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1.  Inverted terminal repeats of adeno-associated virus decrease random integration of a gene targeting fragment in Saccharomyces cerevisiae 
Background
Homologous recombination mediated gene targeting is still too inefficient to be applied extensively in genomics and gene therapy. Although sequence-specific nucleases could greatly stimulate gene targeting efficiency, the off-target cleavage sites of these nucleases highlighted the risk of this strategy. Adeno-associated virus (AAV)-based vectors are used for specific gene knockouts, since several studies indicate that these vectors are able to induce site-specific genome alterations at high frequency. Since each targeted event is accompanied by at least ten random integration events, increasing our knowledge regarding the mechanisms behind these events is necessary in order to understand the potential of AAV-mediated gene targeting for therapy application. Moreover, the role of AAV regulatory proteins (Rep) and inverted terminal repeated sequences (ITRs) in random and homologous integration is not completely known. In this study, we used the yeast Saccharomyces cerevisiae as a genetic model system to evaluate whether the presence of ITRs in the integrating plasmid has an effect on gene targeting and random integration.
Results
We have shown that the presence of ITRs flanking a gene targeting vector containing homology to its genomic target decreased the frequency of random integration, leading to an increase in the gene targeting/random integration ratio. On the other hand, the expression of Rep proteins, which produce a nick in the ITR, significantly increased non-homologous integration of a DNA fragment sharing no homology to the genome, but had no effect on gene targeting or random integration when the DNA fragment shared homology with the genome. Molecular analysis showed that ITRs are frequently conserved in the random integrants, and that they induce rearrangements.
Conclusions
Our results indicate that ITRs may be a useful tool for decreasing random integration, and consequently favor homologous gene targeting.
doi:10.1186/1471-2199-15-5
PMCID: PMC3925961  PMID: 24521444
Yeast; AAV; ITRs; Homologous recombination; Random integration
2.  Cooperative activation of Xenopus rhodopsin transcription by paired-like transcription factors 
Background
In vertebrates, rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/LNrl and Crx/Otx5. The ubiquitous occurrence of their target DNA binding sites throughout rod-specific gene promoters suggests that multiple transcription factor interactions within the promoter are functionally important. Cooperative action by these transcription factors activates rod-specific genes such as rhodopsin. However, a quantitative mechanistic explanation of transcriptional rate determinants is lacking.
Results
We investigated the contributions of various paired-like transcription factors and their cognate cis-elements to rhodopsin gene activation using cultured cells to quantify activity. The Xenopus rhodopsin promoter (XOP) has a bipartite structure, with ~200 bp proximal to the start site (RPP) coordinating cooperative activation by Nrl/LNrl-Crx/Otx5 and the adjacent 5300 bp upstream sequence increasing the overall expression level. The synergistic activation by Nrl/LNrl-Crx/Otx5 also occurred when XOP was stably integrated into the genome. We determined that Crx/Otx5 synergistically activated transcription independently and additively through the two Pax-like cis-elements, BAT1 and Ret4, but not through Ret1. Other Pax-like family members, Rax1 and Rax2, do not synergistically activate XOP transcription with Nrl/LNrl and/or Crx/Otx5; rather they act as co-activators via the Ret1 cis-element.
Conclusions
We have provided a quantitative model of cooperative transcriptional activation of the rhodopsin promoter through interaction of Crx/Otx5 with Nrl/LNrl at two paired-like cis-elements proximal to the NRE and TATA binding site. Further, we have shown that Rax genes act in cooperation with Crx/Otx5 with Nrl/LNrl as co-activators of rhodopsin transcription.
doi:10.1186/1471-2199-15-4
PMCID: PMC3937059  PMID: 24499263
3.  Annual acknowledgement of reviewers 
Contributing reviewers
The editors of BMC Molecular Biology would like to thank all our reviewers who have contributed to the journal in Volume 14 (2013).
doi:10.1186/1471-2199-15-2
PMCID: PMC3902004
4.  Spdef deletion rescues the crypt cell proliferation defect in conditional Gata6 null mouse small intestine 
Background
GATA transcription factors are essential for self-renewal of the small intestinal epithelium. Gata4 is expressed in the proximal 85% of small intestine while Gata6 is expressed throughout the length of small intestine. Deletion of intestinal Gata4 and Gata6 results in an altered proliferation/differentiation phenotype, and an up-regulation of SAM pointed domain containing ETS transcription factor (Spdef), a transcription factor recently shown to act as a tumor suppressor. The goal of this study is to determine to what extent SPDEF mediates the downstream functions of GATA4/GATA6 in the small intestine. The hypothesis to be tested is that intestinal GATA4/GATA6 functions through SPDEF by repressing Spdef gene expression. To test this hypothesis, we defined the functions most likely regulated by the overlapping GATA6/SPDEF target gene set in mouse intestine, delineated the relationship between GATA6 chromatin occupancy and Spdef gene regulation in Caco-2 cells, and determined the extent to which prevention of Spdef up-regulation by Spdef knockout rescues the GATA6 phenotype in conditional Gata6 knockout mouse ileum.
