Exposure to High Dosage of Gold Nanoparticles Accelerates Growth Rate by Modulating Ribosomal Protein Expression.

Gold nanoparticles (AuNPs) have been utilized in various biomedical applications including diagnostics and drug delivery. However, the cellular and metabolic responses of cells to these particles remain poorly characterized. In this study, we used bacteria (Escherichia coli and Bacillus subtilis) and a fungus (Saccharomyces cerevisiae) as model organisms to investigate the cellular and metabolic effects of exposure to different concentrations of citrate-capped spherical AuNPs with diameters of 5 and 10 nm. In different growth media, the synthesized AuNPs displayed stability and microorganisms exhibited uniform levels of uptake. Exposure to a high concentration of AuNPs (1012 particles) resulted in a reduced cell division time and a 2-fold increase in cell density in both bacteria and fungus. The exposed cells exhibited a decrease in average cell size and an increase in the expression of FtsZ protein (cell division marker), further supporting an accelerated growth rate. Notably, exposure to such a high concentration of AuNPs did not induce DNA damage, envelope stress, or a general stress response in bacteria. Differential whole proteome analysis revealed modulation of ribosomal protein expression upon exposure to AuNPs in both E. coli and S. cerevisiae. Interestingly, the accelerated growth observed upon exposure to AuNPs was sensitive to sub-minimum inhibitory concentration (sub-MIC) concentration of drugs that specifically target ribosome assembly and recycling. Based upon these findings, we hypothesize that exposure to high concentrations of AuNPs induces stress on the translation machinery. This leads to an increase in the protein synthesis rate by modulating ribosome assembly, which results in the rapid proliferation of cells.

Live Cells Imaging and Comparative Phosphoproteomics Uncover Proteins from the Mechanobiome in Entamoeba histolytica.

Entamoeba histolytica is a protozoan parasite and the causative agent of amoebiasis in humans. This amoeba invades human tissues by taking advantage of its actin-rich cytoskeleton to move, enter the tissue matrix, kill and phagocyte the human cells. During tissue invasion, E. histolytica moves from the intestinal lumen across the mucus layer and enters the epithelial parenchyma. Faced with the chemical and physical constraints of these diverse environments, E. histolytica has developed sophisticated systems to integrate internal and external signals and to coordinate cell shape changes and motility. Cell signalling circuits are driven by interactions between the parasite and extracellular matrix, combined with rapid responses from the mechanobiome in which protein phosphorylation plays an important role. To understand the role of phosphorylation events and related signalling mechanisms, we targeted phosphatidylinositol 3-kinases followed by live cell imaging and phosphoproteomics. The results highlight 1150 proteins, out of the 7966 proteins within the amoebic proteome, as members of the phosphoproteome, including signalling and structural molecules involved in cytoskeletal activities. Inhibition of phosphatidylinositol 3-kinases alters phosphorylation in important members of these categories; a finding that correlates with changes in amoeba motility and morphology, as well as a decrease in actin-rich adhesive structures.

Elevated inorganic carbon and salinity enhances photosynthesis and ATP synthesis in picoalga Picocystis salinarum as revealed by label free quantitative proteomics.

