Molecular characterisation of the full-length genome of olive latent virus 1 isolated from tomato

Olive latent virus 1 (OLV-1) is a species of the Necrovirus genus. So far, it has been reported to infect olive, citrus tree and tulip. Here, we determined and analysed the complete genomic sequence of an isolate designated as CM1, which was collected from tomato plant in the Wielkopolska region of Poland and represents the prevalent isolate of OLV-1. The CM1 genome consists of monopartite single-stranded positive-sense RNA genome sized 3,699 nt with five open reading frames (ORFs) and small inter-cistronic regions. ORF1 encodes a polypeptide with a molecular weight of 23 kDa and the read-through (RT) of its amber stop codon results in ORF1 RT that encodes the virus RNA-dependent RNA polymerase. ORF2 and ORF3 encode two peptides, with 8 kDa and 6 kDa, respectively, which appear to be involved in cell-to-cell movement. ORF4 is located in the 3′ terminal and encodes a protein with 30 kDa identified as the viral coat protein (CP). The differences in CP region of four OLV-1 isolates whose sequences have been deposited in GenBank were observed. Nucleotide sequence identities of the CP of tomato CM1 isolate with those of olive, citrus and tulip isolates were 91.8%, 89.5% and 92.5%, respectively. In contrast to other OLV-1 isolates, CM1 induced necrotic spots on tomato plants and elicited necrotic local lesions on Nicotiana benthamiana, followed by systemic infection. This is the third complete genomic sequence of OLV-1 reported and the first one from tomato.     

DArT markers tightly linked with the <Emphasis Type="Italic">Rfc1</Emphasis> gene controlling restoration of male fertility in the CMS-C system in cultivated rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

The Rfc1 gene controls restoration of male fertility in rye (Secale cereale L.) with sterility-inducing cytoplasm CMS-C. Two populations of recombinant inbred lines (RIL) were used in this study to identify DArT markers located on the 4RL chromosome, in the close vicinity of the Rfc1 gene. In the population developed from the 541×2020LM intercross, numerous markers tightly linked with the restorer gene were identified. This group contained 91 DArT markers and three SCARs additionally analyzed in the study. All these markers were mapped in the distance not exceeding 6 cM from the gene of interest. In the second mapping population (541×Ot1-3 intercross), only 9 DArT markers located closely to the Rfc1 gene were identified. Five of these DArT markers were polymorphic in both populations.     

Essential Role for the Mnk Pathway in the Inhibitory Effects of Type I Interferons on Myeloproliferative Neoplasm (MPN) Precursors

The mechanisms of generation of the antineoplastic effects of interferons (IFNs) in malignant hematopoietic cells remain to be precisely defined. We examined the activation of type I IFN-dependent signaling pathways in malignant cells transformed by Jak2V617F, a critical pathogenic mutation in myeloproliferative neoplasms (MPNs). Our studies demonstrate that during engagement of the type I IFN receptor (IFNAR), there is activation of Jak-Stat pathways and also engagement of Mnk kinases. Activation of Mnk kinases is regulated by the Mek/Erk pathway and is required for the generation of IFN-induced growth inhibitory responses, but Mnk kinase activation does not modulate IFN-regulated Jak-Stat signals. We demonstrate that for type I IFNs to exert suppressive effects in malignant hematopoietic progenitors from patients with polycythemia vera, induction of Mnk kinase activity is required, as evidenced by studies involving pharmacological inhibition of Mnk or siRNA-mediated Mnk knockdown. Altogether, these findings provide evidence for key and essential roles of the Mnk kinase pathway in the generation of the antineoplastic effects of type I IFNs in Jak2V617F-dependent MPNs.     

Complex patterns of population genetic structure of moose, Alces alces, after recent spatial expansion in Poland revealed by sex-linked markers

In recent years, human activity directly and indirectly influenced the demography of moose in Poland. The species was close to extinction, and only a few isolated populations survived after the Second World War; then, unprecedented demographic and spatial expansions had occurred, possibly generating a very complex pattern of population genetic structure at the present-day margins of the species range in Poland. Over 370 moose from seven populations were collected from Poland, and partial sequences of the mitochondrial control region (mtDNA-cr; 607 bp) were obtained. In addition, the entire mtDNA cytochrome b gene (1,140 bp) and Y-chromosome markers (1,982 bp in total) were studied in a chosen set of individuals. Twelve mtDNA haplotypes that all belonged to the European moose phylogroup were recorded. They could be divided into two distinct clades: Central Europe and the Ural Mountains. The first clade consists of three distinct groups/branches: Biebrza, Polesie, and Fennoscandia. The Biebrza group has experienced spatial and demographic expansion in the recent past. Average genetic differentiation among moose populations in Poland at mtDNA-cr was great and significant (Φ _ST?=?0.407, p?<?0.001). Using mtDNA-cr data, four separate groups of population were recognized using spatial analysis of molecular variance and principal coordinate analysis, including a relict population in Biebrza National Park, a reintroduced Kampinos National Park population, as well as populations that were descendants of moose that colonized Poland from the east (Lithuania, Belarus, and Ukraine) and the north (former East Prussia). Among all the sequenced Y-chromosome markers, polymorphisms were found in the DBY14 marker in three populations only; four haplotypes were recorded in total. No significant differentiation was detected for this Y-linked marker among moose populations in Poland. Our mtDNA study revealed that a variety of different factors—bottleneck, the presence of relict, autochthonous populations, translocations, limited female dispersal, and the colonization from the east and north—are responsible for the observed complex pattern of population genetic structure after demographic and spatial expansion of moose in Poland.     

