Genomics Core FacilityPublications
An efficient method for genome-wide polyadenylation site mapping and RNA quantification.
Wilkening, S., Pelechano, V., Jarvelin, A.I., Tekkedil, M.M., Anders, S., Benes, V. & Steinmetz, L.M.
Nucleic Acids Res. 2013 Mar 1;41(5):e65. doi: 10.1093/nar/gks1249. Epub 2013 Jan7.
The use of alternative poly(A) sites is common and affects the post-transcriptional fate of mRNA, including its stability, subcellular localization and translation. Here, we present a method to identify poly(A) sites in a genome-wide and strand-specific manner. This method, termed 3'T-fill, initially fills in the poly(A) stretch with unlabeled dTTPs, allowing sequencing to start directly after the poly(A) tail into the 3'-untranslated regions (UTR). Our comparative analysis demonstrates that it outperforms existing protocols in quality and throughput and accurately quantifies RNA levels as only one read is produced from each transcript. We use this method to characterize the diversity of polyadenylation in Saccharomyces cerevisiae, showing that alternative RNA molecules are present even in a genetically identical cell population. Finally, we observe that overlap of convergent 3'-UTRs is frequent but sharply limited by coding regions, suggesting factors that restrict compression of the yeast genome.
Disruption of EXOC6B in a patient with developmental delay, epilepsy, and a de novo balanced t(2;8) translocation.
Fruhmesser, A., Blake, J., Haberlandt, E., Baying, B., Raeder, B., Runz, H., Spreiz, A., Fauth, C., Benes, V., Utermann, G., Zschocke, J. & Kotzot, D.
Eur J Hum Genet. 2013 Feb 20. doi: 10.1038/ejhg.2013.18.
Most balanced chromosomal aberrations are not associated with a clinical phenotype, however, in some patients they may disrupt gene structure. With the development of various next-generation sequencing techniques, fast and specific analyses of the breakpoint regions of chromosomal rearrangements are possible. Here, we report on a 19-year-old woman with a de novo balanced translocation t(2;8)(p13.2;q22.1) and a severe clinical phenotype including intellectual disability, epilepsy, behavioral features resembling autism, and minor dysmorphic features. By next-generation sequencing, we defined the breakpoints and found disruption of the exocyst complex component 6B (EXOC6B) gene in intron 1 on chromosome 2p13.2 involving two Alu elements with a homology of 81%. No gene was found at the respective breakpoint on chromosome 8. Expression analysis of the EXOC6B in blood lymphocytes and buccal smear revealed reduced expression in the patient in comparison with the control. Our findings in combination with one recently published case and one other patient listed in DECIPHER v5.1 indicate EXOC6B as a gene relevant for intellectual development and electrophysiological stability.European Journal of Human Genetics advance online publication, 20 February 2013; doi:10.1038/ejhg.2013.18.
Integrative genomic analyses reveal an androgen-driven somatic alteration landscape in early-onset prostate cancer.
Weischenfeldt, J., Simon, R., Feuerbach, L., Schlangen, K., Weichenhan, D., Minner, S., Wuttig, D., Warnatz, H.J., Stehr, H., Rausch, T., Jager, N., Gu, L., Bogatyrova, O., Stutz, A.M., Claus, R., Eils, J., Eils, R., Gerhauser, C., Huang, P.H., Hutter, B., Kabbe, R., Lawerenz, C., Radomski, S., Bartholomae, C.C., Falth, M., Gade, S., Schmidt, M., Amschler, N., Hass, T., Galal, R., Gjoni, J., Kuner, R., Baer, C., Masser, S., von Kalle, C., Zichner, T., Benes, V., Raeder, B., Mader, M., Amstislavskiy, V., Avci, M., Lehrach, H., Parkhomchuk, D., Sultan, M., Burkhardt, L., Graefen, M., Huland, H., Kluth, M., Krohn, A., Sirma, H., Stumm, L., Steurer, S., Grupp, K., Sultmann, H., Sauter, G., Plass, C., Brors, B., Yaspo, M.L., Korbel, J.O. & Schlomm, T.
Cancer Cell. 2013 Feb 11;23(2):159-70. doi: 10.1016/j.ccr.2013.01.002.
Early-onset prostate cancer (EO-PCA) represents the earliest clinical manifestation of prostate cancer. To compare the genomic alteration landscapes of EO-PCA with "classical" (elderly-onset) PCA, we performed deep sequencing-based genomics analyses in 11 tumors diagnosed at young age, and pursued comparative assessments with seven elderly-onset PCA genomes. Remarkable age-related differences in structural rearrangement (SR) formation became evident, suggesting distinct disease pathomechanisms. Whereas EO-PCAs harbored a prevalence of balanced SRs, with a specific abundance of androgen-regulated ETS gene fusions including TMPRSS2:ERG, elderly-onset PCAs displayed primarily non-androgen-associated SRs. Data from a validation cohort of > 10,000 patients showed age-dependent androgen receptor levels and a prevalence of SRs affecting androgen-regulated genes, further substantiating the activity of a characteristic "androgen-type" pathomechanism in EO-PCA.
Df31 Protein and snoRNAs Maintain Accessible Higher-Order Structures of Chromatin.
Schubert, T., Pusch, M.C., Diermeier, S., Benes, V., Kremmer, E., Imhof, A. & Langst, G.
Mol Cell. 2012 Nov 9;48(3):434-44. doi: 10.1016/j.molcel.2012.08.021. Epub 2012Sep 27.
Packaging of DNA into nucleosomes and the formation of higher-order chromatin structures determine DNA accessibility and activity of genome domains. We identified an RNA-dependent mechanism maintaining the open chromatin structure within euchromatic regions in Drosophila cells. The mechanism of reversible chromatin opening, reconstituted in vitro, depends on the Drosophila decondensation factor 31 (Df31) that specifically binds to RNA and localizes to euchromatic regions. Df31 is capable to tether a heterogeneous pool of short, single-stranded RNAs to chromatin. This class of chromatin-associated RNA (caRNA) is stably linked to chromatin and is largely composed of snoRNAs, which are preferentially bound by Df31. We suggest that the Df31-mediated linkage of snoRNAs and chromatin, forms a RNA-chromatin network resulting in the establishment of open chromatin domains. Analysis of caRNAs in human cells also reveals a strong enrichment of snoRNAs, implying a conserved role for these molecules in higher-order structures of chromatin.
A Role for Fkbp6 and the Chaperone Machinery in piRNA Amplification and Transposon Silencing.
Xiol, J., Cora, E., Koglgruber, R., Chuma, S., Subramanian, S., Hosokawa, M., Reuter, M., Yang, Z., Berninger, P., Palencia, A., Benes, V., Penninger, J., Sachidanandam, R. & Pillai, R.S.
Mol Cell. 2012 Sep 28;47(6):970-9. doi: 10.1016/j.molcel.2012.07.019. Epub 2012Aug 16.
Epigenetic silencing of transposons by Piwi-interacting RNAs (piRNAs) constitutes an RNA-based genome defense mechanism. Piwi endonuclease action amplifies the piRNA pool by generating new piRNAs from target transcripts by a poorly understood mechanism. Here, we identified mouse Fkbp6 as a factor in this biogenesis pathway delivering piRNAs to the Piwi protein Miwi2. Mice lacking Fkbp6 derepress LINE1 (L1) retrotransposon and display reduced DNA methylation due to deficient nuclear accumulation of Miwi2. Like other cochaperones, Fkbp6 associates with the molecular chaperone Hsp90 via its tetratricopeptide repeat (TPR) domain. Inhibition of the ATP-dependent Hsp90 activity in an insect cell culture model results in the accumulation of short antisense RNAs in Piwi complexes. We identify these to be byproducts of piRNA amplification that accumulate only in nuage-localized Piwi proteins. We propose that the chaperone machinery normally ejects these inhibitory RNAs, allowing turnover of Piwi complexes for their continued participation in piRNA amplification.
DELLY: structural variant discovery by integrated paired-end and split-read analysis.
Rausch, T., Zichner, T., Schlattl, A., Stutz, A.M., Benes, V. & Korbel, J.O.
Bioinformatics. 2012 Sep 15;28(18):i333-i339.
MOTIVATION: The discovery of genomic structural variants (SVs) at high sensitivity and specificity is an essential requirement for characterizing naturally occurring variation and for understanding pathological somatic rearrangements in personal genome sequencing data. Of particular interest are integrated methods that accurately identify simple and complex rearrangements in heterogeneous sequencing datasets at single-nucleotide resolution, as an optimal basis for investigating the formation mechanisms and functional consequences of SVs. RESULTS: We have developed an SV discovery method, called DELLY, that integrates short insert paired-ends, long-range mate-pairs and split-read alignments to accurately delineate genomic rearrangements at single-nucleotide resolution. DELLY is suitable for detecting copy-number variable deletion and tandem duplication events as well as balanced rearrangements such as inversions or reciprocal translocations. DELLY, thus, enables to ascertain the full spectrum of genomic rearrangements, including complex events. On simulated data, DELLY compares favorably to other SV prediction methods across a wide range of sequencing parameters. On real data, DELLY reliably uncovers SVs from the 1000 Genomes Project and cancer genomes, and validation experiments of randomly selected deletion loci show a high specificity. AVAILABILITY: DELLY is available at www.korbel.embl.de/software.html CONTACT: firstname.lastname@example.org.
RNA-sequencing as useful screening tool in the combat against the misuse of anabolic agents.
Riedmaier, I., Benes, V., Blake, J., Bretschneider, N., Zinser, C., Becker, C., Meyer, H.H. & Pfaffl, M.W.
Anal Chem. 2012 Aug 7;84(15):6863-8. Epub 2012 Jul 13.
The abuse of anabolic substances in animal husbandry is forbidden within the EU and well controlled by detecting substance residues in different matrices. The application of newly designed drugs or substance cocktails represents big problems. Therefore developing sensitive test methods is important. The analysis of physiological changes caused by the use of anabolic agents on the molecular level, for example, by quantifying gene expression response, is a new approach to develop such screening methods. A novel technology for holistic gene expression analysis is RNA sequencing. In this study, the potential of this high-throughput method for the identification of biomarkers was evaluated. The effect of trenbolone acetate plus estradiol on gene expression in liver from Nguni heifers was analyzed with RNA sequencing. The expression of 40 selected candidate genes was verified via RT-qPCR, whereby 20 of these genes were significantly regulated. To extract the intended information from these regulated genes, biostatistical tools for pattern recognition were applied and resulted in a clear separation of the treatment groups. Those candidate genes could be verified in boars and in calves treated with anabolic substances. These results show the potential of RNA sequencing to screen for biomarker candidates to detect the abuse of anabolics. The verification of these biomarkers in boars and calves leads to the assumption that gene expression biomarkers are independent of breed or even species and that biomarkers, identified in farm animals could also act as potential biomarker candidates to detect the abuse of anabolic substances in human sports.
HnRNP L and L-like cooperate in multiple-exon regulation of CD45 alternative splicing.
Preussner, M., Schreiner, S., Hung, L.H., Porstner, M., Jack, H.M., Benes, V., Ratsch, G. & Bindereif, A.
Nucleic Acids Res. 2012 Jul 1;40(12):5666-78. Epub 2012 Mar 8.
CD45 encodes a trans-membrane protein-tyrosine phosphatase expressed in diverse cells of the immune system. By combinatorial use of three variable exons 4-6, isoforms are generated that differ in their extracellular domain, thereby modulating phosphatase activity and immune response. Alternative splicing of these CD45 exons involves two heterogeneous ribonucleoproteins, hnRNP L and its cell-type specific paralog hnRNP L-like (LL). To address the complex combinatorial splicing of exons 4-6, we investigated hnRNP L/LL protein expression in human B-cells in relation to CD45 splicing patterns, applying RNA-Seq. In addition, mutational and RNA-binding analyses were carried out in HeLa cells. We conclude that hnRNP LL functions as the major CD45 splic repressor, with two CA elements in exon 6 as its primary target. In exon 4, one element is targeted by both hnRNP L and LL. In contrast, exon 5 was never repressed on its own and only co-regulated with exons 4 and 6. Stable L/LL interaction requires CD45 RNA, specifically exons 4 and 6. We propose a novel model of combinatorial alternative splicing: HnRNP L and LL cooperate on the CD45 pre-mRNA, bridging exons 4 and 6 and looping out exon 5, thereby achieving full repression of the three variable exons.
Hfe deficiency impairs pulmonary neutrophil recruitment in response to inflammation.
Benesova, K., Vujic Spasic, M., Schaefer, S.M., Stolte, J., Baehr-Ivacevic, T., Waldow, K., Zhou, Z., Klingmueller, U., Benes, V., Mall, M.A. & Muckenthaler, M.U.
PLoS One. 2012;7(6):e39363. doi: 10.1371/journal.pone.0039363. Epub 2012 Jun 21.
Regulation of iron homeostasis and the inflammatory response are tightly linked to protect the host from infection. Here we investigate how imbalanced systemic iron homeostasis in a murine disease model of hereditary hemochromatosis (Hfe(-/-) mice) affects the inflammatory responses of the lung. We induced acute pulmonary inflammation in Hfe(-/-) and wild-type mice by intratracheal instillation of 20 microg of lipopolysaccharide (LPS) and analyzed local and systemic inflammatory responses and iron-related parameters. We show that in Hfe(-/-) mice neutrophil recruitment to the bronchoalveolar space is attenuated compared to wild-type mice although circulating neutrophil numbers in the bloodstream were elevated to similar levels in Hfe(-/-) and wild-type mice. The underlying molecular mechanisms are likely multifactorial and include elevated systemic iron levels, alveolar macrophage iron deficiency and/or hitherto unexplored functions of Hfe in resident pulmonary cell types. As a consequence, pulmonary cytokine expression is out of balance and neutrophils fail to be recruited efficiently to the bronchoalveolar compartment, a process required to protect the host from infections. In conclusion, our findings suggest a novel role for Hfe and/or imbalanced iron homeostasis in the regulation of the inflammatory response in the lung and hereditary hemochromatosis.
Genomics of DNA cytosine methylation in Escherichia coli reveals its role in stationary phase transcription.
Kahramanoglou, C., Prieto, A.I., Khedkar, S., Haase, B., Gupta, A., Benes, V., Fraser, G.M., Luscombe, N.M. & Seshasayee, A.S.
Nat Commun. 2012 Jun 6;3:886. doi: 10.1038/ncomms1878.
DNA cytosine methylation regulates gene expression in mammals. In bacteria, its role in gene expression and genome architecture is less understood. Here we perform high-throughput sequencing of bisulfite-treated genomic DNA from Escherichia coli K12 to describe, for the first time, the extent of cytosine methylation of bacterial DNA at single-base resolution. Whereas most target sites (C(m)CWGG) are fully methylated in stationary phase cells, many sites with an extended CC(m)CWGG motif are only partially methylated in exponentially growing cells. We speculate that these partially methylated sites may be selected, as these are slightly correlated with the risk of spontaneous, non-synonymous conversion of methylated cytosines to thymines. Microarray analysis in a cytosine methylation-deficient mutant of E. coli shows increased expression of the stress response sigma factor RpoS and many of its targets in stationary phase. Thus, DNA cytosine methylation is a regulator of stationary phase gene expression in E. coli.
Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway.
Simpson, J.C., Joggerst, B., Laketa, V., Verissimo, F., Cetin, C., Erfle, H., Bexiga, M.G., Singan, V.R., Heriche, J.K., Neumann, B., Mateos, A., Blake, J., Bechtel, S., Benes, V., Wiemann, S., Ellenberg, J. & Pepperkok, R.
Nat Cell Biol. 2012 Jun 3;14(7):764-74. doi: 10.1038/ncb2510.
The secretory pathway in mammalian cells has evolved to facilitate the transfer of cargo molecules to internal and cell surface membranes. Use of automated microscopy-based genome-wide RNA interference screens in cultured human cells allowed us to identify 554 proteins influencing secretion. Cloning, fluorescent-tagging and subcellular localization analysis of 179 of these proteins revealed that more than two-thirds localize to either the cytoplasm or membranes of the secretory and endocytic pathways. The depletion of 143 of them resulted in perturbations in the organization of the COPII and/or COPI vesicular coat complexes of the early secretory pathway, or the morphology of the Golgi complex. Network analyses revealed a so far unappreciated link between early secretory pathway function, small GTP-binding protein regulation, actin cytoskeleton organization and EGF-receptor-mediated signalling. This work provides an important resource for an integrative understanding of global cellular organization and regulation of the secretory pathway in mammalian cells.
Role of CXCL13 in asthma: novel therapeutic target.
Baay-Guzman, G.J., Huerta-Yepez, S., Vega, M.I., Aguilar-Leon, D., Campillos, M., Blake, J., Benes, V., Hernandez-Pando, R. & Teran, L.M.
Chest. 2012 Apr;141(4):886-94. doi: 10.1378/chest.11-0633. Epub 2011 Oct 20.
