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PubMed2000

Expression profiles of 22 novel molecular markers for organogenetic pathways acting in alfalfa nodule development.

During symbiotic nodule development, a variety of molecular signals of rhizobia and plant origin are likely to be involved in the control of the expression of specific genes in the legume Medicago sativa (alfalfa). Twenty-two new, nodule-associated Expressed Sequence Tags (ESTs, MsNod clones) as well as 16 clones for previously reported alfalfa nodulins were identified by cold-plaque screening. Protein homologs were found for 10 of the 22 MsNod-encoded polypeptides, revealing putative novel functions associated with this symbiosis. Expression of these MsNod genes was investigated in spontaneous nodules (generated in the absence of bacteria), in nodules induced by a Sinorhizobium meliloti wild-type strain and Eps- and Bac- mutant derivatives, as well as in roots inoculated with a Nod- mutant strain. This analysis enabled us to correlate plant gene expression with the different stages of nodule ontogeny and invasion. The effect of phytohormones on MsNod gene expression was analyzed in cytokinin- and auxin-treated alfalfa roots. Cytokinin induced the accumulation of seven MsNod transcripts, four of them were also regulated by the synthetic auxin 2,4-D (2,4-dichlorophenoxyacetic acid). Comparison of MsNod expression profiles in wild-type and transgenic M. truncatula roots overexpressing the early nodulin Enod40 suggested that one clone, the M. sativa L3 ribosomal protein homolog (MsNod377), is a putative component of an Enod40-dependent pathway acting during nodule development. These novel molecular markers may help in the investigation of gene networks and regulatory circuits controlling nodule organogenesis.

Cytokinins↗

PubMed1993

Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins.

The gene that encodes the acyl carrier protein (ACP) of the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2) was replaced with homologs from the granaticin, oxytetracycline, tetracenomycin, and putative frenolicin polyketide synthase gene clusters. All of the replacements led to expression of functional synthases, and the recombinants synthesized aromatic polyketides similar in chromatographic properties to actinorhodin or to shunt products produced by mutants defective in the actinorhodin pathway. Some regions within the ACP were also shown to be interchangeable and allow production of a functional hybrid ACP. Structural analysis of the most abundant polyketide product of one of the recombinants by electrospray mass spectrometry suggested that it is identical to mutactin, a previously characterized shunt product of an actVII mutant (deficient in cyclase and dehydrase activities). Quantitative differences in the product profiles of strains that express the various hybrid synthases were observed. These can be explained, at least in part, by differences in ribosome-binding sites upstream of each ACP gene, implying either that the ACP concentration in some strains is rate limiting to overall PKS activity or that the level of ACP expression also influences the expression of another enzyme(s) encoded by a downstream gene(s) in the same operon as the actinorhodin ACP gene. These results reaffirm the idea that construction of hybrid polyketide synthases will be a useful approach for dissecting the molecular basis of the specificity of PKS-catalyzed reactions. However, they also point to the need for reducing the chemical complexity of the approach by minimizing the diversity of polyketide products synthesized in strains that produce recombinant polyketide synthases.

Acyl Carrier Protein↗

PubMed1996

Functional characterization of a non-AUUUA AU-rich element from the c-jun proto-oncogene mRNA: evidence for a novel class of AU-rich elements.

