THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 5, pp. 2654 2662, February 1, by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
B-cell Receptor Activation Induces BIC/miR-155 Expression through a Conserved AP-1 Element*
Received for publication, October 3, 2007, and in revised form, November 28, 2007 Published, JBC Papers in Press, November 28, 2007, DOI 10.1074/jbc.M708218200
Qinyan Yin, Xia Wang, Jane McBride, Claire Fewell, and Erik Flemington1 From the Department of Pathology, Tulane Health Sciences Center and Tulane Cancer Center, New Orleans, Louisiana, 70112
microRNA-155 is an oncogenic microRNA that has been shown to be critical for B-cell maturation and immunoglobulin production in response to antigen. In line with its function in B-cell activation, miR-155, and its primary transcript, B-cell integration cluster (BIC), is induced by B-cell receptor (BCR) cross-linking. Using pharmacological inhibitors in the human B-cell line, Ramos, we show that activation of BIC and miR-155 expression by BCR signaling occurs through the extracellular signaling-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways but not the p38 pathway. BCR activation results in the induction of c-Fos, FosB, and JunB, and expression of these are suppressed by ERK and JNK inhibitors. Reporter analysis established a key role for a conserved AP-1 site 40 bp upstream from the site of initiation but not an upstream NF-B site or a putative c-Ets located at the site of initiation. Lastly, chromatin immunoprecipitation analysis demonstrated the recruitment of FosB and JunB to the miR-155 promoter following BCR activation. These results identify key determinants of BCR-mediated signaling that lead to the induction of BIC/miR-155.
MicroRNAs (miRNAs)2 have been shown to be key mediators of cell regulatory processes such as those controlling cell growth, differentiation, and development (13). The ability of at least some miRNAs to significantly alter cell processes and cell fate is attributable to their capacity to influence the expression of a large number of target mRNA species. miRNAs function to inhibit translation of mRNAs through specific but imperfect base pairing with their 3-untranslated regions. The binding of miRNA protein complexes to mRNAs results in
* This work was supported by the National Institutes of Health research
Grants GM48045 (to E. K. F.), DE017008 (to E. K. F.), and R01CA124311 (to E. K. F.), a grant from the Lymphoma Research Foundation (to Q. Y.), and a Mentoring a Program in Cancer Genetics National Institutes of Health Center of Biomedical Research Excellence award (to Prescott DeiningerP20 RR020152). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed: Dept. of Pathology, SL79, Tulane Health Sciences Center, New Orleans, LA 70112. Fax: 504-988-5516; E-mail: eflemin@tulane.edu. 2 The abbreviations used are: miRNA, microRNA; BIC, B-cell integration cluster; BCR, B-cell receptor; TLR, Toll-like receptor; ERK, extracellular signaling-regulated kinase; JNK, c-Jun NH2-terminal kinase; MEK, mitogen-activated protein kinase/ERK kinase; TNF, tumor necrosis factor; RT, reverse transcription; qRT, quantitative RT; G3PDH, glyceraldehyde3-phosphate dehydrogenase; RACE, rapid amplification of cDNA ends; RIPA, radioimmune precipitation buffer; EBV, Epstein-Barr virus; EST, expressed sequence tag; IL, interleukin.

localization of the miRNA-protein-mRNA complex to a perinuclear compartment referred to as GW or P bodies, thereby preventing access to ribosomes, and in some cases, leading to the degradation of the respective mRNA (4, 5). The microRNA, miR-155, is processed from a primary transcript, referred to as B-cell integration cluster (BIC), whose upstream region was originally identified as a common site of integration of the avian leukosis virus in lymphomas (6). Transgenic mouse studies demonstrated that B-cell targeted expression of BIC leads to the development of B-cell malignancies (7). Further, a number of miRNA profiling studies have shown elevation of miR-155 in a wide array of cancers including lymphomas (714). To date, miR-155 is one of the most highly implicated microRNAs in cancers. miR-155 has been shown to play a critical role in lymphocyte activation in vivo (15, 16) and is induced by a number of immune cell stimuli including Toll-like receptor (TLR) ligands, tumor necrosis factor- (TNF-), interferon-, and antigen (B-cell receptor (BCR) engagement) (9, 17, 18). The mechanisms through which miR-155 is regulated following TLR and interferon signaling in macrophages has recently come under study (17). In this study, TLR ligand-mediated activation of miR-155 was shown to occur through myeloid differentiation factor 88 (MyD88) and Toll/IL-1 receptor domain-containing adaptor inducing interferon- (TRIF)-dependent pathways (17). Interferon signaling was found to require an autocrine pathway involving TNF-. Lastly, induction of miR-155 by poly(I-C) and TNF- were shown to be inhibited by a Junactivated kinase (JNK) inhibitor, suggesting that this pathway plays a role in induction of miR-155 in these systems. Here we have begun to analyze signaling pathways involved in activating miR-155 following B-cell receptor (BCR) engagement. This analysis identified critical pathways required for BCR-mediated miR-155 activation, some of which likely overlap with pathways activated by TLR and TNF- signaling in macrophages. Together these studies define some of the fundamental miR155 regulatory processes following immune cell activation and lays the groundwork for understanding some of the mechanisms through which BIC/miR-155 is overexpressed in tumors.
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EXPERIMENTAL PROCEDURES Cell Culture and TreatmentsThe EBV-negative human Burkitt lymphoma cell line, Ramos, was cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and penicillin/streptomycin (Invitrogen). B-cell receptor cross-linking experiments were carried out by exposure to anti-human-IgM (Sigma, catalog number I 0759). An equal volume of fresh medium was added to Ramos cells
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AP-1 Activation of BIC/miR-155
(which were at densities of 12 106/ml) 1 day before treatment. On the day of treatment, cells were counted, and cells were added to 10 ml of fresh complete RPMI medium containing 10 M anti-IgM. Inhibitor experiments were carried out as above except that the inhibitors (all purchased from Calbiochem) were added to cells resuspended in fresh medium 30 min prior to the addition of anti-IgM (to a final concentration of 10 M). For RT-PCR analysis, cells were harvested 24 h following the addition of anti-IgM. For Western blot analysis, cells were treated as above except that cells were harvested at the indicated times (in the figures and/or figure legends for Figs. 4 and 5). RNA Preparation and Real-time RT-PCRTotal RNA was prepared using a Qiagen miRNeasy mini prep kit (catalog number 217004) according to the vendors protocol. For primary BIC transcript analysis, 2 g of total RNA was reverse-transcribed to make cDNA using SuperScriptTM III first-strand synthesis system (Invitrogen, catalog number 18080-051). PCR reactions were carried out using the following primers and conditions: BIC forward (BIC-b), 5-CTCTAATGGTGGCACAAA-3; BIC reverse (BIC-c), 5-TGATAAAAACAAACATGGGCTTGAC-3 (14); G3PDH forward, 5-GCCAAGGTCATCCATGACAACTTTGG-3, and G3PDH reverse, 5-GCCTGCTTCACCACCTTCTTGATGTC-3 (19). PCR reactions were performed using the following parameters: 95 C for 3 min followed by 40 cycles of denaturation (95 C 30 s), annealing (62 C 40 s), and extension (72 C 40 s). Real-time PCR was conducted with 2 l of 10-fold diluted cDNA using iQTMSYBR Green Supermix (Bio-Rad catalog number 170-8882) and an iQ5 multicolor real-time PCR detection system (Bio-Rad). Mature miR-155 expression was assessed using a mirVanaTM qRT-PCR miRNA detection kit (Ambion, catalog number 1558) kit with the mirVana qRT-PCR miR-155 primer set (Ambion, catalog number 30302) according to the manufacturers protocol. PCR was carried out using the following conditions: 95 C for 3 min followed by 40 cycles of 95 C, 15 s and 60 C, 30 s. The expression of BIC and miR-155 in experimental samples was determined by the comparative Ct method (2-()()Ct method), in which Ct is the threshold cycle and Ct (Ct BIC) (Ct reference RNA (G3PDH)). BLAST Analysis of miR-155 Promoter versus Mouse Genome 1939 bases of sequence upstream and 562 bases of sequence downstream from the miR-155 start site in humans were used to blast the mouse genome using the National Center for Biotechnology Information (NCBI) Blastn algorithm with default settings. A single homology shown in Fig. 3 was identified as the only hit and corresponds to 8,703,897 8,703,794 and 8,703,618 8,703,488 of the mouse chromosome 11 (NCBI, reference assembly). 5 RACE Analysis5 RACE was carried out using total RNA isolated from the human B-cell line, Ramos, following exposure to anti-IgM, using a SMART RACE cDNA amplification kit (Clontech) according to the manufacturers protocol. The gene-specific primer, 5-CAGCCTACAGCAAGCCTTCAGCACTC-3, which is complementary to the third exon of the human BIC/miR-155 primary transcript was used. One major PCR product with a size of 350 bp was obtained and cloned into the PCR4-TOPO cloning vector by TA cloning. Seven positive clones were sequenced to determine the 5 most end in each

