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1.  Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus) microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86 
BMC Molecular Biology  2009;10:112.
For accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. In most studies on ticks where (semi-)quantitative RT-PCR is employed, normalization occurs with a single reference gene, usually β-actin, without validation of its presumed expression stability. The first goal of this study was to evaluate the expression stability of commonly used reference genes in Rhipicephalus appendiculatus and Rhipicephalus (Boophilus) microplus ticks. To demonstrate the usefulness of these results, an unresolved issue in tick vaccine development was examined. Commercial vaccines against R. microplus were developed based on the recombinant antigen Bm86, but despite a high degree of sequence homology, these vaccines are not effective against R. appendiculatus. In fact, Bm86-based vaccines give better protection against some tick species with lower Bm86 sequence homology. One possible explanation is the variation in Bm86 expression levels between R. microplus and R. appendiculatus. The most stable reference genes were therefore used for normalization of the Bm86 expression profile in all life stages of both species to examine whether antigen abundance plays a role in Bm86 vaccine susceptibility.
The transcription levels of nine potential reference genes: β-actin (ACTB), β-tubulin (BTUB), elongation factor 1α (ELF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glutathione S-transferase (GST), H3 histone family 3A (H3F3A), cyclophilin (PPIA), ribosomal protein L4 (RPL4) and TATA box binding protein (TBP) were measured in all life stages of R. microplus and R. appendiculatus. ELF1A was found to be the most stable expressed gene in both species following analysis by both geNorm and Normfinder software applications, GST showed the least stability. The expression profile of Bm86 in R. appendiculatus and R. microplus revealed a more continuous Bm86 antigen abundance in R. microplus throughout its one-host life cycle compared to the three-host tick R. appendiculatus where large variations were observed between different life stages.
Based on these results, ELF1A can be proposed as an initial reference gene for normalization of quantitative RT-PCR data in whole R. microplus and R. appendiculatus ticks. The observed differences in Bm86 expression profile between the two species alone can not adequately explain the lack of a Bm86 vaccination effect in R. appendiculatus.
PMCID: PMC2809063  PMID: 20040102
2.  Selection of reference genes for expression studies with fish myogenic cell cultures 
BMC Molecular Biology  2009;10:80.
Relatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.). The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation.
Initial results showed several of the candidate genes exhibited stable expression throughout myogenic culture while Sdha was identified as the least stable gene. Further analysis with geNorm, Normfinder and Bestkeeper identified Ef1α, Hprt1, Ppia and RNApolII as stably expressed. Comparison of data normalised with the geometric average obtained from combinations of any three of these genes showed no significant differences, indicating that any combination of these genes is valid.
The geometric average of any three of Hprt1, Ef1α, Ppia and RNApolII is suitable for normalisation of gene expression data in primary myogenic cultures from Atlantic salmon.
PMCID: PMC2736950  PMID: 19664261
3.  Selective control of primer usage in multiplex one-step reverse transcription PCR 
BMC Molecular Biology  2009;10:113.
Multiplex RT-PCR is a valuable technique used for pathogen identification, disease detection and relative quantification of gene expression. The simplification of this protocol into a one-step procedure saves time and reagents. However, intensive PCR optimization is often required to overcome competing undesired PCR primer extension during the RT step.
Herein, we report multiplex one-step RT-PCR experiments in which the PCR primers contain thermolabile phosphotriester modification groups. The presence of these groups minimizes PCR primer extension during the RT step and allows for control of PCR primer extension until the more stringent, elevated temperatures of PCR are reached. Results reveal that the use of primers whose extension can be controlled in a temperature-mediated way provides improved one-step RT-PCR specificity in both singleplex and multiplex reaction formats.
The need for an accurate and sensitive technique to quantify mRNA expression levels makes the described modified primer technology a promising tool for use in multiplex one-step RT-PCR. A more accurate representation of the abundances in initial template sample is feasible with modified primers, as artifacts of biased PCR are reduced because of greater improvements in reaction specificity.
PMCID: PMC2811113  PMID: 20042116
4.  Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium 
BMC Molecular Biology  2009;10:111.
Whole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER), the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood.
We successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences) on the model Rickettsiales ER to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of ER's cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome ER microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from ER microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R2 = 0.7). Moreover, SCOTS method is crucial for microarrays analysis of ER, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively.
We conclude that this SCOTS method has a key importance for the transcriptomic analysis of ER and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both ER pathogenicity and the adaptation of obligate intracellular bacteria to their environment.
PMCID: PMC2806407  PMID: 20034374
5.  TLK1B promotes repair of DSBs via its interaction with Rad9 and Asf1 Caroline Canfield, Justin Rains, and Arrigo De Benedetti 
BMC Molecular Biology  2009;10:110.
The Tousled-like kinases are involved in chromatin assembly, DNA repair, transcription, and chromosome segregation. Previous evidence indicated that TLK1B can promote repair of plasmids with cohesive ends in vitro, but it was inferred that the mechanism was indirect and via chromatin assembly, mediated by its interaction with the chromatin assembly factor Asf1. We recently identified Rad9 as a substrate of TLK1B, and we presented evidence that the TLK1B-Rad9 interaction plays some role in DSB repair. Hence the relative contribution of Asf1 and Rad9 to the protective effect of TLK1B in DSBs repair is not known. Using an adeno-HO-mediated cleavage system in MM3MG cells, we previously showed that overexpression of either TLK1B or a kinase-dead protein (KD) promoted repair and the assembly of Rad9 in proximity of the DSB at early time points post-infection. This established that it is a chaperone activity of TLK1B and not directly the kinase activity that promotes recruitment of 9-1-1 to the DSB. However, the phosphorylation of Rad9(S328) by TLK1B appeared important for mediating a cell cycle checkpoint, and thus, this phosphorylation of Rad9 may have other effects on 9-1-1 functionality.
