remain poorly understood. Using the neuronal TE-671 and placental JEG-3 cells, we showed that the minimal human GnRH-II promoter was located between nucleotide −1124 and −750 (relative to the translation start codon) and that the untranslated exon 1 was important to produce full promoter activity. Two putative E-box binding sites and one Ets-like element were identified within the first exon, and mutational analysis demonstrated that these cis-acting elements functioned cooperatively to stimulate the human GnRH-II gene transcription. EMSAs, UV cross-linking, and Southwestern blot analyses indicated that the basic helix-loop-helix transcription factor AP-4 bound specifically to the two E-box binding sites, whereas an unidentified protein bound to the Ets-like element. The functional importance of AP-4 in controlling human GnRH-II gene transcription was demonstrated by overexpression of sense and antisense full-length AP-4 cDNAs. Taken together, our present data demonstrate a novel mechanism in stimulating basal human GnRH-II gene transcription mediated by cooperative actions of multiple regulatory elements within the untranslated first exon of the gene.

scholarly journals Characterization of Two New Aminopeptidases in Escherichia coli

2005 ◽  
Vol 187 (11) ◽  
pp. 3671-3677 ◽  
Author(s):  
Yu Zheng ◽  
Richard J. Roberts ◽  
Simon Kasif ◽  
Chudi Guan
Keyword(s):  
Escherichia Coli ◽  
Stop Codon ◽  
Bacterial Species ◽  
Start Codon ◽  
Aminopeptidase Activity ◽  
Peptide Substrates ◽  
E Coli ◽  
Terminal Domain ◽  
Methionyl Aminopeptidase

ABSTRACT Two genes in the Escherichia coli genome, ypdE and ypdF, have been cloned and expressed, and their products have been purified. YpdF is shown to be a metalloenzyme with Xaa-Pro aminopeptidase activity and limited methionine aminopeptidase activity. Genes homologous to ypdF are widely distributed in bacterial species. The unique feature in the sequences of the products of these genes is a conserved C-terminal domain and a variable N-terminal domain. Full or partial deletion of the N terminus in YpdF leads to the loss of enzymatic activity. The conserved C-terminal domain is homologous to that of the methionyl aminopeptidase (encoded by map) in E. coli. However, YpdF and Map differ in their preference for the amino acid next to the initial methionine in the peptide substrates. The implication of this difference is discussed. ypdE is the immediate downstream gene of ypdF, and its start codon overlaps with the stop codon of ypdF by 1 base. YpdE is shown to be a metalloaminopeptidase and has a broad exoaminopeptidase activity.

Nucleotide Structure and Characterization of the Murine Gene Encoding Anticoagulant Protein C

1998 ◽  
Vol 79 (02) ◽  
pp. 310-316 ◽  
Author(s):  
Louise Jalbert ◽  
Elliot Rosen ◽  
Ann Lissens ◽  
Peter Carmeliet ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Protein C ◽  
Stop Codon ◽  
Transcriptional Start Site ◽  
Consensus Sequence ◽  
Start Site ◽  
Gene Encoding ◽  
Exon 2 ◽  
Exon 1 ◽  
Translation Stop Codon

SummaryThe 15,160 bp murine gene encoding anticoagulation protein C (PC) was cloned and sequenced, including 414 bp upstream of exon 1 and 80 bp downstream of the translation stop codon. Nine exons and eight introns were identified. The first exon was untranslated and contained the major transcriptional start site, the surrounding nucleotide sequence of which matched reasonably well with the consensus eukaryotic Cap element sequence. The translational initiator methio-nine residue was located in exon 2. The other introns were positioned as splices between the major domain units of the protein. The 5’ untranslated region contained two possible CCAAT sequences and GC boxes, but no TATA box was obvious within the optimal range of distances from the transcription start site. The 3’-flanking nucleotides included a probable polyadenylation site (ATTAAA), beginning 80 nucleotides downstream of the translation stop codon, and a downstream consensus sequence (AGTGTTTC) required for the efficient formation of a 3’ terminus of mRNA. Several high probability transcription factor recognition sequences, including proteins that are enriched in, or specific to, the liver, such as C/EBP, C/EBP, HNF1, and HNF3, have been located in the 5’ region of the gene. These results indicate that all elements are present for liver-based transcription of the gene for murine PC.

scholarly journals Characterization of the gene encoding the human LW blood group protein in LW+ and LW- phenotypes

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2962-2967 ◽  
Author(s):  
P Hermand ◽  
PY Le Pennec ◽  
P Rouger ◽  
JP Cartron ◽  
P Bailly
Keyword(s):  
Blood Group ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
K562 Cells ◽  
Gene Encoding ◽  
Potential Binding ◽  
Exon 1 ◽  
Intercellular Adhesion Molecules ◽  
Group Protein

