scholarly journals Promoter studies of chemically induced BCI-genes in the pathosystem barley – powdery mildew

2017 ◽  
Vol 38 (SI 2 - 6th Conf EFPP 2002) ◽  
pp. 487-488
Author(s):  
U. Geldermann ◽  
G. Langen ◽  
K.-H. Kogel
Keyword(s):  
Powdery Mildew ◽  
Isonicotinic Acid ◽  
Regulatory Elements ◽  
Mobility Shift ◽  
Chemically Induced ◽  
Ef Hand ◽  
Barley Powdery Mildew ◽  
Resistance Inducers ◽  
Gel Mobility Shift ◽  
Promoter Studies

Chemical resistance inducers like BTH (S-methyl benzo (1,2,3)-thiadiazole-7-carbothiate) and DCINA (2,6-dichloro isonicotinic acid) activate resistance in barley against powdery mildew (Blumeria graminis f.sp. hordei). Nine BTH induced genes (Bci, barley chemically induced) have been identified in barley (BESSER et al. 2000) which are not responsive to pathogens in contrast to PR-proteins. From two Bci-genes (Bci3: similar to vsp, Bci4: Ca<sup>2+</sup>-binding EF-hand protein), the promoters were isolated. In transient transformation assays using promoter::GFP and promoter::GUS-constructs the functionality of these chemically induced promoters were studied. To identify the minimal promoter and regions with regulatory elements 5’-deletion constructs were used. Additionally, gel mobility shift assays were performed.

scholarly journals Involvement of PDX-1 in activation of human insulin gene transcription

2006 ◽  
Vol 188 (2) ◽  
pp. 287-294 ◽  
Author(s):  
John Le Lay ◽  
Roland Stein
Keyword(s):  
Cell Lines ◽  
Human Insulin ◽  
Interaction Analysis ◽  
Regulatory Elements ◽  
Insulin Gene ◽  
Β Cell ◽  
Mobility Shift ◽  
Human Insulin Gene ◽  
Synergistic Activation ◽  
Gel Mobility Shift

Islet β cell-specific transcription of the insulin gene is mediated through the binding of the islet-enriched PDX-1, BETA2, and MafA transcription factors to conserved 5′-flanking region regulatory elements. However, additional non-conserved sequences within this region are also significant in regulating expression. Thus, PDX-1 binds to and activates the GG2 element located between nucleotides −145 and −140 of the human gene, while the corresponding, but non-identical, site in the rodent insulin genes are negatively regulated by the Nkx2.2 transcription factor. Here, we show that despite binding PDX-1 approximately 20-fold less effectively than the conserved insulin A3 and A1 sites in gel mobility shift assays, human GG2 appears to be more important for the activation of transfected human insulin enhancer-driven reporter constructs in β cell lines. Furthermore, functional interaction analysis in non-islet cell lines demonstrated that PDX-1 binding to GG2, A1, and A3 contributes to synergistic activation of insulin gene expression with MafA. Our analysis also illustrated the requirement of poorly conserved human sequences between −293 and −251 in mediating activity through the more upstream A3 binding site. Collectively these experiments have revealed distinct features in control of the human and rodent insulin genes by PDX-1, processes that may be involved in regulating insulin expression under both normal and diabetic conditions in humans.

scholarly journals Hepatocyte nuclear factor-4α is a central transactivator of the mouse Ntcp gene

2008 ◽  
Vol 295 (2) ◽  
pp. G226-G233 ◽  
Author(s):  
Andreas Geier ◽  
Ina V. Martin ◽  
Christoph G. Dietrich ◽  
Natarajan Balasubramaniyan ◽  
Sonja Strauch ◽  
...  
Keyword(s):  
Sodium Taurocholate ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Uptake System ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Gel Mobility Shift ◽  
Fold Activation

Sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake system for conjugated bile acids. Deletions of hepatocyte nuclear factor (HNF)-1α and retinoid X receptor-α:retinoic acid receptor-α binding sites in the mouse 5′-flanking region corresponding to putatively central regulatory elements of rat Ntcp do not significantly reduce promoter activity. We hypothesized that HNF-4α, which is increasingly recognized as a central regulator of hepatocyte function, may directly transactivate mouse ( mNtcp). A 1.1-kb 5′-upstream region including the mouse Ntcp promoter was cloned and compared with the rat promoter. In contrast to a moderate 3.5-fold activation of mNtcp by HNF-1α, HNF-4α cotransfection led to a robust 20-fold activation. Deletion analysis of mouse and rat Ntcp promoters mapped a conserved HNF-4α consensus site at −345/−326 and −335/−316 bp, respectively. p-475bp mNtcpLUC is not transactivated by HNF-1α but shows a 50-fold enhanced activity upon cotransfection with HNF-4α. Gel mobility shift assays demonstrated a complex of the HNF-4α-element formed with liver nuclear extracts that was blocked by an HNF-4α specific antibody. HNF-4α binding was confirmed by chromatin immunoprecipitation. Using Hepa 1–6 cells, HNF-4α-knockdown resulted in a significant 95% reduction in NTCP mRNA. In conclusion, mouse Ntcp is regulated by HNF-4α via a conserved distal cis-element independently of HNF-1α.