AU-rich RNA-destabilizing elements (AREs) found in the 3' untranslated regions of many labile mRNAs encoding proto-oncoproteins and cytokines generally contain (i) one or more copies of the AUUUA pentanucleotide and (ii) a high content of uridylate and sometimes also adenylate residues. Recently, we have identified a potent ARE from the 3' untranslated region of c-jun proto-oncogene mRNA that does not contain the AUUUA motif. In an attempt to further our understanding of the general principles underlying mechanisms by which AREs direct rapid and selective mRNA degradation, in this study we have characterized the functionally important structural features and properties of this non-AUUUA ARE. Like AUUUA-containing AREs, this non-AUUUA ARE directs rapid shortening of the poly(A) tail as a necessary first step for mRNA degradation. It can be further dissected into three structurally and functionally distinct regions, designated domains I, II, and III. None of three domains alone is able to significantly destabilize the stable beta-globin mRNA. The two unlinked domains, I and III, together are necessary and sufficient for specifying the full destabilizing function of this non-AUUUA ARE. Domain II appears functionally dispensable but can partially substitute for domain I. Domain swaps made between the c-jun non-AUUUA and the c-fos AUUUA-containing AREs reveal that the two AREs, while sharing no sequence homology, appear to contain sequence domains that are structurally distinct but functionally overlapping and exchangeable. These data support the idea that the ultimate destabilizing function of an individual ARE is determined by its own unique combination of structurally distinct and functionally interdependent domains. Our polysome profile studies show tha the destabilizing function of the c-jun non-AUUUA ARE does not require any active transit by ribosomes of the mRNA bearing it, further corroborating that the destabilizing function of AREs is not tightly coupled to ongoing translation by ribosomes. Moreover, unlike AUUUA-containing AREs, the c-jun ARE is insensitive to blockage of its effects by addition of transcription inhibitors. Thus, our data provide further evidence for the existence of a novel class of ARE with unique properties.

3T3 Cells↗

PubMed2005

Characterization and phylogenetic relationships among microsporidia infecting silkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis.

This study is the first report on the genetic characterization and relationships among different microsporidia infecting the silkworm, Bombyx mori, using inter simple sequence repeat PCR (ISSR-PCR) analysis. Six different microsporidians were distinguished through molecular DNA typing using ISSR-PCR. Thus, ISSR-PCR analysis can be a powerful tool to detect polymorphisms and identify microsporidians, which are difficult to study with microscopy because of their extremely small size. Of the 100 ISSR primers tested, only 28 primers had reproducibility and high polymorphism (93%). A total of 24 ISSR primers produced 55 unique genetic markers, which could be used to differentiate the microsporidians from each other. Among the 28 SSRs tested, the most abundant were (CA)n, (GA)n, and (GT)n repeats. The degree of band sharing was used to evaluate genetic similarity between different microsporidian isolates and to construct a phylogenetic tree using Jaccard's similarity coefficient. The results indicate that the DNA profiles based on ISSR markers can be used as diagnostic tools to identify different microsporidia with considerable accuracy. In addition, the small subunit ribosomal RNA (SSU-rRNA) sequence gene was amplified, cloned, and sequenced from each of the 6 microsporidian isolates. These sequences were compared with 20 other microsporidian SSU-rRNA sequences to develop a phylogenetic tree for the microsporidia isolated from the silkworms. This method was found to be useful in establishing the phylogenetic relationships among the different microsporidians isolated from silkworms. Of the 6 microsporidian isolates, NIK-1s revealed an SSU-rRNA gene sequence similar to Nosema bombycis, indicating that NIK-1s is similar to N. bombycis; the remaining 5 isolates, which differed from each other and from N. bombycis, were considered to be different variants belonging to the species N. bombycis.

Animals↗

PubMed1978

The tubular system of human endometrium.

The so-called "intranuclear" tubular system (TS) of human epithelial endometrial cells has been investigated in 50 specimens and has been found in some instances of secretory endometrium and in one case of hyperplastic endometrium with secretory change. The TS was found both in nucleus and cytoplasm. The intranuclear TS can be associated with the nucleolus, connected to the nuclear envelope or lying free in the karyoplasm. When it is connected with the nuclear envelope an intimately related cytoplasmic invagination containing many ribosomes is frequently found. The cytoplasmic TS is smaller than that found in the nucleus and is usually connected with rough endoplasmic reticulum profiles. These findings allow us to make the suggestion that the TS could have a cytoplasmic origin and subsequently migrate into the nucleus. The function of the TS is unknown.

Cell Movement↗

PubMed2006

Systems analyses reveal two chaperone networks with distinct functions in eukaryotic cells.

