N-methyltransferase Genes Research Articles

Characterization of N-methyltransferase Genes in Coffea liberica var. liberica

Caffeine biosynthesis in Coffea evolved independently through tandem gene duplication and subsequent neofunctionalization of ancestral N-methyltransferases (NMTs). The bulk of our understanding of this process in Coffea is centered on two species: C. arabica and C. canephora. To gain a wider perspective of caffeine biosynthesis in Coffea, this study focused on the less popular but commercially available C. liberica var. liberica, locally known as “kapeng barako.” This study aimed to determine the genomic and coding sequences of the three NMTs – mainly, xanthosine methyltransferase (XMT), 7-methylxanthine-N-methyltransferase (MXMT), and dimethylxanthine methyltransferase (DXMT) – through Sanger sequencing. Relative expression levels of these NMTs in the leaves, flower bud, green fruit, and red fruit stages were also determined through polymerase chain reaction amplification of synthesized cDNA and then gel electrophoresis. Results reveal that the genetic sequences of NMTs of C. liberica were similar to the C. eugenioides-derived NMTs of C. arabica. Results also indicate that there is a possibility that more than one NMT gene for each NMT cluster (XMT, MXMT, DXMT) exists. This study has also identified genomic sequences and transcripts, whose sequences were in between the XMT and MXMT gene clusters. The function of these novel sequences is yet to be determined. NMT expression levels in C. liberica were similar to those observed in C. arabica and C. canephora, wherein expression peaks at the green fruit stage and then diminishes, as the fruit ripens fully. Although the NMTs of C. liberica have a lot of similarities with C. arabica and C. canephora, discovering the function and role in caffeine biosynthesis of these novel genomic sequences and transcripts will help us further understand the evolution of caffeine biosynthesis in Coffea.

Combinatorial Biosynthesis of 3-O-Carbamoylmaytansinol by Rational Engineering of the Tailoring Steps of Ansamitocins.

Currently, most maytansine-containing antibody-drug conjugates (ADCs) in clinical trials are prepared with DM1 or DM4, which in turn is synthesized mainly from ansamitocin P-3 (AP-3), a bacterial maytansinoid, isolated from Actinosynnema pretiosum. However, due to the high self-toxicity of AP-3 to A.pretiosum, the yield of AP-3 has been difficult to improve. Herein, a new maytansinoid with much lower self-toxicity to A.pretiosum, 3-O-carbamoylmaytansinol (CAM, 3), was designed and generated by introducing the 3-O-carbamoyltransferase gene asc21b together with the N-methyltransferase genes from exogenous maytansinoid gene clusters into the 3-O-acyltransferase gene (asm19) deleted mutant HGF052. Meanwhile, two new shunt products, 20-O-demethyl-19-dechloro-N-demethyl-4,5-desepoxy-CAM (4) and 20-O-demethyl-N-demethyl-4,5-desepoxy-CAM (5) were identified from the recombinant strain. Furthermore, by screening of liquid fermentation media, overexpression of bottleneck tailoring enzymes and the pathway-specific activator, the titer of CAM reached 498 mg/L in the engineered strain. Since the 3-O-carbamoyl group of CAM can be removed by chemical cleavage as AP-3 to produce maytansinol, our work suggests that CAM may be a promising alternative to AP-3 in the future development of ADCs.

Discovery and Biochemical Characterization of N-methyltransferase Genes Involved in Purine Alkaloid Biosynthetic Pathway of Camellia gymnogyna Hung T.Chang (Theaceae) from Dayao Mountain

In the present study, purine alkaloid analysis and transcriptome of Camellia gymnogyna Hung T. Chang (Theaceae) from Dayao Mountain were performed by high-performance liquid chromatography (HPLC) and RNA-Seq, respectively. The results showed that the major purine alkaloids accumulated in Camellia gymnogyna Hung T. Chang (Theaceae) were theobromine together with a small amount of theacrine and caffeine. Through polymerase chain reaction (PCR), three types of cDNA encoding N-methyltransferases were isolated from the leaves of Camellia gymnogyna Hung T. Chang (Theaceae) and designated GCS1, GCS2, and GCS3. We subsequently expressed GCS1, GCS2, and GCS3 in Escherichia coli and incubated lysates of the bacterial cells with a variety of xanthine substrates in the presence of S-adenosyl-L-methionine as the methyl donor. We found that the recombinant GCS1 proteins catalyzed 1,3,7-trimethyluric acid to produce theacrine, the recombinant GCS3 proteins catalyzed 7-methylxanthine to produce theobromine, while the recombinant GCS2 proteins did not catalyze any xanthine derivatives. Simultaneous analysis of the expressions of GCS1, GCS2, GCS3, and a caffeine synthase gene (TCS1) in Camellia gymnogyna Hung T. Chang (Theaceae) and other tea plants provided a reference for further research on the functions of these genes.

