Real-time PCR Expression Analysis Research Articles

SIRP Alpha Protein Downregulates in Human Astrocytoma: Presumptive Involvement of Hsa-miR-520d-5p and Hsa-miR-520d-3p.

Astrocytomas are the most common brain tumors with poor survival in malignant forms. Signal regulatory protein alpha (SIRP alpha) is a transmembrane protein expressed on immune cells and macrophages and is reported to modulate tumor cell phagocytosis. In the present study, we investigated the involvement of miR-520d-5p and miR-520d-3p in regulation of SIRP alpha expression. Here, we report mRNA and protein expression profile of SIRP alpha in 39 surgically resected human astrocytoma tissue samples and 14 control brain tissue samples. Transcript expression pattern was studied by real-time PCR while Western blotting and immunohistochemistry were used to evaluate protein expression. Expression profile of miR-520d-5p and miR-520d-3p was studied by real-time PCR. Computational prediction was employed to analyze the binding of miR-520d-5p and miR-520d-3p for SIRP alpha mRNA. It is evident from preliminary investigation that SIRP alpha transcripts are expressed in control brain tissues, increased in low-grade (grade II) tumor tissues, and decreased with further grade progression (P<0.05). SIRP alpha protein was moderately expressed in control brain tissues but under-expressed in low- and high-grade tissue samples (P<0.05). Immunohistochemistry results further confirmed Western blot outcomes. Computational prediction supplemented with 3' and 5'UTR targeting analysis and correlation studies reveals that hsa-miR-520d-5p (P=0.028, R 2=0.94) (95% CI 0.15 to 0.99) and hsa-miR-520d-3p (P=0.027, R 2=0.94) (95% CI 0.17 to 0.99) may be the putative microRNAs involved in regulation of SIRP alpha protein expression. Real-time PCR expression profile depicts that mature form of both miRNAs is significantly overexpressed in low-grade (GII) tumor tissue samples compared to control and high-grade (GIII and GIV) tissue samples. MiR-520d-5p and miR-520d-3p were found with expression pattern similar to SIRP alpha transcripts. We show that SIRP alpha protein is under-expressed in low and high grades of astrocytoma patients' tissue samples. Control brain tissues were found to be positive with SIRP alpha protein expression. Real-time PCR expression analysis confirms that miR-520d-5p and miR-520d-3p expression levels were significantly correlated with SIRP alpha transcripts in control, low-grade, and high-grade tissue samples. Computational prediction further evidenced for binding sites of these miRNAs on 3' and 5'UTR of SIRP alpha transcripts. Taken together, we predict that miR-520d-5p and miR-520d-3p may be having role in the regulation of under-expressed SIRP alpha protein expression.

Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis.

Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but reliable results depend on the use of stable reference genes for proper normalization. This study proposed to test the expression stability of 13 candidate reference genes in Setaria viridis, a monocot species recently proposed as a new C4 model plant. Gene expression stability of these genes was assayed across different tissues and developmental stages of Setaria and under drought or aluminum stress. In general, our results showed Protein Kinase, RNA Binding Protein and SDH as the most stable genes. Moreover, pairwise analysis showed that two reference genes were sufficient to normalize the gene expression data under each condition. By contrast, GAPDH and ACT were the least stably expressed genes tested. Validation of suitable reference genes was carried out to profile the expression of P5CS and GolS during abiotic stress. In addition, normalization of gene expression of SuSy, involved in sugar metabolism, was assayed in the developmental dataset. This study provides a list of reliable reference genes for transcript normalization in S. viridis in different tissues and stages of development and under abiotic stresses, which will facilitate genetic studies in this monocot model plant.