Molecular chaperones assist the folding of newly translated and stress-denatured proteins. In prokaryotes, overlapping sets of chaperones mediate both processes. In contrast, we find that eukaryotes evolved distinct chaperone networks to carry out these functions. Genomic and functional analyses indicate that in addition to stress-inducible chaperones that protect the cellular proteome from stress, eukaryotes contain a stress-repressed chaperone network that is dedicated to protein biogenesis. These stress-repressed chaperones are transcriptionally, functionally, and physically linked to the translational apparatus and associate with nascent polypeptides emerging from the ribosome. Consistent with a function in de novo protein folding, impairment of the translation-linked chaperone network renders cells sensitive to misfolding in the context of protein synthesis but not in the context of environmental stress. The emergence of a translation-linked chaperone network likely underlies the elaborate cotranslational folding process necessary for the evolution of larger multidomain proteins characteristic of eukaryotic cells.

Adenosine Triphosphatases↗

PubMed1985

Ultrastructure of the secretory compartment of the Rana catesbeiana exocrine pancreas.

In the Rana catesbeiana exocrine pancreas 2 types of serozimogenic cells could be observed. They were called light and dark cells. The light cells were observed in a greater number and presented the GER cisterns predominantly located in the basal region of the cell and the vesicular forms of the GER mainly in the supranuclear region. The Golgi complex was well developed and the secretory granules were grouped in the cell apex. The mitochondrion were distributed by the cytoplasm and the nucleous appeared in the basal region of the cell and presented a pale and vesiculous profile that made the nucleolus evident. The dark cells were observed in smaller quantity and in this cell occurred a predominance of GER vesicles. The cytoplasmic matrix presented an electron-dense appearance and a large quantity of free ribosomes. The Golgi complex is poorly developed, the secretory granules and the mitochondria are scarce. We could observe besides the light and dark cells, the centroacinar and the intermediate cells. The centroacinar cells lay in the luminal border of the acinar cells. They presented an electron-lucent cytoplasmic matrix where could be noted a few GER profiles and the secretory granules were absent. The intermediate cells showed, intermingled within the same cell, the zimogenic and the endocrine-like alfa and beta Langerhans granules.

Animals↗

PubMed2006

Abundant transcripts of malting barley identified by serial analysis of gene expression (SAGE).

Serial analysis of gene expression (SAGE) was applied to the major cereal crop barley (Hordeum vulgare) to characterize the transcriptional profile of grain during the malting process. Seven SAGE libraries were generated from seed at different time points during malting, in addition to one library from dry mature seed. A total of 155,206 LongSAGE tags, representing 41,909 unique sequences, was generated. This study reports an in-depth analysis of the most abundant transcripts from each of eight specific time points in a malting barley time course. The 100 most abundant tags from each library were analysed to identify the putative functional role of highly abundant transcripts. The largest functional groups included transcripts coding for stress response and cell defence, ribosomal proteins and storage proteins. The most abundant tag represented B22EL8, a barley metallothionein, which showed significant up-regulation across the malting time course. Considerable changes in the abundance profiles of some of the highly abundant tags occurred at 24 h post-steeping, indicating that it may be an important time point for gene expression changes associated with barley seed germination.

Base Sequence↗

PubMed1982

Analysis of the Escherichia coli ribosome-ribosomal subunit equilibrium using pressure-induced dissociation.

Hydrostatic pressure can be used to perturb the ribosome-ribosomal subunit equilibrium. We have used glutaraldehyde fixation and subsequent sucrose gradient analysis to determine the equilibrium concentrations of Escherichia coli 70 S, 50 S, and 30 S particles at pressures from 1 to 1400 atm. This method is shown to be sufficiently rapid and free of interfering ribosomal aggregation artifacts when performed at Mg2+ concentrations below 8 mM. We show directly that the E. coli ribosome is in equilibrium with its subunits and that the pressure-sensitive reaction is appropriately described by the expression: In Kp = ln K0 + (P delta V/RT), where Kp and K0 are the equilibrium constants at pressure P and 1 atm, respectively, and delta V is the change in molecular volume that occurs during the reaction. The method provides values for K0 under different conditions, and the effects of Mg2+ ion can be readily ascertained. K0 and delta V were also estimated by a method of fitting computer-generated sucrose gradient profiles to experimental profiles. Determination of delta H0, delta S0, and delta V0 at 5 mM Mg2+ are presented. The results are discussed in the context of previous thermodynamic studies of the E. coli ribosome.

