Diverse Mechanisms Regulate the Expression of Genes Coding for C4 Enzymes

1997 ◽  
Vol 24 (4) ◽  
pp. 437 ◽  
Author(s):  
William C. Taylor ◽  
Elke Rosche ◽  
Jerry S. Marshall ◽  
Shahjahan Ali ◽  
Chris J. Chastain ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Dna Sequences ◽  
Single Gene ◽  
Bundle Sheath ◽  
Specific Expression ◽  
Pyruvate Orthophosphate Dikinase ◽  
Expression Of Genes ◽  
Regulatory Dna Sequences ◽  
High Level ◽  
C4 Enzymes

Most of the enzymes of the C4 pathway of photosynthesis are compartmentalised in either mesophyll or bundle sheath cells. We have begun to dissect the mechanisms controlling the cell-specific expression of genes coding for C4 enzymes by locating the regulatory DNA sequences from two genes in the transformable C4 dicot, Flaveria bidentis. We show that chloroplast and cytosolic forms of pyruvate,orthophosphate dikinase are encoded by a single gene, Pdk. By fusing selected regions of the Pdk gene, to the gusA reporter gene we were able to infer promoter activities from measurements of GUS enzyme activity in transgenic F. bidentis plants. We show that the 5´ end of the Pdk gene contains sequences controlling high-level, mesophyll-specific expression of the C4 form of the enzyme. The promoter controlling low-level expression of the cytosolic form was located in the large intron. The C4 isoform of NADP-malic enzyme is encoded by the Me1 gene. Analyses of a series of reporter gene fusions showed that sequences at the 5´ end of the gene control bundle sheath expression but that sequences located near the 3´ end are necessary for high-level expression. The mechanisms regulating the expression of both genes are clearly different and suggest that there is no universal mechanism controlling the expression of genes coding for C4 enzymes.

Promoter Elements of vav Drive Transgene Expression In Vivo Throughout the Hematopoietic Compartment

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 1855-1863 ◽  
Author(s):  
Sarah Ogilvy ◽  
Donald Metcalf ◽  
Leonie Gibson ◽  
Mary L. Bath ◽  
Alan W. Harris ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Transgene Expression ◽  
Cell Types ◽  
Transcription Unit ◽  
Erythroid Cells ◽  
Promoter Elements ◽  
Specific Expression ◽  
Expression Of Genes ◽  
B And T Lymphocytes

To develop a method for targeting expression of genes to the full hematopoietic system, we have used transgenic mice to explore the transcriptional regulation of the vav gene, which is expressed throughout this compartment but rarely outside it. Previously, we showed that a cluster of elements surrounding its promoter could drive hematopoietic-specific expression of a bacterial lacZ reporter gene, but the expression was confined to lymphocytes and was sporadically silenced. Those limitations are ascribed here to the prokaryotic reporter gene. With a human CD4 (hCD4) cell surface reporter, the vav promoter elements drove expression efficiently and stably in virtually all nucleated cells of adult hematopoietic tissues but not notably in nonhematopoietic cell types. In multiple lines, hCD4 appeared on most, if not all, B and T lymphocytes, granulocytes, monocytes, megakaryocytes, eosinophils, and nucleated erythroid cells. Moreover, high levels appeared on both lineage-committed progenitors and the more primitive preprogenitors. In the fetus, expression was evident in erythroid cells of the definitive but not the primitive type. These results indicate that a prokaryotic sequence can inactivate a transcription unit and that the vavpromoter region constitutes a potent transgenic vector for the entire definitive hematopoietic compartment.

A modified pod specific promoter for high level heterologous expression of genes in legumes

2019 ◽  
Author(s):  
Aravind Kumar Konda ◽  
Pallavi Singh ◽  
Khela Ram Soren ◽  
Narendra Pratap Singh
Keyword(s):  
Gene Expression ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Specific Expression ◽  
Tissue Specific ◽  
Cis Acting ◽  
Inducible Promoters ◽  
Expression Of Genes ◽  
Enhanced Expression ◽  
High Level

Promoters are cis-acting regulatory elements that are usually present upstream to the coding sequences and determine the gene expression. Deployment of tissue specific and inducible promoters are constantly increasing for development of successful and stable multiple transgenic plants. To this end, as a strategy for enhanced expression of cis or transgenes, promoter engineering of the native msg promoter from soya bean has been carried out for executing pod specific expression of genes. Cis regulatory elements such as 5’UTR and poly (A) tract have been incorporated for imparting mRNA stability and translational enhancement to generate the modified 1.285 Kb pod specific promoter. Further to attain transcriptional enhancement the modified promoter has been cloned to generate Bi-directional Duplex Promoters (BDDP). The engineered msg promoter gene constructs can be deployed for high level tissue specific gene expression of cis/trans genes along with chosen terminator in chickpea. soybean and other legumes as well.