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case. The major start site of the human BIC transcript was found to be located at nucleotide 12,596,314 of the human chromosome 21 (NCBI, reference assembly). Cloning and Mutagenesis of BIC/miR-155 PromoterThe human BIC/miR-155 promoter extending from 1494 to 228 relative to the start site was isolated from Mutu genomic DNA by PCR using the primers 5-GCAGCTAGCCCAGGGTTGGAACTGAGTTTGA-3 (forward primer) and 5-GCAAAGCTTCAGTTAACCCGGCGGTGA-3 (reverse primer). The isolated fragment was digested with NheI and HindIII and cloned into NheI and HindIII cut pGL3basic (Promega). The entire promoter region was then sequenced, and no discrepancies were identified relative to the GenBankTM genomic sequence. Mutagenesis of the miR-155 reporter plasmid was carried out using a QuikChange II site-directed mutagenesis kit (Stratagene) using the following oligonucleotides: 5-GTAAATTAAGTACTATGCTCGAGCCAGCTCTGACATG-3 and 5CATGTCAGAGCTGGCTCGAGCATAGTACTTAATTTAC-3 (NF-B), 5-CTGGTCGGTTATCTCGAGCAAGTGAGTTAT-3 and 5-ATAACTCACTTGCTCGAGATAACCGACCAG-3 (AP-1), and 5-CGCAGGCGCGGCTCGAGTGTGCGCGGCC-3 and 5-GGCCGCGCACACTCGAGCCGCGCCTGCG-3 (c-Ets). In each case, the core transcription factor binding site was replaced with an XhoI restriction site. Mutations were initially screened by digesting with XhoI and then verified by sequence analysis. Reporter AnalysisFor each reporter plasmid, Ramos cells were distributed into each of two T25 tissue culture flasks containing 10 ml of RPMI 1640 (10% fetal bovine serum, penicillin/streptomycin). 5 g of the respective reporter vector plus 35 g of the carrier plasmid, pUHD10, were mixed with 2.5 ml of Opti-MEM. 2.5 ml of diluted Lipofectamine 2000 (100 l of Lipofectamine plus 2.5 ml of optiMEM) was then added to each set of DNA/opti-MEM mixtures, and the tubes were mixed and incubated at room temperature for 20 min. For each transfection, 2.5 ml of DNA/ Lipofectamine mixture was then added to each of two separate T25 flasks containing cells, and flasks were incubated at 37 C, 5% CO2 for 24 h. At this time, affinity-isolated goat anti human IgM (catalog number I0759, Sigma) was added to a final concentration of 10 M to the second flask for each reporter. Cells were harvested 24 h later and assayed for luciferase activity. Results are presented as the average of three independent experiments. The reporter analyses shown in Fig. 7C were carried out as above except that cells were not treated with anti-IgM and cells were co-transfected with reporters plus either 1 g of a control, JunB, or FosB expression vector or 0.5 g of JunB plus 0.5 g of FosB expression vectors. The reporter analyses of anti-IgM treated Mutu E1dn Cl.3 cells (20) were carried out as described for the anti-IgM analysis in Ramos cells except that cells were transfected by electroporation. Western Blot AnalysisRamos cells were treated as discussed above prior to harvesting for Western blot analysis. For whole cell lysates, cells were suspended in 1 SDS loading buffer and heated for 10 min at 90 C to shear genomic DNA. To generate nuclear extracts, Ramos cells were suspended in 300 l of hypotonic buffer (HEPES (pH 7.9), 10 mM

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KCl, 0.1 mM EDTA, 1 mM dithiothreitol, plus protease and phosphatase inhibitor cocktails), 18 l of 10% Nonidet P-40 was added, and the mixture was vortexed on high for 10 s. The tubes were transferred to a microcentrifuge and spun for 2 min on high. The supernatant was taken off, and 100 l of nuclear extraction buffer (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, plus 1 mM dithiothreitol and protease and phosphatase inhibitor cocktails) was added, and the pellet was suspended immediately by vigorous pipetting. The tubes were put on a rotator for 15 min at 4 C and spun for 5 min in a microcentrifuge (14,000 rpm). The supernatant was transferred to new tubes and quantitated for use in Western blot analysis. Antibodies recognizing phospho-ERK (Cell Signaling (catalog number 9106)), total ERK (Cell Signaling (catalog number 9102)), c-Jun (Cell Signaling (catalog number 2315 and catalog number 9165)), c-Fos (Cell Signaling (catalog number 2250)) and FosB (Cell Signaling catalog number 2251)), JunB (Santa Cruz Biotechnology (sc-8051)), and actin (Sigma (catalog number A4700)) were used for Western blot analysis. Western blotting was carried out using 25 g of total cell lysate of each cell lysate. Signal detection was carried out using an Odyssey infrared imaging system (LI-COR Biosciences). ImmunoprecipitationFor immunoprecipitation, cells were suspended in 500 l of RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 0.1% SDS) and incubated for 1 h at 4 C. Lysates were then centrifuged at 13,000 rpm for 10 min at 4 C, and the supernatants were transferred to new tubes. Cell lysates were precleared with 25 l of protein A (for polyclonal antibody) or protein G (for monoclonal antibody) Sepharose (GE Healthcare) for 1 h. At the same time, 5 l of the FosB antibody (Cell Signaling, catalog number 2251) and 20 l of the JunB antibody (Santa Cruz, catalog number sc-8051) were incubated with 20 l of protein A- or G-Sepharose in 400 l of RIPA buffer for 1 h at 4 C and then washed three times with RIPA buffer. The lysates were then centrifuged briefly, and the supernatants were transferred to the antibody-Sepharose complexes and incubated overnight on a rotator at 4 C. The beads were washed three times with RIPA buffer with 5-min incubations (at 4 C) for each wash. 25 l of 2 SDS page loading buffer was then added to Sepharose bead complexes, and samples were heated for 5 min at 95 C and loaded onto an SDS-PAGE gel for Western blot analysis. Chromatin ImmunoprecipitationRamos cells were cultured for 2 h in the absence or presence of 10 M anti-IgM (Sigma, catalog number I 0759) (cells were used for each immunoprecipitation). Cells were then fixed with 1% formaldehyde for 30 min. Glycine was added to a final concentration of 0.125 M, and cells were spun down. Cells were washed 2 with 1 phosphate-buffered saline. Cell pellets were then suspended in ice-cold RIPA buffer and incubated on ice for 1 h. Cell lysates were then sonicated to obtain DNA fragments averaging 600 bp. Lysates were then spun on high speed in a microcentrifuge at 4 C for 15 min, and supernatant was used for immunoprecipitations. For each immunoprecipitation, 350 l of samples was precleared with 50 l of protein A agarose beads with overnight incubation on a rotator at 4 C. Beads were spun out, and supernatant was transferred to new tubes containing 50 l of protein A agarose beads for a 4-h second preclearing

Downloaded from www.jbc.org by guest, on June 8, 2011 FIGURE 1. Inhibition of BIC and miR-155 expression by MEK and JNK inhibitors following activation of BCR signaling. Ramos cells were pretreated with the indicated inhibitors prior to the addition of anti-IgM. Total RNAs were then analyzed for BIC (A) and miR-155 (B) expression by real-time reverse transcription-PCR. All values are relative to G3PDH expression.
step. Samples were then spun, and supernatant was transferred to a new tube containing the following antibodies: FosB (20 l, Santa Cruz (catalog number sc-48X)), JunB (20 l, Santa Cruz (catalog number sc-73X)), histone H4 (5 l of H4, Abcam (catalog number ab31827)), and acetyl-histone H4 (lys8) (5 l of Ac-H4, Upstate Biochemicals (catalog number 07-328)). Tubes were rotated at 4 C overnight, and 50 l of protein A agarose beads was added for a 2-h incubation on rotator at 4 C. Samples were spun briefly, and supernatant was aspirated off. Beads were washed three times with 1 phosphate-buffered saline. Beads were incubated with 100 l of Tris-EDTA containing 50 g/ml RNase A at 37 C for 1 h. 5 l of 10% SDS plus 2 l of 1 mg/ml proteinase K were added, and beads were incubated for 4 h at 42 C with occasional mixing. Samples were then incubated overnight at 65 C, phenol/chloroform-extracted, ethaVOLUME 283 NUMBER 5 FEBRUARY 1, 2008
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FIGURE 2. Transcript analysis of BIC. The indicated ESTs were identified by BLAST analysis of the EST data base using the miR-155 encoding exon of BIC. The start site in BCR-activated Ramos cells was derived by 5 RACE analysis.
nol-precipitated, and resuspended in 20 l of H2O. 1 l was used for PCR reactions using the following conditions: 95 C for 30 s, C for 30 s, 72 C for 30 s, 95 C for 30 s. Primers used were as follows: BIC promoter primers, BICps, 5-CCTGGTCGGTTATGAGTCAC-3 and BICpas, 5-GAGACTGAAGTCGGCGTACC-3; and BIC intron 1 primers, BICintron1S, 5-CATGGAAGGGTGACAAAACA-3 and BICintron1AS, 5-CGTTTTCCATTTGCCTAACA-3. Electrophoretic Mobility Shift AnalysisFor each binding reaction, 10 g of nuclear extract was combined with 0.1 pmol of IRdye700-labeled probe (LI-COR Biosciences) using standard binding conditions (LI-COR Biosciences (part number 82907910) standard protocol). Labeled AP-1 probe oligonucleotides were purchased from LI-COR Biosciences: AP-1 sense, 5-TCGGTTATGAGTCACAAGTGA-3 and AP-1 antisense 5-TCACTTGTGACTCATAACCGA-3. Unlabeled competitor oligonucleotides were purchased from Integrated DNA Technologies: AP-1wt sense, 5-TCGGTTATGAGTCACAAGTGA-3, AP-1wt antisense, 5-TCACTTGTGACTCATAACCGA-3, AP-1mutant sense, 5-TCGGTTATCTCGAGCAAGTGA-3, and AP-1 mutant antisense, 5-TCACTTGCTCGAGATAACCGA-3. Supershift experiments were carried out using the following antibodies: JunB (Santa Cruz Biotechnology (sc-8051X)), FosB (Santa Cruz Biotechnology (sc-48X)) and normal rabbit IgG (sc-2027). Competition and supershift

experiments were performed by preincubation with extract in binding buffer for 10 min, after which labeled probe was added for a further 20-min incubation at room temperature. Reactions were then loaded onto a pre-run 4% polyacrylamide retardation gel and run for 3 h at 300 V. Gels were scanned using an odyssey infrared imaging system (Li-Cor Biosciences).

