BMC Molecular Biology[JOURNAL]
Sun
200 papers
Lowering the quantification limit of the QubitTM RNA HS assay using RNA spike-in.
Li X, Ben-Dov IZ, Mauro M … +1 more , Williams Z
BMC Mol Biol · 2015 May · PMID 25943882 · Full text
BACKGROUND: RNA quantification is often a prerequisite for most RNA analyses such as RNA sequencing. However, the relatively low sensitivity and large sample consumption of traditional RNA quantification methods such as...
BACKGROUND: RNA quantification is often a prerequisite for most RNA analyses such as RNA sequencing. However, the relatively low sensitivity and large sample consumption of traditional RNA quantification methods such as UV spectrophotometry and even the much more sensitive fluorescence-based RNA quantification assays, such as the Qubit™ RNA HS Assay, are often inadequate for measuring minute levels of RNA isolated from limited cell and tissue samples and biofluids. Thus, there is a pressing need for a more sensitive method to reliably and robustly detect trace levels of RNA without interference from DNA. METHODS: To improve the quantification limit of the Qubit™ RNA HS Assay, we spiked-in a known quantity of RNA to achieve the minimum reading required by the assay. Samples containing trace amounts of RNA were then added to the spike-in and measured as a reading increase over RNA spike-in baseline. We determined the accuracy and precision of reading increases between 1 and 20 pg/μL as well as RNA-specificity in this range, and compared to those of RiboGreen(®), another sensitive fluorescence-based RNA quantification assay. We then applied Qubit™ Assay with RNA spike-in to quantify plasma RNA samples. RESULTS: RNA spike-in improved the quantification limit of the Qubit™ RNA HS Assay 5-fold, from 25 pg/μL down to 5 pg/μL while maintaining high specificity to RNA. This enabled quantification of RNA with original concentration as low as 55.6 pg/μL compared to 250 pg/μL for the standard assay and decreased sample consumption from 5 to 1 ng. Plasma RNA samples that were not measurable by the Qubit™ RNA HS Assay were measurable by our modified method. CONCLUSIONS: The Qubit™ RNA HS Assay with RNA spike-in is able to quantify RNA with high specificity at 5-fold lower concentration and uses 5-fold less sample quantity than the standard Qubit™ Assay.
Nop17 is a key R2TP factor for the assembly and maturation of box C/D snoRNP complex.
Prieto MB, Georg RC, Gonzales-Zubiate FA … +2 more , Luz JS, Oliveira CC
BMC Mol Biol · 2015 Mar · PMID 25888478 · Full text
BACKGROUND: Box C/D snoRNPs are responsible for rRNA methylation and processing, and are formed by snoRNAs and four conserved proteins, Nop1, Nop56, Nop58 and Snu13. The snoRNP assembly is a stepwise process, involving o...
BACKGROUND: Box C/D snoRNPs are responsible for rRNA methylation and processing, and are formed by snoRNAs and four conserved proteins, Nop1, Nop56, Nop58 and Snu13. The snoRNP assembly is a stepwise process, involving other protein complexes, among which the R(2)TP and Hsp90 chaperone. Nop17, also known as Pih1, has been shown to be a constituent of the R(2)TP (Rvb1, Rvb2, Tah1, Pih1) and to participate in box C/D snoRNP assembly by its interaction with Nop58. The molecular function of Nop17, however, has not yet been described. RESULTS: To shed light on the role played by Nop17 in the maturation of snoRNP, here we analyzed the interactions domains of Nop58 - Nop17 - Tah1 and the importance of ATP to the interaction between Nop17 and the ATPase Rvb1/2. CONCLUSIONS: Based on the results shown here, we propose a model for the assembly of box C/D snoRNP, according to which R(2)TP complex is important for reducing the affinity of Nop58 for snoRNA, and for the binding of the other snoRNP subunits.
Wnt antagonist, secreted frizzled-related protein 1, is involved in prenatal skeletal muscle development and is a target of miRNA-1/206 in pigs.
Yang Y, Sun W, Wang R … +4 more , Lei C, Zhou R, Tang Z, Li K
BMC Mol Biol · 2015 Mar · PMID 25888412 · Full text
BACKGROUND: The Wnt signaling pathway is involved in the control of cell proliferation and differentiation during skeletal muscle development. Secreted frizzled-related proteins (SFRPs), such as SFRP1, function as inhibi...