Metabolite Profiling and Transcriptome Analysis Revealed the Conserved Transcriptional Regulation Mechanism of Caffeine Biosynthesis in Tea and Coffee Plants.

Caffeine is a characteristic bioactive compound in tea and coffee plants, which is synthesized and accumulated extensively in leaves and seeds. However, little is known about the regulatory mechanism of caffeine synthesis in plants. This study compared the caffeine metabolite between tea and coffee plants. We found that tea leaves contained significantly higher caffeine than coffee leaves, which is perhaps due to more members of N-methyltransferase (NMT) genes as well as higher expression levels in tea plants. Substantial numbers of transcription factors were predicted to be involved in caffeine biosynthesis regulation, combining weighted gene co-expression network analysis and the cis-element of NMT promoter analysis in tea and coffee plants. Furthermore, analysis of the transcription factors from the caffeine-related modules suggested that the regulatory mechanism of caffeine biosynthesis was probably partly conservative in tea and coffee plants. This study provides an essential resource for the regulatory mechanism of caffeine biosynthesis in plants.

Intracellular Ca2+ accumulation triggered by caffeine provokes resistance against a broad range of biotic stress in rice.

Chemical pesticides are still frequently overused to diminish such crop loss caused by biotic stress despite the threat to humans and the environment. Thus, it is urgent to find safer and more effective defense strategies. In this study, we report that caffeine, implanted through a transgenic approach, enhances resistance against variable biotic stresses in rice without fitness cost. Caffeine-producing rice (CPR) was generated by introducing three N-methyltransferase genes involved in the biosynthesis of caffeine in coffee plants. The CPR plants have no differences in morphology and growth compared to their wild-type counterparts, but they show strongly enhanced resistance to both bacterial leaf blight, rice blast, and attack of white-backed planthoppers. Caffeine acts as a repellent agent against rice pathogens. Moreover, caffeine triggers a series of Ca2+ signalling-like processes to synthesize salicylic acid (SA), a hormone associated with plant resistance. In CPR, phosphodiesterase was inhibited by caffeine, cAMP and cGMP increased, intracellular Ca2+ increased, phenylalanine lyase (PAL) was activated by OsCPK1, and SA synthesis was activated. This finding is a novel strategy to improve resistance against the biotic stresses of crops with a special type of defense inducer.

Metabolite and Transcriptome Profiling on Xanthine Alkaloids-Fed Tea Plant (Camellia sinensis) Shoot Tips and Roots Reveal the Complex Metabolic Network for Caffeine Biosynthesis and Degradation.

While caffeine is one of the most important bioactive metabolites for tea as the most consumed non-alcohol beverage, its biosynthesis and catabolism in tea plants are still not fully understood. Here, we integrated purine alkaloid profiling and transcriptome analysis on shoot tips and roots fed with caffeine, theophylline, or theobromine to gain further understanding of caffeine biosynthesis and degradation. Shoot tips and roots easily took up and accumulated high concentrations of alkaloids, but roots showed much faster caffeine and theophylline degradation rates than shoot tips, which only degraded theophylline significantly but almost did not degrade caffeine. Clearly feedback inhibition on caffeine synthesis or inter-conversion between caffeine, theophylline, and theobromine, and 3-methylxanthine had been observed in alkaloids-fed shoot tips and roots, and these were also evidenced by significant repression of TCS and MXMT genes critical for caffeine biosynthesis. Among these responsively repressed genes, two highly expressed genes TCS-4 and TCS-8 were characterized for their enzyme activity. While we failed to detect TCS-4 activity, TCS-8 displayed N-methyltransferase activities towards multiple substrates, supporting the complex metabolic network in caffeine biosynthesis in tea plants since at least 13 TCS-like N-methyltransferase genes may function redundantly. This study provides new insight into complex metabolic networks of purine alkaloids in tea plants.

