An assessment of the DNA barcodes of Indian freshwater fishes

Freshwater fishes in India are poorly known and plagued by many unresolved cryptic species complexes that masks some latent and endemic species. Limitations in traditional taxonomy have resulted in this crypticism. Hence, molecular approaches like DNA barcoding, are needed to diagnose these latent species. We have analyzed 1383 barcode sequences of 175 Indian freshwater fish species available in the databases, of which 172 sequences of 70 species were generated. The congeneric and conspecific genetic divergences were calculated using Kimura's 2 parameter distance model followed by the construction of a Neighbor Joining tree using the MEGA 5.1. DNA barcoding principle at its first hand approach, led to the straightforward identification of 82% of the studied species with 2.9% (S.E = 0.2) divergence between the nearest congeners. However, after validating some cases of synonymy and mislabeled sequences, 5% more species were found to be valid. Sequences submitted to the database under different names were found to represent single species. On the other hand, some sequences of the species like Barilius barna, Barilius bendelisis and Labeo bata were submitted to the database under a single name but were found to represent either some unexplored species or latent species. Overall, 87% of the available Indian freshwater fish barcodes were diagnosed as true species in parity with the existing checklist and can act as reference barcode for the particular taxa. For the remaining 13% (21 species) the correct species name was difficult to assign as they depicted some erroneous identification and cryptic species complex. Thus, these barcodes will need further assay and inclusion of barcodes of more specimens from same and sister species.     

Molecular characterization and transcriptional analysis of non-mammalian type Toll like receptor (TLR21) from rock bream (Oplegnathus fasciatus)

Toll-like receptors (TLRs) are a large family of pattern recognition receptors, which are involved in triggering host immune responses against various pathogens by detecting their evolutionarily conserved pathogen associated molecular patterns (PAMPs). TLR21 is a non-mammalian type TLR, which recognizes unmethylated CpG DNA, and is considered as a functional homolog of mammalian TLR9. In this study, we attempted to identify and characterize a novel TLR21 counterpart from rock bream (Oplegnathus fasciatus) designated as RbTLR21, at molecular level. The complete coding sequence of RbTLR21 was 2919 bp in length, which encodes a polypeptide of 973 amino acids with a predicted molecular mass of 112 kDa and a theoretical isoelectric point of 8.6. The structure of the deduced RbTLR21 protein is similar to that of the members of typical TLR family, and includes the ectodomain, which consists of 16 leucine rich repeats (LRRs), a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain. According to the pairwise sequence analysis data, RbTLR21 was homologous to that of the orange-spotted grouper (Epinephelus coioides) with 76.9% amino acid identity. Furthermore, our phylogenetic analysis revealed that RbTLR21 is closely related to E. coioides TLR21. The RbTLR21 was ubiquitously expressed in all the tissues tested, but the highest expression was found in spleen. Additionally, upon stimulation with Streptococcus iniae, rock bream iridovirus (RBIV), and Edwardsiella tarda, RbTLR21 mRNA was significantly up-regulated in spleen tissues. Collectively, our findings suggest that RbTLR21 is indeed an ortholog of the TLR21 family and may be important in mounting host immune responses against pathogenic infections.     

2,6-Diaminopurine nucleoside derivative of 9-ethyloxy-2-oxo-1,3-diazaphenoxazine (2-amino-Adap) for recognition of 8-oxo-dG in DNA

8-Oxo-2′-deoxyguanosine (8-oxo-dG) is a nucleoside resulting from oxidative damage and is known to be mutagenic. 8-Oxo-dG has been related to aging and diseases, including neurological disorders and cancer. Recently, we reported that a fluorescent nucleoside derivative, adenosine-1,3-diazaphenoxazine (Adap), forms a stable base pair with 8-oxo-dG in DNA with accompanying efficient quenching. In this study, a new Adap derivative having an additional 2-amino group on the adenosine moiety (2-amino-Adap) was designed with the anticipation of additional hydrogen bonding with the 8-oxo group of 8-oxo-dG. The properties of the ODN containing 2-amino-Adap were evaluated by measuring thermal stability and fluorescence quenching. In contrast to the previously designed Adap, the base-pairing and fluorescence quenching properties of 2-amino-Adap varied depending on the ODN sequence, and there was no clear indication of an additional hydrogen bond with 8-oxo-dG. Instead, the base pairing of 2-amino-Adap with dG was significantly destabilized compared with that of Adap with dG, resulting in improved selectivity for 8-oxo-dG in the human telomere DNA sequence. Thus, the telomere-targeting ODN probe containing 2-amino-Adap displayed selective, sensitive and quantitative detection of 8-oxo-dG in the human telomere DNA sequence in a light-up detection system using SYBR Green.     

Characterization of inosine–uridine nucleoside hydrolase (RihC) from Escherichia coli

A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480 μM. No activity was exhibited against 2′-OH and 3′-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5′-OH group or with the 2′-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.     