BACKGROUND: The Wnt signaling pathway is involved in the control of cell proliferation and differentiation during skeletal muscle development. Secreted frizzled-related proteins (SFRPs), such as SFRP1, function as inhibitors of Wnt signaling. MicroRNA-1/206(miRNA-1/206) is specifically expressed in skeletal muscle and play a critical role in myogenesis. The miRNA-mRNA profiles and bioinformatics study suggested that the SFRP1 gene was potentially regulated by miRNA-1/206 during porcine skeletal muscle development. METHODS: To understand the function of SFRP1 and miRNA-1/206 in swine myogenesis, we first predicted the targets of miRNA-1/206 with the TargetScan and PicTar programs, and analyzed the molecular characterization of the porcine SFRP1 gene. We performed a temporal-spatial expression analysis of SFRP1 mRNA and miRNA-206 in Tongcheng pigs (a Chinese indigenous breed) by quantitative real-time polymerase chain reaction, and conducted the co-expression analyses of SFRP1 and miRNA-1/206. Subsequently, the interaction between SFRP1 and miRNA-1/206 was validated via dual luciferase and Western blot assays. RESULTS: The bioinformatics analysis predicted SFRP1 to be a target of miRNA-1/206. The expression level of the SFRP1 was highly varied across numerous pig tissues and it was down-regulated during porcine skeletal muscle development. The expression level of the SFRP1 was significantly higher in the embryonic skeletal compared with postnatal skeletal muscle, whereas miR-206 showed the inverse pattern of expression. A significant negative correlation was observed between the expression of miR-1/206 and SFRP1 during porcine skeletal muscle development (p <0.05). Dual luciferase assay and Western-blot results demonstrated that SFRP1 was a target of miR-1/206 in porcine iliac endothelial cells. CONCLUSIONS: Our results indicate that the SFRP1 gene is regulated by miR-1/206 and potentially affects skeletal muscle development. These findings increase understanding of the biological functions and the regulation of the SFRP1 gene in mammals.
Pre-amplification in the context of high-throughput qPCR gene expression experiment.
Korenková V, Scott J, Novosadová V … +5 more , Jindřichová M, Langerová L, Švec D, Šídová M, Sjöback R
BMC Mol Biol · 2015 Mar · PMID 25888347 · Full text
BACKGROUND: With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput...
BACKGROUND: With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-amplification reaction and propose the optimal, general setup for gene expression experiment using BioMark instrument (Fluidigm). RESULTS: For evaluating different pre-amplification factors following conditions were combined: four human blood samples from healthy donors and five transcripts having high to low expression levels; each cDNA sample was pre-amplified at four cycles (15, 18, 21, and 24) and five concentrations (equivalent to 0.078 ng, 0.32 ng, 1.25 ng, 5 ng, and 20 ng of total RNA). Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20 ng of total RNA with 15 cycles of pre-amplification, 20x and 40x diluted; and 5 ng and 20 ng of total RNA with 18 cycles of pre-amplification, both 20x and 40x diluted. CONCLUSIONS: We set up upper limits for the bulk gene expression experiment using gene expression Dynamic Array and provided an easy-to-obtain tool for measuring of pre-amplification success. We also showed that variability of the pre-amplification, introduced into the experimental workflow of reverse transcription-qPCR, is lower than variability caused by the reverse transcription step.
MKL1 inhibits cell cycle progression through p21 in podocytes.
Yang S, Liu L, Xu P … +1 more , Yang Z
BMC Mol Biol · 2015 Feb · PMID 25888165 · Full text
BACKGROUND: The glomerular podocyte is a highly specialized cell type with the ability to ultrafilter blood and support glomerular capillary pressure. However, little is known about the genetic programs leading to this f...
BACKGROUND: The glomerular podocyte is a highly specialized cell type with the ability to ultrafilter blood and support glomerular capillary pressure. However, little is known about the genetic programs leading to this functionality or the final phenotype. RESULTS: In the current study, we found that the expression of a myocardin/MKL family member, MKL1, was significantly upregulated during cell cycle arrest induced by a temperature switch in murine podocyte clone 5 (MPC5) cells. Further investigation demonstrated that overexpression of MKL1 led to inhibition of cell proliferation by decreasing the number of cells in S phase of the cell cycle. In contrast, MKL1 knockdown by RNA interference had the opposite effect, highlighting a potential role of MKL1 in blocking G1/S transition of the cell cycle in MPC5 cells. Additionally, using an RT(2) Profiler PCR Array, p21 was identified as a direct target of MKL1. We further revealed that MKL1 activated p21 transcription by recruitment to the CArG element in its promoter, thus resulting in cell cycle arrest. In addition, the expression of MKL1 is positively correlated with that of p21 in podocytes in postnatal mouse kidney and significantly upregulated during the morphological switch of podocytes from proliferation to differentiation. CONCLUSIONS: Our observations demonstrate that MKL1 has physiological roles in the maturation and development of podocytes, and thus its misregulation might lead to glomerular and renal dysfunction.
Fluorescence-based monitoring of ribosome assembly landscapes.
Nikolay R, Schloemer R, Mueller S … +1 more , Deuerling E
BMC Mol Biol · 2015 Feb · PMID 25884162 · Full text
BACKGROUND: Ribosomes and functional complexes of them have been analyzed at the atomic level. Far less is known about the dynamic assembly and degradation events that define the half-life of ribosomes and guarantee thei...