멜론 흰가루병균 및 식물 호르몬 처리하에서 MLO 유전자군의 발현검정

Powdery mildew disease caused by Podosphaera xanthii is a major concern for Cucumis melo production worldwide. Knowledge on genetic behavior of the related genes and their modulating phytohormones often offer the most efficient approach to develop resistance against different diseases. Mildew Resistance Locus O (MLO) genes encode proteins with seven transmembrane domains that have significant function in plant resistance to powdery mildew fungus. We collected 14 MLO genes from ‘Melonomics’ database. Multiple sequence analysis of MLO proteins revealed the existence of both evolutionary conserved cysteine and proline residues. Moreover, natural genetic variation in conserved amino acids and their replacement by other amino acids are also observed. Real-time quantitative PCR expression analysis was conducted for the leaf samples of P. xanthii infected and phytohormones (methyl jasmonate and salicylic acid) treated plants in melon ‘SCNU1154’ line. Upon P. xanthii infection using 7 different races, the melon line showed variable disease reactions with respect to spread of infection symptoms and disease severity. Three out of 14 CmMLO genes were up-regulated and 7 were down-regulated in leaf samples in response to all races. The up- or down-regulation of the other 4 CmMLO genes was race-specific. The expression of 14 CmMLO genes under methyl jasmonate and salicylic acid application was also variable. Eleven CmMLO genes were up-regulated under salicylic acid treatment, and 7 were up-regulated under methyl jasmonate treatments in C. melo L. Taken together, these stress-responsive CmMLO genes might be useful resources for the development of powdery mildew disease resistant C. melo L.

Molecular and biochemical characterization of rice pectin methylesterase inhibitors (OsPMEIs)

Cell wall modifications such as partial degradation and depolymerization by cell wall hydrolases are normal cellular processes and are required for the functionalities of different cell types. Pectin, one of the major cell wall polysaccharides, is predominantly found in primary cell walls and middle lamellae and is subjected to in muro modification, primarily by cell wall-localized pectin methylesterases (PMEs). Molecular biochemical studies have demonstrated that enzymatic activities of PMEs are governed by multiple pectin methylesterase inhibitors (PMEIs), which consequently control the pectin methylesterification status. Although a few studies in Arabidopsis have shown the importance of this PMEI-mediated regulation in the biophysical properties of cell walls, little is known about the molecular physiological functions of rice PMEIs. We found 49 members of the PMEI family in the rice genome. Analysis of their transcript levels by quantitative real-time PCR and meta expression analysis showed that they are regulated spatially and temporally, as well as in response to diverse stresses. Quantification of cell wall-bound methylesters indicated that the degree of pectin methylesterification is developmentally regulated; in particular, higher PMEI activities were detected in cell wall proteins prepared from young leaves. Furthermore, an activity assay demonstrated that two recombinant OsPMEI proteins (OsPMEI8 and 12) were able to inhibit the enzymatic activity of a commercial PME protein. Subcellular localization indicated that OsPMEI8 is targeted to the middle lamella and OsPMEI12 is localized in the plasma membrane and nucleus. Taken together, our findings provide the first molecular and biochemical evidence for functional characterization of PMEIs in rice growth and development.

Expression Analysis of Aldo-Keto Reductase 1 (&amp;lt;i&amp;gt;AKR1&amp;lt;/i&amp;gt;) in Foxtail Millet (&amp;lt;i&amp;gt;Setaria italica&amp;lt;/i&amp;gt; L.) Subjected to Abiotic Stresses

Foxtail millet (Setaria italica L.) is a drought-tolerant millet crop of arid and semi-arid regions. Aldo-keto reductases (AKRs) are significant part of plant defence mechanism, having an ability to confer multiple stress tolerance. In this study, AKR1 gene expression was studied in roots and leaves of foxtail millet subjected to different regimes of PEG- and NaCl-stress for seven days. The quantitative Real-time PCR expression analysis in both root and leaves showed upregulation of AKR1 gene during PEG and salt stress. A close correlation exits between expression of AKR1 gene and the rate of lipid peroxidation along with the retardation of growth. Tissue-specific differences were found in the AKR1 gene expression to the stress intensities studied. The reduction in root and shoot growth under both stress conditions were dependent on stress severity. The level of lipid peroxidation as indicated by MDA formation was significantly increased in roots and leaves along with increased stress levels. Finally, these findings support the early responsive nature of AKR1 gene and seem to be associated at least in part with its ability to contribute in antioxidant defence related pathways which could provide a better protection against oxidative stress under stress conditions.

Fine mapping of a palea defective 1 (pd1), a locus associated with palea and stamen development in rice.

