Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Functional studies with ZWICHEL ( ZWI ), which encodes a Ca(2+)-calmodulin-regulated kinesin, have shown its involvement in trichome morphogenesis and cell division. To identify regulatory regions that control the ZWI expression pattern, we generated transgenic Arabidopsis plants with a GUS reporter driven by different lengths of the ZWI gene 5' region alone or 5' and 3' regions together. The 5' fusions contain varying lengths of the coding and non-coding regions of beta - HYDROXYISOBUTYRYL-CoA HYDROLASE 1 ( CHY1 ), which is upstream of ZWI, and a 162 bp intergenic region. In transgenic plants with 5' 460::GUS, GUS activity was observed primarily in the root hairs whereas transgenic plants with an additional 5' 266 bp region from the CHY1 gene (5' 726::GUS) showed strong GUS accumulation in the entire root including root hairs and root tip, calli and at various developmental stages in trichomes and pollen. However, very little GUS accumulation was detected in roots of dark-grown or root tips of cold-treated seedlings with 5' ZWI constructs. These results were further confirmed by quantifying GUS enzyme activity and transcripts in these seedlings. Calli and pollen transformed with the 5' distal 268 bp fused in antisense orientation to the proximal 460 bp did not show GUS expression. Further, IAA-treated dark-grown seedlings with 726::GUS, but not with 460::GUS, showed high GUS expression in specific regions (outer layer 2a cells) at the base of the lateral roots. The ZWI 3' region (3 kb) did not influence the GUS expression pattern driven by the 5' 726 bp. The absence of CHY1 transcripts in the chy1-2 mutant did not alter either ZWI expression or ZWI-mediated trichome morphogenesis. Thus, our data suggest that the 3' part of the CHY1 gene contains regulatory elements that control ZWI gene expression in dividing cells and other cells that exhibit polarized growth such as root hairs, pollen and trichomes. This is the first evidence that the regulatory regions conferring developmental and cell-specific expression of a gene reside in the introns and exons of its upstream protein-coding gene.
Plant Mol Biol 2004 Jan
PMID:Developmental and cell-specific expression of ZWICHEL is regulated by the intron and exon sequences of its gene. 1515 28

The purpose of this study was to determine if elevated myocardial glycogen content could obviate Ca(2+) overload and subsequent myocardial injury in the setting of low oxygen and diminished exogenous substrate supplies. Isolated harp seal cardiomyocytes, recognized as having large glycogen stores, were incubated under conditions simulating ischemia (oxygen and substrate deprivation) for 1 h. Rat cardiomyocytes were used for comparison. Freshly isolated seal cardiomyocytes contained approximately 10 times more glycogen than those from rats (479 +/- 39 vs. 48 +/- 5 nmol glucose/mg dry weight (dry wt), mean +/- S.E., n = 6), and during ischemia lactate production was significantly greater in seal compared to rat cardiomyocytes (660 +/- 99 vs. 97 +/- 14 nmol/mg dry wt), while glycogen content decreased both in seal (from 479 +/- 39 to 315 +/- 58 nmol glucose/mg dry wt) and rat cardiomyocytes (from 48 +/- 5 to 18 +/- 5 nmol glucose/mg dry wt). Cellular ATP was well maintained in ischemic seal cardiomyocytes, whereas it showed a 65% decline (from 31 +/- 3 to 11 +/- 1 nmol ATP/mg dry wt) in rat cardiomyocytes. Similarly, total seal cardiomyocyte Ca(2+) content was not affected by ischemia, while Ca(2+) increased from 8.5 +/- 2.0 to 13.3 +/- 2.0 nmol/mg dry wt in ischemic rat myocytes. Rat cardiomyocytes also showed a notable decline in the percentage of rod-shaped cells in response to ischemia (from 66 +/- 4% to 30 +/- 3%), and cell morphology was unaffected in seal incubations. Addition of iodoacetate (IAA, an inhibitor of glycolysis) to seal cardiomyocytes, on top of substrate and oxygen deprivation, reduced the cellular content of ATP by 52.9 +/- 4.4% (from 25 +/- 4 to 11 +/- 2 nmol ATP/mg dry wt) and the percentage of rod-shaped myocytes from 51 +/- 3% to 28 +/- 4%, while total Ca(2+) content was unchanged by these conditions. Seal cardiomyocytes thus tolerate low oxygen conditions better than rat cardiomyocytes. This finding is most likely due to a higher glycolysis rate in seals, fueled by larger myocardial glycogen stores.
J Mol Cell Cardiol 2004 Jul
PMID:Endogenous glycogen prevents Ca2+ overload and hypercontracture in harp seal myocardial cells during simulated ischemia. 1524 30

