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PubMed2003

Pleiotropic effects of suppressing deoxyhypusine synthase expression in Arabidopsis thaliana.

A full-length cDNA clone encoding deoxyhypusine synthase (DHS) was isolated from a cDNA expression library prepared from senescing leaves of Arabidopsis thaliana. Southern blot analysis indicated that DHS is encoded by a single-copy gene in Arabidopsis. During leaf development, the abundance of DHS mRNA in the third pair of rosette leaves peaked at days 14 and 35 after emergence coincident with the initiation of bolting and the later stages of leaf senescence, respectively. These changes in DHS expression were paralleled by corresponding changes in transcript abundance for eIF-5A1, one of three isoforms of eIF-5A in Arabidopsis. Levels of DHS transcript also increased in detached leaves coincident with post-harvest senescence. DHS was suppressed in transgenic plants by introducing antisense full-length or 3'-untranslated Arabidopsis DHS cDNA under the regulation of the constitutive cauliflower mosaic virus (CaMV-35S) promoter. Plants expressing the antisense transgenes had reduced levels of leaf DHS protein and, depending on the level of DHS suppression, exhibited delayed natural leaf senescence, delayed bolting, increased rosette leaf and root biomass, and enhanced seed yield. Suppression of DHS also delayed premature leaf senescence induced by drought stress resulting in enhanced survival in comparison with wild-type plants. In addition, detached leaves from DHS-suppressed plants exhibited delayed post-harvest senescence. These pleiotropic effects of DHS suppression indicate that the protein plays a central role in plant development and senescence.

Adaptation, Physiological↗
PubMed1996

Identification of a novel protein containing two C2 domains selectively expressed in the rat brain and kidney.

We have isolated and characterized a rat brain cDNA clone which encodes a new protein of 474 amino acids in length which contains two C2 domains structurally homologous to those present in synaptotagmins. The overall amino acid identity in C2 domains between this protein and the synaptotagmins is 36-44%. This protein also contains 3 putative consensus sequences for phosphorylation by cAMP-dependent protein kinase. RNA blot hybridization revealed a 3.0 kb transcript abundantly expressed only in the rat brain and the kidney. Thus, we called this brain/kidney protein (B/K). In situ hybridization and Northern blot analyses showed that the B/K transcript was found in forebrain including the olfactory bulb, cerebral cortex, hippocampus, and hypothalamus. In the kidney, high levels of B/K transcript were expressed in the papillary region of the inner medulla, the inner stripe of the outer medulla and the cortex. The selective expression in forebrain and kidney suggests that B/K may be involved in similar cAMP-dependent processes at these very different sites.

Amino Acid Sequence↗
PubMed1999

Characterization of closely related delta-TIP genes encoding aquaporins which are differentially expressed in sunflower roots upon water deprivation through exposure to air.

We isolated five sunflower (Helianthus annuus) cDNAs belonging to the TIP (tonoplast intrinsic protein) family. SunRb7 and Sun gammaTIP (partial sequence) are homologous to tobacco TobRb7 and Arabidopsis gamma-TIP, respectively. SunTIP7, 18 and 20 (SunTIPs) are closely related and homologous to Arabidopsis delta-TIP (SunTIP7 and 20 have already been presented in Sarda et al., Plant J. 12 (1997) 1103-1111). As was previously shown for SunTIP7 and 20, expression of SunTIP18 and SunRb7 in Xenopus oocytes caused an increase in osmotic water permeability demonstrating that they are aquaporins. In roots, in situ hybridization revealed that SunTIP7 and 18 mRNAs accumulate in phloem tissues. The expression of TIP-like genes was studied in roots during 24 h water deprivation through exposure to air. During the course of the treatment, each SunTIP gene displayed an individual response: SunTIP7 transcript abundance increased, SunTIP18 decreased whereas that of SunTIP20 was transitorily enhanced. By contrast, SunRb7 and Sun gammaTIP mRNA levels did not fluctuate. Due to the changes in their transcript levels, it is proposed that SUNTIP aquaporins encoded by delta-TIP-like genes play a role in the sunflower response to drought.