Functional overload increases β-MHC promoter activity in rodent fast muscle via the proximal MCAT (βe3) site

2002 ◽  
Vol 282 (3) ◽  
pp. C518-C527 ◽  
Author(s):  
Julia M. Giger ◽  
Fadia Haddad ◽  
Anqi X. Qin ◽  
Kenneth M. Baldwin
Keyword(s):  
Gene Promoter ◽  
Firefly Luciferase ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Type I ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Slow Type ◽  
Responsive Element ◽  
Gel Mobility Shift

Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, β-MHC isoform. Different length rat β-MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of −3,500, −914, −408, and −215 bp promoters increased in response to 1 wk of OL. The smallest −171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the −171 and −408 bp region were performed. The −408 bp promoters containing mutations of the βe1, distal muscle CAT (MCAT; βe2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT (βe3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the βe3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the βe3 site functions as a putative OL-responsive element in the rat β-MHC gene promoter.

scholarly journals Tissue-Selective, Bidirectional Regulation of PEX11α and Perilipin Genes through a Common Peroxisome Proliferator Response Element

2004 ◽  
Vol 24 (3) ◽  
pp. 1313-1323 ◽  
Author(s):  
Makoto Shimizu ◽  
Ayumi Takeshita ◽  
Toshiro Tsukamoto ◽  
Frank J. Gonzalez ◽  
Takashi Osumi
Keyword(s):  
Gene Transfection ◽  
Regulatory Elements ◽  
Common Element ◽  
Peroxisome Proliferator ◽  
Mobility Shift ◽  
Response Element ◽  
Peroxisome Proliferator Activated Receptor ◽  
Bidirectional Regulation ◽  
Peroxisome Proliferator Response Element ◽  
Gel Mobility Shift

ABSTRACT Most cis-acting regulatory elements have generally been assumed to activate a single nearby gene. However, many genes are clustered together, raising the possibility that they are regulated through a common element. We show here that a single peroxisome proliferator response element (PPRE), located between the mouse PEX11α and perilipin genes, confers on both genes activation by peroxisome proliferator-activated receptor alpha (PPARα) and PPARγ. A functional PPRE 8.4 kb downstream of the promoter of PEX11α, a PPARα target gene, was identified by a gene transfection study. This PPRE was positioned 1.9 kb upstream of the perilipin gene and also functioned with the perilipin promoter. In addition, this PPRE, when combined with the natural promoters of the PEX11α and perilipin genes, conferred subtype-selective activation by PPARα and PPARγ2. The PPRE sequence specifically bound to the heterodimer of RXRα and PPARα or PPARγ2, as assessed by electrophoretic gel mobility shift assays. Furthermore, tissue-selective binding of PPARα and PPARγ to the PPRE was demonstrated in hepatocytes and adipocytes, respectively, by chromatin immunoprecipitation assay. Hence, the expression of these genes is induced through the same PPRE in the liver and adipose tissue, where the two PPAR subtypes are specifically expressed.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

scholarly journals Dissimilarity of barley powdery mildew resistances Lomerit and Heils Hanna

2011 ◽  
Vol 47 (No. 3) ◽  
pp. 95-100 ◽  
Author(s):  
A. Dreiseitl
Keyword(s):  
Powdery Mildew ◽  
Agricultural Research ◽  
Blumeria Graminis ◽  
The Other ◽  
Japanese Isolate ◽  
Agricultural Research Institute ◽  
Reaction Type ◽  
Barley Cultivars ◽  
Barley Powdery Mildew ◽  
Old Japanese

&nbsp; The resistance Heils Hanna (HH) was postulated in several tens of 471 previously tested winter barley cultivars. In this paper, new tests on 29 of these cultivars are reported. Thirty-two reference isolates of Blumeria graminis f.sp. hordei held in the pathogen genebank at the Agricultural Research Institute in Kromeriz, Ltd. including a Japanese isolate and five Israeli isolates were used for response tests. However, the resistance HH conferred by the gene Mla8 and herein characterised by reaction type 0 to an old Japanese isolate known as Race I was now postulated only in four cultivars. In the other 25 cultivars another resistance, characterised by reaction type 0 to Race I and also to two Israeli isolates, was detected. In addition to the two mentioned resistances, eight known (Bw, Dr2, Ha, IM9, Ln, Lv, Ra and Sp) resistances were found in the set examined. Lomerit was the only registered cultivar tested here in which the newly detected resistance was present alone, therefore, it is recommended that this resistance be designated Lo.

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