KEY MESSAGE : pd1, a genetic factor in a 69 kb region between RM11239 and RM11245 on rice chromosome 1, controls stamen number and palea development. Spikelets are important organs that store photosynthetic products in rice. Spikelet development directly affects grain yield and rice quality. Here, we report a palea defective (pd1) mutant identified from selfing progenies of indica cv. 93-11 after (60)Co γ ray treatment. pd1 mutant flowers only had four stamens (wild-type has six), but pollen fertility was not affected. Compared with 93-11 palea, pd1 mutant palea showed smaller and flatter leaf, which caused the lemma to bend excessively inward. pd1 mutants had only 46% seed setting rate and 21.6 g 1000-grain weight, which led to two-thirds loss of grain yield. Scanning electron microscope analysis revealed that pd1 mutants had reduced epidermal cell size and reduced numbers of fibrous sclerenchyma cells in both palea and lemma. To analyze the genetic factors involved, we crossed pd1 mutants with three japonica cultivars and generated F1 and F2 populations. The F1 phenotype and F2 segregation ratio indicated that a recessive gene controlled the mutant traits. Using the F2 population, we found that pd1 mapped between the simple sequence repeat markers RM11236 and RM11280 on rice chromosome 1. From a segregating population of 2836 plants, 77 recombinants were screened by RM11236 and RM11280. High-resolution linkage analysis narrowed the pd1 locus to a 69 kb region between RM11239 and RM11245 that contained 10 open reading frames (ORFs). Sequence alignment and quantitative real-time PCR expression analysis of these ORFs between 93-11 and pd1 mutant plants found no unequivocal evidence to identify the pd1 gene.

Identification and characterization of a PutCu/Zn-SOD gene from Puccinellia tenuiflora (Turcz.) Scribn. et Merr.

The role of superoxide dismutase (SOD) in organismal stress conditions has been widely reported. However, little is known concerning the function of superoxide dismutase in Puccinellia tenuiflora (Turcz.) Scribn. et Merr. In this study, PutCu/Zn-SOD, a cDNA clone that encodes a copper–zinc superoxide dismutase gene, was derived from a cDNA library of P. tenuiflora treated with NaHCO3. Nucleotide sequence analysis revealed that the PutCu/Zn-SOD gene cDNA clone consisted of 615 bp encoding a protein with 204 amino acids. In transformed Arabidopsis protoplasts, a PutCu/Zn-SOD protein was located in the chloroplasts as revealed by a green fluorescent protein. Real-time quantitative PCR expression analysis revealed that the PutCu/Zn-SOD gene was expressed in leaves and roots. PutCu/Zn-SOD gene expression was inducible by 60 mM NaHCO3, 150 mM NaCl, and 1.8 mM H2O2. The differential mRNA expression of PutCu/Zn-SOD was observed for leaves and roots exposed to salinity, alkalinity or oxidative stress. The overexpression of PutCu/Zn-SOD gene in transgenic yeast and Arabidopsis plants increased resistance to multiple abiotic stresses. Transgenic Arabidopsis seedlings exhibited elevated total SOD activity. This study suggested that PutCu/Zn-SOD gene can play an important role in multiple stress.

Epigenetic inactivation of ST6GAL1 in human bladder cancer.

BackgroundPosttranslational protein modifications are known to modulate key biological processes like proliferation and apoptosis. Accumulating evidence shows that ST6GAL1, an enzyme that catalyzes the transfer of sialic acid onto galactose-containing substrates, is aberrantly expressed in various cancers and may affect cell motility and invasion. This is the first study to describe ST6GAL1 expression and regulation in human bladder cancer.MethodsST6GAL1 mRNA expression levels in human cell lines (UROtsa, RT4, RT112 and J82) and tissue samples (n = 15 normal urothelium (NU), n = 13 papillary non-invasive tumors (pTa), n = 12 carcinoma in situ (CIS), n = 26 muscle invasive tumors (pT2-4)) were assessed using real-time PCR. In addition, ST6GAL1 protein expression was evaluated using immunohistochemistry. Promoter methylation analysis was performed using methylation-specific PCR (MSP) in cell lines (n = 4) and patient samples (n = 23 NU, n = 12 CIS, n = 29 pTa, n = 41 pT2-4). Epigenetic ST6GAL1 gene silencing was confirmed by in vitro demethylation of bladder cell lines. Data were validated by analysis of an independent bladder tumor data set (n = 184) based on The Cancer Genome Atlas (TCGA) portal.ResultsSemi-quantitative ST6GAL1 real-time PCR expression analysis showed two distinct trends: In muscle-invasive tumors ST6GAL1 expression was downregulation by 2.7-fold, while papillary non-invasive tumors showed an increased ST6GAL1 mRNA expression compared to normal urothelium. ST6GAL1 loss in muscle-invasive tumors was associated with increasing invasiveness. On the protein level, 69.2% (n = 45/65) of all tumors showed a weak ST6GAL1 protein staining (IRS ≤ 4) while 25.6% (16/65) exhibited a complete loss (IRS = 0) of ST6GAL1 protein. Tumor-specific DNA methylation of the ST6GAL1 promoter region was frequently found in pT2-4 tumors (53.6% (22/41)), whereas only 13.8% (4/29) of pTa tumors showed ST6GAL1 promoter methylation. Normal urothelium remained unmethylated. Importantly, we significantly revealed an inverse correlation between ST6GAL1 mRNA expression and ST6GAL1 promoter merthylation in primary bladder cancer. These findings were clearly verified by the TCGA public data set and in vitro demethylation assays functionally confirmed ST6GAL1 promoter methylation as a potential regulatory factor for ST6GAL1 gene silencing.ConclusionsOur study characterizes for the first time ST6GAL1 expression loss caused by aberrant ST6GAL1 promoter methylation potentially indicating a tumor suppressive role in bladder carcinogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-901) contains supplementary material, which is available to authorized users.