RESULTS

Induction of BIC/miR-155 Transcription following BCR Activation Requires the ERK and JNK Signaling PathwaysTo identify pathways involved in mediating BIC promoter activation following BCR activation, the human B-cell line, Ramos, was treated with anti-IgM in the presence of ERK, JNK, or p38 pathway inhibitors. Cells were treated with either Me2SO or inhibitors of the upstream ERK effector, mitogen-activated protein kinase kinase (MEK (1/2)), PD98059 or U0126, the JNK inhibitor, SP600125, or the p38 inhibitor, SB203580, for 0.5 h prior to the addition of anti-IgM. 24 h later, cells were harvested, RNA was prepared, and the RNA was subjected to realtime RT-PCR to assess the levels of BIC and miR-155 transcripts. As shown in Fig. 1, the MEK inhibitors and the JNK inhibitor, SP600125 suppressed induction of BIC (Fig. 1A) as well as mature miR-155 (Fig. 1B), whereas the p38 inhibitor, SB203580, has little influence. These data demonstrate that
there may be differential promoter usage in different tissues. We therefore carried out 5 RACE using RNA from Ramos cells following exposure to anti-IgM. Seven independent clones were sequenced, and none of these contained the upstream exon sequences (Fig. 2). This analysis revealed that at least in activated B-cells, the BIC transcript is composed of three exons with the major initiation site located at nucleotide 12,596,314 of chromosome 21 (NCBI reference assembly), inline with most of the ESTs identified as well as the previously reported cDNA structure (21). Further, a classical TATA box sequence is located 24 bp upstream from the start site (Fig. 2A), which is within the 20 30-bp spacing that generally separates these two transcriptional features in TATA-containing promoters. Evolutionary Conservation of BIC Promoter SequencesTo gain insights into possible conserved regulatory elements in the BIC promoter, a BLAST search against the mouse genomic data base was carried out using sequences surrounding the human BIC start site (1939 bp upstream to 562 bp downstream). A single 75 nucleotide homology was identified that is roughly centered around the TATA box (Fig. 3A). Importantly, the homologous sequences are located on mouse chromosome kb upstream from FIGURE 3. The conserved AP-1 element is important for mediating activation of BIC transcription following BCR activation. A shows the core homology region of the BIC promoter. B shows schematic representation the mouse miR-155. This location is of human versus mouse promoter. C, analysis of wild type (wt) versus mutant (mNF-B, mAP-1, and mEts) positionally similar to the 11,835- bp reporters in response to BCR signaling in Ramos cells and Mutu E1dn Cl.3 (an EBV-negative derivative of the cell spacing between the human miR-155 line, Mutu (20)). sequence and the human sequences indicated in the promoter alignment. The human BIC promoter was then analyzed for potential activation of BIC transcription is mediated through the ERK transcription factor binding sites using the program and JNK pathways. Identification of the BIC Transcription Initiation Site in BCR- TFSEARCH (22). This analysis identified an AP-1 site and a activated Ramos CellsBIC/miR-155 expression is highly reg- c-Ets site with high probability scores within the human/mouse ulated at the transcription level. Based on reported cDNA homology region (Fig. 3A). Also notable is a high scoring NF-B sequences, BIC has been previously identified as a three-exon site located 1150 bp upstream from the human BIC transcripgene with the miR-155 sequence located in exon 3 (21). Never- tion start site (Fig. 3B). Although the AP-1 and c-Ets sites are theless, an analysis of the EST data base unexpectedly identified conserved in mouse, the NF-B site is not positionally conan EST (BI821816) that contained all three exons plus an addi- served. Instead, there is a putative NF-B site located more tional exon located 134 bp upstream from the 5 most extend- proximal to the human/mouse homology region (264 bases ing EST, CR99368 (Fig. 2). Moreover, the sequences for the upstream from the c-Ets site (Fig. 3B)). splice donor and acceptor of this transcript match well with The AP-1 Site Is Required for Activation of BIC Transcription consensus donor/acceptor consensus sequences. This raised following BCR EngagementThe putative NF-B, AP-1, and concerns that BIC may be composed of four exons and/or that c-Ets sites were considered to be good candidates for mediating

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FIGURE 4. The AP-1 family members, JunB, c-Fos, and FosB are induced by BCR cross-linking. Time course analysis shows early activation of phosphoERK (p-ERK) followed by induction of JunB, c-Fos, and FosB between 1 and 2 h after induction in Ramos cells. t-ERK, total ERK. FIGURE 5. Induction of JunB, c-Fos, and FosB is inhibited by ERK and JNK inhibitors. Ramos cells were pretreated with the indicated inhibitors prior to the addition of anti-IgM. Cells were lysed and subjected to Western blot analysis as described under Experimental Procedures. MEK1/2 Inh., MEK1/2 inhibition; JNK Inh., JNK inhibition; p38 Inh., p38 inhibition; DMSO, Me2SO; p-ERK, phospho-ERK.
or contributing to the activation of BIC following activation of the BCR. To assess the possible contribution of these elements in mediating BIC regulation, the human BIC promoter was cloned upstream from a luciferase reporter gene, and mutants were generated at each of these promoter elements. Wild type or mutant reporters were transfected into Ramos cells or an EBV-negative derivative of the cell line, Mutu (20), and were either left untreated or stimulated with an anti-IgM antibody. As shown in Fig. 3C, mutation of the NF-B site had little influence on promoter activation, and mutation of the c-Ets site had only a marginal influence on promoter activity. In contrast, mutation of the AP-1 site decreased basal promoter activity and substantially impaired response to BCR activation. These results indicate that the AP-1 site plays a central role in BIC promoter activity following activation of B-cell receptor signaling. Induction of the AP-1 Family Members, Jun B, c-Fos, and FosB, Requires the ERK and JNK Signaling PathwaysWe first analyzed members of the AP-1 family of transcription factors that may be responsible for induction of the BIC promoter through the AP-1 site by assessing c-Jun, JunB, c-Fos, and FosB expression by Western blot analysis. Using either whole cell lysates or nuclear extracts, we were consistently unable to detect c-Jun using two antibodies that recognize total c-Jun (Cell Signaling (L70B11 and 60A8)) or the phospho-specific antibodies recognizing phospho-c-Jun (Ser-63) or phospho-cJun (Ser-73). In contrast, Jun B levels were readily detected starting at 2 h after induction. Robust induction of both c-Fos and FosB were observed at 2 h and persisted through 6 h (Fig. 4). The expression levels of Jun B, c-Fos, and FosB were then analyzed in the presence of MEK, JNK, and p38 inhibitors. As shown in Fig. 5, c-Fos induction is significantly inhibited by the MEK inhibitor, U0126, and slightly less well inhibited by the MEK inhibitor, PD98059, and the JNK inhibitor, SP600125.

FosB expression was also lower in the presence of each of these inhibitors. The slowest migrating form of JunB was decreased by the MEK and JNK inhibitors, whereas the faster migrating form was only moderately affected. In contrast, the p38 inhibitor, SB202190, had little detectable influence on the expression of any of these AP-1 family members. These results show that the MEK/ERK and JNK pathways mediate induced expression of the AP-1 family members, JunB, c-Fos, and/or FosB, suggesting that these factors may play a role in the induction of BIC transcription. JunB and FosB Are Recruited to the BIC Promoter following B-cell Receptor EngagementAs expected, co-immunoprecipitation experiments readily demonstrated interactions between Jun B and either FosB or c-Fos (Fig. 6, AC), indicating that heterodimerization between these family members likely occurs following BCR activation in this system. The binding of JunB and FosB to the BIC promoter following B-cell receptor engagement was then assessed by chromatin immunoprecipitation analysis. As positive controls, antibodies that recognize histone H4 or the acetylated form of histone H4 were used. Although the level of overall histone H4 precipitated was not effected by BCR activation, elevated levels of the acetylated form were precipitated from cells exposed to anti-IgM (Fig. 6D). This provided confidence in the chromatin immunoprecipitation assay and provided evidence that the local chromatin environment at the BIC promoter is activated following BCR activation. Using the JunB- and FosB-specific antibodies, enhanced precipitation of the BIC promoter was similarly observed, indicating that JunB and FosB bind to the BIC proJOURNAL OF BIOLOGICAL CHEMISTRY
To further establish that JunB and FosB bind directly to the AP-1 site of the BIC promoter, electrophoretic mobility shift analysis was carried out using a 20-mer oligonucleotide probe spanning the AP-1 site. An intense shifted band is observed using extracts from Ramos cells treated with anti-Ig that is not observed in extracts from uninduced Ramos cells (Fig. 7A). This band is competed by excess unlabeled wild type competitor oligonucleotide but not a competitor oligonucleotide containing the AP-1 site mutation used in the reporter assay shown in Fig. 3C. Nearly 100% of this band is super-shifted using a JunB antibody, and this band is partially supershifted using an antibody against FosB (Fig. 7B). FIGURE 6. AC, JunB co-precipitates with c-Fos and FosB following BCR activation. H.C. refers to the immuno- These data indicate that this globulin heavy chain signal. Cntl Ab, control antibody; IP Ab, immunoprecipitation antibody. D, chromatin induced band is composed of immunoprecipitation analysis shows that FosB and JunB bind to the BIC promoter (BICp) but not the first intron JunB, which is likely heterodimerof the BIC gene following BCR activation. Quantitation was carried out using ImageJ software. ized with FosB. Whether the lack of a complete supershift of this band with the FosB antibody is due to incomplete binding of the antibody or due to a portion of this band representing JunB heterodimerized to another AP-1 family member such as c-Fos is not clear at this time. Nevertheless, these data indicate that JunB is a major component of this complex and that FosB is a contributor. Lastly, co-transfection experiments using either a wild type BIC reporter or the AP-1 mutant with control, JunB, FosB, or JunB plus FosB expression vectors were carried out to establish that the binding of these factors to the AP-1 site is capable of mediating promoter activation. Transfection of JunB or FosB FIGURE 7. JunB and FosB bind and activate the BIC promoter through the AP-1 element. A and B, electro- expression vectors alone moderphoretic mobility shift analysis was carried out using the BIC AP-1 promoter element and extracts from uninduced and anti-IgM induced Ramos cells. Competitor oligonucleotides sequences and antibodies used are ately activates the wild type prodescribed under Experimental Procedures. control Ab, control antibody. C, Ramos cells were transfected with moter, whereas co-transfection of either the wild type BIC reporter (wt) or the AP-1 site mutant BIC reporter (mAP-1) along either with 1 g of control, JunB, or FosB expression vectors or with 0.5 g of JunB plus 0.5 g of FosB expression vector. All values the JunB and FosB expression vectors together results in a more are relative to wild type promoter activity plus control expression vectors. CNTL, control. robust induction (Fig. 7C). No moter following activation of the B-cell receptor. In contrast, induction was observed using the AP-1 site mutant, indicating no signal was detected in FosB or JunB immunoprecipitates that activation likely occurs through the direct binding to the using primers that amplify a 162-bp fragment located in intron conserved AP-1 element. 1, 6 kb downstream from the site of transcription initiation (Fig. 6D). Therefore, FosB and JunB bind specifically to the BIC pro- DISCUSSION moter following BCR activation and likely play a role in the activation of BIC expression. The 5 RACE analysis described here identified the major start sites and a three-exon structure for BIC in a BCR-activated

