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author:("Ren, dinghu")
1.  Epithelial and stromal expression of miRNAs during prostate cancer progression 
Global microRNA (miRNA) profile may predict prostate cancer (PCa) behaviors. In this study, we examined global miRNA expression by miRNA profiling as well as specific miRNA expression levels in PCa epithelium and stroma by in situ hybridization (ISH) and correlated with various clinicopathological features. We first performed comprehensive miRNA profiling on 27 macrodissected cases of PCa by miRNA microarray. A total of 299 miRNAs were significantly dysregulated in high grade and advanced stage PCa. We demonstrated that PCa can be readily classified into high grade/stage and low-grade/stage groups by its global miRNA expression profile. Next, we examined the expression of several selected dysregulated miRNAs, including let-7c, miR-21, miR-27a, miR-30c, and miR-219, in PCa by ISH. The levels of miRNA expression in epithelial and stromal cells were scored semiquantitatively and compared with clinicopathological features, including age, race, Gleason score, stage, PSA recurrence, metastasis, hormone resistance and survival. We found that the expression of miR-30c and miR-219 were significantly down-regulated in PCa. miR-21 and miR-30c were significantly down-regulated in PCa in African Americans compared to Caucasian Americans. In addition, down-regulation of let-7c, miR-21, miR-30c, and miR-219 are associated with metastatic disease. Furthermore, down-regulation of miR-30c and let-7c are significantly associated with androgen-dependent PCa. In PCa stromal cells, let-7c downregulation is significantly associated with extraprostatic extension. Our data suggest that selected miRNAs may serve as potential biomarkers to predict cancer progression.
PMCID: PMC4113495  PMID: 25075250
miRNA; prostate cancer progression
2.  Expression of Androgen Receptor and its Phosphorylated Forms in Breast Cancer Progression 
Cancer  2013;119(14):10.1002/cncr.28092.
Background
Androgen receptor (AR) expression in breast cancers may serve as a prognostic and predictive marker. We examined the expression pattern of AR and its phosphorylated forms, Ser-213 (AR-Ser(P)-213) and Ser-650 (AR-Ser(P)-650), in breast cancer and evaluated their association with clinicopathological parameters.
Methods
Immunohistochemistry was performed on primary and distant metastatic breast cancers and benign breast tissue using antibodies against AR, AR-Ser(P)-213, and AR-Ser(P)-650. The levels of cytoplasmic and nuclear expression were scored semiquantitatively using a histoscore.
Results
Nuclear staining of AR was observed in all benign breast tissue and 67% of cancer cases. Nuclear and cytoplasmic AR-Ser(P)-213 was increased in breast cancers 2-fold (p=0.0014 ) and 1.7-fold ( p= 0.05), respectively, compared to benign controls, whereas nuclear and cytoplasmic AR-Ser(P)-650 expression was decreased in tumors by 1.9-fold and 1.7-fold (both p<0.0001), respectively. Increased expression of nuclear or cytoplasmic AR-Ser(P)-213 was observed in metastatic breast cancers (1.3-fold, p=0.05), ER-negative (2.6-fold, p=0.001) and invasive ductal carcinoma (6.8-fold, p=0.04). AR-Ser(P)-650 expression is downregulated in lymph node-positive (1.4-fold, p=0.02) breast cancers, but is upregulated in invasive ductal carcinomas (3.2-fold, p<0.0001) and metastases (1.5-fold, p=0.003). Moreover, in ER-negative breast cancers nuclear AR-Ser(P)-650 was decreased (1.4-fold, p=0.005) and cytoplasmic ARSer(P)-650 was increased (1.4-fold, p=0.003).
Conclusion
AR and its phosphorylation at serines 213 and 650 are differentially expressed in breast cancer tumorigenesis and progression. Phosphorylation of AR at serines 213 and 650 is increased in ER-negative, ductal carcinomas, and metastases and may have predictive value in breast cancer prognosis.
doi:10.1002/cncr.28092
PMCID: PMC3874891  PMID: 23605249
breast; breast neoplasms; androgen; androgen receptor; phosphorylation
3.  Regulation of a Novel Androgen Receptor Target Gene, the Cyclin B1 Gene, through Androgen-Dependent E2F Family Member Switching 
Molecular and Cellular Biology  2012;32(13):2454-2466.