Amino Acid Sequence↗
PubMed2003

Quantitative expression analysis of a Glyptapanteles indiensis polydnavirus protein tyrosine phosphatase gene in its natural lepidopteran host, Lymantria dispar.

In the present study, expression of a newly identified Glyptapanteles indiensis polydnavirus (GiPDV) gene encoding a putative protein tyrosine phosphatase (PDVPTP) was monitored in vivo in the parasitized host, L. dispar, using one step RT-PCR. Expression levels of the PDVPTP transcript were also evaluated in various host tissues at different times post parasitization (pp) using RT quantitative competitive PCR (RT-qcPCR). Expression levels varied, with the most abundant transcript detected in host haemolymph 2 h pp. The high expression level in host haemolymph at an early stage of parasitization suggested a potential role for viral PDVPTP in disruption of the host immune system and protection of the endoparasitoid egg from encapsulation. Additionally, the PDVPTP gene or its homolog(s) mapped to more than one GiPDV genomic DNA segment, which may account for its increased level of expression in the absence of virus replication.

Animals↗
PubMed2005

Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets.

The polyubiquitin receptor Rpn10 targets ubiquitylated Sic1 to the 26S proteasome for degradation. In contrast, turnover of at least one ubiquitin-proteasome system (UPS) substrate, CPY*, is impervious to deletion of RPN10. To distinguish whether RPN10 is involved in the turnover of only a small set of cell cycle regulators that includes Sic1 or plays a more general role in the UPS, we sought to develop a general method that would allow us to survey the spectrum of ubiquitylated proteins that selectively accumulate in rpn10Delta cells. Polyubiquitin conjugates from yeast cells that express hexahistidine-tagged ubiquitin (H6-ubiquitin) were first enriched on a polyubiquitin binding protein affinity resin. This material was then denatured and subjected to IMAC to retrieve H6-ubiquitin and proteins to which it may be covalently linked. Using this approach, we identified 127 proteins that are candidate substrates for the 26S proteasome. We then sequenced ubiquitin conjugates from cells lacking Rpn10 (rpn10Delta) and identified 54 proteins that were uniquely recovered from rpn10Delta cells. These include two known targets of the UPS, the cell cycle regulator Sic1 and the transcriptional activator Gcn4. Our approach of comparing the ubiquitin conjugate proteome in wild-type and mutant cells has the resolving power to identify even an extremely in abundant transcriptional regulatory protein and should be generally applicable to mapping enzyme substrate networks in the UPS.

Carrier Proteins↗
PubMed2003

Effect of oxidized low-density lipoprotein on differential gene expression in primary human endothelial cells.

Oxidative modification of low-density lipoprotein (LDL) plays an important role in the initiation and progression of atherosclerosis. It has been proposed that the biological action of oxidized LDL (ox-LDL) may be partially attributed to its effect on a shift of the pattern of gene expression in endothelial cells. To examine the transcriptional response to ox-LDL, we applied cDNA array technology to cultured primary human endothelial cells challenged with oxidized human LDL. A twofold or greater difference in the expression of a particular gene was considered a significant difference in transcript abundance. Seventy-eight of the 588 genes analyzed were differentially expressed in response to the treatment. Ox-LDL significantly affected the expression of genes encoding for transcription factors, cell receptors, growth factors, adhesion molecules, extracellular matrix proteins, and enzymes involved in cholesterol metabolism. The alteration of the expression pattern of several genes was substantiated post hoc using RT-PCR. The experimental strategy identified several novel ox-LDL-sensitive genes associated with a "response to injury" providing a conceptual background to be utilized for future studies addressing the molecular basis of the early stages of atherogenesis.

