TaqMan REAL-Time PCR-based approach for differentiation between Globodera rostochiensis (golden nematode) and Globodera artemisiae species.

Cyst nematodes from the genus Globodera are common, and widely distributed parasites of Solaraceae. Intact cysts persist in soil even up to 10 years without detriment. Out of two Globodera species occurring in Poland, Globodera rostochiensis is considered by European and Mediterranean Plant Protection Organization (EPPO) as a quarantine pest, while Globodera artemisiae is not. Therefore, the distinction between these two species is crucial. Classic methods of detection and differentiation are laborious and time-consuming. Instead, application of molecular biology techniques allows obtaining of rapid and reliable results. The aim of this study was to establish detection and differentiation method of two species, G. rostochiensis and G. artemisiae, based upon real-time polymerase chain reaction with the use of TaqMan probes. In reaction with primers and probes specific for the nematodes' ribosomal DNA (rDNA), the samples used were DNAs isolated from the two species, alone or in mixture, as well as crushed single cysts. Applied probes enable not only to identify the species in DNA mixtures but also in a single cyst. The use of a crushed cyst eliminates long-lasting procedure of DNA isolation and reduces costs of analysis.

Structural bioinformatics analysis of enzymes involved in the biosynthesis pathway of the hypermodified nucleoside ms(2)io(6)A37 in tRNA.

TRNAs from all organisms contain posttranscriptionally modified nucleosides, which are derived from the four canonical nucleosides. In most tRNAs that read codons beginning with U, adenosine in the position 37 adjacent to the 3' position of the anticodon is modified to N(6)-(Delta(2)-isopentenyl) adenosine (i(6)A). In many bacteria, such as Escherichia coli, this residue is typically hypermodified to N(6)-isopentenyl-2-thiomethyladenosine (ms(2)i(6)A). In a few bacteria, such as Salmonella typhimurium, ms(2)i(6)A can be further hydroxylated to N(6)-(cis-4-hydroxyisopentenyl)-2-thiomethyladenosine (ms(2)io(6)A). Although the enzymes that introduce the respective modifications (prenyltransferase MiaA, methylthiotransferase MiaB, and hydroxylase MiaE) have been identified, their structures remain unknown and sequence-function relationships remain obscure. We carried out sequence analysis and structure prediction of MiaA, MiaB, and MiaE, using the protein fold-recognition approach. Three-dimensional models of all three proteins were then built using a new modeling protocol designed to overcome uncertainties in the alignments and divergence between the templates. For MiaA and MiaB, the catalytic core was built based on the templates from the P-loop NTPase and Radical-SAM superfamilies, respectively. For MiaB, we have also modeled the C-terminal TRAM domain and the newly predicted N-terminal flavodoxin-fold domain. For MiaE, we confidently predict that it shares the three-dimensional fold with the ferritin-like four-helix bundle proteins and that it has a similar active site and mechanism of action to diiron carboxylate enzymes, in particular, methane monooxygenase (E.C.1.14.13.25) that catalyses the biological hydroxylation of alkanes. Our models provide the first structural platform for enzymes involved in the biosynthesis of i(6)A, ms(2)i(6)A, and ms(2)io(6)A, explain the data available from the literature and will help to design further experiments and interpret their results.

The sequence and model structure analysis of three Polish peanut stunt virus strains.

Peanut stunt virus (PSV) belongs to the Cucumovirus genus of the family Bromoviridae and is widely distributed worldwide, also in Poland. PSV is a common pathogen of a wide range of economically important plants. Its coat protein (CP), similarly as in other viruses, plays an important role in many processes during viral life cycle and has great impact on the infectivity. In this study, we present the results of sequence-structure analysis of CP derived from three Polish strains of PSV: PSV Ag, G, and P. Sequences were determined using RT-PCR amplification followed by sequencing and compared with each other and also with CP from other known PSV viruses. We analyzed their phylogenetic relationship, based on CP sequence, using bioinformatic tools as well as their spatial model using homology-modeling approach with combination of ROSETTA algorithm for de novo modeling. We compared our model with those recently obtained for other cucumoviruses including PSV-Er. Our results have shown that all Polish strains probably belong to the first subgroup of PSV viruses. Homology level between strains Ag and G proved very high. Using theoretical modeling approach we obtained a model very similar to the one resolved previously with the differences caused by slightly different amino acid sequence. We have also undertaken an attempt to analyze its distant regions; however, results are not unequivocal. Analysis of symptoms and their correlation with specific amino acid position was also performed on the basis of results published elsewhere. The definite interpretation is impeded by the presence of satellite RNAs in Ag and P strains modulating symptoms' severity, though.