The malignant transformation of human prostatic epithelium is associated with the loss of androgen receptor (AR) in the surrounding stroma. However, the function and mechanisms of AR signaling in prostate cancer (PCa) stroma remain elusive. Here we report, by using proteomics pathway array analysis (PPAA), that androgen and its receptor inhibit the proliferation of prostate stromal cells through transcriptional suppression of cyclin B1, and we confirmed our findings at mRNA and protein levels using AR-negative or -positive primary prostate stromal cells. Furthermore, AR showed a negative correlation with cyclin B1 expression in stroma of human PCa samples in vivo. Mechanistically, we identify cyclin B1 as a bona fide AR target gene in prostate stromal cells. The negative regulation of cyclin B1 by AR is mediated through switching between E2F1 and E2F4 on the promoter of cyclin B1. E2F1 binds to the cyclin B1 promoter and maintains its expression and subsequent cell cycle progression in AR-negative stromal cells or AR-positive stromal cells when androgens are depleted. Upon stimulation with androgen in AR-positive stromal cells, E2F1 is displaced from the binding site by AR and replaced with E2F4, leading to the recruitment of the silencing mediator for retinoid and thyroid hormone receptor (SMRT)/histone deacetylase 3 (HDAC3) corepressor complex and repression of cyclin B1 at the chromatin level. The switch between E2F1 and E2F4 at the E2F binding site of the cyclin B1 promoter coincides with an androgen-dependent interaction between AR and E2F1 as well as the cytoplasmic-to-nuclear translocation of E2F4. Thus, we identified a novel mechanism for E2F factors in the regulation of cell cycle gene expression and cell cycle progression under the control of AR signaling.
doi:10.1128/MCB.06663-11
PMCID: PMC3434485  PMID: 22508987
4.  CD163 versus CD68 in tumor associated macrophages of classical hodgkin lymphoma 
Diagnostic Pathology  2012;7:12.
Classical Hodgkin lymphoma (CHL) is a B-cell lymphoproliferative disorder with a relatively good prognosis. A small but significant percentage of patients, however, will respond poorly to therapy. A recent gene expression profiling study has identified a macrophage signature which has been correlated with primary treatment failure, and immunohistochemical tissue microarray for CD68 was shown to reflect the gene signature as a potentially clinically useful marker to predict adverse prognosis.
We examined 44 cases of CHL, mostly nodular sclerosis subtype, in which the immunohistochemical stains for the histiocytic markers CD68 and CD163 were performed. The staining intensity was graded for each stain (< 5, 5-25, and > 25 percent of cells positive in the Hodgkin cell (HC) rich nodules) and background staining characteristics were recorded.
CD163 staining was lower than CD68 in HC rich nodules, with lower background staining (p 0.03). There was no significant difference between either CD68 or CD163 and disease recurrence in a subset (N = 41) of cases.
In conclusion, we demonstrate that CD163 staining is lower than CD68, with less non-specific staining of background inflammatory cells and Hodgkin cells, therefore is a better marker for tumor associated macrophages. However, we did not identify a correlation between staining for CD68 or CD163 and recurrence of disease.
Virtual slides
The virtual slide(s) for this article can be found here:
http://www.diagnosticpathology.diagnomx.eu/vs/1460518258831620
doi:10.1186/1746-1596-7-12
PMCID: PMC3281786  PMID: 22289504
5.  The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions 
Merchant, Sabeeha S. | Prochnik, Simon E. | Vallon, Olivier | Harris, Elizabeth H. | Karpowicz, Steven J. | Witman, George B. | Terry, Astrid | Salamov, Asaf | Fritz-Laylin, Lillian K. | Maréchal-Drouard, Laurence | Marshall, Wallace F. | Qu, Liang-Hu | Nelson, David R. | Sanderfoot, Anton A. | Spalding, Martin H. | Kapitonov, Vladimir V. | Ren, Qinghu | Ferris, Patrick | Lindquist, Erika | Shapiro, Harris | Lucas, Susan M. | Grimwood, Jane | Schmutz, Jeremy | Cardol, Pierre | Cerutti, Heriberto | Chanfreau, Guillaume | Chen, Chun-Long | Cognat, Valérie | Croft, Martin T. | Dent, Rachel | Dutcher, Susan | Fernández, Emilio | Ferris, Patrick | Fukuzawa, Hideya | González-Ballester, David | González-Halphen, Diego | Hallmann, Armin | Hanikenne, Marc | Hippler, Michael | Inwood, William | Jabbari, Kamel | Kalanon, Ming | Kuras, Richard | Lefebvre, Paul A. | Lemaire, Stéphane D. | Lobanov, Alexey V. | Lohr, Martin | Manuell, Andrea | Meier, Iris | Mets, Laurens | Mittag, Maria | Mittelmeier, Telsa | Moroney, James V. | Moseley, Jeffrey | Napoli, Carolyn | Nedelcu, Aurora M. | Niyogi, Krishna | Novoselov, Sergey V. | Paulsen, Ian T. | Pazour, Greg | Purton, Saul | Ral, Jean-Philippe | Riaño-Pachón, Diego Mauricio | Riekhof, Wayne | Rymarquis, Linda | Schroda, Michael | Stern, David | Umen, James | Willows, Robert | Wilson, Nedra | Zimmer, Sara Lana | Allmer, Jens | Balk, Janneke | Bisova, Katerina | Chen, Chong-Jian | Elias, Marek | Gendler, Karla | Hauser, Charles | Lamb, Mary Rose | Ledford, Heidi | Long, Joanne C. | Minagawa, Jun | Page, M. Dudley | Pan, Junmin | Pootakham, Wirulda | Roje, Sanja | Rose, Annkatrin | Stahlberg, Eric | Terauchi, Aimee M. | Yang, Pinfen | Ball, Steven | Bowler, Chris | Dieckmann, Carol L. | Gladyshev, Vadim N. | Green, Pamela | Jorgensen, Richard | Mayfield, Stephen | Mueller-Roeber, Bernd | Rajamani, Sathish | Sayre, Richard T. | Brokstein, Peter | Dubchak, Inna | Goodstein, David | Hornick, Leila | Huang, Y. Wayne | Jhaveri, Jinal | Luo, Yigong | Martínez, Diego | Ngau, Wing Chi Abby | Otillar, Bobby | Poliakov, Alexander | Porter, Aaron | Szajkowski, Lukasz | Werner, Gregory | Zhou, Kemin | Grigoriev, Igor V. | Rokhsar, Daniel S. | Grossman, Arthur R.