BACKGROUND: B cells play an important role in allergic asthma. However, the mechanisms by which these cells are activated in the airways remain poorly understood. METHODS: We used a mouse model of ovalbumin (OVA)-induced allergic inflammation to study CXCL13 and to investigate the concentration of this chemokine in the BAL fluid derived from asthmatic and normal control subjects. RESULTS: We found that OVA-challenged mice upregulate the CXCL13/CXCR5 axis, which is associated with several changes in their airways, including recruitment of B and CD4(+) cells, development of bronchial-associated lymphoid tissue, and airway inflammation. Treating sensitized mice with an anti-CXCL13 antibody reduced cell recruitment, bronchial-associated lymphoid tissue formation, and airways inflammation. Interestingly, measurements of CXCL13 using enzyme-linked immunosorbent assay showed that levels of this cytokine were significantly elevated in BAL fluid from subjects with asthma compared with control subjects (median, 162 [range, 120-296] vs 31 [range, 120-156] pg/mL; P = .005). CONCLUSIONS: All together, these findings suggest that CXCL13 is involved in the allergic airway inflammatory process, and targeting this chemokine may constitute a novel approach in asthma.
Direct regulation of microRNA biogenesis and expression by estrogen receptor beta in hormone-responsive breast cancer.
Paris, O., Ferraro, L., Grober, O.M., Ravo, M., De Filippo, M.R., Giurato, G., Nassa, G., Tarallo, R., Cantarella, C., Rizzo, F., Di Benedetto, A., Mottolese, M., Benes, V., Ambrosino, C., Nola, E. & Weisz, A.
Oncogene. 2012 Jan 9. doi: 10.1038/onc.2011.583.
Estrogen effects on mammary epithelial and breast cancer (BC) cells are mediated by the nuclear receptors ERalpha and ERbeta, transcription factors that display functional antagonism with each other, with ERbeta acting as oncosuppressor and interfering with the effects of ERalpha on cell proliferation, tumor promotion and progression. Indeed, hormone-responsive, ERalpha+ BC cells often lack ERbeta, which when present associates with a less aggressive clinical phenotype of the disease. Recent evidences point to a significant role of microRNAs (miRNAs) in BC, where specific miRNA expression profiles associate with distinct clinical and biological phenotypes of the lesion. Considering the possibility that ERbeta might influence BC cell behavior via miRNAs, we compared miRNome expression in ERbeta+ vs ERbeta- hormone-responsive BC cells and found a widespread effect of this ER subtype on the expression pattern of these non-coding RNAs. More importantly, the expression pattern of 67 miRNAs, including 10 regulated by ERbeta in BC cells, clearly distinguishes ERbeta+, node-negative, from ERbeta-, metastatic, mammary tumors. Molecular dissection of miRNA biogenesis revealed multiple mechanisms for direct regulation of this process by ERbeta+ in BC cell nuclei. In particular, ERbeta downregulates miR-30a by binding to two specific sites proximal to the gene and thereby inhibiting pri-miR synthesis. On the other hand, the receptor promotes miR-23b, -27b and 24-1 accumulation in the cell by binding in close proximity of the corresponding gene cluster and preventing in situ the inhibitory effects of ERalpha on pri-miR maturation by the p68/DDX5-Drosha microprocessor complex. These results indicate that cell autonomous regulation of miRNA expression is part of the mechanism of action of ERbeta in BC cells and could contribute to establishment or maintenance of a less aggressive tumor phenotype mediated by this nuclear receptor.Oncogene advance online publication, 9 January 2012; doi:10.1038/onc.2011.583.
Genome Sequencing of Pediatric Medulloblastoma Links Catastrophic DNA Rearrangements with TP53 Mutations.
Rausch, T., Jones, D.T., Zapatka, M., Stutz, A.M., Zichner, T., Weischenfeldt, J., Jager, N., Remke, M., Shih, D., Northcott, P.A., Pfaff, E., Tica, J., Wang, Q., Massimi, L., Witt, H., Bender, S., Pleier, S., Cin, H., Hawkins, C., Beck, C., von Deimling, A., Hans, V., Brors, B., Eils, R., Scheurlen, W., Blake, J., Benes, V., Kulozik, A.E., Witt, O., Martin, D., Zhang, C., Porat, R., Merino, D.M., Wasserman, J., Jabado, N., Fontebasso, A., Bullinger, L., Rucker, F.G., Dohner, K., Dohner, H., Koster, J., Molenaar, J.J., Versteeg, R., Kool, M., Tabori, U., Malkin, D., Korshunov, A., Taylor, M.D., Lichter, P., Pfister, S.M. & Korbel, J.O.
Cell. 2012 Jan 20;148(1-2):59-71.
Genomic rearrangements are thought to occur progressively during tumor development. Recent findings, however, suggest an alternative mechanism, involving massive chromosome rearrangements in a one-step catastrophic event termed chromothripsis. We report the whole-genome sequencing-based analysis of a Sonic-Hedgehog medulloblastoma (SHH-MB) brain tumor from a patient with a germline TP53 mutation (Li-Fraumeni syndrome), uncovering massive, complex chromosome rearrangements. Integrating TP53 status with microarray and deep sequencing-based DNA rearrangement data in additional patients reveals a striking association between TP53 mutation and chromothripsis in SHH-MBs. Analysis of additional tumor entities substantiates a link between TP53 mutation and chromothripsis, and indicates a context-specific
Kaposi's Sarcoma Herpesvirus microRNAs Target Caspase 3 and Regulate Apoptosis.
Suffert, G., Malterer, G., Hausser, J., Viiliainen, J., Fender, A., Contrant, M., Ivacevic, T., Benes, V., Gros, F., Voinnet, O., Zavolan, M., Ojala, P.M., Haas, J.G. & Pfeffer, S.
PLoS Pathog. 2011 Dec;7(12):e1002405. Epub 2011 Dec 8.
Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.
Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by iron regulatory proteins.
Sanchez, M., Galy, B., Schwanhaeusser, B., Blake, J., Bahr-Ivacevic, T., Benes, V., Selbach, M., Muckenthaler, M.U. & Hentze, M.W.
Blood. 2011 Nov 24;118(22):e168-79. Epub 2011 Sep 22.
Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metabolism by binding to conserved RNA motifs called iron-responsive elements (IREs). The currently known IRP-binding mRNAs encode proteins involved in iron uptake, storage, and release as well as heme synthesis. To systematically define the IRE/IRP regulatory network on a transcriptome-wide scale, IRP1/IRE and IRP2/IRE messenger ribonucleoprotein complexes were immunoselected, and the mRNA composition was determined using microarrays. We identify 35 novel mRNAs that bind both IRP1 and IRP2, and we also report for the first time cellular mRNAs with exclusive specificity for IRP1 or IRP2. To further explore cellular iron metabolism at a system-wide level, we undertook proteomic analysis by pulsed stable isotope labeling by amino acids in cell culture in an iron-modulated mouse hepatic cell line and in bone marrow-derived macrophages from IRP1- and IRP2-deficient mice. This work investigates cellular iron metabolism in unprecedented depth and defines a wide network of mRNAs and proteins with iron-dependent regulation, IRP-dependent regulation, or both.
Nuclear hormone 1alpha,25-dihydroxyvitamin D3 elicits a genome-wide shift in the locations of VDR chromatin occupancy.
Heikkinen, S., Vaisanen, S., Pehkonen, P., Seuter, S., Benes, V. & Carlberg, C.
Nucleic Acids Res. 2011 Nov 1;39(21):9181-93. Epub 2011 Aug 16.
A global understanding of the actions of the nuclear hormone 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) and its vitamin D receptor (VDR) requires a genome-wide analysis of VDR binding sites. In THP-1 human monocytic leukemia cells we identified by ChIP-seq 2340 VDR binding locations, of which 1171 and 520 occurred uniquely with and without 1alpha,25(OH)(2)D(3) treatment, respectively, while 649 were common. De novo identified direct repeat spaced by 3 nucleotides (DR3)-type response elements (REs) were strongly associated with the ligand-responsiveness of VDR occupation. Only 20% of the VDR peaks diminishing most after ligand treatment have a DR3-type RE, in contrast to 90% for the most growing peaks. Ligand treatment revealed 638 1alpha,25(OH)(2)D(3) target genes enriched
Exome sequencing identifies MRPL3 mutation in mitochondrial cardiomyopathy.
Galmiche, L., Serre, V., Beinat, M., Assouline, Z., Lebre, A.S., Chretien, D., Nietschke, P., Benes, V., Boddaert, N., Sidi, D., Brunelle, F., Rio, M., Munnich, A. & Rotig, A.
Hum Mutat. 2011 Nov;32(11):1225-31. doi: 10.1002/humu.21562. Epub 2011 Sep14.
By combining exome sequencing in conjunction with genetic mapping, we have identified the first mutation in large mitochondrial ribosomal protein MRPL3 in a family of four sibs with hypertrophic cardiomyopathy, psychomotor retardation, and multiple respiratory chain deficiency. Affected sibs were compound heterozygotes for a missense MRPL3 mutation (P317R) and a large-scale deletion, inherited from the mother and the father, respectively. These mutations were shown to alter ribosome assembly and cause a mitochondrial translation deficiency in cultured skin fibroblasts resulting in an abnormal assembly of several complexes of the respiratory chain. This observation gives support to the view that exome sequencing combined with genetic mapping is a powerful approach for the identification of new genes of mitochondrial disorders.
Primer Sequence Disclosure: A Clarification of the MIQE Guidelines.
Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J. & Wittwer, C.T.
Clin Chem. 2011 Jun;57(6):919-21. Epub 2011 Mar 18. PubMed
RNA-Seq analysis in mutant zebrafish reveals role of U1C protein in alternative splicing regulation.
Rosel, T.D., Hung, L.H., Medenbach, J., Donde, K., Starke, S., Benes, V., Ratsch, G. & Bindereif, A.
EMBO J. 2011 May 18;30(10):1965-76. Epub 2011 Apr 5.
Precise 5' splice-site recognition is essential for both constitutive and regulated pre-mRNA splicing. The U1 small nuclear ribonucleoprotein particle (snRNP)-specific protein U1C is involved in this first step of spliceosome assembly and important for stabilizing early splicing complexes. We used an embryonically lethal U1C mutant zebrafish, hi1371, to investigate the potential genomewide role of U1C for splicing regulation. U1C mutant embryos contain overall stable, but U1C-deficient U1 snRNPs. Surprisingly, genomewide RNA-Seq analysis of mutant versus wild-type embryos revealed a large set of specific target genes that changed their alternative splicing patterns in the absence of U1C. Injection of ZfU1C cRNA into mutant embryos and in vivo splicing experiments in HeLa cells after siRNA-mediated U1C knockdown confirmed the U1C dependency and specificity, as well as the functional conservation of the effects observed. In addition, sequence motif analysis of the U1C-dependent 5' splice sites uncovered an association with downstream intronic U-rich elements. In sum, our findings provide evidence for a new role of a general snRNP protein, U1C, as a mediator of alternative splicing regulation.
The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice.
Castoldi, M., Vujic Spasic, M., Altamura, S., Elmen, J., Lindow, M., Kiss, J., Stolte, J., Sparla, R., D'Alessandro, L.A., Klingmuller, U., Fleming, R.E., Longerich, T., Grone, H.J., Benes, V., Kauppinen, S., Hentze, M.W. & Muckenthaler, M.U.
J Clin Invest. 2011 Apr;121(4):1386-96. doi: 10.1172/JCI44883.
Systemic iron homeostasis is mainly controlled by the liver through synthesis of the peptide hormone hepcidin (encoded by Hamp), the key regulator of duodenal iron absorption and macrophage iron release. Here we show that the liver-specific microRNA miR-122 is important for regulating Hamp mRNA expression and tissue iron levels. Efficient and specific depletion of miR-122 by injection of a locked-nucleic-acid-modified (LNA-modified) anti-miR into WT mice caused systemic iron deficiency, characterized by reduced plasma and liver iron levels, mildly impaired hematopoiesis, and increased extramedullary erythropoiesis in the spleen. Moreover, miR-122 inhibition increased the amount of mRNA transcribed by genes that control systemic iron levels, such as hemochromatosis (Hfe), hemojuvelin (Hjv), bone morphogenetic protein receptor type 1A (Bmpr1a), and Hamp. Importantly, miR-122 directly targeted the 3' untranslated region of 2 mRNAs that encode activators of hepcidin expression, Hfe and Hjv. These data help to explain the increased Hamp mRNA levels and subsequent iron deficiency in mice with reduced miR-122 levels and establish a direct mechanistic link between miR-122 and the regulation of systemic iron metabolism.
Differentially expressed genes associated with human limbal epithelial phenotypes: new molecules that potentially facilitate selection of stem cell-enriched populations.
Takacs, L., Toth, E., Losonczy, G., Szanto, A., Bahr-Ivacevic, T., Benes, V., Berta, A. & Vereb, G.
Invest Ophthalmol Vis Sci. 2011 Mar 10;52(3):1252-60. doi: 10.1167/iovs.10-5242.
PURPOSE: The aim of this study was to identify differentially expressed genes in the human limbal epithelium by microarray analysis. METHODS: Total RNA isolates of human limbal and central corneal epithelia were used after transcription for hybridization on whole human genome expression microarrays. A set of differentially expressed genes detected by both microarrays was established. In the case of eight selected molecules, microarray results were confirmed by qRT-PCR, and protein expression in the cornea was examined by confocal immunofluorescence microscopy. Colocalization with the putative stem cell marker C/EBPdelta was also examined. RESULTS: The authors established a database of 126 limbal overexpressed genes. qRT-PCR confirmed microarray results in all examined cases (SPON1, IFITM1, ITM2A, PHLDA1, CXCR4, FZD7, DCT, DKK4). Limbal localization of the protein product of SPON1, IFITM1, ITM2A, CXCR4, and DKK4 was shown with confocal immunofluorescence microscopy. SPON1, IFITM1, and ITM2A signals mostly colocalized with C/EBPdelta-positive putative resting limbal stem cells. CONCLUSIONS: By detecting several new differentially expressed genes in the human corneal limbus, this study further expands current knowledge on the molecular signature of limbal epithelial stem cells. Plasma membrane localization of IFITM1 and ITM2A suggests their potential usefulness as targets to select stem cell-enriched populations from the limbal epithelium.
Direct and indirect effects of H-NS and Fis on global gene expression control in Escherichia coli.
Kahramanoglou, C., Seshasayee, A.S., Prieto, A.I., Ibberson, D., Schmidt, S., Zimmermann, J., Benes, V., Fraser, G.M. & Luscombe, N.M.
Nucleic Acids Res. 2011 Mar;39(6):2073-91. doi: 10.1093/nar/gkq934. Epub 2010 Nov21.
Nucleoid-associated proteins (NAPs) are global regulators of gene expression in Escherichia coli, which affect DNA conformation by bending, wrapping and bridging the DNA. Two of these--H-NS and Fis--bind to specific DNA sequences and structures. Because of their importance to global gene expression, the binding of these NAPs to the DNA was previously investigated on a genome-wide scale using ChIP-chip. However, variation in their binding profiles across the growth phase and the genome-scale nature of their impact on gene expression remain poorly understood. Here, we present a genome-scale investigation of H-NS and Fis binding to the E. coli chromosome using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq). By performing our experiments under multiple time-points during growth in rich media, we show that the binding regions of the two proteins are mutually exclusive under our experimental conditions. H-NS binds to significantly longer tracts of DNA than Fis, consistent with the linear spread of H-NS binding from high- to surrounding lower-affinity sites; the length of binding regions is associated with the degree of transcriptional repression imposed by H-NS. For Fis, a majority of binding events do not lead to differential expression of the proximal gene; however, it has a significant indirect effect on gene expression partly through its effects on the expression of other transcription factors. We propose that direct transcriptional regulation by Fis is associated with the interaction of tandem arrays of Fis molecules to the DNA and possible DNA bending, particularly at operon-upstream regions. Our study serves as a proof-of-principle for the use of ChIP-seq for global DNA-binding proteins in bacteria, which should become significantly more economical and feasible with the development of multiplexing techniques.
Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation.
Grober, O.M., Mutarelli, M., Giurato, G., Ravo, M., Cicatiello, L., De Filippo, M.R., Ferraro, L., Nassa, G., Papa, M.F., Paris, O., Tarallo, R., Luo, S., Schroth, G.P., Benes, V. & Weisz, A.
BMC Genomics. 2011 Jan 14;12:36. doi: 10.1186/1471-2164-12-36.
BACKGROUND: Estrogen receptors alpha (ERalpha) and beta (ERbeta) are transcription factors (TFs) that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer (BC). The two receptors can be found co-expressed and play specific, often opposite, roles, with ERbeta being able to modulate the effects of ERalpha on gene transcription and cell proliferation. ERbeta is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease. The identification of the genomic targets of ERbeta in hormone-responsive BC cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology. RESULTS: Expression of full-length ERbeta in hormone-responsive, ERalpha-positive MCF-7 cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. By ChIP-Seq we identified 9702 ERbeta and 6024 ERalpha binding sites in estrogen-stimulated cells, comprising sites occupied by either ERbeta, ERalpha or both ER subtypes. A search for TF binding matrices revealed that the majority of the binding sites identified comprise one or more Estrogen Response Element and the remaining show binding matrixes for other TFs known to mediate ER interaction with chromatin by tethering, including AP2, E2F and SP1. Of 921 genes differentially regulated by estrogen in ERbeta+ vs ERbeta- cells, 424 showed one or more ERbeta site within 10 kb. These putative primary ERbeta target genes control cell proliferation, death, differentiation, motility and adhesion, signal transduction and transcription, key cellular processes that might explain the biological and clinical phenotype of tumors expressing this ER subtype. ERbeta binding in close proximity of several miRNA genes and in the mitochondrial genome, suggests the possible involvement of this receptor in small non-coding RNA biogenesis and mitochondrial genome functions. CONCLUSIONS: Results indicate that the vast majority of the genomic targets of ERbeta can bind also ERalpha, suggesting that the overall action of ERbeta on the genome of hormone-responsive BC cells depends mainly on the relative concentration of both ERs in the cell.