Here we present direct evidence that TLK1B can promote repair of linearized plasmids with incompatible ends that require processing prior to ligation. Immunodepletion of Rad9 indicated that Rad9 was important for processing the ends preceding ligation, suggesting that the interaction of TLK1B with Rad9 is a key mediator for this type of repair. Ligation of incompatible ends also required DNA-PK, as addition of wortmannin or immunodepletion of Ku70 abrogated ligation. Depletion of Ku70 prevented the ligation of the plasmid but did not affect stimulation of the fill-in of the ends by added TLK1B, which was attributed to Rad9. From experiments with the HO-cleavage system, we now show that Rad17, a subunit of the "clamp loader", associates normally with the DSB in KD-overexpressing cells. However, the subsequent release of Rad17 and Rad9 upon repair of the DSB was significantly slower in these cells compared to controls or cells expressing wt-TLK1B.
TLKs play important roles in DNA repair, not only by modulation of chromatin assembly via Asf1, but also by a more direct function in processing the ends of a DSB via interaction with Rad9. Inhibition of Rad9 phosphorylation in KD-overexpressing cells may have consequences in signaling completion of the repair and cell cycle re-entry, and could explain a loss of viability from DSBs in these cells.
PMCID: PMC2803485  PMID: 20021694
6.  Transpositionally active episomal hAT elements 
BMC Molecular Biology  2009;10:108.
hAT elements and V(D)J recombination may have evolved from a common ancestral transposable element system. Extrachromosomal, circular forms of transposable elements (referred to here as episomal forms) have been reported yet their biological significance remains unknown. V(D)J signal joints, which resemble episomal transposable elements, have been considered non-recombinogenic products of V(D)J recombination and a safe way to dispose of excised chromosomal sequences. V(D)J signal joints can, however, participate in recombination reactions and the purpose of this study was to determine if hobo and Hermes episomal elements are also recombinogenic.
Up to 50% of hobo/Hermes episomes contained two intact, inverted-terminal repeats and 86% of these contained from 1-1000 bp of intercalary DNA. Episomal hobo/Hermes elements were recovered from Musca domestica (a natural host of Hermes), Drosophila melanogaster (a natural host of hobo) and transgenic Drosophila melanogaster and Aedes aegypti (with autonomous Hermes elements). Episomal Hermes elements were recovered from unfertilized eggs of M. domestica and D. melanogaster demonstrating their potential for extrachromosomal, maternal transmission. Reintegration of episomal Hermes elements was observed in vitro and in vivo and the presence of Hermes episomes resulted in lower rates of canonical Hermes transposition in vivo.
Episomal hobo/Hermes elements are common products of element excision and can be maternally transmitted. Episomal forms of Hermes are capable of integration and also of influencing the transposition of canonical elements suggesting biological roles for these extrachromosomal elements in element transmission and regulation.
PMCID: PMC2803484  PMID: 20003420
7.  Nuclear factor I-A represses expression of the cell adhesion molecule L1 
BMC Molecular Biology  2009;10:107.
The neural cell adhesion molecule L1 plays a crucial role in development and plasticity of the nervous system. Neural cells thus require precise control of L1 expression.
We identified a full binding site for nuclear factor I (NFI) transcription factors in the regulatory region of the mouse L1 gene. Electrophoretic mobility shift assay (EMSA) showed binding of nuclear factor I-A (NFI-A) to this site. Moreover, for a brain-specific isoform of NFI-A (NFI-A bs), we confirmed the interaction in vivo using chromatin immunoprecipitation (ChIP). Reporter gene assays showed that in neuroblastoma cells, overexpression of NFI-A bs repressed L1 expression threefold.
Our findings suggest that NFI-A, in particular its brain-specific isoform, represses L1 gene expression, and might act as a second silencer of L1 in addition to the neural restrictive silencer factor (NRSF).
PMCID: PMC2805660  PMID: 20003413
8.  Characterization of the transcripts and protein isoforms for cytoplasmic polyadenylation element binding protein-3 (CPEB3) in the mouse retina 
BMC Molecular Biology  2009;10:109.
Cytoplasmic polyadenylation element binding proteins (CPEBs) regulate translation by binding to regulatory motifs of defined mRNA targets. This translational mechanism has been shown to play a critical role in oocyte maturation, early development, and memory formation in the hippocampus. Little is known about the presence or functions of CPEBs in the retina. The purpose of the current study is to investigate the alternative splicing isoforms of a particular CPEB, CPEB3, based on current databases, and to characterize the expression of CPEB3 in the retina.