Promoter Elements of vav Drive Transgene Expression In Vivo Throughout the Hematopoietic Compartment

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 1855-1863 ◽  
Author(s):  
Sarah Ogilvy ◽  
Donald Metcalf ◽  
Leonie Gibson ◽  
Mary L. Bath ◽  
Alan W. Harris ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Transgene Expression ◽  
Cell Types ◽  
Transcription Unit ◽  
Erythroid Cells ◽  
Promoter Elements ◽  
Specific Expression ◽  
Expression Of Genes ◽  
B And T Lymphocytes

Abstract To develop a method for targeting expression of genes to the full hematopoietic system, we have used transgenic mice to explore the transcriptional regulation of the vav gene, which is expressed throughout this compartment but rarely outside it. Previously, we showed that a cluster of elements surrounding its promoter could drive hematopoietic-specific expression of a bacterial lacZ reporter gene, but the expression was confined to lymphocytes and was sporadically silenced. Those limitations are ascribed here to the prokaryotic reporter gene. With a human CD4 (hCD4) cell surface reporter, the vav promoter elements drove expression efficiently and stably in virtually all nucleated cells of adult hematopoietic tissues but not notably in nonhematopoietic cell types. In multiple lines, hCD4 appeared on most, if not all, B and T lymphocytes, granulocytes, monocytes, megakaryocytes, eosinophils, and nucleated erythroid cells. Moreover, high levels appeared on both lineage-committed progenitors and the more primitive preprogenitors. In the fetus, expression was evident in erythroid cells of the definitive but not the primitive type. These results indicate that a prokaryotic sequence can inactivate a transcription unit and that the vavpromoter region constitutes a potent transgenic vector for the entire definitive hematopoietic compartment.

scholarly journals Targeting the Hprt locus in mice reveals differential regulation of Tie2 gene expression in the endothelium

2000 ◽  
Vol 2 (2) ◽  
pp. 67-75 ◽  
Author(s):  
VALERIE EVANS ◽  
ANTONIS HATZOPOULOS ◽  
WILLIAM C. AIRD ◽  
HELEN B. RAYBURN ◽  
ROBERT D. ROSENBERG ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cell ◽  
Dna Sequences ◽  
Single Copy ◽  
Differential Regulation ◽  
Lacz Gene ◽  
Specific Expression ◽  
Regulatory Dna Sequences ◽  
Hprt Locus

Evans, Valerie, Antonis Hatzopoulos, William C. Aird, Helen B. Rayburn, Robert D. Rosenberg, and Jan Albert Kuivenhoven. Targeting the Hprt locus in mice reveals differential regulation of Tie2 gene expression in the endothelium. Physiol Genomics 2: 67–75, 2000.—To study the in vivo expression of the murine Tie2 gene, we have targeted the hypoxanthine phosphoribosyltransferase ( Hprt) gene locus to generate two single-copy transgenic mice: T1, containing the 2,100-bp Tie2 promoter upstream from the β-galactosidase ( LacZ) gene, and T5, which also included an enhancing element originating from the first intron of the Tie2 gene. Comparing T1 and T5 embryos at day E10.5 revealed differential endothelial cell-specific expression of LacZ, whereas colocalization analyses showed that the expression was confined to endothelial cells. Moderate reporter gene activity was observed in the brain and kidney of T1 adults, whereas extensive LacZ gene expression was seen in the vasculature of most organs of the T5 adults. This study demonstrates the feasibility of targeting the Hprt locus with endothelial cell-specific sequences to analyze the spatial-temporal expression of transgenes. Of particular importance is the observation that the analysis of a single transgene copy in a defined locus allows for an accurate and rapid comparison of transcriptional activity among regulatory DNA sequences.

Transcription factor/DNA interactions visualized by electron spectroscopic imaging

1992 ◽  
Vol 50 (1) ◽  
pp. 450-451
Author(s):  
David P. Bazett-Jones ◽  
Mark L. Brown
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Phosphorus Content ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
Dna Backbone ◽  
Two Parameters ◽  
High Level

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.

scholarly journals A Method for Rapid Demineralization of Teeth and Bones

2010 ◽  
Vol 4 (1) ◽  
pp. 223-229 ◽  
Author(s):  
Andrew Cho ◽  
Shigeki Suzuki ◽  
Junko Hatakeyama ◽  
Naoto Haruyama ◽  
Ashok B Kulkarni
Keyword(s):  
Reporter Gene ◽  
Gene Expression Pattern ◽  
Cell Lineage ◽  
Careful Analysis ◽  
Lacz Gene ◽  
Bone Specimen ◽  
Specific Expression ◽  
Temporal Gene Expression ◽  
Gene And Protein Expression ◽  
Tissue Specimens

