This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ http://www.biomedcentral.com/bmcmolbiol/mostviewed/ Most viewed articles in last 30 days from BMC Molecular Biology (ISSN 1471-2199) published by BioMed Central The discovery of microRNAs (miRNAs) has added an extra level of intricacy to the already complex system underlying gene regulation. These single-stranded RNA molecules, 18-25 nucleotides in length, negatively regulate gene expression through translational inhibition or mRNA cleavage. The discovery that aberrant expression of specific miRNAs contributes to human disease has fueled great interest in profiling the expression of these molecules. Real-time quantitative PCR (RQ-PCR) is a sensitive and reproducible gene expression quantitation technique which is now being used to profile miRNA expression in cells and tissues. To correct for systematic variables such as amount of starting template, RNA quality and enzymatic efficiencies, RQ-PCR data is commonly normalised to an endogenous control (EC) gene, which ideally, is stably-expressed across the test sample set. A universal endogenous control suitable for every tissue type, treatment and disease stage has not been identified and is unlikely to exist, so, to avoid introducing further error in the quantification of expression data it is necessary that candidate ECs be validated in the samples of interest. While ECs have been validated for quantification of mRNA expression in various experimental settings, to date there is no report of the validation of miRNA ECs for expression profiling. In this study, the expression of five miRNAs and three small nucleolar RNAs was examined across malignant, benign and normal breast tissues to determine the most appropriate ECs. This is the first study to identify reliable ECs for analysis of miRNA by RQ-PCR in human breast tissue. http://www.biomedcentral.com/1471-2199/9/76 Pamela A Davoren, Roisin E McNeill, Aoife J Lowery, Michael J Kerin and Nicola Miller BMC Molecular Biology 2008, 9:76 Number of accesses: 887 2008-08-21 doi:10.1186/1471-2199-9-76 BMC Molecular Biology 1471-2199 9 76 2008-08-21 Background: Methylation of CpG dinucleotides is a fundamental mechanism of epigenetic regulation in eukaryotic genomes. Development of methods for rapid genome wide methylation profiling will greatly facilitate both hypothesis and discovery driven research in the field of epigenetics. In this regard, a single molecule approach to methylation profiling offers several unique advantages that include elimination of chemical DNA modification steps and PCR amplification. Results: A single molecule approach is presented for the discernment of methylation profiles, based on optical mapping. We report results from a series of pilot studies demonstrating the capabilities of optical mapping as a platform for methylation profiling of whole genomes. Optical mapping was used to discern the methylation profile from both an engineered and wild type Escherichia coli. Furthermore, the methylation status of selected loci within the genome of human embryonic stem cells was profiled using optical mapping. Conclusion: The optical mapping platform effectively detects DNA methylation patterns. Due to single molecule detection, optical mapping offers significant advantages over other technologies. This advantage stems from obviation of DNA modification steps, such as bisulfite treatment, and the ability of the platform to assay repeat dense regions within mammalian genomes inaccessible to techniques using array-hybridization technologies. http://www.biomedcentral.com/1471-2199/9/68 Gene E Ananiev, Steve Goldstein, Rod Runnheim, Dan K Forrest, Shiguo Zhou, Konstantinos Potamousis, Chris P Churas, Veit Bergendahl, James A Thomson and David C Schwartz BMC Molecular Biology 2008, 9:68 Number of accesses: 644 2008-07-30 doi:10.1186/1471-2199-9-68 BMC Molecular Biology 1471-2199 9 68 2008-07-30 Background: Housekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes. Results: The expression of the potential reference genes was examined in matched malignant and non-malignant tissue specimens from 25 patients with clear cell renal cell carcinoma. Quality assessment of isolated RNA performed with a 2100 Agilent Bioanalyzer showed a mean RNA integrity number of 8.7 for all samples. The between-run variations related to the crossing points of PCR