Touroutine, D. et al. (2019) International Worm Meeting "Superselective primers - a powerful molecular tool with broad application to detect SNPs."

Touroutine, D., Tanis, J.

[International Worm Meeting, 2019]

With wide spread use of single nucleotide variants generated through mutagenesis screens, the million mutation project, and genome editing technologies there is pressing need for an efficient and low-cost strategy to detect SNPs. Current SNP-discriminating methods involve PCR amplification of the variant containing region followed by either restriction digest if there is a SNP induced change in a restriction site or direct sequencing. These methods are labor intensive, requiring extra steps beyond PCR, and expensive, demanding purchase of unique restriction enzymes or sequencing reactions. Other strategies, including simple allele-discriminating PCR and ARMS-PCR require extensive troubleshooting to identify primers and conditions that can accurately distinguish variants. We have developed a rapid and cheap method for detection of point mutants through optimization of superselective (SS) primers [1] for end PCR. A SS primer consists of three sections: an anchor, bridge and foot. The anchor region, which anneals to the template, is followed by a bridge containing non-specific sequence and then a short foot which anneals to the template to distinguish mismatches. We explored how length, stability and sequence composition of each segment affected primer selectivity in order to develop simple rules for primer design that allow for discrimination between any mismatches in various genetic contexts over a broad range of annealing temperatures. Based on our design, SS primers can easily detect even (A/T-G/C) transitions, which are primarily generated by the EMS mutagen and greatly tolerated by Taq polymerase. We have demonstrated that SS primers are capable of identifying a desired mutation in the presence of a thousand fold excess wild-type template, allowing for pooling screening strategies for detection of genome editing events. Further, SS primers can be utilized for colony PCR to identify successful site-directed mutagenesis constructs. In summary, we adapted and optimized SS primers to enable greater flexibility in design and demonstrated their utility for various laboratory applications. Vargas DY, Kramar FR, Tyagi S, Marras S (2016) Multiplex real-time PCR Assays that measure the abundance of extremely rare mutations associated with cancer. PLoS One, 11(5):e0156546

Touroutine D et al. (2020) Genetics "A Rapid, Super-Selective Method for Detection of Single Nucleotide Variants in Caenorhabditis elegans."

Tanis JE, Touroutine D

[Genetics, 2020]

With the widespread use of single nucleotide variants generated through mutagenesis screens and genome editing technologies, there is pressing need for an efficient and low-cost strategy to genotype single nucleotide substitutions. We have developed a rapid and inexpensive method for detection of point mutants through optimization of SuperSelective (SS) primers for end point PCR in <i>Caenorhabditis</i><i>elegans</i> Each SS primer consists of a 5' &quot;anchor&quot; that hybridizes to the template, followed by a non-complementary &quot;bridge,&quot; and a &quot;foot&quot; corresponding to the target allele. The foot sequence is short, such that a single mismatch at the terminal 3' nucleotide destabilizes primer binding and prevents extension, enabling discrimination of different alleles. We explored how length and sequence composition of each SS primer segment affected selectivity and efficiency in various genetic contexts in order to develop simple rules for primer design that allow for differentiation between alleles over a broad range of annealing temperatures. Manipulating bridge length affected amplification efficiency, while modifying the foot sequence altered discriminatory power. Changing the anchor position enabled SS primers to be used for genotyping in regions with sequences that are challenging for standard primer design. After defining primer design parameters, we demonstrated the utility of SS primers for genotyping crude <i>C. elegans</i> lysates, suggesting that this approach could also be used for SNP mapping and screening of CRISPR mutants. Further, since SS primers reliably detect point mutations, this method has potential for broad application in all genetic systems.

Sakoguchi H et al. (2016) Biosci Biotechnol Biochem "Screening of biologically active monosaccharides: growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode Caenorhabditis elegans."

Izumori K, Yoshihara A, Sakoguchi H, Sato M

[Biosci Biotechnol Biochem, 2016]

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars d-allose (d-All), d-talose (d-Tal), and l-idose (l-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-d-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by d-All.

Sakoguchi H et al. (2016) Bioorg Med Chem Lett "Growth inhibitory effect of d-arabinose against the nematode Caenorhabditis elegans: Discovery of a novel bioactive monosaccharide."

Shintani T, Izumori K, Sato M, Sakoguchi H, Okuma K, Yoshihara A

[Bioorg Med Chem Lett, 2016]

Biological activities of unusual monosaccharides (rare sugars) have largely remained unstudied until recently. We compared the growth inhibitory effects of aldohexose stereoisomers against the animal model Caenorhabditis elegans cultured in monoxenic conditions with Escherichia coli as food. Among these stereoisomers, the rare sugar d-arabinose (d-Ara) showed particularly strong growth inhibition. The IC50 value for d-Ara was estimated to be 7.5mM, which surpassed that of the potent glycolytic inhibitor 2-deoxy-d-glucose (19.5mM) used as a positive control. The inhibitory effect of d-Ara was also observed in animals cultured in axenic conditions using a chemically defined medium; this excluded the possible influence of E. coli. To our knowledge, this is the first report of biological activity of d-Ara. The d-Ara-induced inhibition was recovered by adding either d-ribose or d-fructose, but not d-glucose. These findings suggest that the inhibition could be induced by multiple mechanisms, for example, disturbance of d-ribose and d-fructose metabolism.

