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PubMed2003

STAT3 is enriched in nuclear bodies.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is involved in a variety of biological functions. It is essential for the signal transduction of interleukin-6 (IL-6) and related cytokines. In response to IL-6 stimulation STAT3 becomes phosphorylated and translocates into the nucleus where it binds to enhancer sequences of target genes. We found that activated STAT3 is enriched in dot-like structures within the nucleus, which we termed STAT3 nuclear bodies. To examine the dynamics of STAT3 nuclear body formation, a fusion protein of STAT3 and yellow fluorescent protein (YFP) was constructed. Studies in living cells have shown that the appearance of STAT3 nuclear bodies is transient, correlating with the timecourse of tyrosine-phosphorylation of STAT3. Furthermore, we show by fluorescence recovery after photobleaching (FRAP) analysis that STAT3 within nuclear bodies consists of a highly mobile and an immobile fraction. Colocalization studies provided evidence that these bodies are accompanied with CREB binding protein (CBP) and acetylated histone H4, which are markers for transcriptionally active chromatin. Moreover, STAT3 nuclear bodies in HepG2 cells are not colocalized with promyelocytic leukemia oncoprotein (PML)-containing bodies; neither is a sumoylation of activated STAT3 detectable. Taken together, our data suggest that STAT3 nuclear bodies are either directly involved in active gene transcription or they serve as reservoirs of activated STAT3.

Animals↗
PubMed2003

Viviparous1 alters global gene expression patterns through regulation of abscisic acid signaling.

Maize (Zea mays) Viviparous1 (VP1) and Arabidopsis ABI3 are orthologous transcription factors that regulate key aspects of plant seed development and ABA signaling. To understand VP1-regulated gene expression on a global scale, we have performed oligomicroarray analysis of transgenic Arabidopsis carrying 35S::VP1 in an abi3 null mutant background. We have identified 353 VP1/ABA-regulated genes by GeneChip analysis. Seventy-three percent of the genes were affected by both VP1 and ABA in vegetative tissues, indicating a tight coupling between ABA signaling and VP1 function. A large number of seed-specific genes were ectopically expressed in vegetative tissue of 35S::VP1 plants consistent with evidence that VP1 and ABI3 are key determinants of seed-specific expression. ABI5, a positive regulator of ABA signaling, was activated by VP1, indicating conservation of the feed-forward pathway mediated by ABI3. ABA induction of ABI1 and ABI2, negative regulators of ABA signaling, was strongly inhibited by VP1, revealing a second pathway of feed-forward regulation. These results indicate that VP1 strongly modifies ABA signaling through feed-forward regulation of ABI1/ABI5-related genes. Of the 32 bZIP transcription factors represented on the GeneChip, genes in the ABI5 clade were specifically coregulated by ABA and VP1. Statistical analysis of 5' upstream sequences of the VP1/ABA-regulated genes identified consensus abscisic responsive elements as an enriched element, indicating that many of the genes could be direct targets of the ABI5-related bZIPs. The Sph element is an enriched sequence motif in promoters of genes co-activated by ABA and VP1 but not in promoters of genes activated by ABA alone. This analysis reveals that distinct combinatorial patterns of promoter elements distinguish subclasses of VP1/ABA coregulated genes.

Abscisic Acid↗
PubMed2002

Combinatorial selection of RNA ligands for complex cellular targets : the RNA liagands-based proteomics.

This study explores the selection of high affinity RNA ligands for the complex cellular targets present in crude HeLa nuclear extract through directed evolution and deconvolution. RNA ligands for the mixed nuclear targets were selected from around 6 x 10(14) RNA sequences through an iterated enrichment process. RNA ligands for various gene products of the extract were simultaneously selected and were shown to specifically interact with their target molecules. The target molecules were isolated from the nuclear extract by affinity chromatography using columns tagged with the RNA ligands, resolved on two-dimensional gels, and identified by mass spectrometry. These RNA ligands may be useful in characterizing novel functions of cellular proteins and modulating complex molecular events.

