scholarly journals Cross-Competition in Transgenic Chloroplasts Expressing Single Editing Sites Reveals Shared cis Elements

2002 ◽  
Vol 22 (24) ◽  
pp. 8448-8456 ◽  
Author(s):  
Anne-Laure Chateigner-Boutin ◽  
Maureen R. Hanson
Keyword(s):  
Editing Site ◽  
Competition Effect ◽  
High Level Expression ◽  
Consensus Sequences ◽  
Endogenous Genes ◽  
High Level ◽  
Cross Competition ◽  
Selection Of

ABSTRACT RNA editing in organelles of angiosperm plants results in alteration of Cs to Us in transcripts. In most editing sites analyzed in vitro or in vivo, sequences within approximately 30 nucleotides (nt) 5′ and 10 nt 3′ of the edited C have been found to be required for selection of the correct C editing target and for editing efficiency, but no consensus sequences have been identified. The effect of high-level expression of two different minigenes carrying either the rpoB-2 or the ndhF-2 editing site on editing was determined for all 31 known edited Cs in two transgenic tobacco lines. The editing efficiencies of both the corresponding rpoB and ndhF editing sites in the endogenous genes' transcripts and in several other genes' transcripts were reduced in the chloroplast transgenic plants. Conserved nucleotides could be identified in the sequences immediately 5′ of each overexpressed editing site and in the sites in the additional genes that experienced a competition effect, though the conserved nucleotides differ 5′ of rpoB-2 and 5′ of ndhF-2. Inspection of sequences surrounding chloroplast and mitochondrial editing sites reveals that they can be grouped into clusters which carry conserved nucleotides within 30 nt 5′ of the C target of editing. The data are consistent with a model in which the same trans factor recognizes several chloroplast or mitochondrial editing sites, depending on the particular sequence 5′ of the edited C.

scholarly journals Thyroid hormone induces constitutive keratin gene expression during Xenopus laevis development.

1989 ◽  
Vol 9 (5) ◽  
pp. 1823-1831 ◽  
Author(s):  
P M Mathisen ◽  
L Miller
Keyword(s):  
Thyroid Hormone ◽  
Xenopus Laevis ◽  
Constitutive Expression ◽  
High Level Expression ◽  
Keratin Gene ◽  
Explant Cultures ◽  
Extensive Cell ◽  
High Level

We have used in vitro explant cultures of Xenopus laevis skin to investigate the role that the thyroid hormone triiodothyronine (T3) plays in activating the 63-kilodalton (kDa) keratin genes. The activation of these genes in vivo requires two distinct steps, one independent of T3 and one dependent on T3. In this report we have shown that the same two steps are required to fully activate the 63-kDa keratin genes in skin explant cultures, and we have characterized the T3-mediated step in greater detail. Unlike the induction of transcription by T3 or steroid hormones in adult tissues, there was a long latent period of approximately 2 days between the addition of T3 to skin cultures and an increase in concentration of keratin mRNA. While the T3 induction of 63-kDa keratin gene transcription cannot occur until age 48, a short transient exposure of stage 40 skin cultures to T3 resulted in high-level expression of these genes 5 days later, when normal siblings had reached stage 48. This result indicates that T3 induces a stable change in epidermal cells which can be expressed much later, after extensive cell proliferation has occurred in the absence of T3. Once the 63-kDa keratin genes were induced, they were stably expressed, and by the end of metamorphosis T3 had no further effect on their expression. The results suggest that T3 induces constitutive expression of the 63-kDa keratin genes during metamorphosis.