In this study, we have characterized CPEB3, whose putative role is to regulate the translation of GluR2 mRNA. We identify the presence of multiple alternative splicing isoforms of CPEB3 transcripts and proteins in the current databases. We report the presence of eight alternative splicing patterns of CPEB3, including a novel one, in the mouse retina. All but one of the patterns appear to be ubiquitous in 13 types of tissue examined. The relative abundance of the patterns in the retina is demonstrated. Experimentally, we show that CPEB3 expression is increased in a time-dependent manner during the course of postnatal development, and CPEB3 is localized mostly in the inner retina, including retinal ganglion cells.
The level of CPEB3 was up-regulated in the retina during development. The presence of multiple CPEB3 isoforms indicates remarkable complexity in the regulation and function of CPEB3.
PMCID: PMC2807433  PMID: 20003455
9.  Quantitative TaqMan® real-time PCR assays for gene expression normalisation in feline tissues 
BMC Molecular Biology  2009;10:106.
Gene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. Co-analysis of suitable reference genes is crucial for accurate expression normalisation. Reference gene expression may vary, e.g., among species or tissues; thus, candidate genes must be tested prior to use in expression studies. The domestic cat is an important study subject in both medical research and veterinary medicine. The aim of the present study was to develop TaqMan® real-time PCR assays for eight potential reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from healthy cats, and neoplastic tissues from FeLV-infected cats.
RNA extraction from tissues was optimised for minimal genomic DNA (gDNA) contamination without use of a DNase treatment. Real-time PCR assays were established and optimised for v-abl Abelson murine leukaemia viral oncogene homolog (ABL), β-actin (ACTB), β-2-microglobulin (B2M), β-glucuronidase (GUSB), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein S7 (RPS7), and tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ). The presence of pseudogenes was confirmed for four of the eight investigated genes (ACTB, HPRT, RPS7, and YWHAZ). The assays were tested together with previously developed TaqMan® assays for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the universal 18S rRNA gene. Significant differences were found among the expression levels of the ten candidate reference genes, with a ~106-fold expression difference between the most abundant (18S rRNA) and the least abundant genes (ABL, GUSB, and HMBS). The expression stability determined by the geNorm and NormFinder programs differed significantly. Using the ANOVA-based NormFinder program, RPS7 was the most stable gene in the tissues studied, followed by ACTB and ABL; B2M, HPRT, and the 18S rRNA genes were the least stable ones.
The reference gene expression stability varied considerably among the feline tissues investigated. No tested gene was optimal for normalisation in all tissues. For the majority of the tissues, two to three reference genes were necessary for accurate normalisation. The present study yields essential information on the correct choice of feline reference genes depending on the tissues analysed.
PMCID: PMC2803789  PMID: 20003366
10.  Sequence-dependent DNA helical rise and nucleosome stability 
BMC Molecular Biology  2009;10:105.
Nucleosomes are the basic structural units of eukaryotic chromatin and play a key role in regulation of gene expression. After resolution of the nucleosome structure, the bipartite nature of this particle has revealed itself and has disclosed the presence, on the histone surface, of a symmetric distribution of positive charges, able to interact with their negative DNA phosphate counterpart.
We analyzed helical steps in known nucleosomal DNA sequences, observing a significant relationship between their symmetric distribution and nucleosome stability. Synthetic DNA sequences able to form stable nucleosomes were used to compare distances on the left and on the right side of the nucleosomal dyad axis, where DNA phosphates and charged residues of the (H3H4)2-tetramer interact. We observed a linear relationship between coincidence of distances and nucleosome stability, i. e., the more symmetric these distances the more stable the nucleosome.
Curves related to this symmetric distribution along the DNA sequence identify preferential sites for positioning of the dyad axis, which we termed palinstases. The comparison of our data with known nucleosome positions in archaeal and eukaryotic sequences shows many coincidences of location. Sequences that impair nucleosome formation and DNase I hypersensitive sites yield curves with a lower degree of symmetry. Analysis performed on DNA tracts of promoters close to the transcription start and termination sites identified peculiar patterns: in particular low affinity for nucleosome binding at the transcription start site and a high affinity exactly at the transcription termination site, suggesting a major role of nucleosomes in the termination of transcription.
PMCID: PMC2788551  PMID: 19943916
11.  Arsenic transport by zebrafish aquaglyceroporins 
BMC Molecular Biology  2009;10:104.
Arsenic is one of the most ubiquitous toxins and endangers the health of tens of millions of humans worldwide. It is a mainly a water-borne contaminant. Inorganic trivalent arsenic (AsIII) is one of the major species that exists environmentally. The transport of AsIII has been studied in microbes, plants and mammals. Members of the aquaglyceroporin family have been shown to actively conduct AsIII and its organic metabolite, monomethylarsenite (MAsIII). However, the transport of AsIII and MAsIII in in any fish species has not been characterized.
In this study, five members of the aquaglyceroporin family from zebrafish (Danio rerio) were cloned, and their ability to transport water, glycerol, and trivalent arsenicals (AsIII and MAsIII) and antimonite (SbIII) was investigated. Genes for at least seven aquaglyceroporins have been annotated in the zebrafish genome project. Here, five genes which are close homologues to human AQP3, AQP9 and AQP10 were cloned from a zebrafish cDNA preparation. These genes were named aqp3, aqp3l, aqp9a, aqp9b and aqp10 according to their similarities to the corresponding human AQPs. Expression of aqp9a, aqp9b, aqp3, aqp3l and aqp10 in multiple zebrafish organs were examined by RT-PCR. Our results demonstrated that these aquaglyceroporins exhibited different tissue expression. They are all detected in more than one tissue. The ability of these five aquaglyceroporins to transport water, glycerol and the metalloids arsenic and antimony was examined following expression in oocytes from Xenopus leavis. Each of these channels showed substantial glycerol transport at equivalent rates. These aquaglyceroporins also facilitate uptake of inorganic AsIII, MAsIII and SbIII. Arsenic accumulation in fish larvae and in different tissues from adult zebrafish was studied following short-term arsenic exposure. The results showed that liver is the major organ of arsenic accumulation; other tissues such as gill, eye, heart, intestine muscle and skin also exhibited significant ability to accumulate arsenic. The zebrafish larvae also accumulate considerable amounts of arsenic.
