Acid phosphatase-1.1, a molecular marker tightly linked to root-knot nematode resistance in tomato

Root knot nematode resistance in tomato is a genetic trait which is determined by a single dominant gene ( Mi ) on chromosome 6 of tomato. Information about the mRNA or protein product is completely lacking, which precludes the cloning of Mi by conventional strategies based on gene expression. However, an acid phosphatase-1 allozyme marker ( Aps-1 1 ) is known, which shows tight genetic linkage to the root knot nematode resistance trait. With a view to isolating Mi nucleotide sequences by a positional cloning approach, we have developed a molecular probe for the Aps-1 1 gene, using the polymerase chain reaction (PCR) and a pair of primers, corresponding to amino acid sequence information from the protein encoded by the Aps-1 1 gene.The Aps-1 1 gene product (APS-1 1) was purified from hydroponic tomato roots and tomato suspension cells using conventional low pressure column chromatographic techniques. The most striking purification was achieved by concanavalin A (Con A)-Sepharose 4B affinity column chromatography, which constituted the fourth step in our purification procedure and produced virtually pure APS-1 1. Unfortunately, however, this step caused contamination of the APS-1 1preparation with Con A released form the column. Therefore, a final Mono Q-FPLC purification step was added to remove the Con A-contamination. The resulting APS-1 1preparation was homogeneous in denaturing and non-denaturing polyacrylamide gel electrophoresis. In addition, the purified protein showed co-electrophoresis and co-elution with the APS-1 1enzymatic activity in non-denaturing PAGE and gel filtration column chromatography, respectively, thereby demonstrating its APS-1 1identity. The yield of our purification protocol was a few μg of APS-1 1protein per kg of tomato suspension cells or roots. No major loss of APS-1 1activity was observed in any of the purification steps, indicating that the low yield attained is truly reflecting the very low expression level of the Aps-1 1gene.The purified APS-1 1preparation was treated with CNBr and trypsin to produce APS-1 1peptides. Following purification by HPLC, amino acid sequence analysis of two CNBr and seven trypsin cleavage products revealed 61 residues of APS-1 1amino acid sequence.The amino acid sequence information from two APS-1 1peptides consisting of 8 and 14 amino acids respectively, allowed the synthesis of PCR primer pools to direct the amplification of a 2.4 kb Aps-1 1 fragment using a genomic DNA template. Crucial for the effectivity of these pools was the limitation of the number of different primers used to account for codon degeneracy. Restriction of the complexity of the primer pools was achieved by incorporating deoxyinosine or the most probable nucleotide(s) at ambiguous codon positions in the 5' part of the primers. On the other hand, efficient primer elongation was assured by including in the primer pool every possible combination of the three 3'-terminal codons. The 2.4 kb fragment was only amplified from a genomic template carrying the Aps-1 1 allele and was not found in using a template that carried the Aps-1 3 or Aps-1 1 + allele. Moreover, the 2.4 kb amplification product was found to reveal RFLPs between a pair of nematode resistant and sensitive nearly isogenic lines, which only differed for the Aps-1 1/Mi region. The specific amplification under the direction of the particular Aps-1 allele from which the primers had been derived and its genetic map position provide evidence showing, that the 2.4 kb PCR product represents the Aps- 1 1 target fragment. Using cDNA as a template and the same primers that directed the synthesis of the 2.4 kb genomic PCR product, a 490 bp Aps-1 1 fragment was obtained. An overlapping Aps-1 1 cDNA sequence of 550 bp was amplified using the same upstream primers but a different pool of downstream primers corresponding to a peptide that turned out to represent the C- terminus of APS-1 1. The amount of these cDNA-directed amplification products synthesized in 30 cycles of PCR was so low, that their production was only detectable by Southern blot hybridization using the 2.4 kb genomic PCR product as a probe, which provides another demonstration of the very low expression level of the Aps-1 1 gene.In addition to the 2.4 kb genomic Aps-1 1 sequence, another PCR product of about 115 bp was obtained using a different pair of primers and either a genomic or a cDNA template. This product was found to comprise several related nucleotide sequences of similar size. Because of the poor performance of the cloned products as a probe, it was not possible to establish their genetic map position by RFLP analysis.Screening of a cDNA library with the 2.4 kb putative Aps-1 1 sequence identified two clones of related nucleotide sequences, one of which apparently represented an Aps-1 1 cDNA clone, as it encoded three APS-1 1peptides (together 30 amino acids) in the orientation predicted by the PCR results.'Me nucleotide sequence of this Aps-1 1 cDNA clone revealed a stretch of 69 residues of tomato APS-1 1amino acid sequence starting from the C-terminus, and showed that peptide VII is the C-terminal tryptic peptide. Another stretch of putative APS-1 1amino acid sequence was deduced from the nucleotide sequence adjacent to the upstream primer in the 2.4 kb genomic PCR product, and comprised 56 amino acids sequence information starting with peptide IX at the N-terminal end. The amino acid sequence of tomato APS-1 1elucidated so far, did not present major sequence homology with the sequences of any other (acid) phosphatase in the GenBank or EMBL data bases. A striking sequence homology was found, however, with a vegetative storage protein from soybean, VSP-β, that accumulates to very high levels in leaves after depodding of the plants.The detection of an Aps-1 1 -related cDNA clone using the 2.4 kb genomic Aps-1 1 sequence as a probe, and furthermore, the sequence heterogeneity among the 115 bp PCR product points to the existence of a family of Aps-1 1 -related nucleotide sequences within the tomato genome. Upon comparison of four different nucleotide sequences present in the 115 bp PCR product with the Aps-1 1 cDNA sequence, it was found, that one of them corresponded with the Aps-1 1 sequence. Neither of the other three, nor the Aps-1 1 -related cDNA clone showed a higher homology at the amino acid level with soybean VSP-βthan with tomato APS-1 1, which argues against one of these sequences representing the tomato homolog of soybean VSP-β.In the isolation of an Aps-1 1 cDNA clone, we have shown that it is possible to design highly specific degenerate PCR primer pools. Therefore, whenever going from protein to gene, we recommend to try PCR first in order to obtain a highly selective probe, before turning to library screening using degenerate oligonucleotides. Furthermore, since an Aps-1 1 cDNA clone is available now, a possible starting point or a useful landmark has been provided for a chromosomal walk towards the nematode resistance gene Mi