CRX ChIP-seq reveals the cis-regulatory architecture of mouse photoreceptors.
Corbo, J.C., Lawrence, K.A., Karlstetter, M., Myers, C.A., Abdelaziz, M., Dirkes, W., Weigelt, K., Seifert, M., Benes, V., Fritsche, L.G., Weber, B.H. & Langmann, T.
Genome Res. 2010 Nov;20(11):1512-25. Epub 2010 Aug 6.
Approximately 98% of mammalian DNA is noncoding, yet we understand relatively little about the function of this enigmatic portion of the genome. The cis-regulatory elements that control gene expression reside in noncoding regions and can be identified by mapping the binding sites of tissue-specific transcription factors. Cone-rod homeobox (CRX) is a key transcription factor in photoreceptor differentiation and survival, but its in vivo targets are largely unknown. Here, we used chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) on CRX to identify thousands of cis-regulatory regions around photoreceptor genes in adult mouse retina. CRX directly regulates downstream photoreceptor transcription factors and their target genes via a network of spatially distributed regulatory elements around each locus. CRX-bound regions act in a synergistic fashion to activate transcription and contain multiple CRX binding sites which interact in a spacing- and orientation-dependent manner to fine-tune transcript levels. CRX ChIP-seq was also performed on Nrl(-/-) retinas, which represent an enriched source of cone photoreceptors. Comparison with the wild-type ChIP-seq data set identified numerous rod- and cone-specific CRX-bound regions as well as many shared elements. Thus, CRX combinatorially orchestrates the transcriptional networks of both rods and cones by coordinating the expression of photoreceptor genes including most retinal disease genes. In addition, this study pinpoints thousands of noncoding regions of relevance to both Mendelian and complex retinal disease.
Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available.
Benes, V. & Castoldi, M.
Methods. 2010 Apr;50(4):244-9. doi: 10.1016/j.ymeth.2010.01.026. Epub 2010 Jan28.
We review different methodologies to estimate the expression levels of microRNAs (miRNAs) using real-time quantitative PCR (qPCR). As miRNA analysis is a fast changing research field, we have introduced novel technological approaches and compared them to existing qPCR profiling methodologies. qPCR also remains the method of choice for validating results obtained from whole-genome screening (e.g. with microarray). In contrast to presenting a stepwise description of different platforms, we discuss expression profiling of mature miRNAs by qPCR in four sequential sections: (1) cDNA synthesis; (2) primer design; (3) detection of amplified products; and (4) data normalization. We address technical challenges associated with each of these and outline possible solutions.
Thyrotroph embryonic factor regulates light-induced transcription of repair genes in zebrafish embryonic cells.
Gavriouchkina, D., Fischer, S., Ivacevic, T., Stolte, J., Benes, V. & Dekens, M.P.
PLoS One. 2010 Sep 7;5(9):e12542.
Numerous responses are triggered by light in the cell. How the light signal is detected and transduced into a cellular response is still an enigma. Each zebrafish cell has the capacity to directly detect light, making this organism particularly suitable for the study of light dependent transcription. To gain insight into the light signalling mechanism we identified genes that are activated by light exposure at an early embryonic stage, when specialised light sensing organs have not yet formed. We screened over 14,900 genes using micro-array GeneChips, and identified 19 light-induced genes that function primarily in light signalling, stress response, and DNA repair. Here we reveal that PAR Response Elements are present in all promoters of the light-induced genes, and demonstrate a pivotal role for the PAR bZip transcription factor Thyrotroph embryonic factor (Tef) in regulating the majority of light-induced genes. We show that tefbeta transcription is directly regulated by light while transcription of tefalpha is under circadian clock control at later stages of development. These data leads us to propose their involvement in light-induced UV tolerance in the zebrafish embryo.
Analysis of microribonucleic acid expression by quantitative real-time polymerase chain reaction.
Benes, V., Stolte, J., Ibberson, D., Castoldi, M., Muckenthaler, M.
In The PCR Revolution: Basic Technologies and Applications (S. Bustin, Ed.) Cambridge Univ Press (2010), ISBN: 978-0-521-88231-6.
An assessment of air as a source of DNA contamination encountered when performing PCR.
Witt, N., Rodger, G., Vandesompele, J., Benes, V., Zumla, A., Rook, G.A. & Huggett, J.F.
J Biomol Tech. 2009 Dec;20(5):236-40.
Sensitive molecular methods, such as the PCR, can detect low-level contamination, and careful technique is required to reduce the impact of contaminants. Yet, some assays that are designed to detect high copy-number target sequences appear to be impossible to perform without contamination, and frequently, personnel or laboratory environment are held responsible as the source. This complicates diagnostic and research analysis when using molecular methods. To investigate the air specifically as a source of contamination, which might occur during PCR setup, we exposed tubes of water to the air of a laboratory and clean hood for up to 24 h. To increase the chances of contamination, we also investigated a busy open-plan office in the same way. All of the experiments showed the presence of human and rodent DNA contamination. However, there was no accumulation of the contamination in any of the environments investigated, suggesting that the air was not the source of contamination. Even the air from a busy open-plan office was a poor source of contamination for all of the DNA sequences investigated (human, bacterial, fungal, and rodent). This demonstrates that the personnel and immediate laboratory environment are not necessarily to blame for the observed contamination.
Widespread estrogen-dependent repression of micrornas involved in breast tumor cell growth.
Maillot, G., Lacroix-Triki, M., Pierredon, S., Gratadou, L., Schmidt, S., Benes, V., Roche, H., Dalenc, F., Auboeuf, D., Millevoi, S. & Vagner, S.
Cancer Res. 2009 Nov 1;69(21):8332-40. Epub 2009 Oct 13.
Altered expression of microRNAs (miRNA), an abundant class of small nonprotein-coding RNAs that mostly function as negative regulators of protein-coding gene expression, is common in cancer. Here, we analyze the regulation of miRNA expression in response to estrogen, a steroid hormone that is involved in the development and progression of breast carcinomas and that is acting via the estrogen receptors (ER) transcription factors. We set out to thoroughly describe miRNA expression, by using miRNA microarrays and real-time reverse transcription-PCR (RT-PCR) experiments, in various breast tumor cell lines in which estrogen signaling has been induced by 17beta-estradiol (E(2)). We show that the expression of a broad set of miRNAs decreases following E(2) treatment in an ER-dependent manner. We further show that enforced expression of several of the repressed miRNAs reduces E(2)-dependent cell growth, thus linking expression of specific miRNAs with estrogen-dependent cellular response. In addition, a transcriptome analysis revealed that the E(2)-repressed miR-26a and miR-181a regulate many genes associated with cell growth and proliferation, including the progesterone receptor gene, a key actor in estrogen signaling. Strikingly, miRNA expression is also regulated in breast cancers of women who had received antiestrogen neoadjuvant therapy. Overall, our data indicate that the extensive alterations in miRNA regulation upon estrogen signaling pathway play a key role in estrogen-dependent functions and highlight the utility of considering miRNA expression in the understanding of antiestrogen resistance of breast cancer.
Inhibition of aurora kinases for tailored risk-adapted treatment of multiple myeloma.
Hose, D., Reme, T., Meissner, T., Moreaux, J., Seckinger, A., Lewis, J.D., Benes, V., Benner, A., Hundemer, M., Hielscher, T., Shaughnessy JD, J.r, Barlogie, B., Neben, K., Kramer, A., Hillengass, J., Bertsch, U., Jauch, A., De Vos, J., Rossi, J.F., Mohler, T., Blake, J., Zimmermann, J., Klein, B. & Goldschmidt, H.
Blood. 2009 Apr 30;113(18):4331-40. Epub 2009 Jan 26.
Genetic instability and cellular proliferation have been associated with aurora kinase expression in several cancer entities, including multiple myeloma. Therefore, the expression of aurora-A, -B, and -C was determined by Affymetrix DNA microarrays in 784 samples including 2 independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive interphase fluorescence in situ hybridization and proliferation of primary myeloma cells by propidium iodine staining. We found aurora-A and -B to be expressed at varying frequencies in primary myeloma cells of different patient cohorts, but aurora-C in testis cell samples only. Myeloma cell samples with detectable versus absent aurora-A expression show a significantly higher proliferation rate, but neither a higher absolute number of chromosomal aberrations (aneuploidy), nor of subclonal aberrations (chromosomal instability). The clinical aurora kinase inhibitor VX680 induced apoptosis in 20 of 20 myeloma cell lines and 5 of 5 primary myeloma cell samples. Presence of aurora-A expression delineates significantly inferior event-free and overall survival in 2 independent cohorts of patients undergoing high-dose chemotherapy, independent from conventional prognostic factors. Using gene expression profiling, aurora kinase inhibitors as a promising therapeutic option in myeloma can be tailoredly given to patients expressing aurora-A, who in turn have an adverse prognosis.
The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.
Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J. & Wittwer, C.T.
Clin Chem. 2009 Apr;55(4):611-22. Epub 2009 Feb 26.
BACKGROUND: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments. CONTENT: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. SUMMARY: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.
Unreliable real-time PCR analysis of human endogenous retrovirus-W (HERV-W) RNA expression and DNA copy number in multiple sclerosis.
Garson, J.A., Huggett, J.F., Bustin, S.A., Pfaffl, M.W., Benes, V., Vandesompele, J. & Shipley, G.L.
AIDS Res Hum Retroviruses. 2009 Mar;25(3):377-8; author reply 379-81. PubMed
Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.
Weinmann, L., Hock, J., Ivacevic, T., Ohrt, T., Mutze, J., Schwille, P., Kremmer, E., Benes, V., Urlaub, H. & Meister, G.
Cell. 2009 Feb 6;136(3):496-507. Epub 2009 Jan 22.
Small regulatory RNAs including small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide Argonaute (Ago) proteins to specific target RNAs leading to mRNA destabilization or translational repression. Here, we report the identification of Importin 8 (Imp8) as a component of miRNA-guided regulatory pathways. We show that Imp8 interacts with Ago proteins and localizes to cytoplasmic processing bodies (P bodies), structures involved in RNA metabolism. Furthermore, we detect Ago2 in the nucleus of HeLa cells, and knockdown of Imp8 reduces the nuclear Ago2 pool. Using immunoprecipitations of Ago2-associated mRNAs followed by microarray analysis, we further demonstrate that Imp8 is required for the recruitment of Ago protein complexes to a large set of Ago2-associated target mRNAs, allowing for efficient and specific gene silencing. Therefore, we provide evidence that Imp8 is required for cytoplasmic miRNA-guided gene silencing and affects nuclear localization of Ago proteins.
Aging and replicative senescence have related effects on human stem and progenitor cells.
Wagner, W., Bork, S., Horn, P., Krunic, D., Walenda, T., Diehlmann, A., Benes, V., Blake, J., Huber, F.X., Eckstein, V., Boukamp, P. & Ho, A.D.
PLoS One. 2009 Jun 9;4(6):e5846.
The regenerative potential diminishes with age and this has been ascribed to functional impairments of adult stem cells. Cells in culture undergo senescence after a certain number of cell divisions whereby the cells enlarge and finally stop proliferation. This observation of replicative senescence has been extrapolated to somatic stem cells in vivo and might reflect the aging process of the whole organism. In this study we have analyzed the effect of aging on gene expression profiles of human mesenchymal stromal cells (MSC) and human hematopoietic progenitor cells (HPC). MSC were isolated from bone marrow of donors between 21 and 92 years old. 67 genes were age-induced and 60 were age-repressed. HPC were isolated from cord blood or from mobilized peripheral blood of donors between 27 and 73 years and 432 genes were age-induced and 495 were age-repressed. The overlap of age-associated differential gene expression in HPC and MSC was moderate. However, it was striking that several age-related gene expression changes in both MSC and HPC were also differentially expressed upon replicative senescence of MSC in vitro. Especially genes involved in genomic integrity and regulation of transcription were age-repressed. Although telomerase activity and telomere length varied in HPC particularly from older donors, an age-dependent decline was not significant arguing against telomere exhaustion as being causal for the aging phenotype. These studies have demonstrated that aging causes gene expression changes in human MSC and HPC that vary between the two different cell types. Changes upon aging of MSC and HPC are related to those of replicative senescence of MSC in vitro and this indicates that our stem and progenitor cells undergo a similar process also in vivo.
MicroRNA signatures predict oestrogen receptor, progesterone receptor and HER2/neu receptor status in breast cancer.
Lowery, A.J., Miller, N., Devaney, A., McNeill, R.E., Davoren, P.A., Lemetre, C., Benes, V., Schmidt, S., Blake, J., Ball, G. & Kerin, M.J.
Breast Cancer Res. 2009;11(3):R27. Epub 2009 May 11.
INTRODUCTION: Breast cancer is a heterogeneous disease encompassing a number of phenotypically diverse tumours. Expression levels of the oestrogen, progesterone and HER2/neu receptors which characterize clinically distinct breast tumours have been shown to change during disease progression and in response to systemic therapies. Mi(cro)RNAs play critical roles in diverse biological processes and are aberrantly expressed in several human neoplasms including breast cancer, where they function as regulators of tumour behaviour and progression. The aims of this study were to identify miRNA signatures that accurately predict the oestrogen receptor (ER), progesterone receptor (PR) and HER2/neu receptor status of breast cancer patients to provide insight into the regulation of breast cancer phenotypes and progression. METHODS: Expression profiling of 453 miRNAs was performed in 29 early-stage breast cancer specimens. miRNA signatures associated with ER, PR and HER2/neu status were generated using artificial neural networks (ANN), and expression of specific miRNAs was validated using RQ-PCR. RESULTS: Stepwise ANN analysis identified predictive miRNA signatures corresponding with oestrogen (miR-342, miR-299, miR-217, miR-190, miR-135b, miR-218), progesterone (miR-520g, miR-377, miR-527-518a, miR-520f-520c) and HER2/neu (miR-520d, miR-181c, miR-302c, miR-376b, miR-30e) receptor status. MiR-342 and miR-520g expression was further analysed in 95 breast tumours. MiR-342 expression was highest in ER and HER2/neu-positive luminal B tumours and lowest in triple-negative tumours. MiR-520g expression was elevated in ER and PR-negative tumours. CONCLUSIONS: This study demonstrates that ANN analysis reliably identifies biologically relevant miRNAs associated with specific breast cancer phenotypes. The association of specific miRNAs with ER, PR and HER2/neu status indicates a role for these miRNAs in disease classification of breast cancer. Decreased expression of miR-342 in the therapeutically challenging triple-negative breast tumours, increased miR-342 expression in the luminal B tumours, and downregulated miR-520g in ER and PR-positive tumours indicates that not only is dysregulated miRNA expression a marker for poorer prognosis breast cancer, but that it could also present an attractive target for therapeutic intervention.
RNA degradation compromises the reliability of microRNA expression profiling.
Ibberson, D., Benes, V., Muckenthaler, M.U. & Castoldi, M.
BMC Biotechnol. 2009 Dec 21;9:102.
BACKGROUND: MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression and their expression is frequently altered in human diseases, including cancer. To correlate clinically relevant parameters with microRNA expression, total RNA is frequently prepared from samples that were archived for various time periods in frozen tissue banks but, unfortunately, RNA integrity is not always preserved in these frozen tissues. Here, we investigate whether experimentally induced RNA degradation affects microRNA expression profiles. RESULTS: Tissue samples were maintained on ice for defined time periods prior to total RNA extraction, which resulted in different degrees of RNA degradation. MicroRNA expression was then analyzed by microarray analysis (miCHIP) or microRNA-specific real-time quantitative PCR (miQPCR). Our results demonstrate that the loss of RNA integrity leads to in unpredictability of microRNA expression profiles for both, array-based and miQPCR assays. CONCLUSION: MicroRNA expression cannot be reliably profiled in degraded total RNA. For the profiling of microRNAs we recommend use of RNA samples with a RNA integrity number equal to or above seven.
Cyclical DNA methylation of a transcriptionally active promoter.
Metivier, R., Gallais, R., Tiffoche, C., Le Peron, C., Jurkowska, R.Z., Carmouche, R.P., Ibberson, D., Barath, P., Demay, F., Reid, G., Benes, V., Jeltsch, A., Gannon, F. & Salbert, G.
Nature. 2008 Mar 6;452(7183):45-50.
