scholarly journals 058 Cloning of a Defensin-related Gene and Its Expression during Dormancy and Fruit Development in Peach [Prunus persica (L.) Batsch]

HortScience ◽  
1999 ◽  
Vol 34 (3) ◽  
pp. 451B-451
Author(s):  
M. Wisniewski ◽  
T. Artlip ◽  
R. Webb ◽  
C. Bassett ◽  
A. Callahan
Keyword(s):  
Protease Inhibitor ◽  
Fruit Development ◽  
Blot Analysis ◽  
Prunus Persica ◽  
Extracellular Proteins ◽  
Full Length ◽  
Amino Terminal ◽  
Partial Clone ◽  
Extracellular Transport ◽  
Rna Blot Analysis

During the past several years we have been involved in identifying seasonally regulated proteins and genes from peach bark. In the present study, we describe the cloning of a protease inhibitor from a cDNA library made from winter bark tissues. A partial clone obtained from the library was extended to full length by 5' RACE. The full-length cDNA clone (final3b) is 613 bp in length, not including the poly A+ tail. The open reading frame of 237 bp codes for a 79 amino acid protease inhibitor related to the defensin family of proteins. This family of small, cysteine-rich, extracellular proteins play a role in the plantís defense response through their antifungal properties. Sequence comparison of the encoded protein using BLAST analysis revealed significant homology to protease inhibitors from Glycine max, Arabidopsis thaliana, and a defensin protein from bell pepper (Capsicum annuum). Similar to these other cysteine-rich proteins, the peach defensin contains a consensus cys arrangement and is predicted to have an amino terminal signal peptide, presumably targeting it for extracellular transport. RNA-blot analysis indicated that the gene is seasonally expressed in bark tissues of 1-year-old shoots. Transcript abundance of final3b increased in the fall, reached a peak in midwinter and then decreased. The gene was also expressed during early stages of fruit development. RNA-blot analysis of the gene in other tissues, and in response to environmental stress and wounding, is in progress.

scholarly journals Expression of a Truncated Yeast Ccc1 Vacuolar Transporter Increases the Accumulation of Endogenous Iron

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1120
Author(s):  
Raquel Sorribes-Dauden ◽  
María Teresa Martínez-Pastor ◽  
Sergi Puig
Keyword(s):  
Iron Homeostasis ◽  
Sufficient Conditions ◽  
Constitutive Expression ◽  
Iron Bioavailability ◽  
Full Length ◽  
Yeast Cells ◽  
Deficiency Anemia ◽  
Functional Protein ◽  
Amino Terminal ◽  
In Cells

Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox cofactor in multiple metabolic processes. Iron bioavailability is highly restricted due to the low solubility of its oxidized form, frequently leading to iron deficiency anemia. The baker’s yeast Saccharomyces cerevisiae is used as a model organism for iron homeostasis studies, but also as a food supplement and fermentative microorganism in the food industry. Yeast cells use the vacuolar Ccc1 transporter to detoxify and store excess iron in the vacuoles. Here, we modulate CCC1 expression and properties to increase iron extraction from the environment. We show that constitutive expression of full-length CCC1 is toxic, whereas deletion of its cytosolic amino-terminal (Nt) domain (NtDCCC1) rescues this phenotype. Toxicity is exacerbated in cells lacking AFT1 transcription factor. Further characterization of NtDCcc1 protein suggests that it is a partially functional protein. Western blot analyses indicate that deletion of Ccc1 Nt domain does not significantly alter GFP-Ccc1 protein stability. A functional full-length GFP-Ccc1 protein localized to particular regions of the vacuolar membrane, whereas GFP-NtDCcc1 protein was evenly distributed throughout this endogenous membrane. Interestingly, expression of NtDCCC1 increased the accumulation of endogenous iron in cells cultivated under iron-sufficient conditions, a strategy that could be used to extract iron from media that are not rich in iron.

scholarly journals Herpes Simplex Virus UL12.5 Targets Mitochondria through a Mitochondrial Localization Sequence Proximal to the N Terminus

2009 ◽  
Vol 83 (6) ◽  
pp. 2601-2610 ◽  
Author(s):  
Jennifer A. Corcoran ◽  
Holly A. Saffran ◽  
Brett A. Duguay ◽  
James R. Smiley
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Mitochondrial Genome ◽  
Nuclear Localization ◽  
Full Length ◽  
Mitochondrial Localization ◽  
Amino Terminal ◽  
The Matrix ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The herpes simplex virus type 1 (HSV-1) gene UL12 encodes a conserved alkaline DNase with orthologues in all herpesviruses. The HSV-1 UL12 gene gives rise to two separately promoted 3′ coterminal mRNAs which encode distinct but related proteins: full-length UL12 and UL12.5, an amino-terminally truncated form that initiates at UL12 codon 127. Full-length UL12 localizes to the nucleus where it promotes the generation of mature viral genomes from larger precursors. In contrast, UL12.5 is predominantly mitochondrial and acts to trigger degradation of the mitochondrial genome early during infection. We examined the basis for these very different subcellular localization patterns. We confirmed an earlier report that the amino-terminal region of full-length UL12 is required for nuclear localization and provide evidence that multiple nuclear localization determinants are present in this region. In addition, we demonstrate that mitochondrial localization of UL12.5 relies largely on sequences located between UL12 residues 185 and 245 (UL12.5 residues 59 to 119). This region contains a sequence that resembles a typical mitochondrial matrix localization signal, and mutations that reduce the positive charge of this element severely impaired mitochondrial localization. Consistent with matrix localization, UL12.5 displayed a detergent extraction profile indistinguishable from that of the matrix protein cyclophilin D. Mitochondrial DNA depletion required the exonuclease activity of UL12.5, consistent with the idea that UL12.5 located within the matrix acts directly to destroy the mitochondrial genome. These results clarify how two highly related viral proteins are targeted to different subcellular locations with distinct functional consequences.

Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits

1994 ◽  
Vol 14 (1) ◽  
pp. 42-49
Author(s):  
K H Holt ◽  
L Olson ◽  
W S Moye-Rowley ◽  
J E Pessin
Keyword(s):  
Gene Fusion ◽  
Kinase Activity ◽  
Expression System ◽  
Sh2 Domain ◽  
Full Length ◽  
Transactivation Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Sh2 Domains ◽  
Amino Terminal ◽  
Phosphatidylinositol 3

Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110.

scholarly journals Mutational activation of c-raf-1 and definition of the minimal transforming sequence.

1990 ◽  
Vol 10 (6) ◽  
pp. 2503-2512 ◽  
Author(s):  
G Heidecker ◽  
M Huleihel ◽  
J L Cleveland ◽  
W Kolch ◽  
T W Beck ◽  
...  
Keyword(s):  
Structural Features ◽  
Kinase Domain ◽  
Full Length ◽  
Wild Type ◽  
Amino Terminal ◽  
Raf Kinase ◽  
Rich Domain ◽  
Binding Residue ◽  
Transforming Activity ◽  
Mutational Activation

A series of wild-type and mutant raf genes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine- and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and CR2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.

scholarly journals Compositional Changes during the Fruit Development of Two Peach Cultivars Differing in Juice Acidity

1998 ◽  
Vol 123 (5) ◽  
pp. 770-775 ◽  
Author(s):  
Annick Moing ◽  
Laurence Svanella ◽  
Dominique Rolin ◽  
Monique Gaudillère ◽  
Jean-Pierre Gaudillère ◽  
...  
Keyword(s):  
Citric Acid ◽  
Fruit Development ◽  
Prunus Persica ◽  
Sucrose Concentration ◽  
Titratable Acidity ◽  
The Difference ◽  
Normal Acidity ◽  
Vacuolar Ph ◽  
C Nmr

Changes in metabolites were studied during the fruit development of two greenhouse grown peach [Prunus persica (L.) Batsch] cultivars with low acidity (`Jalousia') or normal acidity (`Fantasia'). Both cultivars had the same sucrose concentration in fruit mesocarp at maturity. In the fruit juice, pH was higher and titratable acidity was lower for `Jalousia' than for `Fantasia' from 80 days after bloom to maturity. At four different times during fruit development, in vivo 13C NMR spectroscopy was used to measure the vacuolar pH of fruit mesocarp. At 55 days after bloom, the vacuolar pH of fruit mesocarp was not significantly different between `Jalousia' and `Fantasia', whereas the juice pH was different between cultivars. The three major organic acids in fruit mesocarp were malic, citric, and quinic acids for both cultivars. Citric acid concentrations were similar in both cultivars until ≈85 days after bloom and then became significantly higher in `Fantasia'. A significantly higher concentration in malic acid in `Fantasia' than in `Jalousia' was observed from the end of the first growth phase to maturity. At maturity, `Fantasia' fruit had two and five times more malic and citric acid, respectively, than `Jalousia' fruit. The differences observed between `Jalousia' and `Fantasia' fruit for malic and citric acid concentrations accounted for the difference in titratable acidity. The differences in acid concentration appeared during the plateau between the two rapid growth phases of the fruit, i.e., far before the onset of maturation. The three major amino acids were asparagine, glutamic acid, and proline for both cultivars. Their concentration followed similar patterns in acid and low-acid fruit.

scholarly journals The Ubiquitin-associated Domain of hPLIC-2 Interacts with the Proteasome

2003 ◽  
Vol 14 (9) ◽  
pp. 3868-3875 ◽  
Author(s):  
Maurits F. Kleijnen ◽  
Rodolfo M. Alarcón ◽  
Peter M. Howley
Keyword(s):  
Proteasomal Degradation ◽  
Binding Domain ◽  
Full Length ◽  
Degradation Pathways ◽  
Wild Type ◽  
Amino Terminal ◽  
Uba Domain ◽  
Carboxyl Terminal ◽  
Protein Ligases