Cell Adhesion↗
PubMed2006

Moderate increase of mean daily temperature adversely affects fruit set of Lycopersicon esculentum by disrupting specific physiological processes in male reproductive development.

BACKGROUND AND AIMS: Global warming is gaining significance as a threat to natural and managed ecosystems since temperature is one of the major environmental factors affecting plant productivity. Hence, the effects of moderate temperature increase on the growth and development of the tomato plant (Lycopersicon esculentum) were investigated. METHODS: Plants were grown at 32/26 degrees C as a moderately elevated temperature stress (METS) treatment or at 28/22 degrees C (day/night temperatures) as a control with natural light conditions. Vegetative growth and reproductive development as well as sugar content and metabolism, proline content and translocation in the androecium were investigated. KEY RESULTS: METS did not cause a significant change in biomass, the number of flowers, or the number of pollen grains produced, but there was a significant decrease in the number of fruit set, pollen viability and the number of pollen grains released. Glucose and fructose contents in the androecium (i.e. all stamens from one flower) were generally higher in the control than METS, but sucrose was higher in METS. Coincidently, the mRNA transcript abundance of acid invertase in the androecium was decreased by METS. Proline contents in the androecium were almost the same in the control and METS, while the mRNA transcript level of proline transporter 1, which expresses specifically at the surface of microspores, was significantly decreased by METS. CONCLUSIONS: The research indicated that failure of tomato fruit set under a moderately increased temperature above optimal is due to the disruption of sugar metabolism and proline translocation during the narrow window of male reproductive development.

Amino Acid Transport Systems, Neutral↗
PubMed1991

Identification of a novel murine IAP-promoted placenta-expressed gene.

We have cloned and characterized a novel cellular gene that is promoted by an intracisternal A-particle (IAP) LTR and expressed in the mouse placenta (mouse IAP promoted placental gene, MIPP). A 1067bp cDNA clone containing an IAP LTR U5 region duplicated in its 5' terminus and an ORF coding for a potential 202 amino acids protein was isolated from an 8.5 day old mouse embryo cDNA library. Sequence analysis of the 5' region of a genomic clone revealed the presence of a solo IAP LTR with the same U5 duplication, and primer extension analysis confirmed that transcription of the MIPP gene is under the control of the IAP LTR. Expression of the MIPP gene parallels that of IAP genes in normal mouse tissues with abundant transcripts present in the placenta and also in the myeloma MOPC-315. The MIPP-encoded protein is composed of four 48-amino acid repeat units and shares homology with a vaccinia virus gene product. MIPP-related sequences were also detected in higher eukaryotic genomes including human.

Amino Acid Sequence↗
PubMed2006

High-resolution dynamics of the transcriptional response to nutrition in Drosophila: a key role for dFOXO.

A high-resolution time series of transcript abundance was generated to describe global expression dynamics in response to nutrition in Drosophila. Nonparametric change-point statistics revealed that within 7 h of feeding upon yeast, transcript levels changed significantly for approximately 3,500 genes or 20% of the Drosophila genome. Differences as small as 15% were highly significant, and 80% of the changes were <1.5-fold. Notably, transcript changes reflected rapid downregulation of the nutrient-sensing insulin and target of rapamycin pathways, shifting of fuel metabolism from lipid to glucose oxidation, and increased purine synthesis, TCA-biosynthetic functions and mitochondria biogenesis. To investigate how nutrition coordinates these transcriptional changes, feeding-induced expression changes were compared with those induced by the insulin-regulated transcription factor dFOXO in Drosophila S2 cells. Remarkably, 28% (995) of the nutrient-responsive genes were regulated by activated dFOXO, including genes of mitochondrial biogenesis and a novel homolog of mammalian peroxisome proliferator-gamma coactivator-1 (PGC-1), a transcriptional coactivator implicated in controlling mitochondrial gene expression in mammals. These data implicate dFOXO as a major coordinator of the transcriptional response to nutrients downstream of insulin and suggest that mitochondria biogenesis is linked to insulin signaling via dFOXO-mediated repression of a PGC-1 homolog.