Processes that regulate gene transcription are directly under the influence of the genome organization. The epigenome contains additional information that is not brought by DNA sequence, and generates spatial and functional constraints that complement genetic instructions. DNA methylation on CpGs constitutes an epigenetic mark generally correlated with transcriptionally silent condensed chromatin. Replication of methylation patterns by DNA methyltransferases maintains genome stability through cell division. Here we present evidence of an unanticipated dynamic role for DNA methylation in gene regulation in human cells. Periodic, strand-specific methylation/demethylation occurs during transcriptional cycling of the pS2/TFF1 gene promoter on activation by oestrogens. DNA methyltransferases exhibit dual actions during these cycles, being involved in CpG methylation and active demethylation of 5mCpGs through deamination. Inhibition of this process precludes demethylation of the pS2 gene promoter and its subsequent transcriptional activation. Cyclical changes in the methylation status of promoter CpGs may thus represent a critical event in transcriptional achievement.
Transient cyclical methylation of promoter DNA.
Kangaspeska, S., Stride, B., Metivier, R., Polycarpou-Schwarz, M., Ibberson, D., Carmouche, R.P., Benes, V., Gannon, F. & Reid, G.
Nature. 2008 Mar 6;452(7183):112-5.
Methylation of CpG dinucleotides is generally associated with epigenetic silencing of transcription and is maintained through cellular division. Multiple CpG sequences are rare in mammalian genomes, but frequently occur at the transcriptional start site of active genes, with most clusters of CpGs being hypomethylated. We reported previously that the proximal region of the trefoil factor 1 (TFF1, also known as pS2) and oestrogen receptor alpha (ERalpha) promoters could be partially methylated by treatment with deacetylase inhibitors, suggesting the possibility of dynamic changes in DNA methylation. Here we show that cyclical methylation and demethylation of CpG dinucleotides, with a periodicity of around 100 min, is characteristic for five selected promoters, including the oestrogen (E2)-responsive pS2 gene, in human cells. When the pS2 gene is actively transcribed, DNA methylation occurs after the cyclical occupancy of ERalpha and RNA polymerase II (polII). Moreover, we report conditions that provoke methylation cycling of the pS2 promoter in cell lines in which pS2 expression is quiescent and the proximal promoter is methylated. This coincides with a low-level re-expression of ERalpha and of pS2 transcripts.
Diverse roles of hnRNP L in mammalian mRNA processing: a combined microarray and RNAi analysis.
Hung, L.H., Heiner, M., Hui, J., Schreiner, S., Benes, V. & Bindereif, A.
RNA. 2008 Feb;14(2):284-96. Epub 2007 Dec 11.
Alternative mRNA splicing patterns are determined by the combinatorial control of regulator proteins and their target RNA sequences. We have recently characterized human hnRNP L as a global regulator of alternative splicing, binding to diverse C/A-rich elements. To systematically identify hnRNP L target genes on a genome-wide level, we have combined splice-sensitive microarray analysis and an RNAi-knockdown approach. As a result, we describe 11 target genes of hnRNP L that were validated by RT-PCR and that represent several new modes of hnRNP L-dependent splicing regulation, involving both activator and repressor functions: first, intron retention; second, inclusion or skipping of cassette-type exons; third, suppression of multiple exons; and fourth, alternative poly(A) site selection. In sum, this approach revealed a surprising diversity of splicing-regulatory processes as well as poly(A) site selection in which hnRNP L is involved.
Transcriptome profiling of estrogen-regulated genes in human primary osteoblasts reveals an osteoblast-specific regulation of the insulin-like growth factor binding protein 4 gene.
Denger, S., Bahr-Ivacevic, T., Brand, H., Reid, G., Blake, J., Seifert, M., Lin, C.Y., May, K., Benes, V., Liu, E.T. & Gannon, F.
Mol Endocrinol. 2008 Feb;22(2):361-79. Epub 2007 Oct 25.
Estradiol (E2) is believed to modulate physiological functions relevant to osteoblast biology through the actions of estrogen receptors (ERs) that in turn regulate the expression of target genes. The molecular effects of estrogen action in bone remain to be fully elucidated. This study reports a genome-wide molecular and computational analysis of the interaction between ER and regulatory elements on the DNA of target genes in human primary osteoblasts. Of approximately 54,000 gene probes surveyed in this study, a total of 375 genes were up-regulated and 418 genes were down-regulated on exposure to E2, with only 46 of these being direct target genes after 24 h, as determined by concomitant cycloheximide treatment. Computational analysis discovered several pathways where E2 coregulates multiple functionally linked components. Examination of the genomic sequence of IGF binding protein 4 located ER response elements within the first intron. Using by chromatin immunoprecipitation, we show a site- and cell-specific recruitment of transcription factors to this newly identified regulatory region. Transient transfection studies revealed that this intronic region acts as a functional promoter in human osteoblasts. Taken together, this analysis provides a comprehensive gene transcription profile and identifies several genes of potential physiological importance in controlling estrogen-mediated signaling in primary osteoblasts.
Replicative senescence of mesenchymal stem cells: a continuous and organized process.
Wagner, W., Horn, P., Castoldi, M., Diehlmann, A., Bork, S., Saffrich, R., Benes, V., Blake, J., Pfister, S., Eckstein, V. & Ho, A.D.
PLoS ONE. 2008 May 21;3(5):e2213.
Mesenchymal stem cells (MSC) comprise a promising tool for cellular therapy. These cells are usually culture expanded prior to their application. However, a precise molecular definition of MSC and the sequel of long-term in vitro culture are yet unknown. In this study, we have addressed the impact of replicative senescence on human MSC preparations. Within 43 to 77 days of cultivation (7 to 12 passages), MSC demonstrated morphological abnormalities, enlargement, attenuated expression of specific surface markers, and ultimately proliferation arrest. Adipogenic differentiation potential decreased whereas the propensity for osteogenic differentiation increased. mRNA expression profiling revealed a consistent pattern of alterations in the global gene expression signature of MSC at different passages. These changes are not restricted to later passages, but are continuously acquired with increasing passages. Genes involved in cell cycle, DNA replication and DNA repair are significantly down-regulated in late passages. Genes from chromosome 4q21 were over-represented among differentially regulated transcripts. Differential expression of 10 genes has been verified in independent donor samples as well as in MSC that were isolated under different culture conditions. Furthermore, miRNA expression profiling revealed an up-regulation of hsa-mir-371, hsa-mir-369-5P, hsa-mir-29c, hsa-mir-499 and hsa-let-7f upon in vitro propagation. Our studies indicate that replicative senescence of MSC preparations is a continuous process starting from the first passage onwards. This process includes far reaching alterations in phenotype, differentiation potential, global gene expression patterns, and miRNA profiles that need to be considered for therapeutic application of MSC preparations.
miChip: an array-based method for microRNA expression profiling using locked nucleic acid capture probes.
Castoldi, M., Schmidt, S., Benes, V., Hentze, M.W. & Muckenthaler, M.U.
Nat Protoc. 2008;3(2):321-9.
MicroRNAs (miRNAs) represent a class of short (22 nt) noncoding RNAs that control gene expression post-transcriptionally. Microarray technology is frequently applied to monitor miRNA expression levels but is challenged by (i) the short length of miRNAs that offers little sequence for appending detection molecules; (ii) low copy number of some miRNA; and (iii) a wide range of predicted melting temperatures (Tm) versus their DNA complementary sequences. We recently developed a microarray platform for genome-wide profiling of miRNAs (miChip) by applying locked nucleic acid (LNA)-modified capture probes. Here, we provide detailed protocols for the generation of the miChip microarray platform, the preparation and fluorescent labeling of small RNA containing total RNA, its hybridization to the immobilized LNA-modified capture probes and the post-hybridization handling of the microarray. Starting from the intact tissue sample, the entire protocol takes approximately 3 d to yield highly accurate and sensitive data about miRNA expression levels.
miChip: a microarray platform for expression profiling of microRNAs based on locked nucleic acid (LNA) oligonucleotide capture probes.
Castoldi, M., Benes, V., Hentze, M.W. & Muckenthaler, M.U.
Methods. 2007 Oct;43(2):146-52.
As key regulators of post-transcriptional gene expression, it is important to monitor the expression of microRNAs (miRNA) in diverse physiological and pathophysiological processes. Here, we describe a method for sensitive and accurate microarray-based expression profiling of miRNAs. The protocol focuses on the use of locked nucleic acid (LNA)-modified capture probes. LNAs are bicyclic nucleotide analogues that significantly increase the melting temperature (T(m)) of hybrids with miRNAs. Mixed LNA/DNA capture probes thus can be designed for equal T(m)s for all miRNAs, which naturally cover a range between 45 and 74 degrees C. The protocols established are easy to apply, as they do not require RNA size selection and/or amplification of miRNAs. Moreover, they enable high affinity hybridizations yielding accurate signals that discriminate between single nucleotide differences and hence closely related miRNA family members.
Life cycle transcriptome of the malaria mosquito Anopheles gambiae and comparison with the fruitfly Drosophila melanogaster.
Koutsos, A.C., Blass, C., Meister, S., Schmidt, S., MacCallum, R.M., Soares, M.B., Collins, F.H., Benes, V., Zdobnov, E., Kafatos, F.C. & Christophides, G.K.
Proc Natl Acad Sci U S A. 2007 Jul 3;104(27):11304-9. Epub 2007 Jun 11.
The African mosquito Anopheles gambiae is the major vector of human malaria. We report a genome-wide survey of mosquito gene expression profiles clustered temporally into developmental programs and spatially into adult tissue-specific patterns. Global expression analysis shows that genes that belong to related functional categories or that encode the same or functionally linked protein domains are associated with characteristic developmental programs or tissue patterns. Comparative analysis of our data together with data published from Drosophila melanogaster reveal an overall strong and positive correlation of developmental expression between orthologous genes. The degree of correlation varies, depending on association of orthologs with certain developmental programs or functional groups. Interestingly, the similarity of gene expression is not correlated with the coding sequence similarity of orthologs, indicating that expression profiles and coding sequences evolve independently. In addition to providing a comprehensive view of temporal and spatial gene expression during the A. gambiae life cycle, this large-scale comparative transcriptomic analysis has detected important evolutionary features of insect transcriptomes.
Pax-Six-Eya-Dach network during amphioxus development: conservation in vitro but context specificity in vivo.
Kozmik, Z., Holland, N.D., Kreslova, J., Oliveri, D., Schubert, M., Jonasova, K., Holland, L.Z., Pestarino, M., Benes, V. & Candiani, S.
Dev Biol. 2007 Jun 1;306(1):143-59. Epub 2007 Mar 13.
The Drosophila retinal determination gene network occurs in animals generally as a Pax-Six-Eyes absent-Dachshund network (PSEDN). For amphioxus, we describe the complete network of nine PSEDN genes, four of which-AmphiSix1/2, AmphiSix4/5, AmphSix3/6, and AmphiEya-are characterized here for the first time. For amphioxus, in vitro interactions among the genes and proteins of the network resemble those of other animals, except for the absence of Dach-Eya binding. Amphioxus PSEDN genes are expressed in highly stage- and tissue-specific patterns (sometimes conspicuously correlated with the local intensity of cell proliferation) in the gastrular organizer, notochord, somites, anterior central nervous system, peripheral nervous system, pharyngeal endoderm, and the likely homolog of the vertebrate adenohypophysis. In this last tissue, the anterior region expresses all three amphioxus Six genes and is a zone of active cell proliferation, while the posterior region expresses only AmphiPax6 and is non-proliferative. In summary, the topologies of animal PSEDNs, although considerably more variable than originally proposed, are conserved enough to be recognizable among species and among developing tissues; this conservation may reflect indispensable involvement of PSEDNs during the critically important early phases of embryology (e.g. in the control of mitosis, apoptosis, and cell/tissue motility).
Adhesion of hematopoietic progenitor cells to human mesenchymal stem cells as a model for cell-cell interaction.
Wagner, W., Wein, F., Roderburg, C., Saffrich, R., Faber, A., Krause, U., Schubert, M., Benes, V., Eckstein, V., Maul, H. & Ho, A.D.
Exp Hematol. 2007 Feb;35(2):314-25.
OBJECTIVE: The significant role of direct contact between hematopoietic progenitor cells (HPC) and the cellular microenvironment for maintaining "stemness" has been demonstrated. Human mesenchymal stem cell (MSC) feeder layers represent a surrogate model for this interaction. Specific adhesion molecules are responsible for this cell-cell contact. METHODS: To define cell-cell contact between HPC and MSC, we have studied adhesive interaction of various fractions of HPC by using a novel assay based on gravitational force upon inversion. Adherent and nonadherent cells were separated and further analyzed with regard to gene expression and long-term hematopoietic culture initiating cell (LTC-IC) frequency. RESULTS: HPC subsets with higher self-renewing capacity demonstrated significantly higher adherence to human MSC (CD34(+) vs CD34(-), CD34(+)/CD38(-) vs CD34(+)/CD38(+), slow dividing fraction vs fast dividing fraction). LTC-IC frequency was significantly higher in the adherent fraction than in the nonadherent fraction. Furthermore, genes coding for adhesion proteins and extracellular matrix were higher expressed in the adherent subsets of CD34(+) cells (fibronectin 1, cadherin 11, vascular cell adhesion molecule-1, connexin 43, integrin beta-like 1, and TGFBI). CONCLUSION: In this study we have demonstrated that primitive subsets of HPC have higher affinity to human MSC. The essential role of specific junction proteins for stabilization of cell-cell contact is indicated by their significant higher expression.
Molecular composition of cell-cell contact between hematopoietic stem cells and mesenchymal stem cells.
Wagner, W; Wein, F; Roderburg, C; Benes, V.; Frankhauser, M; Wuchter, P; Eckstein, V; Ho, AD
Biology of blood and marrow transplantation February 2007 13(2)
Modular Proteins from the Drosophila sallimus (sls) Gene and their Expression in Muscles with Different Extensibility.
Burkart, C., Qiu, F., Brendel, S., Benes, V., Haag, P., Labeit, S., Leonard, K. & Bullard, B.
J Mol Biol. 2007 Jan 27;.
The passive elasticity of the sarcomere in striated muscle is determined by large modular proteins, such as titin in vertebrates. In insects, the function of titin is divided between two shorter proteins, projectin and sallimus (Sls), which are the products of different genes. The Drosophila sallimus (sls) gene codes for a protein of 2 MDa. The N-terminal half of the protein is largely made up of immunoglobulin (Ig) domains and unique sequence; the C-terminal half has two stretches of sequence similar to the elastic PEVK region of titin, and at the end of the molecule there is a region of tandem Ig and fibronectin domains. We have investigated splicing pathways of the sls gene and identified isoforms expressed in different muscle types, and at different stages of Drosophila development. The 5' half of sls codes for zormin and kettin; both proteins contain Ig domains and can be expressed as separate isoforms, or as larger proteins linked to sequence downstream. There are multiple splicing pathways between the kettin region of sls and sequence coding for the two PEVK regions. All the resulting protein isoforms have sequence derived from the 3' end of the sls gene. Splicing of exons varies at different stages of development. Kettin RNA is predominant in the embryo, and longer transcripts are expressed in larva, pupa and adult. Sls isoforms in the indirect flight muscle (IFM) are zormin, kettin and Sls(700), in which sequence derived from the end of the gene is spliced to kettin RNA. Zormin is in both M-line and Z-disc. Kettin and Sls(700) extend from the Z-disc to the ends of the thick filaments, though, Sls(700) is only in the myofibril core. These shorter isoforms would contribute to the high stiffness of IFM. Other muscles in the thorax and legs have longer Sls isoforms with varying amounts of PEVK sequence; all span the I-band to the ends of the thick filaments. In muscles with longer I-bands, the proportion of PEVK sequence would determine the extensibility of the sarcomere. Alternative Sls isoforms could regulate the stiffness of the many fibre types in Drosophila muscles.
A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA).
Castoldi, M., Schmidt, S., Benes, V., Noerholm, M., Kulozik, A.E., Hentze, M.W. & Muckenthaler, M.U.
RNA. 2006 May;12(5):913-20. Epub 2006 Mar 15.
MicroRNAs represent a class of short (approximately 22 nt), noncoding regulatory RNAs involved in development, differentiation, and metabolism. We describe a novel microarray platform for genome-wide profiling of mature miRNAs (miChip) using locked nucleic acid (LNA)-modified capture probes. The biophysical properties of LNA were exploited to design probe sets for uniform, high-affinity hybridizations yielding highly accurate signals able to discriminate between single nucleotide differences and, hence, between closely related miRNA family members. The superior detection sensitivity eliminates the need for RNA size selection and/or amplification. MiChip will greatly simplify miRNA expression profiling of biological and clinical samples.
The molecular elasticity of the insect flight muscle proteins projectin and kettin.
Bullard, B., Garcia, T., Benes, V., Leake, M.C., Linke, W.A. & Oberhauser, A.F.
Proc Natl Acad Sci U S A. 2006 Mar 21;103(12):4451-6. Epub 2006 Mar 14.