The LW blood group is carried by a 42-kD glycoprotein that belongs to the family of intercellular adhesion molecules. The LW gene is organized into three exons spanning an HindIII fragment of approximately 2.65 kb. The exon/intron architecture correlates to the structural domains of the protein and resembles that of other Ig superfamily members except that the signal peptide and the first Ig- like domain are encoded by the first exon. The 5′UT region (nucleotides -289 to +9) includes potential binding sites for various transcription factors (Ets, CACC, SP1, GATA-1, AP2) and exhibited a significant transcriptional activity after transfection in the erythroleukemic K562 cells. No obvious abnormality of the LW gene, including the 5′UT region, has been detected by sequencing polymerase chain reaction- amplified genomic DNA from RhD+ or RhD- donors and from an Rhnull variant that lacks the Rh and LW proteins on red blood cells. However, a deletion of 10 bp in exon 1 of the LW gene was identified in the genome of an LW (a- b-) individual (Big) deficient for LW antigens but carrying a normal Rh phenotype. The 10-bp deletion generates a premature stop codon and encodes a truncated protein without transmembrane and cytoplasmic domain. No detectable abnormality of the LW gene or transcript could be detected in another LW(a- b-) individual (Nic), suggesting the heterogeneity of these phenotypes.

Molecular Characterization of Two Homozygous Factor VII Variants Associated with Intracranial Bleeding

2021 ◽  
Author(s):  
Elisabeth Andersen ◽  
Maria Eugenia Chollet ◽  
Marit Sletten ◽  
Benedicte Stavik ◽  
Ellen Skarpen ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Molecular Mechanisms ◽  
Stop Codon ◽  
High Titer ◽  
Premature Stop Codon ◽  
Homozygous Deletion ◽  
Factor Vii ◽  
Mammalian Cell Lines ◽  
Exon 1

Clinical parameters have been extensively studied in factor (F) VII deficiency, but the knowledge of molecular mechanisms of this disease is scarce. We report on three probands with intracranial bleeds at early age, one of which had concomitant high titer of FVII inhibitor. The aim of the present study was to identify the causative mutations and to elucidate the underlying molecular mechanisms. All nine F7 exons were sequenced in the probands and the closest family members. A homozygous deletion in exon 1, leading to a frame shift and generation of a premature stop codon (p.C10Pfs*16), was found in proband 1. Proband 2 and 3 (siblings) were homozygous for a missense mutation in exon 8, resulting in a glycine (G) to arginine (R) substitution at amino acid 240 (p.G240R). All probands had severely reduced FVII activity (FVII:C < 1 IU/dL). Treatment consisted of recombinant FVIIa and/or plasma concentrate, and proband 1 developed a FVII inhibitor shortly after initiation of treatment. The FVII variants were overexpressed in mammalian cell lines. No FVII protein was produced in cells expressing the p.C10Pfs*16 variant, and the inhibitor development in proband 1 was likely linked to the complete absence of circulating FVII. Structural analysis suggested that the G to R substitution in FVII found in probands 2 and 3 would destabilize the protein structure, and cell studies demonstrated a defective intracellular transport and increased endoplasmic reticulum stress. The molecular mechanism underlying the p.G240R variant could be reduced secretion caused by protein destabilization and misfolding.

scholarly journals Phenotypic Characterization of pncAMutants of Mycobacterium tuberculosis

2000 ◽  
Vol 44 (9) ◽  
pp. 2291-2295 ◽  
Author(s):  
Glenn P. Morlock ◽  
Jack T. Crawford ◽  
W. Ray Butler ◽  
Suzanne E. Brim ◽  
David Sikes ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleotide Sequence ◽  
Susceptibility Testing ◽  
Start Codon ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
The Third ◽  
Regulatory Mutations ◽  
Type Sequence

ABSTRACT We examined the correlation of mutations in the pyrazinamidase (PZase) gene (pncA) with the pyrazinamide (PZA) resistance phenotype with 60 Mycobacterium tuberculosis isolates. PZase activity was determined by the method of Wayne (L. G. Wayne, Am. Rev. Respir. Dis. 109:147–151, 1974), and the entirepncA nucleotide sequence, including the 74 bp upstream of the start codon, was determined. PZA susceptibility testing was performed by the method of proportions on modified Middlebrook and Cohn 7H10 medium. The PZA MICs were ≥100 μg/ml for 37 isolates, 34 of which had alterations in the pncA gene. These mutations included missense substitutions for 24 isolates, nonsense substitutions for 3 isolates, frameshifts by deletion for 4 isolates, a three-codon insertion for 1 isolate, and putative regulatory mutations for 2 isolates. Among 21 isolates for which PZA MICs were <100 μg/ml, 3 had the same mutation (Thr47→Ala) and 18 had the wild-type sequence. For the three Thr47→Ala mutants PZA MICs were 12.5 μg/ml by the method of proportions on 7H10 agar; two of these were resistant to 100 μg of PZA per ml and the third was resistant to 800 μg of PZA per ml by the BACTEC method. In all, 30 different pncA mutations were found among the 37 pncA mutants. No PZase activity was detected in 35 of 37 strains that were resistant to ≥100 μg of PZA per ml or in 34 of 37 pncA mutants. Reduced PZase activity was found in the three mutants with the Thr47→Ala mutation. This study demonstrates that mutations in the pncA gene may serve as a reliable indicator of resistance to ≥100 μg of PZA per ml.