reactions of a control material ranged from 0.17% to 0.38%. The expression of all genes did not depend on age, sex, and tumour stage. Except the genes TATA box binding protein (TBP) and peptidylprolyl isomerase A (PPIA), all genes showed significant differences in expression between malignant and non-malignant pairs. The expression stability of the candidate reference genes was additionally controlled using the software programs geNorm and NormFinder. TBP and PPIA were validated as suitable reference genes by normalizing the target gene ADAM9 using these two most stably expressed genes in comparison with up- and down-regulated housekeeping genes of the panel. Conclusion: Our study demonstrated the suitability of the two housekeeping genes PPIA and TBP as endogenous reference genes when comparing malignant tissue samples with adjacent normal tissue samples from clear cell renal cell carcinoma. Both genes are recommended as reference genes for relative gene quantification in gene profiling studies either as single gene or preferably in combination. http://www.biomedcentral.com/1471-2199/8/47 Monika Jung, Azizbek Ramankulov, Jan Roigas, Manfred Johannsen, Martin Ringsdorf, Glen Kristiansen and Klaus Jung BMC Molecular Biology 2007, 8:47 Number of accesses: 630 2007-06-08 doi:10.1186/1471-2199-8-47 BMC Molecular Biology 1471-2199 8 47 2007-06-08 Background: The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. Results: Here, we present a USER Friendly cloning based technique that allows simultaneous cloning of the two required homologous recombination sequences into different sites of a recipient vector in a single cloning step. The advantages include a simple experimental design, independence of restriction enzymes, speed and user convenience. The vectors are intented for Agrobacterium tumefaciens and protoplast based transformation technologies. The system has been tested by the construction of vectors for targeted replacement and overexpression of 17 genes in Fusarium graminearum. The results show that four fragment vectors can be constructed in a single cloning step with an average efficiency of 84 % for gene replacement and 80 % for targeted overexpression. Conclusion: The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in F. graminearum. http://www.biomedcentral.com/1471-2199/9/70 Rasmus J.N. Frandsen, Jens A. Andersson, Matilde B. Kristensen and Henriette Giese BMC Molecular Biology 2008, 9:70 Number of accesses: 544 2008-08-01 doi:10.1186/1471-2199-9-70 BMC Molecular Biology 1471-2199 9 70 2008-08-01 Background: Methyl-DNA binding proteins help to translate epigenetic information encoded by DNA methylation into covalent histone modifications. MBD2/3 is the only candidate gene in the Drosophila genome with extended homologies to mammalian MBD2 and MBD3 proteins, which represent a co-repressor and an integral component of the Nucleosome Remodelling and Deacetylase (NuRD) complex, respectively. An association of Drosophila MBD2/3 with the Drosophila NuRD complex has been suggested previously. We have now analyzed the molecular interactions between MBD2/3 and the NuRD complex in greater detail. Results: The two MBD2/3 isoforms precisely cofractionated with NuRD proteins during gel filtration of extracts derived from early and late embryos. In addition, we demonstrate that MBD2/3 forms multimers, and engages in specific interactions with the p55 and MI-2 subunits of the Drosophila NuRD complex. Conclusion: Our data provide novel insights into the association between Drosophila MBD2/3 and NuRD proteins. Additionally, this work provides a first analysis of the architecture of the Drosophila NuRD complex. http://www.biomedcentral.com/1471-2199/5/20 Joachim Marhold, Alexander Brehm and Katja Kramer BMC Molecular Biology 2004, 5:20 Number of accesses: 432 2004-10-29 doi:10.1186/1471-2199-5-20 BMC Molecular Biology 1471-2199 5 20 2004-10-29 Background: RNA polymerase (pol) III transcription is specifically elevated in a variety of cancers and is a target of regulation by a variety of tumor suppressors and oncogenes. Accurate initiation by RNA pol III is dependent on TFIIIB. In higher eukaryotes, two forms of TFIIIB have been characterized. TFIIIB required for proper initiation from gene internal RNA pol III promoters is comprised of TBP, Bdp1, and Brf1. Proper initiation from gene external RNA pol III