The sloughing of root cap cells from the root tip is important because it assists the growing root in penetrating the soil. Using a promoter-reporter (GUS) and RT-PCR analysis, we identified an endo-beta-1,4-glucanase (AtCel5) of Arabidopsis thaliana that is expressed exclusively in root cap cells of both primary and secondary roots. Expression is inhibited by high concentrations of IAA, both exogenous and internal, as well as by ABA. AtCel5 expression begins once the mature tissue pattern is established and continues for 3 weeks. GUS staining is observed in both root cap cells that are still attached and cells that have already been shed. Using AtCel5-GUS as a marker, we observed that the root cap cells begin to separate at the sides of the tip while the cells of the central region of the tip separate last. Separation involves sequential tiers of intact cells that separate from the periphery of the root tip. A homozygous T-DNA insertion mutant that does not express AtCel5 forms the root cap and sheds root cap cells but sloughing is less efficient compared to wild type. The reduction in sloughing in the mutant does not affect the overall growth performance of the plant in loose media. The modest effect of abolishing AtCel5 expression suggests that there are multiple redundant genes regulating the process of sloughing of the root cap, including AtCel3/At1g71380, the paralog of the AtCel5 gene that is also expressed in the root cap cells. Thus, these two endo-1,4-beta-D-glucanases may have a role in the sloughing of border cells from the root tip. We propose that AtCel5, provides a new molecular marker to further analyze the process of root cap cell separation and a root cap specific promoter for targeting to the environment genes with beneficial properties for plant growth.
Plant Mol Biol 2004 Sep
PMID:Root cap specific expression of an endo-beta-1,4-D-glucanase (cellulase): a new marker to study root development in Arabidopsis. 1560 46

Previously, we characterized 92 Arabidopsis genes (AtSFLs) similar to the S-locus F-box genes involved in S-RNase-based self-incompatibility and found that they likely play diverse roles in Arabidopsis. In this study, we investigated the role of one of these genes, CEGENDUO (CEG, AtSFL61), in the lateral root formation. A T-DNA insertion in CEG led to an increased lateral root production, which was complemented by transformation of the wild-type gene. Its downregulation by RNAi also produced more lateral roots in transformed Arabidopsis plants whereas its overexpression generated less lateral roots compared to wild-type, indicating that CEG acts as a negative regulator for the lateral root formation. It was found that CEG was expressed abundantly in vascular tissues of the primary root, but not in newly formed lateral root primordia and the root meristem, and induced by exogenous auxin NAA (alpha-naphthalene acetic acid). In addition, the ceg mutant was hyposensitive to NAA, IAA (indole-3-acetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid), as well as the auxin transport inhibitor TIBA (3,3,5-triiodobenzoic acid), showing that CEG is an auxin-inducible gene. Taken together, our results show that CEG is a novel F-box protein negatively regulating the auxin-mediated lateral root formation in Arabidopsis.
Plant Mol Biol 2006 Mar
PMID:An auxin-inducible F-box protein CEGENDUO negatively regulates auxin-mediated lateral root formation in Arabidopsis. 1652 94

It was well known that auxin is critical for anther/pollen grain development, however, the clear distribution and detailed effects of auxin during floral development are still unclear. We have shown here that, through analyzing GUS activities of Arabidopsis lines harboring auxin response elements DR5-GUS, auxin was mainly accumulated in the anther during flower stages 10-12. Further studies employing the indoleacetic acid-lysine synthetase (iaaL) coding gene from Pseudomonas syringae subsp. savastanoi under control of the promoter region of Arabidopsis phosphatidylinositol monophosphate 5-kinase 1 gene, which conducts the anther filament-specific expression, showed that block of auxin flow of filaments resulted in shortened filaments and significantly defective pollen grains. Similar phenotype was observed in tobacco plants transformed with the same construct, confirming the effects of auxin flow in filaments on anther development. Detailed studies further revealed that the meiosis process of pollen grain was normal while the mitosis at later stage was significantly defected, indicating the effects of auxin flow in filaments on pollen grain mitosis process. Analysis employing [(14)C]IAA, as well as the observation on the expression of AtPIN1, coding for auxin efflux carrier, demonstrated the presence of polar auxin transport in anther filaments and pollen grains.
Plant Mol Biol 2006 May
PMID:Auxin flow in anther filaments is critical for pollen grain development through regulating pollen mitosis. 1678 2