Integrated assessment by multiple gene expression analysis of quercetin bioactivity on anticancer-related mechanisms in colon cancer cells in vitro

Background Many different mechanisms are involved in nutrient¿related prevention of colon cancer. In this study, a comprehensive assessment of the spectrum of possible biological actions of the bioactive compound quercetin is made using multiple gene expression analysis. Quercetin is a flavonoid that can inhibit proliferation of tumor cells and reduce the number of aberrant crypt foci, although increase of number of colon tumors was also reported. Aim of the study In order to elucidate possible mechanisms involved in its mode of action the effect of quercetin on expression of 4000 human genes in Caco¿2 cells was studied and related to functional effects. Methods Caco¿2 cells were exposed to 5 or 50 µM quercetin for 48 hours, differential expression of 4000 human genes was studied using microarrays and related to functional effects. Differentially expressed genes were categorized in seven functional groups: cell cycle and differentiation, apoptosis, tumor suppressor genes and oncogenes, cell adhesion and cell¿cell interaction, transcription, signal transduction and energy metabolism. Also, cell proliferation and cell cycle distribution were measured. Results Quercetin (5µM) downregulated expression of cell cycle genes (for example CDC6, CDK4 and cyclin D1), downregulated cell proliferation and induced cell cycle arrest in Caco¿2 cells. After exposure to 50 µM quercetin cell proliferation decreased to 51.3% of control, and further decrease of the percentage of cells in the G1 phase coincided with an increase of the percentage of cells in the sub¿G1 phase. Quercetin upregulated expression of several tumor suppressor genes. In addition, genes involved in signal transduction pathways like beta catenin/TCF signalling and MAPK signal transduction were influenced by quercetin. Conclusions This study shows that large¿scale gene expression analysis in combination with functional assays yields a considerable amount of information on (anti¿)carcinogenic potential of food components like querceti

From biofuel to bioproduct: is bioethanol a suitable fermentation feedstock for synthesis of bulk chemicals?