Saline soda lakes are of immense ecological value as they niche some of the most exclusive haloalkaliphilic communities dominated by bacterial and archaeal domains, with few eukaryotic algal representatives. A handful reports describe Picocystis as a key primary producer with great production rates in extremely saline alkaline habitats. An extremely haloalkaliphilic picoalgal strain, Picocystis salinarum SLJS6 isolated from hypersaline soda lake Sambhar, Rajasthan, India, grew robustly in an enriched soda lake medium containing mainly Na2CO3, 50 g/l; NaHCO3, 50 g/l, NaCl, 50 g/l (salinity ≈150‰) at pH 10. To elucidate the molecular basis of such adaptation to high inorganic carbon and NaCl concentrations, a high-throughput label-free quantitation based quantitative proteomics approach was applied. Out of the total 383 proteins identified in treated samples, 225 were differentially abundant proteins (DAPs), of which 150 were statistically significant (p < 0.05) including 70 upregulated and 64 downregulated proteins after 3 days of growth in highly saline-alkaline medium. Most DAPs were involved in photosynthesis, oxidative phosphorylation, glucose metabolism and ribosomal structural components envisaging that photosynthesis and ATP synthesis were central to the salinity-alkalinity response. Key components of photosynthetic machinery like photosystem reaction centres, adenosine triphosphate (ATP) synthase ATP, Rubisco, Fructose-1,6-bisphosphatase, Fructose-bisphosphate aldolase were highly upregulated. Enzymes peptidylprolyl isomerases (PPIase), important for correct protein folding showed remarkable marked-up regulation along with other chaperon proteins indicating their role in osmotic adaptation. Enhanced photosynthetic activity exhibited by P. salinarum in highly saline-alkaline condition is noteworthy as photosynthesis is suppressed under hyperosmotic conditions in most photosynthetic organisms. The study provided the first insights into the proteome of extremophilic alga P. salinarum exhibiting extraordinary osmotic adaptation and proliferation in polyextreme conditions prevailing in saline sodic ecosystems, potentially unraveling the basis of resilience in this not so known organism and paves the way for a promising future candidate for biotechnological applications and model organism for deciphering the molecular mechanisms of osmotic adaptation. The mass spectrometry proteomics data is available at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD037170.

Multi-omics analysis to characterize molecular adaptation of Entamoeba histolytica during serum stress.

Entamoeba histolytica is responsible for dysentery and extraintestinal disease in humans. To establish successful infection, it must generate adaptive response against stress due to host defense mechanisms. We have developed a robust proteomics workflow by combining miniaturized sample preparation, low flow-rate chromatography, and ultra-high sensitivity mass spectrometry, achieving increased proteome coverage, and further integrated proteomics and RNA-seq data to decipher regulation at translational and transcriptional levels. Label-free quantitative proteomics led to identification of 2344 proteins, an improvement over the maximum number identified in E. histolytica proteomic studies. In serum-starved cells, 127 proteins were differentially abundant and were associated with functions including antioxidant activity, cytoskeleton, translation, catalysis, and transport. The virulence factor, Gal/GalNAc-inhibitable lectin subunits, was significantly altered. Integration of transcriptomic and proteomic data revealed that only 30% genes were coordinately regulated at both transcriptional and translational levels. Some highly expressed transcripts did not change in protein abundance. Conversely, genes with no transcriptional change showed enhanced protein abundance, indicating post-transcriptional regulation. This multi-omics approach enables more refined gene expression analysis to understand the adaptive response of E. histolytica during growth stress.

Intrinsic bias at non-canonical, ß-arrestin-coupled seven transmembrane receptors.

G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMRs), typically interact with two distinct signal-transducers, i.e., G proteins and ß-arrestins (ßarrs). Interestingly, there are some non-canonical 7TMRs that lack G protein coupling but interact with ßarrs, although an understanding of their transducer coupling preference, downstream signaling, and structural mechanism remains elusive. Here, we characterize two such non-canonical 7TMRs, namely, the decoy D6 receptor (D6R) and the complement C5a receptor subtype 2 (C5aR2), in parallel with their canonical GPCR counterparts. We discover that D6R and C5aR2 efficiently couple to ßarrs, exhibit distinct engagement of GPCR kinases (GRKs), and activate non-canonical downstream signaling pathways. We also observe that ßarrs adopt distinct conformations for D6R and C5aR2, compared to their canonical GPCR counterparts, in response to common natural agonists. Our study establishes D6R and C5aR2 as ßarr-coupled 7TMRs and provides key insights into their regulation and signaling with direct implication for biased agonism.

Switch to Autophagy the Key Mechanism for Trabecular Meshwork Death in Severe Glaucoma.