Base Sequence↗
PubMed2026

Integrated phytochemical and bioactivity profiling of Xanthium strumarium fruits from Korea and China: Implications for origin-specific quality specification.

BACKGROUND: Geographic origin influences the phytochemical composition and biological activities of medicinal plant resources. Xanthium strumarium L. (XS) fruit is widely used in East Asian traditional medicine. However, current pharmacopeial standards primarily recognize Chinese-derived material, despite the availability and traditional use of XS in Korea. To address this gap and support origin-informed quality specification, we compared fruits from Korea (XS-K) and China (XS-C) using chloroplast genome sequencing, targeted phytochemical profiling (high-performance liquid chromatography (HPLC) for selected phenolics and gas chromatography-flame ionization detection (GC-FID) for fatty acids and phytosterols, and multivariate chemometric analysis. RESULTS: Chloroplast genome analysis revealed high overall similarity but localized divergence around the rpoC2 locus and a greater mutation burden in XS-C, supporting origin-associated genomic differentiation. Phytochemical profiling revealed distinct origin-dependent metabolic signatures. XS-K showed higher levels of phytosterols, chlorogenic acid, 4,5-dicaffeoylquinic acid (4,5-DCQ), and xanthatin was detected only in XS-K, whereas XS-C exhibited greater abundance of total fatty acids, particularly oleic acid. Unsupervised clustering and log2 fold-change ranking confirmed clear compositional separation, and variable importance in projection (VIP) analysis identified chlorogenic acid, β-sitosterol, oleic acid, 4,5-DCQ, and xanthatin as major discriminators between origins. Bioactivity assays demonstrated that XS-K exerted stronger antioxidant effects in ABTS, DPPH and FRAP assays, stronger skin-related enzyme inhibition, and greater antibacterial activity against Staphylococcus aureus, consistent with its enriched phenolic and sterol profile. CONCLUSION: Together, chloroplast sequence variation, targeted metabolite quantification, and screening bioassays consistently distinguished XS-K from XS-C. These findings support the use of candidate markers for the origin-based authentication and quality control of XS fruit-derived ingredients. © 2026 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Fruit↗
PubMed2003

An alpha-1,3-mannosyltransferase of Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic fungus, distinguished by an elaborate polysaccharide capsule that is essential for its virulence. As part of an effort to understand the biosynthesis of this important structure, we initiated purification of an alpha-1,3-mannosyltransferase with appropriate specificity for a role in building the main capsule polysaccharide, glucuronoxylomannan. A pool of proteins that was 5,000-fold enriched in this activity included several polypeptides, which acted potentially as the catalytic protein. These were analyzed using sequence information and double-stranded RNA interference. Interference that targeted a sequence corresponding to part of a 46 kDa protein in the enriched fraction abolished the activity of interest and reduced the capsule on the affected cells. This gene was cloned and expressed in active form in Saccharomyces cerevisiae to confirm function, and was termed CMT1, for cryptococcal mannosyltransferase 1. CMT1 has no confirmed homologs in GenBank other than CAP59, a cryptococcal gene encoding a protein of unknown function that is required for capsule synthesis and virulence. The Cmt1p protein also co-purifies with a homolog of CAP64, a gene whose product has similarly been implicated in capsule synthesis and virulence. A strain disrupted in CMT1 was generated in C. neoformans; this had no effect on virulence in an animal model of cryptococcosis.

Amino Acid Sequence↗
PubMed1996

The transcriptional activator hepatocyte nuclear factor 6 regulates liver gene expression.