BACKGROUND: Ribosomes and functional complexes of them have been analyzed at the atomic level. Far less is known about the dynamic assembly and degradation events that define the half-life of ribosomes and guarantee their quality control. RESULTS: We developed a system that allows visualization of intact ribosomal subunits and assembly intermediates (i.e. assembly landscapes) by convenient fluorescence-based analysis. To this end, we labeled the early assembly ribosomal proteins L1 and S15 with the fluorescent proteins mAzami green and mCherry, respectively, using chromosomal gene insertion. The reporter strain harbors fluorescently labeled ribosomal subunits that operate wild type-like, as shown by biochemical and growth assays. Using genetic and chemical perturbations by depleting genes encoding the ribosomal proteins L3 and S17, respectively, or using ribosome-targeting antibiotics, we provoked ribosomal subunit assembly defects. These defects were readily identified by fluorometric analysis after sucrose density centrifugation in unprecedented resolution. CONCLUSION: This strategy is useful to monitor and characterize subunit specific assembly defects caused by ribosome-targeting drugs that are currently used and to characterize new molecules that affect ribosome assembly and thereby constitute new classes of antibacterial agents.
An Rb1-dependent amplification loop between Ets1 and Zeb1 is evident in thymocyte differentiation and invasive lung adenocarcinoma.
Dean KC, Huang L, Chen Y … +2 more , Lu X, Liu Y
BMC Mol Biol · 2015 Mar · PMID 25880398 · Full text
BACKGROUND: Ras pathway mutation leads to induction and Erk phosphorylation and activation of the Ets1 transcription factor. Ets1 in turn induces cyclin E and cyclin dependent kinase (cdk) 2 to drive cell cycle progressi...
BACKGROUND: Ras pathway mutation leads to induction and Erk phosphorylation and activation of the Ets1 transcription factor. Ets1 in turn induces cyclin E and cyclin dependent kinase (cdk) 2 to drive cell cycle progression. Ets1 also induces expression of the epithelial-mesenchymal transition (EMT) transcription factor Zeb1, and thereby links Ras mutation to EMT, which is thought to drive tumor invasion. Ras pathway mutations are detected by the Rb1 tumor suppression pathway, and mutation or inactivation of the Rb1 pathway is required for EMT. RESULTS: We examined linkage between Rb1, Ets1 and Zeb1. We found that an Rb1-E2F complex binds the Ets1 promoter and constitutively limits Ets1 expression. But, Rb1 repression of Zeb1 provides the major impact of Rb1 on Ets1 expression. We link Rb1 repression of Zeb1 to induction of miR-200 family members, which in turn target Ets1 mRNA. These findings suggest that Ets1 and Zeb1 comprise an amplification loop that is dependent upon miR-200 and regulated by Rb1. Thus, induction of Ets1 when the Rb1 pathway is lost may contribute to deregulated cell cycle progression through Ets1 induction of cyclin E and cdk2. Consistent with such an amplification loop, we correlate expression of Ets1 and Zeb1 in mouse and human lung adenocarcinoma. In addition we demonstrate that Ets1 expression in thymocytes is also dependent upon Zeb1. CONCLUSIONS: Taken together, our results provide evidence of an Rb1-dependent Ets1-Zeb1 amplification loop in thymocyte differentiation and tumor invasion.
Mechanical stimulation of human tendon stem/progenitor cells results in upregulation of matrix proteins, integrins and MMPs, and activation of p38 and ERK1/2 kinases.
Popov C, Burggraf M, Kreja L … +3 more , Ignatius A, Schieker M, Docheva D
BMC Mol Biol · 2015 Mar · PMID 25880261 · Full text
BACKGROUND: Tendons are dense connective tissues subjected periodically to mechanical stress upon which complex responsive mechanisms are activated. These mechanisms affect not only the development of these tissues but a...
BACKGROUND: Tendons are dense connective tissues subjected periodically to mechanical stress upon which complex responsive mechanisms are activated. These mechanisms affect not only the development of these tissues but also their healing. Despite of the acknowledged importance of the mechanical stress for tendon function and repair, the mechanotransduction mechanisms in tendon cells are still unclear and the elucidation of these mechanisms is a key goal in tendon research. Tendon stem/progenitor cells (TSPC) possess common adult stem cell characteristics, and are suggested to actively participate in tendon development, tissue homeostasis as well as repair. This makes them an important cell population for tendon repair, and also an interesting research target for various open questions in tendon cell biology. Therefore, in our study we focused on TSPC, subjected them to five different mechanical protocols, and investigated the gene expression changes by using semi-quantitative, quantitative PCR and western blotting technologies. RESULTS: Among the 25 different genes analyzed, we can convincingly report that the tendon-related genes - fibromodulin, lumican and versican, the collagen I-binding integrins - α1, α2 and α11, the matrix metalloproteinases - MMP9, 13 and 14 were strongly upregulated in TSPC after 3 days of mechanical stimulation with 8% amplitude. Molecular signaling analyses of five key integrin downstream kinases suggested that mechanical stimuli are mediated through ERK1/2 and p38, which were significantly activated in 8% biaxial-loaded TSPC. CONCLUSIONS: Our results demonstrate the positive effect of 8% mechanical loading on the gene expression of matrix proteins, integrins and matrix metalloproteinases, and activation of integrin downstream kinases p38 and ERK1/2 in TSPC. Taken together, our study contributes to better understanding of mechanotransduction mechanisms in TPSC, which in long term, after further translational research between tendon cell biology and orthopedics, can be beneficial to the management of tendon repair.