scholarly journals High-yield Heterologous Expression of Wild Type and Mutant Ca2+ ATPase: Characterization of Ca2+ Binding Sites by Charge Transfer

2009 ◽  
Vol 391 (5) ◽  
pp. 858-871 ◽  
Author(s):  
Yueyong Liu ◽  
Rajendra Pilankatta ◽  
David Lewis ◽  
Giuseppe Inesi ◽  
Francesco Tadini-Buoninsegni ◽  
...  
Keyword(s):  
Charge Transfer ◽  
Heterologous Expression ◽  
Binding Sites ◽  
High Yield ◽  
Wild Type

scholarly journals Molecular cloning, sequencing and functional study of the promoter region of the human α2C4-adrenergic receptor gene

1997 ◽  
Vol 328 (2) ◽  
pp. 431-438 ◽  
Author(s):  
Stéphane SCHAAK ◽  
Jean-Christophe DEVEDJIAN ◽  
Cécile CAYLA ◽  
Yolande SENDER ◽  
Hervé PARIS
Keyword(s):  
Binding Sites ◽  
Promoter Region ◽  
Stop Codon ◽  
Receptor Gene ◽  
Tata Box ◽  
Start Codon ◽  
Functional Study ◽  
Coding Region ◽  
Rnase Protection ◽  
Flanking Region

Screening of a human foetal brain genomic DNA library allowed us to isolate an EcoRI-EcoRI fragment containing 6 kb of the 5ʹ-flanking region, the open reading frame and 4 kb of the 3ʹ-flanking region of the α2C4 gene. Analysis of the sequenced region (4850 bp) revealed that the first 900 bp 5ʹ to the start codon are very rich in GC (84%), contain several Sp1-binding sites and lack a consensus TATA box. The 5ʹ- and 3ʹ-ends of the α2C4 transcript were determined by RNase-protection assays carried out with a series of antisense probes. The data obtained with cellular RNA from HepG2 cells demonstrated that transcription is initiated 891 bases upstream of the translation-start site and that the polyadenylation site is located 550 bases downstream of the stop codon. These results are consistent with the existence of a non-conventional TATA box (TTAGAAA) and the presence of a unique polyadenylation signal (AATAAA). They also fit with the size of α2C4-RNA found by Northern-blot analysis (2.9 kb). The transcriptional activity of the α2C4 promoter region was investigated by transfecting several cell types with chimaeric constructs containing various fragments of the 5ʹ-non-coding region and luciferase as a reporter gene. The activity of the construct containing the entire 5ʹ-non-coding region appeared to depend on the host cell. Removal of the 5ʹ-untranslated region resulted in loss of cell specificity and a concomitant increase in luciferase activity. Transfection of HepG2 and SK-N-MC cells with constructs deleted of additional 5ʹ-flanking fragments permitted the definition of a minimal 200 bp promoter fragment containing the pseudo-TATA box and two putative SP1-binding sites.

Characterization of Thromboxane A2/Prostaglandin H2 Receptors in Porcine Coronary Artery -The Inhibitory Effect of a Novel Dibenzoxepin Derivative, KW-3635

1992 ◽  
Vol 67 (05) ◽  
pp. 582-584 ◽  
Author(s):  
Ichiro Miki ◽  
Akio Ishii
Keyword(s):  
Coronary Artery ◽  
Binding Sites ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Single Class ◽  
Porcine Coronary Artery ◽  
Equilibrium Binding ◽  
H2 Receptors

SummaryWe characterized the thromboxane A2/prostaglandin H2 receptors in porcine coronary artery. The binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to coronary arterial membranes was saturable and displaceable. Scatchard analysis of equilibrium binding showed a single class of high affinity binding sites with a dissociation constant of 18.5 ±1.0 nM and the maximum binding of 80.7 ± 5.2 fmol/mg protein. [3H]SQ 29,548 binding was concentration-dependently inhibited by thromboxane A2 antagonists such as SQ 29,548, BM13505 and BM13177 or the thromboxane A2 agonists such as U46619 and U44069. KW-3635, a novel dibenzoxepin derivative, concentration-dependently inhibited the [3H]SQ 29,548 binding to thromboxane A2/prosta-glandin H2 receptors in coronary artery with an inhibition constant of 6.0 ± 0.69 nM (mean ± S.E.M.).

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