The hepatocyte nuclear factor 3(alpha) (HNF-3(alpha)), -3(beta), and -3(gamma) proteins share homology in the winged-helix/fork head DNA binding domain and mediate hepatocyte-enriched transcription of numerous genes whose expression is necessary for organ function. In this work, we identify a liver-enriched transcription factor, HNF-6, which recognizes the -138 to -126 region of the HNF-3(beta) promoter and binds the original HNF-3 site of the transthyretin promoter (-94 to -106). We show that HNF-6 and HNF-3 possess different DNA binding specificities by competition and methylation interference studies and are immunologically distinct. Site-directed mutagenesis of the HNF-6 sites in the HNF-3(beta) and transthyretin promoters diminishes reporter gene expression, suggesting that HNF-6 activates transcription of these promoters. Using the HNF-6 binding sequence DHWATTGAYTWWD (where W = A or T, Y = T or C, H is not G, and D is not C) determined by sequence comparison and methylation interference, we predicted that HNF-6 will bind to 22 additional hepatocyte-enriched genes. Of these potential target genes, we selected seven of the HNF-6 binding sequences and demonstrated that they bind the HNF-6 protein. These include promoter sequences from alpha-2 urinary globulin, alpha-1 antitrypsin, cytochrome P-450 2C13, L-type 6-phosphofructo-2-kinase, mouse major urinary protein, tryptophan oxygenase, and alpha-fetoprotein genes. HNF-6 binding activity was also found in the intestinal epithelial cell line HT29, and potential HNF-6 binding sites were present in intestinal sucrase isomaltase, cdx-2 homeodomain protein, and intestinal fatty acid binding protein promoter regions. These studies suggest that HNF-6 may regulate hepatocyte-specific genes and may play a role in epithelial cell differentiation of gut endoderm via regulation of HNF-3(beta).

Amino Acid Sequence↗
PubMed2005

Identification of direct serum-response factor gene targets during Me2SO-induced P19 cardiac cell differentiation.

Serum-response factor (SRF) is an obligatory transcription factor, required for the formation of vertebrate mesoderm leading to the origin of the cardiovascular system. Protein A-TEV-tagged chromatin immunoprecipitation technology was used to collect direct SRF-bound gene targets from pluripotent P19 cells, induced by Me2SO treatment into an enriched cardiac cell population. From 242 sequenced DNA fragments, we identified 188 genomic DNA fragments as potential direct SRF targets that contain CArG boxes and CArG-like boxes. Of the 92 contiguous genes that were identified, a subgroup of 43 SRF targets was then further validated by co-transfection assays with SRF. Expression patterns of representative candidate genes were compared with the LacZ reporter expression activity of the endogenous SRF gene. According to the Unigene data base, 84% of the SRF target candidates were expressed, at least, in the heart. In SRF null embryonic stem cells, 81% of these SRF target candidates were greatly affected by the absence of SRF. Among these SRF-regulated genes, Raf1, Map4k4, and Bicc1 have essential roles in mesoderm formation. The 12 regulated SRF target genes, Mapk10 (JNK3), Txnl2, Azi2, Tera, Sema3a, Lrp4, Actc1, Myl3, Hspg2, Pgm2, Hif3a, and Asb5, have been implicated in cardiovascular formation, and the Ski and Hes6 genes have roles in muscle differentiation. SRF target genes related to cell mitosis and cycle, E2f5, Npm1, Cenpb, Rbbp6, and Scyl1, expressed in the heart tissue were differentially regulated in SRF null ES cells.

Amino Acid Motifs↗
PubMed2026

Yes-Associated Protein (YAP)1 and β-Catenin Immunohistochemistry as a Surrogate Marker for GTF2I-Mutant Type A/AB Thymomas.