Efficient 5'-3' DNA end resection by HerA and NurA is essential for cell viability in the crenarchaeon Sulfolobus islandicus.
Huang Q, Liu L, Liu J … +3 more , Ni J, She Q, Shen Y
BMC Mol Biol · 2015 Feb · PMID 25880130 · Full text
BACKGROUND: ATPase/Helicases and nucleases play important roles in homologous recombination repair (HRR). Many of the mechanistic details relating to these enzymes and their function in this fundamental and complicated D...
BACKGROUND: ATPase/Helicases and nucleases play important roles in homologous recombination repair (HRR). Many of the mechanistic details relating to these enzymes and their function in this fundamental and complicated DNA repair process remain poorly understood in archaea. Here we employed Sulfolobus islandicus, a hyperthermophilic archaeon, as a model to investigate the in vivo functions of the ATPase/helicase HerA, the nuclease NurA, and their associated proteins Mre11 and Rad50. RESULTS: We revealed that each of the four genes in the same operon, mre11, rad50, herA, and nurA, are essential for cell viability by a mutant propagation assay. A genetic complementation assay with mutant proteins was combined with biochemical characterization demonstrating that the ATPase activity of HerA, the interaction between HerA and NurA, and the efficient 5'-3' DNA end resection activity of the HerA-NurA complex are essential for cell viability. NurA and two other putative HRR proteins: a PIN (PilT N-terminal)-domain containing ATPase and the Holliday junction resolvase Hjc, were co-purified with a chromosomally encoded N-His-HerA in vivo. The interactions of HerA with the ATPase and Hjc were further confirmed by in vitro pull down. CONCLUSION: Efficient 5'-3' DNA end resection activity of the HerA-NurA complex contributes to necessity of HerA and NurA in Sulfolobus, which is crucial to yield a 3'-overhang in HRR. HerA may have additional binding partners in cells besides NurA.
Molecular characterization of the piggyBac-like element, a candidate marker for phylogenetic research of Chilo suppressalis (Walker) in China.
Luo GH, Li XH, Han ZJ … +7 more , Guo HF, Yang Q, Wu M, Zhang ZC, Liu BS, Qian L, Fang JC
BMC Mol Biol · 2014 Dec · PMID 25515331 · Full text
BACKGROUND: Transposable elements (TEs, transposons) are mobile genetic DNA sequences. TEs can insert copies of themselves into new genomic locations and they have the capacity to multiply. Therefore, TEs have been cruci...
BACKGROUND: Transposable elements (TEs, transposons) are mobile genetic DNA sequences. TEs can insert copies of themselves into new genomic locations and they have the capacity to multiply. Therefore, TEs have been crucial in the shaping of hosts' current genomes. TEs can be utilized as genetic markers to study population genetic diversity. The rice stem borer Chilo suppressalis Walker is one of the most important insect pests of many subtropical and tropical paddy fields. This insect occurs in all the rice-growing areas in China. This research was carried out in order to find diversity between C. suppressalis field populations and detect the original settlement of C. suppressalis populations based on the piggyBac-like element (PLE). We also aim to provide insights into the evolution of PLEs in C. suppressalis and the phylogeography of C. suppressalis. RESULTS: Here we identify a new piggyBac-like element (PLE) in the rice stem borer Chilo suppressalis Walker, which is called CsuPLE1.1 (GenBank accession no. JX294476). CsuPLE1.1 is transcriptionally active. Additionally, the CsuPLE1.1 sequence varied slightly between field populations, with polymorphic indels (insertion/deletion) and hyper-variable regions including the identification of the 3' region outside the open reading frame (ORF). CsuPLE1.1 insertion frequency varied between field populations. Sequences variation was found between CsuPLE1 copies and varied within and among field populations. Twenty-one different insertion sites for CsuPLE1 copies were identified with at least two insertion loci found in all populations. CONCLUSIONS: Our results indicate that the initial invasion of CsuPLE1 into C. suppressalis occurred before C. suppressalis populations spread throughout China, and suggest that C. suppressalis populations have a common ancestor in China. Additionally, the lower reaches of the Yangtze River are probably the original settlement of C. suppressalis in China. Finally, the CsuPLE1 insertion site appears to be a candidate marker for phylogenetic research of C. suppressalis.
Néstor-Guillermo Progeria Syndrome: a biochemical insight into Barrier-to-Autointegration Factor 1, alanine 12 threonine mutation.
Paquet N, Box JK, Ashton NW … +7 more , Suraweera A, Croft LV, Urquhart AJ, Bolderson E, Zhang SD, O'Byrne KJ, Richard DJ
BMC Mol Biol · 2014 Dec · PMID 25495845 · Full text
BACKGROUND: Premature aging syndromes recapitulate many aspects of natural aging and provide an insight into this phenomenon at a molecular and cellular level. The progeria syndromes appear to cause rapid aging through d...