The participation of phytocystatin TrcC-4 in the activity regulation of EP8, the main prolamin degrading cysteine endopeptidase in triticale seeds

Phytocystatins (PCs) are protein inhibitors of endogenous plant endopeptidases and exogenous pathogen proteinases. We have previously described the protein inhibitor TrcC-4, which is probably involved in the control of protein degradation during triticale seeds germination. The occurrence of the LARFAVXEHN motif supports the TrcC-4 designation as a PC. In this paper TrcC-4 was expressed in Escherichia coli using the pET28 expression vector. TrcC-4(6×His) was purified by affinity chromatography with a single step of purification. Western blot analysis showed the presence of TrcC-4 in both developing and germinating triticale seeds. TrcC-4 protein level was higher both in scutellum of germinating seeds and in developing grains of the triticale cultivar more resistant to pre-harvest sprouting (Zorro) than in a less resistant one (Disco). Furthermore it was demonstrated that the activity of EP8, cysteine endopeptidase responsible for the mass hydrolysis of prolamin during germination, is inhibited by TrcC-4(6×His), as confirmed by native PAGE with gliadin as a substrate. These results suggest that phytocystatin TrcC-4 controls the activity of cysteine endopeptidases involved in germination and, thus, is potentially involved in pre-harvest sprouting.     

Experience with Saccharomyces boulardii Probiotic in Oncohaematological Patients

Probiotics and Antimicrobial Proteins - Very few reports have been published to date on the bloodstream infections caused by Saccharomyces spp. in oncohaematological patients, and there are no...     

Struggle to survive: aphid—plant relationships under low-light stress. A case of <Emphasis Type="Italic">Acyrthosiphon pisum</Emphasis> (Harris) and <Emphasis Type="Italic">Pisum sativum</Emphasis> L.

Light is primary source of energy and also plays signaling and regulatory roles in developmental processes and defense responses of plants. The aim of the study was to determine the performance, settling preferences, probing, and feeding behavior of Acyrthosiphon pisum on Pisum sativum grown in complete darkness (NL), with light at minimum level required for photoperiodic reaction (LL) and under full-light (FL) conditions. The effect of A. pisum infestation on metabolic status and defense responses of peas under FL, LL, and NL conditions was also determined. The population growth rate was limited on LL and NL pea plants as compared to FL plants. The reproductive period of aphids on LL and NL plants was eight times shorter than on plants growing in FL. In contrast to aphids on FL plants, the majority of A. pisum rejected LL and NL plants during settling. Aphid probing activities were not impeded on LL and NL plants but the probes were significantly shorter than on FL plants and consisted mainly of non-phloem activities. The analysis of tolerance of P. sativum to A. pisum showed that on FL plants, the number of aphids was nearly five times higher than on plants growing in low light (LL) at the end of the 2-week experiment but the tolerance index of FL plants was higher than that of LL plants. The contents of chlorophyll a, chlorophyll b, carotenoids, saccharides, and phenolics and the activity of β-d-glucosidase were notably lower in LL and NL plants than in FL plants. The increase in light intensity from complete darkness to the minimum level required for photoperiodic reaction did not stimulate evident changes in the measured plant biochemical parameters. These trends occurred in aphid-free (AF) and aphid-infested (AI) plants. However, under FL conditions, β-d-glucosidase activity and the content of saccharides were lower in AI plants than in AF plants. No differences in the measured plant biochemical parameters between AI and AF plants occurred under LL and NL conditions. The low β-d-glucosidase activity and low content of phenolics in the light-deprived plants that have reduced ability to photosynthesize show that under the biotic stress of aphid infestation plants invest in supporting basic metabolism rather than in defense against herbivores.     

Fusion of DNA-binding domain of Pyrococcus furiosus ligase with TaqStoffel DNA polymerase as a useful tool in PCR with difficult targets

Applied Microbiology and Biotechnology - The DNA coding sequence of TaqStoffel polymerase was fused with the DNA-binding domain of Pyrococcus furiosus ligase. The resulting novel recombinant gene...     

Mechanism of Peptide Binding and Cleavage by the Human Mitochondrial Peptidase Neurolysin

Proteolysis plays an important role in mitochondrial biogenesis, from the processing of newly imported precursor proteins to the degradation of mitochondrial targeting peptides. Disruption of peptide degradation activity in yeast, plant and mammalian mitochondria is known to have deleterious consequences for organism physiology, highlighting the important role of mitochondrial peptidases. In the present work, we show that the human mitochondrial peptidase neurolysin (hNLN) can degrade mitochondrial presequence peptides as well as other fragments up to 19 amino acids long. The crystal structure of hNLN^E475Q in complex with the products of neurotensin cleavage at 2.7 Å revealed a closed conformation with an internal cavity that restricts substrate length and highlighted the mechanism of enzyme opening/closing that is necessary for substrate binding and catalytic activity. Analysis of peptide degradation in vitro showed that hNLN cooperates with presequence protease (PreP or PITRM1) in the degradation of long targeting peptides and amyloid-β peptide, Aβ1–40, associated with Alzheimer disease, particularly cleaving the hydrophobic fragment Aβ35–40. These findings suggest that a network of proteases may be required for complete degradation of peptides localized in mitochondria.     

The characterization of arabms1: an Arabidopsis thaliana sequence homologous to bacteriophage M13 repeat element

A genomic DNA fragment was isolated from the genome of Arabidopsisthaliana via hybridization with bacteriophage M13 protein IIIrepeat element. A 45 bp region of the A. thaliana DNA fragmenthas a repeating 12 bp structure that shows sequence homologyto both the M13 repeat and to a rice minisatellite-like element.Hybridization of digests of A. thaliana genomic DNA with theminisatellite DNA generates a multilocus DNA fingerprint withlow polymorphism. Key words: Minisatellite, M13 repeat element, Arabidopsis thaliana