promoters requires TBP, Bdp1, and Brf2. We hypothesized that deregulation of RNA polymerase III transcription in cancer may be a consequence of altered TFIIIB expression Results: Here, we report: (1) the TFIIIB subunits Brf1 and Brf2 are differentially expressed in a variety of cancer cell lines: (2) the Brf1 and Brf2 promoters differ in activity in cancer cell lines, and (3) VAI transcription is universally elevated, as compared to U6, in breast, prostate and cervical cancer cells. Conclusions: Deregulation of TFIIIB-mediated transcription may be an important step in tumor development. We demonstrate that Brf1 and Brf2 mRNA are differentially expressed in a variety of cancer cells and that the Brf2 promoter is more active than the Brf1 promoter in all cell lines tested. We also demonstrate, that Brf1-dependent VAI transcription was significantly higher than the Brf2-dependent U6 snRNA transcription in all cancer cell lines tested. The data presented suggest that Brf2 protein expression levels correlate with U6 promoter activity in the breast, cervical and prostate cell lines tested. Interestingly, the Brf1 protein levels did not vary considerably in HeLa, MCF-7 and DU-145 cells, yet Brf1 mRNA expression varied considerably in breast, prostate and cervical cancer cell lines tested. Thus, Brf1 promoter activity and Brf1 protein expression levels did not correlate well with Brf1-dependent transcription levels. Taken together, we reason that deregulation of Brf1 and Brf2 expression could be a key mechanism responsible for the observed deregulation of RNA pol III transcription in cancer cells. http://www.biomedcentral.com/1471-2199/9/74 Stephanie Cabarcas, Joby Jacob, Ingrid Veras and Laura Schramm BMC Molecular Biology 2008, 9:74 Number of accesses: 417 2008-08-12 doi:10.1186/1471-2199-9-74 BMC Molecular Biology 1471-2199 9 74 2008-08-12 Background: The integrity of RNA molecules is of paramount importance for experiments that try to reflect the snapshot of gene expression at the moment of RNA extraction. Until recently, there has been no reliable standard for estimating the integrity of RNA samples and the ratio of 28S:18S ribosomal RNA, the common measure for this purpose, has been shown to be inconsistent. The advent of microcapillary electrophoretic RNA separation provides the basis for an automated high-throughput approach, in order to estimate the integrity of RNA samples in an unambiguous way. Methods: A method is introduced that automatically selects features from signal measurements and constructs regression models based on a Bayesian learning technique. Feature spaces of different dimensionality are compared in the Bayesian framework, which allows selecting a final feature combination corresponding to models with high posterior probability. Results: This approach is applied to a large collection of electrophoretic RNA measurements recorded with an Agilent 2100 bioanalyzer to extract an algorithm that describes RNA integrity. The resulting algorithm is a user-independent, automated and reliable procedure for standardization of RNA quality control that allows the calculation of an RNA integrity number (RIN). Conclusion: Our results show the importance of taking characteristics of several regions of the recorded electropherogram into account in order to get a robust and reliable prediction of RNA integrity, especially if compared to traditional methods. http://www.biomedcentral.com/1471-2199/7/3 Andreas Schroeder, Odilo Mueller, Susanne Stocker, Ruediger Salowsky, Michael Leiber, Marcus Gassmann, Samar Lightfoot, Wolfram Menzel, Martin Granzow and Thomas Ragg BMC Molecular Biology 2006, 7:3 Number of accesses: 404 2006-01-31 doi:10.1186/1471-2199-7-3 BMC Molecular Biology 1471-2199 7 3 2006-01-31 Background: Noncoding RNAs (ncRNAs) play important roles in a variety of cellular processes. Characterizing the transcriptional activity of ncRNA promoters is therefore a critical step toward understanding the complex cellular roles of ncRNAs. Results: Here we present an in vivo transcriptional analysis of three C. elegans ncRNA upstream motifs (UM1-3). Transcriptional activity of all three motifs has been demonstrated, and mutational analysis revealed differential contributions of different parts of each motif. We showed that upstream motif 1 (UM1) can drive the expression of green fluorescent protein (GFP), and utilized this for detailed analysis of temporal and spatial expression patterns of 5 SL2 RNAs. Upstream motifs 2 and 3 do not drive GFP expression, and termination at consecutive T runs suggests transcription by RNA polymerase III. The UM2 sequence resembles the tRNA promoter, and is actually embedded within its own short-lived, primary transcript. This is a structure which is also found at a few plant and yeast loci, and may indicate an evolutionarily very old dicistronic transcription pattern in which a tRNA serves as a promoter for an adjacent snoRNA. Conclusion: The study has demonstrated that the three upstream motifs UM1-3 have promoter activity. The UM1 sequence can drive expression of GFP, which allows for the use of UM1::GFP fusion constructs to study temporal-spatial expression patterns of UM1 ncRNA loci. The UM1 loci appear to act in concert with other upstream sequences, whereas the transcriptional activities of the UM2 and UM3 are confined to the motifs themselves. http://www.biomedcentral.com/1471-2199/9/71 Tiantian Li, Housheng He, Yunfei Wang, Haixia Zheng, Geir Skogerbø and Runsheng Chen BMC Molecular Biology 2008, 9:71 Number of accesses: 390 2008-08-05 doi:10.1186/1471-2199-9-71 BMC Molecular Biology 1471-2199 9 71 2008-08-05 Background: Circadian oscillation of clock-controlled gene expression is mainly regulated at the transcriptional level. Heterodimers of CLOCK and BMAL1 act as activators of target gene transcription; however, interactions of PER and CRY proteins with the heterodimer abolish its transcriptional activation capacity. PER and CRY are therefore referred to as negative regulators of the circadian clock. To further elucidate the mechanism how positive and negative components of the clock interplay, we characterized the interactions of PER2, CRY1 and CRY2 with BMAL1 and CLOCK using a mammalian two-hybrid system and co-immunoprecipitation assays. Results: Both PER2 and the CRY proteins were found to interact with BMAL1 whereas only PER2 interacts with CLOCK. CRY proteins seem to have a higher affinity to BMAL1 than PER2. Moreover, we provide evidence that PER2, CRY1 and CRY2 bind to different domains in the BMAL1 protein. Conclusion: The regulators of clock-controlled transcription PER2, CRY1 and CRY2 differ in their capacity to interact with each single component of the BMAL1-CLOCK heterodimer and, in the case of BMAL1, also in their interaction sites. Our data supports the hypothesis that CRY proteins, especially CRY1, are stronger repressors than PER proteins. http://www.biomedcentral.com/1471-2199/9/41 Sonja Langmesser, Tiziano Tallone, Alain Bordon, Sandro Rusconi and Urs Albrecht BMC Molecular Biology 2008, 9:41 Number of accesses: 357 2008-04-22 doi:10.1186/1471-2199-9-41 BMC Molecular Biology 1471-2199 9 41 2008-04-22 Background: The piggyBac transposable element is a popular tool for germ-line transgenesis of eukaryotes. Despite this, little is known about the mechanism of transposition or the transposase itself. A thorough understanding of just how piggyBac works may lead to more effective use of this important mobile element. A PSORTII analysis of the transposase amino acid sequence predicts a bipartite nuclear localization signal (NLS) near the c-terminus, just upstream of a putative zinc finger. Results: We fused the piggyBac transposase upstream of and in-frame with the enhanced yellow fluorescent protein (EYFP) in the Drosophila melanogaster inducible metallothionein protein. Using Drosophila Schneider 2 (S2) cells and the deep red fluorescent nuclear stain Draq5, we were able to track the pattern of piggyBac localization with a scanning confocal microscope 48 hours after induction with copper sulfate. Conclusions: Through n and c-terminal truncations, targeted internal deletions, and specific amino acid mutations of the piggyBac transposase open reading frame, we found that not only is the PSORTII predicted NLS required for the transposase to enter the nucleus of S2 cells, but there are additional requirements for negatively charged amino acids a short length upstream of this signal for proper function of the NLS. http://www.biomedcentral.com/1471-2199/9/72 James H Keith, Tresa S Fraser and Malcolm J Fraser BMC Molecular Biology 2008, 9:72 Number of accesses: 287 2008-08-11 doi:10.1186/1471-2199-9-72 BMC Molecular Biology 1471-2199 9 72 2008-08-11