BACKGROUND: Premature aging syndromes recapitulate many aspects of natural aging and provide an insight into this phenomenon at a molecular and cellular level. The progeria syndromes appear to cause rapid aging through disruption of normal nuclear structure. Recently, a coding mutation (c.34G > A [p.A12T]) in the Barrier to Autointegration Factor 1 (BANF1) gene was identified as the genetic basis of Néstor-Guillermo Progeria syndrome (NGPS). This mutation was described to cause instability in the BANF1 protein, causing a disruption of the nuclear envelope structure. RESULTS: Here we demonstrate that the BANF1 A12T protein is indeed correctly folded, stable and that the observed phenotype, is likely due to the disruption of the DNA binding surface of the A12T mutant. We demonstrate, using biochemical assays, that the BANF1 A12T protein is impaired in its ability to bind DNA while its interaction with nuclear envelope proteins is unperturbed. Consistent with this, we demonstrate that ectopic expression of the mutant protein induces the NGPS cellular phenotype, while the protein localizes normally to the nuclear envelope. CONCLUSIONS: Our study clarifies the role of the A12T mutation in NGPS patients, which will be of importance for understanding the development of the disease.
Efficient isolation of specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous DNA-binding proteins.
Fujita T, Fujii H
BMC Mol Biol · 2014 Nov · PMID 25428274 · Full text
BACKGROUND: Comprehensive understanding of mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. We previously developed the insertional chromatin immun...
BACKGROUND: Comprehensive understanding of mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. We previously developed the insertional chromatin immunoprecipitation (iChIP) technology to isolate specific genomic regions retaining molecular interactions and identify their associated molecules. iChIP consists of locus-tagging and affinity purification. The recognition sequences of an exogenous DNA-binding protein such as LexA are inserted into a genomic region of interest in the cell to be analyzed. The exogenous DNA-binding protein fused with a tag(s) is expressed in the cell and the target genomic region is purified with antibody against the tag(s). In this study, we developed the iChIP system using recombinant DNA-binding proteins to make iChIP more straightforward than the conventional iChIP system using expression of the exogenous DNA-binding proteins in the cells to be analyzed. RESULTS: In this system, recombinant 3xFNLDD-D (r3xFNLDD-D) consisting of the 3xFLAG-tag, a nuclear localization signal (NLS), the DNA-binding domain plus the dimerization domain of the LexA protein, and the Dock-tag is used for isolation of specific genomic regions. r3xFNLDD-D was expressed using a silkworm-baculovirus expression system and purified by affinity purification. iChIP using r3xFNLDD-D could efficiently isolate the single-copy chicken Pax5 (cPax5) locus, in which LexA binding elements were inserted, with negligible contamination of other genomic regions. In addition, we could detect RNA associated with the cPax5 locus using this form of the iChIP system combined with RT-PCR. CONCLUSIONS: The iChIP system using r3xFNLDD-D can isolate specific genomic regions retaining molecular interactions without expression of the exogenous DNA-binding protein in the cell to be analyzed. iChIP using r3xFNLDD-D would be more straightforward and useful for analysis of specific genomic regions to elucidate their functions as compared to the previously published iChIP protocol.
Downregulation of microRNA-100 protects apoptosis and promotes neuronal growth in retinal ganglion cells.
Kong N, Lu X, Li B
BMC Mol Biol · 2014 Nov · PMID 25406880 · Full text
BACKGROUND: Retinal ganglion cells (RGCs) are preferentially lost in glaucoma or optic neuritis. In the present study, we investigated the protective effect of mircoRNA 100 (miR-100) against oxidative stress induced apop...
BACKGROUND: Retinal ganglion cells (RGCs) are preferentially lost in glaucoma or optic neuritis. In the present study, we investigated the protective effect of mircoRNA 100 (miR-100) against oxidative stress induced apoptosis in RGC-5 cells. RESULTS: Rat RGC-5 cells were cultured in plates and H2O2 was added to induce oxidative stress. TUNEL assay and qRT-PCR showed H2O2 induced apoptosis and up-regulated miR-100 in a dose-dependent manner in RGC-5 cells. Conversely, lentiviral-mediated miR-100 down-regulation protected H2O2 induced apoptosis, promoted neurite growth and activated AKT/ERK and TrkB pathways through phosphorylation. Luciferase assay confirmed that IGF1R was directly regulated by miR-100 in RGC-5 cells, and siRNA-mediated IGF1R knockdown activated AKT protein through phosphorylation, down-regulated miR-100, therefore exerted a protective effect on RGC-5 apoptosis. CONCLUSION: Down-regulating miR-100 is an effective method to protect H2O2 induced apoptosis in RGC-5 cells, possible associated with IGF1R regulation.
Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells.
Jacob F, Hitchins MP, Fedier A … +5 more , Brennan K, Nixdorf S, Hacker NF, Ward R, Heinzelmann-Schwarz VA
BMC Mol Biol · 2014 Oct · PMID 25294702 · Full text
BACKGROUND: The GBGT1 gene encodes the globoside alpha-1,3-N-acetylgalactosaminyltransferase 1. This enzyme catalyzes the last step in the multi-step biosynthesis of the Forssman (Fs) antigen, a pentaglycosyl ceramide of...