Projectin and kettin are titin-like proteins mainly responsible for the high passive stiffness of insect indirect flight muscles, which is needed to generate oscillatory work during flight. Here we report the mechanical properties of kettin and projectin by single-molecule force spectroscopy. Force-extension and force-clamp curves obtained from Lethocerus projectin and Drosophila recombinant projectin or kettin fragments revealed that fibronectin type III domains in projectin are mechanically weaker (unfolding force, F(u) approximately 50-150 pN) than Ig-domains (F(u) approximately 150-250 pN). Among Ig domains in Sls/kettin, the domains near the N terminus are less stable than those near the C terminus. Projectin domains refolded very fast [85% at 15 s(-1) (25 degrees C)] and even under high forces (15-30 pN). Temperature affected the unfolding forces with a Q(10) of 1.3, whereas the refolding speed had a Q(10) of 2-3, probably reflecting the cooperative nature of the folding mechanism. High bending rigidities of projectin and kettin indicated that straightening the proteins requires low forces. Our results suggest that titin-like proteins in indirect flight muscles could function according to a folding-based-spring mechanism.
Adhesion of hematopoietic progenitor cells to hurnan mesenchymal stromal cells as a model for interaction between stem cells and their niche.
Wagner, Wolfgang; Wein, Frederik; Roderburg, Christoph; Benes, V., Diehlmann, Anke; Krause, Ulf; Frankhauser, Maria; Wuchter, Patrick; Volker, Eckstein; Ho, Anthony D.
Blood November 2006 108(11)
Vertebrate-type intron-rich genes in the marine annelid Platynereis dumerilii.
Raible, F., Tessmar-Raible, K., Osoegawa, K., Wincker, P., Jubin, C., Balavoine, G., Ferrier, D., Benes, V., de Jong, P., Weissenbach, J., Bork, P. & Arendt, D.
Science 2005 Nov 25;310(5752):1325-6.
Previous genome comparisons have suggested that one important trend in vertebrate evolution has been a sharp rise in intron abundance. By using genomic data and expressed sequence tags from the marine annelid Platynereis dumerilii, we provide direct evidence that about two-thirds of human introns predate the bilaterian radiation but were lost from insect and nematode genomes to a large extent. A comparison of coding exon sequences confirms the ancestral nature of Platynereis and human genes. Thus, the urbilaterian ancestor had complex, intron-rich genes that have been retained in Platynereis and human.
Multiple mechanisms induce transcriptional silencing of a subset of genes, including oestrogen receptor alpha, in response to deacetylase inhibition by valproic acid and trichostatin A.
Reid, G., Metivier, R., Lin, C.Y., Denger, S., Ibberson, D., Ivacevic, T., Brand, H., Benes, V., Liu, E.T. & Gannon, F.
Oncogene 2005 Jul 21;24(31):4894-907.
Valproate (VPA) and trichostatin A (TSA), inhibitors of zinc-dependent deacetylase activity, induce reduction in the levels of mRNA encoding oestrogen receptor-alpha (ERalpha), resulting in subsequent clearance of ERalpha protein from breast and ovarian cell lines. Inhibition of oestrogen signalling may account for the endocrine disorders, menstrual abnormalities, osteoporosis and weight gain that occur in a proportion of women treated with VPA for epilepsy or for bipolar mood disorder. Transcriptome profiling revealed that VPA and TSA also modulate the expression of, among others, key regulatory components of the cell cycle. Meta-analysis of genes directly responsive to oestrogen indicates that VPA and TSA have a generally antioestrogenic profile in ERalpha positive cells. Concomitant treatment with cycloheximide prevented most of these changes in gene expression, including downregulation of ERalpha mRNA, indicating that a limited number of genes signal a hyperacetylated state within cells. Three members of the NAD-dependent deacetylases, the sirtuins, are upregulated by VPA and by TSA and sirtuin activity contributes to loss of ERalpha expression. However, prolonged inhibition of the sirtuins by sirtinol also induces loss of ERalpha from cells. Mechanistically, we show that VPA invokes reversible promoter shutoff of the ERalpha, pS2 and cyclin D1 promoters, by inducing recruitment of methyl cytosine binding protein 2 (MeCP2) with concomitant exclusion of the maintenance methylase DNMT1. Furthermore, we demonstrate that, in the presence of VPA, local DNA methylation, deacetylation and demethylation of activated histones and recruitment of inhibitory complexes occurs on the pS2 promoter.Oncogene (2005) 24, 4894-4907. doi:10.1038/sj.onc.1208662; published online 2 May 2005.
Quantitative real-time RT-PCR - a perspective.
Bustin, S.A., Benes, V., Nolan, T. & Pfaffl, M.W.
J Mol Endocrinol 2005 Jun;34(3):597-601.
The real-time reverse transcription polymerase chain reaction (RT-PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction. This combines the nucleic acid amplification and detection steps into one homogeneous assay and obviates the need for gel electrophoresis to detect amplification products. Use of appropriate chemistries and data analysis eliminates the need for Southern blotting or DNA sequencing for amplicon identification. Its simplicity, specificity and sensitivity, together with its potential for high throughput and the ongoing introduction of new chemistries, more reliable instrumentation and improved protocols, has made real-time RT-PCR the benchmark technology for the detection and/or comparison of RNA levels.
Alternative splicing microarrays reveal functional expression of neuron-specific regulators in Hodgkin lymphoma cells.
Relogio, A., Ben-Dov, C., Baum, M., Ruggiu, M., Gemund, C., Benes, V., Darnell, R.B. & Valcarcel, J.
J Biol Chem 2005 Feb 11;280(6):4779-84. Epub 2004 Nov 16.
Alternative splicing provides a versatile mechanism of gene regulation, which is often subverted in disease. We have used customized oligonucleotide microarrays to interrogate simultaneously the levels of expression of splicing factors and the patterns of alternative splicing of genes involved in tumor progression. Analysis of RNAs isolated from cell lines derived from Hodgkin lymphoma tumors indicate that the relative abundance of alternatively spliced isoforms correlates with transformation and tumor grade. Changes in expression of regulators were also detected, and a subset sample was confirmed at the protein level. Ectopic expression of neuron-specific splicing regulatory proteins of the Nova family was observed in some cell lines and tumor samples, correlating with expression of a neuron-specific mRNA isoform of JNK2 kinase. This microarray design can help assess the role of alternative splicing in a variety of biological and medical problems and potentially serve as a diagnostic tool.
Transcriptional complexes engaged by apo-estrogen receptor-alpha isoforms have divergent outcomes.
Metivier, R., Penot, G., Carmouche, R.P., Hubner, M.R., Reid, G., Denger, S., Manu, D., Brand, H., Kos, M., Benes, V. & Gannon, F.
EMBO J 2004 Sep 15;23(18):3653-66. Epub 2004 Sep 02.
Unliganded (apo-) estrogen receptor alpha (ERalpha, NR3A1) is classically considered as transcriptionally unproductive. Reassessing this paradigm demonstrated that apo-human ERalpha (ERalpha66) and its N-terminally truncated isoform (ERalpha46) are both predominantly nuclear transcription factors that cycle on the endogenous estrogen-responsive pS2 gene promoter in vivo. Importantly, isoform-specific consequences occur in terms of poising the promoter for transcription, as evaluated by determining (i) the engagement of several cofactors and the resulting nucleosomal organization; and (ii) the CpG methylation state of the pS2 promoter. Although transcriptionally unproductive, cycling of apo-ERalpha66 prepares the promoter to respond to ligand, through sequentially targeting chromatin remodeling complexes and general transcription factors. Additionally, apo-ERalpha46 recruits corepressors, following engagement of cofactors identical to those recruited by apo-ERalpha66. Together, these data describe differential activities of ERalpha isoforms. Furthermore, they depict the maintenance of a promoter in a repressed state as a cyclical process that is intrinsically dependent on initial poising of the promoter.
Characterization of an Escherichia coli elaC deletion mutant.
Schilling, O., Ruggeberg, S., Vogel, A., Rittner, N., Weichert, S., Schmidt, S., Doig, S., Franz, T., Benes, V., Andrews, S.C., Baum, M. & Meyer-Klaucke, W.
Biochem Biophys Res Commun 2004 Aug 6;320(4):1365-73.
The elaC gene of Escherichia coli encodes a binuclear zinc phosphodiesterase (ZiPD). ZiPD homologs from various species act as3' tRNA processing endoribonucleases, and although the homologous gene in Bacillus subtilis is essential for viability [EMBO J. 22(2003) 4534], the physiological function of E. coli ZiPD has remained enigmatic. In order to investigate the function of E. coli ZiPDwe generated and characterized an E. coli elaC deletion mutant. Surprisingly, the E. coli elaC deletion mutant was viable and had wild-type like growth properties. Microarray-based transcriptional analysis indicated expression of the E. coli elaC gene at basal levels during aerobic growth. The elaC gene deletion had no effect on the expression of genes coding for RNases or amino-acyl tRNA synthetases or any other gene among a total of > 1300 genes probed. 2D-PAGE analysis showed that the elaC mutation, like-wise, had no effect on the proteome. These results strengthen doubts about the involvement of E. coli ZiPD in tRNA maturation and suggest functional diversity within the ZiPD/ElaC1 protein family. In addition to these unexpected features of the E. coli elaC deletion mutant, a sequence comparison of ZiPD (ElaC1) proteins revealed specific regions for either enterobacterial or mammalian ZiPD (ElaC1) proteins.
Targeted mutagenesis of the murine IRP1 and IRP2 genes reveals context-dependent RNA processing differences in vivo.
Galy, B., Ferring, D., Benesova, M., Benes, V. & Hentze, M.W.
RNA 2004 Jul;10(7):1019-25.
We report the targeted mutagenesis of the murine iron regulatory protein (IRP)-1 and IRP2 genes, respectively, with a classical gene trap construct. Insertion of the targeting cassette into the second intron of either gene by homologous recombination interrupts their open reading frames near the N termini. Mice that are homozygous for the correctly modified IRP1 or IRP2 alleles, respectively, display a strong reduction (90%, IRP1(-/-)) or nondetectable levels (IRP2(-/-)) of the targeted proteins. Interestingly, the pre-mRNAs transcribed from the identical targeting cassettes are processed differently within the two different contexts. Detailed analysis of the respective products identifies the choice of alternative splice and 3' end processing sites in the same tissues in vivo. We discuss the implications for the understanding of RNA processing and for targeting strategies for functional genomics in the mouse.
Diversity of vertebrate splicing factor U2AF35: identification of alternatively spliced U2AF1 mRNAS.
Pacheco, T.R., Gomes, A.Q., Barbosa-Morais, N.L., Benes, V., Ansorge, W., Wollerton, M., Smith, C.W., Valcarcel, J. & Carmo-Fonseca, M.
J Biol Chem. 2004 Jun 25;279(26):27039-49. Epub 2004 Apr 19.
U2 small nuclear ribonucleoprotein auxiliary factor small subunit (U2AF(35)) is encoded by a conserved gene designated U2AF1. Here we provide evidence for the existence of alternative vertebrate transcripts encoding different U2AF(35) isoforms. Three mRNA isoforms (termed U2AF(35)a-c) were produced by alternative splicing of the human U2AF1 gene. U2AF(35)c contains a premature stop codon that targets the resulting mRNA to nonsense-mediated mRNA decay. U2AF(35)b differs from the previously described U2AF(35)a isoform in 7 amino acids located at the atypical RNA Recognition Motif involved in dimerization with U2AF(65). Biochemical experiments indicate that isoform U2AF(35)b, which has been highly conserved from fish to man, maintains the ability to interact with U2AF(65), stimulates U2AF(65) binding to a pre-mRNA, and promotes U2AF splicing activity in vitro. Real time, quantitative PCR analysis indicates that U2AF(35)a is the most abundant isoform expressed in murine tissues, although the ratio between U2AF(35)a and U2AF(35)b varies from 10-fold in the brain to 20-fold in skeletal muscle. We propose that post-transcriptional regulation of U2AF1 gene expression may provide a mechanism by which the relative cellular concentration and availability of U2AF(35) protein isoforms are modulated, thus contributing to the finely tuned control of splicing events in different tissues.
Developmentally regulated switching of titin size alters myofibrillar stiffness in the perinatal heart.
Opitz, C.A., Leake, M.C., Makarenko, I., Benes, V. & Linke, W.A.
Circ Res. 2004 Apr 16;94(7):967-75. Epub 2004 Feb 26.
Before birth, the compliance of the heart is limited predominantly by extracardiac constraint. Reduction of this constraint at birth requires that myocardial compliance be determined mainly by the heart's own constituents. Because titin is a principal contributor to ventricular passive tension (PT), we studied the expression and mechanics of cardiac-titin isoforms during perinatal rat heart development. Gel electrophoresis and immunoblotting revealed a single, 3.7-MDa, N2BA isoform present 6 days before birth and an additional, also previously unknown, N2BA isoform of 3.5 to 3.6 MDa expressed in the near-term fetus. These large isoforms rapidly disappear after birth and are replaced by a small N2B isoform (3.0 MDa) predominating in 1-week-old and adult rats. In addition, neonatal pig hearts showed large N2BA-titin isoforms distinct from those present in the adult porcine myocardium. By quantitative reverse transcriptase-polymerase chain reaction, developmentally expressed titin-mRNA species were detected in rat heart. Titin-based PT was much lower (approximately 15 times) in fetal than adult rat cardiomyocytes, and measured PT levels were readily predictable with a model of worm-like chain titin elasticity. Immunofluorescence microscopy showed the extensibility of the differentially spliced molecular spring regions of fetal/neonatal titin isoforms in isolated rat cardiomyofibrils. Whereas the titin-isoform shift by 700 kDa ensures high passive stiffness of the postnatal cardiac myofibrils, the expression of specific fetal/neonatal cardiac-titin isoforms may also have important functions for contractile properties, myofibril assembly or turnover, and myocardial signaling during perinatal heart development.
BAC engineering for the generation of ES cell-targeting constructs and mouse transgenes.
Testa, G., Vintersten, K., Zhang, Y., Benes, V., Muyrers, J.P. & Stewart, A.F.
Methods Mol Biol. 2004;256:123-39. PubMed
ET recombination: DNA engineering using homologous recombination in E. coli.
Muyrers, J.P., Zhang, Y., Benes, V., Testa, G., Rientjes, J.M. & Stewart, A.F.
Methods Mol Biol 2004;256:107-21. PubMed
Microcontact printing of DNA molecules
Lange S, Benes V, Kern DP, Hoerber JKH, Bernard A
AnalyticalBiochem. (2004), 76, 1641-1647, doi: 10.1021/ac035127w.
Standardization of protocols in cDNA microarray analysis.
Benes, V. & Muckenthaler, M.
Trends Biochem Sci 2003 May;28(5):244-9.
Systematic variations can occur at various steps of a cDNA microarray experiment and affect the measurement of gene expression levels. Accepted standards integrated into every cDNA microarray analysis can assess these variabilities and aid the interpretation of cDNA microarray experiments from different sources. A universally applicable approach to evaluate parameters such as input and output ratios, signal linearity, hybridization specificity and consistency across an array, as well as normalization strategies, is the utilization of exogenous control genes as spike-in and negative controls. We suggest that the use of such control sets, together with a sufficient number of experimental repeats, in-depth statistical analysis and thorough data validation should be made mandatory for the publication of cDNA microarray data.
Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles.
Testa, G., Zhang, Y., Vintersten, K., Benes, V., Pijnappel, W.W., Chambers, I., Smith, A.J., Smith, A.G. & Stewart, A.F.
Nat Biotechnol 2003 Apr;21(4):443-7.
The mouse is the leading vertebrate model because its genome can be altered by both random transgenesis and homologous recombination with targeting constructs. Both approaches have been hindered by the size and site limitations implicit in conventional Escherichia coli DNA-engineering methods. Homologous recombination in E. coli, or 'recombineering', has overcome these limitations for bacterial artificial chromosome (BAC) transgenesis. Here we applied Red/ET recombineering (using the lambda Redalpha/Redbeta recombinase pair) to generate a 64 kilobase targeting construct that carried two selectable cassettes permitting the simultaneous mutation of the target gene, Mll, at sites 43 kb apart in one round of mouse embryonic stem (ES) cell targeting. The targeting frequency after dual selection was 6%. Because the two selectable cassettes were flanked by FRT or loxP sites, three more alleles can be generated by site-specific recombination. Our approach represents a simple way to introduce changes at two or more sites in a genetic locus, and thereafter generate allele combinations. The size of BAC templates offers new freedom for the design of targeting constructs. Combined with the use of two selectable cassettes placed far apart, BAC-based targeting constructs may be applicable to tasks such as regional exchanges, deletions, and insertions.
Cloning and developmental expression of amphioxus Dachschund.
Candiani, S., Kreslova, J., Benes, V., Oliveri, D., Castagnola, P., Pestarino, M. & Kozmik, Z.
Gene Expr Patterns 2003 Mar;3(1):65-9.
The nuclear factor dachshund (dac) is a key regulator of eye and leg development in Drosophila. We have cloned a Dachshund homologue from an invertebrate chordate amphioxus (Branchiostoma floridae). Sequence comparison reveals a high degree of similarity of amphioxus Dachshund (AmphiDach) to the known vertebrate and Drosophila dachshund genes. AmphiDach is first expressed in the prospective paraxial mesoderm at the gastrula stage. At the early neurula stage, expression is detected in developing somites and anterior endoderm, but in late neurula transcripts are present exclusively in the most posterior region of the cerebral vesicle and the anterior pharynx endoderm. Then, in larva, AmphiDach is localized in photoreceptive neurons of the frontal eye, infundibular organ, and endostyle as well as in Hesse organs and in nerve cells scattered along the nerve cord. Comparison of Dach expression in amphioxus and vertebrates suggests that such patterns are relatively similar (because they are expressed in somites, photoreceptor cells and CNS), even if expression of AmphiDach in the endostyle has no counterparts in its vertebrate homolog, the thyroid.