Yoshihara A et al. (2019) Bioorg Med Chem Lett "Growth inhibition by 1-deoxy-d-allulose, a novel bioactive deoxy sugar, screened using Caenorhabditis elegans assay."

Sakoguchi H, Fleet GWJ, Izumori K, Shintani T, Sato M, Yoshihara A

[Bioorg Med Chem Lett, 2019]

The biological activities of deoxy sugars (deoxy monosaccharides) have remained largely unstudied until recently. We compared the growth inhibition by all 1-deoxyketohexoses using the animal model Caenorhabditis elegans. Among the eight stereoisomers, 1-deoxy-d-allulose (1d-d-Alu) showed particularly strong growth inhibition. The 50% inhibition of growth (GI₅₀) concentration by 1d-d-Alu was estimated to be 5.4mM, which is approximately 10 times lower than that of d-allulose (52.7mM), and even lower than that of the potent glycolytic inhibitor, 2-deoxy-d-glucose (19.5mM), implying that 1d-d-Alu has a strong growth inhibition. In contrast, 5-deoxy- and 6-deoxy-d-allulose showed no growth inhibition of C. elegans. The inhibition by 1d-d-Alu was alleviated by the addition of d-ribose or d-fructose. Our findings suggest that 1d-d-Alu-mediated growth inhibition could be induced by the imbalance in d-ribose metabolism. To our knowledge, this is the first report of biological activity of 1d-d-Alu which may be considered as an antimetabolite drug candidate.

Katane M et al. (2020) Biochim Biophys Acta Proteins Proteom "Biochemical characterization of d-aspartate oxidase from Caenorhabditis elegans: Its potential use in the determination of free D-glutamate in biological samples."

Katane M, Sekine M, Nakayama K, Homma H, Kuwabara H, Saitoh Y, Miyamoto T

[Biochim Biophys Acta Proteins Proteom, 2020]

d-Aspartate oxidase (DDO) is a flavin adenine dinucleotide (FAD)-containing flavoprotein that stereospecifically acts on acidic D-amino acids (i.e., free d-aspartate and D-glutamate). Mammalian DDO, which exhibits higher activity toward d-aspartate than D-glutamate, is presumed to regulate levels of d-aspartate in the body and is not thought to degrade D-glutamate in vivo. By contrast, three DDO isoforms are present in the nematode Caenorhabditis elegans, DDO-1, DDO-2, and DDO-3, all of which exhibit substantial activity toward D-glutamate as well as d-aspartate. In this study, we optimized the Escherichia coli culture conditions for production of recombinant C. elegans DDO-1, purified the protein, and showed that it is a flavoprotein with a noncovalently but tightly attached FAD. Furthermore, C. elegans DDO-1, but not mammalian (rat) DDO, efficiently and selectively degraded D-glutamate in addition to d-aspartate, even in the presence of various other amino acids. Thus, C. elegans DDO-1 might be a useful tool for determining these acidic D-amino acids in biological samples.

Shintani T et al. (2019) J Appl Glycosci (1999) "D-Allose, a Stereoisomer of D-Glucose, Extends the Lifespan of Caenorhabditis elegans via Sirtuin and Insulin Signaling."

Izumori K, Sakoguchi H, Yoshihara A, Shintani T, Sato M

[J Appl Glycosci (1999), 2019]

D-Allose (D-All), C-3 epimer of D-glucose, is a rare sugar known to suppress reactive oxygen species generation and prevent hypertension. We previously reported that D-allulose, a structural isomer of D-All, prolongs the lifespan of the nematode Caenorhabditis elegans. Thus, D-All was predicted to affect longevity. In this study, we provide the first empirical evidence that D-All extends the lifespan of C. elegans. Lifespan assays revealed that a lifespan extension was induced by 28 mM D-All. In particular, a lifespan extension of 23.8 % was achieved (p< 0.0001). We further revealed that the effects of D-All on lifespan were dependent on the insulin gene daf-16 and the longevity gene sir-2.1, indicating a distinct mechanism from those of other hexoses, such as D-allulose, with previously reported antiaging effects.

Sato M et al. (2008) J Nat Med "Potential anthelmintic: D-psicose inhibits motility, growth and reproductive maturity of L1 larvae of Caenorhabditis elegans."