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human B-cell system. Nevertheless, the splice junctions of the first intron of the BI821816 transcript match reasonably well to consensus splice donor acceptor sites, providing some evidence that this EST represents a bona fide transcript. BI821816 was derived from a pool of brain, lung, and testis RNAs, raising the possibility that there may be differential promoter usage in different tissues, suggesting added complexity to BIC regulation. Nevertheless, usage of the promoter mapped here in the BCRactivated B-cell line, Ramos, is not specific to this particular cell line. 5 RACE analysis of another B-cell line that expresses high constitutive levels of BIC (due to the presence of EBV) revealed the same start site.3 It is therefore likely that this promoter is commonly used in B-cells and possibly other lymphoid tissues. As shown in Fig. 5, inhibition of either the ERK or the JNK pathways inhibits the level of c-Fos and FosB induction. This likely occurs through ERK- and JNK-mediated phosphorylation of the T-cell factor family member, Elk-1, which is a critical determinant of c-Fos and FosB promoter activation (through the binding to the Ets motif of serum-response elements) (23 25). In addition, there is evidence for ERK-dependent phosphorylation of FosB (26), which may also contribute to the activation of FosB function. Inhibition of the ERK or JNK pathways primarily had an effect on the levels of the slower migrating JunB band, suggesting a possible role in influencing a modified form of JunB. It is possible that in addition to the lower overall levels of c-Fos and/or FosB, this change in the slower migrating form of JunB may also contribute to the inhibition of overall AP-1 activity by ERK and JNK inhibitors. Together, these results provide evidence for a key role in ERK- and JNK-mediated activation of FosB (and probably c-Fos) and JunB in the induction of BIC expression following BCR activation. A number of previous studies have demonstrated robust expression of BIC in EBV-infected cells expressing the full repertoire of EBV latency genes (27, 28). This implicates (but does not prove) that EBV latency genes are responsible for the induction of BIC. JunB is a well established downstream target of EBV latency genes that is induced by EBV infection (29). It is therefore possible that EBV induces high level BIC expression at least in part through the induction of JunB. The data shown in Fig. 3 indicate that the AP-1 site is important for both basal transcription and activated transcription, indicating that it is a crucial element in facilitating BIC transcription. Nevertheless, we still see a measurable response to BCR activation with the AP-1 site mutant (2.8-fold induction), suggesting that other promoter elements may contribute and/or cooperate with AP-1 family members to activate BIC transcription. This is unlikely to be due to incomplete ablation of the AP-1 site since the six core nucleotides of this element were mutated (wild type ATGAGTCAC, mutant ATctcgagC). A small but measurable decrease in response was observed when the c-Ets site located at the start site was mutated, suggesting the possibility that this site may contribute to activation, possibly through the binding of the Ets family member, Elk-1. In addition, there is a consensus Elk-1 site (25) located at 310 to 300 (GTTTCCTTTT), which could play a role in facilitating activation in response to BCR and perhaps TLR activation. We anticipate that although the AP-1 site is an important determinant of promoter activity, other elements are also likely to contribute to promoter function and play a role in BIC promoter regulation under these or other activating conditions. OConnell et al. (17) have shown that the JNK pathway is required for activation of BIC expression following the activation of certain TLR signaling pathways; however, the involvement of ERK or the FosB, c-Fos, or JunB AP-1 family members is not yet clear. Moreover, although it is likely that the BIC AP-1 site is important for TLR-mediated induction of BIC transcription, this has not to our knowledge been tested. Further studies will reveal how extensive the overlap is between these pathways in the activation of BIC/miR-155 expression. AP-1 signaling plays a key role in mediating inflammatory responses in the immune system and is important for the induction of regulatory cytokines. In addition, AP-1 activation plays a role in immune cell activation and differentiation. For example, Carrozza et al. (30) showed that transgenic mice expressing the naturally occurring FosB dominant negative, -FosB, under the direction of a T-cell-specific promoter resulted in impaired T-cell development. Interestingly, BIC/ miR-155 knock-out mice showed a defect in immune cell activation (15, 16). It is therefore likely that the induction of BIC/miR-155 plays a key role in facilitating at least some of the phenotypic effects of AP-1 activation. Similarly, the association between AP-1 proteins and cancer is well established, and AP-1 family members support tumor growth, survival, and metastasis through a wide array of downstream genes. The highly implicated nature of miR-155 in tumorigenesis suggests that induction of BIC/miR-155 may be a critical component of AP-1 signaling that contributes to AP-1 oncogenic signaling.

AcknowledgmentsWe thank Dr. Jennifer Cameron for helpful insights throughout the course of this project. We also thank Dr. Matt Burow for the advice and Dr. Dexing Fang and Dr. Huichen Wang for excellent technical advice on immunoprecipitation and chromatin immunoprecipitation assays.

REFERENCES

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X. Wang and E. Flemington, unpublished data.
11. Tam, W., Hughes, S. H., Hayward, W. S., and Besmer, P. (2002) J. Virol. 76, 12. Yanaihara, N., Caplen, N., Bowman, E., Seike, M., Kumamoto, K., Yi, M., Stephens, R. M., Okamoto, A., Yokota, J., Tanaka, T., Calin, G. A., Liu, C. G., Croce, C. M., and Harris, C. C. (2006) Cancer Cell 9, 13. Iorio, M. V., Ferracin, M., Liu, C. G., Veronese, A., Spizzo, R., Sabbioni, S., Magri, E., Pedriali, M., Fabbri, M., Campiglio, M., Menard, S., Palazzo, J. P., Rosenberg, A., Musiani, P., Volinia, S., Nenci, I., Calin, G. A., Querzoli, P., Negrini, M., and Croce, C. M. (2005) Cancer Res. 65, 70657070 14. Eis, P. S., Tam, W., Sun, L., Chadburn, A., Li, Z., Gomez, M. F., Lund, E., and Dahlberg, J. E. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 36273632 15. Thai, T. H., Calado, D. P., Casola, S., Ansel, K. M., Xiao, C., Xue, Y., Murphy, A., Frendewey, D., Valenzuela, D., Kutok, J. L., Schmidt-Supprian, M., Rajewsky, N., Yancopoulos, G., Rao, A., and Rajewsky, K. (2007) Science 316, 16. Rodriguez, A., Vigorito, E., Clare, S., Warren, M. V., Couttet, P., Soond, D. R., van Dongen, S., Grocock, R. J., Das, P. P., Miska, E. A., Vetrie, D., Okkenhaug, K., Enright, A. J., Dougan, G., Turner, M., and Bradley, A. (2007) Science 316, 17. OConnell, R. M., Taganov, K. D., Boldin, M. P., Cheng, G., and Baltimore, D. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 18. Taganov, K. D., Boldin, M. P., Chang, K. J., and Baltimore, D. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1248112486 19. Joseph, A. M., Babcock, G. J., and Thorley-Lawson, D. A. (2000) J. Virol. 74, 20. Cameron, J., Yin, Q., Fewell, C., Lacey, M., McBride, J., Wang, X., Lin, Z., Schaefer, B., and Flemington, E. (2007) J. Virol., in press 21. Tam, W., Ben-Yehuda, D., and Hayward, W. S. (1997) Mol. Cell. Biol. 17, 22. Heinemeyer, T., Wingender, E., Reuter, I., Hermjakob, H., Kel, A. E., Kel, O. V., Ignatieva, E. V., Ananko, E. A., Podkolodnaya, O. A., Kolpakov, F. A., Podkolodny, N. L., and Kolchanov, N. A. (1998) Nucleic Acids Res. 26, 362367 23. Lazo, P. S., Dorfman, K., Noguchi, T., Mattei, M. G., and Bravo, R. (1992) Nucleic Acids Res. 20, 343350 24. Yordy, J. S., and Muise-Helmericks, R. C. (2000) Oncogene 19, 25. Treisman, R., Marais, R., and Wynne, J. (1992) EMBO J. 11, 26. Rosenberger, S. F., Finch, J. S., Gupta, A., and Bowden, G. T. (1999) J. Biol. Chem. 274, 27. Jiang, J., Lee, E. J., and Schmittgen, T. D. (2006) Genes Chromosomes Cancer 45, 103106 28. Kluiver, J., Haralambieva, E., de Jong, D., Blokzijl, T., Jacobs, S., Kroesen, B. J., Poppema, S., and van den Berg, A. (2006) Genes Chromosomes Cancer 45, 147153 29. Cahir-McFarland, E. D., Carter, K., Rosenwald, A., Giltnane, J. M., Henrickson, S. E., Staudt, L. M., and Kieff, E. (2004) J. Virol. 78, 30. Carrozza, M. L., Jacobs, H., Acton, D., Verma, I., and Berns, A. (1997) Oncogene 14, 10831091