Animal Nutritional Physiological Phenomena↗
PubMed2004

Serial Analysis of Gene Expression: Applications in Human Studies.

Serial analysis of gene expression (SAGE) is a powerful tool, which provides quantitative and comprehensive expression profile of genes in a given cell population. It works by isolating short fragments of genetic information from the expressed genes that are present in the cell being studied. These short sequences, called SAGE tags, are linked together for efficient sequencing. The frequency of each SAGE tag in the cloned multimers directly reflects the transcript abundance. Therefore, SAGE results in an accurate picture of gene expression at both the qualitative and the quantitative levels. It does not require a hybridization probe for each transcript and allows new genes to be discovered. This technique has been applied widely in human studies and various SAGE tags/SAGE libraries have been generated from different cells/tissues such as dendritic cells, lung fibroblast cells, oocytes, thyroid tissue, B-cell lymphoma, cultured keratinocytes, muscles, brain tissues, sciatic nerve, cultured Schwann cells, cord blood-derived mast cells, retina, macula, retinal pigment epithelial cells, skin cells, and so forth. In this review we present the updated information on the applications of SAGE technology mainly to human studies.

Journal Article↗
PubMed1988

Cloning of the RNA8 gene of Saccharomyces cerevisiae, detection of the RNA8 protein, and demonstration that it is essential for nuclear pre-mRNA splicing.

Strains of Saccharomyces cerevisiae that bear the temperature-sensitive mutation rna8-1 are defective in nuclear pre-mRNA splicing at the restrictive temperature (36 degrees C), suggesting that the RNA8 gene encodes a component of the splicing machinery. The RNA8 gene was cloned by complementation of the temperature-sensitive growth defect of an rna8-1 mutant strain. Integrative transformation and gene disruption experiments confirmed the identity of the cloned DNA and demonstrated that the RNA8 gene encodes an essential function. The RNA8 gene was shown to be represented once per S. cerevisiae haploid genome and to encode a low-abundance transcript of approximately 7.4 kilobases. By using antisera raised against beta-galactosidase-RNA8 fusion proteins, the RNA8 gene product was identified in S. cerevisiae cell extracts as a low-abundance protein of approximately 260 kilodaltons. Immunodepletion of the RNA8 protein specifically abolished the activity of S. cerevisiae in vitro splicing extracts, confirming that RNA8 plays an essential role in splicing.

Animals↗
PubMed2005

Reliability and reproducibility issues in DNA microarray measurements.

DNA microarrays enable researchers to monitor the expression of thousands of genes simultaneously. However, the current technology has several limitations. Here we discuss problems related to the sensitivity, accuracy, specificity and reproducibility of microarray results. The existing data suggest that for relatively abundant transcripts the existence and direction (but not the magnitude) of expression changes can be reliably detected. However, accurate measurements of absolute expression levels and the reliable detection of low abundance genes are difficult to achieve. The main problems seem to be the sub-optimal design or choice of probes and some incorrect probe annotations. Well-designed data-analysis approaches can rectify some of these problems.

Animals↗
PubMed2002

Transcriptional analysis of genes encoding enzymes of the folate pathway in the human malaria parasite Plasmodium falciparum.