Thioester-containing proteins of the tick Ixodes ricinus: Gene expression, response to microbial challenge and their role in phagocytosis of the yeast Candida albicans

The ability of ticks to act as vectors for a wide range of serious human and animal infectious diseases is apparently linked to the insufficiency of the tick immune system to effectively eliminate pathogens they transmit. At the tick-pathogen interface, an important role is presumably played by components of an ancient complement system that includes a repertoire of thioester-containing proteins (TEPs), which in Ixodes sp. comprises three α2-macroglobulins (A2M), three C3 complement component-related molecules (C3), two macroglobulin complement-related (Mcr) and one insect-type TEPs (Tep). In order to assess the function of TEPs in tick immunity, a quantitative real-time PCR expression analysis of tick TEPs was performed at various developmental stages of Ixodes ricinus, and in tissues dissected from adult females. Expression of TEP genes was mostly tissue specific; IrA2M1, IrC3-1, IrC3-3 were found to be expressed in cells of tick fat body adjacent to the tracheal trunks, IrA2M2 in hemocytes, IrTep in ovaries, IrMcr1 in salivary glands and only IrA2M3, IrC3-2 and IrMcr2 mRNAs were present in multiple organs. Expression of tick TEPs was further examined in response to injection of model microbes representing Gram-negative, Gram-positive bacteria and yeast. The greatest expression induction was observed for IrA2M1 and IrC3-1 after challenge with the yeast Candida albicans. Phagocytosis of the yeast was strongly dependent on an active thioester bond and the subsequent silencing of individual tick TEPs by RNA interference demonstrated the involvement of IrC3-1 and IrMcr2. This result suggests the existence of a distinct complement-like pathway, different from that leading to phagocytosis of Gram-negative bacteria. Understanding of the tick immune response against model microbes should provide new concepts for investigating interactions between ticks and relevant tick-borne pathogens.

Identification and characterization of nuclear genes involved in photosynthesis in Populus.

BackgroundThe gap between the real and potential photosynthetic rate under field conditions suggests that photosynthesis could potentially be improved. Nuclear genes provide possible targets for improving photosynthetic efficiency. Hence, genome-wide identification and characterization of the nuclear genes affecting photosynthetic traits in woody plants would provide key insights on genetic regulation of photosynthesis and identify candidate processes for improvement of photosynthesis.ResultsUsing microarray and bulked segregant analysis strategies, we identified differentially expressed nuclear genes for photosynthesis traits in a segregating population of poplar. We identified 515 differentially expressed genes in this population (FC ≥ 2 or FC ≤ 0.5, P < 0.05), 163 up-regulated and 352 down-regulated. Real-time PCR expression analysis confirmed the microarray data. Singular Enrichment Analysis identified 48 significantly enriched GO terms for molecular functions (28), biological processes (18) and cell components (2). Furthermore, we selected six candidate genes for functional examination by a single-marker association approach, which demonstrated that 20 SNPs in five candidate genes significantly associated with photosynthetic traits, and the phenotypic variance explained by each SNP ranged from 2.3% to 12.6%. This revealed that regulation of photosynthesis by the nuclear genome mainly involves transport, metabolism and response to stimulus functions.ConclusionsThis study provides new genome-scale strategies for the discovery of potential candidate genes affecting photosynthesis in Populus, and for identification of the functions of genes involved in regulation of photosynthesis. This work also suggests that improving photosynthetic efficiency under field conditions will require the consideration of multiple factors, such as stress responses.