PURPOSE: The key differences in cell death mechanisms in the trabecular meshwork (TM) in adult moderate and severe primary glaucoma remain still unanswered. This study explored key differences in cell death mechanisms in the trabecular meshwork (TM) in adult moderate and severe primary glaucoma. DESIGN: In-vitro laboratory study on surgical specimens and primary cell lines. METHODS: Select cell death-related proteins differentially expressed on mass spectrometric analysis in ex-vivo dissected TM specimens patients with severe adult primary open-angle (POAG) or angle-closure glaucoma (PACG) compared to controls (cadaver donor cornea) were validated for temporal changes in cell death-related gene expression on in-vitro primary human TM cell culture after 48 hours (moderate) or 72 hours (severe) oxidative stress with H2O2 (400-1000 uM concentration). These were compared with histone modifications after oxidative stress in human TM (HTM) culture and peripheral blood of patients with moderate and severe glaucoma. RESULTS: Autophagy-related proteins seemed to be the predominant cell-death mechanism over apoptosis in ex-vivo dissected TM specimens in severe glaucoma. Analyzing HTM cell gene expression at 48 hours and 72 hours of oxidative stress, autophagy genes were up-regulated at 48-72 hours of exposure in contrast to apoptosis-related genes, showing down-regulation at 72 hours. There was associated increased expression of H3K14ac in HTM after 72 hours of oxidative stress and in peripheral blood of severe POAG and PACG. CONCLUSION: A preference of autophagy over apoptosis may underlie stage transition from moderate to severe glaucoma in the trabecular meshwork or peripheral blood, which may be tightly regulated by epigenetic modulators.

Identification of hub genes associated with RNAi-induced silencing of XIAP through targeted proteomics approach in MCF7 cells.

BACKGROUND: The X-linked inhibitor of apoptosis protein (XIAP) is the most potent caspase inhibitor of the IAP family in apoptosis pathway. This study aims to identify the molecular targets of XIAP in human breast cancer cells exposed to XIAP siRNA by proteomics screening. The expression of XIAP was reduced in MCF-7 breast cancer cells by siRNA. Cell viability and the mRNA expression level of this gene were evaluated by MTS and quantitative real-time PCR procedures, respectively. Subsequently, the XIAP protein level was visualized by Western blotting and analyzed by two-dimensional (2D) electrophoresis and LC-ESI-MS/MS. RESULTS: Following XIAP silencing, cell proliferation was reduced in XIAP siRNA transfected cells. The mRNA transcription and protein expression of XIAP were decreased in cells exposed to XIAP siRNA than si-NEG. We identified 30 proteins that were regulated by XIAP, of which 27 down-regulated and 3 up-regulated. The most down-regulated proteins belonged to the Heat Shock Proteins family. They participate in cancer related processes including apoptosis and MAPK signaling pathway. Reduced expression of HSP90B1 was associated with apoptosis induction by androgen receptor and prostate specific antigen. Suppression of XIAP resulted in the enhancement of GDIB, ENO1, and CH60 proteins expression. The network analysis of XIAP-regulated proteins identified HSPA8, HSP90AA1, ENO1, and HSPA9 as key nodes in terms of degree and betweenness centrality methods. CONCLUSIONS: These results suggested that XIAP may have a number of biological functions in a diverse set of non-apoptotic signaling pathways and may provide an insight into the biomedical significance of XIAP over-expression in MCF-7 cells.

Proteomics evaluation of MDA-MB-231 breast cancer cells in response to RNAi-induced silencing of hPTTG.