Folate metabolism in Plasmodium falciparum is essential for cell growth and replication, and the target of important antimalarial agents. The pathway comprises a series of enzymes that convert GTP to derivatives of tetrahydrofolate, which are cofactors in one-carbon transfer reactions. We investigated the expression of five of the genes encoding these enzymes by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using a threshold detection technique. We followed changes in mRNA levels as parasites progress through the erythrocytic cell cycle and examined this process in two cloned lines of diverse origins, as well as under stress conditions, induced by either removal of important metabolites or challenge by folate enzyme inhibitors. Although conventionally regarded as performing housekeeping functions, these genes show disparate levels of and changes in expression through the cell cycle, but respond quite uniformly to folate pathway-specific stress factors, with no evidence of feedback at the transcriptional level. Overall, the two genes involved in the thymidylate cycle (encoding dihy-drofolate reductase-thymidylate synthase, dhfr-ts, and serine hydroxymethyltransferase, shmt) gave the most abundant transcripts. However, only the latter showed major variation across the cell cycle, with a peak around the time of onset of DNA replication, possibly indicative of a regulatory function.

Animals↗
PubMed1996

Suppression of stellate cell type I collagen gene expression involves AP-2 transmodulation of nuclear factor-1-dependent gene transcription.

The regulation of collagen gene expression was studied in culture-activated rat hepatic stellate cells, the fibrogenic effector cell involved in hepatic fibrogenesis. Treatment of cells with a 5-lipoxygenase-specific inhibitor caused a reduction in alphaI(I) collagen mRNA transcript abundance, which suggested that leukotriene production was involved in maintaining the activated cell's high level of collagen mRNA production. The underlying mechanism involved a decrease in collagen gene transcription. Suppression of gene transcription was localized to an nuclear factor-1 (NF-1) binding domain in the proximal promoter and an AP-2 binding domain adjacent to it. Gel retardation assays demonstrated that an increase in AP-2 binding adjacent to the NF-1 site was likely to be the transmodulator responsible for the suppression of the NF-1-dependent gene expression. The data suggest that post-translational alterations in AP-2 activity are responsible for this unappreciated mechanism of regulating the collagen gene.

Animals↗
PubMed1988

Isolation, sequence analysis, and transcriptional studies of the flavodoxin gene from Anacystis nidulans R2.

The nonheme, iron-sulfur protein ferredoxin is the terminal constituent of the photosynthetic electron transport chain. Under conditions of iron stress, many cyanobacteria and eucaryotic algae replace ferredoxin with the flavoprotein flavodoxin. The gene for flavodoxin was cloned from the cyanobacterium Anacystis nidulans R2 by using three mixed oligonucleotide probes derived from the partial Synechococcus sp. strain PCC 6301 amino acid sequence. Nucleotide sequence analysis revealed a 513-base-pair open reading frame with a deduced amino acid sequence having homology to other long-chain flavodoxins. Assuming proteolytic cleavage of the initial methionine residue, the molecular weight of the A. nidulans R2 flavodoxin is 18,609. Southern blot hybridization under conditions of reduced stringency detected only one copy of the flavodoxin sequence in the A. nidulans R2 genome. Northern (RNA) blot hybridization analyses by using cloned flavodoxin gene probes indicated that no transcripts are detectable under conditions of iron saturation. However, under iron-deficient growth conditions the flavodoxin gene appeared to be transcribed as part of a larger operon. The operon yielded at least three transcripts. The first was of approximately 1,100 bases (designated RNA 1) and terminated immediately upstream from the 5' end of the flavodoxin open reading frame. A second, less abundant transcript of approximately 1,900 bases (designated RNA 2) encoded all of RNA 1 as well as the flavodoxin polypeptide. Analysis indicated that both transcripts initiate in close proximity to each other. A third, minor transcript of approximately 1,100 bases (designated RNA 3) was detectable downstream of the flavodoxin gene sequence. Addition of iron-stressed A. nidulans R2 cells resulted in almost total loss of detectable mRNA transcripts within 60 min of the addition. The ferredoxin gene transcript has previously been characterized as a monocistronic message of approximately 430 bases (M. E. Reith, D. E. Laudenbach, and N. A. Straus, J. Bacteriol. 168: 1319-1324, 1986). Here we show that the ferredoxin message is detectable under all iron regimes tested is quantitatively unaffected by decreases in iron availability to the cells.

Amino Acid Sequence↗
PubMed2006

Serial analysis of gene expression in eukaryotic pathogens.

