The MerE protein encoded by transposon Tn21 is a broad mercury transporter in Escherichia coli

In order to clarify the physiological role of the merE gene of transposon Tn21, a pE4 plasmid that contained the merR gene of plasmid pMR26 from Pseudomonas strain K-62, and the merE gene of Tn21 from the Shigella flexneri plasmid NR1 (R100) was constructed. Bacteria with plasmid pE4 (merR-o/p-merE) were more hypersensitive to CH₃Hg(I) and Hg(II), and took up significantly more CH₃Hg(I) and Hg(II), than the isogenic strain. The MerE protein encoded by pE4 was localized in the membrane cell fraction, but not in the soluble fraction. Based on these experimental results, we suggest for the first time that the merE gene is a broad mercury transporter mediating the transport of both CH₃Hg(I) and Hg(II) across the bacterial membrane.     

荧光转基因斑马鱼Tg(ttn.2:EGFP)的构建

为了建立一种用于研究肌肉和心脏发育及其相关疾病的绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因斑马鱼品系,本研究使用斑马鱼ttn.2基因编码区上游启动子序列和绿色荧光蛋白基因编码序列构建了重组表达载体,并将该载体和Tol2转座酶的加帽mRNA显微共注射入斑马鱼1-细胞期胚胎,通过荧光检测、遗传杂交筛选和分子鉴定等方法,成功建立了能稳定遗传的Tg(ttn.2:EGFP)转基因斑马鱼品系。荧光表达分析及原位杂交分析结果表明,绿色荧光信号在斑马鱼肌肉和心脏组织中特异表达模式与ttn.2基因的mRNA表达一致。通过反向PCR鉴定转基因表达载体在F1代斑马鱼品系中的随机整合位点,结果表明:No.33转基因品系的EGFP基因整合在斑马鱼的4号和11号染色体上,No.34转基因品系则整合在1号染色体上。该荧光转基因斑马鱼品系Tg(ttn.2:EGFP)的成功构建为肌肉和心脏发育以及相关疾病研究提供了一个新的理想实验模型。此外,绿色荧光强烈表达的斑马鱼品系还可以作为一种新的观赏鱼。     

Molecular analysis of SMN1, SMN2, NAIP, GTF2H2, and H4F5 genes in 157 Chinese patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is a common and lethal autosomal recessive neurodegenerative disorder, which is caused by mutations of the survival motor neuron 1 (SMN1) gene. Additionally, the phenotype is modified by several genes nearby SMN1 in the 5q13 region. In this study, we analyzed mutations in SMN1 and quantified the modifying genes, including SMN2, NAIP, GTF2H2, and H4F5 by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), multiplex ligation-dependent probe amplification (MLPA), TA cloning, allele-specific long-range PCR, and Sanger sequencing in 157 SMA patients. Most SMA patients (94.90%) possessed a homozygous SMN1 deletion, while 10 patients demonstrated only the absence of exon 7, but the presence of exon 8. Two missense mutations (c.689 C > T and c.844 C > T) were identified in 2 patients who both carried a single copy of SMN1. We found inverse correlations between SMN2, the NAIP copy number, and the clinical severity of the disease. Furthermore, 7 severe type I patients possessed large-scale deletions, including SMN1, NAIP, and GTF2H2. We conclude that SMN1 gene conversion, SMN1 subtle mutations, SMN2 copy number, and the extent of deletion in the 5q13 region should all be considered in the genotype–phenotype analysis of SMA.     

Loss of Trx-2 enhances oxidative stress-dependent phenotypes in Drosophila

Overexpression of thioredoxin (TRX) confers oxidative stress resistance and extends lifespan in mammals and insects. However, less is known about phenotypes associated with loss of TRX. We investigated loss-of-function phenotypes of Trx-2 in Drosophila, and found that the mutant flies are hyper-susceptible to paraquat, a free radical generator, but not to hydrogen peroxide. They contain a high amount of protein carbonyl, which dramatically increases with age. Trx-2 mutants express high levels of anti-oxidant genes, such as superoxide dismutase, catalase, and glutathione synthetase. This is the first demonstration of biochemical and physiological consequences caused by loss of Trx-2 in Drosophila.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.     