Results
Using publicly available profiling data, we found that 83% of GATA6-regulated genes are also regulated by SPDEF, and that proliferation/cancer is the function most likely to be modulated by this overlapping gene set. In human Caco-2 cells, GATA6 knockdown results in an up-regulation of Spdef gene expression, modeling our mouse Gata6 knockout data. GATA6 occupies a genetic locus located 40 kb upstream of the Spdef transcription start site, consistent with direct regulation of Spdef gene expression by GATA6. Prevention of Spdef up-regulation in conditional Gata6 knockout mouse ileum by the additional deletion of Spdef rescued the crypt cell proliferation defect, but had little effect on altered lineage differentiation or absorptive enterocytes gene expression.
Conclusion
SPDEF is a key, immediate downstream effecter of the crypt cell proliferation function of GATA4/GATA6 in the small intestine.
doi:10.1186/1471-2199-15-3
PMCID: PMC3917371  PMID: 24472151
GATA6; SPDEF; Crypt cell proliferation; Intestinal differentiation
5.  MicroRNA miR-378 promotes BMP2-induced osteogenic differentiation of mesenchymal progenitor cells 
Background
MicroRNAs (miRNAs) are a family of small, non-coding single-stranded RNA molecules involved in post-transcriptional regulation of gene expression. As such, they are believed to play a role in regulating the step-wise changes in gene expression patterns that occur during cell fate specification of multipotent stem cells. Here, we have studied whether terminal differentiation of C2C12 myoblasts is indeed controlled by lineage-specific changes in miRNA expression.
Results
Using a previously generated RNA polymerase II (Pol-II) ChIP-on-chip dataset, we show differential Pol-II occupancy at the promoter regions of six miRNAs during C2C12 myogenic versus BMP2-induced osteogenic differentiation. Overexpression of one of these miRNAs, miR-378, enhances Alp activity, calcium deposition and mRNA expression of osteogenic marker genes in the presence of BMP2.
Conclusions
Our results demonstrate a previously unknown role for miR-378 in promoting BMP2-induced osteogenic differentiation.
doi:10.1186/1471-2199-15-1
PMCID: PMC3905160  PMID: 24467925
6.  Dissecting domains necessary for activation and repression of splicing by muscleblind-like protein 1 
BMC Molecular Biology  2013;14:29.
Background
Alternative splicing contributes to the diversity of the proteome, and provides the cell with an important additional layer of regulation of gene expression. Among the many RNA binding proteins that regulate alternative splicing pathways are the Muscleblind-like (MBNL) proteins. MBNL proteins bind YGCY motifs in RNA via four CCCH zinc fingers arranged in two tandem arrays, and play a crucial role in the transition from embryonic to adult muscle splicing patterns, deregulation of which leads to Myotonic Dystrophy. Like many other RNA binding proteins, MBNL proteins can act as both activators or repressors of different splicing events.
Results
We used targeted point mutations to interfere with the RNA binding of MBNL1 zinc fingers individually and in combination. The effects of the mutations were tested in assays for splicing repression and activation, including overexpression, complementation of siRNA-mediated knockdown, and artificial tethering using MS2 coat protein. Mutations were tested in the context of both full length MBNL1 as well as a series of truncation mutants. Individual mutations within full length MBNL1 had little effect, but mutations in ZF1 and 2 combined were more detrimental than those in ZF 3 and 4, upon splicing activation, repression and RNA binding. Activation and repression both required linker sequences between ZF2 and 3, but activation was more sensitive to loss of linker sequences.
Conclusions
Our results highlight the importance of RNA binding by MBNL ZF domains 1 and 2 for splicing regulatory activity, even when the protein is artificially recruited to its regulatory location on target RNAs. However, RNA binding is not sufficient for activity; additional regions between ZF 2 and 3 are also essential. Activation and repression show differential sensitivity to truncation of this linker region, suggesting interactions with different sets of cofactors for the two types of activity.
doi:10.1186/1471-2199-14-29
PMCID: PMC3880588  PMID: 24373687
7.  Differential regulation of the rainbow trout (Oncorhynchus mykiss) MT-A gene by nuclear factor interleukin-6 and activator protein-1 
BMC Molecular Biology  2013;14:28.
Background
Previously we have identified a distal region of the rainbow trout (Oncorhynchus mykiss) metallothionein-A (rtMT-A) enhancer region, being essential for free radical activation of the rtMT-A gene. The distal promoter region included four activator protein 1 (AP1) cis-acting elements and a single nuclear factor interleukin-6 (NF-IL6) element. In the present study we used the rainbow trout hepatoma (RTH-149) cell line to further examine the involvement of NF-IL6 and AP1 in rtMT-A gene expression following exposure to oxidative stress and tumour promotion.