This is the first molecular identification of fish arsenite transport systems and we propose that the extensive expression of the fish aquaglyceroporins and their ability to transport metalloids suggests that aquaglyceroporins are the major pathways for arsenic accumulation in a variety of zebrafish tissues. Uptake is one important step of arsenic metabolism. Our results will contribute to a new understanding of aquatic arsenic metabolism and will support the use of zebrafish as a new model system to study arsenic associated human diseases.
PMCID: PMC2788550  PMID: 19939263
12.  Identification of an exonic splicing silencer in exon 6A of the human VEGF gene 
BMC Molecular Biology  2009;10:103.
The different isoforms of vascular endothelial growth factor (VEGF) play diverse roles in vascular growth, structure and function. Alternative splicing of the VEGF gene results in the expression of three abundant isoforms: VEGF121, VEGF165 and VEGF189. The mRNA for VEGF189 contains the alternatively spliced exon 6A whereas the mRNA for VEGF165 lacks this exon. The objective of this study was to identify the cis elements that control utilization of exon 6A. A reporter minigene was constructed (pGFP-E6A) containing the coding sequence for GFP whose translation was dependent on faithful splicing for removal of the VEGF exon 6A. To identify cis-acting splicing elements, sequential deletions were made across exon 6A in the pGFP-E6A plasmid.
A candidate cis-acting exonic splicing silencer (ESS) comprising nucleotides 22-30 of exon 6A sequence was identified corresponding to the a silencer consensus sequence of AAGGGG. The function of this sequence as an ESS was confirmed in vivo both in the context of the reporter minigene as a plasmid and in the context of a longer minigene with VEGF exon 6A in its native context in an adenoviral gene transfer vector. Further mutagenesis studies resulted in the identification of the second G residue of the putative ESS as the most critical for function.
This work establishes the identity of cis sequences that regulate alternative VEGF splicing and dictate the relative expression levels of VEGF isoforms.
PMCID: PMC2784459  PMID: 19922608
13.  The sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCPx) is involved in cholesterol uptake in the midgut of Spodoptera litura: gene cloning, expression, localization and functional analyses 
BMC Molecular Biology  2009;10:102.
Sterol carrier protein-2/3-oxoacyl-CoA thiolase (SCPx) gene has been suggested to be involved in absorption and transport of cholesterol. Cholesterol is a membrane component and is a precursor of ecdysteroids, but cannot be synthesized de novo in insects. However, a direct association between SCPx gene expression, cholesterol absorption and development in lepidopteran insects remains to be experimentally demonstrated.
An SCPx cDNA (SlSCPx) cloned from the common cutworm, Spodoptera litura, was characterized. The SlSCPx cDNA encoded a 535-amino acid protein consisting of a 3-oxoacyl-CoA thiolase (SCPx-t) domain and a SCP-2 (SCPx-2) domain. SlSCPx mRNA was expressed predominately in the midgut, while SlSCPx-2 mRNA was detected in the midgut, fat body and epidermis and no SlSCPx-t mRNA was detected. A 58-kDa full-length SCPx protein and a 44-kDa SCPx-t protein were detected in the midgut of sixth instar larvae when the anti-SlSCPx-t antibody was used in western blotting analysis; a 16-kDa SCP-2 protein was detected when anti-SlSCPx-2 antibody was used. SlSCPx protein was post-translationally cleaved into two smaller proteins, SCPx-t and SCPx-2. The gene appeared to be expressed into two forms of mRNA transcripts, which were translated into the two proteins, respectively. SlSCPx-t and SlSCPx-2 proteins have distinct and different locations in the midgut of sixth instar larvae. SlSCPx and SlSCPx-t proteins were detected predominately in the cytoplasm, whereas SlSCPx-2 protein was detected in the cytoplasm and nuclei in the Spli-221 cells. Over-expression of SlSCPx and SlSCPx-2 proteins enhanced cholesterol uptake into the Spli-221 cells. Knocking-down SlSCPx transcripts by dsRNA interference resulted in a decrease in cholesterol level in the hemolymph and delayed the larval to pupal transition.
Spatial and temporal expression pattern of this SlSCPx gene during the larval developmental stages of S. litura showed its specific association with the midgut at the feeding stage. Over-expression of this gene increased cholesterol uptake and interference of its transcript decreased cholesterol uptake and delayed the larval to pupal metamorphosis. All of these results taken together suggest that this midgut-specific SlSCPx gene is important for cholesterol uptake and normal development in S. litura.
PMCID: PMC2779813  PMID: 19912624
14.  Functional characterization of two CITED3 homologs (gcCITED3a and gcCITED3b) in the hypoxia-tolerant grass carp, Ctenopharyngodon idellus 
BMC Molecular Biology  2009;10:101.