Thyroid hormone induces constitutive keratin gene expression during Xenopus laevis development

1989 ◽  
Vol 9 (5) ◽  
pp. 1823-1831
Author(s):  
P M Mathisen ◽  
L Miller
Keyword(s):  
Thyroid Hormone ◽  
Xenopus Laevis ◽  
Constitutive Expression ◽  
High Level Expression ◽  
Keratin Gene ◽  
Explant Cultures ◽  
Extensive Cell ◽  
High Level

We have used in vitro explant cultures of Xenopus laevis skin to investigate the role that the thyroid hormone triiodothyronine (T3) plays in activating the 63-kilodalton (kDa) keratin genes. The activation of these genes in vivo requires two distinct steps, one independent of T3 and one dependent on T3. In this report we have shown that the same two steps are required to fully activate the 63-kDa keratin genes in skin explant cultures, and we have characterized the T3-mediated step in greater detail. Unlike the induction of transcription by T3 or steroid hormones in adult tissues, there was a long latent period of approximately 2 days between the addition of T3 to skin cultures and an increase in concentration of keratin mRNA. While the T3 induction of 63-kDa keratin gene transcription cannot occur until age 48, a short transient exposure of stage 40 skin cultures to T3 resulted in high-level expression of these genes 5 days later, when normal siblings had reached stage 48. This result indicates that T3 induces a stable change in epidermal cells which can be expressed much later, after extensive cell proliferation has occurred in the absence of T3. Once the 63-kDa keratin genes were induced, they were stably expressed, and by the end of metamorphosis T3 had no further effect on their expression. The results suggest that T3 induces constitutive expression of the 63-kDa keratin genes during metamorphosis.

scholarly journals Constitutive High Expression of Chromosomal β-Lactamase in Pseudomonas aeruginosa Caused by a New Insertion Sequence (IS1669) Located in ampD

2002 ◽  
Vol 46 (11) ◽  
pp. 3406-3411 ◽  
Author(s):  
Niels Bagge ◽  
Oana Ciofu ◽  
Morten Hentzer ◽  
Joan I. A. Campbell ◽  
Michael Givskov ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Insertion Sequence ◽  
Enterobacter Cloacae ◽  
High Level Expression ◽  
Dramatic Decrease ◽  
Strain Pao1 ◽  
Genetic Changes ◽  
High Level

ABSTRACT The expression of chromosomal AmpC β-lactamase in Pseudomonas aeruginosa is negatively regulated by the activity of an amidase, AmpD. In the present study we examined resistant clinical P. aeruginosa strains and several resistant variants isolated from in vivo and in vitro biofilms for mutations in ampD to find evidence for the genetic changes leading to high-level expression of chromosomal β-lactamase. A new insertion sequence, IS1669, was found located in the ampD genes of two clinical P. aeruginosa isolates and several biofilm-isolated variants. The presence of IS1669 in ampD resulted in the expression of high levels of AmpC β-lactamase. Complementation of these isolates with ampD from the reference P. aeruginosa strain PAO1 caused a dramatic decrease in the expression of AmpC β-lactamase and a parallel decrease of the MIC of ceftazidime to a level comparable to that of PAO1. One highly resistant, constitutive β-lactamase-producing variant contained no mutations in ampD, but a point mutation was observed in ampR, resulting in an Asp-135→Asn change. An identical mutation of AmpR in Enterobacter cloacae has been reported to cause a 450-fold higher AmpC expression. However, in many of the isolates expressing high levels of chromosomal β-lactamase, no changes were found in either ampD, ampR, or in the promoter region of ampD, ampR, or ampC. Our results suggest that multiple pathways may exist leading to increased antimicrobial resistance due to chromosomal β-lactamase.

scholarly journals Stable High-Level Expression of Heterologous Genes In Vitro and In Vivo by Noncytopathic DNA-Based Kunjin Virus Replicon Vectors

2000 ◽  
Vol 74 (9) ◽  
pp. 4394-4403 ◽  
Author(s):  
Andrei N. Varnavski ◽  
Paul R. Young ◽  
Alexander A. Khromykh
Keyword(s):  
Host Cells ◽  
Mouse Lung ◽  
Lung Epithelium ◽  
High Level Expression ◽  
Mammalian Cell Lines ◽  
Rna Transfection ◽  
Intramuscular Inoculation ◽  
High Level

