Cloning of a Picea abies monosaccharide transporter gene and expression – analysis in plant tissues and ectomycorrhizas

Ectomycorrhizas are formed between certain soil fungi and fine roots of woody plants. An important feature of this symbiosis is the supply of photoassimilates to the fungus. Hexoses, formed from sucrose in the common apoplast at the root/fungus interface, can be taken up by both plant and fungal monosaccharide transporters. Recently we characterised a monosaccharide transporter from the ectomycorrhizal fungus Amanita muscaria. This transporter was up-regulated in mycorrhizas, thus increasing the hexose uptake capacity of the fungal partner in symbiosis. In order to characterise host (Picea abies) root monosaccharide transporters, degenerate oligonucleotide primers, designed to match conserved regions from known plant hexose transporters, were used to isolate a cDNA fragment of a transporter by PCR. This fragment was used to identify a presumably full length clone (PaMST1) in a P. abies/A. muscaria mycorrhizal cDNA library. The entire cDNA code for an open reading frame of 513 amino acids, revealing best homology to H⁺/monosaccharide transporters from Ara- bidopsis, Saccharum and Ricinus. PaMST1 was highly expressed in the hypocotyl and in roots of P. abies seedlings, but not in needles. Mycorrhiza formation led to a slight reduction of PaMST1 expression. The results are discussed with special reference to carbon allocation in ectomycorrhizas. Received: 9 October 1999 / Accepted: 22 December 1999     

181 Integrative analysis of gene expression and protein–protein interaction networks in Glioblastoma

Glioblastoma multiforme (GBM) is the most malignant of all the brain tumors with very low median survival time of one year, as per Central Brain Tumor Registry of the USA, 2001. Efforts are ongoing to understand this disease pathogenesis in complete details. Global gene expression changes in GBM pathogenesis have been studied by several groups using microarray technology (e.g. Carro et al., 2010). One of the many approaches to ‘understand the control mechanisms underlying the observed changes in the activity of a biological process’ (Cline et al., 2007) is integration of gene expression and protein–protein interactions (PPI) datasets. Among several examples, aberrant activation of Wnt/β-catenin signaling pathway as well as sonic hedgehog (SHH) signaling pathway is reported in GBMs (Klaus & Birchmeier, 2008). Further, these two pathways are also involved in proliferation and clonogenicity of glioma cancer stem cells (Li et al., 2009), which are thought to play a role in glioma initiation, proliferation, and invasion, and are one of the important points of intervention. Hedgehog–Gli1 signaling is also found to regulate the expression of stemness genes. In this paper, analyses of the relationship between the significant differential expression of these and other genes and the connectivity as well as topological features of a PPI network would be discussed. This way, genes potentially overlooked when relying solely on expression profiles may be identified which can be biologically relevant as possible drug target/s or disease biomarker/s.     

Culture–gene coevolution of individualism–collectivism and the serotonin transporter gene

Culture–gene coevolutionary theory posits that cultural values have evolved, are adaptive and influence the social and physical environments under which genetic selection operates. Here, we examined the association between cultural values of individualism–collectivism and allelic frequency of the serotonin transporter functional polymorphism (5-HTTLPR) as well as the role this culture–gene association may play in explaining global variability in prevalence of pathogens and affective disorders. We found evidence that collectivistic cultures were significantly more likely to comprise individuals carrying the short (S) allele of the 5-HTTLPR across 29 nations. Results further show that historical pathogen prevalence predicts cultural variability in individualism–collectivism owing to genetic selection of the S allele. Additionally, cultural values and frequency of S allele carriers negatively predict global prevalence of anxiety and mood disorder. Finally, mediation analyses further indicate that increased frequency of S allele carriers predicted decreased anxiety and mood disorder prevalence owing to increased collectivistic cultural values. Taken together, our findings suggest culture–gene coevolution between allelic frequency of 5-HTTLPR and cultural values of individualism–collectivism and support the notion that cultural values buffer genetically susceptible populations from increased prevalence of affective disorders. Implications of the current findings for understanding culture–gene coevolution of human brain and behaviour as well as how this coevolutionary process may contribute to global variation in pathogen prevalence and epidemiology of affective disorders, such as anxiety and depression, are discussed.     