Escherichia coli↗

PubMed2006

cDNA cloning and gene expression of ribosomal S9 protein gene in the mollusk Corbicula fluminea: a new potential biomarker of metal contamination up-regulated by cadmium and repressed by zinc.

Biological indicators can be used to assess polluted sites, but their success depends on their specificity. The aim of the present study was to identify a specific cadmium biomarker of the freshwater bivalve Corbicula fluminea. Differential display technique was used to identify new molecular indicators for cadmium exposure. Gene expression of C. fluminea transplanted into a cadmium- and zinc-contaminated river was compared to gene expression of bivalves from a reference site. One differentially expressed band was identified, and the entire cDNA sequence coding for the ribosomal protein S9 (rpS9) was cloned. The rpS9 gene expression was studied by real-time reverse transcription-polymerase chain reaction. After controlled cadmium (15 microg/L) and/or zinc (1 mg/L) laboratory contaminations of the bivalves, this gene was shown to be up-regulated by cadmium and down-regulated by zinc. A 48-d transplantation of animals into a polymetallic field contamination in the Lot river basin (Midi-Pyrénées region, France) showed that rpS9 gene regulation was correlated with the fluctuating cadmium and zinc water contents. To our knowledge, the existence of a gene that is up-regulated by cadmium and repressed by zinc is demonstrated here for the first time.

Amino Acid Sequence↗

PubMed1971

Further characterization of the cellular DNA hybridizing with the RNA of Rous sarcoma virus.

Both 70S and 4S RNAs of Rous sarcoma virus (RSV) contain nucleotide sequences homologous to those in cellular DNA. Preparations of these RNAs were found to include some adventitious cellular sRNA. Nevertheless, both 70S and 4S RSV-RNAs were found by competition techniques to contain sequences distinct from those in cellular ribosomal RNA and 4S RNA. Competition experiments also reveal substantial homology between 70S and 4SRSV-RNA. In hybridization experiments performed in formamide at low temperatures, the melting profiles of hybrids between 70S RSV-RNA and cellular DNA, and 4S RSV-RNA and cellular DNA, are essentially superimposable. It is suggested that there are at least two origins of 4S RSV-RNA in RSV-RNA preparations: degradation of 70S RSV-RNA and inclusion of adventitious cellular 4S RNA. The possibility of a specific viral 4S RNA is not excluded.

Avian Sarcoma Viruses↗

PubMed1999

Phenotypic and genotypic heterogeneity among cultivable pathogen-related oral spirochetes and Treponema vincentii.

Recent findings challenge the assumption that pathogen-related oral spirochetes (PROS) are related to Treponema pallidum. Treponema vincentii, grown in OMIZ-Pat media, cross-reacted with monoclonal antibody H9-2 against T. pallidum, and cultivable PROS had 16S rRNA gene sequences similar to those of T. vincentii (C.-B. Choi, C. Wyss, and U. B. Göbel. J. Clin. Microbiol. 34:1922-1925, 1996). Aims of the present study were to determine whether antigen phenotypes of oral treponemas were influenced by growth conditions and to evaluate the genetic relatedness of cultivable PROS to T. pallidum and T. vincentii. Results show that three T. pallidum monoclonal antibodies (H9-1, H9-2, and F5) cross-reacted with whole cells from four Treponema species grown in modified OMIZ-Pat medium, but not with treponemas grown in NOS medium. Only H9-2 reacted in immunoblots with reduced proteins from cultivable PROS and T. vincentii. Three of five PROS isolates were amplified by T. vincentii-specific PCR, and one was amplified by Treponema medium-specific PCR. None were amplified by T. pallidum-specific PCR. Three of five PROS isolates had 16S ribosomal DNA restriction fragment length polymorphism patterns identical to that of T. vincentii, and the patterns of two isolates resembled that of T. medium. Arbitrarily primed-PCR profiles from whole genomic DNA were distinct among five PROS isolates and two T. vincentii strains. Thus, PROS isolates represent a heterogeneous group of treponemas that share some 16S rRNA gene sequences with T. vincentii and T. medium, but not with T. pallidum. It is proposed that the PROS nomenclature be dropped.