Izumori K, Yamasaki T, Kurose H, Sato M

[J Nat Med, 2008]

No anthelmintic sugars have yet been identified. Eight ketohexose stereoisomers (D- and L-forms of psicose, fructose, tagatose and sorbose), along with D-galactose and D-glucose, were examined for potency against L1 stage Caenorhabditis elegans fed Escherichia coli. Of the sugars, D-psicose specifically inhibited the motility, growth and reproductive maturity of the L1 stage. D-Psicose probably interferes with the nematode nutrition. The present results suggest that D-psicose, one of the rare sugars, is a potential anthelmintic.

Yasuaki Saitoh et al. (2010) East Asia Worm Meeting "Study on physiological functions of D-amino acid degradative enzymes in nematode Caenorhabditis elegans"

Yousuke Seida, Kazuhiro Maeda, Tomonori Kawata, Masumi Katane, Hiroyuki Kobuna, Takao Inoue, Yasuaki Saitoh, Hiroyuki Arai, Yasuhito Nakagawa, Masae Sekine, Taro Sakamoto, Hiroshi Homma, Takemitsu Furuchi

[East Asia Worm Meeting, 2010]

Among free D-amino acids existing in living organisms, D-serine (D-Ser) and D-aspartate (D-Asp) are the most actively studied. D-Ser has been proposed as a neuromodulator that regulates L-glutamate-mediated activation of the N-methyl-D-Asp (NMDA) receptor by acting as a co-agonist. On the other hand, several lines of evidence suggest that D-Asp plays important roles in regulating developmental processes, hormone secretion and steroidogenesis. D-Amino acid oxidase (DAO) and D-Asp oxidase (DDO) are known as stereospecific degradative enzymes that catalyze the oxidative deamination of D-amino acids. DAO displays broad substrate specificity and acts on several neutral and basic D-amino acids, while DDO is highly specific for acidic D-amino acids. DAO and DDO are presumed to regulate endogenous D-Ser and D-Asp levels, respectively, as well as mediate the elimination of accumulated exogenous D-amino acids in various organs. Previously, we demonstrated that nematode Caenorhabditis elegans, a multicellular model animal has at least one active DAO gene and three active DDO genes, while it had been thought that most organisms bear only one copy of each DAO and DDO gene. In addition, our previous study revealed that the spatiotemporal distributions of these enzymes in the body of C. elegans are different from one another. In this study, to elucidate the physiological roles of the C. elegans DAO and DDOs, we characterized several phenotypes of four C. elegans mutants in which each gene is partially deleted and inactivated. We also determined free D-amino acid contents in several worm samples using high-performance liquid chromatography (HPLC) techniques. We will report the phenotypes of the C. elegans mutants in comparison with those of wild-type C. elegans, as well as alterations in D-amino acid levels within the body.

Saitoh, Yasuaki et al. (2013) International Worm Meeting "D-Aspartate oxidase is involved in the caloric restriction-induced lifespan extension in C. elegans."

Inoue, Takao, Sekine, Masae, Saitoh, Yasuaki, Arai, Hiroyuki, Furuchi, Takemitsu, Sakamoto, Taro, Okutsu, Mari, Homma, Hiroshi, Katane, Masumi

[International Worm Meeting, 2013]

Among free D-amino acids existing in living organisms, D-serine (D-Ser) and D-aspartate (D-Asp) are the most intensively studied. In mammals, D-Ser has been proposed as a neuromodulator that regulates L-glutamate (L-Glu)-mediated activation of the N-methyl-D-Asp (NMDA) receptor by acting as a co-agonist. On the other hand, several lines of evidence suggest that D-Asp plays important roles in regulating hormone secretion and steroidogenesis. D-Amino acid oxidase (DAO) and D-Asp oxidase (DDO) are known as stereospecific degradative enzymes that catalyze the oxidative deamination of D-amino acids. Mammalian DAO and DDO are presumed to regulate endogenous D-Ser and D-Asp levels, respectively. Previously, we demonstrated that D-Ser, D-Asp, D-Glu and D-alanine (D-Ala) are present in nematode Caenorhabditis elegans, a multicellular model animal. We also found that C. elegans has at least one active DAO gene and three active DDO genes (DDO-1, DDO-2 and DDO-3), and that the spatiotemporal distributions of these enzymes in the body of C. elegans differ from one another. Furthermore, our previous study showed that alterations of brood size and hatching rate are observed in four C. elegans mutants lacking each gene for the DAO and DDOs. Interestingly, lifespan extension was observed in the DDO-3 mutant. To characterize the mechanism of lifespan extension in the DDO-3 mutant, we performed genetic epistasis experiments to test interactions between the DDO-3 gene and other known longevity pathways. The results suggest that DDO-3 is involved in caloric restriction-induced lifespan extension but not in insulin/IGF signaling pathway, NAD/sir2 pathway nor mitochondrial electron transport system. We also found that D-Glu and L-tryptophan (L-Trp) accumulate throughout life in the DDO-3 mutant. Now we are investigating the relationship between aging and the accumulations of D-Glu and L-Trp.