AIMS: Breast cancer is the most common cancer in women worldwide. Several genes are up-regulated in breast cancer such as human pituitary tumor transforming gene (hPTTG). This study aims to evaluate cell proliferation and the downstream expression pattern of hPTTG1 gene at the mRNA and protein levels after specific down-regulation of hPTTG1 by siRNA. MAIN METHODS: The human breast cancer MDA-MB-231 cell line was transfected with siRNA against hPTTG1. The mRNA and protein expression levels were examined by Real-time PCR and Western blot, respectively. The cell proliferation was assayed by MTS. To investigate the pattern of protein expression, total cellular protein was analyzed by 2D gel electrophoresis and mass spectroscopy. Subsequently, the possible biological consequences were determined by the bioinformatics databases. KEY FINDINGS: Subsequent of hPTTG1 silencing in the MDA_MB-231 cells, the proliferation of cells decreased obviously. In response to hPTTG1 silencing, the levels mRNA and protein were effectively down-regulated 80% and 50%, respectively, at 48 h post-transfection. The proteomics evidenced that PTTG1 increased the expression of 5 proteins. The reduced expression of PTTG1 was functionally involved in hypoxia (NPM1, ENO1), cell proliferation and apoptosis (ENO1, NPM1, NME1, STMN1), and metastasis (NPM1, NME1). SIGNIFICANCE: We identified the hPTTG1-regulated proteins and its molecular mechanism in pathogenesis of breast cancer. Further study emphasis is to understand the association of hPTTG1 with other genes in cancer progression. This novel modality might also consider for identification of targeted drugs, prognosis and follow up in breast cancer gene therapy.

New insights into the roles of the FOXO3 and P27Kip1 genes in signaling pathways.

Background: The forkhead box O3 (FOXO3) and p27Kip1 are two important genes in breast cancer progression. In the present study we analyzed the effect of simultaneous FOXO3 silencing and p27Kip1 activation on breast cancer cell survival and the potential targets of these changes in cancer molecular pathways. Materials and methods: The present study involved the cloning of FOXO3a shRNA and p27Kip1 genes under the control of the bidirectional survivin promoter to down- and up-regulate FOXO3 and p27Kip1 genes, respectively. After transfection of the recombinant expression vector into the breast cancer cell line, the inhibition of cell growth was assessed by MTS and flow cytometry assays. Following the extraction of total mRNA and protein, the expression of target genes was evaluated by qPCR and Western blotting in both treated and untreated cell lines. Then, the downstream protein responses were examined by 2 D electrophoresis. The differentially expressed proteins were also identified by mass spectrometry. Results: Rates of cell proliferation were significantly inhibited in the transfected cell line 72 h post-transfection. Proteomic profiling of the cell line resulted in the identification of seven novel protein markers in breast cancer responsive to these changes in expression of FOXO3 and p27Kip1. The changes in expression of these markers suggested that certain signaling pathways contribute to the development of breast cancer. Conclusion: Simultaneous silencing of FOXO3 and activation of p27Kip1 in MDA-MB-231 cells caused alterations in the expression level of several genes involved in apoptosis, cell proliferation, cell cycle control, tissue invasion, drug resistance, and metastasis. It seems that the identified genes might serve as useful biomarkers for breast cancer.

LipidII interaction with specific residues of Mycobacterium tuberculosis PknB extracytoplasmic domain governs its optimal activation.

The Mycobacterium tuberculosis kinase PknB is essential for growth and survival of the pathogen in vitro and in vivo. Here we report the results of our efforts to elucidate the mechanism of regulation of PknB activity. The specific residues in the PknB extracytoplasmic domain that are essential for ligand interaction and survival of the bacterium are identified. The extracytoplasmic domain interacts with mDAP-containing LipidII, and this is abolished upon mutation of the ligand-interacting residues. Abrogation of ligand-binding or sequestration of the ligand leads to aberrant localization of PknB. Contrary to the prevailing hypothesis, abrogation of ligand-binding is linked to activation loop hyperphosphorylation, and indiscriminate hyperphosphorylation of PknB substrates as well as other proteins, ultimately causing loss of homeostasis and cell death. We propose that the ligand-kinase interaction directs the appropriate localization of the kinase, coupled to stringently controlled activation of PknB, and consequently the downstream processes thereof.

Acetylation is the most abundant actin modification in Entamoeba histolytica and modifications of actin's amino-terminal domain change cytoskeleton activities.