Determination of MDR1 gene expression for prediction of chemotherapy tolerance and treatment outcome in dogs with lymphoma

The multidrug resistance gene 1(MDR1) expression levels were analysed in 27 dogs with different types of malignant lymphomas receiving a standard chemotherapy protocol. Blood samples were used for MDR1 real-time PCR expression analysis. Treatment tolerance and outcome were evaluated on a regular basis by clinical examination and client questioning. Dogs developing severe adverse effects under treatment showed significantly lower basal MDR1 gene expression levels when compared with those who tolerated the drugs well. In the longitudinal MDR1 gene expression analysis during treatment, four dogs showed a greater than two-fold MDR1 up-regulation, compared to baseline expression. All four of these dogs, but none of the others, showed disease progression. In conclusion, basal and follow-up MDR1 gene expression levels could be of predictive value for the occurrence of severe adverse drug reactions and/or the development of MDR during chemotherapy for lymphoma in dogs.

Identification and real-time expression analysis of selected Toxoplasma gondii in-vivoinduced antigens recognized by IgG and IgM in sera of acute toxoplasmosis patients

BackgroundToxoplasma gondii is an obligate intracellular zoonotic parasite of the phylum Apicomplexa which infects a wide range of warm-blooded animals, including humans. In this study in-vivo induced antigens of this parasite was investigated using in-vivo induced antigen technology (IVIAT) and pooled sera from patients with serological evidence of acute infection.MethodsThe pooled sera was first pre-absorbed against three different preparations of antigens from in-vitro-grown cells of each T. gondii and E. coli XL1-Blue MRF’, subsequently it was used to screen T. gondii cDNA phage expression library. Positive clones from each group were subjected to quantitative real-time PCR expression analysis on mRNA of in-vivo and in-vitro grown parasites.ResultsA total of 29 reactive clones from each IgM and IgG immunoscreenings were found to have high homology to T. gondii genes. Quantitative real-time PCR expression analysis showed that 20 IgM-detected genes and 11 IgG-detected genes were up-regulated in-vivo relative to their expression levels in-vitro. These included genes encoding micronemes, sterol-regulatory element binding protein site, SRS34A, MIC2-associated protein M2AP, nucleoredoxin, protein phosphatase 2C and several hypothetical proteins. A hypothetical protein (GenBank accession no. 7899266) detected by IgG had the highest in-vivo over in-vitro fold change of 499.86; while another up-regulated hypothetical protein (GenBank accession no. 7898829) recognized by IgM showed high sensitivity (90%) and moderate specificity (70%) in detecting T. gondii antibodies when tested with 20 individual serum samples.ConclusionThe highly up-regulated genes and the corresponding proteins, in particular the hypothetical proteins, may be useful in further studies on understanding the disease pathogenesis and as potential vaccine candidates.

The DNA repair protein ALKBH2 mediates temozolomide resistance in human glioblastoma cells

Glioblastoma multiforme (GBM; World Health Organization astrocytoma grade IV) is the most frequent and most malignant primary brain tumor in adults. Despite multimodal therapy, all such tumors practically recur during the course of therapy, causing a median survival of only 14.6 months in patients with newly diagnosed GBM. The present study was aimed at examining the expression of the DNA repair protein AlkB homolog 2 (ALKBH2) in human GBM and determining whether it could promote resistance to temozolomide chemotherapy. ALKBH2 expression in GBM cell lines and in human GBM was determined by quantitative real-time PCR (qRT-PCR) and gene expression analysis, respectively. Drug sensitivity was assessed in GBM cells overexpressing ALKBH2 and in cells in which ALKBH2 expression was silenced by small-interfering (si)RNA. ALKBH2 expression following activation of the p53 pathway was examined by western blotting and qRT-PCR. ALKBH2 was abundantly expressed in established GBM cell lines and human GBM, and temozolomide exposure increased cellular ALKBH2 expression levels. Overexpression of ALKBH2 in the U87 and U251 GBM cell lines enhanced resistance to the methylating agents temozolomide and methyl methanesulfonate but not to the nonmethylating agent doxorubicin. Conversely, siRNA-mediated knockdown of ALKBH2 increased sensitivity of GBM cells to temozolomide and methyl methanesulfonate but not to doxorubicin or cisplatin. Nongenotoxic activation of the p53 pathway by the selective murine double minute 2 antagonist nutlin-3 caused a significant decrease in cellular ALKBH2 transcription levels. Our findings identify ALKBH2 as a novel mediator of temozolomide resistance in human GBM cells. Furthermore, we place ALKBH2 into a new cellular context by showing its regulation by the p53 pathway.