CITED proteins belong to a family of non-DNA-binding transcriptional co-regulators that are characterized by a conserved ED-rich domain at the C-terminus. This family of genes is involved in the regulation of a variety of transcriptional responses through interactions with the CBP/p300 integrators and various transcription factors. In fish, very little is known about the expression and functions of CITEDs.
We have characterized two closely related but distinct CITED3 genes, gcCited3a and gcCited3b, from the hypoxia-tolerant grass carp. The deduced gcCITED3a and gcCITED3b proteins share 72% amino acid identity, and are highly similar to the CITED3 proteins of both chicken and Xenopus. Northern blot analysis indicates that the mRNA expression of gcCited3a and gcCited3b is strongly induced by hypoxia in the kidney and liver, respectively. Luciferase reporter assays demonstrated that both gene promoters are activated by gcHIF-1. Further, ChIP assays comparing normal and hypoxic conditions reveal differential in vivo binding of gcHIF-1 to both gene promoters in kidney and liver tissues. HRE-luciferase reporter assays demonstrated that both gcCITED3a and gcCITED3b proteins inhibit gcHIF-1 transcriptional activity, and GST pull-down assays confirmed that both proteins bind specifically to the CH1 domain of the grass carp p300 protein.
The grass carp gcCITED3a and gcCITED3b genes are differentially expressed and regulated in different fish organs in response to hypoxic stress. This is the first report demonstrating in vivo regulation of two closely-related CITED3 isogenes by HIF-1, as well as CITED3 regulation of HIF-1 transcriptional activity in fish. Overall, our findings suggest that unique molecular mechanisms operate through these two gcCITED3 isoforms that likely play an important regulatory role in the hypoxic response in the grass carp.
PMCID: PMC2780419  PMID: 19883516
15.  Evaluation of real-time PCR endogenous control genes for analysis of gene expression in bovine endometrium 
BMC Molecular Biology  2009;10:100.
Quantitative real-time PCR gene expression results are generally normalised using endogenous control genes. These reference genes should be expressed at a constant level across all sample groups in a study, and should not be influenced by study treatments or conditions. There has been no systematic investigation of endogenous control genes for bovine endometrium to date. The suitability of both commonly used and novel endogenous control genes was evaluated in this study, with the latter being selected from stably expressed transcripts identified through microarray analysis of bovine endometrium. Fifteen candidate endogenous control genes were assessed across different tissue subtypes in pregnant and cycling Holstein-Friesian dairy cows from two divergent genetic backgrounds.
The expression profiles of five commonly used endogenous control genes (GAPDH, PPIA, RPS9, RPS15A, and UXT) and 10 experimentally derived candidate endogenous control genes (SUZ12, C2ORF29, ZNF131, ACTR1A, HDAC1, SLC30A6, CNOT7, DNAJC17, BBS2, and RANBP10) were analysed across 44 samples to determine the most stably expressed gene. Gene stability was assessed using the statistical algorithms GeNorm and Normfinder. All genes presented with low overall variability (0.87 to 1.48% CV of Cq). However, when used to normalise a differentially expressed gene (oxytocin receptor - OXTR) in the samples, the reported relative gene expression levels were significantly affected by the control gene chosen. Based on the results of this analysis, SUZ12 is proposed as the most appropriate control gene for use in bovine endometrium during early pregnancy or the oestrus cycle.
This study establishes the suitability of novel endogenous control genes for comparing expression levels in endometrial tissues of pregnant and cycling bovines, and demonstrates the utility of microarray analysis as a method for identifying endogenous control gene candidates.
PMCID: PMC2774697  PMID: 19878604
16.  Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae 
BMC Molecular Biology  2009;10:99.
Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae.
From public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, ALG9, TAF10, TFC1 and UBC6 turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with ACT1. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for GPH1 and 20-fold for GSY2 between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, SGA1 expression was independent of the carbon source and increased by 3-fold in stationary phase.
In this work, we provided a set of genes that are suitable reference genes for quantitative gene expression analysis by real-time RT-PCR in yeast biological samples covering a large panel of physiological states. In contrast, we invalidated and discourage the use of ACT1 as well as other commonly used reference genes (PDA1, TDH3, RDN18, etc) as internal controls for quantitative gene expression analysis in yeast.
PMCID: PMC2776018  PMID: 19874630
17.  RAD51 paralogs promote homology-directed repair at diversifying immunoglobulin V regions 
BMC Molecular Biology  2009;10:98.
Gene conversion depends upon the same factors that carry out more general process of homologous recombination, including homologous gene targeting and recombinational repair. Among these are the RAD51 paralogs, conserved factors related to the key recombination factor, RAD51. In chicken and other fowl, gene conversion (templated mutation) diversifies immunoglobulin variable region sequences. This allows gene conversion and recombinational repair to be studied using the chicken DT40 B cell line, which carries out constitutive gene conversion and provides a robust and physiological model for homology-directed repair in vertebrate cells.
We show that DT40 contains constitutive nuclear foci of the repair factors RAD51D and XRCC2, consistent with activated homologous recombination. Single-cell imaging of a DT40 derivative in which the rearranged and diversifying immunoglobulin λR light chain gene is tagged with polymerized lactose operator, DT40 PolyLacO-λR, showed that RAD51D and XRCC2 localize to the diversifying λR gene. Colocalizations correlate both functionally and physically with active immunoglobulin gene conversion. Ectopic expression of either RAD51D or XRCC2 accelerated the clonal rate of gene conversion, and conversion tracts were significantly longer in RAD51D than XRCC2 transfectants.