Actin is one of the most conserved, abundant, and ubiquitous proteins in all eukaryotes characterised to date. Posttranslation modifications of actin modify the organisation of the actin-rich cytoskeleton. In particular, chemical modifications of actin's amino-terminal region determine how filamentous actin is organised into scaffolds. After assuming that protein modifications account for the multiple functional activities exerted by the single actin in Entamoeba histolytica, we profiled posttranslational modifications of this protein. Acetylation (on 21 different amino acids) was the most abundant modification, followed by phosphorylation. Furthermore, the glycine residue at Position 2 in E. histolytica's actin (Gly2, not found in most other eukaryotic actins) was found to be acetylated. The impact of Gly2 on the amoeba's life cycle and pathogenicity was then assessed in mutagenesis experiments. We found that Gly2 was necessary for cell morphology and division, parasite-host cell adhesion, and host invasion in an in vitro model of amoebic human infection.

Comparative Proteomic Analyses of Avirulent, Virulent, and Clinical Strains of Mycobacterium tuberculosis Identify Strain-specific Patterns.

Mycobacterium tuberculosis is an adaptable intracellular pathogen, existing in both dormant as well as active disease-causing states. Here, we report systematic proteomic analyses of four strains, H37Ra, H37Rv, and clinical isolates BND and JAL, to determine the differences in protein expression patterns that contribute to their virulence and drug resistance. Resolution of lysates of the four strains by liquid chromatography, coupled to mass spectrometry analysis, identified a total of 2161 protein groups covering ∼54% of the predicted M. tuberculosis proteome. Label-free quantification analysis of the data revealed 257 differentially expressed protein groups. The differentially expressed protein groups could be classified into seven K-means cluster bins, which broadly delineated strain-specific variations. Analysis of the data for possible mechanisms responsible for drug resistance phenotype of JAL suggested that it could be due to a combination of overexpression of proteins implicated in drug resistance and the other factors. Expression pattern analyses of transcription factors and their downstream targets demonstrated substantial differential modulation in JAL, suggesting a complex regulatory mechanism. Results showed distinct variations in the protein expression patterns of Esx and mce1 operon proteins in JAL and BND strains, respectively. Abrogating higher levels of ESAT6, an important Esx protein known to be critical for virulence, in the JAL strain diminished its virulence, although it had marginal impact on the other strains. Taken together, this study reveals that strain-specific variations in protein expression patterns have a meaningful impact on the biology of the pathogen.

Identification of EhTIF-IA: The putative E. histolytica orthologue of the human ribosomal RNA transcription initiation factor-IA.

Initiation of rDNA transcription requires the assembly of a specific multi-protein complex at the rDNA promoter containing the RNA Pol I with auxiliary factors. One of these factors is known as Rrn3P in yeast and Transcription Initiation Factor IA (TIF-IA) in mammals. Rrn3p/TIF-IA serves as a bridge between RNA Pol I and the pre-initiation complex at the promoter. It is phosphorylated at multiple sites and is involved in regulation of rDNA transcription in a growth-dependent manner. In the early branching parasitic protist Entamoeba histolytica, the rRNA genes are present exclusively on circular extra chromosomal plasmids. The protein factors involved in regulation of rDNA transcription in E. histolytica are not known. We have identified the E. histolytica equivalent of TIF-1A (EhTIF-IA) by homology search within the database and was further cloned and expressed. Immuno-localization studies showed that EhTIF-IA co-localized partially with fibrillarin in the peripherally localized nucleolus. EhTIF-IA was shown to interact with the RNA Pol I-specific subunit RPA12 both in vivo and in vitro. Mass spectroscopy data identified RNA Pol I-specific subunits and other nucleolar proteins to be the interacting partners of EhTIF-IA. Our study demonstrates for the first time a conserved putative RNA Pol I transcription factor TIF-IA in E. histolytica.

Cellular and proteomics analysis of the endomembrane system from the unicellular Entamoeba histolytica.