BACKGROUND: The GBGT1 gene encodes the globoside alpha-1,3-N-acetylgalactosaminyltransferase 1. This enzyme catalyzes the last step in the multi-step biosynthesis of the Forssman (Fs) antigen, a pentaglycosyl ceramide of the globo series glycosphingolipids. While differential GBGT1 mRNA expression has been observed in a variety of human tissues being highest in placenta and ovary, the expression of GBGT1 and the genes encoding the glycosyltransferases and glycosidases involved in the biosynthesis of Fs as well as the possible involvement of DNA methylation in transcriptional regulation of GBGT1 expression have not yet been investigated. RESULTS: RT-qPCR profiling showed high GBGT1 expression in normal ovary surface epithelial (HOSE) cell lines and low GBGT1 expression in all (e.g. A2780, SKOV3) except one (OVCAR3) investigated ovarian cancer cell lines, a finding that was confirmed by Western blot analysis. Hierarchical cluster analysis showed that GBGT1 was even the most variably expressed gene of Fs biosynthesis-relevant glycogenes and among the investigated cell lines, whereas NAGA which encodes the alpha-N-acetylgalactosaminidase hydrolyzing Fs was not differentially expressed. Bisulfite- and COBRA-analysis of the CpG island methylation status in the GBGT1 promoter region demonstrated high or intermediate levels of GBGT1 DNA methylation in all ovarian cancer cell lines (except for OVCAR3) but marginal levels of DNA methylation in the two HOSE cell lines. The extent of DNA methylation inversely correlated with GBGT1 mRNA and protein expression. Bioinformatic analysis of GBGT1 in The Cancer Genome Atlas ovarian cancer dataset demonstrated that this inverse correlation was also found in primary ovarian cancer tissue samples confirming our cell line-based findings. Restoration of GBGT1 mRNA and protein expression in low GBGT1-expressing A2780 cells was achieved by 5-aza-2'-deoxycytidine treatment and these treated cells exhibited increased helix pomatia agglutinin-staining, reflecting the elevated presence of Fs disaccharide on these cells. CONCLUSIONS: GBGT1 expression is epigenetically silenced through promoter hypermethylation in ovarian cancer. Our findings not only suggest an involvement of DNA methylation in the synthesis of Fs antigen but may also explain earlier studies showing differential GBGT1 expression in various human tissue samples and disease stages.
Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung.
Ohno M, Kida Y, Sakaguchi M … +2 more , Sugahara Y, Oyama F
BMC Mol Biol · 2014 Oct · PMID 25294623 · Full text
BACKGROUND: Mice and humans produce chitinase-like proteins (CLPs), which are highly homologous to chitinases but lack chitinolytic activity. Mice express primarily three CLPs, including breast regression protein-39 (BRP...
BACKGROUND: Mice and humans produce chitinase-like proteins (CLPs), which are highly homologous to chitinases but lack chitinolytic activity. Mice express primarily three CLPs, including breast regression protein-39 (BRP-39) [chitinase 3-like-1 (Chi3l1) or 38-kDa glycoprotein (gp38k)], Ym1 (Chi3l3) and Ym2 (Chi3l4). Recently, CLPs have attracted considerable attention due to their increased expression in a number of pathological conditions, including asthma, allergies, rheumatoid arthritis and malignant tumors. Although the exact functions of CLPs are largely unknown, the significance of their increased expression levels during pathophysiological states needs to be determined. The quantification of BRP-39, Ym1 and Ym2 is an important step in gaining insight into the in vivo regulation of the CLPs. METHODS: We constructed a standard DNA for quantitative real-time PCR (qPCR) by containing three CLPs target fragments and five reference genes cDNA in a one-to-one ratio. We evaluated this system by analyzing the eight target cDNA sequences. Tissue cDNAs obtained by reverse transcription from total RNA from four embryonic stages and eight adult tissues were analyzed using the qPCR system with the standard DNA. RESULTS: We established a qPCR system detecting CLPs and comparing their expression levels with those of five reference genes using the same scale in mouse tissues. We found that BRP-39 and Ym1 were abundant in the mouse lung, whereas Ym2 mRNA was abundant in the stomach, followed by lung. The expression levels of BRP-39 and Ym1 in the mouse lung were higher than those of two active chitinases and were comparable to glyceraldehyde-3-phosphate dehydrogenase, a housekeeping gene which is constitutively expressed in all tissues. CONCLUSION: Our results indicate that catalytically inactive BRP-39 and Ym1 are constitutive genes in normal mouse lung.
Chromatin structure is distinct between coding and non-coding single nucleotide polymorphisms.
Liu H, Zhai J, Luo K … +1 more , Liu L
BMC Mol Biol · 2014 Oct · PMID 25282079 · Full text
BACKGROUND: Previous studies suggested that nucleosomes are enriched with single nucleotide polymorphisms (SNPs) in humans and that the occurrence of mutations is closely associated with CpG dinucleotides. We aimed to de...