These results demonstrate direct functions of RAD51D and XRCC2 in immunoglobulin gene conversion, and also suggest that modulation of levels of repair factors may be a useful strategy to promote gene correction in other cell types.
PMCID: PMC2774322  PMID: 19863810
18.  Preliminary molecular characterization of the human pathogen Angiostrongylus cantonensis 
BMC Molecular Biology  2009;10:97.
Human angiostrongyliasis is an emerging food-borne public health problem, with the number of cases increasing worldwide, especially in mainland China. Angiostrongylus cantonensis is the causative agent of this severe disease. However, little is known about the genetics and basic biology of A. cantonensis.
A cDNA library of A. cantonensis fourth-stage larvae was constructed, and ~1,200 clones were sequenced. Bioinformatic analyses revealed 378 cDNA clusters, 54.2% of which matched known genes at a cutoff expectation value of 10-20. Of these 378 unique cDNAs, 168 contained open reading frames encoding proteins containing an average of 238 amino acids. Characterization of the functions of these encoded proteins by Gene Ontology analysis showed enrichment in proteins with binding and catalytic activity. The observed pattern of enzymes involved in protein metabolism, lipid metabolism and glycolysis may reflect the central nervous system habitat of this pathogen. Four proteins were tested for their immunogenicity using enzyme-linked immunosorbent assays and histopathological examinations. The specificity of each of the four proteins was superior to that of crude somatic and excretory/secretory antigens of larvae, although their sensitivity was relatively low. We further showed that mice immunized with recombinant cystatin, a product of one of the four cDNA candidate genes, were partially protected from A. cantonensis infection.
The data presented here substantially expand the available genetic information about the human pathogen A. cantonensis, and should be a significant resource for angiostrongyliasis researchers. As such, this work serves as a starting point for molecular approaches for diagnosing and controlling human angiostrongyliasis.
PMCID: PMC2774698  PMID: 19852860
19.  Dual effect of a single nucleotide polymorphism in the first intron of the porcine Secreted phosphoprotein 1 gene: allele-specific binding of C/EBP beta and activation of aberrant splicing 
BMC Molecular Biology  2009;10:96.
Secreted phosphoprotein 1 (SPP1 or Osteopontin, OPN) is a multifunctional matricellular glycoprotein involved in development and regeneration of skeletal muscle. Previously, we have demonstrated that porcine SPP1 shows breed-related differential mRNA expression during myogenesis. With the aim to identify putative contributing cis-regulatory DNA variation we resequenced the 5' upstream region of the gene in the respective breeds Pietrain and Duroc. We found two single nucleotide polymorphisms (SNP; [GenBank:M84121]: g.1804C>T and g.3836A>G). We focused our investigation on the SNP g.3836A>G, because in silico analysis and knowledge about the regulation of SPP1 suggested an effect of this SNP on a CCAAT/enhancer binding protein beta (C/EBPβ) responsive transcriptional enhancer.
Using electrophoretic mobility shift assay we demonstrated that, similar to human SPP1, the 3' terminal end of the first intron of porcine SPP1 harbors a C/EBPβ binding site and showed that this binding site is negatively affected by the mutant G allele. Genotyping of 48 fetuses per breed revealed that the G allele segregated exclusively in Duroc fetuses with a frequency of 57 percent. Using real-time quantitative PCR we showed that, consistent with its negative effect on a transcriptional enhancer element, the G allele tends to decrease mRNA abundance of SPP1 in the fetal musculus longissimus dorsi (~1.3 fold; P ≥ 0.1).
Moreover, we showed that the SNP g.3836A>G leads to ubiquitous aberrant splicing of the first intron by generating a de novo and activating a cryptic splice acceptor site. Aberrantly spliced transcripts comprise about half of the SPP1 messages expressed by the G allele. Both aberrant splice variants differ from the native transcript by insertions in the leader sequences which do not change the reading frame of SPP1.
At the 3' terminal end of the first intron of the porcine SPP1 we identified a unique, dually functional SNP g.3836A>G. This SNP affects the function of the SPP1 gene at the DNA level by affecting a C/EBPβ binding site and at the RNA level by activating aberrant splicing of the first intron, and thus represents an interesting DNA-marker to study phenotypic effects of SPP1 DNA-variation.
PMCID: PMC2773772  PMID: 19845957
20.  A small intergenic region drives exclusive tissue-specific expression of the adjacent genes in Arabidopsis thaliana 
BMC Molecular Biology  2009;10:95.
Transcription initiation by RNA polymerase II is unidirectional from most genes. In plants, divergent genes, defined as non-overlapping genes organized head-to-head, are highly represented in the Arabidopsis genome. Nevertheless, there is scarce evidence on functional analyses of these intergenic regions. The At5g06290 and At5g06280 loci are head-to-head oriented and encode a chloroplast-located 2-Cys peroxiredoxin B (2CPB) and a protein of unknown function (PUF), respectively. The 2-Cys peroxiredoxins are proteins involved in redox processes, they are part of the plant antioxidant defence and also act as chaperons. In this study, the transcriptional activity of a small intergenic region (351 bp) shared by At5g06290 and At5g06280 in Arabidopsis thaliana was characterized.