Tooth and bone specimen require extensive demineralization for careful analysis of cell morphology, as well as gene and protein expression levels. The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate. This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest. To analyze LacZ activity, and the expression of other genes and their protein products in teeth and bones, it is necessary to carry out a complete demineralization of the specimen before cutting sections. However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase. Therefore, most protocols currently use mild acids such as 0.1 M ethylene diamine tetra-acetic acid (EDTA) for demineralization of tooth and bone specimen, which require a longer period of treatment for complete demineralization. A method by which hard tissue specimens such as teeth and bones can be rapidly, but gently, decalcified is necessary to save time and effort. Here, we report a suitable method for rapid demineralization of mouse teeth in 0.1M EDTA at 42˚C without any loss of ß-galactosidase activity.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

scholarly journals Molecular and physiological characterization of the effects of auxin-enriched rootstock on grafting

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Longmei Zhai ◽  
Xiaomin Wang ◽  
Dan Tang ◽  
Qi Qi ◽  
Huseyin Yer ◽  
...  
Keyword(s):  
Callus Formation ◽  
Ethylene Biosynthesis ◽  
Union Formation ◽  
Biosynthetic Gene ◽  
Graft Union ◽  
Specific Expression ◽  
Physiological Characterization ◽  
Graft Healing ◽  
Expression Of Genes ◽  
Or Gene

AbstractsGrafting is a highly useful technique, and its success largely depends on graft union formation. In this study, we found that root-specific expression of the auxin biosynthetic gene iaaM in tobacco, when used as rootstock, resulted in more rapid callus formation and faster graft healing. However, overexpression of the auxin-inactivating iaaL gene in rootstocks delayed graft healing. We observed increased endogenous auxin levels and auxin-responsive DR5::GUS expression in scions of WT/iaaM grafts compared with those found in WT/WT grafts, which suggested that auxin is transported upward from rootstock to scion tissues. A transcriptome analysis showed that auxin enhanced graft union formation through increases in the expression of genes involved in graft healing in both rootstock and scion tissues. We also observed that the ethylene biosynthetic gene ACS1 and the ethylene-responsive gene ERF5 were upregulated in both scions and rootstocks of the WT/iaaM grafts. Furthermore, exogenous applications of the ethylene precursor ACC to the junction of WT/WT grafts promoted graft union formation, whereas application of the ethylene biosynthesis inhibitor AVG delayed graft healing in WT/WT grafts, and the observed delay was less pronounced in the WT/iaaM grafts. These results demonstrated that elevated auxin levels in the iaaM rootstock in combination with the increased auxin levels in scions caused by upward transport/diffusion enhanced graft union formation and that ethylene was partially responsible for the effects of auxin on grafting. Our findings showed that grafting success can be enhanced by increasing the auxin levels in rootstocks using transgenic or gene-editing techniques.

Activation of octamer-containing promoters by either octamer-binding transcription factor 1 (OTF-1) or OTF-2 and requirement of an additional B-cell-specific component for optimal transcription of immunoglobulin promoters

1990 ◽  
Vol 10 (12) ◽  
pp. 6204-6215
Author(s):  
A Pierani ◽  
A Heguy ◽  
H Fujii ◽  
R G Roeder
Keyword(s):  
B Cell ◽  
Regulatory Element ◽  
Immunoglobulin Gene ◽  
Mobility Shift ◽  
Specific Expression ◽  
Tissue Specific ◽  
Activation Domains ◽  
Cell Extracts ◽  
Octamer Motif ◽  
High Level

Several distinct octamer-binding transcription factors (OTFs) interact with the sequence ATTTGCAT (the octamer motif), which acts as a transcription regulatory element for a variety of differentially controlled genes. The ubiquitous OTF-1 plays a role in expression of the cell cycle-regulated histone H2b gene as well as several other genes, while the tissue-specific OTF-2 has been implicated in the tissue-specific expression of immunoglobulin genes. In an attempt to understand the apparent transcriptional selectivity of these factors, we have investigated the physical and functional characteristics of OTF-1 purified from HeLa cells and both OTF-1 and OTF-2 purified from B cells. High-resolution footprinting and mobility shift-competition assays indicated that these factors were virtually indistinguishable in binding affinities and DNA-protein contacts on either the H2b or an immunoglobulin light-chain (kappa) promoter. In addition, each of the purified factors showed an equivalent intrinsic capacity to activate transcription from either immunoglobulin promoters (kappa and heavy chain) or the H2b promoter in OTF-depleted HeLa and B-cell extracts. However, with OTF-depleted HeLa extracts, neither factor could restore immunoglobulin gene transcription to the relatively high level observed in unfractionated B-cell extracts. Restoration of full immunoglobulin gene activity appears to require an additional B-cell regulatory component which interacts with the OTFs. The additional B-cell factor could act either by facilitating interaction of OTF activation domains with components of the general transcriptional machinery or by contributing a novel activation domain.

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