Induced production of tropane alkaloids, and expression of hyoscyamine 6β-hydroxylase (h6h) and putrescine N-methyl transferase (pmt2) genes in hairy roots and propagated plantlets of Atropa belladonna L. elicited by methyl jasmonate

Atropa belladonna L. is an important medicinal plant due to the biosynthesis of hyoscyamine and scopolamine; two important tropane alkaloids, mostly well known for their anticholinergic properties acting on the parasympathetic nervous system. This study aimed to analyze the effect of methyl jasmonate (MJ), at different concentrations, on hyoscyamine and scopolamine production from in vitro hairy root culture and propagated plantlets of A. belladonna L. Gene expression analysis of two key enzymes PMT and H6H responsible for the biosynthesis of hyoscyamine and scopolamine, respectively, was also carried out under MJ application. MJ decreased the growth parameters in both plantlets and hairy root cultures. In shoots of the plantlets, MJ increased significantly the hyoscyamine and scopolamine contents to the highest value of 3.32 mg g−1 DW and 0.5 mg g−1 DW, respectively. Moreover, MJ up-regulated pmt2 and h6h gene expression in root of the plantlets, which is the site of biosynthesis of hyoscyamine and scopolamine, while the accumulation occurs in the aerial parts. In hairy root culture, the hyoscyamine content increased as the MJ concentration increased, and reached its highest level of 2.1 mg g−1 DW at 0.04 mM MJ, showing the same pattern as for pmt2 gene expression. Nevertheless, the scopolamine content decreased by increasing the MJ concentration as it also showed a reverse pattern compared to the h6h gene expression, presumably due to the negative effect of hyoscyamine accumulation on H6H enzyme activity.

Targeted gene knockdown validates the essential role of lactate dehydrogenase in Cryptosporidium parvum

Cryptosporidium parvum is a zoonotic protozoan that can cause a life-threatening gastrointestinal syndrome in children and in immunocompromised adults. Currently, the only approved drug for treatment of Cryptosporidium infections in humans is nitazoxanide, but it is not effective in immunocompromised individuals or in children with malnutrition. This is compounded by the lack of genetic methods for studying and validating potential drug targets in the parasite. Therefore, in this study, we endeavoured to adapt the use of a phosphorodiamidate morpholino oligomer (morpholino) antisense approach to develop a targeted gene knockdown assay for use in C. parvum. We show that morpholinos, at non-toxic concentrations, are rapidly internalised by both C. parvum and host cells (HCT-8), and distribute diffusely throughout the cytosol. Using morpholinos to separately target C. parvum lactate dehydrogenase and putative arginine n-methyltransferase genes, within 36h of in vitro culture, we achieved over 10-fold down-regulation of the respective encoded proteins in C. parvum. Pursuant to this, we observed that knockdown of C. parvum lactate dehydrogenase produced a dramatic reduction in intracellular growth and development of C. parvum by 56h of culture. On the other hand, C. parvum putative arginine n-methyltransferase knockdown did not appear to have any effect on parasite growth, but nevertheless provided the proof-of-principle that the morpholino knockdown assay in C. parvum was consistent. Together, our findings present a gene regulation approach for interrogating gene function in C. parvum in vitro, and further provide genetic evidence for the essential role of C. parvum lactate dehydrogenase in fueling the growth and development of intracellular C. parvum.

Biosynthetic Pathways of Purine and Pyridine Alkaloids in Coffee Plants

Caffeine (1,3,7-N-trimethylxanthine) and trigonelline (IN-methylnicotinic acid) are major alkaloids in coffee plants. The key enzymes involved-in the biosyntliesis of these compounds are very closely related N-methyltransferases belonging to the motif B' family of methyltransferases. The major biosynthetic pathways of caffeine and trigonelline are summarized in this review, including new evidence obtained from recombinant enzymes. In addition, precursor supply pathways are discussed with newly obtained results. Transgenic plants produced by the modification of the expression of N-methyltransferase genes are also introduced.