Science (New York, N.Y.)  2007;318(5848):245-250.
Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.
doi:10.1126/science.1143609
PMCID: PMC2875087  PMID: 17932292
6.  The Complete Genome Sequence of Haloferax volcanii DS2, a Model Archaeon 
PLoS ONE  2010;5(3):e9605.
Background
Haloferax volcanii is an easily culturable moderate halophile that grows on simple defined media, is readily transformable, and has a relatively stable genome. This, in combination with its biochemical and genetic tractability, has made Hfx. volcanii a key model organism, not only for the study of halophilicity, but also for archaeal biology in general.
Methodology/Principal Findings
We report here the sequencing and analysis of the genome of Hfx. volcanii DS2, the type strain of this species. The genome contains a main 2.848 Mb chromosome, three smaller chromosomes pHV1, 3, 4 (85, 438, 636 kb, respectively) and the pHV2 plasmid (6.4 kb).
Conclusions/Significance
The completed genome sequence, presented here, provides an invaluable tool for further in vivo and in vitro studies of Hfx. volcanii.
doi:10.1371/journal.pone.0009605
PMCID: PMC2841640  PMID: 20333302
7.  The 2008 update of the Aspergillus nidulans genome annotation: a community effort 
Wortman, Jennifer Russo | Gilsenan, Jane Mabey | Joardar, Vinita | Deegan, Jennifer | Clutterbuck, John | Andersen, Mikael R. | Archer, David | Bencina, Mojca | Braus, Gerhard | Coutinho, Pedro | von Döhren, Hans | Doonan, John | Driessen, Arnold J.M. | Durek, Pawel | Espeso, Eduardo | Fekete, Erzsébet | Flipphi, Michel | Estrada, Carlos Garcia | Geysens, Steven | Goldman, Gustavo | de Groot, Piet W.J. | Hansen, Kim | Harris, Steven D. | Heinekamp, Thorsten | Helmstaedt, Kerstin | Henrissat, Bernard | Hofmann, Gerald | Homan, Tim | Horio, Tetsuya | Horiuchi, Hiroyuki | James, Steve | Jones, Meriel | Karaffa, Levente | Karányi, Zsolt | Kato, Masashi | Keller, Nancy | Kelly, Diane E. | Kiel, Jan A.K.W. | Kim, Jung-Mi | van der Klei, Ida J. | Klis, Frans M. | Kovalchuk, Andriy | Kraševec, Nada | Kubicek, Christian P. | Liu, Bo | MacCabe, Andrew | Meyer, Vera | Mirabito, Pete | Miskei, Márton | Mos, Magdalena | Mullins, Jonathan | Nelson, David R. | Nielsen, Jens | Oakley, Berl R. | Osmani, Stephen A. | Pakula, Tiina | Paszewski, Andrzej | Paulsen, Ian | Pilsyk, Sebastian | Pócsi, István | Punt, Peter J. | Ram, Arthur F.J. | Ren, Qinghu | Robellet, Xavier | Robson, Geoff | Seiboth, Bernhard | Solingen, Piet van | Specht, Thomas | Sun, Jibin | Taheri-Talesh, Naimeh | Takeshita, Norio | Ussery, Dave | vanKuyk, Patricia A. | Visser, Hans | van de Vondervoort, Peter J.I. | de Vries, Ronald P. | Walton, Jonathan | Xiang, Xin | Xiong, Yi | Zeng, An Ping | Brandt, Bernd W. | Cornell, Michael J. | van den Hondel, Cees A.M.J.J. | Visser, Jacob | Oliver, Stephen G. | Turner, Geoffrey
Fungal genetics and biology : FG & B  2008;46(Suppl 1):S2-13.
The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.
doi:10.1016/j.fgb.2008.12.003
PMCID: PMC2826280  PMID: 19146970
Aspergillus nidulans; aspergilli; genome; annotation; fungal community; assembly; transcription factors; CADRE
8.  Complete Genome Sequence of the Multiresistant Taxonomic Outlier Pseudomonas aeruginosa PA7 
PLoS ONE  2010;5(1):e8842.