Antibodies, Monoclonal↗

PubMed2005

Differential display analysis of gene expression in brains from hepatitis C-infected patients.

OBJECTIVES: Hepatitis C virus (HCV) infection is often associated with cognitive dysfunction, fatigue and depression. The current study was undertaken to determine whether HCV infection affects gene expression in brain tissue. DESIGN: We analysed the gene expression pattern in brain tissue in a group of HCV-infected patients compared with HCV-negative controls. METHODS: Brain tissue samples were obtained at autopsy from three HCV-positive patients and three HCV-negative control patients. The analysis of gene expression was conducted using differential display and reverse Northern hybridization. Only those genes that were up or downregulated more than 1.8 times were considered to be differentially expressed. RESULTS: Altogether, 29 differentially expressed genes were identified by differential display and subsequently confirmed by reverse Northern hybridization. A prominent finding was the downregulation of mitochondrial oxidative phosphorylation genes in HCV-infected patients. The impairment of brain oxidative/energy metabolism has previously been suggested to be the proximate cause of many disorders that impair mentation. Another finding was the downregulation of some ribosomal protein genes and several genes involved in transcription regulation, perhaps reflecting reduced metabolic activities. CONCLUSION: Our findings suggest for the first time that there may be a biological basis for the neuropsychiatric symptoms and cognitive impairment associated with HCV infection.

Aged↗

PubMed2005

Development of a coral cDNA array to examine gene expression profiles in Montastraea faveolata exposed to environmental stress.

The development of a cDNA array of coral genes and its application to investigate changes in coral gene expression associated with stressful conditions is described. The array includes both well-characterized and previously unidentified coral genes from Acropora cervicornis and Montastraea faveolata. Corals were exposed to either natural or anthropogenic stressors to elicit the expression of stress genes for isolation and incorporation onto the array. A total of 32 genes involved in protein synthesis, apoptosis, cell signaling, metabolism, cellular defense and inflammation were included on the array. Labeled cDNA from coral (Montastraea faveolata) exposed to elevated seawater temperature, salinity and ultraviolet light was tested against the microarray to determine patterns of gene expression associated with each stressor. Carbonic anhydrase, thioredoxin, a urokinase plasminogen activator receptor (uPAR) and three ribosomal genes demonstrated differential expression across all replicates on the array and between replicate colonies. Specific gene expression patterns produced in response to different stressors demonstrate the potential for gene expression profiling in characterizing the coral stress response.

Animals↗

PubMed2004

Renal toxicogenomic response to chronic uranyl nitrate insult in mice.

Although the nephrotoxicity of uranium has been established through numerous animal studies, relatively little is known about the effects of long-term environmental uranium exposure. Using a combination of conventional biochemical studies and serial analysis of gene expression (SAGE), we examined the renal responses to uranyl nitrate (UN) chronic exposure. Renal uranium levels were significantly increased 4 months after ingestion of uranium in drinking water. Creatinine levels in serum were slightly but significantly increased compared with those in controls. Although no further significant differences in other parameters were noted, substantial molecular changes were observed in toxicogenomic profiles. UN induced dramatic alterations in expression levels of more than 200 genes, mainly up-regulated, including oxidative-response-related genes, genes encoding for cellular metabolism, ribosomal proteins, signal transduction, and solute transporters. Seven differentially expressed transcripts were confirmed by real-time quantitative polymerase chain reaction. In addition, significantly increased peroxide levels support the implication of oxidative stress in UN toxicant response. This report highlights the potential of SAGE for the discovery of novel toxicant-induced gene expression alterations. Here, we present, for the first time, a comprehensive view of renal molecular events after uranium long-term exposure.

Administration, Oral↗

PubMed1982

Rapid alterations in initiation rate and recruitment of inactive RNA are temporally correlated with S6 phosphorylation.