Entamoeba histolytica is the protozoan parasite agent of amoebiasis, an infectious disease of the human intestine and liver. Specific active pathogenic factors are secreted toward the external milieu upon interaction of the parasite with human tissue. Trafficking dynamics and secretion of these factors is not known and characterization of the dynamics interplay of subcellular compartments such as the ER or Golgi apparatus is still pending. In this work, we took advantage of cell fractionation and a wide proteomic analysis to search for principal components of the endomembrane system in E. histolytica. Over 1500 proteins were identified and the two top categories contained components of trafficking machinery and GTPases. Trafficking related proteins account for over 100 markers from the ER, Golgi, MVB, and retromers. The lack of important components supporting Golgi polarization was also highlighted. The data further describe principal components of the endosomal traffic highlighting Alix in isolated vesicles and during parasite division. BIOLOGICAL SIGNIFICANCE: This work represents the first in-depth proteomics analysis of subcellular compartments in E. histolytica and allows a detailed map of vesicle traffic components in an ancient single-cell organism that lacks a stereotypical ER and Golgi apparatus to be established.

Phosphorylation of nucleoporin Tpr governs its differential localization and is required for its mitotic function.

A major constituent of the nuclear basket region of the nuclear pore complex (NPC), nucleoporin Tpr, plays roles in regulating multiple important processes. We have previously established that Tpr is phosphorylated in both a MAP-kinase-dependent and MAP-kinase-independent manner, and that Tpr acts as both a substrate and as a scaffold for ERK2 (also known as MAPK1). Here, we report the identification of S2059 and S2094 as the major novel ERK-independent phosphorylation sites and T1677, S2020, S2023 and S2034 as additional ERK-independent phosphorylation sites found in the Tpr protein in vivo. Our results suggest that protein kinase A phosphorylates the S2094 residue and that the site is hyperphosphorylated during mitosis. Furthermore, we find that Tpr is phosphorylated at the S2059 residue by CDK1 and the phosphorylated form distinctly localizes with chromatin during telophase. Abrogation of S2059 phosphorylation abolishes the interaction of Tpr with Mad1, thus compromising the localization of both Mad1 and Mad2 proteins, resulting in cell cycle defects. The identification of novel phosphorylation sites on Tpr and the observations presented in this study allow better understanding of Tpr functions.

Data set for the proteomics analysis of the endomembrane system from the unicellular Entamoeba histolytica.

Entamoeba histolytica is the protozoan parasite agent of amebiasis, an infectious disease of the human intestine and liver. This parasite contact and kills human cells by an active process involving pathogenic factors. Cellular traffic and secretion activities are poorly characterized in E. histolytica. In this work, we took advantage of a wide proteomic analysis to search for principal components of the endomembrane system in E. histolytica. A total of 5683 peptides matching with 1531 proteins (FDR of 1%) were identified which corresponds to roughly 20% of the total amebic proteome. Bioinformatics investigations searching for domain homologies (Smart and InterProScan programs) and functional descriptions (KEGG and GO terms) allowed this data to be organized into distinct categories. This data represents the first in-depth proteomics analysis of subcellular compartments in E. histolytica and allows a detailed map of vesicle traffic components in an ancient single-cell organism that lacks a stereotypical ER and Golgi apparatus to be established. The data are related to [1].

Substrate-bound crystal structures reveal features unique to Mycobacterium tuberculosis N-acetyl-glucosamine 1-phosphate uridyltransferase and a catalytic mechanism for acetyl transfer.