Altered expression of genes involved in progesterone biosynthesis, metabolism and action in endometrial cancer

Endometrial cancer (EC) is one of the most common gynecological malignancies worldwide. It is associated with prolonged exposure to estrogens that is unopposed by the protective effects of progesterone, which suggests that altered progesterone biosynthesis, metabolism and actions might be implicated in the development of EC. Our aim was to evaluate these processes through quantitative real-time PCR expression analysis in up to 47 pairs of EC tissue and adjacent control endometrium. First, we examined the expression of genes encoding proteins associated with progesterone biosynthesis: steroidogenic acute regulatory protein (STAR); a side chain cleavage enzyme (CYP11A1); and 3β-hydroxysteroid dehydrogenase/ketosteroid isomerase (HSD3B). There were 1.9- and 10.0-fold decreased expression of STAR and CYP11A1, respectively, in EC versus adjacent control endometrium, with no significant differences in the expression of HSD3B1 and HSD3B2. Next, we examined expression of genes encoding five progesterone metabolizing enzymes: the 3-keto and 20-ketosteroid reductases (AKR1C1–AKR1C3) and 5α-reductases (SRD5A1 and SRD5A2); and the opposing 20α-hydroxysteroid dehydrogenase (HSD17B2). These genes are expressed in EC and adjacent control endometrium. No statistically significant differences were seen in mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1. Expression of HSD17B2 was 3.0-fold increased, and expression of SRD5A2 was 3.7-fold decreased, in EC versus adjacent control endometrium. We also examined mRNA levels of progesterone receptors A and B (PGR), and separately the expression of progesterone receptor B (PR-B). Here we saw 1.8- and 2.0-fold lower mRNA levels of PGR and PR-B, respectively, in EC versus adjacent control endometrium. This down-regulation of STAR, CYP11A1 and PGR in endometrial cancer may lead to decreased progesterone biosynthesis and actions although the effects on progesterone levels should be further studied.

Cloning and expression analysis of MuNAC4 transcription factor protein from horsegram (Macrotyloma uniflorum (Lam.) Verdc.) conferred salt stress tolerance in Escherichia coli

The NAC gene family encodes plant-specific transcription factors which play diverse roles in abiotic stress responses of plants. NAC TFs reported to be involved in the regulatory pathway of multiple abiotic stresses. In the present study, a salt stress-inducible NAC gene, named MuNAC4 (Macrotyloma uniflorum NAC4) from horsegram was isolated, cloned, characterized and studied its expression. Real-time PCR expression analysis showed up-regulation of MuNAC4 in horsegram across salinity, cold, drought and dehydration stress conditions. However, salt stress resulted a sixfold increase in MuNAC4 transcript levels. The involvement of MuNAC4 in abiotic stress tolerance was investigated by cloning MuNAC4 gene in expression vector pET28a and transformation in Escherichia coli BL21 (DH3). The apparent molecular weight of the recombinant protein was found to be 38.3 kDa as evident from SDS-PAGE analysis. The functional role of MuNAC4 in Escherichia coli confers the tolerance against salt (6 % NaCl), heavy metal (100 mM CuSO4) and water stresses (6 % PEG). The E. coli cells transformed with pET28a + MuNAC4 was showed better survival ratio and higher growth rates under NaCl stress than other abiotic stress conditions when compared to control cells (BL21/pET28a). This provides the experimental evidence that MuNAC4 protein enhance salt stress tolerance of E. coli cells suggesting their suitability as candidates for genetic manipulations for enhanced crop tolerance to salt stress and other abiotic stresses.