Thymomas are rare thymic epithelial tumors classified by the World Health Organization into type A/AB thymomas, which commonly harbor GTF2I mutations and behave indolently, and type B thymomas and thymic carcinomas, in which these mutations are less common. Type A and AB thymomas are uniquely enriched for a recurrent somatic hotspot mutation in GTF2I p. L424H; yet, this gene is rarely included in clinical sequencing panels, limiting its diagnostic utility. Yes-associated protein (YAP)1, the principal effector of the Hippo signaling pathway, and β-catenin, the central transcriptional effector of the Wnt pathway, have emerging roles in thymoma biology; however, their relationship to GTF2I mutation status and histologic subtype has not been systematically characterized. We analyzed The Cancer Genome Atlas thymoma data set and an institutional cohort of 38 thymic epithelial tumors to evaluate YAP1 and β-catenin immunohistochemistry (IHC) as surrogate markers for GTF2I mutation status and histologic classification. In The Cancer Genome Atlas data set, YAP1 and CTNNB1 mRNA expression were markedly elevated in type A/AB thymomas relative to type B and carcinoma subtypes, and GTF2I-mutant tumors exhibited significantly higher YAP1 mRNA expression than GTF2I-wildtype tumors. Targeted next-generation sequencing of our institutional cohort confirmed enrichment of the canonical GTF2I p. L424H hotspot in indolent subtypes. By IHC, both nuclear YAP1 positivity and cytoplasmic β-catenin localization were significantly more frequent in indolent thymomas. Cytoplasmic β-catenin demonstrated high specificity (94%) for indolent histology, supporting its use in diagnostically challenging cases such as type A versus type B3 distinction on small biopsies. YAP1 IHC showed a high negative predictive value for GTF2I mutations, such that a YAP1-negative result reliably excludes a GTF2I-mutant tumor. These findings implicate crosstalk between Hippo and Wnt signaling in GTF2I-mutant thymomas and position YAP1 and β-catenin IHC as accessible, cost-effective surrogates for molecular subtyping in a tumor where standard sequencing panels have limited coverage.

GTF2I↗
PubMed1989

Positive genetic selection for gene disruption in mammalian cells by homologous recombination.

Efficient modification of genes in mammalian cells by homologous recombination has not been possible because of the high frequency of nonhomologous recombination. An efficient method for targeted gene disruption has been developed. Cells with substitution of exogenous sequences into a chromosomal locus were enriched, by a factor of 100, using a positive genetic selection that specifically selects for homologous recombination at the targeted site. The selection is based on the conditional expression of a dominant selectable marker by virtue of in-frame gene fusion with the target gene. The dominant selectable marker was derived by modification of the Escherichia coli neo gene so that it retains significant activity in mammalian cells after in-frame fusion with heterologous coding sequences. In the example presented here, homologous recombinants were efficiently recovered from a pool in which the targeted gene was disrupted in 1 per 10,000 cells incorporating exogenous DNA.

Animals↗
PubMed2001

Sensitivity and performance characteristics of a direct PCR with stool samples in comparison to conventional techniques for diagnosis of Shigella and enteroinvasive Escherichia coli infection in children with acute diarrhoea in Calcutta, India.

As the sensitivity of the conventional techniques for identifying Shigella spp. and enteroinvasive Escherichia coli (EIEC) causing dysentery cases is low, a PCR assay was evaluated in this study. Analytical sensitivity (2 x 10(2) cfu) of the PCR technique was obtained by artificially spiking negative stool samples with a standard strain of S. flexneri type 2, then determining the detection limit. Specificity (100%) of the method was determined by testing a number of known Shigella and EIEC strains and organisms other than Shigella spp. A total of 300 stool samples collected from children with acute diarrhoea was plated on to two selective agar media after enrichment in Luria broth. Shigella spp. were isolated from 7.7% (23 of 300) and EIEC from 1% (3 of 300) patients. All enriched stool samples were subjected to PCR to amplify the target sequence of invasive plasmid antigen (ipa)H locus, a multicopy element found on the chromosome and invasion plasmid. The stool PCR was positive in 24 of the 26 culture-positive and in 22 culture-negative stools, thus detecting the presence of Shigella spp. or EIEC in 15.3% (46 of 300) of diarrhoea cases. When an ial probe was used for colony hybridistion with enriched stool cultures blotted on to membranes, 9.6% (29 of 300) of dysentery cases were identified as being caused by Shigella spp. or EIEC. Thus the sensitivity of enriched stool culture, colony hybridisation and enriched stool PCR was found to be 54%, 60% and 96%, respectively, when each of the methods was compared to the total microbiologically confirmed cases of dysentery. It was also observed that only 38% (48 of 126) of acute bloody dysentery cases actually had shigella or EIEC infection, as confirmed by laboratory methods. Moreover, this PCR assay could identify a number of untypable Shigella strains (Sh OUT), which would have remained undiagnosed had this assay not been used.