BioCentrum-DTU

Annual Report 2005
Annual Report 2005 Maj 2006
ISBN 87-91494-17-6 BioCentrum-DTU Technical University of Denmark, DTU Sltofts Plads, Building 221 DK-2800 Kgs. Lyngby Denmark www.biocentrum.dtu.dk
Chief editor Editorial team Text Photos Design/Layout Print
Ole Filtenborg Kasper Antonsen, Anne Birgitte Hansen Mogens Bisgaard, KONKRET PR & kommunikation Emil Rnn Andersen, Kasper Antonsen Kasper Antonsen Salogruppen
FOR LIFE AND SCIENCE BIOCENTRUM-DTU IN 2005
Commitment is the cornerstone Education Research Innovation the BiC way Public Relations Organisation and HR Seeing life as a system Cracking the code of life Central dogma: The cornerstone of life The cell in four dimensions Hi-tech biology Safe living in a globalised world Return of The great white plague At the root of Alzheimers Systems Biology - Not only medicine Drugs from the abyss
FEATURE - SYSTEMS BIOLOGY

INDUSTRIAL BIOTECHNOLOGY

Green Chemistry fundamentals Natural compounds from microscopic factories Going over the edge: High-gravity fermentation Nosing around: Identifying fungi from volatiles Making a super bug for antibiotics production Approaching disease from all angles Drugs united: New hope for Cystic brosis patients In the matrix: From organism to biolm Quorum sensing inhibitors on prescription Quality and health from gene to product Statistics in the bakery New sources for natural food colours Pulp facts Food quality: a question of hygiene Sustainable energy for the world Energy for the future Gas from garbage Science improves environment

MEDICINE

ENERGY
PUBLICATIONS IN 2005 PH.D. GRADUATES IN 2005

-FOR LIFE AND SCIENCE

Our motto For Life and Science is a common denominator and goal for the research and education activities at BioCentrum-DTU. It describes our field of work as life sciences in a technological setting and fixes the end goal of our research activities: improving the quality of life by research in sustainable, technological solutions. In 2005, we focused our research in four strategic areas: Energy, medicine, industrial biotechnology and food. This means that at the centre of our activities are: Creating sustainable alternatives to fossil energy sources, providing medicine that is non-resistance inducing, replacing traditional chemical industry with a less polluting green chemistry, and reducing the effect of life style related diseases by improving the quality of food. BiC is the largest biotechnological research and education facility in Northern Europe. We believe it is worth fighting for this status. We must continuously establish ourselves as unique in order to be competitive for public and industrial research funding. Copying other research centres or universities will bring us nowhere. Over the years, we have developed our own understanding and application of life sciences, contributing to the high technological profile of the Technical University of Denmark. Application of new technology is the driving force behind our work. However, when it comes to biotechnology, the development of new technology goes with the curiosity and scientific urge of science. This is the main incentive for our engineers, systems biologists, geneticists and biochemists.
Commitment is the cornerstone We demand commitment from our employees in the education of students as well as in research. We encourage the interest of our students by introducing them to biotechnology from day one. It gives them an ideal opportunity immediately to find out for themselves if biotechnology is exciting and the right study. The head of departments role is comparable to that of the coach in a sports field, as the communication between the coach and the team leader must be utmost efficient. The team leader in turn should understand how to inspire the rest of the team to perform at its best. The crucial attraction of talents to the team is dependent on outstanding players and team leaders. In this spirit, the annual report 2005 does not focus on projects in the individual research groups. Instead, we underline the teams collaborative efforts within the four strategic areas. The feature theme of this years annual report is systems biology, an area that illustrates our capability to develop world-class methods, applicable to a wide range of research areas. The feature theme is also an excellent example of a research field that attracts talents and attention to BioCentrum-DTU. Systems biology, along with its natural companion bioinformatics, has been a key focus area at BioCentrum-DTU and DTU since 1993, when a grant from the Danish National Research Foundation made it possible to establish the Center for Biological Sequence Analysis (CBS). Today, CBS is regarded one of the foremost public bioinformatics research centers in the world and has proven to be a very attractive environment for talented scientists. Information technology has become an increasingly important part of all sciences including biotechnology. Modern, applied biotechnology is about fine-tuning cell factories, predicting and preventing microbial invasion, discovering new species, and much more. This requires a system-level understanding of cells, organisms, or even biotopes and is

SPENDING 101 mio.
Once again a paper from Center for Biological Sequence Analysis at BiC was on the ISI Biology Hot paper list top ten. The publication is Jannick Bendtsens paper, Improved prediction of signal peptides: SignalP 3.0, by J.D. Bendtsen, H. Nielsen, G. von Heijne, S. Brunak, J. Mol. Biol. 340:783-795, July, 2004. As per 1 December 2005 it has 200 ISI citations in total.

28 mio. 13 mio.

18 mio.
THOMSON SCIENTIFICS HONOUR
Salary Other expenditures Capital investment Overhead fee charged on external funding
In May 2005 Thomson Scientic honoured, along with the Danish National Library Association, professor Sren Molin as one of Denmarks most-cited authors for his publications in microbiology.
INNO V A T I O N T HE B I C W A Y
Transferring scientific results into commercial end products can be a long and cumbersome process. To assist our scientists in this process, BiC has appointed an innovation consultant, namely associate professor Per Vggemose. Nielsen. He will work closely together with the DTU office for Research and Innovation on commercialising inventions made at BiC. Potential inventors will be inspired to consider commercialisation and IPR (Intellectual Property Rights) at a very early stage. Once an idea has matured, we can assist in organising workshops and providing access to innovation and project management tools, Per explains. It is also
important to create and stimulate strong, local innovation networks: We will set up user groups, organise seminars and courses on different aspects of innovation from idea to implementation, he continues. Making the innovation and inventions done at BiC-DTU available to the public means disseminating BiCs results. At the moment we are planning two initiatives: First, we will promote our competencies via our homepage - www. biocentrum.dtu.dk - and by publishing information folders. Secondly, we will arrange a BiC Innovation Day in the autumn of 2006, when key areas of commercial interest will be dealt with. The event will be public in that we will invite companies with interests in BioCentrum-DTUs field of
BIC RESEARCHER OF THE YEAR
Ulrik de Lichtenberg was chosen as the 'Researcher of the Year' on basis of an outstanding high-ranking publication record considering his young age.
OUTSTANDING FOOD RESEARCH
An international evaluation of the Centre for Advanced Food Studies shows outstanding Food research at BioCentrum-DTU: The center has taken a signicant step forward to become one of the prominent centres of research and academic education in food science and nutrition.

PIONEERS OF GENETIC RESEARCH
The words gene, genotype and phenotype were coined by Danish pharmacist Wilhelm Johannsen (1857-1927), in his 1909-paper on inherited traits in princess beans and thus rediscovered Gregor Mendels Elements of inheritance, introduced in 1866. The DNA molecule was discovered by Swiss biochemist Friedrich Miescher in 1869. In 1872, Walther Flemming described mitosis and chromosomes. The double helical structure of the DNA-molecule was described by Francis Crick and James D. Watson in Cambridge, 1953.
glycosylation or phosphorylation sites. Part of the study is devoted to predictive bioinformatics. This includes prediction of capsase cleavage, which is an early event in the development of Alzheimers disease, and prediction of propeptide cleavage. Both have industrial signicance, as they are important events in the cellular formation of insulin and many antibiotics. Among the most signicant, posttranslational modications are phosphorylation processes, which add a phosphate group to a protein, rendering the protein polar and hydrophilic. This is often responsible for switching the functionality of proteins and enzymes on and off.
BUD9 YRO2 BEM1 ELO1 TPK3 SED1 YLR040C SDL1 YIL177C TPS2 TPS3 TPK2 BNI4 YRF1-4 YJL181W CAC2 MSI1 JEM1 PKH1 MRP8 PIL1 HO PHO8 YPL267W TOS2 MCM3 PHO11 TAF2 ORC6 BNS1 YDL038C MCM6 NUM1 CDC6 VPS5 VPS17 BOP1 RDH54 YJL217W PRY3 YNL208W YHR149C CDC9 STB1 YDL241W DBF4 PRM7 KIN3 YER139C YLR254C ORC4 CDC7 ORC2 ORC1 ORC3 RTT106 CTF4 YDL010W DSE1 STB2 SPH1 YBR089W YGR151C POL1 PRI2 YBR070C TOF1 PDS5 PRI1 POL12 GSY1 YOR385W YCL042W WTM2 TPO4 GLG2 YOL019W POL32 RFC3 RFC2 YPL208W DSE2 ASF2 GSY2 ELG1 BIO2 RFC1 CSM3 CRH1 POL30 RAD27 YPS3 YOX1 RTT109 RSR1 ERP3 RAD54 PDR16 HUA2 ESC8 SEN34 SUR1 QRI1 BAT2 YDL163W MRC1 YPR174C RAD5 CTS1 LPP1 MUM2 RLF2 EXO1 OCH1 CUE4 IME4 GIC2 BCY1 SPT21 YNL310C

ERV29 PHO12 RGT2

MCM/ORC

CDC54 CDC46 TAF8 CDC47

TPS1 TSL1

Regulation of meiosis

MCM2 YGR146C

Protein kinase A

CDC45 PAC1 ORC5
UTH1 FUN26 YHL026C YMR031C WTM1
Glycogen synthesis Nucleosome/ bud formation
GIN4 KCC4 POL2 ESP1 NIS1 YOL070C AMN1 PAF1 HTZ1 NAP1

DNA replication

FIG2 PHO3 PHD1 BSC1 YNL057W KRE6
APC11 APC4 CDC20 APC2 APC5 CDH1

Mitotic exit

PDS1 SPT16

DNA polymerase

HYS2 CDC2 YNL134C
YIL141W MCD4 HEM15 BCP1 CDC21

YLR297W MRH1 KTR2

DPB2 YLR236C DPB3 MET16 HIF1 ERP5 OGG1 YIF1
CDC23 DBF2 CDC27 DOC1 SPO12 APC1 KAR5 MOB1 YCK3 APC9 CDC26
RNR3 HAT2 YDL009C YOL007C AXL2 DUN1