Results
Using enhancer deletion studies we observed strong paraquat (PQ)-induced rtMT-A activation via NF-IL6 while the AP1 cis-elements showed a weak but significant activation. In contrast to mammals the metal responsive elements were not activated by oxidative stress. Electrophoretic mobility shift assay (EMSA) mutation analysis revealed that the two most proximal AP1 elements, AP11,2, exhibited strong binding to the AP1 consensus sequence, while the more distal AP1 elements, AP13,4 were ineffective. Phorbol-12-myristate-13-acetate (PMA), a known tumor promoter, resulted in a robust induction of rtMT-A via the AP1 elements alone. To determine the conservation of regulatory functions we transfected human Hep G2 cells with the rtMT-A enhancer constructs and were able to demonstrate that the cis-elements were functionally conserved. The importance of NF-IL6 in regulation of teleost MT is supported by the conservation of these elements in MT genes from different teleosts. In addition, PMA and PQ injection of rainbow trout resulted in increased hepatic rtMT-A mRNA levels.
Conclusions
These studies suggest that AP1 primarily is involved in PMA regulation of the rtMT-A gene while NF-IL6 is involved in free radical regulation. Taken together this study demonstrates the functionality of the NF-IL6 and AP-1 elements and suggests an involvement of MT in protection during pathological processes such as inflammation and cancer.
doi:10.1186/1471-2199-14-28
PMCID: PMC3867414  PMID: 24341438
Rainbow trout; Metallothionein-A promoter; Nuclear factor interleukin-6; Activator protein-1; Oxidative stress
8.  An aryl hydrocarbon receptor induces VEGF expression through ATF4 under glucose deprivation in HepG2 
BMC Molecular Biology  2013;14:27.
Background
Aryl hydrocarbon receptor (AhR) not only regulates drug-metabolizing enzyme expression but also regulates cancer malignancy. The steps to the development of malignancy include angiogenesis that is induced by tumor microenvironments, hypoxia, and nutrient deprivation. Vascular endothelial growth factor (VEGF) plays a central role in the angiogenesis of cancer cells, and it is induced by activating transcription factor 4 (ATF4).
Results
Recently, we identified that glucose deprivation induces AhR translocation into the nucleus and increases CYP1A1 and 1A2 expression in HepG2 cells. Here, we report that the AhR pathway induces VEGF expression in human hepatoblastoma HepG2 cells under glucose deprivation, which involves ATF4. ATF4 knockdown suppressed VEGF expression under glucose deprivation. Moreover, AhR knockdown suppressed VEGF and ATF4 expression under glucose deprivation at genetic and protein levels.
Conclusions
The AhR-VEGF pathway through ATF4 is a novel pathway in glucose-deprived liver cancer cells that is related to the microenvironment within a cancer tissue affecting liver cancer malignancy.
doi:10.1186/1471-2199-14-27
PMCID: PMC3866938  PMID: 24330582
Aryl hydrocarbon receptor (AhR); Vascular endothelial growth factor (VEGF); Angiogenesis; Activating transcription factor 4 (ATF4); Human hepatocellular liver carcinoma cell
9.  Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries 
BMC Molecular Biology  2013;14:25.
Background
Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions.
Results
Through microarray analysis of exponential- and stationary-phase cultures with low and high domoic acid production, respectively, we identified candidate reference genes whose transcripts did not vary across conditions. We tested eleven potential reference genes for stability using RT-qPCR and GeNorm analyses. Our results indicated that transcripts encoding JmjC, dynein, and histone H3 proteins were the most suitable for normalization of expression data under conditions of silicon-limitation, in late-exponential through stationary phase. The microarray studies identified a number of genes that were up- and down-regulated under toxin-producing conditions. RT-qPCR analysis, using the validated controls, confirmed the up-regulation of transcripts predicted to encode a cycloisomerase, an SLC6 transporter, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, a small heat shock protein, and an aldo-keto reductase, as well as the down-regulation of a transcript encoding a fucoxanthin-chlorophyll a-c binding protein, under these conditions.
Conclusion
Our results provide a strong basis for further studies of RNA expression levels in Ps-n, which will contribute to our understanding of genes involved in the production and release of domoic acid, an important neurotoxin that affects human health as well as ecosystem function.
doi:10.1186/1471-2199-14-25
PMCID: PMC3832940  PMID: 24180290
Gene expression; Gene regulation; cDNA microarray; RT-qPCR; Normalization; Reference gene; Domoic acid; Pseudo-nitzschia multiseries; Bacillariophyceae; Diatom
10.  Identification of an NF-κB p50/p65-responsive site in the human MIR155HG promoter 
BMC Molecular Biology  2013;14:24.