HeLa cells propagated in spinner culture for 3-4 days without replenishing medium or serum progressively decrease the amount of mRNA and rRNA in polysomes, as well as the elongation rate. Treatment of these cells with low doses of cycloheximide shifts at least two thirds of the subpolysomal ribosomal particles into polysomes, indicating that the rate of ribosome attachment limits translation in these cells. Transfer of serum factor-depleted cells to fresh medium containing 10% calf serum likewise results in an extensive translocation of mRNA and rRNA into polysomes. Polysome absorbance profiles and sizes suggest that serum stimulation causes these changes by enhancing initiation rate. Newly recruited mRNAs derive from both subpolysomal translocation and recent nuclear RNA export, and contain a greater proportion of poly(A)-deficient mRNA molecules than the pre-stimulated polysomal mRNA population. Kinetic measurements show that these events occur principally within 20 min after serum addition, suggesting rapid modifications of preexisting components are involved. The phosphorylation kinetics of ribosomal protein S6, which closely parallel the alterations in translational activity, suggest that this modification may influence ribosome function.

Culture Media↗

PubMed2002

Diagnosis of pelvic actinomycosis by 16S ribosomal RNA gene sequencing and its clinical significance.

Traditional ways of identification of anaerobic Gram-positive non-sporulating bacilli by isolation of the organism and studying it phenotypically by elucidation of its morphologic and biochemical characteristics and metabolic end products are associated with a need for special equipment and expertise, and strains that are "unidentified" because of ambiguous biochemical profiles. In this study, an anaerobic Gram-positive non-sporulating bacterium was isolated from the intrauterine contraceptive device of a 36-year old woman with pyosalpinx. The Vitek system (ANI) showed that it was 99% Propionibacterium granulosum; whereas the API system (20A) showed that it was 78% Actinomyces meyeri/odontolyticus. The 16S ribosomal RNA gene of the strain was amplified and sequenced. There was 0 base difference between the isolate and A. odontolyticus (GenBank Accession no. AJ234047), indicating the isolate most closely resembled a strain of A. odontolyticus. Identification of the organism in this study was important because the duration of antibiotic therapy would be entirely different. In the present case, identification of the bacterium as A. odontolyticus inferred that the patient suffered from an intermediate form of pelvic actinomycosis. A prolonged course of antibiotics would be more desirable, as the relapse rate of actinomycosis after a short course of antibiotics is high.

Actinomyces↗

PubMed2003

XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response.

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). We have investigated here the contribution of the UPR transcription factors XBP-1, ATF6alpha, and ATF6beta to UPR target gene expression. Gene profiling of cell lines lacking these factors yielded several XBP-1-dependent UPR target genes, all of which appear to act in the ER. These included the DnaJ/Hsp40-like genes, p58(IPK), ERdj4, and HEDJ, as well as EDEM, protein disulfide isomerase-P5, and ribosome-associated membrane protein 4 (RAMP4), whereas expression of BiP was only modestly dependent on XBP-1. Surprisingly, given previous reports that enforced expression of ATF6alpha induced a subset of UPR target genes, cells deficient in ATF6alpha, ATF6beta, or both had minimal defects in upregulating UPR target genes by gene profiling analysis, suggesting the presence of compensatory mechanism(s) for ATF6 in the UPR. Since cells lacking both XBP-1 and ATF6alpha had significantly impaired induction of select UPR target genes and ERSE reporter activation, XBP-1 and ATF6alpha may serve partially redundant functions. No UPR target genes that required ATF6beta were identified, nor, in contrast to XBP-1 and ATF6alpha, did the activity of the UPRE or ERSE promoters require ATF6beta, suggesting a minor role for it during the UPR. Collectively, these results suggest that the IRE1/XBP-1 pathway is required for efficient protein folding, maturation, and degradation in the ER and imply the existence of subsets of UPR target genes as defined by their dependence on XBP-1. Further, our observations suggest the existence of additional, as-yet-unknown, key regulators of the UPR.

Activating Transcription Factor 6↗