ABSTRACT Primary features of the flavivirus Kunjin (KUN) subgenomic replicons include continuous noncytopathic replication in host cell cytoplasm and the ability to be encapsidated into secreted virus-like particles (VLPs). Previously we reported preparation of RNA-based KUN replicon vectors and expression of heterologous genes (HG) in cell culture after RNA transfection or after infection with recombinant KUN VLPs (A. N. Varnavski and A. A. Khromykh, Virology 255:366–375, 1999). In this study we describe the development of the next generation of KUN replicon vectors, which allow synthesis of replicon RNA in vivo from corresponding plasmid DNAs. These DNA-based vectors were able to direct stable expression of β-galactosidase (β-Gal) in several mammalian cell lines, and expression remained high (∼150 pg per cell) throughout cell passaging. The applicability of these vectors in vivo was demonstrated by β-Gal expression in the mouse lung epithelium for at least 8 weeks after intranasal inoculation and induction of anti-β-Gal antibody response after intramuscular inoculation of the β-Gal-encoding KUN replicon DNA. The noncytopathic nature of DNA-based KUN replicon vectors combined with high-level and stability of HG expression in a broad range of host cells should prove them to be useful in a variety of applications in vitro and in vivo.

High-Level Expression of Mpl in Platelets and Megakaryocytes Is Independent of Thrombopoietin

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2859-2866 ◽  
Author(s):  
Karine Cohen-Solal ◽  
Natacha Vitrat ◽  
Monique Titeux ◽  
William Vainchenker ◽  
Françoise Wendling
Keyword(s):  
Major Effect ◽  
Hematopoietic Growth Factor ◽  
High Level Expression ◽  
Rnase Protection ◽  
Platelet Production ◽  
Ectopic Secretion ◽  
High Level ◽  
Circulating Levels

Thrombopoietin (TPO) is a hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production through binding to its receptor, Mpl, encoded by the c-mpl proto-oncogene. Circulating levels of TPO are regulated by receptor-mediated uptake and degradation. To better understand this mode of TPO regulation, we examined whether expression of Mpl was regulated by its ligand. Using RNase protection analysis, we found no differences in the levels ofc-mpl transcripts in megakaryocytes (MKs) produced in vitro either in the presence or absence of TPO and in platelets (PLTs) obtained from mice hyperstimulated in vivo by ectopic secretion of TPO. Similarly, Western blot analysis of MKs produced in the presence or absence of TPO showed no difference in Mpl levels. Levels of Mpl, GpIIb, or P-selectin were virtually identical in platelet lysates obtained from normal, TPO knockout and mildly TPO-stimulated mice. In contrast, the expression of Mpl was significantly reduced in PLTs from severely thrombocythemic mice. These results show that TPO does not have a major effect on the transcription or translation of Mpl. However, they do suggest that an excess of circulating TPO can lead to the disappearance of Mpl from PLTs via catabolism.

scholarly journals Rad23 Promotes the Targeting of Proteolytic Substrates to the Proteasome

2002 ◽  
Vol 22 (13) ◽  
pp. 4902-4913 ◽  
Author(s):  
Li Chen ◽  
Kiran Madura
Keyword(s):  
Cellular Proteins ◽  
Regulatory Factor ◽  
High Level Expression ◽  
Ubiquitinated Proteins ◽  
Efficient Delivery ◽  
Catalytically Active ◽  
High Level ◽  
First Time

ABSTRACT Rad23 contains a ubiquitin-like domain (UbLR23) that interacts with catalytically active proteasomes and two ubiquitin (Ub)-associated (UBA) sequences that bind Ub. The UBA domains can bind Ub in vitro, although the significance of this interaction in vivo is poorly understood. Rad23 can interfere with the assembly of multi-Ub chains in vitro, and high-level expression caused stabilization of proteolytic substrates in vivo. We report here that Rad23 interacts with ubiquitinated cellular proteins through the synergistic action of its UBA domains. Rad23 plays an overlapping role with Rpn10, a proteasome-associated multi-Ub chain binding protein. Mutations in the UBA domains prevent efficient interaction with ubiquitinated proteins and result in poor suppression of the growth and proteolytic defects of a rad23Δ rpn10Δ mutant. High-level expression of Rad23 revealed, for the first time, an interaction between ubiquitinated proteins and the proteasome. This increase was not observed in rpn10Δ mutants, suggesting that Rpn10 participates in the recognition of proteolytic substrates that are delivered by Rad23. Overexpression of UbLR23 caused stabilization of a model substrate, indicating that an unregulated UbLR23-proteasome interaction can interfere with the efficient delivery of proteolytic substrates by Rad23. Because the suppression of a rad23Δ rpn10Δ mutant phenotype required both UbLR23 and UBA domains, our findings support the hypothesis that Rad23 encodes a novel regulatory factor that translocates ubiquitinated substrates to the proteasome.