Background
MicroRNA-155 (miR-155) is the diced product of the MIR155HG gene. miR-155 regulates the expression of many immune-specific transcripts, is overexpressed in many human lymphomas, and has oncogenic activity in mouse transgenic models. MIR155HG has been proposed to be a target gene for transcription factor NF-κB largely due to the positive correlation between high nuclear NF-κB activity and increased miR-155 expression following treatment with NF-κB inducers or in subsets of hematopoietic cancers. Nevertheless, direct regulation of the human MIR155HG promoter by NF-κB has not been convincingly demonstrated previously.
Results
This report shows that induction of NF-κB activity rapidly leads to increased levels of both primary MIR155HG mRNA and mature miR-155 transcripts. We have mapped an NF-κB-responsive element to a position approximately 178 nt upstream of the MIR155HG transcription start site. The -178 site is specifically bound by the NF-κB p50/p65 heterodimer and is required for p65-induced reporter gene activation. Moreover, the levels of miR-155 in nine human B-lymphoma cell lines generally correlate with increased nuclear NF-κB proteins.
Conclusion
Overall, the identification of an NF-κB-responsive site in the MIR155HG proximal promoter suggests that MIR155HG is a direct NF-κB target gene in vivo. Understanding NF-κB-mediated regulation of miR-155 could lead to improved immune cell-related diagnostic tools and targeted therapies.
doi:10.1186/1471-2199-14-24
PMCID: PMC3849010  PMID: 24059932
miR-155; MIR155HG; BIC; NF-kappaB; Promoter; Transcriptional activation
11.  3′ terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing 
BMC Molecular Biology  2013;14:23.
Background
Post-transcriptional 3′ end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3′ RACE coupled with high-throughput sequencing to characterize the 3′ terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes.
Results
The 3′ terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3′ terminus of an in vitro transcribed MRP RNA control and the differing 3′ terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR).
Conclusions
3′ RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3′ terminal sequences of noncoding RNAs.
doi:10.1186/1471-2199-14-23
PMCID: PMC3849073  PMID: 24053768
RNase MRP RNA; 3′ RACE deep sequencing; Oligo(U); Oligo(A); Telomerase RNA
12.  VprBP (DCAF1): a promiscuous substrate recognition subunit that incorporates into both RING-family CRL4 and HECT-family EDD/UBR5 E3 ubiquitin ligases 
BMC Molecular Biology  2013;14:22.
The terminal step in the ubiquitin modification system relies on an E3 ubiquitin ligase to facilitate transfer of ubiquitin to a protein substrate. The substrate recognition and ubiquitin transfer activities of the E3 ligase may be mediated by a single polypeptide or may rely on separate subunits. The latter organization is particularly prevalent among members of largest class of E3 ligases, the RING family, although examples of this type of arrangement have also been reported among members of the smaller HECT family of E3 ligases. This review describes recent discoveries that reveal the surprising and distinctive ability of VprBP (DCAF1) to serve as a substrate recognition subunit for a member of both major classes of E3 ligase, the RING-type CRL4 ligase and the HECT-type EDD/UBR5 ligase. The cellular processes normally regulated by VprBP-associated E3 ligases, and their targeting and subversion by viral accessory proteins are also discussed. Taken together, these studies provide important insights and raise interesting new questions regarding the mechanisms that regulate or subvert VprBP function in the context of both the CRL4 and EDD/UBR5 E3 ligases.
doi:10.1186/1471-2199-14-22
PMCID: PMC3847654  PMID: 24028781
VprBP; DCAF1; DDB1; Cul4; CRL4; EDD; UBR5; Dyrk2; Merlin; Katanin; UNG2; LGL2; Mcm10; Histone H3; RORα; Methyl degron; p53; TERT; telomerase; RAG1; V(D)J recombination; HIV; Vpr; Vpx; UL35; Ubiquitin; E3 ubiquitin ligase; RING; HECT; WD40 repeat
13.  Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells 
BMC Molecular Biology  2013;14:21.
Background
Selecting stably expressed reference genes is essential for proper reverse transcription quantitative polymerase chain reaction gene expression analysis. However, this choice is not always straightforward. In the case of differentiating human embryonic stem (hES) cells, differentiation itself introduces changes whereby reference gene stability may be influenced.
Results
In this study, we evaluated the stability of various references during retinoic acid-induced (2 microM) differentiation of hES cells. Out of 12 candidate references, beta-2-microglobulin, ribosomal protein L13A and Alu repeats are found to be the most stable for this experimental set-up.
Conclusions
Our results show that some of the commonly used reference genes are actually not amongst the most stable loci during hES cell differentiation promoted by retinoic acid. Moreover, a novel normalization strategy based on expressed Alu repeats is validated for use in hES cell experiments.
doi:10.1186/1471-2199-14-21
PMCID: PMC3848990  PMID: 24028740
Reverse transcription quantitative PCR; Normalization; Reference genes; Alu repeats; Human embryonic stem cells; Stem cell differentiation
14.  Pseudo attP sites in favor of transgene integration and expression in cultured porcine cells identified by streptomyces phage phiC31 integrase 
BMC Molecular Biology  2013;14:20.
Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. We show that PhiC31 integrase can mediate site-specific transgene integration into the genome of pig kidney PK15 cells. Intramolecular recombination in pig PK15 cell line occurred at maximum frequency of 82% with transiently transfected attB- and attP-containing plasmids. An optimal molar ratio of pCMV-Int to pEGFP-N1-attB at 5:1 was observed for maximum number of cell clones under drug selection. Four candidate pseudo attP sites were identified by TAIL-PCR from those cell clones with single-copy transgene integration. Two of them gave rise to higher integration frequency occurred at 33%. 5′ and 3′ junction PCR showed that transgene integration mediated by PhiC31 integrase was mono-allelic. Micro- deletion and insertion were observed by sequencing the integration border, indicating that double strand break was induced by the recombination. We then constructed rescue reporter plasmids by ABI-REC cloning of the four pseudo attP sites into pBCPB + plasmid. Transfection of these rescue plasmids and pCMV-Int resulted in expected intramolecular recombination between attB and pseudo attP sites. This proved that the endogenous pseudo attP sites were functional substrates for PhiC31 integrase-mediated site-specific recombination. Two pseudo attP sites maintained robust extracellular and intracellular EGFP expression. Alamar blue assay showed that transgene integration into these specific sites had little effect on cell proliferation. This is the first report to document the potential use of PhiC31 integrase to mediate site-specific recombination in pig cells. Our work established an ideal model to study the position effect of identical transgene located in diverse chromosomal contexts. These findings also form the basis for targeted pig genome engineering and may be used to produce genetically modified pigs for agricultural and biomedical uses.
doi:10.1186/1471-2199-14-20
PMCID: PMC3844521  PMID: 24010979
PhiC31 integrase; Pig; Pseudo attP site; TAIL-PCR
15.  Molecular cloning and RNA interference-mediated functional characterization of a Halloween gene spook in the white-backed planthopper Sogatella furcifera 
BMC Molecular Biology  2013;14:19.
Background
Ecdysteroid hormones ecdysone and 20-hydroxyecdysone play fundamental roles in insect postembryonic development and reproduction. Five cytochrome P450 monooxygenases (CYPs), encoded by Halloween genes, have been documented to be involved in the ecdysteroidogenesis in insect species of diverse orders such as Diptera, Lepidoptera and Orthoptera. Up to now, however, the involvement of the Halloween genes in ecdysteroid synthesis has not been confirmed in hemipteran insect species.
Results
In the present paper, a Halloween gene spook (Sfspo, Sfcyp307a1) was cloned in the hemipteran Sogatella furcifera. SfSPO has three insect conserved P450 motifs, i.e., Helix-K, PERF and heme-binding motifs. Temporal and spatial expression patterns of Sfspo were evaluated by qPCR. Sfspo showed three expression peaks in late second-, third- and fourth-instar stages. In contrast, the expression levels were lower and formed three troughs in the newly-molted second-, third- and fourth-instar nymphs. On day 3 of the fourth-instar nymphs, Sfspo clearly had a high transcript level in the thorax where PGs were located. Dietary introduction of double-stranded RNA (dsRNA) of Sfspo into the second instars successfully knocked down the target gene, and greatly reduced expression level of ecdysone receptor (EcR) gene. Moreover, knockdown of Sfspo caused lethality and delayed development during nymphal stages. Furthermore, application of 20-hydroxyecdysone on Sfspo-dsRNA-exposed nymphs did not increase Sfspo expression, but could almost completely rescue SfEcR expression, and relieved the negative effects on nymphal survival and development.
Conclusion
In S. furcifera, Sfspo was cloned and the conservation of SfSPO is valid. Thus, SfSPO is probably also involved in ecdysteroidogenesis for hemiptera.
doi:10.1186/1471-2199-14-19
PMCID: PMC3766648  PMID: 24007644
Sogatella furcifera; Halloween gene; Ecdysteroidogenesis; RNA interference; Lethality; Development
16.  A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector 
BMC Molecular Biology  2013;14:18.
Background
Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous.
Results
We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells.
Conclusions
We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins.
doi:10.1186/1471-2199-14-18
PMCID: PMC3765358  PMID: 23957834
Gateway cloning; Fusion protein; Combinatorial; Fluorescent protein; Epitope tag
17.  Three-stage biochemical selection: cloning of prototype class IIS/IIC/IIG restriction endonuclease-methyltransferase TsoI from the thermophile Thermus scotoductus 
BMC Molecular Biology  2013;14:17.
