http://www.biomedcentral.com/bmcmolbiol/ The latest research articles published by BMC Molecular Biology 2009-11-25T00:00:00Z This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ Background: 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. Trivalent arsenic (AsIII) is one of the major species that exists environmentally. The transport of AsIII has been studied in microbes, plant and mammals. Members of aquaglyceroporins family have been shown to actively conduct AsIII and its organic metabolite, monomethylarsenite (MAsIII). However, the transport in any fish species has not been characterized. Results: 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 uptake at equivalent rates. These aquaglyceroporins also facilitate uptake of inorganic trivalent arsenite (AsIII), organic monomethylarsenite (MAsIII) and trivalent antimonite (SbIII) in a mercury regulatory manner. 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. Conclusion: 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. http://www.biomedcentral.com/1471-2199/10/104 Mohamad Hamdi Marco Sanchez Lauren Beene Qianyong Liu Scott Landfear Barry Rosen Zijuan Liu BMC Molecular Biology 2009, 10:104 2009-11-25T00:00:00Z doi:10.1186/1471-2199-10-104 BMC Molecular Biology 1471-2199 10 104 2009-11-25T00:00:00Z PDF Background: The different isoforms of vascular endothelial growth factor (VEGF) play diverseroles in vascular growth, structure and function. Alternative splicing of the VEGF gene results inthe expression of three abundant isoforms: VEGF121, VEGF165 and VEGF189. The mRNA forVEGF189 contains the alternatively spliced exon 6A whereas the mRNA for VEGF165 lacksthis exon. The objective of this study was to identify the cis elements that control utilization ofexon 6A. A reporter minigene was constructed (pGFP-E6A) containing the coding sequence forGFP whose translation was dependent on faithful splicing for removal of the VEGF exon 6A. Toidentify cis-acting splicing elements, sequential deletions were made across exon 6A in thepGFP-E6A plasmid. Results: A candidate cis-acting exonic splicing silencer (ESS) comprising nucleotides 22-30 ofexon 6A sequence was identified corresponding to the a silencer consensus sequence ofAAGGGG. The function of this sequence as an ESS was confirmed in vivo both in the context ofthe reporter minigene as a plasmid and in the context of a longer minigene with VEGF exon 6Ain its native context in an adenoviral gene transfer vector. Further mutagenesis studies resulted inthe identification of the second G residue of the putative ESS as the most critical for function. Conclusion: This work establishes the identity of cis sequences that regulate alternative VEGFsplicing and dictate the relative expression levels of VEGF isoforms. http://www.biomedcentral.com/1471-2199/10/103 Rui Wang Ronald Crystal Neil Hackett BMC Molecular Biology 2009, 10:103 2009-11-17T00:00:00Z doi:10.1186/1471-2199-10-103 BMC Molecular Biology 1471-2199 10 103 2009-11-17T00:00:00Z PDF Background: 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. Results: 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. Conclusion: 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. http://www.biomedcentral.com/1471-2199/10/102 Xing-Rong Guo Si-Chun Zheng Lin Liu Qi-Li Feng BMC Molecular Biology 2009, 10:102 2009-11-13T00:00:00Z doi:10.1186/1471-2199-10-102 BMC Molecular Biology 1471-2199 10 102 2009-11-13T00:00:00Z XML Background: 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. Results: 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. Conclusion: 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. http://www.biomedcentral.com/1471-2199/10/101 Patrick Ng Sung-Kay Chiu Theresa Kwong Richard Yu Minnie Wong Richard Kong BMC Molecular Biology 2009, 10:101 2009-11-03T00:00:00Z doi:10.1186/1471-2199-10-101 BMC Molecular Biology 1471-2199 10 101 2009-11-03T00:00:00Z XML Background: 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. Results: 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. Conclusion: 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. http://www.biomedcentral.com/1471-2199/10/100 Caroline Walker Susanne Meier Murray Mitchell John Roche Mathew Littlejohn BMC Molecular Biology 2009, 10:100 2009-11-01T00:00:00Z doi:10.1186/1471-2199-10-100 BMC Molecular Biology 1471-2199 10 100 2009-11-01T00:00:00Z XML Background: 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. Results: 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. Conclusion: 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. http://www.biomedcentral.com/1471-2199/10/99 Marie-Ange Teste Manon Duquenne Jean Francois Jean-Luc Parrou BMC Molecular Biology 2009, 10:99 2009-10-30T00:00:00Z doi:10.1186/1471-2199-10-99 BMC Molecular Biology 1471-2199 10 99 2009-10-30T00:00:00Z XML Background: 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. Results: 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. Conclusion: 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. http://www.biomedcentral.com/1471-2199/10/98 Ellen Ordinario Munehisa Yabuki Priya Handa W. Jason Cummings Nancy Maizels BMC Molecular Biology 2009, 10:98 2009-10-28T00:00:00Z doi:10.1186/1471-2199-10-98 BMC Molecular Biology 1471-2199 10 98 2009-10-28T00:00:00Z XML Background: 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. Results: 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. Conclusion: 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. http://www.biomedcentral.com/1471-2199/10/97 Hualiang He Mei Cheng Xiao Yang Jinxiu Meng Ai He Xiaoying Zheng Zhuoya Li Pengjuan Guo Zhihua Pan Ximei Zhan BMC Molecular Biology 2009, 10:97 2009-10-25T00:00:00Z doi:10.1186/1471-2199-10-97 BMC Molecular Biology 1471-2199 10 97 2009-10-25T00:00:00Z XML Background: 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. Results: 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. Conclusion: 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. http://www.biomedcentral.com/1471-2199/10/96 Eduard Murani Siriluck Ponsuksili Hans-Martin Seyfert Xuanming Shi Klaus Wimmers BMC Molecular Biology 2009, 10:96 2009-10-21T00:00:00Z doi:10.1186/1471-2199-10-96 BMC Molecular Biology 1471-2199 10 96 2009-10-21T00:00:00Z XML Background: 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. Results: 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. Conclusion: 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. http://www.biomedcentral.com/1471-2199/10/95 Hernan Bondino Estela Valle BMC Molecular Biology 2009, 10:95 2009-10-16T00:00:00Z doi:10.1186/1471-2199-10-95 BMC Molecular Biology 1471-2199 10 95 2009-10-16T00:00:00Z XML