High-Level Expression of Mpl in Platelets and Megakaryocytes Is Independent of Thrombopoietin

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2859-2866 ◽  
Author(s):  
Karine Cohen-Solal ◽  
Natacha Vitrat ◽  
Monique Titeux ◽  
William Vainchenker ◽  
Françoise Wendling
Keyword(s):  
Major Effect ◽  
Hematopoietic Growth Factor ◽  
High Level Expression ◽  
Rnase Protection ◽  
Platelet Production ◽  
Ectopic Secretion ◽  
High Level ◽  
Circulating Levels

Abstract Thrombopoietin (TPO) is a hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production through binding to its receptor, Mpl, encoded by the c-mpl proto-oncogene. Circulating levels of TPO are regulated by receptor-mediated uptake and degradation. To better understand this mode of TPO regulation, we examined whether expression of Mpl was regulated by its ligand. Using RNase protection analysis, we found no differences in the levels ofc-mpl transcripts in megakaryocytes (MKs) produced in vitro either in the presence or absence of TPO and in platelets (PLTs) obtained from mice hyperstimulated in vivo by ectopic secretion of TPO. Similarly, Western blot analysis of MKs produced in the presence or absence of TPO showed no difference in Mpl levels. Levels of Mpl, GpIIb, or P-selectin were virtually identical in platelet lysates obtained from normal, TPO knockout and mildly TPO-stimulated mice. In contrast, the expression of Mpl was significantly reduced in PLTs from severely thrombocythemic mice. These results show that TPO does not have a major effect on the transcription or translation of Mpl. However, they do suggest that an excess of circulating TPO can lead to the disappearance of Mpl from PLTs via catabolism.

Endothelial progenitor cell homing: prominent role of the IGF2-IGF2R-PLCβ2 axis

Blood ◽  
2009 ◽  
Vol 113 (1) ◽  
pp. 233-243 ◽  
Author(s):  
Yong-Sun Maeng ◽  
Hyun-Jung Choi ◽  
Ja-Young Kwon ◽  
Yong-Won Park ◽  
Kyu-Sil Choi ◽  
...  
Keyword(s):  
Postnatal Life ◽  
Protein Signaling ◽  
Vascular Networks ◽  
High Level Expression ◽  
Endothelial Progenitor ◽  
Hypoxic Conditions ◽  
High Level

Abstract Homing of endothelial progenitor cells (EPCs) to the neovascular zone is now considered to be an essential step in the formation of vascular networks during embryonic development and also for neovascularization in postnatal life. We report here the prominent role of the insulin-like growth factor 2 (IGF2)/IGF2 receptor (IGF2R) system in promoting EPC homing. With high-level expression of IGF2R in EPCs, IGF2-induced hypoxic conditions stimulated multiple steps of EPC homing in vitro and promoted both EPC recruitment and incorporation into the neovascular area, resulting in enhanced angiogenesis in vivo. Remarkably, all IGF2 actions were exerted predominantly through IGF2R-linked G(i) protein signaling and required intracellular Ca2+ mobilization induced by the β2 isoform of phospholipase C. Together, these findings indicate that locally generated IGF2 at either ischemic or tumor sites may contribute to postnatal vasculogenesis by augmenting the recruitment of EPCs. The utilization of the IGF2/IGF2R system may therefore be useful for the development of novel means to treat angiogenesis-dependent diseases.

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