Genome-wide investigation of gene–environment interactions in colorectal cancer

Colorectal cancer (CRC), one of the most frequent neoplasias worldwide, has both genetic and environmental causes. As yet, however, gene–environment (G × E) interactions in CRC have been studied mostly for a small number of candidate genes only. Therefore, we investigated the possible interaction, in CRC etiology, between single-nucleotide polymorphisms (SNPs) on the one hand, and overweight, smoking and alcohol consumption on the other, at a genome-wide level. To this end, we adopted a two-tiered approach comprising a case-only screening stage I (314 cases) and a case–control validation stage II (259 cases, 1,002 controls). Interactions with the smallest p value in stage I were verified in stage II using multiple logistic regression analysis adjusted for sex and age. In addition, we specifically studied known CRC-associated SNPs for possible G × E interactions. Upon adjustment for sex and age, and after allowing for multiple testing, however, only a single SNP (rs1944511) was found to be involved in a statistically significant interaction, namely with overweight (multiplicity-corrected p = 0.042 in stage II). Several other G × E interactions were nominally significant but failed correction for multiple testing, including a previously reported interaction between rs9929218 and alcohol consumption that also emerged in our candidate SNP study (nominal p = 0.008). Notably, none of the interactions identified in our genome-wide analysis was with a previously reported CRC-associated SNP. Our study therefore highlights the potential of an “agnostic” genome-wide approach to G × E analysis.     

Molecular and expression analysis of manganese superoxide dismutase (Mn–SOD) gene under temperature and starvation stress in rotifer Brachionus calyciflorus

Superoxide dismutase (SOD) is an important antioxidant enzyme that protects organs from damage by reactive oxygen species. We cloned cDNA encoding SOD activated with manganese (Mn–SOD) from the rotifer Brachionus calyciflorus Pallas. The full-length cDNA of Mn–SOD was 1,016 bp and had a 669 bp open reading frame encoding 222 amino acids. The deduced amino acid sequence of B. calyciflorus Mn–SOD showed 89.1, 71.3, and 62.1 % similarity with the Mn–SOD of the marine rotifer Brachionus plicatilis, the nematode Caenorhabditis elegans, and the fruit fly Drosophila melanogaster, respectively. The phylogenetic tree constructed based on the amino acid sequences of Mn–SODs from B. calyciflorus and other organisms revealed that this rotifer is closely related to nematodes. Analysis of the mRNA expression of Mn–SOD under different conditions revealed that expression was enhanced 5.6-fold (p < 0.001) at 30 °C after 2 h, however, low temperature (15 °C) promoted Mn SOD temporarily (2.5-fold, p < 0.001) and then decreased to normal level (p > 0.05). Moderate starvation promoted Mn–SOD mRNA expression (p ₁₂ < 0.01, p ₃₆ < 0.05), which reached a maximum value (15.3 times higher than control, p ₂₄ < 0.01) at 24 h. SOD and CAT activities also elevated at the 12 h–starved group. These results indicate that induction of Mn–SOD expression by stressors likely plays an important role in aging of B. calyciflorus.     

Genome-wide copy number variation study and gene expression analysis identify ABI3BP as a susceptibility gene for Kashin–Beck disease

Kashin–Beck disease (KBD) is a chronic osteochondropathy. In this study, we conducted the first genome-wide copy number variation study (GCNVS) of KBD totally involving 2,743 Chinese Han adults. GCNVS was first performed using Affymetrix Human SNP6.0 Arrays. The identified copy number variations (CNVs) were then replicated in an independent Chinese Han sample containing 1,026 subjects. SNP genotyping, CNV identification and quality control were implemented by Birdsuite. STRUCTURE and EIGENSTRAT were applied for controlling potential population stratification in the GCNVS. Association analysis was conducted using PLINK. Microarray and qRT-PCR were also conducted to compare the expression levels of the genes overlapping with identified CNVs between KBD patients and healthy controls. GCNVS found that CNV452 (P value = 7.78 × 10^?5) overlapping with ABI3BP gene was significantly associated with KBD. Replication association study observed that rs9850273 (P value = 0.008) and rs7613610 (P value = 0.021) in ABI3BP gene were significantly associated with KBD. Gene expression analysis also found that ABI3BP was up-regulated in KBD patients compared to healthy controls. Our results suggest that ABI3BP was a novel susceptibility gene for KBD.     