Characterization and Expression of a Phytochelatin Synthase Gene in Birch-leaf Pear (Pyrus betulaefolia Bunge)

Phytochelatins play a major role in metal detoxification in plants. However, the molecular mechanisms of heavy metal detoxification remain unknown, and birch-leaf pear genes related to metal detoxification have not yet been identified. Here, we report the isolation of cDNA and genomic DNA sequences encoding a phytochelatin synthase (PCS) from birch-leaf pear (Pyrus betulaefolia Bunge). The PbPCS1 cDNA sequence contained 1,965 nucleotides of a 1,494 bp open reading frame (ORF), which encodes a 497-amino-acid protein with a molecular weight of 55.00 kDa. Its corresponding genomic DNA sequence consists of 3,820 nucleotides and eight exons separated by seven introns. The deduced amino acid sequence of PbPCS1 contains five Cys residues (56, 90, 91, 109 and 113 amino acids) that are highly conserved in the plant PCS1 family, and 12 cysteine residues putatively involved in heavy metal binding sites, which included three adjacent Cys–Cys components (331–332, 351–352 and 369–370 amino acids) in the C-terminal variable domain. Homology analysis of the deduced PbPCS1 amino acid sequence revealed that it shares a high sequence similarity amongst N-terminal amino acids and low similarity with C-terminal amino acids with plant PCS1 proteins deposited with NCBI. PbPCS1 was most related to PCS1 from legume plants Lotus japonicus (GenBank accession no. AAT80342) and soybean (Glycine max L.; AAL78384) as they were clustered into the same clade in a neighbor-joining phylogenetic tree. Quantitative real-time PCR (qPCR) expression analysis revealed that PbPCS1 had a very low basal expression level in untreated whole seedlings, and levels were higher in roots than in leaves and stems. After 24 h of exposure to 20 μM CdSO4, CuSO4 or ZnSO4, PbPCS1 expression increased significantly in different organs. In addition, L-buthionine-sulfo-ximine (BSO) can inhibit PbPCS1 expression in roots, stems and leaves, while L-glutathionereduced (GSH) stimulates PbPCS1 expression in different organs of birch-leaf pear.

Molecular cloning and characterization of two differentially expressed Cellulose synthase gene isoforms in &amp;lt;i&amp;gt;Leucaena leucocephala&amp;lt;/i&amp;gt;: A pulp yielding tree species

Leucaena leucocephala is fast growing leguminous tree species, acclimatized to variety of soil and climatic conditions. It is widely used for pulp production in India. Pulp mainly consists of cellulose, which is a simple polymer of unbranched β-1, 4-linked glucan chains. The polymerization of glucose residues into a β-1, 4-linked backbone is catalysed by the enzyme cellulose synthase (CesA). Here, cDNAs encoding CesA genes from Leucaena were isolated and characterized. The two complete cDNAs of 3.228 kb and 3.222 kb encoding CesA gene from L. leucocephala were designated as Ll-7CesA (FJ871987) and Ll-8CesA (GQ267555) respectively. In-silico studies showed that Ll-7CesA has 95.2% identities and Ll- 8CesA has 95.8% identities with Acacia mangium CesA2. Phylogenetic analysis revealed significant similarity with known dicot CesA genes. The deduced amino acid sequence of both CesA genes contained the conserved D, D, D, QxxRW motif, eight membrane spanning regions and a putative zinc binding domain, which are characteristic of glycosyltransferases. DNA blot analysis suggested, CesA gene to be in multiple copies in Leucaena genome. Semi quantitative and quantitative real-time PCR expression analysis of Ll-7CesA gene showed more expression in stem than leaf and not detected in root where as Ll-8CesA gene was expressed more in stem than leaf and root. Overall Ll-8CesA was expressed in all tested tissues and could be involved in active cellulose biosynthesis.

A multilocus, temperature stress‐related gene expression profile assay in Acropora millepora, a dominant reef‐building coral

We report an accurate multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay, capable of reproducing gene expression profiles from 16 target genes [12 genes of interest (GOIs) and four reference genes (RGs)] in Acropora millepora, a common reef-building model coral species. The 12 GOIs have known or putative roles in the coral bleaching response, yet the method is not restricted to this particular assay and gene set. The procedure is based on the Beckman Coulter (Fullerton, CA, USA) GenomeLab™ GeXP Genetic Analysis System and bridges the gap between quantitative real-time PCR (qPCR) expression analysis of a single or a small number of genes and microarray gene expression surveys of thousands of genes. Despite large variation among biological replicates, the majority of GOIs were up-regulated (up to 4000%) in most colonies during a laboratory-based thermal stress experiment. Two genes, Nf-kβ2 and MnSod, were consistently up-regulated in all colonies tested, and we therefore propose these as candidate markers useful for population-level evaluations of thermal stress. Our assay provides an important new tool for coral bleaching studies; because of the lower cost, labour and amount of cDNA required compared with singleplex qPCR, population-level studies with large biological replication are feasible.