Salicylic acid and methyljasmonate restore the transcription of caffeine biosynthetic N-methyltransferases from a transcription inhibition noticed during late endosperm maturation in coffee

Transcripts of the three N-methyltransferase (NMT) genes involved in the core caffeine biosynthesis of coffee are repressed during the early stages of endosperm maturation, coincident with the onset of dry weight accumulation and seed desiccation. Previously, we noticed that the exogenous application of salicylic acid (SA) and methyljasmonate (MeJ) to Coffea canephora var. robusta cv. S-274 overexpresses transcripts of the three NMTs in young leaves. Thus, transcriptional regulation of caffeine biosynthetic genes may play a vital role in caffeine accumulation. We carried out this study to find if salicylic acid (SA) (50μM and 500μM concentrations) and MeJ treatment of the maturing fruits is capable of restoring the transcription activity of NMT genes. MeJ leads to overexpression of the first two NMTs (XMTs and MXMTs) whereas the effect of SA appeared to be concentration-dependent. Though SA (500μM) de-repressed all three NMTs, SA (50μM) restored transcription of only the second NMT. Neither SA (50μM) nor MeJ could restore transcription of the third and final NMT (DXMTs) of the pathway. Biochemical estimation of methylxanthines indicates a slight but significant increase in theobromine content (11.8% increase) in endosperms treated with 50μM SA. Caffeine showed a statistically significant increase in both MeJ (14.4% increase) and SA (50μM) (14.8% increase) treatments. Since de-repression in DXMTs (caffeine synthase) was not observed, theobromine to caffeine conversion in MeJ and SA (50μM) treated fruits is probably catalyzed by the basal or the extant DXMT gene product. Caffeine levels remained constant in SA (500μM) treatments indicating a possible post-transcriptional regulation. These results are indicative of cross talk between the cascades induced by SA and MeJ and the maturation-induced regulation of caffeine accumulation and may be helpful in studying the interaction between these pathways.

Co-expression of ApGSMT and ApDMT promotes biosynthesis of glycine betaine in rice (Oryza sativa L.) and enhances salt and cold tolerance

Glycine betaine (GB) is an osmoprotectant that protects plants from abiotic stress. GB is biosynthesized by choline dehydrogenation/oxidation and glycine methylation. The latter pathway involves glycine as a substrate and produces GB via successive methylation of glycine by two different N-methyltransferase enzymes; glycine sarcosine methyltransferase (GSMT), and sarcosine dimethylglycine methyltransferase (SDMT). In this study, Agrobacterium-mediated gene transformation was used to produce transgenic rice (Oryza sativa L., cv. Nipponbare) plants containing ApGSMT and ApDMT genes isolated from Aphanothece halophytica. The co-expression of both ApGSMT and ApDMT transgenes resulted in a significant increase of GB biosynthesis and enhanced tolerance to salt and cold stresses in the transgenic rice plants. These results demonstrate the potential of bioengineering for glycine N-methyltransferase genes in crop plants tolerance to abiotic stress.

Identification of splice variants, expression analysis and single nucleotide polymorphisms of the PRMT2 gene in dairy cattle

Protein arginine N-methyltransferase 2 (PRMT2), also named HRMT1L1, belongs to the Bovine Protein arginine N-methyltransferase (PRMT) genes which are involved in the immune response. To explore the variability of the PRMT2 gene and resistance to mastitis in cows, splice variant (SV), and single nucleotide polymorphisms (SNPs) were identified in this study. A SV (PRMT2-SV) lacking exon 7 (98-bp) of the PRMT2 gene was found in healthy and mastitis-infected mammary gland tissues. Two of four SNPs were significantly associated with bovine milk yield and protein content. Further, we estimated the relative expression of PRMT2-SV in the mammary gland tissue of dairy cattle by using quantitative real-time polymerase chain reaction. The result showed that expression of the PRMT2-SV mRNA was significantly upregulated 4.02-fold (p<0.05) in infected mammary tissues (n=5) compared to healthy tissues (n=5). Our findings reveal that PRMT2-SV may play an important role in mastitis resistance in dairy cattle. The SNPs may be used as a possible candidate SNPs for marker-assisted selection and management in Chinese Holstein cattle.

Evaluating the effect and effectiveness of different constructs with a conserved sequence for silencing of Coffea canephora N-methyltransferases