The ubiquitin-like hPLIC proteins can associate with proteasomes, and hPLIC overexpression can specifically interfere with ubiquitin-mediated proteolysis ( Kleijnen et al., 2000 ). Because the hPLIC proteins can also interact with certain E3 ubiquitin protein ligases, they may provide a link between the ubiquitination and proteasomal degradation machineries. The amino-terminal ubiquitin-like (ubl) domain is a proteasome-binding domain. Herein, we report that there is a second proteasome-binding domain in hPLIC-2: the carboxyl-terminal ubiquitin-associated (uba) domain. Coimmunoprecipitation experiments of wild-type and mutant hPLIC proteins revealed that the ubl and uba domains each contribute independently to hPLIC-2–proteasome binding. There is specificity for the interaction of the hPLIC-2 uba domain with proteasomes, because uba domains from several other proteins failed to bind proteasomes. Furthermore, the binding of uba domains to polyubiquitinated proteins does not seem to be sufficient for the proteasome binding. Finally, the uba domain is necessary for the ability of full-length hPLIC-2 to interfere with the ubiquitin-mediated proteolysis of p53. The PLIC uba domain has been reported to bind and affect the functions of proteins such as GABAAreceptor and presenilins. It is possible that the function of these proteins may be regulated or mediated through proteasomal degradation pathways.

Disruption of dynamic cell surface architecture of NIH3T3 fibroblasts by the N-terminal domains of moesin and ezrin: in vivo imaging with GFP fusion proteins

1999 ◽  
Vol 112 (1) ◽  
pp. 111-125 ◽  
Author(s):  
M.R. Amieva ◽  
P. Litman ◽  
L. Huang ◽  
E. Ichimaru ◽  
H. Furthmayr
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Full Length ◽  
Cell Behavior ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, >90% of N-moesin and >60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1–320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6–8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.

BMP-binding modules in chordin: a model for signalling regulation in the extracellular space

Development ◽  
2000 ◽  
Vol 127 (4) ◽  
pp. 821-830 ◽  
Author(s):  
J. Larrain ◽  
D. Bachiller ◽  
B. Lu ◽  
E. Agius ◽  
S. Piccolo ◽  
...  
Keyword(s):  
Dissociation Constants ◽  
Extracellular Proteins ◽  
Full Length ◽  
Dorsoventral Patterning ◽  
C Elegans ◽  
Growth Factor Signalling ◽  
Bmp Antagonist ◽  
Nanomolar Range ◽  
Shed Light ◽  
Novel Protein

A number of genetic and molecular studies have implicated Chordin in the regulation of dorsoventral patterning during gastrulation. Chordin, a BMP antagonist of 120 kDa, contains four small (about 70 amino acids each) cysteine-rich domains (CRs) of unknown function. In this study, we show that the Chordin CRs define a novel protein module for the binding and regulation of BMPs. The biological activity of Chordin resides in the CRs, especially in CR1 and CR3, which have dorsalizing activity in Xenopus embryo assays and bind BMP4 with dissociation constants in the nanomolar range. The activity of individual CRs, however, is 5- to 10-fold lower than that of full-length Chordin. These results shed light on the molecular mechanism by which Chordin/BMP complexes are regulated by the metalloprotease Xolloid, which cleaves in the vicinity of CR1 and CR3 and would release CR/BMP complexes with lower anti-BMP activity than intact Chordin. CR domains are found in other extracellular proteins such as procollagens. Full-length Xenopus procollagen IIA mRNA has dorsalizing activity in embryo microinjection assays and the CR domain is required for this activity. Similarly, a C. elegans cDNA containing five CR domains induces secondary axes in injected Xenopus embryos. These results suggest that CR modules may function in a number of extracellular proteins to regulate growth factor signalling.

Multiple elements and auto-repression regulate Rox1, a repressor of hypoxic genes in Saccharomyces cerevisiae.

Genetics ◽  
1995 ◽  
Vol 139 (3) ◽  
pp. 1149-1158 ◽  
Author(s):  
J Deckert ◽  
R Perini ◽  
B Balasubramanian ◽  
R S Zitomer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Blot Analysis ◽  
Lacz Fusion ◽  
Upstream Region ◽  
Fusion Construct ◽  
Gel Retardation ◽  
Gal1 Promoter ◽  
Rna Blot Analysis

Abstract The ROX1 gene encodes a heme-induced repressor of hypoxic genes in yeast. Using RNA blot analysis and a ROX1/lacZ fusion construct that included the ROX1 upstream region and only the first codon, we discovered that Rox1 represses its own expression. Gel-retardation experiments indicated that Rox1 was capable of binding to its own upstream region. Overexpression of Rox1 from the inducible GAL1 promoter was found to be inhibitory to cell growth. Also, we found that, as reported previously, Hap1 is partially responsible for heme-induction of ROX1, but, in addition, it also may play a role in ROX1 repression in the absence of heme. There is a second repressor of anaerobic ROX1 expression that requires the general repressor Tup1/Ssn6 for its function.

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