Acute Disease↗
PubMed1997

Gli family members are differentially expressed during the mitotic phase of spermatogenesis.

The Gli family of DNA binding proteins has been implicated in multiple neoplasias and developmental abnormalities, suggesting a primary involvement in cell development and differentiation. However, to date their specific roles and mechanisms of action remain obscure, and a drawback has been the lack of a model system in which to study their normal function. Here we demonstrate that Gli family members are differentially expressed during spermatogenesis in mice. Specifically, Gli and Gli3 mRNAs were detected in mouse germ cells, while Gli2 was not. Further, both Gli and Gli3 exhibited stage-dependent patterns of expression selectively in type A and B spermatogonia. Gli expression was somewhat higher in type B spermatogonia while the abundance of Gli3 transcripts was similar in type A and B cells. Gel-shift analyses also demonstrated the enrichment of DNA binding activity specific for the Gli target sequence in spermatogonial cells. These results indicate a selective role for Gli and Gli3 during mitotic stages of male germ cell development. Spermatogenesis may thus provide a unique opportunity to identify downstream targets and explore the normal function of Gli family proteins.

Animals↗
PubMed1994

Enrichment for RNA molecules that bind a Diels-Alder transition state analog.

RNA molecules that bind a transition state analog for a Diels-Alder reaction (Kd = 0.35 +/- 0.05 mM) were isolated from a starting pool of approximately 10(14) sequences by affinity chromatography. After the initial rise and plateau of the amount of RNA that eluted with soluble analog, a step gradient elution was used to further enrich the pool for sequences with higher affinities for the target. To our knowledge, the isolation of RNA molecules that bind either a nonplanar or a hydrophobic ligand has not been reported previously. A conserved nucleotide sequence and secondary structure present in many of the RNA molecules are necessary but not sufficient for binding the analog. No catalysts of the targeted Diels-Alder reaction were found among the binders. The absence of catalysis contrasts with previous successful experiments with antibodies and suggests that other strategies may be needed to identify oligonucleotides with diverse catalytic activities.

Base Sequence↗
PubMed1989

Sequence and structure of the yeast galactose transporter.

The previously cloned GAL2 gene of the Saccharomyces cerevisiae galactose transporter has been sequenced. The nucleotide sequence predicts a protein with 574 amino acids (Mr, 63,789). Hydropathy plots suggest that there are 12 membrane-spanning segments. The galactose transporter shows both sequence and structural homology with a superfamily of sugar transporters which includes the human HepG2-erythrocyte and fetal muscle glucose transporters, the rat brain and liver glucose transporters, the Escherichia coli xylose and arabinose permeases, and the S. cerevisiae glucose, maltose, and galactose transporters. Sequence and structural motifs at the N-terminal and C-terminal regions of the proteins support the view that the genes of this superfamily arose by duplication of a common ancestral gene. In addition to the sequence homology and the presence of the 12 membrane-spanning segments, the members of the superfamily show characteristic lengths and distributions of the charged, hydrophilic connecting loops. There is indirect evidence that the transporter is an N-glycoprotein. However, its only N-glycosylation site occurs in a charged, hydrophilic segment. This could mean that this segment is part of a hydrophilic channel in the membrane. The transporter has a substrate site for the cyclic AMP-dependent protein kinase which may be a target of catabolite inactivation. The transporter lacks a strong sequence enriched for proline (P), glutamate (E), aspartate, serine (S), and threonine (T) and flanked by basic amino acids (PEST sequence) even though it has a short half-life. Mechanisms for converting the poor PEST to a possible PEST sequence are considered. Like the other members of the superfamily, the galactose transporter lacks a signal sequence.