Activity of the intergenic region in both orientations was analyzed by driving the β-glucuronidase (GUS) reporter gene during the development and growth of Arabidopsis plants under physiological and stressful conditions. Results have shown that this region drives expression either of 2cpb or puf in photosynthetic or vascular tissues, respectively. GUS expression driven by the promoter in 2cpb orientation was enhanced by heat stress. On the other hand, the promoter in both orientations has shown similar down-regulation of GUS expression under low temperatures and other stress conditions such as mannitol, oxidative stress, or fungal elicitor.
The results from this study account for the first evidence of an intergenic region that, in opposite orientation, directs GUS expression in different spatially-localized Arabidopsis tissues in a mutually exclusive manner. Additionally, this is the first demonstration of a small intergenic region that drives expression of a gene whose product is involved in the chloroplast antioxidant defence such as 2cpb. Furthermore, these results contribute to show that 2cpb is related to the heat stress defensive system in leaves and roots of Arabidopsis thaliana.
PMCID: PMC2772851  PMID: 19835620
21.  Validation of suitable house keeping genes for hypoxia-cultured human chondrocytes 
BMC Molecular Biology  2009;10:94.
Hypoxic culturing of chondrocytes is gaining increasing interest in cartilage research. Culturing of chondrocytes under low oxygen tension has shown several advantages, among them increased synthesis of extracellular matrix and increased redifferentiation of dedifferentiated chondrocytes. Quantitative gene expression analyses such as quantitative real-time PCR (qRT-PCR) are powerful tools in the investigation of underlying mechanisms of cell behavior and are used routinely for differentiation and phenotype assays. However, the genes used for normalization in normoxic cell-cultures might not be suitable in the hypoxic environment. The objective of this study was to determine hypoxia-stable housekeeping genes (HKG) for quantitative real-time PCR (qRT-PCR) in human chondrocytes cultured in 21%, 5% and 1% oxygen by geNorm and NormFinder analyses.
The chondrocytic response to the hypoxic challange was validated by a significant increase in expression of the hypoxia-inducible gene ankyrin repeat 37 as well as SOX9 in hypoxia. When cultured on the 3-dimentional (3D) scaffold TATA-binding protein (TBP) exhibited the highest expression stability with NormFinder while Ribosomal protein L13a (RPL13A) and beta2-microglobulin (B2M) were the most stable using geNorm analysis. In monolayer RPL13A were the most stable gene using NormFinder, while geNorm assessed RPL13A and human RNA polymerase II (RPII) as most stable. When examining the combination of (3D) culturing and monolayer RPL13A and B2M showed the highest expression stability from geNorm analysis while RPL13A also showed the highest expression stability using NormFinder. Often used HKG such as beta actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were the most unstable genes investigated in all comparisons. The pairwise variations for the two most stable HKG in each group were all below the cut-off value of 0.15, suggesting that the two most stable HKG from geNorm analysis would be sufficient for qRT-PCR.
All data combined we recommend RPL13A, B2M and RPII as the best choice for qRT-PCR analyses when comparing normoxic and hypoxic cultured human chondrocytes although other genes might also be suitable. However, the matching of HKG to target genes by means of a thorough investigation of the stability in each study would always be preferable.
PMCID: PMC2764705  PMID: 19818117
22.  Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR 
BMC Molecular Biology  2009;10:93.
Real-time quantitative reverse transcription PCR (RT-qPCR) data needs to be normalized for its proper interpretation. Housekeeping genes are routinely employed for this purpose, but their expression level cannot be assumed to remain constant under all possible experimental conditions. Thus, a systematic validation of reference genes is required to ensure proper normalization. For soybean, only a small number of validated reference genes are available to date.
A systematic comparison of 14 potential reference genes for soybean is presented. These included seven commonly used (ACT2, ACT11, TUB4, TUA5, CYP, UBQ10, EF1b) and seven new candidates (SKIP16, MTP, PEPKR1, HDC, TIP41, UKN1, UKN2). Expression stability was examined by RT-qPCR across 116 biological samples, representing tissues at various developmental stages, varied photoperiodic treatments, and a range of soybean cultivars. Expression of all 14 genes was variable to some extent, but that of SKIP16, UKN1 and UKN2 was overall the most stable. A combination of ACT11, UKN1 and UKN2 would be appropriate as a reference panel for normalizing gene expression data among different tissues, whereas the combination SKIP16, UKN1 and MTP was most suitable for developmental stages. ACT11, TUA5 and TIP41 were the most stably expressed when the photoperiod was altered, and TIP41, UKN1 and UKN2 when the light quality was changed. For six different cultivars in long day (LD) and short day (SD), their expression stability did not vary significantly with ACT11, UKN2 and TUB4 being the most stable genes. The relative gene expression level of GmFTL3, an ortholog of Arabidopsis FT (FLOWERING LOCUS T) was detected to validate the reference genes selected in this study.
None of the candidate reference genes was uniformly expressed across all experimental conditions, and the most suitable reference genes are conditional-, tissue-specific-, developmental-, and cultivar-dependent. Most of the new reference genes performed better than the conventional housekeeping genes. These results should guide the selection of reference genes for gene expression studies in soybean.