YJL018W DPM1 RHC1

Cdc28-cyclin
YPL014W SIC1 CLN3 CLB6 CLB2
RAD53 TOS4 HMLALPHA1 HTA1 HTB2 SWI4
RNR1 RNH203 YER152C YLR050C YOR114W YKL066W PMT3

YCK2 YNL058C NRG2 YCK1

TBF1 HHF1/2 HTB1

Histones

HI- T EC H B I O L O GY

Successful systems biology approaches rely heavily on advances in laboratory equipment, computer technology and open source software. Systems biology is a fusion of the dry science mathematics, physics, and computer science with the wet science cell biology, molecular biology, and biochemistry. Within both the wet and dry science, only the technological advances in equipment and technology may full the fundamental intentions of the human genome projects. Among the technological advances is the creation of advanced analysis methods such as GC-MS, in which gas chromatography is combined with mass spectrometry in order to separate the individual peptides and subsequently quantify them. Another example is the MALDI-TOF, which allows for identication of individual peptides and ngerprinting of unknown proteins by dissolving the large molecules in a matrix and subsequently analysing their mass using mass spectrometry. In order to obtain enough DNA to analyse a gene, the DNA strand must be replicated. Today, this is most often done with PCR-technology, a polymerase chain reaction, in which known sequences anking the gene act as target sequences for corresponding primers, created by a DNA-synthesiser, thus allowing for clone less replication of the whole gene sequence. PCR technology also enables the reconstruction of a gene, corresponding with a particular protein. High throughput analysis methods allow for analysis of gene expression. Microarrays or DNA chips are utilised to identify gene sequences. DNA from the cell or tissue that the scientists wish to study is labelled with uorescent dyes, and when they hybridise with genes sequences spotted on a glass chip, colour changes reveal the presence of the gene. BiC-scientists apply PCR-technology and DNA-chips in the search of new microorganisms with a potential as production organisms in the manufacture of e.g. bioethanol or medicine. Examples of such projects are described in the chapters Searching the extremes and Drugs from the abyss. Open source software is frequently used to store and present data in systems biology and bioinformatics. Open source means that the source code may be distributed and altered freely or with a few restrictions. This enables computer scientists to create tailor-made applications to perform very advanced calculations and algorithms on-line.

UNDERSTANDING YEAST

Yeasts have monetary importance in the food and medical industry and mechanisms in the yeast cell are very similar to mechanisms in the human cell, which is why yeast may be used as a model organism. Scientists at the Center for Microbial Biotechnology (CMB) have developed a method involving a GC-MS platform optimised for the purpose, to analyse the metabolome of yeast. The result is a complete image of the cells transformation of nutritional compounds into cell mass. The method provides insight into specic metabolic pathways and lays the groundwork for integrated transcriptionmetabolism studies of yeasts.

A JUNGLE OF DATABASES

The publicly accessible systems biology databases include primary sequences, protein sequences, carbohydrates, 3D-structures, RNA, genomes, model organisms, proteins, protein families, proteomes, structures, pathways, microarrays, 2 D-structures, and more. The search options are legion: searches may be text-based, structure-based, motif-based, or mass based and there are many other options. On top of this, scientists have a large range of on-line tools for dataanalysis to choose between.
The use of open source software implies that many people are contributing to the software and are developing new applications. The limitations of the open source software are the many standards and interfaces being developed. Efforts are made, however, to coordinate the access to databases and to provide standards for access e.g. in the EU funded Networks of Excellence ENFIN! and EMBRACE. Finally - bioinformatics sources at your fingertips The acronym ENFIN! French for nally stands for Experimental Network for Functional Integration. ENFIN! is a virtual institute of Systems Biology comprising 20 research groups across 17 institutions in 13 countries. The Commission of the European Union has awarded 9 million over ve years to the network. One of the groups in the network is the Center for Biological Sequence Analysis (CBS) at BiC. In an ENFIN!-context, CBS will focus mainly on the development of tools for data-analysis. ENFIN! was established to give scientists in the participating research group a roadmap to all the biological information made available in the last decade. On the internet, countless databases contain information about almost every type of biological data, and the ways of accessing and processing information are almost as many. The consequences are that many scientists often utilise only a fraction of the available information. The purpose of ENFIN! is to enable scientists to combine public data with their own unpublished data, and thus perform integrated analyses, using data from different types of experiments. Apart from creating an infrastructure that will be made freely available to scientists all over the world, ENFIN! will place a strong experimental focus on understanding the regulation and deregulation of cell division. Deregulated cell division is believed to cause a number of diseases most prominently cancer. Applying ENFIN! to cancer research may contribute to solving the mystery of cancer. Embracing bioinformatics Better and more coordinated access to biological information is also the aim of the EMBRACE Network of Excellence. Funded by a 8.3 million grant from the Commission of the European Union, the network encompasses computational biologists from 17 institutes (including CBS) in 11 countries. The European Bioinformatics Institute in Hinxton, UK, coordinates the project. The main purpose of the EMBRACE network is to simplify and standardise the way in which biological information is made available to scientists. Today, database access often requires special software or special skills, as different data providers use different methods of presentation. A scientist might have to search ten or more different data providers using different search methods in order to nd all information pertaining to a particular set of candidate genes.

The scientists found specic volatile release prole for each of the investigated species: In general, P. carneum isolates produced the lowest number of sesquiterpenes, all of which were unique for P. carneum within the series Roqueforti. P. roqueforti and P. paneum produced a larger variety of volatile metabolites, some of which they have in common and some of which are unique for the two species.
Pharmaceutical and industrial potential from metabolic engineering of antibiotics-producing bacteria are at the core of Irina Borodinas research. To Irina Borodina, a PhD-student at the Center for Microbial Biotechnology, the pharmaceutical and industrial potential of the research is highly motivating. I dream about discovering a new antibiotic or a new cure for a life-threatening disease, so naturally, the functional genomics of the Streptomyces is the subject of my PhD-thesis, she says. Approximately 40% of all the antibiotics currently used in the world are produced by different varieties of Streptomyces. In the research group, the focus is on improving the properties of the organisms by over expression or deletion of certain genes. The trick is to nd out which genes to change, and we use metabolic models to help us with predictions, Irina says, adding: The genome sequences of still more Streptomyces species
MAKING A SUPER BUG FOR ANTIBIOTICS PRODUCTION
become available; now our task is to understand how the genes function and how we can use the information. Metabolic models can help us do it. Irina explains that she has been fascinated by biotechnology since her rst encounter with the subject: In the tenth grade, I found a brochure about recombinant insulin production left in the school library, she says. The school system in Lithuania, where I was brought up, was not quite up to date, but with the inspiration and support from my parents, who are both engineers, I ended up studying chemical engineering at KTU, the technical university of Kaunas. When she became aware of an international exchange programme nanced by the Danish government, she decided to apply well aware of the fact that she had to compete with PhD-students
for the eleven scholarships: I fortunately got a ve month scholarship at DTU and nalised my BSc here at BioCentrum the facilities are great, and the research environment here at CMB and BioCentrum-DTU is very friendly and inspiring. So for me, it was natural to continue my studies and write my MSc and PhDthesis here as well, she says. The scientists at CMB aim at developing new genetically modied Streptomyces strains for antibiotics production. The industrial strains applied today are normally selected from chemically or UV-mutagenised strains, a relatively slow and inaccurate process. We are developing models of the bacterias metabolic network, in order to predict the production of antibiotics. The scientists make use of the Streptomyces coelicolor bacterium as a model organism to test the theories. The antibiotics are very colourful and the model organisms

a grid. The matrix also comprises technology, production, marketing and communication issues. As BiC is a biotechnological department at a technical university, the focus is on the biological and technological aspects of food from gene to product. For the scientists at BiC, food quality starts with the genes. Fine-tuning the genes in lactic acid bacteria regulates the homolactic fermentation and affects the aroma of cheese. High-tech analysis methods improve the understanding of the raw material characteristics allowing for selection of varieties with the best technological quality. Also, the design of food processing technology that allows for a high hygienic standard, while at the same time implementing modern management theory, creates the basis for a truly innovative food industry. Finally, understanding the complex interaction of food components with each other
and with the human immune system reects the nutritional approach. The gene-to-product approach also forms the basis of a new initiative at DTU and BiC a nutrigenomics research platform that joins scientists within nutritional immunology, bioinformatics, and functional genomics. Utilising postgenomic analysis methods in a combination with classic laboratory disciplines will create a new perception of the relationship between the human genome and food. In time, this will be of invaluable importance to medicine, dietary advisors and food engineers, enabling them to develop both traditional food with high nutritional standard and functional food with improved health properties.
S TATIS T I C S I N T H E B AK ER Y
Modern, statistical methods help improve bread as scientists use rapid analysis methods to determine end-use quality of wheat one month before harvest. Gaining knowledge about wheat quality at the earliest possible stage and the ability to plan ahead of harvest would mean economic advantages in the whole value chain, from primary producers to consumers. Scientists in the Biochemistry and Nutrition Group have developed a technique allowing for prediction of enduse quality one month before harvest. Baking quality of wheat depends on its content of glutenin and gliadin, responsible for gluten-formation in dough when it is kneaded. Gluten gives structure to bread, as its elasticity allows the dough to expand and hold the carbon dioxide (CO2), produced by the yeast during fermentation. The method involves MALDI-TOF (matrix assisted laser desorption / ionisation time ight mass spectrometry) and multivariate data analysis. The scientists examined two different varieties of wheat over a period between 15 days after blooming and 45 days after blooming one with a low content of proteins and the other with a high content of proteins. In each case, the scientists were able to predict the end-use quality of the grains and identify the varieties. The gluten proteins were extracted from the wheat grains. After mass spectrometry, the scientists applied multivariate data analysis. The new and fast technique showed the potential to replace traditional, time-consuming analysis methods, such as 2-D gel electrophoresis.