Automatisierte DNA-Extraktion durch Kombination von Liquid Handling und
Zimmermann, J., Zinn, T., Benes, V., Ibberson, D., Boulin, C., Griebel, R., Lomax, P., Mausel, M., Schubell, U. & Gunther-Eberle, K.
Laborwelt 2003 4(5) 41-42
Small is beautiful: Flexible Automatisierunginsel in der genomischen Forschung.
Zimmermann, J., Schmidt, S., Benes, V., Boulin, C., Griebel, R., Lomax, P., Zinn, T., Mausel, M., Schubell, U. & Gunther-Eberle, K.
Biospektrum 2003 9 764-766
Large BAC-based targeting constructs present new options for genomic engineering.
Testa G, Zhang Y, Vintersten K, Benes V, Chambers I, Pim Pijnappel WWM, Smith AJH, Smith AA and Stewart AF
Nature Biotech. (2003), 21, 443-7.
The genome sequence of the malaria mosquito Anopheles gambiae.
Holt, R.A., Subramanian, G.M., Halpern, A., Sutton, G.G., Charlab, R., Nusskern, D.R., Wincker, P., Clark, A.G., Ribeiro, J.M., Wides, R., Salzberg, S.L., Loftus, B., Yandell, M., Majoros, W.H., Rusch, D.B., Lai, Z., Kraft, C.L., Abril, J.F., Anthouard, V., Arensburger, P., Atkinson, P.W., Baden, H., de Berardinis, V., Baldwin, D., Benes, V., Biedler, J., Blass, C., Bolanos, R., Boscus, D., Barnstead, M., Cai, S., Center, A., Chatuverdi, K., Christophides, G.K., Chrystal, M.A., Clamp, M., Cravchik, A., Curwen, V., Dana, A., Delcher, A., Dew, I., Evans, C.A., Flanigan, M., Grundschober-Freimoser, A., Friedli, L., Gu, Z., Guan, P., Guigo, R., Hillenmeyer, M.E., Hladun, S.L., Hogan, J.R., Hong, Y.S., Hoover, J., Jaillon, O., Ke, Z., Kodira, C., Kokoza, E., Koutsos, A., Letunic, I., Levitsky, A., Liang, Y., Lin, J.J., Lobo, N.F., Lopez, J.R., Malek, J.A., McIntosh, T.C., Meister, S., Miller, J., Mobarry, C., Mongin, E., Murphy, S.D., O'Brochta, D.A., Pfannkoch, C., Qi, R., Regier, M.A., Remington, K., Shao, H., Sharakhova, M.V., Sitter, C.D., Shetty, J., Smith, T.J., Strong, R., Sun, J., Thomasova, D., Ton, L.Q., Topalis, P., Tu, Z., Unger, M.F., Walenz, B., Wang, A., Wang, J., Wang, M., Wang, X., Woodford, K.J., Wortman, J.R., Wu, M., Yao, A., Zdobnov, E.M., Zhang, H., Zhao, Q., Zhao, S., Zhu, S.C., Zhimulev, I., Coluzzi, M., della Torre, A., Roth, C.W., Louis, C., Kalush, F., Mural, R.J., Myers, E.W., Adams, M.D., Smith, H.O., Broder, S., Gardner, M.J., Fraser, C.M., Birney, E., Bork, P., Brey, P.T., Venter, J.C., Weissenbach, J., Kafatos, F.C., Collins, F.H. & Hoffman, S.L.
Science 2002 Oct 4;298(5591):129-49.
Anopheles gambiae is the principal vector of malaria, a disease that afflicts more than 500 million people and causes more than 1 million deaths each year. Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs. A total of 91% of the genome was organized in 303 scaffolds; the largest scaffold was 23.1 million base pairs. There was substantial genetic variation within this strain, and the apparent existence of two haplotypes of approximately equal frequency ("dual haplotypes") in a substantial fraction of the genome likely reflects the outbred nature of the PEST strain. The sequence produced a conservative inference of more than 400,000 single-nucleotide polymorphisms that showed a markedly bimodal density distribution. Analysis of the genome sequence revealed strong evidence for about 14,000 protein-encoding transcripts. Prominent expansions in specific families of proteins likely involved in cell adhesion and immunity were noted. An expressed sequence tag analysis of genes regulated by blood feeding provided insights into the physiological adaptations of a hematophagous insect.
A genomic switch at the transition from cell proliferation to terminal differentiation in the Drosophila eye.
Jasper, H., Benes, V., Atzberger, A., Sauer, S., Ansorge, W. & Bohmann, D.
Dev Cell 2002 Oct;3(4):511-21.
Organogenesis involves cell proliferation followed by complex determination and differentiation events that are intricately controlled in time and space. The instructions for these different steps are, to a large degree, implicit in the gene expression profiles of the cells that partake in organogenesis. Combining fluorescence-activated cell sorting and SAGE, we analyzed genomic expression patterns in the developing eye of Drosophila melanogaster. Genomic activity changes as cells pass from an uncommitted proliferating progenitor state through determination and differentiation steps toward a specialized cell fate. Analysis of the upstream sequences of genes specifically expressed during the proliferation phase of eye development implicates the transcription factor DREF and its inhibitor dMLF in the control of cell growth in this organ.
Functional equivalency of amphioxus and vertebrate Pax258 transcription factors suggests that the activation of mid-hindbrain specific genes in vertebrates occurs via the recruitment of Pax regulatory elements.
Krelova, J., Holland, L.Z., Schubert, M., Burgtorf, C., Benes, V. & Kozmik, Z.
Gene 2002 Jan 9;282(1-2):143-50.
Pax genes encode transcription factors that control key developmental decisions in various animal phyla. The Pax2/5/8 subfamily plays a key role in specification and/or maintenance of vertebrate mid-hindbrain boundary (MHB) region by directly regulating expression of other genes, most notably En2. In the invertebrate chordate amphioxus, expression of AmphiPax2/5/8 is found in many sites that are homologous to the regions of the vertebrate embryo expressing orthologous genes Pax2, Pax5 or Pax8. However, no co-expression of AmphiPax2/5/8 and AmphiEn is detected in the region of the neural tube that might correspond to the vertebrate MHB. Based on this observation and the absence of AmphiWnt expression in this region it appears that amphioxus does not have a MHB. Here we investigated the possibility that the AmphiPax2/5/8, as a key component of MHB development, has lost some of the properties of its vertebrate counterparts. We have analyzed both the DNA-binding and transactivation properties of AmphiPax2/5/8 as well as its ability to interact with the groucho co-repressor. In all these assays AmphiPax2/5/8 is indistinguishable from the human Pax5. In addition, we found two alternatively spliced AmphiPax2/5/8 isoforms that function similarly to the alternatively spliced isoforms of human Pax8. Analysis of the AmphiEn regulatory region provided no evidence for AmphiPax2/5/8 binding and transactivation. Therefore, in amphioxus, AmphiPax2/5/8, although capable of performing all the necessary functions has not been recruited for a developmental mechanism which usually sets up MHB development in vertebrates.
The genomic response of the Drosophila embryo to JNK signaling.
Jasper, H., Benes, V., Schwager, C., Sauer, S., Clauder-Munster, S., Ansorge, W. & Bohmann, D.
Dev Cell 2001 Oct;1(4):579-86.
During Drosophila development, the Jun N-terminal kinase signal transduction pathway regulates morphogenetic tissue closure movements that involve cell shape changes and reorganization of the actin cytoskeleton. We analyzed the genome-wide transcriptional response to activation of the JNK pathway in the Drosophila embryo by serial analysis of gene expression (SAGE) and identified loci encoding cell adhesion molecules and cytoskeletal regulators as JNK responsive genes. The role of one of the upregulated genes, chickadee (chic), encoding a Drosophila profilin, in embryogenesis was analyzed genetically. chic-deficient embryos fail to execute the JNK-mediated cytoskeletal rearrangements during dorsal closure. This study demonstrates a transcriptional mechanism of cytoskeletal regulation and establishes SAGE as an advantageous approach for genomic experiments in the fruitfly.
Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana.
Salanoubat, M., Lemcke, K., Rieger, M., Ansorge, W., Unseld, M., Fartmann, B., Valle, G., Blocker, H., Perez-Alonso, M., Obermaier, B., Delseny, M., Boutry, M., Grivell, L.A., Mache, R., Puigdomenech, P., De Simone, V., Choisne, N., Artiguenave, F., Robert, C., Brottier, P., Wincker, P., Cattolico, L., Weissenbach, J., Saurin, W., Quetier, F., Schafer, M., Muller-Auer, S., Gabel, C., Fuchs, M., Benes, V., Wurmbach, E., Drzonek, H., Erfle, H., Jordan, N., Bangert, S., Wiedelmann, R., Kranz, H., Voss, H., Holland, R., Brandt, P., Nyakatura, G., Vezzi, A., D'Angelo, M., Pallavicini, A., Toppo, S., Simionati, B., Conrad, A., Hornischer, K., Kauer, G., Lohnert, T.H., Nordsiek, G., Reichelt, J., Scharfe, M., Schon, O., Bargues, M., Terol, J., Climent, J., Navarro, P., Collado, C., Perez-Perez, A., Ottenwalder, B., Duchemin, D., Cooke, R., Laudie, M., Berger-Llauro, C., Purnelle, B., Masuy, D., de Haan, M., Maarse, A.C., Alcaraz, J.P., Cottet, A., Casacuberta, E., Monfort, A., Argiriou, A., flores, M., Liguori, R., Vitale, D., Mannhaupt, G., Haase, D., Schoof, H., Rudd, S., Zaccaria, P., Mewes, H.W., Mayer, K.F., Kaul, S., Town, C.D., Koo, H.L., Tallon, L.J., Jenkins, J., Rooney, T., Rizzo, M., Walts, A., Utterback, T., Fujii, C.Y., Shea, T.P., Creasy, T.H., Haas, B., Maiti, R., Wu, D., Peterson, J., Van Aken, S., Pai, G., Militscher, J., Sellers, P., Gill, J.E., Feldblyum, T.V., Preuss, D., Lin, X., Nierman, W.C., Salzberg, S.L., White, O., Venter, J.C., Fraser, C.M., Kaneko, T., Nakamura, Y., Sato, S., Kato, T., Asamizu, E., Sasamoto, S., Kimura, T., Idesawa, K., Kawashima, K., Kishida, Y., Kiyokawa, C., Kohara, M., Matsumoto, M., Matsuno, A., Muraki, A., Nakayama, S., Nakazaki, N., Shinpo, S., Takeuchi, C., Wada, T., Watanabe, A., Yamada, M., Yasuda, M. & Tabata, S.
Nature 2000 Dec 14;408(6814):820-2.
Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.
Point mutation of bacterial artificial chromosomes by ET recombination.
Muyrers, J.P., Zhang, Y., Benes, V., Testa, G., Ansorge, W. & Stewart, A.F.
EMBO Rep. 2000 Sep;1(3):239-43.
Bacterial artificial chromosomes (BACs) offer many advantages for functional studies of large eukaryotic genes. To utilize the potential applications of BACs optimally, new approaches that allow rapid and precise engineering of these large molecules are required. Here, we describe a simple and flexible two-step approach based on ET recombination, which permits point mutations to be introduced into BACs without leaving any other residual change in the recombinant product. Introduction of other modifications, such as small insertions or deletions, is equally feasible. The use of ET recombination to achieve site-directed mutagenesis opens access to a powerful use of BACs and is extensible to DNA molecules of any size in Escherichia coli, including the E. coli chromosome.
A member of a gene family on Xp22.3, VCX-A, is deleted in patients with X-linked nonspecific mental retardation.
Fukami, M., Kirsch, S., Schiller, S., Richter, A., Benes, V., Franco, B., Muroya, K., Rao, E., Merker, S., Niesler, B., Ballabio, A., Ansorge, W., Ogata, T. & Rappold, G.A.
Am J Hum Genet 2000 Sep;67(3):563-73.
X-linked nonspecific mental retardation (MRX) has a frequency of 0.15% in the male population and is caused by defects in several different genes on the human X chromosome. Genotype-phenotype correlations in male patients with a partial nullisomy of the X chromosome have suggested that at least one locus involved in MRX is on Xp22.3. Previous deletion mapping has shown that this gene resides between markers DXS1060 and DXS1139, a region encompassing approximately 1.5 Mb of DNA. Analyzing the DNA of 15 males with Xp deletions, we were able to narrow this MRX critical interval to approximately 15 kb of DNA. Only one gene, VCX-A (variably charged, X chromosome mRNA on CRI-S232A), was shown to reside in this interval. Because of a variable number of tandem 30-bp repeats in the VCX-A gene, the size of the predicted protein is 186-226 amino acids. VCX-A belongs to a gene family containing at least four nearly identical paralogues on Xp22.3 (VCX-A, -B, -B1, and -C) and two on Yq11.2 (VCY-D, VCY-E), suggesting that the X and Y copies were created by duplication events. We have found that VCX-A is retained in all patients with normal intelligence and is deleted in all patients with mental retardation. There is no correlation between the presence or absence of VCX-B1, -B, and VCX-C and mental status in our patients. These results suggest that VCX-A is sufficient to maintain normal mental development.
An estimate of large-scale sequencing accuracy.
Hill, F., Gemund, C., Benes, V., Ansorge, W. & Gibson, T.J.
EMBO Rep. 2000 Jul;1(1):29-31.
The accuracy of large-scale DNA sequencing is difficult to estimate without redundant effort. We have found that the mobile genetic element IS10, a component of the transposon Tn10, has contaminated a significant number of clones in the public databases, as a result of the use of the transposon in bacterial cloning strain construction. These contaminations need to be annotated as such. More positively, by defining the range of sequence variation in IS10, we have been able to determine that the rate of sequencing errors is very low, most likely surpassing the stated aim of one error or less in ten thousand bases.
Anopheles gambiae pilot gene discovery project: identification of mosquito innate immunity genes from expressed sequence tags generated from immune-competent cell lines.
Dimopoulos, G., Casavant, T.L., Chang, S., Scheetz, T., Roberts, C., Donohue, M., Schultz, J., Benes, V., Bork, P., Ansorge, W., Soares, M.B. & Kafatos, F.C.
Proc Natl Acad Sci U S A 2000 Jun 6;97(12):6619-24
Together with AIDS and tuberculosis, malaria is at the top of the list of devastating infectious diseases. However, molecular genetic studies of its major vector, Anopheles gambiae, are still quite limited. We have conducted a pilot gene discovery project to accelerate progress in the molecular analysis of vector biology, with emphasis on the mosquito's antimalarial immune defense. A total of 5,925 expressed sequence tags were determined from normalized cDNA libraries derived from immune-responsive hemocyte-like cell lines. The 3,242 expressed sequence tag-containing cDNA clones were grouped into 2,380 clone clusters, potentially representing unique genes. Of these, 1,118 showed similarities to known genes from other organisms, but only 27 were identical to previously known mosquito genes. We identified 38 candidate genes, based on sequence similarity, that may be implicated in immune reactions including antimalarial defense; 19 of these were shown experimentally to be inducible by bacterial challenge, lending support to their proposed involvement in mosquito immunity.
Genetic diversity of Borrelia burgdorferi sensu lato in ticks from mainland Portugal.
De Michelis, S., Sewell, H.S., Collares-Pereira, M., Santos-Reis, M., Schouls, L.M., Benes, V., Holmes, E.C. & Kurtenbach, K.
J Clin Microbiol 2000 Jun;38(6):2128-33.
To date Borrelia lusitaniae is the only genospecies of Borrelia burgdorferi sensu lato isolated from Ixodes ricinus ticks collected in Portugal and Tunisia. This suggests that the genospecies diversity of B. burgdorferi sensu lato decreases toward the southwestern margin of its Old World subtropical range. In order to further explore the genetic diversity of B. burgdorferi sensu lato from this region, 55 I. ricinus and 27 Hyalomma marginatum questing adults, collected during the spring of 1998 from a sylvatic habitat south of Lisbon, Portugal, were analyzed. Infection prevalences of 75% in I. ricinus ticks and 7% in H. marginatum ticks were detected by a nested PCR that targets the rrf (5S)-rrl (23S) spacer of B. burgdorferi sensu lato. Restriction fragment length polymorphism (RFLP) analysis of the I. ricinus-derived amplicons showed that the sequences in the majority of samples were similar to those of B. lusitaniae type strains (76% for strain PotiB1, 5% for strain PotiB3). Two novel RFLP patterns were obtained from 12% of the samples. The remaining 7% of samples gave mixed RFLP patterns. Phylogenetic analysis of rrf-rrl spacer sequences revealed a diverse population of B. lusitaniae in questing adult I. ricinus ticks (the sequences did not cluster with those of any other genospecies). This population consisted of 10 distinct sequence types, suggesting that multiple strains of B. lusitaniae were present in the local I. ricinus population. We hypothesize that B. lusitaniae has a narrow ecological niche that involves host species restricted to the Mediterranean Basin.