Amino Acid Sequence↗
PubMed1993

Targeted cloning of a subfamily of LINE-1 elements by subfamily-specific LINE-1-PCR.

Subfamily-specific LINE-1 PCR (SSL1-PCR) is the targeted amplification and cloning of defined subfamilies of LINE-1 elements and their flanking sequences. The targeting is accomplished by incorporating a subfamily-specific sequence difference at the 3' end of a LINE-1 PCR primer and pairing it with a primer to an anchor ligated within the flanking region. SSL1-PCR was demonstrated by targeting amplification of a Mus spretus-specific LINE-1 subfamily. The amplified fragments were cloned to make an SSL1-PCR library, which was found to be 100-fold enriched for the targeted elements. PCR primers were synthesized based on the sequence flanking the LINE-1 element of four different clones. Three of the clones were recovered from Mus spretus DNA. A fourth clone was recovered from a congenic mouse containing both Mus spretus and Mus domesticus DNA. Amplification between these flanking primers and LINE-1 PCR primers produced a product in Mus spretus and not in Mus domesticus. These dimorphisms were further verified to be due to insertion of Mus spretus-specific LINE-1 elements into Mus spretus DNA and not into Mus domesticus DNA.

Animals↗
PubMed1998

Use of subtractive hybridization to design habitat-based oligonucleotide probes for investigation of natural bacterial communities.

We describe a rapid oligonucleotide probe design strategy based on subtractive hybridization which yields probes for 16S rRNA or rRNA genes of individual members of microbial communities that are specific within the context of those communities. This strategy circumvents the need to sequence many similar or identical clones of dominant members of a community. Radioactively labeled subfragments of a cloned 16S rRNA gene sequence for which a probe is required (target) were hybridized with biotinylated total 16S ribosomal DNA (rDNA) amplified from the microbial community, and the hybrids formed were subsequently discarded. The remaining enriched fragments were used to screen a library consisting of cloned subfragments of the target sequence by colony hybridization in order to identify the variable regions of the 16S rRNA gene with the required specificity. The sequencing of random clones in one 16S rDNA library demonstrated that only those clones with 100% sequence identity with the probe fragment were detected by it. Moreover, sequencing of other, randomly selected, probe-positive clones revealed 100% sequence identity with the probe. Probes developed in this way tended to correspond to more variable regions of the 16S rRNA if the target sequences were similar to the sequences of other clones in the library and to less variable regions if the target sequences were phylogenetically isolated within the clone library. Although the absolute specificity of the latter probes, as assessed by comparison with available database sequences, was lower than the absolute specificity of the probes from the more variable regions, they were specific within the context of the environmental samples from which they were derived.

Bacteria↗
PubMed2006

Identification of potential pharmacological and toxicological targets differentiating structural analogs by a combination of transcriptional profiling and promoter analysis in LS-180 and Caco-2 adenocarcinoma cell lines.