USEFUL BY-PRODUCTS

Secondary metabolites is the common name for compounds that are created during the metabolism of fungi and other microorganisms but not utilised in the metabolism. Secondary metabolites from fungi include e.g. Penicillin and other antibiotics. In science, secondary metabolites are used to recognise species of fungi or discover new ones, by analysing the chemical prole in a metabolome analysis.

FUNGAL FOOD COLOURS

Li Shizhen (1518-1593) has provided the rst written description of Ang Kak Chinese for red rice and of its use in the production of rice wine, sh paste, and red soybean curd, as well as in the treatment of e.g. indigestion and dysentery. The red colour in the rice is due to the fungus Monascus purpureus, which produces a variety of pigments, ranging from purple to yellow. Today, food colours produced by Monascus rubeus are sold all over the world and the therapeutical effect of Monascus-metabolites is utilised in e.g. blood pressure lowering drugs.

P U LP F A C T S

Potato bres extracted from potato pulp may become a key ingredient in tomorrows low fat food and the source of ingredients for health-promoting, functional foods. Functional foods i.e. food items with health-promoting properties are currently in focus in research and in food industry. Functional foods that full both consumer expectations and legislation requirements will undoubtedly become an important business expansion area to future food producers. Recent experiments have shown that soluble dietary bres are promising additives in low fat products and that they possess other health promoting properties. Added bres may very well increase the health properties of a wide range of food products such as meat products, bread or soft drinks. Dietary bres occur naturally in vegetables, fruit and pulses. One of the best Danish sources of dietary bre is the potato and by-products from the industrial utilisation of this versatile tuber mainly potato pulp.
Scientists in FBE compared ve low fat pig liver pates, made by adding a commercially available potato bre product, dry potato pulp and three different enzymatically extracted potato bres. The use of potato bres with a low content of lignin and cellulose and a high content of soluble non-starch polysaccharides resulted in a pate, which had desirable consumer quality whereas the use of commercial potato starch or bres with high lignin content resulted in a harder, discoloured liver pate. This makes potato bre from potato pulp, which is a byproduct from the production of potato starch, an interesting source of future food ingredients. Denmark is one of Europes leading manufacturers of potato starch: The annual production is 160,000 tonnes approx 10% of the total European production. Potato starch is used in the food and pharmaceutical industries and in the graphical industry. The by-product, 90,000-100,000 tonnes of potato pulp per year, is sold to farmers, who use it as animal feed. Scientists in FBE have developed an enzymatic process, utilising an enzyme, which is produced by fungi of the species Aspergillus aculeatus and manufactured commercially by Novozymes A/S under the name Viscozyme. The enzyme is also utilised in the production of bioethanol from grain. The enzymatic potato starch process, originally invented at BioCentrum-DTU in the 90s, was re-initiated in 2003, when it was proven that desirable

Bioethanol production requires very specic properties, which may be found in microorganisms from hot springs. Scientists of EMAB have developed a method that enables isolation of single microorganisms from a mixture of cells, harvested from a hot spring. The system is based on an inverted microscope, a micro-injector and a micro-manipulator. The isolated cell is captured in a micro-capillary from a volume of 400 l and transferred to an appropriate growth medium. The system has been used to isolate and cultivate the thermophilic Archaeon Metallosphaera sedula and the hyperthermophilic Archaeon Sulfolobus solfataricus from enriched hot spring samples. The high efciency of single-cell isolation and cultivation has been demonstrated over a range of temperatures in different environments. This is probably due to the elimination of osmotic stress and limitation of temperature uctuations during the isolation process, as a result of the large sample volume from which the cells are isolated.

G AS F RO M GA R B A GE

When utilised in a simultaneous methane-bioethanol production, municipal waste problems are solved while at the same time bioethanol production becomes environmentally sounder. The waste depots of the past have gone. Today, most municipal waste ends up in incinerators connected to a power plant. Scientists of EMAB have investigated the possibility of proting from the waste by utilising the organic fraction of municipal solid waste in a biomethane production. In the bioethanol plants currently being developed at the Danish Center for Biofuels, the production is integrated with biomethane production. Utilising municipal waste in these plants will further increase the sustainability of future energy production. The yield of biogas from the biogas plant is approx. 640790 millilitres methane per
gram solid organic waste the yield of a full-scale biogas plant will be 180-220 m3 biogas pr tonne of municipal waste. The system developed by EMAB scientists for anaerobic digestion of municipal waste includes two reactors. One operates at a temperature of 55 C (thermophilic) and is the primary fermentor, while the other reactor operates at a temperature of 68 C (hyper-thermophilic), and is implemented as a post-treatment step. The second reactor increases hydrolysis of the recalcitrant organic matter, improves sanitation and eases stripping of ammonia from the reactor.
The volatile solids of the waste are reduced by 78-89% during the process, and the biogas yield is 640-790 ml / g volatile solid. The reduction of volatile solids in the combined system is up to 7% higher than in the single-stage treatment, but the combination does not increase the yield of methane. The time the waste spends in the reactor is an important factor. Shifting the hydraulic retention time of the hyper-thermophilic reactor from ve to three days, results in a drop in the methanogenic activity in the hydrolysis reactor to a minimum. Operation of the hyper-thermophilic reactor (68C) at a hydraulic retention time of 2448 hours is sufcient to achieve a high conversion of volatile solids into volatile fatty acids. Furthermore, the removal of pathogens is enhanced by the hyper-thermophilic post-treatment. With a ow of headspace gas through the reactor equivalent to four times the biogas ow produced in the thermophilic reactor, 7% of the ammonia load is removed in the hyper-thermophilic reactor. Gradually, the pig manure is completely replaced with municipal waste. This causes the pH in the fermentor to rise, which might eventually inhibit the methane-producing bacteria. However, the production is stabilised by the recirculation of process water from the simultaneous bioethanol production, and the high biogas yield is maintained. The volatile solids of the waste fraction are reduced by 69-74% during the process. It is possible to mix the manure in a 50-50 ratio without compromising the biogas yield.

SCIENCE IMPROVES ENVIRONMENT
ferent mutant HY10 strains, with focus on preventing by products like acetate and lactate. They have therefore gained important knowledge about the metabolism of the HY10. One of the strains has been tested thoroughly on different substrates in continuous reactor systems with very
promising results. We are scaling up the process in the pilot development unit in the Maxifuels-Project where the modied organisms will be used to produce ethanol in 0.5 m3 reactors, and the commercial value of the process can be proved.

PUBLICATIONS IN 2005

Articles in journals, Scientic, Peer reviewed and ISI-indexed.
Andersen, B.; Hansen, M.E.; Smedsgaard, J.: Automated and unbiased image analyses as tools in phenotypic classication of small-spored Alternaria species. Phytopathology, vol:95 (9), 1021-1029. Bendtsen, J.D.; Binnewies, T.T.; Hallin, P.F.; Sicheritz-Ponten, T.; Ussery, D.W.: Genome update: prediction of secreted proteins in 225 bacterial proteomes. MICROBIOLOGY-SGM, vol.: 151, 1725-1727. Bendtsen, J.D.; Binnewies, T.T.; Hallin, P.F.; Ussery, D.W.: Genome update: Prediction of membrane proteins in prokaryotic genomes. MICROBIOLOGY-SGM, vol.: 151, 2119-2121. Bendtsen, JD; Kiemer, L; Fausboll, A; Brunak, S.: Non-classical protein secretion in bacteria. BMC MICROBIOLOGY, vol.: 5, ar 58. Bendtsen, J.D.; Nielsen, H.; Widdick, D.; Palmer, T.; Brunak, S.: Prediction of twinarginine signal peptides. BMC BIOINFORMATICS, vol.: 6, ar 167. Binnewies, T.T.; Bendtsen, J.D.; Hallin, P.F.; Nielsen, N.; Wassenaar, T.M.; Pedersen, M.B.; Klemm, P.; Ussery, D.: Genome Update: Protein secretion systems in 225 bacterial genomes. Microbiology, 1013-1016. Binnewies, T.T.; Hallin, P.F.; Staerfeldt, H.H.; Ussery, D.W.: Genome update: proteome comparisons. MICROBIOLOGY-SGM, vol.: 151, 14. Bjarnsholt, T.; Jensen, P.; Rasmussen, T.B.; Christophersen, L.; Calum, H.; Hentzer, M.; Hougen, H-P; Rygaard, J.; Moser, C.; Eberl, L.; Hoiby, N.; Givskov, M.C.: Garlic blocks quorum sensing and promotes rapid clearing of pulmonary Pseudomonas aeruginosa infections. Microbiology, 3873-3880. Blunt, J.W.; Dalsgaard, P.W.; Munro, M.H.G.; Frisvad, J.C.; Christophersen, C.: Communesin G and H, new alkaloids from the psychrotolerant fungus Penicillium rivulum. Journal of Natural Products, vol:68, 258-261. Bok, J. W.; Balajee, S. A.; Marr, K.A.; Andes, D.; Nielsen, K.F.; Frisvad, J.C.: LaeA - a regulator of morphogenetic fungal virulence factors. Eukaryotic Cell, vol:4, 1574-1582. Borodina, I.; Krabben, P.; Nielsen, J.: Genome-scale analysis of Streptomyces coelicolor A3 (2) metabolism. Genome Research, vol:15, 820-829. Borodina, I.; Nielsen, J.: From genomes to in silico cells via metabolic networks. Current Opinion in Biotechnology, vol:16, 1-6. Borodina, I.; Schller, C.E.G.; Eliasson Lantz, A.; Nielsen, J.: Metabolic network analysis of Streptomyces tenebrarius, Streptomyces with Entner-Doudoroff pathway. Applied Enviromental Microbioogy, vol:71, 2294-2302. 44