The tag-based method of serial analysis of gene expression (SAGE) has been used to measure mRNA abundance and differential expression in a variety of organisms including several parasites and fungal pathogens. SAGE is based on the collection of short sequence tags as a measure of transcript abundance and the method provides an alternative, and in some instances, complementary approach to array-based methods of measuring differential gene expression. These methods are being used to improve our molecular understanding of the pathogenesis of eukaryotic microbes and SAGE in particular presents valuable opportunities for gene discovery and genome annotation. For eukaryotic pathogens, the SAGE method has been employed for the parasites Plasmodium falciparum, Toxoplasma gondii and Giardia lamblia, as well as fungal pathogens of plants (Magnaporthe grisea, Blumeria graminis, Ustilago maydis) and humans (Cryptococcus neoformans, Coccidiodes posadasii, Trichophyton rubrum). The accumulating information promises to speed the identification of key pathogen functions for virulence and proliferation in the host with the hope that some of these will represent important targets for drug and vaccine development.

Animals↗
PubMed2002

Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants.

PURPOSE: The retinal pigment epithelium (RPE) and choroid comprise a functional unit of the eye that is essential to normal retinal health and function. Here we describe expressed sequence tag (EST) analysis of human RPE/choroid as part of a project for ocular bioinformatics. METHODS: A cDNA library (cs) was made from human RPE/choroid and sequenced. Data were analyzed and assembled using the program GRIST (GRouping and Identification of Sequence Tags). Complete sequencing, Northern and Western blots, RH mapping, peptide antibody synthesis and immunofluorescence (IF) have been used to examine expression patterns and genome location for selected transcripts and proteins. RESULTS: Ten thousand individual sequence reads yield over 6300 unique gene clusters of which almost half have no matches with named genes. One of the most abundant transcripts is from a gene (named "alpha") that maps to the BBS1 region of chromosome 11. A number of tissue preferred transcripts are common to both RPE/choroid and iris. These include oculoglycan/opticin, for which an alternative splice form is detected in RPE/choroid, and "oculospanin" (Ocsp), a novel tetraspanin that maps to chromosome 17q. Antiserum to Ocsp detects expression in RPE, iris, ciliary body, and retinal ganglion cells by IF. A newly identified gene for a zinc-finger protein (TIRC) maps to 19q13.4. Variant transcripts of several genes were also detected. Most notably, the predominant form of Bestrophin represented in cs contains a longer open reading frame as a result of splice junction skipping. CONCLUSIONS: The unamplified cs library gives a view of the transcriptional repertoire of the adult RPE/choroid. A large number of potentially novel genes and splice forms and candidates for genetic diseases are revealed. Clones from this collection are being included in a large, nonredundant set for cDNA microarray construction.

Aged↗
PubMed2002

The Kaposi's sarcoma-associated herpesvirus K12 transcript from a primary effusion lymphoma contains complex repeat elements, is spliced, and initiates from a novel promoter.

Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects KS tumors, primary effusion lymphomas (PELs), and PEL cell lines. K12 (T0.7) is the most abundant transcript expressed in latent KSHV infection. We characterize here the K12 transcript from a PEL tumor prior to passage in cell culture. The PEL tumor KSHV K12 transcript contained additional complex nucleotide repeat elements compared to the previously described K12 message of the BCBL-1 PEL cell line. The PEL tumor lacked kaposin B, the predominant BCBL-1 K12 protein product, but encoded kaposins A and C. The K12 transcript was spliced and the splicing event occurred in all KSHV isolates tested. The 5' end of the K12 transcript was mapped by 5' RACE (rapid amplification of cDNA ends) and S1 nuclease protection assays and was at the site of an active promoter. This work demonstrates that the K12 transcript contains variable, complex repeat elements, is spliced and is expressed from a novel KSHV promoter.

Amino Acid Sequence↗