Caffeine biosynthesis involves sequential methylation of the purine ring by the transfer of methyl group from the methyl donor S-adenosyl methionine. Genes that code for the individual methyl transferase have a high degree of sequence homology. To achieve decaffeination by genetic engineering of coffee, the effect and efficiency of different post-transcriptional gene silencing (PTGS) constructs were evaluated. Sense, antisense and invert repeat constructs were developed using a 339 bp fragment from the conserved region of the coffee N-methyltransferase genes involved in caffeine biosynthesis. The silencing constructs were mobilized into Agrobacterium tumefaciens (EHA101). Sonication assisted Agrobacterium-mediated transformation of the somatic embryos of C.canephora was performed, followed by regeneration of the transformants. Molecular analysis and identification of flanking regions confirmed the stable integration of the transgenes in the transformants and their integration in non coding regions of the genome, respectively. Effectiveness of the silencing constructs was evaluated by transcript analysis of the targeted genes by RT-PCR and northern blotting. The silencing efficiency was further evaluated by estimation of the purine alkaloid content in the transgenic lines using HPLC. The three constructs differed in their silencing efficiencies and specificity. Though the constructs were not specific to a single N-methyltransferase, transformants obtained were mainly affected in one of the N-methyltransferases. Results show that the use of homologous coding sequence in the PTGS constructs results in a much higher efficiency in silencing the caffeine biosynthetic pathway. Further, the lowering of caffeine content should be the preferred over complete decaffeination due to extremely low survival rate of transgenic plants with extremely low levels of caffeine.

Involvement of a novel intronic microRNA in cross regulation of N-methyltransferase genes involved in caffeine biosynthesis in Coffea canephora

There are numerous reports on intronic miRNAs from plants, most of which are involved in the regulation of unrelated genes. Some of the target genes are antagonistic to the host genes. Intronic miRNAs in animal systems, however, are known to have synergistic effects. This article is the first to report a similar regulatory effect of a miRNA originating from an intron in plants. NMT genes involved in caffeine biosynthesis were silenced to obtain transformants with reduced caffeine. Transcript analysis revealed the accumulation of transcripts for a related NMT gene (CaMTL1) in transformants bearing either antisense or RNAi constructs. The altered expression was assumed to relate to the silencing of the NMT genes. Bioinformatics analysis of the genes involved in biosynthesis revealed the presence of an intronic miRNA originating from the intron of the theobromine synthase gene targeting CaMTL1. The putative miRNA was cloned and sequenced. Modified 5′-RLM-RACE mapping of the cleavage site and subsequent Northern blotting experimentally demonstrated the presence and activity of such a miRNA in Coffea canephora. This novel regulatory mechanism previously unreported in plants will shed more light onto the evolution of multigene families and the role of introns in this process.

The freshwater cyanobacterium Anabaena doliolum transformed with ApGSMT-DMT exhibited enhanced salt tolerance and protection to nitrogenase activity, but became halophilic

Glycine betaine (GB) is an important osmolyte synthesized in response to different abiotic stresses, including salinity. The two known pathways of GB synthesis involve: 1) two step oxidation of choline (choline → betaine aldehyde → GB), generally found in plants, microbes and animals; and 2) three step methylation of glycine (glycine → sarcosine → dimethylglycine → GB), mainly found in halophilic archaea, sulphur bacteria and the cyanobacterium Aphanothece (Ap.) halophytica. Here, we transformed a salt-sensitive freshwater diazotrophic filamentous cyanobacterium Anabaena (An.) doliolum with N-methyltransferase genes (ApGSMT-DMT) from Ap. halophytica using the triparental conjugation method. The transformed An. doliolum synthesized and accumulated GB in cells, and showed increased salt tolerance and protection to nitrogenase activity. The salt responsiveness of the transformant was also apparent as GB synthesis increased with increasing concentrations of NaCl in the nutrient solution, and maximal [12.92 µmol (g dry weight)(-1)] in cells growing at 0.5 M NaCl. Therefore, the transformed cyanobacterium has changed its behaviour from preferring freshwater to halophily. This study may have important biotechnological implications for the development of stress tolerant nitrogen-fixing cyanobacteria as biofertilizers for sustainable agriculture.

Progress in Sequencing Human Cancer Genomes: Advancements in Childhood Medulloblastoma