Genome-wide analysis of the phospholipase D family in Oryza sativa and functional characterization of PLDβ1 in seed germination

Phospholipase D （PLD） plays a critical role in plant growth and development, as well as in hormone and stress responses. PLD encoding genes constitute a large gene family that are present in higher plants. There are 12 members of the PLD family in Arabidopsis thaliana and several of them have been functionally characterized; however, the members of the PLD family in Oryza sativa remain to be fully described. Through genome-wide analysis, 17 PLD members found in different chromosomes have been identified in rice. Protein domain structural analysis reveals a novel subfamily, besides the C2-PLDs and PXPH-PLDs, that is present in rice - the SP-PLD. SP-PLD harbors a signal peptide instead of the C2 or PXPH domains at the N-terminus. Expression pattern analysis indicates that most PLD-encoding genes are differentially expressed in various tissues, or are induced by hormones or stress conditions, suggesting the involvement of PLD in multiple developmental processes. Transgenic studies have shown that the suppressed expression office PLDβ1 results in reduced sensitivity to exogenous ABA during seed germination. Further analysis of the expression of ABA signaling-related genes has revealed that PLDβ1 stimulates ABA signaling by activating SAPK, thus repressing GAmyb exoression and inhibiting seed germination.     

Molecular characterization and functional analysis of “fruit-weight2.2-like” gene family in rice

Tomato fruit-weight 2.2 (FW2.2) was reported to control up to 30 % fruit weight. Recent studies demonstrated that FW2.2-like (FWL) genes also play important roles in plant growth and development. For instance, a maize homolog of FW2.2, named cell number regulator 1 (CNR1), negatively regulates plant and organ size. However, FWL genes in rice have not been characterized yet. In this study, eight FWL genes were identified in rice genome and designated as OsFWL1-8. The chromosome location, gene structure, protein motif, and phylogenetic relationship of OsFWL genes were analyzed. RT-PCR result and microarray data revealed that OsFWL genes exhibited diverse expression patterns and the detailed expression patterns of OsFWL5, 6, and 7 negatively correlated with leaf growth activity. Rice protoplast transient transformation experiment showed that most OsFWL proteins locate at cell membrane but OsFWL8 is present in the nucleus. In addition, the functions of OsFWL genes were investigated by analyzing two T-DNA insertion lines for OsFWL3 and 5. Compared with wild type, the grain weight of osfwl3 mutant and the plant height of osfwl5 mutant were increased by 5.3 and 12.5 %, respectively. We also found that the increase in grain length of osfwl3 mutant was due chiefly to incremental cell number, not cell size and the expression of OsFWL3 negatively correlated with glume growth activity. These results provide a comprehensive foundation for further study of OsFWL functions in rice.     

Molecular cloning and structural analysis of human norepinephrine transporter gene（NETHG）

A cDNA molecule encoding a major part of the human Norepinephrine transporter(hNET) was synthesized by means of Polymerase Chain Reaction(PCR) technique and used as a probe for selecting the human genomic NET gene.A positive clone harbouring the whole gene was obtained from a human lymphocyte genomic library through utilizing the “genomic walking” technique.The clone,designated as phNET,harbours a DNA fragment of about 59 kd in length inserted into BamH I site in cosmid pWE15.The genomic clone contains 14 exons encoding all amino acid residues in the protein.A single exon encodes a distinct transmembrane domain,except for transmembrane domain 10 and 11,which are encoded by part of two exons respectively,and exon 12,which encodes part of domain 11 and all of domain 12.These results imply that there is a close relationship between exon splicing of a gene and structureal domains of the protein,as is the case for the human γ－aminobutyric acid transporter(hGAT) and a number of other membrane proteins.     

Characterization and expression profiling of selected microRNAs in tomato (Solanum lycopersicon) ‘Jiangshu14’

Presence of selected tomato (Solanum lycopersicon) microRNAs (sly-miRNAs) was validated and their expression profiles established in roots, stems, leaves, flowers and fruits of tomato variety Jiangshu14 by quantitative RT-PCR (qRT-PCR). In addition conservation characteristics these sly-miRNAs were analyzed and target genes predicted bioinformatically. Results indicate that some of these miRNAs are specific to tomato while most are conserved in other plant species. Predicted sly-miRNA targets genes were shown to be targeted by either by a single or more miRNAs and are involved in diverse processes in tomato plant growth and development. All the 36 miRNAs were present in the cDNA of mixed tissues and qRT-PCR revealed that some of these sly-miRNAs are ubiquitous in tomato while others have tissue-specific expression. The experimental validation and expression profiling as well target gene prediction of these miRNAs in tomato as done in this study can add to the knowledge on the important roles played by these sly-miRNAs in the growth and development, environmental stress tolerance as well as pest and disease resistance in tomatoes and related species. In addition these findings broaden the knowledge of small RNA-mediated regulation in S. lycopersicon. It is recommended that experimental validation of the target genes be done so as to give a much more comprehensive information package on these miRNAs in tomato and specifically in the selected variety.