BACKGROUND: Previous studies suggested that nucleosomes are enriched with single nucleotide polymorphisms (SNPs) in humans and that the occurrence of mutations is closely associated with CpG dinucleotides. We aimed to determine if the chromatin organization is genomic locus specific around SNPs, and if newly occurring mutations are associated with SNPs. RESULTS: Here, we classified SNPs according their loci and investigated chromatin organization in both CD4+ T cell and lymphoblastoid cell in humans. We calculated the SNP frequency around somatic mutations. The results indicated that nucleosome occupancy is different around SNPs sites in different genomic loci. Coding SNPs are mainly enriched at nucleosomes and associated with repressed histone modifications (HMs) and DNA methylation. Contrastingly, intron SNPs occur in nucleosome-depleted regions and lack HMs. Interestingly, risk-associated non-coding SNPs are also enriched at nucleosomes with HMs but associated with low GC-content and low DNA methylation level. The base-transversion allele frequency is significantly low in coding-synonymous SNPs (P < 10⁻¹¹). Another finding is that at the -1 and +1 positions relative to the somatic mutation sites, the SNP frequency was significantly higher (P < 3.2 × 10⁻⁵). CONCLUSIONS: The results suggested chromatin structure is different around coding SNPs and non-coding SNPs. New mutations tend to occur at the -1 and +1 position immediately near the SNPs.
Pax8 modulates the expression of Wnt4 that is necessary for the maintenance of the epithelial phenotype of thyroid cells.
Filippone MG, Di Palma T, Lucci V … +1 more , Zannini M
BMC Mol Biol · 2014 Sep · PMID 25270402 · Full text
BACKGROUND: The transcription factor Pax8 is expressed during thyroid development and is involved in the morphogenesis of the thyroid gland and maintenance of the differentiated phenotype. In particular, Pax8 has been sh...
BACKGROUND: The transcription factor Pax8 is expressed during thyroid development and is involved in the morphogenesis of the thyroid gland and maintenance of the differentiated phenotype. In particular, Pax8 has been shown to regulate genes that are considered markers of thyroid differentiation. Recently, the analysis of the gene expression profile of FRTL-5 differentiated thyroid cells after the silencing of Pax8 identified Wnt4 as a novel target. Like the other members of the Wnt family, Wnt4 has been implicated in several developmental processes including regulation of cell fate and patterning during embryogenesis. To date, the only evidence on Wnt4 in thyroid concerns its down-regulation necessary for the progression of thyroid epithelial tumors. RESULTS: Here we demonstrate that Pax8 is involved in the transcriptional modulation of Wnt4 gene expression directly binding to its 5'-flanking region, and that Wnt4 expression in FRTL-5 cells is TSH-dependent. Interestingly, we also show that in thyroid cells a reduced expression of Wnt4 correlates with the alteration of the epithelial phenotype and that the overexpression of Wnt4 in thyroid cancer cells is able to inhibit cellular migration. CONCLUSIONS: We have identified and characterized a functional Pax8 binding site in the 5'-flanking region of the Wnt4 gene and we show that Pax8 modulates the expression of Wnt4 in thyroid cells. Taken together, our results suggest that in thyroid cells Wnt4 expression correlates with the integrity of the epithelial phenotype and is reduced when this integrity is perturbed. In the end, we would like to suggest that the overexpression of Wnt4 in thyroid cancer cells is able to revert the mesenchymal phenotype.
Wingless gene cloning and its role in manipulating the wing dimorphism in the white-backed planthopper, Sogatella furcifera.
Yu JL, An ZF, Liu XD
BMC Mol Biol · 2014 Sep · PMID 25266639 · Full text
BACKGROUND: Wingless gene (Wg) plays a fundamental role in regulating the segment polarity and wing imaginal discs of insects. The rice planthoppers have an obvious wing dimorphism, and the long- and short-winged forms e...
BACKGROUND: Wingless gene (Wg) plays a fundamental role in regulating the segment polarity and wing imaginal discs of insects. The rice planthoppers have an obvious wing dimorphism, and the long- and short-winged forms exist normally in natural populations. However, the molecular characteristics and functions of Wg in rice planthoppers are poorly understood, and the relationship between expression level of Wg and wing dimorphism has not been clarified. RESULTS: In this study, wingless gene (Wg) was cloned from three species of rice planthopper, Sogatella furcifera, Laodelphgax striatellus and Nilaparvata lugens, and its characteristics and role in determining the wing dimorphism of S. furcifera were explored. The results showed that only three different amino acid residuals encoded by Wg were found between S. furcifera and L. striatellus, but more than 10 residuals in N. lugens were different with L. striatellus and S. furcifera. The sequences of amino acids encoded by Wg showed a high degree of identity between these three species of rice planthopper that belong to the same family, Delphacidae. The macropterous and brachypterous lineages of S. furcifera were established by selection experiment. The Wg mRNA expression levels in nymphs were significantly higher in the macropterous lineage than in the brachypterous lineage of S. furcifera. In macropterous adults, the Wg was expressed mainly in wings and legs, and less in body segments. Ingestion of 100 ng/μL double-stranded RNA of Wg from second instar nymphs led to a significant decrease of expression level of Wg during nymphal stage and of body weight of subsequent adults. Moreover, RNAi of Wg resulted in significantly shorter and deformative wings, including shrunken and unfolded wings. CONCLUSION: Wg has high degree of identity among three species of rice planthopper. Wg is involved in the development and growth of wings in S. furcifera. Expression level of Wg during the nymphal stage manipulates the size and pattern of wings in S. furcifera.