The first pilot-scale factories for the production of bioethanol from lignocellulose have been installed, indicating that we are on the brink of overcoming most hurdles for an economically feasible process. When bioethanol is competitive as biofuel with fuels originating from petrochemical resources, it will also become interesting to use lignocellulose as a feedstock for the fermentative synthesis of bulk chemicals. Lignocellulose hydrolysates, however, are highly complex and viscous media, posing challenges to oxygen transfer, product formation at low sugar concentration, product recovery, etc. Bioethanol is an exceptional product in this respect because it can be produced anaerobically, at low sugar concentrations, and can be easily removed from the broth by distillation. For products that do not have these benefits, another approach may be interesting, in which lignocellulose is first converted to bioethanol, which in turn serves as a substrate for a second conversion into the desired product, similar to the traditional production of acetic acid in vinegar. In this perspective, we compare these one-stage and two-stage conversions with respect to overall yield and productivity of the fermentation process and the differences that occur in product removal. © 2011 Society of Chemical Industry and John Wiley & Sons, Lt

Evolution of hominin detoxification: Neanderthal and modern human Ah receptor respond similarly to TCDD

In studies of hominin adaptations to fire use, the role of the aryl hydrocarbon receptor (AHR) in the evolution of detoxification has been highlighted, including statements that the modern human AHR confers a significantly better capacity to deal with toxic smoke components than the Neanderthal AHR. To evaluate this, we compared the AHR-controlled induction of cytochrome P4501A1 (CYP1A1) mRNA in HeLa human cervix epithelial adenocarcinoma cells transfected with an Altai-Neanderthal or a modern human reference AHR expression construct, and exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We compared the complete AHR mRNA sequences including the untranslated regions (UTRs), maintaining the original codon usage. We observe no significant difference in CYP1A1 induction by TCDD between Neanderthal and modern human AHR, whereas a 150–1,000 times difference was previously reported in a study of the AHR coding region optimized for mammalian codon usage and expressed in rat cells. Our study exemplifies that expression in a homologous cellular background is of major importance to determine (ancient) protein activity. The Neanderthal and modern human dose–response curves almost coincide, except for a slightly higher extrapolated maximum for the Neanderthal AHR, possibly caused by a 5′-UTR G-variant known from modern humans (rs7796976). Our results are strongly at odds with a major role of the modern human AHR in the evolution of hominin detoxification of smoke components and consistent with our previous study based on 18 relevant genes in addition to AHR, which concluded that efficient detoxification alleles are more dominant in ancient hominins, chimpanzees, and gorillas than in modern humans

Dual effects of N-acetyl-l-cysteine dependent on NQO1 activity: Suppressive or promotive of 9,10-phenanthrenequinone-induced toxicity

A typical antioxidant, N-acetyl-L-cysteine (NAC) generally protects cells from oxidative damage induced by reactive oxygen species (ROS). 9,10-Phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust particles, produces ROS in redox cycling following two-electron reduction by NAD(P)H:quinone oxidoreductase 1 (NQO1), which has been considered as a cause of its cyto- and genotoxicity. In this study, we show that NAC unexpectedly augments the toxicity of 9,10-PQ in cells with low NQO1 activity. In four human skin cell lines, the expression and the activity of NQO1 were lower than in human adenocarcinoma cell lines, A549 and MCF7. In the skin cells, the cytotoxicity of 9,10-PQ was significantly enhanced by addition of NAC. The formation of DNA double strand breaks accompanying phosphorylation of histone H2AX, was also remarkably augmented. On the other hand, the cyto- and genotoxicity were suppressed by addition of NAC in the adenocarcinoma cells. Two contrasting experiments: overexpression of NQO1 in CHO-K1 cells which originally expressed low NQO1 levels, and knock-down of NQO1 in the adenocarcinoma cell line A549 by transfection of RNAi, also showed that NAC suppressed 9,10-PQ-induced toxicity in cell lines expressing high NQO1 activity and enhanced it in cell lines with low NQO1 activity. The results suggested that dual effects of NAC on the cyto- and genotoxicity of 9,10-PQ were dependent on tissue-specific NQO1 activity

Activation of EpRE-mediated gene transcription by quercetin glucuronides depends on their deconjugation

Quercetin is a flavonoid reported to have health-promoting properties. Due to its extensive metabolism to glucuronides in vivo, questions were raised if studies conducted with quercetin aglycone, stating its health-promoting activity, are of actual relevance. Here we show that glucuronides of quercetin, and its methylated forms isorhamnetin and tamarixetin, can induce EpRE-mediated gene expression up to 5-fold. Furthermore, evidence is presented that EpRE-mediated gene induction by these glucuronides involves their deglucuronidation. This indicates that although quercetin-derived glucuronides are the major metabolites present in the systemic circulation, deglucuronidated derivatives are the active compounds responsible for their beneficial EpRE-mediated gene expression effects