Background
In continuing our research into the new family of bifunctional restriction endonucleases (REases), we describe the cloning of the tsoIRM gene. Currently, the family includes six thermostable enzymes: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI, TsoI, isolated from various Thermus sp. and two thermolabile enzymes: RpaI and CchII, isolated from mesophilic bacteria Rhodopseudomonas palustris and Chlorobium chlorochromatii, respectively. The enzymes have several properties in common. They are large proteins (molecular size app. 120 kDa), coded by fused genes, with the REase and methyltransferase (MTase) in a single polypeptide, where both activities are affected by S-adenosylmethionine (SAM). They recognize similar asymmetric cognate sites and cleave at a distance of 11/9 nt from the recognition site. Thus far, we have cloned and characterised TaqII, Tth111II, TthHB27I, TspGWI and TspDTI.
Results
TsoI REase, which originate from thermophilic Thermus scotoductus RFL4 (T. scotoductus), was cloned in Escherichia coli (E. coli) using two rounds of biochemical selection of the T. scotoductus genomic library for the TsoI methylation phenotype. DNA sequencing of restriction-resistant clones revealed the common open reading frame (ORF) of 3348 bp, coding for a large polypeptide of 1116 aminoacid (aa) residues, which exhibited a high level of similarity to Tth111II (50% identity, 60% similarity). The ORF was PCR-amplified, subcloned into a pET21 derivative under the control of a T7 promoter and was subjected to the third round of biochemical selection in order to isolate error-free clones. Induction experiments resulted in synthesis of an app. 125 kDa protein, exhibiting TsoI-specific DNA cleavage. Also, the wild-type (wt) protein was purified and reaction optima were determined.
Conclusions
Previously we identified and cloned the Thermus family RM genes using a specially developed method based on partial proteolysis of thermostable REases. In the case of TsoI the classic biochemical selection method was successful, probably because of the substantially lower optimal reaction temperature of TsoI (app. 10-15°C). That allowed for sufficient MTase activity in vivo in recombinant E. coli. Interestingly, TsoI originates from bacteria with a high optimum growth temperature of 67°C, which indicates that not all bacterial enzymes match an organism’s thermophilic nature, and yet remain functional cell components. Besides basic research advances, the cloning and characterisation of the new prototype REase from the Thermus sp. family enzymes is also of practical importance in gene manipulation technology, as it extends the range of available DNA cleavage specificities.
doi:10.1186/1471-2199-14-17
PMCID: PMC3751577  PMID: 23919831
18.  APOBEC3 inhibits DEAD-END function to regulate microRNA activity 
BMC Molecular Biology  2013;14:16.
The RNA binding protein DEAD-END (DND1) is one of the few proteins known to regulate microRNA (miRNA) activity at the level of miRNA-mRNA interaction. DND1 blocks miRNA interaction with the 3′-untranslated region (3′-UTR) of specific mRNAs and restores protein expression. Previously, we showed that the DNA cytosine deaminase, APOBEC3 (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide like 3), interacts with DND1. APOBEC3 has been primarily studied for its role in restricting and inactivating retroviruses and retroelements. In this report, we examine the significance of DND1-APOBEC3 interaction. We found that while human DND1 inhibits miRNA-mediated inhibition of P27, human APOBEC3G is able to counteract this repression and restore miRNA activity. APOBEC3G, by itself, does not affect the 3′-UTR of P27. We found that APOBEC3G also blocks DND1 function to restore miR-372 and miR-206 inhibition through the 3′-UTRs of LATS2 and CX43, respectively. In corollary experiments, we tested whether DND1 affects the viral restriction function or mutator activity of APOBEC3. We found that DND1 does not affect APOBEC3 inhibition of infectivity of exogenous retrovirus HIV (ΔVif) or retrotransposition of MusD. In addition, examination of Ter/Ter;Apobec3−/− mice, lead us to conclude that DND1 does not regulate the mutator activity of APOBEC3 in germ cells. In summary, our results show that APOBEC3 is able to modulate DND1 function to regulate miRNA mediated translational regulation in cells but DND1 does not affect known APOBEC3 function.
doi:10.1186/1471-2199-14-16
PMCID: PMC3729616  PMID: 23890083
DND1; APOBEC3G; APOBEC3; microRNA; P27
19.  Loss of CARM1 is linked to reduced HuR function in replicative senescence 
BMC Molecular Biology  2013;14:15.
Background
The co-activator-associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of HuR. However, the functional impact of this modification is not fully understood. Here, we investigated the influence of HuR methylation by CARM1 upon the turnover of HuR target mRNAs encoding senescence-regulatory proteins.
Results
Changing the methylation status of HuR in HeLa cells by either silencing CARM1 or mutating the major methylation site (R217K) greatly diminished the effect of HuR in regulating the turnover of mRNAs encoding cyclin A, cyclin B1, c-fos, SIRT1, and p16. Although knockdown of CARM1 or HuR individually influenced the expression of cyclin A, cyclin B1, c-fos, SIRT1, and p16, joint knockdown of both CARM1 and HuR did not show further effect. Methylation by CARM1 enhanced the association of HuR with the 3′UTR of p16 mRNA, but not with the 3′UTR of cyclin A, cyclin B1, c-fos, or SIRT1 mRNAs. In senescent human diploid fibroblasts (HDFs), reduced CARM1 was accompanied by reduced HuR methylation. In addition, knockdown of CARM1 or mutation of the major methylation site of HuR in HDF markedly impaired the ability of HuR to regulate the expression of cyclin A, cyclin B1, c-fos, SIRT1, and p16 as well to maintain a proliferative phenotype.