Progress in Arabidopsis genome sequencing and functional genomics.
Wambutt, R., Murphy, G., Volckaert, G., Pohl, T., Dusterhoft, A., Stiekema, W., Entian, K.D., Terryn, N., Harris, B., Ansroge, W., Brandt, P., Grivell, L., Rieger, M., Weichselgartner, M., de Simone, V., Obermaier, B., Mache, R., Muller, M., Kreis, M., Delseny, M., Puigdomenech, P., Watson, M., Schmidtheini, T., Reichert, B., Portatelle, D., Perez-Alonso, M., Bountry, M., Bancroft, I., Vos, P., Hoheisel, J., Zimmermann, W., Wedler, H., Ridley, P., Langham, S.A., McCullagh, B., Bilham, L., Robben, J., Van der Schueren, J., Grymonprez, B., Chuang, Y.J., Vandenbussche, F., Braeken, M., Weltjens, I., Voet, M., Bastiens, I., Aert, R., Defoor, E., Weitzenegger, T., Bothe, G. & Rose, M.
J Biotechnol 2000 Mar 31;78(3):281-92.
Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.
Sequence and expression of the kettin gene in drosophila melanogaster and caenorhabditis elegans.
Kolmerer, B., Clayton, J., Benes, V., Allen, T., Ferguson, C., Leonard, K., Weber, U., Knekt, M., Ansorge, W., Labeit, S. & Bullard, B.
J Mol Biol 2000 Feb 18;296(2):435-48
Kettin is a large modular protein associated with thin filaments in the Z-disc region of insect muscles. The sequence of a 21.3 kb contig of the Drosophila gene has been determined. The corresponding protein sequence has 35 immunoglobulin-like (Ig) domains which are separated by shorter linker sequences, except near the N and C termini of the molecule where linker sequences are short or missing. This confirms a model in which each Ig domain binds to an actin protomer. The Drosophila kettin gene is at 62C 1-3 on the third chromosome. Two P- element insertions, l(3)j1D7 and l(3)rL182 are in the kettin gene, and complementation tests showed that existing l(3)dre8 mutations are in the same gene. The RNA was detected in wild-type Drosophila embryos at stage 11, first in the gut invagination region of the mesoderm, and by stage 13 in both visceral and somatic mesoderm. Somatic mesoderm expression became segmental at stage 13. RNA expression was greatly reduced in embryos of P-element homozygotes but normal in heterozygotes. The structure of the flight muscle in all the heterozygous mutants was normal, including the myofibril-cuticle connections, and they were able to fly. Kettin sequence homologous to the Drosophila protein, was identified in the Caenorhabditis elegans genome database. The RNA was detected in pharyngeal, body wall and anal depressor muscles of larvae and adult worms, as well as in the male gonad. Antibody to insect kettin labelled the pharyngeal, body wall, anal depressor and proximal gonadal muscles in adult worms. Body wall muscles were labelled in an obliquely striated pattern consistent with the Z-disc localisation in insect muscle. The relationship of kettin to D-titin, which has been assigned to the same chromosomal locus in Drosophila, is discussed. Copyright 2000 Academic Press.
BAC trimming: Minimizing clone overlaps.
Hill, F., Benes, V., Thomasova, D., Stewart, A.F., Kafatos, F.C. & Ansorge, W.
Genomics 2000 Feb 15;64(1):111-113
Bacterial vectors containing large inserts of genomic DNA are now the standard substrates for large-scale genomic sequencing. Long overlaps between some clones lead to considerable redundant effort. A method for deleting defined regions from bacterial artificial chromosome (BAC) inserts, using homologous recombination, was applied to minimize the overlap between successive BAC clones. This procedure, called trimming, was carried out in the recA(-) BAC host. We have precisely deleted up to 70 kb of DNA from BACs that were to be sequenced. This method requires minimal prior characterization of the clones: collections of BAC end sequences or STS-based maps will accelerate the process. BAC trimming will be useful in both small and large genome sequencing projects and will be of particular utility for gap closure in finishing phases. Copyright 2000 Academic Press.
Genomic locus and promoter region of rat Smad7, an important antagonist of TGFbeta signaling.
Stopa, M., Benes, V., Ansorge, W., Gressner, A.M. & Dooley, S.
Mamm Genome 2000 Feb;11(2):169-76
SMAD proteins are essential components of the intracellular signaling pathways utilized by members of the transforming growth factor beta (TGFbeta) superfamily of growth factors. Certain SMAD proteins (Smad1, 2, 3, and 5) can act as regulated transcriptional activators. This process involves phosphorylation of these proteins by activated TGFbeta receptors. Recently, Smad6 and Smad7 were identified; they antagonize TGFbeta signaling by preventing the activation of signal-transducing SMAD complexes. TGFbeta rapidly induces the expression of Smad7 mRNA, suggesting participation of Smad7 in a negative feedback loop to control TGFbeta responses. Similarly, epidermal growth factor (EGF) and interferon gamma (IFN-gamma) have been reported to induce Smad7 expression. In a rat model system of liver fibrosis, TGFbeta inducibility of Smad7 is abrogated during transformation of hepatic stellate cells (HSC), indicating an important switch in transcriptional regulation of the gene. With the detailed characterization of the rat Smad7 genomic organization including the promoter region, we present the first identified Smad7 gene so far. The gene is composed of four exons separated by three introns covering a DNA region of about 30 kilobases (kb) in total. The major transcription start site is conserved between rat and mouse, and two polyadenylation signals were detected. In the promoter region, a potential CAGA box, a signal transducer and activator of transcription (STAT) factor-related recognition site, and different AP1 sites were identified, which could be the targets of TGFbeta, IFN-gamma, and EGF-dependent Smad7 transcription initiation.
Sequence and analysis of chromosome 4 of the plant Arabidopsis thaliana.
Mayer, K., Schuller, C., Wambutt, R., Murphy, G., Volckaert, G., Pohl, T., Dusterhoft, A., Stiekema, W., Entian, K.D., Terryn, N., Harris, B., Ansorge, W., Brandt, P., Grivell, L., Rieger, M., Weichselgartner, M., de Simone, V., Obermaier, B., Mache, R., Muller, M., Kreis, M., Delseny, M., Puigdomenech, P., Watson, M., McCombie, W.R. & et, al
Nature 1999 Dec 16;402(6763):769-77
The higher plant Arabidopsis thaliana (Arabidopsis) is an important model for identifying plant genes and determining their function. To assist biological investigations and to define chromosome structure, a coordinated effort to sequence the Arabidopsis genome was initiated in late 1996. Here we report one of the first milestones of this project, the sequence of chromosome 4. Analysis of 17.38 megabases of unique sequence, representing about 17% of the genome, reveals 3,744 protein coding genes, 81 transfer RNAs and numerous repeat elements. Heterochromatic regions surrounding the putative centromere, which has not yet been completely sequenced, are characterized by an increased frequency of a variety of repeats, new repeats, reduced recombination, lowered gene density and lowered gene expression. Roughly 60% of the predicted protein-coding genes have been functionally characterized on the basis of their homology to known genes. Many genes encode predicted proteins that are homologous to human and Caenorhabditis elegans proteins.
Sec61beta, a subunit of the protein translocation channel, is required during Drosophila development.
Valcarcel, R., Weber, U., Jackson, D.B., Benes, V., Ansorge, W., Bohmann, D. & Mlodzik, M.
J Cell Sci 1999 Dec;112 ( Pt 23):4389-96
We have identified and isolated mutations in the first Drosophila gene encoding a subunit of the Sec61 protein translocation channel, DSec61beta. While neither the Saccharomyces cerevisiae Sec61beta nor its functional Escherichia coli homologue are essential for viability or for protein translocation, we show that DSec61beta is essential for embryonic development. Homozygous mutant embryos die at the end of embryogenesis and are impaired in the secretion of cuticle proteins from the epidermis. DSec61beta germ line clones, result in defects in dorso-ventral patterning of the egg and are consistent with affected secretion of the protein Gurken from the oocyte to the follicle cells. Clonal analyses in the imaginal discs reveal defects in adult structures, including rhabdomere morphogenesis and a reduction of the size of tarsal segments in the leg. This is the first in vivo study of a component of the protein translocation machinery in higher eukaryotes, and illustrates how a protein that has an inessential, kinetic function in single-cell organisms can become critical for the complex development of a multicellular organism.
DNA binding and transactivating properties of the paired and homeobox protein Pax4.
Kalousova, A., Benes, V., Paces, J., Paces, V. & Kozmik, Z.
Biochem Biophys Res Commun 1999 Jun 16;259(3):510-8.
Transcription factors Pax-4 and Pax-6 are known to be key regulators of pancreatic cell differentiation and development. We report on the cloning of a mouse Pax-4 gene, which contains 10 exons, spanning a 4. 7-kbp region. The gene-targeting experiments revealed that Pax-4 and Pax-6 cannot substitute for each other in tissue with overlapping expression of both genes. We identified DNA-binding specificities of Pax-4 paired domain and paired-type homeodomain. Despite the different Pax-4 amino acid residues in positions responsible for Pax-6 paired-domain specificity, the DNA-binding specificities of Pax-4 and Pax-6 are similar. The Pax-4 homeodomain was shown to preferentially dimerize on DNA sequences consisting of an inverted TAAT motif, separated by 4-nucleotide spacing. The Pax-4 transactivation domain was localized within its C-terminal region, which transactivated GAL-based reporter 2.5-fold less than the C-terminal region of Pax-6. We believe that Pax-4 can act as a Pax-6 "repressor," due to the competition for binding sites and lower transactivation potential of Pax-4.
Dynamics of gene expression revealed by comparison of serial analysis of gene expression transcript profiles from yeast grown on two different carbon sources.
Kal, A.J., van Zonneveld, A.J., Benes, V., van den Berg, M., Koerkamp, M.G., Albermann, K., Strack, N., Ruijter, J.M., Richter, A., Dujon, B., Ansorge, W. & Tabak, H.F.
Mol Biol Cell 1999 Jun;10(6):1859-72
We describe a genome-wide characterization of mRNA transcript levels in yeast grown on the fatty acid oleate, determined using Serial Analysis of Gene Expression (SAGE). Comparison of this SAGE library with that reported for glucose grown cells revealed the dramatic adaptive response of yeast to a change in carbon source. A major fraction (>20%) of the 15,000 mRNA molecules in a yeast cell comprised differentially expressed transcripts, which were derived from only 2% of the total number of approximately 6300 yeast genes. Most of the mRNAs that were differentially expressed code for enzymes or for other proteins participating in metabolism (e.g., metabolite transporters). In oleate- grown cells, this was exemplified by the huge increase of mRNAs encoding the peroxisomal beta-oxidation enzymes required for degradation of fatty acids. The data provide evidence for the existence of redox shuttles across organellar membranes that involve peroxisomal, cytoplasmic, and mitochondrial enzymes. We also analyzed the mRNA profile of a mutant strain with deletions of the PIP2 and OAF1 genes, encoding transcription factors required for induction of genes encoding peroxisomal proteins. Induction of genes under the immediate control of these factors was abolished; other genes were up-regulated, indicating an adaptive response to the changed metabolism imposed by the genetic impairment. We describe a statistical method for analysis of data obtained by SAGE.
Bacteriophage phi29 early protein p17 is conditionally required for the first rounds of viral DNA replication.
Crucitti, P., Lazaro, J.M., Benes, V. & Salas, M.
Gene 1998 Nov 26;223(1-2):135-42.
The gene 17 of the Bacillus subtilis phage phi29 is known to be involved in the viral DNA replication in vivo. In this paper, we show that the presence of protein p17 is required when phage infection occurs at a low multiplicity of infection (moi), which is probably the natural condition for infection, but is dispensable at a high moi. Gene 17 has been cloned in an Escherichia coli expression vector and protein p17 purified. A stimulatory effect of protein p17 was demonstrated under in vitro conditions required to amplify phi29 DNA, starting with a low amount of input DNA. We propose that p17, which is synthesized early after infection, is required at the very beginning of the phage amplification, conditions in which a low number of viral DNA molecules enter the host cell, possibly to recruit the limiting amount of initiation factors at the replication origins. Once the infection process is established and the other replication proteins reach optimal concentration, p17 becomes dispensable.
Systematic gain-of-function genetics in Drosophila.
Rorth, P., Szabo, K., Bailey, A., Laverty, T., Rehm, J., Rubin, G.M., Weigmann, K., Milan, M., Benes, V., Ansorge, W. & Cohen, S.M.
Development 1998 Mar;125(6):1049-57.
A modular misexpression system was used to carry out systematic gain-of-function genetic screens in Drosophila. The system is based on inducible expression of genes tagged by insertion of a P-element vector carrying a GAL4-regulated promoter oriented to transcribe flanking genomic sequences. To identify genes involved in eye and wing development, the 2300 independent lines were screened for dominant phenotypes. Among many novel genes, the screen identified known genes, including hedgehog and decapentaplegic, implicated in these processes. A genetic interaction screen for suppressors of a cell migration defect in a hypomorphic slow border cells mutant identified known genes with likely roles in tyrosine kinase signaling and control of actin cytoskeleton, among many novel genes. These studies demonstrate the ability of the modular misexpression system to identify developmentally important genes and suggest that it will be generally useful for genetic interaction screens.
Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana
Bevan, M., Bancroft, I., Bent, E., Love, K., Goodman, H., Dean, C., Bergkamp, R., Dirkse, W., Van Staveren, M., Stiekema, W., Drost, L., Ridley, P., Hudson, S.A., Patel, K., Murphy, G., Piffanelli, P., Wedler, H., Wedler, E., Wambutt, R., Weitzenegger, T., Pohl, T.M., Terryn, N., Gielen, J., Villarroel, R., Chalwatzis, N. & et, al
Nature 1998 Jan 29 391(6666) 485-488
The plant Arabidopsis thaliana (Arabidopsis) has become an important model species for the study of many aspects of plant biology. The relatively small size of the nuclear genome and the availability of extensive physical maps of the five chromosomes provide a feasible basis for initiating sequencing of the five chromosomes. The YAC (yeast artificial chromosome)-based physical map of chromosome 4 was used to construct a sequence-ready map of cosmid and BAC (bacterial artificial chromosome) clones covering a 1.9-megabase (Mb) contiguous region, and the sequence of this region is reported here. Analysis of the sequence revealed an average gene density of one gene every 4.8 kilobases (kb), and 54% of the predicted genes had significant similarity to known genes. Other interesting features were found, such as the sequence of a disease-resistance gene locus, the distribution of retroelements, the frequent occurrence of clustered gene families, and the sequence of several classes of genes not previously encountered in plants.
Strawberry vein banding virus--definitive member of the genus Caulimovirus.
Petrzik, K., Benes, V., Mraz, I., Honetslegrova-Franova, J., Ansorge, W. & Spak, J.
Virus Genes 1998 16(3) 303-305
The complete DNA sequence (7876 nucleotides) of strawberry vein banding virus (SVBV) has been determined. Seven open reading frames are detected that potentially code for proteins of calculated weight 37.8; 18.3; 16.6; 56.0; 81.1; 59.0 and 12.6 kDa, respectively. Their position on the viral genome is the same as that of the corresponding proteins on the cauliflower mosaic virus (CaMV) genome. Phylogenetic analysis based on the amino acid sequence of this protein shows a closer relationship of SVBV with CaMV, figwort mosaic virus and carnation etched ring virus than with other caulimoviruses.
Bacteriophage B103: complete DNA sequence of its genome and relationship to other Bacillus phages.
Pecenkova, T., Benes, V., Paces, J., Vlcek, C. & Paces, V.
Gene 1997 Oct 15;199(1-2):157-63.
The genome of Bacillus subtilis bacteriophage B103 consists of double-stranded linear DNA 18,630 bp long. The DNA was sequenced, and the sequence was compared with DNA sequences of closely related phages, namely the members of the phage phi29 family. Among them, phage Nf was shown to be the most closely related to B103. Comparisons of several open reading frames (ORFs) among the family members helped to identify genes 1 and 5. A cluster of ORFs between genes 16 and 17 contains two ORFs with partial homology with two phi29 ORFs located in the same region. There are three more ORFs in this region of B103 with good ribosome binding sites (RBS) and optimal codon usage that are not homologous to any of the phi29 ORFs. The function of these five ORFs remains unexplained. It was shown that major promoters characterized in phi29 are retained in B103. Where many substitutions occur in the vicinity of a promoter, at least the -10 and -35 boxes are conserved.
Automated cycle sequencing with Taquenase: protocols for internal labeling, dye primer and "doublex" simultaneous sequencing.
Voss, H., Nentwich, U., Duthie, S., Wiemann, S., Benes, V., Zimmermann, J. & Ansorge, W.