Pseudomonas aeruginosa PA7 is a non-respiratory human isolate from Argentina that is multiresistant to antibiotics. We first sequenced gyrA, gyrB, parC, parE, ampC, ampR, and several housekeeping genes and found that PA7 is a taxonomic outlier. We report here the complete sequence of the 6,588,339 bp genome, which has only about 95% overall identity to other strains. PA7 has multiple novel genomic islands and a total of 51 occupied regions of genomic plasticity. These islands include antibiotic resistance genes, parts of transposons, prophages, and a pKLC102-related island. Several PA7 genes not present in PAO1 or PA14 are putative orthologues of other Pseudomonas spp. and Ralstonia spp. genes. PA7 appears to be closely related to the known taxonomic outlier DSM1128 (ATCC9027). PA7 lacks several virulence factors, notably the entire TTSS region corresponding to PA1690-PA1725 of PAO1. It has neither exoS nor exoU and lacks toxA, exoT, and exoY. PA7 is serotype O12 and pyoverdin type II. Preliminary proteomic studies indicate numerous differences with PAO1, some of which are probably a consequence of a frameshift mutation in the mvfR quorum sensing regulatory gene.
doi:10.1371/journal.pone.0008842
PMCID: PMC2809737  PMID: 20107499
9.  Comparative genomics of the neglected human malaria parasite Plasmodium vivax 
Nature  2008;455(7214):757-763.
The human malaria parasite Plasmodium vivax is responsible for 25-40% of the ~515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated in the laboratory except in non-human primates. We determined the genome sequence of P. vivax in order to shed light on its distinctive biologic features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternate invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance scientific investigation into this neglected species.
doi:10.1038/nature07327
PMCID: PMC2651158  PMID: 18843361
10.  Three Genomes from the Phylum Acidobacteria Provide Insight into the Lifestyles of These Microorganisms in Soils▿ †  
The complete genomes of three strains from the phylum Acidobacteria were compared. Phylogenetic analysis placed them as a unique phylum. They share genomic traits with members of the Proteobacteria, the Cyanobacteria, and the Fungi. The three strains appear to be versatile heterotrophs. Genomic and culture traits indicate the use of carbon sources that span simple sugars to more complex substrates such as hemicellulose, cellulose, and chitin. The genomes encode low-specificity major facilitator superfamily transporters and high-affinity ABC transporters for sugars, suggesting that they are best suited to low-nutrient conditions. They appear capable of nitrate and nitrite reduction but not N2 fixation or denitrification. The genomes contained numerous genes that encode siderophore receptors, but no evidence of siderophore production was found, suggesting that they may obtain iron via interaction with other microorganisms. The presence of cellulose synthesis genes and a large class of novel high-molecular-weight excreted proteins suggests potential traits for desiccation resistance, biofilm formation, and/or contribution to soil structure. Polyketide synthase and macrolide glycosylation genes suggest the production of novel antimicrobial compounds. Genes that encode a variety of novel proteins were also identified. The abundance of acidobacteria in soils worldwide and the breadth of potential carbon use by the sequenced strains suggest significant and previously unrecognized contributions to the terrestrial carbon cycle. Combining our genomic evidence with available culture traits, we postulate that cells of these isolates are long-lived, divide slowly, exhibit slow metabolic rates under low-nutrient conditions, and are well equipped to tolerate fluctuations in soil hydration.
doi:10.1128/AEM.02294-08
PMCID: PMC2663196  PMID: 19201974
11.  Genome Degradation in Brucella ovis Corresponds with Narrowing of Its Host Range and Tissue Tropism 
PLoS ONE  2009;4(5):e5519.
Brucella ovis is a veterinary pathogen associated with epididymitis in sheep. Despite its genetic similarity to the zoonotic pathogens B. abortus, B. melitensis and B. suis, B. ovis does not cause zoonotic disease. Genomic analysis of the type strain ATCC25840 revealed a high percentage of pseudogenes and increased numbers of transposable elements compared to the zoonotic Brucella species, suggesting that genome degradation has occurred concomitant with narrowing of the host range of B. ovis. The absence of genomic island 2, encoding functions required for lipopolysaccharide biosynthesis, as well as inactivation of genes encoding urease, nutrient uptake and utilization, and outer membrane proteins may be factors contributing to the avirulence of B. ovis for humans. A 26.5 kb region of B. ovis ATCC25840 Chromosome II was absent from all the sequenced human pathogenic Brucella genomes, but was present in all of 17 B. ovis isolates tested and in three B. ceti isolates, suggesting that this DNA region may be of use for differentiating B. ovis from other Brucella spp. This is the first genomic analysis of a non-zoonotic Brucella species. The results suggest that inactivation of genes involved in nutrient acquisition and utilization, cell envelope structure and urease may have played a role in narrowing of the tissue tropism and host range of B. ovis.
doi:10.1371/journal.pone.0005519
PMCID: PMC2677664  PMID: 19436743
12.  Complete Genome Sequence of the Aerobic CO-Oxidizing Thermophile Thermomicrobium roseum 
PLoS ONE  2009;4(1):e4207.