Ectopic expression of FaesAP3, a Fagopyrum esculentum (Polygonaceae) AP3 orthologous gene rescues stamen development in an Arabidopsis ap3 mutant

Arabidopsis thaliana APETALA3 (AP3) and Antirrhinum majus DEFICIENS (DEF) MADS box genes are required to specify petal and stamen identity. AP3 and DEF are members of the euAP3 lineage, which arose by gene duplication coincident with radiation of the core eudicots. In order to investigate the molecular mechanisms underlying organ development in early diverging clades of core eudicots, we isolated and identified an AP3 homolog, FaesAP3, from Fagopyrum esculentum (buckwheat, Polygonaceae), a multi-food-use pseudocereal with healing benefits. Protein sequence alignment and phylogenetic analyses revealed that FaesAP3 grouped into the euAP3 lineage. Expression analysis showed that FaesAP3 was transcribed only in developing stamens, and differed from AP3 and DEF, which expressed in developing petals and stamens. Moreover, ectopic expression of FaesAP3 rescued stamen development without complementation of petal development in an Arabidopsis ap3 mutant. Our results suggest that FaesAP3 is involved in the development of stamens in buckwheat. These results also suggest that FaesAP3 holds some potential for biotechnical engineering to create a male sterile line of F. esculentum.     

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H₂O₂) through the plasma membrane decreases during adaptation to H₂O₂ by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H₂O₂ the anisotropy of the plasma membrane increases. Adaptation to H₂O₂ was studied at several times (15min up to 90min) by applying the steady-state H₂O₂ delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H₂O₂ was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H₂O₂ adaptation. H₂O₂ diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H₂O₂ permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H₂O₂ is mediated by modulation of the biophysical properties of the plasma membrane.     

Retinoic acid signaling in perioptic mesenchyme represses Wnt signaling via induction of Pitx2 and Dkk2

Morphogenesis during eye development requires retinoic acid (RA) receptors plus RA-synthesizing enzymes, and loss of RA signaling leads to ocular disorders associated with loss of Pitx2 expression in perioptic mesenchyme. Several Wnt signaling components are expressed in ocular tissues during eye development including Dkk2, encoding an inhibitor of Wnt/β-catenin signaling, which was previously shown to be induced by Pitx2 in the perioptic mesenchyme. Here, we investigated potential cross-talk between RA and Wnt signaling during ocular development. Genetic studies using Raldh1/Raldh3 double null mice deficient for ocular RA synthesis demonstrated that Pitx2 and Dkk2 were both down-regulated in perioptic mesenchyme. Chromatin immunoprecipitation and gel mobility shift studies demonstrated the existence of a DR5 RA response element upstream of Pitx2 that binds all three RA receptors in embryonic eye. Axin2, an endogenous readout of Wnt/β-catenin signaling, was up-regulated in cornea and perioptic mesenchyme of RA deficient embryos. Also, expression of Wnt5a was expanded in perioptic mesenchyme of RA deficient eyes. Our findings demonstrate excessive activation of Wnt signaling in the perioptic mesenchyme of RA deficient mice which may be responsible for abnormal development leading to defective optic cup, cornea, and eyelid morphogenesis.     

The identification of microRNAs in the whitespotted bamboo shark (Chiloscyllium plagiosum) liver by Illumina sequencing

MicroRNAs are indispensable players in the regulation of a broad range of biological processes. Here, we report the first deep sequencing of the whitespotted bamboo shark (Chiloscyllium plagiosum) liver. We mapped 91 miRNAs in the Callorhinchus milii genome that have previously been described in the Danio rerio, Fugu rubripes, Oryzias latipes, Xenopus laevis, Xenopus tropicalis, Homo sapiens, and Mus musculus. In addition, 156 new putative candidate (PC) C. plagiosum miRNAs were identified. From these 247 miRNAs, 39 miRNA clusters were identified, and the expression of these clustered miRNAs was observed to vary significantly. A total of 7 candidate miRNAs were selected for expression confirmation by stem-loop RT-PCR. This study resulted in the addition of a significant number of novel miRNA sequences to GenBank and laid the foundation for further understanding of the function of miRNAs in the regulation of C. plagiosum liver development.     