‘Click’ chemistry synthesis and capillary electrophoresis study of 1,4‐linked 1,2,3‐triazole AZT‐systemin conjugate

The Cu(I) catalyzed Huisgen 1,3‐dipolar azide‐alkyne cycloaddition (CuAAC) was applied for a nucleoside‐peptide bioconjugation. Systemin (Sys), an 18‐aa plant signaling peptide naturally produced in response to wounding or pathogen attack, was chemically synthesized as its N‐propynoic acid functionalized analog (Prp‐Sys) using the SPPS. Next, CuAAC was applied to conjugate Prp‐Sys with 3′‐azido‐2′,3′‐dideoxythymidine (AZT), a model cargo molecule. 1,4‐Linked 1,2,3‐triazole AZT‐Sys conjugate was designed to characterize the spreading properties and ability to translocate of cargo molecules of systemin. CuAAC allowed the synthesis of the conjugate in a chemoselective and regioselective manner, with high purity and yield. The presence of Cu(I) ions generated in situ drove the CuAAC reaction to completion within a few minutes without any by‐products. Under typical separation conditions of phosphate ‘buffer’ at low pH and uncoated fused bare‐silica capillary, an increasing peak intensity assigned to triazole‐linked AZT‐Sys conjugate was observed using capillary electrophoresis (CE) during CuAAC. CE analysis showed that systemin peptides are stable in tomato leaf extract for up to a few hours. CE‐ESI‐MS revealed that the native Sys and its conjugate with AZT are translocated through the tomato stem and can be directly detected in stem exudates. The results show potential application of systemin as a transporter of low molecular weight cargo molecules in tomato plant and of CE method to characterize a behavior of plant peptides and its analogs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Exploring the Biological and Chemical Complexity of the Ligases

Using a novel method to map and cluster chemical reactions, we have re-examined the chemistry of the ligases [Enzyme Commission (EC) Class 6] and their associated protein families in detail. The type of bond formed by the ligase can be automatically extracted from the equation of the reaction, replicating the EC subclass division. However, this subclass division hides considerable complexities, especially for the C–N forming ligases, which fall into at least three distinct types. The lower levels of the EC classification for ligases are somewhat arbitrary in their definition and add little to understanding their chemistry or evolution. By comparing the multi-domain architecture of the enzymes and using sequence similarity networks, we examined the links between overall reaction and evolution of the ligases. These show that, whilst many enzymes that perform the same overall chemistry group together, both convergent (similar function, different ancestral lineage) and divergent (different function, common ancestor) evolution of function are observed. However, a common theme is that a single conserved domain (often the nucleoside triphosphate binding domain) is combined with ancillary domains that provide the variation in substrate binding and function.     

Arabidopsis SOI33／AtENT8 Gene Encodes a Putative Equilibrative Nucleoside Transporter That Is Involved in Cytokinin Transport In Planta

The plant phytohormone cytokinin plays an important role in many facets of plant growth and development by regulating cell division and differentiation. Recent studies have shed significant light into the mechanisms of cytokinin metabolism and signaling. However, little is known about how the hormone is transported in planta, although it has been proposed that the hormone is presumably transported in nucleoside-conjugated forms. Here, we report the identification and characterization of cytokinin transporters in Arabidopsis. We previously reported that a gain-of-function mutation in the PGA22/AtlPT8 gene caused overproduction of cytokinins in planta. In an effort to screen for suppressor of pga22/atipt8 (soi) mutants, we identified a mutant soi33-1. Molecular and genetic analyses indicated that S0133 encodes a putative equilibrative nucleoside transporter (ENT), previously designated as AtENT8. Members of this small gene family are presumed to be involved in the transport of nucleosides in eukaryodc cells. Under conditions of nitrogen starvation, loss-of-function mutations in SOI33/AtENT8 or in a related gene AtENT3 cause a reduced sensitivity to the nucleoside-type cytokinins isopentenyladenine riboside (iPR) and transzeatin riboside (tZR), but display a normal response to the free base-type cytokinins isopentenyladenine (iP) and trans-zeatin (tZ). Conversely, overexpression of SOI33/AtENT8 renders transgenic plants hypersensitive to iPR but not to iP. An in planta measurement experiment indicated that uptake efficiency of^3Hlabeled iPR was reduced more than 40% in soi33 and atent3 mutants. However, a mutation in AtENT1 had no substantial effect on the cytokinin response and iPR uptake efficiency. Our results suggest that SOI33/AtENT8 and AtENT3 are involved in the transport of nucleoside-type cytokinins in Arabidopsis.     

Synthesis of peptide nucleic acid oligomers carrying 5-methylcytosine derivatives by postsynthetic substitution

The preparation of the thymine peptide nucleicacid (PNA) monomer carrying a 2-nitrophenyl group in position4 is described. This monomer is incorporated into PNAoligomers and reacted with amines to yield PNA oligomerscarrying 5-methylcytosine derivatives. During thedeprotection-modification step two side reactions weredetected: degradation of PNA oligomer from the N-terminal residue and modification of N ⁴-tert-butylbenzoyl cytosine residue. Protection of the N-terminal position and the use of N ⁴-acetyl group for the protection of cytosine eliminate these side reactions.     

枯草芽孢杆菌嘌呤核苷磷酸化酶酶学性质研究及在利巴韦林酶法合成中的应用

利用PCR技术从枯草芽孢杆菌基因组DNA中扩增出其编码嘌呤核苷磷酸化酶的两种基因deoD和punA,构建工程菌并采用金属螯合层析纯化PNP₇₀₂和PNP₈₁₆,酶学性质研究表明:二者具有一致的最适反应温度(60℃)和最适反应pH值(7~8),PNP₈₁₆磷酸解肌苷的催化效率(k_cat/K_m)比PNP₇₀₂高出11.12倍。底物特异性试验表明:PNP₇₀₂为高分子量的六聚体,而PNP₈₁₆为低分子量的三聚体。分别以纯化酶和工程菌菌体为酶源,以肌苷或鸟苷为核糖基供体,TCA(1,2,4-三氮唑-3-甲酰胺)为底物,酶法合成核苷类抗病毒药物利巴韦林,PNP₈₁₆和工程菌XL-Blue(pPNP₈₁₆)较PNP₇₀₂和工程菌XL-Blue(pPNP₇₀₂)具有更高的催化速度和底物转化率,表明来源于微生物的低分子量的三聚体PNP在核苷类药物和中间体微生物酶法合成中具有更高的应用价值。