Conclusion
CARM1 represses replicative senescence by methylating HuR and thereby enhancing HuR’s ability to regulate the turnover of cyclin A, cyclin B1, c-fos, SIRT1, and p16 mRNAs.
doi:10.1186/1471-2199-14-15
PMCID: PMC3718661  PMID: 23837869
CARM1; HuR methylation; mRNA turnover; Replicative senescence
20.  Tip110 interacts with YB-1 and regulates each other’s function 
BMC Molecular Biology  2013;14:14.
Background
Tip110 plays important roles in tumor immunobiology, pre-mRNA splicing, expression regulation of viral and host genes, and possibly protein turnover. It is clear that our understanding of Tip110 biological function remains incomplete.
Results
Herein, we employed an immunoaffinity-based enrichment approach combined with protein mass spectrometry and attempted to identify Tip110-interacting cellular proteins. A total of 13 major proteins were identified to be complexed with Tip110. Among them was Y-box binding protein 1 (YB-1). The interaction of Tip110 with YB-1 was further dissected and confirmed to be specific and involve the N-terminal of both Tip110 and YB-1 proteins. A HIV-1 LTR promoter-driven reporter gene assay and a CD44 minigene in vivo splicing assay were chosen to evaluate the functional relevance of the Tip110/YB-1 interaction. We showed that YB-1 potentiates the Tip110/Tat-mediated transactivation of the HIV-1 LTR promoter while Tip110 promotes the inclusion of the exon 5 in CD44 minigene alternative splicing.
Conclusions
Tip110 and YB-1 interact to form a complex and mutually regulate each other’s biological functions.
doi:10.1186/1471-2199-14-14
PMCID: PMC3716619  PMID: 23822148
HIV-1 Tat; Tip110; YB-1; Alternative Splicing; CD44; Transcription
21.  How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population 
BMC Molecular Biology  2013;14:13.
Background
Flower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population.
Results
An accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H.
Conclusions
Currently in plant research, validated and qualitative RT-qPCR protocols are still rare. The protocol in this study can be implemented on all plant species to assure accurate quantification of gene expression. We have been able to correlate flower colour to the combined regulation of structural genes, both in the early and late branch of the pathway. This allowed us to differentiate between flower colours in a broader genetic background as was done so far in flower colour studies. These data will now be used for eQTL mapping to comprehend even more the regulation of this pathway.
doi:10.1186/1471-2199-14-13
PMCID: PMC3698002  PMID: 23800303
RT-qPCR; Flower colour; RNA quality; noRT; Standard curves; Reference genes; Gene expression; Pink
22.  Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element 
BMC Molecular Biology  2013;14:12.
Background
Ribosomal protein L30 belongs to the L7Ae family of RNA-binding proteins, which recognize diverse targets. L30 binds to kink-turn motifs in the 28S ribosomal RNA, L30 pre-mRNA, and mature L30 mRNA. L30 has a noncanonical function as a component of the UGA recoding machinery that incorporates selenocysteine (Sec) into selenoproteins during translation. L30 binds to a putative kink-turn motif in the Sec Insertion Sequence (SECIS) element in the 3’ UTR of mammalian selenoprotein mRNAs. The SECIS also interacts with SECIS-binding protein 2 (SBP2), an essential factor for Sec incorporation. Previous studies showed that L30 and SBP2 compete for binding to the SECIS in vitro. The SBP2:SECIS interaction has been characterized but much less is known about how L30 recognizes the SECIS.
Results
Here we use enzymatic RNA footprinting to define the L30 binding site on the SECIS. Like SBP2, L30 protects nucleotides in the 5’ side of the internal loop, the 5’ side of the lower helix, and the SECIS core, including the GA tandem base pairs that are predicted to form a kink-turn. However, L30 has additional determinants for binding as it also protects nucleotides in the 3’ side of the internal loop, which are not protected by SBP2. In support of the competitive binding model, we found that purified L30 repressed UGA recoding in an in vitro translation system, and that this inhibition was rescued by SBP2. To define the amino acid requirements for SECIS-binding, site-specific mutations in L30 were generated based on published structural studies of this protein in a complex with its canonical target, the L30 pre-mRNA. We identified point mutations that selectively inhibited binding of L30 to the SECIS, to the L30 pre-mRNA, or both RNAs, suggesting that there are subtle differences in how L30 interacts with the two targets.