Requirements for resuming translation in chimeric transfer-messenger RNAs of Escherichia coli and Mycobacterium tuberculosis.
Wower IK, Zwieb C, Wower J
BMC Mol Biol · 2014 Sep · PMID 25220282 · Full text
BACKGROUND: Trans-translation is catalyzed by ribonucleprotein complexes composed of SmpB protein and transfer-messenger RNA. They release stalled ribosomes from truncated mRNAs and tag defective proteins for proteolytic...
BACKGROUND: Trans-translation is catalyzed by ribonucleprotein complexes composed of SmpB protein and transfer-messenger RNA. They release stalled ribosomes from truncated mRNAs and tag defective proteins for proteolytic degradation. Comparative sequence analysis of bacterial tmRNAs provides considerable insights into their secondary structures in which a tRNA-like domain and an mRNA-like region are connected by a variable number of pseudoknots. Progress toward understanding the molecular mechanism of trans-translation is hampered by our limited knowledge about the structure of tmRNA:SmpB complexes. RESULTS: Complexes consisting of M. tuberculosis tmRNA and E. coli SmpB tag truncated proteins poorly in E. coli. In contrast, the tagging activity of E. coli tmRNA is well supported by M. tuberculosis SmpB that is expressed in E. coli. To investigate this incompatibility, we constructed 12 chimeric tmRNA molecules composed of structural features derived from both E. coli and M. tuberculosis. Our studies demonstrate that replacing the hp5-pk2-pk3-pk4 segment of E. coli tmRNA with the equivalent segment of M. tuberculosis tmRNA has no significant effect on the tagging efficiency of chimeric tmRNAs in the presence of E. coli SmpB. Replacing either helices 2b-2d, the single-stranded part of the ORF, pk1, or residues 79-89 of E. coli tmRNA with the equivalent features of M. tuberculosis tmRNA yields chimeric tmRNAs that are tagged at 68 to 88 percent of what is observed with E. coli tmRNA. Exchanging segments composed of either pk1 and the single-stranded segment upstream of the ORF or helices 2b-2d and pk1 results in markedly impaired tagging activity. CONCLUSION: Our observations demonstrate the existence of functionally important but as yet uncharacterized structural constraints in the segment of tmRNA that connects its TLD to the ORF used for resuming translation. As trans-translation is important for the survival of M. tuberculosis, our work provides a new target for pharmacological intervention against multidrug-resistant tuberculosis.
Insight into the cellular involvement of the two reverse gyrases from the hyperthermophilic archaeon Sulfolobus solfataricus.
Couturier M, Bizard AH, Garnier F … +1 more , Nadal M
BMC Mol Biol · 2014 Sep · PMID 25200003 · Full text
BACKGROUND: Reverse gyrases are DNA topoisomerases characterized by their unique DNA positive-supercoiling activity. Sulfolobus solfataricus, like most Crenarchaeota, contains two genes each encoding a reverse gyrase. We...
BACKGROUND: Reverse gyrases are DNA topoisomerases characterized by their unique DNA positive-supercoiling activity. Sulfolobus solfataricus, like most Crenarchaeota, contains two genes each encoding a reverse gyrase. We showed previously that the two genes are differently regulated according to temperature and that the corresponding purified recombinant reverse gyrases have different enzymatic characteristics. These observations suggest a specialization of functions of the two reverse gyrases. As no mutants of the TopR genes could be obtained in Sulfolobales, we used immunodetection techniques to study the function(s) of these proteins in S. solfataricus in vivo. In particular, we investigated whether one or both reverse gyrases are required for the hyperthermophilic lifestyle. RESULTS: For the first time the two reverse gyrases of S. solfataricus have been discriminated at the protein level and their respective amounts have been determined in vivo. Actively dividing S. solfataricus cells contain only small amounts of both reverse gyrases, approximately 50 TopR1 and 125 TopR2 molecules per cell at 80°C. S. solfataricus cells are resistant at 45°C for several weeks, but there is neither cell division nor replication initiation; these processes are fully restored upon a return to 80°C. TopR1 is not found after three weeks at 45°C whereas the amount of TopR2 remains constant. Enzymatic assays in vitro indicate that TopR1 is not active at 45°C but that TopR2 exhibits highly positive DNA supercoiling activity at 45°C. CONCLUSIONS: The two reverse gyrases of S. solfataricus are differently regulated, in terms of protein abundance, in vivo at 80°C and 45°C. TopR2 is present both at high and low temperatures and is therefore presumably required whether cells are dividing or not. By contrast, TopR1 is present only at high temperature where the cell division occurs, suggesting that TopR1 is required for controlling DNA topology associated with cell division activity and/or life at high temperature. Our findings in vitro that TopR1 is able to positively supercoil DNA only at high temperature, and TopR2 is active at both temperatures are consistent with them having different functions within the cells.
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