In order to enrich the phylogenetic diversity represented in the available sequenced bacterial genomes and as part of an “Assembling the Tree of Life” project, we determined the genome sequence of Thermomicrobium roseum DSM 5159. T. roseum DSM 5159 is a red-pigmented, rod-shaped, Gram-negative extreme thermophile isolated from a hot spring that possesses both an atypical cell wall composition and an unusual cell membrane that is composed entirely of long-chain 1,2-diols. Its genome is composed of two circular DNA elements, one of 2,006,217 bp (referred to as the chromosome) and one of 919,596 bp (referred to as the megaplasmid). Strikingly, though few standard housekeeping genes are found on the megaplasmid, it does encode a complete system for chemotaxis including both chemosensory components and an entire flagellar apparatus. This is the first known example of a complete flagellar system being encoded on a plasmid and suggests a straightforward means for lateral transfer of flagellum-based motility. Phylogenomic analyses support the recent rRNA-based analyses that led to T. roseum being removed from the phylum Thermomicrobia and assigned to the phylum Chloroflexi. Because T. roseum is a deep-branching member of this phylum, analysis of its genome provides insights into the evolution of the Chloroflexi. In addition, even though this species is not photosynthetic, analysis of the genome provides some insight into the origins of photosynthesis in the Chloroflexi. Metabolic pathway reconstructions and experimental studies revealed new aspects of the biology of this species. For example, we present evidence that T. roseum oxidizes CO aerobically, making it the first thermophile known to do so. In addition, we propose that glycosylation of its carotenoids plays a crucial role in the adaptation of the cell membrane to this bacterium's thermophilic lifestyle. Analyses of published metagenomic sequences from two hot springs similar to the one from which this strain was isolated, show that close relatives of T. roseum DSM 5159 are present but have some key differences from the strain sequenced.
doi:10.1371/journal.pone.0004207
PMCID: PMC2615216  PMID: 19148287
13.  Draft Genome of the Filarial Nematode Parasite Brugia malayi 
Science (New York, N.Y.)  2007;317(5845):1756-1760.
Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the ~90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict ~11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during ~350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.
doi:10.1126/science.1145406
PMCID: PMC2613796  PMID: 17885136
14.  High-Throughput Phenotypic Characterization of Pseudomonas aeruginosa Membrane Transport Genes 
PLoS Genetics  2008;4(10):e1000211.
The deluge of data generated by genome sequencing has led to an increasing reliance on bioinformatic predictions, since the traditional experimental approach of characterizing gene function one at a time cannot possibly keep pace with the sequence-based discovery of novel genes. We have utilized Biolog phenotype MicroArrays to identify phenotypes of gene knockout mutants in the opportunistic pathogen and versatile soil bacterium Pseudomonas aeruginosa in a relatively high-throughput fashion. Seventy-eight P. aeruginosa mutants defective in predicted sugar and amino acid membrane transporter genes were screened and clear phenotypes were identified for 27 of these. In all cases, these phenotypes were confirmed by independent growth assays on minimal media. Using qRT-PCR, we demonstrate that the expression levels of 11 of these transporter genes were induced from 4- to 90-fold by their substrates identified via phenotype analysis. Overall, the experimental data showed the bioinformatic predictions to be largely correct in 22 out of 27 cases, and led to the identification of novel transporter genes and a potentially new histamine catabolic pathway. Thus, rapid phenotype identification assays are an invaluable tool for confirming and extending bioinformatic predictions.
Author Summary
Genome sequencing has led to the identification of literally millions of new genes, for which there is no experimental evidence concerning their function. This limits our knowledge of these genes to computational predictions; however, the accuracy of such bioinformatic predictions is essentially unknown. We have focused on investigating the accuracy of bioinformatic predictions for a specific class of genes—those encoding membrane transporters. Our approach used Biolog phenotype MicroArrays to screen transporter gene knockout mutants in the bacterium P. aeruginosa for the ability to metabolize hundreds of different compounds. We were able to identify functions for 27 out of 78 genes, all of which were confirmed through independent growth assays. For 80% of these genes, the computationally predicted and experimentally determined functions were either identical or generically similar. Additionally, this led to the discovery of entirely new types of transporters and a novel potential histamine metabolic pathway.
doi:10.1371/journal.pgen.1000211
PMCID: PMC2542419  PMID: 18833300
15.  Genome of the Epsilonproteobacterial Chemolithoautotroph Sulfurimonas denitrificans▿ † 
Sulfur-oxidizing epsilonproteobacteria are common in a variety of sulfidogenic environments. These autotrophic and mixotrophic sulfur-oxidizing bacteria are believed to contribute substantially to the oxidative portion of the global sulfur cycle. In order to better understand the ecology and roles of sulfur-oxidizing epsilonproteobacteria, in particular those of the widespread genus Sulfurimonas, in biogeochemical cycles, the genome of Sulfurimonas denitrificans DSM1251 was sequenced. This genome has many features, including a larger size (2.2 Mbp), that suggest a greater degree of metabolic versatility or responsiveness to the environment than seen for most of the other sequenced epsilonproteobacteria. A branched electron transport chain is apparent, with genes encoding complexes for the oxidation of hydrogen, reduced sulfur compounds, and formate and the reduction of nitrate and oxygen. Genes are present for a complete, autotrophic reductive citric acid cycle. Many genes are present that could facilitate growth in the spatially and temporally heterogeneous sediment habitat from where Sulfurimonas denitrificans was originally isolated. Many resistance-nodulation-development family transporter genes (10 total) are present; of these, several are predicted to encode heavy metal efflux transporters. An elaborate arsenal of sensory and regulatory protein-encoding genes is in place, as are genes necessary to prevent and respond to oxidative stress.
doi:10.1128/AEM.01844-07
PMCID: PMC2258580  PMID: 18065616
16.  Complete Genome Sequence of the N2-Fixing Broad Host Range Endophyte Klebsiella pneumoniae 342 and Virulence Predictions Verified in Mice 
PLoS Genetics  2008;4(7):e1000141.