PMCID: PMC2761916  PMID: 19785741
23.  From sequence to dynamics: the effects of transcription factor and polymerase concentration changes on activated and repressed promoters 
BMC Molecular Biology  2009;10:92.
The fine tuning of two features of the bacterial regulatory machinery have been known to contribute to the diversity of gene expression within the same regulon: the sequence of Transcription Factor (TF) binding sites, and their location with respect to promoters. While variations of binding sequences modulate the strength of the interaction between the TF and its binding sites, the distance between binding sites and promoters alter the interaction between the TF and the RNA polymerase (RNAP).
In this paper we estimated the dissociation constants (Kd) of several E. coli TFs in their interaction with variants of their binding sequences from the scores resulting from aligning them to Positional Weight Matrices. A correlation coefficient of 0.78 was obtained when pooling together sites for different TFs. The theoretically estimated Kd values were then used, together with the dissociation constants of the RNAP-promoter interaction to analyze activated and repressed promoters. The strength of repressor sites -- i.e., the strength of the interaction between TFs and their binding sites -- is slightly higher than that of activated sites. We explored how different factors such as the variation of binding sequences, the occurrence of more than one binding site, or different RNAP concentrations may influence the promoters' response to the variations of TF concentrations. We found that the occurrence of several regulatory sites bound by the same TF close to a promoter -- if they are bound by the TF in an independent manner -- changes the effect of TF concentrations on promoter occupancy, with respect to individual sites. We also found that the occupancy of a promoter will never be more than half if the RNAP concentration-to-Kp ratio is 1 and the promoter is subject to repression; or less than half if the promoter is subject to activation. If the ratio falls to 0.1, the upper limit of occupancy probability for repressed drops below 10%; a descent of the limits occurs also for activated promoters.
The number of regulatory sites may thus act as a versatility-producing device, in addition to serving as a source of robustness of the transcription machinery. Furthermore, our results show that the effects of TF concentration fluctuations on promoter occupancy are constrained by RNAP concentrations.
PMCID: PMC2761915  PMID: 19772633
24.  Quadruple 9-mer-based protein binding microarray with DsRed fusion protein 
BMC Molecular Biology  2009;10:91.
The interaction between a transcription factor and DNA motif (cis-acting element) is an important regulatory step in gene regulation. Comprehensive genome-wide methods have been developed to characterize protein-DNA interactions. Recently, the universal protein binding microarray (PBM) was introduced to determine if a DNA motif interacts with proteins in a genome-wide manner.
We facilitated the PBM technology using a DsRed fluorescent protein and a concatenated sequence of oligonucleotides. The PBM was designed in such a way that target probes were synthesized as quadruples of all possible 9-mer combinations, permitting unequivocal interpretation of the cis-acting elements. The complimentary DNA strands of the features were synthesized with a primer and DNA polymerase on microarray slides. Proteins were labeled via N-terminal fusion with DsRed fluorescent protein, which circumvents the need for a multi-step incubation. The PBM presented herein confirmed the well-known DNA binding sequences of Cbf1 and CBF1/DREB1B, and it was also applied to elucidate the unidentified cis-acting element of the OsNAC6 rice transcription factor.
Our method demonstrated PBM can be conveniently performed by adopting: (1) quadruple 9-mers may increase protein-DNA binding interactions in the microarray, and (2) a one-step incubation shortens the wash and hybridization steps. This technology will facilitate greater understanding of genome-wide interactions between proteins and DNA.
PMCID: PMC2754467  PMID: 19761621
25.  Characterization of bovine miRNAs by sequencing and bioinformatics analysis 
BMC Molecular Biology  2009;10:90.
MicroRNAs (miRNAs) are a family of ~22 nucleotide small RNA molecules which regulate gene expression by fully or partially binding to their complementary sequences in mRNAs or promoters. A large number of miRNAs and their expression patterns have been reported in human, mouse and rat. However, miRNAs and their expression patterns in live stock species such as beef cattle are not well studied.
We constructed and sequenced small-RNA libraries to yield a total of 13,541 small-RNA sequences from 11 bovine tissues including brain, subcutaneous fat, muscle, liver, kidney, spleen and thymus. In total, 228 miRNAs including 29 novel miRNA candidates were identified. Of the 199 miRNAs, 101 have been previously reported as bovine miRNAs and the other 98 are bovine orthologs of known miRNAs that have been identified in at least one other mammalian species. Of the 29 novel miRNA candidates, 17 appeared at this point in time to be bovine specific, while the remaining 12 had evidence of evolutionary conservation in other mammalian species. Five miRNAs (miR-23a, -23b, -99a, -125b and -126-5p) were very abundant across the 11 tissues, accounting for 44.3% of all small RNA sequences. The expression analysis of selected miRNAs using qRT-PCR also showed that miR-26a and -99a were highly expressed in all tissues, while miR-122 and miR-133a were predominantly expressed in liver and muscle, respectively.
The miRNA expression patterns among 11 tissues from beef cattle revealed that most miRNAs were ubiquitously expressed in all tissues, while only a few miRNAs were tissue specific. Only 60% miRNAs in this study were found to display strand bias, suggesting that there are some key factors for mature miRNA selection other than internal stability. Most bovine miRNAs are highly conserved in other three mammalian species, indicating that these miRNAs may have a role in different species that are potential molecular markers for evolution.
PMCID: PMC2761914  PMID: 19758457

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