Improvements in technology and sequencing of the human genome have provided a template for polymerase chain reaction primers to amplify and sequence coding exons of large protein-coding genes. This has lead to genome-wide analyses of human cancers and sequencing of all protein-encoding genes in more than 80 human cancers. Medulloblastomas (MBs) are the most common malignant brain tumor in children and are diagnosed in approximately 1 in 200 000 children less than 15 years old each year.1 Prior research has identified 2 subgroups of MBs associated with either alterations in genes of the Hedgehog pathway and those mutated in the Wnt pathway.2,3 Alterations in TP53 (8), MYC, and OTX2 have also been uncovered.4,5 Although these alterations have helped define genetic subtypes of MDs, most patients do not have these mutations. In a recent study by Parsons et al, copy alterations were assessed using microarrays and all known protein-coding genes microRNA genes were sequenced.6 The authors found that each tumor had 11 gene alterations, 5 to 10 times fewer than that of other solid tumors sequenced to date. Mutations in known pathways of medulloblastoma were confirmed including Wnt and Hedgehog pathways. Additionally, a number of new mutations were uncovered including histone-lysine N-methyltransferase genes MLL2 or MLL3 in 16% of MB patients. These involved genes were found to be tumor suppressor genes, being responsible for chromatin remodeling and transcriptional regulation. In contrast to germline mutations present in the fertilized egg from which individuals develop, somatic mutations occur in normal cells as tissues progress during postnatal life. Somatic mutations in tumors either have no effect on tumor growth (passenger mutations) or confer an advantage allowing it to expand, invade local tissue, and/or metastasize (driver mutation). Passenger mutations provide a record of the number of divisions a cell has undergone during both normal development and tumor progression. Although most adult cancers such as breast, ovary, colorectal, and pancreas, and glioma7,8 have 1000 to 10 000 somatic mutations, some cancers particularly prone to environmental carcinogens such as melanoma and lung cancer may have more than 100 000.7,9-11 The low number of mutations in medulloblastomas, along with key mutations in developmental pathways, may be key differences differentiating adult from childhood cancers. An improved genetic classification for MB could be beneficial both in terms of defining prognosis as well as developing specific therapies. Currently, large cell/anaplastic MBs associated with MYC amplification have a worse prognosis than desmoplastic MBs that are often associated with PTCH1 or other Hedgehog pathway alterations.3,12,13 The classic MB subtype does not have a defining molecular alterations, and the MLL2/MLL3 mutations do not fit into a definable subtype.6 Furthermore, some cases show no mutations in cancer genes or only one alteration of any gene. Clearly, there are far more alterations to identify. As our understanding of malignant brain tumor biology has progressed, so too has the goal to develop more effective, less toxic, rationally conceived therapeutics. The standard of care for adjuvant therapies are relatively uniform, consisting of a combination of radiation and cytotoxic chemotherapy. As molecular profiling continues to evolve, however, the opportunities for more targeted, individualized intervention are rapidly increasing. Moving forward, under the International Cancer Genome Consortium, large-scale sequencing of each major cancer subtype is underway.14 With improvement in technology and declines in cost, it is likely that whole genome sequencing will be the methodology of the future rather than limiting exploration to sequencing the DNA of exons of protein-coding genes or analysis of transcriptomes. Complete sequencing will have a role on detection, diagnosis, treatment, and prevention, while further defining pathogenesis, progression, and mechanisms of drug resistance. Although each cancer type is likely defined by overlapping sets of mutated genes, a single genomic screening test may become an integral element of treatment planning of newly diagnosed patients and the design of clinical trials. To this end, initial stratification of brain tumors patients into molecularly determined treatment groups has already begun. The 2 best examples are promoter methylation and transcriptional silencing of O-6-methlylguanine-DNA methyltransferase (MGMT), which leads to improved GMB chemosensitivity, and eGFR vIII mutation, which may allow response to the eGFR inhibitor erlotinib. These findings represent a paradigm shift—one that stratifies patients by molecular determinants into treatment groups, thereby guiding personalized, biologically grounded therapies.Figure 1: Axial MRI with and without contrast depicting a classic medulloblastoma.Ricardo J. Komotar Robert M. Starke E. Sander Connolly Michael B. Sisti

Homocysteine homeostasis in the rat is maintained by compensatory changes in cystathionine β-synthase, betaine-homocysteine methyltransferase, and phosphatidylethanolamine N-methyltransferase gene transcription occurring in response to maternal protein and folic acid intake during pregnancy and fat intake after weaning