We report here the sequencing and analysis of the genome of the nitrogen-fixing endophyte, Klebsiella pneumoniae 342. Although K. pneumoniae 342 is a member of the enteric bacteria, it serves as a model for studies of endophytic, plant-bacterial associations due to its efficient colonization of plant tissues (including maize and wheat, two of the most important crops in the world), while maintaining a mutualistic relationship that encompasses supplying organic nitrogen to the host plant. Genomic analysis examined K. pneumoniae 342 for the presence of previously identified genes from other bacteria involved in colonization of, or growth in, plants. From this set, approximately one-third were identified in K. pneumoniae 342, suggesting additional factors most likely contribute to its endophytic lifestyle. Comparative genome analyses were used to provide new insights into this question. Results included the identification of metabolic pathways and other features devoted to processing plant-derived cellulosic and aromatic compounds, and a robust complement of transport genes (15.4%), one of the highest percentages in bacterial genomes sequenced. Although virulence and antibiotic resistance genes were predicted, experiments conducted using mouse models showed pathogenicity to be attenuated in this strain. Comparative genomic analyses with the presumed human pathogen K. pneumoniae MGH78578 revealed that MGH78578 apparently cannot fix nitrogen, and the distribution of genes essential to surface attachment, secretion, transport, and regulation and signaling varied between each genome, which may indicate critical divergences between the strains that influence their preferred host ranges and lifestyles (endophytic plant associations for K. pneumoniae 342 and presumably human pathogenesis for MGH78578). Little genome information is available concerning endophytic bacteria. The K. pneumoniae 342 genome will drive new research into this less-understood, but important category of bacterial-plant host relationships, which could ultimately enhance growth and nutrition of important agricultural crops and development of plant-derived products and biofuels.
Author Summary
Bacterial endophytes are capable of inhabiting the living tissues of plants without causing them significant harm. Klebsiella pneumoniae 342 (Kp342) is a model for this plant host-bacterial association, in part due to its capacity to colonize in high numbers the interior of plants including wheat and maize, two of the most important crops in the world. Kp342 possesses the ability to capture atmospheric nitrogen gas and turn it into an organic form (a process known as nitrogen fixation), of which part may be used as fertilizer by its plant host. Here, we describe the genome sequence and analysis of this model endophyte. When the Kp342 genome is compared to the genome of a closely related pathogenic relative, we can begin to surmise that its preference to engage in a harmonious relationship with plants is a result of many interacting factors. These include differences in its protein secretion systems, the manner in which its genes are regulated, and its ability to sense and respond to its environment. The study of endophytes is increasing in intensity due to the roles they may play in multiple biotechnological applications, including enhancing crop growth and nutrition, bioremediation, and development of plant-derived products and biofuels.
doi:10.1371/journal.pgen.1000141
PMCID: PMC2453333  PMID: 18654632
17.  Draft Genome Sequence of the Sexually Transmitted Pathogen Trichomonas vaginalis 
Science (New York, N.Y.)  2007;315(5809):207-212.
We describe the genome sequence of the protist Trichomonas vaginalis, a sexually transmitted human pathogen. Repeats and transposable elements comprise about two-thirds of the ~160-megabase genome, reflecting a recent massive expansion of genetic material. This expansion, in conjunction with the shaping of metabolic pathways that likely transpired through lateral gene transfer from bacteria, and amplification of specific gene families implicated in pathogenesis and phagocytosis of host proteins may exemplify adaptations of the parasite during its transition to a urogenital environment. The genome sequence predicts previously unknown functions for the hydrogenosome, which support a common evolutionary origin of this unusual organelle with mitochondria.
doi:10.1126/science.1132894
PMCID: PMC2080659  PMID: 17218520
18.  Genome Sequence of Babesia bovis and Comparative Analysis of Apicomplexan Hemoprotozoa 
PLoS Pathogens  2007;3(10):e148.