The actin multigene family in Populus: organization, expression and phylogenetic analysis

Despite the significance of actin in plant growth and development, little is known of the structure, expression and evolution of the actin gene family in woody plants. In this study, we systematically examined the diversification of the actin gene family in Populus by integrating genomic organization, expression, and phylogeny data. Genome-wide analysis of the Populus genome indicated that actin is a multigene family consisting of eight members, all predicted to encode 377-amino acid polypeptides that share high sequence homology ranging from 94.2 to 100% identity. Microarray and real-time PCR expression analysis showed that the PtrACT family members are differentially expressed in different tissues, exhibiting overlapping and unique expression patterns. Of particular interest, all PtrACT genes have been found to be preferentially expressed in the stem phloem and xylem, suggesting that poplar PtrACTs are involved in the wood formation. Gene structural and phylogenetic analyses revealed that the PtrACT family is composed of two main subgroups that share an ancient common ancestor. Extremely high intraspecies synonymous nucleotide diversity of pi(syn) = 0.01205 was detected, and the pi(non-syn)/pi(syn) ratio was significantly less than 1; therefore, the PtACT1 appears to be evolving in Populus, primarily under purifying selection. We demonstrated that the actin gene family in Populus is divided into two distinct subgroups, suggesting functional divergence. The results reported here will be useful in conducting future functional genomics studies to understand the detailed function of actin genes in tree growth and development.

Frequent aberrant DNA methylation of ABCB1, FOXC1, PPP2R2B and PTEN in ductal carcinoma in situ and early invasive breast cancer

IntroductionDuctal carcinoma in situ (DCIS) is a non-invasive lesion of the breast that is frequently detected by mammography and subsequently removed by surgery. However, it is estimated that about half of the detected lesions would never have progressed into invasive cancer. Identifying DCIS and invasive cancer specific epigenetic lesions and understanding how these epigenetic changes are involved in triggering tumour progression is important for a better understanding of which lesions are at risk of becoming invasive.MethodsQuantitative DNA methylation analysis of ABCB1, CDKN2A/p16INK4a, ESR1, FOXC1, GSTP1, IGF2, MGMT, MLH1, PPP2R2B, PTEN and RASSF1A was performed by pyrosequencing in a series of 27 pure DCIS, 28 small invasive ductal carcinomas (IDCs), 34 IDCs with a DCIS component and 5 normal breast tissue samples. FOXC1, ABCB1, PPP2R2B and PTEN were analyzed in 23 additional normal breast tissue samples. Real-Time PCR expression analysis was performed for FOXC1.ResultsAberrant DNA methylation was observed in all three diagnosis groups for the following genes: ABCB1, FOXC1, GSTP1, MGMT, MLH1, PPP2R2B, PTEN and RASSF1A. For most of these genes, methylation was already present at the DCIS level with the same frequency as within IDCs. For FOXC1 significant differences in methylation levels were observed between normal breast tissue and invasive tumours (P < 0.001). The average DNA methylation levels were significantly higher in the pure IDCs and IDCs with DCIS compared to pure DCIS (P = 0.007 and P = 0.001, respectively). Real-time PCR analysis of FOXC1 expression from 25 DCIS, 23 IDCs and 28 normal tissue samples showed lower gene expression levels of FOXC1 in both methylated and unmethylated tumours compared to normal tissue (P < 0.001). DNA methylation levels of FOXC1, GSTP1, ABCB1 and RASSF1A were higher in oestrogen receptor (ER) positive vs. ER negative tumours; whereas methylation levels of FOXC1, ABCB1, PPP2R2B and PTEN were lower in tumours with a TP53 mutation.ConclusionsQuantitative methylation analysis identified ABCB1, FOXC1, PPP2R2B and PTEN as novel genes to be methylated in DCIS. In particular, FOXC1 showed a significant increase in the methylation frequency in invasive tumours. Low FOXC1 gene expression in both methylated and unmethylated DCIS and IDCs indicates that the loss of its expression is an early event during breast cancer progression.