BmICE-2 is a novel pro-apoptotic caspase involved in apoptosis in the silkworm, Bombyx mori

In this study we identified a potential pro-apoptotic caspase gene, Bombyx mori(B. mori)ICE-2 (BmICE-2) which encoded a polypeptide of 284 amino acid residues, including a ¹⁶⁹QACRG¹⁷³ sequence which surrounded the catalytic site and contained a p20 and a p10 domain. BmICE-2 expressed in Escherichia coli (E. coli) exhibited high proteolytic activity for the synthetic human initiator caspase-9 substrates Ac-LEHD-pNA, but little activity towards the effector caspase-3 substrates Ac-DEVD-pNA. When BmICE-2 was transiently expressed in BmN-SWU1 silkworm B. mori cells, we found that the high proteolytic activity for Ac-LEHD-pNA triggered caspase-3-like protease activity resulting in spontaneous cleavage and apoptosis in these cells. This effect was not replicated in Spodoptera frugiperda 9 cells. In addition, spontaneous cleavage of endogenous BmICE-2 in BmN-SWU1 cells could be induced by actinomycin D. These results suggest that BmICE-2 may be a novel pro-apoptotic gene with caspase-9 activity which is involved apoptotic processes in BmN-SWU1 silkworm B. mori cells.     

Upregulation of PHLDA2 in Dicer knockdown HEK293 cells

It has been reported that RNAi-dependent chromatin silencing in vertebrates is not restricted to the centromeres. To address whether RNAi machinery could regulate the chromatin structure of imprinted genes, we knocked down Dicer in HEK293 cells and found that the expression of PHLDA2, one of the several genes in the imprinted gene domain of 11p15.5, was specifically upregulated. This was accompanied by a shift towards more activated chromatin at PHLDA2 locus as indicated by change in H3K9 acetylation, however, the methylation state at this locus was not affected. Furthermore, we found that PHLDA2 was downregulated in growth-arrested HEK293 cells induced by either serum deprivation or contact inhibition. This suggests that PHLDA2 upregulation might be a direct result of Dicer depletion rather than the consequence of growth arrest induced by Dicer knockdown. Considering the reports that there is consistent placental outgrowth in PHLDA2 knockout mice and that PHLDA2 overexpression in mice causes growth inhibition, we speculate that PHLDA2 may be a candidate for contributing to the reduced growth rate of Dicer-deficient cells and the very early embryonic lethality in Dicer knockout mice.     

Allele frequency distribution of CYP2C9*2 and CYP2C9*3 polymorphisms in six Mexican populations

Allele frequency differences of functional CYP2C9 polymorphisms are responsible for some of the variation in drug response observed in human populations. The most relevant CYP2C9 functional variants are CYP2C9*2 (rs1799853) and CYP2C9*3 (rs1057910). These polymorphisms show variation in allele frequencies among different population groups. The present study aimed to analyze these polymorphisms in 947 Mexican-Mestizo from Mexico City and 483 individuals from five indigenous Mexican populations: Nahua, Teenek, Tarahumara, Purepecha and Huichol. The CYP2C9*2 allele frequencies in the Mestizo, Nahua and Teenek populations were 0.051, 0.007 and 0.005, respectively. As for CYP2C9*3, the allelic frequencies in the Mestizo, Nahua and Teenek populations were 0.04, 0.005 and 0.005, respectively. The CYP2C9*2 and CYP2C9*3 alleles were not observed in the Tarahumara, Purepecha and Huichol populations. These findings are in agreement with previous studies reporting very low allele frequencies for these polymorphisms in American Indigenous populations.     

A cis-encoded sRNA controls the expression of fabH2 in Yersinia

YsrH is a novel cis-encoded sRNA located on the opposite strand to fabH2, which is essential for fatty acid biosynthesis in bacteria. In this study, YsrH-mediated regulation of fabH2 expression was investigated in Yersinia pseudotuberculosis. Constitutive and inducible over-expression of YsrH decreased the mRNA level of fabH2, while expression of downstream fabD and fabG remained unaffected. Polynucleotide phosphorylase (PNPase) also played an important role in this regulation process by mediating YsrH decay in the exponential phase. Thus, our data defines a cis-encoded sRNA that regulates fatty acid synthesis via a regulatory mechanism also involving PNPase.