Applied and Environmental Microbiology, vol:71, 2113-2120. Frisvad, J.C.; Lund, F.; Elmholt, S.: Ochratoxin A producing Penicillium verrucosum isolates from cereals reveal large AFLP ngerprinting variability. Journal of Applied Microbiology, vol:98, 684-692. Frisvad, J.C.; Samson, R.A.: comparison of three different groups of aatoxin producers and a new efcient producer of aatoxin B1, sterigmatocystin and 3-O-methylsterigmatocystin, Aspergillus rambellii sp. nov. Systematic and Applied Microbiology -in press, vol:28. Fukuda K.; Jensen, M.H.; Aghajari, N.; Haser, R.; Svensson, B.: Rational engineering of the N-terminal region in barley -amylase 2 to enhance secretory expression in Saccharomyces cerevisiae by using Degenerate Oligonucleotide Gene Shuffling. Prot. Eng. Design Select. 18, 515-526. Gavala, H.N.; Skiadas, I.V.; Ahring, B.K.; Lyberatos, G.: Potential for biohydrogen and methane production from olive pulp. Water Science and Technology, vol:52 (1-2), 209-215. Ghirado A.; Srensen, H.A.; Petersen, M.K.; Jacobsen, S.; Sndergaard, I.: Determination of wheat quality during the grain development using matrix-assisted laser desorption/ionisation time-of-ight mass spectrometry and multivariate data analysis. Rapid Communication in Mass Spectrometry, vol:19, 525-532. Gjermansen, M.; Ragas, P.C.; Sternberg, C.; Molin, S; Tolker-Nielsen, T.: Characterization of starvation-induced dispersion in Pseudomonas putida biolms. Environmental Microbiology, 894-904. Gonalves, B.; Silva, A.P.; MoutinhoPereira, J.; Bacelar, E.; Rosa, E.; Meyer, A.B.S.: Effect of ripeness and postharvest storage on the evolution of color and anthocyanins in cherries (Prunus avium L.). Food Chem. Grotkjr, T.; Christakopoulos, P.; Nielsen, J.; Olsson, L.: Comparative metabolic network analysis of two xylose fermenting recombinant Saccharomyses cerevisiae strains. Metabolic Engineering, Published on-line. Guo, Z.: Enzymatic modication of phospholipids forfunctional applications and human nutrition. Biotechnology Advances, vol:23, 203-259. Gttsche, J.; Nielsen, N.S.; Nielsen, H.H.; Mu, H.: Lipolysis of Different Oils using Crude Enzyme Isolate from the Intestinal Tract of Rainbow Trout, Oncorhynchus mykiss. Lipids, vol:40, 1273-1279. Haack, M.B.; Olsson, L.; Hansen, K.; Eliasson Lantz, A.: Change in hyphal morphology of Aspergillus oryzae during fed-batch cultivation. Applied Microbiology and Biotechnology. Published on-line. Hallin, P.F.; Nielsen, N.; Devine, K.M.; Binnewies, T.T.; Willenbrock, H.; Ussery, D.W.: Genome Update: base skews in 200+ bacterial chromosomes. MICROBIOLOGY-SGM, vol.: 151, 633-637. Hansen, E. H.; Schafer, T.; Molin, S.; Gram, L.: Effect of environmental and physiological factors on the antibacterial activity of Curvularia haloperoxidase system against Escherichia coli. J. Appl. Microbiol., 581-588.

Nielsen, J.; Kjelleberg, S.; Givskov, M.C.: The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors. Micobiology, 3589-3602. Koebmann, B.; Solem, C.; Jensen, P.R.: Control analysis as a tool to understand the formation of the las operon in Lactococcus lactis. FEBS Journal, vol:272, 2292-2303. ISSN: 1742-4658 Kramhft, B.; Bak-Jensen, K.S.; Mori, H.; Juge, N.; Nhr, J.; Svensson, B.: Multiple attack, kinetic parameters, and product profiles in amylose hydrolysis by barley -amylase 1 variants. Biochemistry 44, 1824-1832. Kristensen, E.F.; Elmholt, S.; Thrane, U.: High-temperature treatment for efcient drying of bread rye and reduction of fungal contaminants. Biosystems Engineering, vol:92, 183-195. Kristensen, J.B.; Xu, X.; Mu, H.: Process optimization using response surface design and pilot plant production of dietary diacylglycerols by lipase-catalyzed glycerolysis. J. Agric. Food Chem., vol:53, 7059-7066. Kristensen, J.B.; Xu, X.; Mu, H.: Diacylglycerol synthesis by lipase-catalyzed glycerolysis. J. Am. Oil Chem. Soc., vol:82, 329-334. Kristiansen, K.; Blom, N.: Prediction of caspase cleavage sites. FEBS JOURNAL, vol.: 272, 156-157. Kristiansen, T.B.; Pedersen, A.G.; Eugen-Olsen, J; Katzenstein, T.L.; Lundgren, J.D.: Genetic evolution of HIV in patients remaining on a stable HAART regimen despite insufcient viral suppression. SCANDINAVIAN JOURNAL OF INFECTIOUS DISEASES, vol.: 37, 890-901. Kvist, T.; Mengewein, A.; Manzei, S.; Ahring, B.K.; Westermann, P.: Diversity of thermophilic and non-thermophilic Crenarchaeota at 80C. FEMS Microbiol. Lett., vol:244, 61-68. La Cour Petersen, M.; Hejgaard, J.; Thompson, G.A.; Schulz, A.: Cucurbit phloem serpins are graft-transmissible and appear to be resistant to turnover in the sieve element-companion cell complex. J. Exp. Bot., 56, 3111-3120. Lage, K.: Identication of disease genes in genetically heterogeneous disorders through bioinformatic data integration. FEBS JOURNAL, vol.: 272, 127-127. Lambertsen, L.; Molin, S.; Kroer, N.; T., C.M.: Transcriptional regulation of pWW0 transfer genes in Pseudomonas putida KT2440., 169-181. Lange, M.; Westermann, P.; Ahring, B.K.: Archaea in Protozoa and metazoan. Applied Microbiology and Biotechnology, vol:66, 465-474. Larsen, M.V.; Lundegaard, C.; Lamberth, K.; Buus, S.; Brunak, S.; Lund, O.; Nielsen, M.: An integrative approach to CTL epitope prediction: A combined algorithm integrating MHC class I binding, TAP transport efciency, and proteasomal cleavage predictions. EUROPEAN JOURNAL OF IMMUNOLOGY, vol.: 35, 2295-2303. Larsen, T.O.; Petersen, B.O.; Srensen, D.; Duus, J.; Frisvad, J.C.; Hansen, M.E.: Discovery of novel natural products by application of X-hitting, a novel algorithm for automated comparison of full

Tsitsigiannis, D. I.; Bok, J.-W.; Andes, D.; Nielsen, K.F.; Frisvad, J.C.; Keller, N. P.: Aspergillus cyclooxygenase-like enzymes are associated with prostaglandin production and virulence. Infection and Immunity, vol:73, 4548-4559. Uhlig, S.; Gutleb, A. C.; Thrane, U.; Flyen, A.: Identication of cytotoxic principles from Fusarium avenaceum using bioassay-guided fractionation. Toxicon, vol:46, 150-159. Vestergaard, C.S.; Risum, J.; AdlerNissen, J.: 23Na-MRI quantication of sodium and water mobility in pork during brine curing. Meat Science, vol:69, 663672. Vestergaard, C.S.; Erbou, S.G.; Thauland, T.; Berg, P.; Adler-Nissen, J.: Salt distribution in dry-cured ham measured by computed tomography and image analysis. Meat Science, vol:69, 9-15. Vikbjerg, A.F.; Mu, H.; Xu, X.: Lipasecatalyzed acyl exchange of soybean phosphatidylcholine in n-hexane: a critical evaluation of both acyl incorporation and product recovery. Biotechnol. Prog., vol:21, 397-404. Vikbjerg, A.F.; Mu, H.; Xu, X.: Monitoring of monooctanoyl phosphatidylcholine synthesis by enzymatic acidolysis between soybean phosphatidylcholine and caprylic acid by thin-layer chromatography with a ame ionization detector. J. Agric. Food Chem., vol:53, 3937-3942. Vikbjerg, A.F.; Peng, L.; Mu, H.; Xu, X.: Continuous Production of Structured Phospholipids in a Packed Bed Reactor with Lipase from Thermomyces lanuginosa. J. Am. Oil Chem. Soc., vol:82 (4), 237-242. Vikbjerg, A.F.; Mu, H.; Xu, X.: Parameters affecting incorporation and by-product formation during the production of structured phospholipids by lipase-catalyzed acidolysis in solvent free system. J. Mol. Catal. B, vol:36, 14-21. Villas-Bas, S.G.; Moxley, J.F.; kesson, M.F.; Stephanopoulos, G.; Nielsen, J.: High-throughput metabolic state analysis: The missing link in integrated functional genomics of yeasts. Biochemical Journal, vol:388, 669-677. Villas-Bas, S.G.; kesson, M.F.; Nielsen, J.: Biosynthesis of glyoxylate from glycine in Saccharomyces cerevisiae. FEMS Yeast Research, vol:5, 703-709. Villas-Bas, S.G.; Mas, S.; kesson, M.F.; Smedsgaard, J.; Nielsen, J.: Metabolome analysis by mass spectrometry. Mass Spectrometry Review, vol:24, 613-646. Wernersson, R.: FeatureExtract - extraction of sequence annotation made easy. NUCLEIC ACIDS RESEARCH, vol.: 33 , 567-569. Wernersson, R.; Nielsen, H.B.: OligoWiz 2.0 - integrating sequence feature annotation into the design of microarray probes. NUCLEIC ACIDS RESEARCH, vol.: 33, 611-615. Wernersson, R.; Schierup, M.H.; Jorgensen, F.G.; Gorodkin, J.; Panitz, F.; Staerfeldt, H.H.; Christensen, O.F.; Mailund, T.; Hornshoj, H.; Klein, A.; Wang, J.; Liu, B.; Hu, S.N.; Dong, W.; Li, W.; Wong, G.K.S.; Yu, J.; Wang, J.; Bendixen, C.; Fredholm, M.; Brunak, S.;