Conclusions
This study establishes that L30 and SBP2 bind to overlapping but non-identical sites on the SECIS. The amino acid requirements for the interaction of L30 with the SECIS differ from those that mediate binding to the L30 pre-mRNA. Our results provide insight into how L7Ae family members recognize their cognate RNAs.
doi:10.1186/1471-2199-14-12
PMCID: PMC3706390  PMID: 23777426
RNA-binding protein; L30; SECIS-binding protein 2; Selenocysteine; Selenoprotein
23.  Characterization of Stra8 in Southern catfish (Silurus meridionalis): evidence for its role in meiotic initiation 
BMC Molecular Biology  2013;14:11.
Background
RA (retinoic acid) signal pathway has been proved to be required for germ cell meiotic initiation in mammals, aves and amphibians. Stra8 (Stimulated by retinoic acid gene 8) is an important factor in RA signal pathway. However, the role of RA and Stra8 in germ cell meiotic initiation in teleosts is poorly characterized.
Results
In this study, the full length cDNA of Stra8 was cloned from Southern catfish (Silurus meridionalis), and its spatio-temporal expression profiles were analyzed. The Stra8 cDNA (1606 bp) includes 163 bp 5’-UTR (untranslated region), 456 bp 3’-UTR, and an ORF (open reading frame) of 987 bp, encoding a polypeptide of 328 aa. Phylogenetic analysis revealed its existence in some primitive teleosts, such as Siluriformes and Salmoniformes. Tissue distribution analysis by RT-PCR showed that Stra8 is specifically expressed in gonads. By real-time PCR, in situ hybridization and immunohistochemistry, the highest expression level of Stra8/Stra8 was detected in 50 and 130 dah (day after hatching), the premeiotic stage of germ cells in XX and XY gonads, respectively.
Conclusions
Our results suggest that Stra8 might be involved in germ cell meiotic initiation in S. meridionalis as it did in tetrapods.
doi:10.1186/1471-2199-14-11
PMCID: PMC3668142  PMID: 23697400
Stra8; mRNA and protein; Expression profile; Meiotic initiation; Silurus meridionalis
24.  Screening and analysis of PoAkirin1 and two related genes in response to immunological stimulants in the Japanese flounder (Paralichthys olivaceus) 
BMC Molecular Biology  2013;14:10.
A member of the NF-κB signaling pathway, PoAkirin1, was cloned from a full-length cDNA library of Japanese flounder (Paralichthys olivaceus). The full-length cDNA comprises a 5′UTR of 202 bp, an open reading frame of 564 bp encoding a 187-amino-acid polypeptide and a 521-bp 3′UTR with a poly (A) tail. The putative protein has a predicted molecular mass of 21 kDa and an isoelectric point (pI) of 9.22. Amino acid sequence alignments showed that PoAkirin1 was 99% identical to the Scophthalmus maximus Akirin protein (ADK27484). Yeast two-hybrid assays identified two proteins that interact with PoAkirin1: PoHEPN and PoC1q. The cDNA sequences of PoHEPN and PoC1q are 672 bp and 528 bp, respectively. Real-time quantitative reverse-transcriptase polymerase chain reaction analysis showed that bacteria could induce the expressions of PoAkirin1, PoHEPN and PoC1q. However, the responses of PoHEPN and PoC1q to the bacterial challenge were slower than that of PoAkirin1. To further study the function of PoAkirin1, recombinant PoAkirin1 and PoHEPN were expressed in Escherichia coli and would be used to verify the PoAkirin1-PoHEPN binding activity. These results identified two proteins that potentially interact with PoAkirin1 and that bacteria could induce their expression.
doi:10.1186/1471-2199-14-10
PMCID: PMC3700861  PMID: 23651673
Akirin; Japanese flounder; NF-κB; Yeast two-hybrid assay; Immunity; HEPN; C1q
25.  Human single-stranded DNA binding proteins are essential for maintaining genomic stability 
The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. However, there are various instances where single-stranded DNA is exposed, such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. In these instances, single-stranded DNA binding proteins are essential for the sequestration and processing of single-stranded DNA. In order to bind single-stranded DNA, these proteins utilise a characteristic and evolutionary conserved single-stranded DNA-binding domain, the oligonucleotide/oligosaccharide-binding (OB)-fold. In the current review we discuss a subset of these proteins involved in the direct maintenance of genomic stability, an important cellular process in the conservation of cellular viability and prevention of malignant transformation. We discuss the central roles of single-stranded DNA binding proteins from the OB-fold domain family in DNA replication, the restart of stalled replication forks, DNA damage repair, cell cycle-checkpoint activation, and telomere maintenance.
doi:10.1186/1471-2199-14-9
PMCID: PMC3626794  PMID: 23548139
Single-stranded DNA binding proteins (SSBs); Oligonucleotide/oligosaccharide binding (OB)-fold; Double-strand DNA break (DSB) repair; Homology-directed repair (HDR); Translesion synthesis; Nucleotide excision repair (NER); Replication fork restart; Cell cycle checkpoint activation; Telomere maintenance

Results 1-25 (705)