Biotechniques 1997 Aug;23(2):312-8
This paper describes automated cycle sequencing protocols for internal labeling, dye primer and "doublex" simultaneous sequencing using Taquenase, a new genetically modified DNA polymerase with increased thermostability. Sequencing performance both with labeled and unlabeled primer yields uniform unambiguous signals up to the resolution limit of the sequencing gels. Primer walking with internal labeling was successfully performed on Pl-derived artificial chromosome (PAC) constructs with 130-kb inserts. Taquenase, a commercially available modified thermostable sequencing enzyme (delta 280, F667Y Taq DNA polymerase), incorporates a variety of fluorescent dNTPs carrying fluorescein isothiocyanate, TexasRed or Cy5 labels during the cycle- sequencing process with higher efficiency than other thermostable DNA polymerases. Comparison to other modified Taq DNA polymerases suggests that the particular N-terminal deletion of Taquenase rather than the presence of the F667Y mutation is responsible for the efficient incorporation and extension of labeled dNTPs. Taquenase makes feasible highly accurate "doublex" simultaneous cylce sequencing on both strands of template DNA with two internal labels or two dye-labeled primers in combination with the EMBL-2-dye DNA sequencing system, ARAKIS, or with two commercial DNA sequencers. It allows up to 2000 bases at > 99% accuracy to be determined in a single reaction.
Direct primer walking on P1 plasmid DNA.
Benes, V., Kilger, C., Voss, H., Paabo, S. & Ansorge, W.
Biotechniques 1997 Jul;23(1):98-100 PubMed
DNA sequencing and analysis of 130 kb from yeast chromosome XV.
Voss, H., Benes, V., Andrade, M.A., Valencia, A., Rechmann, S., Teodoru, C., Schwager, C., Paces, V., Sander, C. & Ansorge, W.
Yeast 1997 Jun 15;13(7):655-72
We have determined the nucleotide sequence of 129,524 bases of yeast (Saccharomyces cerevisiae) chromosome XV. Sequence analysis revealed the presence of 59 non-overlapping open reading frames (ORFs) of length > 300 bp, three tRNA genes, four delta elements and one Ty-element. Among the 21 previously known yeast genes (36% of all ORFs in this fragment) were nucleoporin (NUP1), ras protein (RAS1), RNA polymerase III (RPC1) and elongation factor 2 (EF2). Further, 31 ORFs (53% of the total) were found to be homologous to known protein or DNA sequences, or sequence patterns. For seven ORFs (11% of the total) no homology was found. Among the most interesting protein identification in this DNA fragment are an inositol polyphosphatase, the second gene of this type found in yeast (homologous to the human OCRL gene involved in Lowe's syndrome), a new ADP ribosylation factor of the arf6 subfamily, the first protein containing three C2 domains, and an ORF similar to a Bacillus subtilis cell-cycle related protein. For each ORF detailed sequence analysis was carried out, with a full consideration of its biological function and pointing out key regions of interest for further functional analysis.
Simultaneous loading of 200 sample lanes for DNA sequencing on vertical and horizontal, standard and ultrathin gels.
Erfle, H., Ventzki, R., Voss, H., Rechmann, S., Benes, V., Stegemann, J. & Ansorge, W.
Nucleic Acids Res 1997 Jun 1;25(11):2229-30
We have developed a simple and efficient technique for automated parallel loading of >/=200 lanes on a 30 cm-wide gel in automated DNA sequencing, using porous filter materials and an associated manual or robotic system. The samples are loaded onto the teeth of a comb made of the porous material. The comb, with samples, is inserted directly above the straight edge of the polymerized gel. The samples are driven from the comb into the gel by the applied electrical field. A particularly advantageous aspect of this method is the elimination of the thin gel walls separating the sample wells in the standard gel loading technique. The time for sample loading is significantly reduced to a few minutes. The loading technique is applicable to horizontal or vertical systems, with standard or ultrathin gels.
The nucleotide sequence of Saccharomyces cerevisiae chromosome XII.
Johnston, M., Hillier, L., Riles, L., Albermann, K., Andre, B., Ansorge, W., Benes, V., Bruckner, M., Delius, H., Dubois, E., Dusterhoft, A., Entian, K.D., Floeth, M., Goffeau, A., Hebling, U., Heumann, K., Heuss-Neitzel, D., Hilbert, H., Hilger, F., Kleine, K., Kotter, P., Louis, E.J., Messenguy, F., Mewes, H.W., Hoheisel, J.D. & et, al
Nature 1997 May 29;387(6632 Suppl):87-90
The yeast Saccharomyces cerevisiae is the pre-eminent organism for the study of basic functions of eukaryotic cells. All of the genes of this simple eukaryotic cell have recently been revealed by an international collaborative effort to determine the complete DNA sequence of its nuclear genome. Here we describe some of the features of chromosome XII.
The nucleotide sequence of Saccharomyces cerevisiae chromosome XV.
Dujon, B., Albermann, K., Aldea, M., Alexandraki, D., Ansorge, W., Arino, J., Benes, V., Bohn, C., Bolotin-Fukuhara, M., Bordonne, R., Boyer, J., Camasses, A., Casamayor, A., Casas, C., Cheret, G., Cziepluch, C., Daignan-Fornier, B., Dang, D.V., de Haan, M., Delius, H., Durand, P., Fairhead, C., Feldmann, H., Gaillon, L., Kleine, K. & et, al
Nature 1997 May 29;387(6632 Suppl):98-102
Chromosome XV was one of the last two chromosomes of Saccharomyces cerevisiae to be discovered. It is the third-largest yeast chromosome after chromosomes XII and IV, and is very similar in size to chromosome VII. It alone represents 9% of the yeast genome (8% if ribosomal DNA is included). When systematic sequencing of chromosome XV was started, 93 genes or markers were identified, and most of them were mapped. However, very little else was known about chromosome XV which, in contrast to shorter chromosomes, had not been the object of comprehensive genetic or molecular analysis. It was therefore decided to start sequencing chromosome XV only in the third phase of the European Yeast Genome Sequencing Programme, after experience was gained on chromosomes III, XI and II. The sequence of chromosome XV has been determined from a set of partly overlapping cosmid clones derived from a unique yeast strain, and physically mapped at 3.3-kilobase resolution before sequencing. As well as numerous new open reading frames (ORFs) and genes encoding tRNA or small RNA molecules, the sequence of 1,091,283 base pairs confirms the high proportion of orphan genes and reveals a number of ancestral and successive duplications with other yeast chromosomes.
The nucleotide sequence of Saccharomyces cerevisiae chromosome XVI.
Bussey, H., Storms, R.K., Ahmed, A., Albermann, K., Allen, E., Ansorge, W., Araujo, R., Aparicio, A., Barrell, B., Badcock, K., Benes, V., Botstein, D., Bowman, S., Bruckner, M., Carpenter, J., Cherry, J.M., Chung, E., Churcher, C., Coster, F., Davis, K., Davis, R.W., Dietrich, F.S., Delius, H., DiPaolo, T., Hani, J. & et, al
Nature 1997 May 29;387(6632 Suppl):103-5
The nucleotide sequence of the 948,061 base pairs of chromosome XVI has been determined, completing the sequence of the yeast genome. Chromosome XVI was the last yeast chromosome identified, and some of the genes mapped early to it, such as GAL4, PEP4 and RAD1 (ref. 2) have played important roles in the development of yeast biology. The architecture of this final chromosome seems to be typical of the large yeast chromosomes, and shows large duplications with other yeast chromosomes. Chromosome XVI contains 487 potential protein-encoding genes, 17 tRNA genes and two small nuclear RNA genes; 27% of the genes have significant similarities to human gene products, and 48% are new and of unknown biological function. Systematic efforts to explore gene function have begun.
Molecular characterization of two high affinity sulfate transporters in Saccharomyces cerevisiae.
Cherest, H., Davidian, J.C., Thomas, D., Benes, V., Ansorge, W. & Surdin-Kerjan, Y.
Genetics 1997 Mar;145(3):627-35
Strains resistant to the toxic analogues of sulfate, selenate and chromate have been isolated. Their genetic analysis allowed us to identify four genes. One, called MET28, encodes a transcriptional factor. The three other genes, called SUL1, SUL2 and SUL3, encode proteins involved in sulfate transport. The sequence of Sul1p and Sul2p indicate that they are integral membrane proteins exhibiting, respectively, 11 and 10 transmembrane domains. Moreover, Sul1p and Sul2p share a high degree of similarity. Sulfate transport kinetic studies made with parental and mutant strains show that, as expected from genetic results, Saccharomyces cerevisiae has two high affinity sulfate transport systems. Sul3p has been shown to be involved in the transcriptional regulation of the SUL2 gene.
System for simultaneous sequencing of 240 clones with 5 dyes, 1300 bases readings (up to 5000 bases per sequence reaction) with novel loading technique for vertical and horizontal standard or ultrathin gels.
Stegemann, J., Ventzki, R., Schwager, C., Erfle, H., Benes, V., Voss, H., & Ansorge, W.
FASEB J. 1997 11(9)
Sequencing and analysis of 51 kb on the right arm of chromosome XV from Saccharomyces cerevisiae reveals 30 open reading frames.
Wiemann, S., Rechmann, S., Benes, V., Voss, H., Schwager, C., Vlcek, C., Stegemann, J., Zimmermann, J., Erfle, H., Paces, V. & Ansorge, W.
Yeast 1996 Mar 15;12(3):281-8
We have sequenced a region of 51 kb of the right arm from chromosome XV of Saccharomyces cerevisiae. The sequence contains 30 open reading frames (ORFs) of more than 100 amino acid residues. Thirteen new genes have been identified. Thirteen ORFs correspond to known yeast genes. One delta element and one tRNA gene were identified. Upstream of the RPO31 gene, encoding the largest subunit of RNA polymerase III, lies a Abf1p binding site. The nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDBJ nucleotide sequence databases under the Accession Number X90518.
"Doublex" fluorescent DNA sequencing: two independent sequences obtained simultaneously in one reaction with internal labeling and unlabeled primers.
Wiemann, S., Stegemann, J., Zimmermann, J., Voss, H., Benes, V. & Ansorge, W.
Anal Biochem 1996 Feb 15;234(2):166-74
The novel "doublex" DNA sequencing technique that makes it possible to obtain simultaneously two independent sequences from one sequencing reaction with the use of unlabeled primers and internal labeling is described. The different sequencing products are labeled in parallel with fluorescein-15-dATP and Texas red-5-dCTP present in the same tube. The characteristics of T7 DNA polymerase are exploited to ensure that only either of the labeled dNTPs is incorporated into the corresponding sequencing products. Specificity of labeling is ensured by the selection of primers. One of the unlabeled primers is chosen to be followed by an "A," the other by a "C" to be incorporated immediately downstream from the primer binding site. The doublex sequencing technique is applicable to the simultaneous sequencing of either the same DNA template/strand or a mixture of different templates. Combinations of unlabeled and labeled primers in the same sequencing reaction are also possible. The two sequences can be determined in parallel and on-line in the same lanes of a gel with a novel automated DNA sequencer, which was previously described for use with labeled primers.
Sequencing 39,350 bp of Saccharomyces cerevisiae chromosome XII utilizing ordered shotgun libraries.
Scholler, P., Benes, V., Voss, H., Ansorge, W. & Hoheisel, J.D.
DNA Seq 1996;6(5):257-62
A 39,350 bp cosmid containing DNA of Saccharomyces cerevisiae chromosome XII was sequenced by making use of ordered sub-clones of 1 kb insert-length selected from a physical clone map. In a first analysis, 96 clones were sequenced from both ends (10 gels) with two standard sequencing primers covering 91% of the total sequence (49% double-stranded). After selection of another eight clones six gaps of a total of 1.8 kb and several single-stranded stretches remained. These gaps were closed by 86 primer walks leading to an overall redundancy of 4.4 per base and a total of 292 sequencing reactions. The number of walking primers can be reduced significantly by more uniform clone lengths and longer sequencing reads, thus, the total amount of sequencing reactions can approach the minimum value achieved with primer walking strategies, with only very few walking primers needed for gap closure.
Nucleotide sequence analysis of a 30-kb region of the bovine herpesvirus 1 genome which exhibits a colinear gene arrangement with the UL21 to UL4 genes of herpes simplex virus.
Vlcek, C., Benes, V., Lu, Z., Kutish, G.F., Paces, V., Rock, D., Letchworth, G.J. & Schwyzer, M.
Virology. 1995 Jun 20;210(1):100-8.
We report the nucleotide sequence of the 19-kb HindIII fragment B of bovine herpesvirus 1 (BHV-1) DNA and adjacent parts of the HindIII A and L fragments, which together span a still completely uncharted 30-kb region located between the glycoprotein H gene and the right end of the unique long segment. The analysis revealed 17 complete open reading frames (ORFs) and 2 ORFs that were interrupted by potential splice donor and acceptor sites. All of these ORFs exhibited strong amino acid sequence homology to the gene products of other alphaherpesviruses. The BHV-1 ORFs were arranged colinearly with the prototype sequence of herpes simplex virus 1 in the range of the UL21 to UL4 genes. Colinearity was also observed with the genes of betaherpesviruses and gamma herpesviruses, although not all ORFs exhibited clear sequence homology. The possible functions of the proteins encoded within the sequenced region are assessed and features found are discussed. Unexpected findings include the following: high amino acid sequence conservation among alphaherpesviruses despite large differences in G + C content, ranging from 45% for varicella zoster virus to 72% for BHV-1; high similarity with other UL20 proteins at the predicted structural level in spite of relatively low amino acid homology; and a 2-kb open reading frame overlapping UL19 in the opposite sense and exhibiting high amino acid similarity to the same area of pseudorabies virus.
Protein-induced bending or flexing at the 5'-end of the duck beta A-globin promoter.
Matlova, R., Horska, K., Benes, V., Cvekl, A. & Sponar, J.
Gene. 1995 Apr 24;156(2):277-81.
The 5'-end of the duck beta A-globin promoter contains a protein binding site BS-3/Sp1, the A + T-rich part of which could be involved in DNA bending. Plasmids were constructed using the pBend2 plasmid containing BS-3/Sp1. Circular permutation analysis of the fragments cut out from the plasmids using various restriction endonucleases, in the presence of a partially purified protein extract from embryonic duck erythrocytes, was performed. The results indicate that a rather complicated change in the fragment shape takes place upon protein binding, which is best explained as an induction of two points of bending or flexure within the fragment. Analogical points of flexure may exist at the protein-binding sites of the duck and chicken beta A-globin promoters in spite of differing DNA sequences.
M13 and pUC vectors with new unique restriction sites for cloning.
Benes, V., Hostomsky, Z., Arnold, L. & Paces, V.
Gene. 1993 Aug 16;130(1):151-2.
Several vectors based on the widely used phage M13 and plasmid pUC were constructed. The vectors contain polylinkers (MCS) for DNA insertions with several new restriction sites (e.g., ApaI, NotI, StuI, SacII). Moreover, the NarI site in the nonessential part of the vector molecule was changed into a BssHII site, so that the NarI site in the MCS became unique.
Isolation, biochemical characterization and crystallization of the p15gag proteinase of myeloblastosis associated virus expressed in E. coli.
Pichova, I., Strop, P., Sedlacek, J., Kapralek, F., Benes, V., Travnicek, M., Pavlickova, L., Soucek, M., Kostka, V. & Foundling, S.
Int J Biochem. 1992 Feb;24(2):235-42.
1. The p15gag proteinase responsible for the processing of the polyprotein precursor of the myeloblastosis associated virus was obtained by a recombinant technique in an E. coli expression system. The massive expression of the intentionally truncated precursor (Pr25lac-delta gag) was accompanied by its structurally correct processing. 2. Three procedures for the purification of the recombinant proteinase from both the cytoplasmic fraction and the inclusion bodies were developed. 3. The purified proteinase was compared with the authentic proteinase isolated from MAV virions by N-terminal sequence analysis and amino acid analysis, molecular weight determination, reverse-phase HPLC and FPLC elution profiles, electrophoretic mobility and isoelectric point determination, and activity assays with proteins and synthetic substrates. The identity of both enzymes was shown. 3. Contrary to reported data, the amino acid sequence of the p15gag proteinase differs from the sequence of the homologous Rous sarcoma virus proteinase in one residue only, as follows from cDNA sequencing. 4. Crystallization of the proteinase from a citrate-phosphate buffer at pH 5.6 afforded hexagonal crystals which diffracted well as 2.3 A without deterioration.
Nucleotide sequence of the right early region of Bacillus phage phi 15 and comparison with related phages: reorganization of gene 17 during evolution.
Benes, V., Arnold, L., Smrt, J. & Paces, V.
Gene. 1989 Feb 20;75(2):341-7.
The rightmost 2016 bp of the Bacillus subtilis phage phi 15 genome were sequenced. The nucleotide sequence was compared with the homologous regions of the related phages PZA and phi 29. There are six open reading frames (ORFs) in this region of the phi 15 genome; all of them are present in the PZA and phi 29 genomes. One of the ORFs was assigned to gene 17, which is involved in the replication of the phage DNA. Gene 17 has undergone reorganization during the evolution of this phage family. Comparison of the nucleotide sequence of its mRNA-like strand in phi 15, PZA and phi 29 showed that deletions in its central and 3'-end-proximal parts are tolerated and do not interfere with the gene 17 product function. It seems that the only portion of gene 17 that has to be conserved to encode the functional product is its 5'-end-proximal part.
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