Babesia bovis is an apicomplexan tick-transmitted pathogen of cattle imposing a global risk and severe constraints to livestock health and economic development. The complete genome sequence was undertaken to facilitate vaccine antigen discovery, and to allow for comparative analysis with the related apicomplexan hemoprotozoa Theileria parva and Plasmodium falciparum. At 8.2 Mbp, the B. bovis genome is similar in size to that of Theileria spp. Structural features of the B. bovis and T. parva genomes are remarkably similar, and extensive synteny is present despite several chromosomal rearrangements. In contrast, B. bovis and P. falciparum, which have similar clinical and pathological features, have major differences in genome size, chromosome number, and gene complement. Chromosomal synteny with P. falciparum is limited to microregions. The B. bovis genome sequence has allowed wide scale analyses of the polymorphic variant erythrocyte surface antigen protein (ves1 gene) family that, similar to the P. falciparum var genes, is postulated to play a role in cytoadhesion, sequestration, and immune evasion. The ∼150 ves1 genes are found in clusters that are distributed throughout each chromosome, with an increased concentration adjacent to a physical gap on chromosome 1 that contains multiple ves1-like sequences. ves1 clusters are frequently linked to a novel family of variant genes termed smorfs that may themselves contribute to immune evasion, may play a role in variant erythrocyte surface antigen protein biology, or both. Initial expression analysis of ves1 and smorf genes indicates coincident transcription of multiple variants. B. bovis displays a limited metabolic potential, with numerous missing pathways, including two pathways previously described for the P. falciparum apicoplast. This reduced metabolic potential is reflected in the B. bovis apicoplast, which appears to have fewer nuclear genes targeted to it than other apicoplast containing organisms. Finally, comparative analyses have identified several novel vaccine candidates including a positional homolog of p67 and SPAG-1, Theileria sporozoite antigens targeted for vaccine development. The genome sequence provides a greater understanding of B. bovis metabolism and potential avenues for drug therapies and vaccine development.
Author Summary
Vector-transmitted blood parasites cause some of the most widely distributed, serious, and poorly controlled diseases globally, including the most severe form of human malaria caused by Plasmodium falciparum. In livestock, tick-transmitted blood parasites include the protozoa Theileria parva, the cause of East Coast fever and Babesia bovis, the cause of tick fever, to which well over half of the world's cattle population are at risk. There is a critical need to better understand the mechanisms by which these parasites are transmitted, persist, and cause disease in order to optimize methods for control, including development of vaccines. This manuscript presents the genome sequence of B. bovis, and provides a whole genome comparative analysis with P. falciparum and T. parva. Genome-wide characterization of the B. bovis antigenically variable ves1 family reveals interesting differences in organization and expression from the related P. falciparum var genes. The second largest gene family (smorf) in B. bovis was newly discovered and may itself be involved in persistence, highlighting the utility of this approach in gene discovery. Organization and structure of the B. bovis genome is most similar to that of Theileria, and despite common features in clinical outcome is limited to microregional similarity with P. falciparum. Comparative gene analysis identifies several previously unknown proteins as homologs of vaccine candidates in one or more of these parasites, and candidate genes whose expression might account for unique properties such as the ability of Theileria to reversibly transform leukocytes.
doi:10.1371/journal.ppat.0030148
PMCID: PMC2034396  PMID: 17953480
19.  Phosphorylation of the SQ H2A.X Motif Is Required for Proper Meiosis and Mitosis in Tetrahymena thermophila▿  
Molecular and Cellular Biology  2007;27(7):2648-2660.
Phosphorylation of the C terminus SQ motif that defines H2A.X variants is required for efficient DNA double-strand break (DSB) repair in diverse organisms but has not been studied in ciliated protozoa. Tetrahymena H2A.X is one of two similarly expressed major H2As, thereby differing both from mammals, where H2A.X is a quantitatively minor component, and from Saccharomyces cerevisiae where it is the only type of major H2A. Tetrahymena H2A.X is phosphorylated in the SQ motif in both the mitotic micronucleus and the amitotic macronucleus in response to DSBs induced by chemical agents and in the micronucleus during prophase of meiosis, which occurs in the absence of a synaptonemal complex. H2A.X is phosphorylated when programmed DNA rearrangements occur in developing macronuclei, as for immunoglobulin gene rearrangements in mammals, but not during the DNA fragmentation that accompanies breakdown of the parental macronucleus during conjugation, correcting the previous interpretation that this process is apoptosis-like. Using strains containing a mutated (S134A) SQ motif, we demonstrate that phosphorylation of this motif is important for Tetrahymena cells to recover from exogenous DNA damage and is required for normal micronuclear meiosis and mitosis and, to a lesser extent, for normal amitotic macronuclear division; its absence, while not lethal, leads to the accumulation of DSBs in both micro- and macronuclei. These results demonstrate multiple roles of H2A.X phosphorylation in maintaining genomic integrity in different phases of the Tetrahymena life cycle.
doi:10.1128/MCB.01910-06
PMCID: PMC1899910  PMID: 17242195
20.  Genome Sequence of Aeromonas hydrophila ATCC 7966T: Jack of All Trades▿  
Journal of Bacteriology  2006;188(23):8272-8282.