Detecting and understanding the potential for off-target pharmacological effects is critical in the optimization of lead compounds in drug discovery programs. Compound-mediated activation of the pregnane X receptor (PXR; NR1I2), a key regulator for drug metabolism genes, is often monitored to avoid potential drug-drug interactions. Two structural analogs, MRL-1 and MRL-2, were determined to be equivalent PXR activators in trans-activation assays. To differentiate these two PXR activators, their transcriptional effects were examined in PXR-sufficient (LS180) and PXR-deficient (Caco-2) adenocarcinoma cell lines. Both compounds regulated drug-management genes (e.g. CYP3A4, CYP2B6, UGT1A1 and ABCB1) in LS180 cells, but not in PXR-deficient Caco-2 cells. The potency of MRL-1 and MRL-2 on PXR activation was again equivalent as revealed by a set of 113 genes that were regulated by four prototypical PXR agonists (rifampicin, ritonavir, troglitazone and dexamethasone) in the LS180 cells. The specificity of the PXR signature genes was supported by the enrichment of putative PXR binding sites uncovered by sequence-based promoter analyses. Interestingly, an additional off-target activity of MRL-2 was suggested where sterol response element binding protein binding sites were found enriched in a subset of PXR signature genes. These genes, involved in cholesterol and fatty acid synthesis, were significantly regulated by ritonavir, chlorpromazine and MRL-2, which were linked to the manifestation of phospholipidosis. The present study demonstrates the utility of our approach in the differentiation and selection of lead compounds for drug development.

Benzopyrans↗
PubMed1997

Nramp1 locus encodes a 65 kDa interferon-gamma-inducible protein in murine macrophages.

The murine Nramp1 (natural-resistance-associated macrophage protein) locus, formerly known as Ity/Lsh/Bcg, was isolated previously on the basis of chromosomal location, and as conferring natural resistance to infection against intracellular macrophage pathogens. The gene encodes a transporter molecule of unknown function. We have prepared polyclonal antisera against the C-terminal 35 amino acids of murine Nramp1. This serum is reactive towards a 65 kDa protein, expressed in murine macrophage cells from resistant or susceptible mice stimulated with interferon-gamma and lipopolysaccharide, but not in non-macrophage cells. Evidence indicates that Nramp1 is localized in a subcellular membrane rather than at the cell surface. This evidence includes: the identification of conserved endocytic targeting motifs following inspection of human and murine Nramp sequences; the enrichment of Nramp1, following magnetic selection of phagolysosomal vesicles from activated macrophages that were allowed to phagocytose magnetic, IgG-coated beads; confocal microscopy. These studies place Nramp1 on a membrane in close proximity to obligate intracellular pathogens. A link between Nramp1 and divalent-cation transport is suggested by sequence similarity with yeast SMF1. Evidence showing modulation of Nramp1 protein levels by iron chelation provides a direct link with Nramp1 function and divalent-cation metabolism.

Amino Acid Sequence↗
PubMed2002

Transgenic barley grain overexpressing thioredoxin shows evidence that the starchy endosperm communicates with the embryo and the aleurone.

Homozygous lines of barley overexpressing a wheat thioredoxin h transgene (up to 30-fold) were generated earlier by using a B(1)-hordein promoter with a signal peptide sequence for targeting to the protein body and found to be enriched in starch debranching enzyme (pullulanase). Here, we describe the effect of biochemically active, overexpressed thioredoxin h on germination and the onset of alpha-amylase activity. Relative to null segregant controls lacking the transgene, homozygotes overexpressing thioredoxin h effected (i) an acceleration in the rate of germination and appearance of alpha-amylase activity with a 1.6- to 2.8-fold increase in gibberellin A(1) (GA(1)) content; (ii) a similar acceleration in the appearance of the alpha-amylase activity in deembryonated transgenic grain incubated with gibberellic acid; (iii) a 35% increase in the ratio of relative reduction (abundance of SH) of the propanol soluble proteins (hordein I fraction); and (iv) an increase in extractable and soluble protein of 5-12% and 11-35%, respectively. Thioredoxin h, which was highly reduced in the dry grain, was degraded in both the null segregant and homozygote after imbibition. The increase in alpha-amylase activity and protein reduction status was accompanied by a shift in the distribution of protein from the insoluble to the soluble fraction. The results provide evidence that thioredoxin h of the starchy endosperm communicates with adjoining tissues, thereby regulating their activities, notably by accelerating germination of the embryo and the appearance of alpha-amylase released by the aleurone.

Desiccation↗