N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme involved in bacterial cell wall synthesis is exclusive to prokaryotes. GlmU, now recognized as a promising target to develop new antibacterial drugs, catalyzes two key reactions: acetyl transfer and uridyl transfer at two independent domains. Hitherto, we identified GlmU from Mycobacterium tuberculosis (GlmU(Mtb)) to be unique in possessing a 30-residue extension at the C terminus. Here, we present the crystal structures of GlmU(Mtb) in complex with substrates/products bound at the acetyltransferase active site. Analysis of these and mutational data, allow us to infer a catalytic mechanism operative in GlmU(Mtb). In this S(N)2 reaction, His-374 and Asn-397 act as catalytic residues by enhancing the nucleophilicity of the attacking amino group of glucosamine 1-phosphate. Ser-416 and Trp-460 provide important interactions for substrate binding. A short helix at the C-terminal extension uniquely found in mycobacterial GlmU provides the highly conserved Trp-460 for substrate binding. Importantly, the structures reveal an uncommon mode of acetyl-CoA binding in GlmU(Mtb); we term this the U conformation, which is distinct from the L conformation seen in the available non-mycobacterial GlmU structures. Residues, likely determining U/L conformation, were identified, and their importance was evaluated. In addition, we identified that the primary site for PknB-mediated phosphorylation is Thr-418, near the acetyltransferase active site. Down-regulation of acetyltransferase activity upon Thr-418 phosphorylation is rationalized by the structures presented here. Overall, this work provides an insight into substrate recognition, catalytic mechanism for acetyl transfer, and features unique to GlmU(Mtb), which may be exploited for the development of inhibitors specific to GlmU.

The nucleolus in Entamoeba histolytica and Entamoeba invadens is located at the nuclear periphery.

The ribosomal RNA genes in the human parasite Entamoeba histolytica and its reptilian counterpart Entamoeba invadens are located on extrachromosomal circles. The expression of rRNA genes generally takes place in a specialized nuclear compartment-the nucleolus. In Entamoeba species the nuclear space that may be called the nucleolus has yet to be defined. Previous studies showed that the rDNA circles are located at the nuclear periphery. Here we have raised antibodies against the E. histolytica homologue of fibrillarin, a highly conserved protein known to be a marker for nucleolus. These antibodies cross-reacted preferentially with the nuclear periphery, forming a peripheral ring. There was complete colocalization of fibrillarin with the signal obtained by antibodies against E. histolytica RNA polymerase I (but not polymerase II and III), strongly suggesting that the nucleolus in E. histolytica is indeed located at the nuclear periphery. The dynamic nature of the nucleolus was evident when cells were subjected to a variety of growth stresses. Although the peripheral nucleolar structure was retained, stress was accompanied by significant cytoplasmic localization of RNA polymerase I, and to some extent fibrillarin. The nucleolus in E. invadens was also located at the nuclear periphery. When these cells were induced to encyst the nucleolar ring structure was lost, giving way to small, fragmented foci. This study gives the first clear insight into nucleolar structure in Entamoeba.

Promoter analysis of palindromic transcription units in the ribosomal DNA circle of Entamoeba histolytica.

rRNA genes of Entamoeba histolytica are organized as palindromic ribosomal DNA (rDNA) units (I and II) in a 24.5-kb circle. Although the two rDNAs are identical in sequence, their upstream spacers are completely different. Since the intergenic sequences (IGS) of all rDNA copies in other organisms are conserved and contain transcription regulatory sequences, the lack of sequence conservation in the IGS prompted the question of whether both rDNAs are indeed transcriptionally active. We mapped the transcriptional start points (tsp's) and promoters of the two rDNAs. A 51-bp sequence immediately upstream of the tsp's was highly conserved in both units. In addition, both units had an A+T-rich stretch upstream of the 51-bp core. Analysis of reporter gene transcription showed promoter activity to reside in the regions from positions -86 to +123 (rDNA I) and positions -101 to +140 (rDNA II). The promoter-containing fragments from both units could bind and compete with each other for protein(s) from nuclear extracts. Protein binding was especially dependent on the A+T-rich region upstream of the 51-bp core (positions -53 to -68). The requirement of >80 bp downstream of the tsp was striking. Although this sequence was not conserved in the two units, it could potentially fold into very long stem-loops. Both rDNAs transcribed with comparable efficiency, as measured by nuclear runon. Thus, both rDNAs share very similar organization of promoter sequences, and in exponential culture both rDNAs are transcribed. It remains to be seen whether the different IGS affect the regulation of the two units under adverse conditions.