The reactions of the methionine/homocysteine pathway are mediated by several enzymes, including phosphatidylethanolamine N-methyltransferase, cystathionine β-synthase, and betaine-homocysteine methyltransferase. Homocysteine homeostasis is regulated by these enzymes. We hypothesized here that the protein and folic acid content in the maternal diet affects methionine/homocysteine metabolism in the progeny. To test this hypothesis, pregnant rats were fed a diet with normal protein and normal folic acid levels (a modified casein-based AIN-93G diet), a protein-restricted and normal folic acid diet, a protein-restricted and folic acid-supplemented diet, or a normal protein and folic acid-supplemented diet. The progeny were fed either the modified AIN-93G diet or a high-fat lard-based diet. Progeny were analyzed for expression of the phosphatidylethanolamine N-methyltransferase, cystathionine β-synthase, and betaine-homocysteine methyltransferase genes in the liver and for serum homocysteine concentration. Interactions between prenatal and postnatal nutrition were also determined. The progeny of the dams fed the diets supplemented with folic acid showed decreased expression of all 3 genes (P < .001). An interaction effect between the protein and folic acid content in the maternal diet contributed to this down-regulation (P < .001), and the postweaning diet modified these effects. Serum homocysteine concentrations were approximately 15% higher in the male rats (P < .01), but neither prenatal nutrition nor the postweaning diet affected it significantly. We conclude that maternal diet during gestation has an important effect on the transcription level of these 3 genes, but changes in gene expression were not associated with significant changes in progeny homocysteine concentrations.

The genetic landscape of the childhood cancer medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor of children. To identify the genetic alterations in this tumor type, we searched for copy number alterations using high-density microarrays and sequenced all known protein-coding genes and microRNA genes using Sanger sequencing in a set of 22 MBs. We found that, on average, each tumor had 11 gene alterations, fewer by a factor of 5 to 10 than in the adult solid tumors that have been sequenced to date. In addition to alterations in the Hedgehog and Wnt pathways, our analysis led to the discovery of genes not previously known to be altered in MBs. Most notably, inactivating mutations of the histone-lysine N-methyltransferase genes MLL2 or MLL3 were identified in 16% of MB patients. These results demonstrate key differences between the genetic landscapes of adult and childhood cancers, highlight dysregulation of developmental pathways as an important mechanism underlying MBs, and identify a role for a specific type of histone methylation in human tumorigenesis.

Generation of tobacco lines with widely different reduction in nicotine levels via RNA silencing approaches

Issues related to the nicotine content of tobacco have been public concerns. Several reports have described decreasing nicotine levels by silencing the putrescine N-methyltransferase (PMT) genes, but the reported variations of nicotine levels among transgenic lines are relatively low in general. Here we describe the generation in tobacco (Nicotiana tabacum) lines with widely different, reduced nicotine levels using three kinds of RNA-silencing approaches. The relative efficacies of suppression were compared among the three approaches regarding the aspect of nicotine level in tobacco leaves. By suppressing expression of the PMT genes, over 200 transgenic lines were obtained with nicotine levels reduced by 9.1-96.7%. RNA interference (RNAi) was the most efficient method of reducing the levels of nicotine,whereas cosuppression and antisense methods were less effective. This report gives clues to the efficient generation of plants with a variety of metabolite levels, and the results demonstrate the relative efficiencies of various RNA-silencing methods.

Physiogenomic Comparison of Human Fat Loss in Response to Diets Restrictive of Carbohydrate or Fat

Genetic factors that predict responses to diet may ultimately be used to individualize recommendations. We used physiogenomics to explore associations among polymorphisms in candidate genes and changes in relative body fat (Δ%BF) to low fat (LFD) and low carbohydrate (LCD) diets. We assessed Δ%BF with DXA in 93 healthy adults who consumed a LCD (12% carbohydrate) and in 70, a LFD (25% fat). Fifty‐three single nucleotide polymorphisms (SNPs) from 28 candidate genes involved in food intake, energy homeostasis and adipocyte regulation were ranked according to probability of association with Δ%BF using multiple linear regression. Dieting reduced %BF by 3.0 ± 2.6% for LCD and 1.9 ± 1.7% for LFD (NS). SNPs in 9 genes were significantly associated with Δ%BF, with 4 significant after correction for multiple statistical testing: rs322695 near the retinoic acid receptor beta (RARB) (p&lt;0.005), rs2838549 in the hepatic phosphofructokinase (PFKL) and rs3100722 in the histamine N‐methyl transferase (HNMT) genes (both p&lt;0.041) due to LFD; and the rs5950584 SNP in the angiotensin receptor Type II (AGTR2) gene due to LCD (p&lt;0.021). Fat loss with LCD and LFD appears to have distinct mechanisms, with PFKL and HNMT and RARB involved in fat restriction; and AGTR2 involved in carbohydrate restriction. These discoveries could provide clues to important physiologic mechanisms underlying the Δ%BF to LCD and LFD.