The complete genome of Aeromonas hydrophila ATCC 7966T was sequenced. Aeromonas, a ubiquitous waterborne bacterium, has been placed by the Environmental Protection Agency on the Contaminant Candidate List because of its potential to cause human disease. The 4.7-Mb genome of this emerging pathogen shows a physiologically adroit organism with broad metabolic capabilities and considerable virulence potential. A large array of virulence genes, including some identified in clinical isolates of Aeromonas spp. or Vibrio spp., may confer upon this organism the ability to infect a wide range of hosts. However, two recognized virulence markers, a type III secretion system and a lateral flagellum, that are reported in other A. hydrophila strains are not identified in the sequenced isolate, ATCC 7966T. Given the ubiquity and free-living lifestyle of this organism, there is relatively little evidence of fluidity in terms of mobile elements in the genome of this particular strain. Notable aspects of the metabolic repertoire of A. hydrophila include dissimilatory sulfate reduction and resistance mechanisms (such as thiopurine reductase, arsenate reductase, and phosphonate degradation enzymes) against toxic compounds encountered in polluted waters. These enzymes may have bioremediative as well as industrial potential. Thus, the A. hydrophila genome sequence provides valuable insights into its ability to flourish in both aquatic and host environments.
doi:10.1128/JB.00621-06
PMCID: PMC1698176  PMID: 16980456
21.  Comparative Genomic Evidence for a Close Relationship between the Dimorphic Prosthecate Bacteria Hyphomonas neptunium and Caulobacter crescentus 
Journal of Bacteriology  2006;188(19):6841-6850.
The dimorphic prosthecate bacteria (DPB) are α-proteobacteria that reproduce in an asymmetric manner rather than by binary fission and are of interest as simple models of development. Prior to this work, the only member of this group for which genome sequence was available was the model freshwater organism Caulobacter crescentus. Here we describe the genome sequence of Hyphomonas neptunium, a marine member of the DPB that differs from C. crescentus in that H. neptunium uses its stalk as a reproductive structure. Genome analysis indicates that this organism shares more genes with C. crescentus than it does with Silicibacter pomeroyi (a closer relative according to 16S rRNA phylogeny), that it relies upon a heterotrophic strategy utilizing a wide range of substrates, that its cell cycle is likely to be regulated in a similar manner to that of C. crescentus, and that the outer membrane complements of H. neptunium and C. crescentus are remarkably similar. H. neptunium swarmer cells are highly motile via a single polar flagellum. With the exception of cheY and cheR, genes required for chemotaxis were absent in the H. neptunium genome. Consistent with this observation, H. neptunium swarmer cells did not respond to any chemotactic stimuli that were tested, which suggests that H. neptunium motility is a random dispersal mechanism for swarmer cells rather than a stimulus-controlled navigation system for locating specific environments. In addition to providing insights into bacterial development, the H. neptunium genome will provide an important resource for the study of other interesting biological processes including chromosome segregation, polar growth, and cell aging.
doi:10.1128/JB.00111-06
PMCID: PMC1595504  PMID: 16980487
23.  The Genome of Deep-Sea Vent Chemolithoautotroph Thiomicrospira crunogena XCL-2 
PLoS Biology  2006;4(12):e383.
Presented here is the complete genome sequence of Thiomicrospira crunogena XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 base pairs), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of coding sequences (CDSs) encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety of options for acquiring these substrates from the environment. Thiom. crunogena XCL-2 is unusual among obligate sulfur-oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome.
Secrets of the deep are revealed from the genome sequence ofThiomicrospira crunogena XCL-2, a chemolithoautotrophic sulfur-oxidizing gammaproteobacterium isolated from deep-sea hydrothermal vents.
doi:10.1371/journal.pbio.0040383
PMCID: PMC1635747  PMID: 17105352
24.  TransportDB: a comprehensive database resource for cytoplasmic membrane transport systems and outer membrane channels 
Nucleic Acids Research  2006;35(Database issue):D274-D279.
TransportDB () is a comprehensive database resource of information on cytoplasmic membrane transporters and outer membrane channels in organisms whose complete genome sequences are available. The complete set of membrane transport systems and outer membrane channels of each organism are annotated based on a series of experimental and bioinformatic evidence and classified into different types and families according to their mode of transport, bioenergetics, molecular phylogeny and substrate specificities. User-friendly web interfaces are designed for easy access, query and download of the data. Features of the TransportDB website include text-based and BLAST search tools against known transporter and outer membrane channel proteins; comparison of transporter and outer membrane channel contents from different organisms; known 3D structures of transporters, and phylogenetic trees of transporter families. On individual protein pages, users can find detailed functional annotation, supporting bioinformatic evidence, protein/DNA sequences, publications and cross-referenced external online resource links. TransportDB has now been in existence for over 10 years and continues to be regularly updated with new evidence and data from newly sequenced genomes, as well as having new features added periodically.
doi:10.1093/nar/gkl925
PMCID: PMC1747178  PMID: 17135193
25.  Macronuclear Genome Sequence of the Ciliate Tetrahymena thermophila, a Model Eukaryote 
PLoS Biology  2006;4(9):e286.
The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.
The macronuclear genome ofTetrahymena thermophila is sequenced and analyzed. Conservation in this single-celled ciliate of some features normally observed in only multicellular organisms sheds light on early eukaryotic evolution.
doi:10.1371/journal.pbio.0040286
PMCID: PMC1557398  PMID: 16933976

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