Based upon the phenotype of young, dark-grown seedlings, a cytokinin-resistant mutant, cnr1, has been isolated, which displays altered cytokinin- and auxin-induced responses. The mutant seedlings possess short hypocotyls and open apical hooks (in dark), and display agravitropism, hyponastic cotyledons, reduced shoot growth, compact rosettes and short roots with increased adventitious branching and reduced number of root hairs. A number of these features invariably depend upon auxin/cytokinin ratio but the cnr1 mutant retains normal sensitivity towards auxin as well as auxin polar transport inhibitor, TIBA, although upregulation of primary auxin-responsive Aux/IAA genes is reduced. The mutant shows resistance towards cytokinin in hypocotyl/root growth inhibition assays, displays reduced regeneration in tissue cultures (cytokinin response) and decreased sensitivity to cytokinin for anthocyanin accumulation. It is thus conceivable that due to reduced sensitivity to cytokinin, the cnr1 mutant also shows altered auxin response. Surprisingly, the mutant retains normal sensitivity to cytokinin for induction of primary response genes, the type-A Arabidopsis response regulators, although the basal level of their expression was considerably reduced as compared to the wild-type. The zeatin and zeatin riboside levels, as estimated by HPLC, and the cytokinin oxidase activity were comparable in the cnr1 mutant and the wild-type. The hypersensitivity to red light (in hypocotyl growth inhibition assay), partial photomorphogenesis in dark, and hypersensitivity to sugars, are some other features displayed by the cnr1 mutant. The lesion in the cnr1 mutant has been mapped to the top of chromosome 1 where no other previously known cytokinin-resistant mutant has been mapped, indicating that the cnr1 mutant defines a novel locus involved in hormone, light and sugar signalling.
Plant Mol Biol 2006 Oct
PMID:Arabidopsis cytokinin-resistant mutant, cnr1, displays altered auxin responses and sugar sensitivity. 1691 15

The moss Physcomitrella patens has two life cycles, filamentous protonema and leafy gametophore. A modified from of suppression subtractive hybridization (SSH), mirror orientation selection (MOS), was applied to screen genes differentially expressed in the P. patens protonema. Using reverse Northern blot analysis, differentially expressed clones were identified. The identified genes were involved mainly in metal binding and detoxification. One of these genes was an AP2 (APETALA2) domain-containing protein (PpACP1), which was highly up-regulated in the protonema. Alignment with other AP2/EREBPs (Ethylene Responsive Element Binding Proteins) revealed significant sequence homology of the deduced amino acid sequence in the AP2/EREBP DNA binding domain. Northern analysis under various stress conditions showed that PpACP1 was induced by ethephon, cadmium, copper, ABA, IAA, and cold. In addition, it was highly expressed in suspension-cultured protonema. We suggest that PpACP1 is involved in responses to metals, and that suspension culture enhance the expression of genes responding to metals.
Mol Cells 2007 Feb 28
PMID:Modified suppression subtractive hybridization identifies an AP2-containing protein involved in metal responses in Physcomitrella patens. 1746 18

Fusion proteins are commonly used as a source of antigen for producing antibodies and in many cases can be useful for biochemical analyses. This unit describes two widely used expression systems for producing large amounts of proteins in E. coli. One system expresses lacZ fusions using the pUR series of vectors and the other expresses trpE fusions using the pATH vectors. The gene of interest is first subcloned into either a pUR or pATH vector in the correct reading frame. The correct transformant is selected, grown, and then induced with either IPTG or IAA. Sonication of cells in the presence of protease inhibitors is used to prepare extracts containing both types of fusion proteins, as well as other types of proteins overexpressed in E. coli. The extracts are checked for the presence of fusion protein on an SDS-polyacrylamide gel.
Curr Protoc Mol Biol 2001 May
PMID:Expression and purification of lacZ and trpE fusion proteins. 1826 32

Elucidation of the genetic basis of the control of leaf shape could be of use in the manipulation of crop traits, leading to more stable and increased crop production. To improve our understanding of the process controlling leaf shape, we identified a mutant gene in rice that causes a significant decrease in the width of the leaf blade, termed narrow leaf 7 (nal7). This spontaneous mutation of nal7 occurred during the process of developing advanced back-crossed progeny derived from crosses of rice varieties with wild type leaf phenotype. While the mutation resulted in reduced leaf width, no significant morphological changes at the cellular level in leaves were observed, except in bulli-form cells. The NAL7 locus encodes a flavin-containing monooxygenase, which displays sequence homology with YUCCA. Inspection of a structural model of NAL7 suggests that the mutation results in an inactive enzyme. The IAA content in the nal7 mutant was altered compared with that of wild type. The nal7 mutant overexpressing NAL7 cDNA exhibited overgrowth and abnormal morphology of the root, which was likely to be due to auxin overproduction. These results indicate that NAL7 is involved in auxin biosynthesis.
Mol Genet Genomics 2008 May
PMID:NARROW LEAF 7 controls leaf shape mediated by auxin in rice. 1829 11

Hormonal signals are transduced (and sometimes perceived) by protein-protein interactions. Understanding these interactions is therefore crucial to understanding the network of signalling components as a whole. Often, genetic analysis serves up a selection of players that may or may not interact directly to carry the signal in question and further insight inevitably leads to some sort of biochemistry. Using the example of the auxin-regulated interaction between the auxin receptor TIR1 and the Aux/IAA repressor proteins, this chapter deals with some of this biochemistry, describing a very simple assay for looking at which proteins are interacting, and if and how those interactions are regulated.
Methods Mol Biol 2009
PMID:Pull-down assays for plant hormone research. 1908 46


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