Intragenic suppression: Stalker,a retrovirus-like transposable element,can compensate for a deficiency at the cut locus of Drosophila melanogaster

A number of mutations at the cut locus were induced by non-precise exision of a silent P-element insertion which resulted in deletions at the regulatory region of the locus. Unexpectedly, a reversion of one of these mutations was found, which appears as a result of insertion of Stalker (a retrovirus-like mobile element) near the 1.3 kb deletion. Thus an insertion of a retrovirus-like mobile element can suppress the deficiency at the regulatory region of a gene.     

Pinjata ruminata gen. et sp. n.—A New Member of Centrohelid Family Yogsothothidae (Haptista: Centroplasthelida) from the Brackish River

A new genus and species of centrohelid heliozoan Pinjata ruminata from the Tuzlukkol’ River (Orenburg Region of Russia) and Gor?koe Lake (Chelyabinsk Region of Russia) is studied with light‐ and electron microscopy. Pinjata ruminata has two types of plate scales, partially running up the sides of the axopodia. Inner plate scales (3.2–4.9 × 1.5–2.6 μm) are flat, ovate‐oblong and have a broad axial thickening and a thin electron‐dense border. Outer plate scales (4.2–6.7 × 1.5–3.0 μm) are concave, elongated, of irregular shape, often curved, and broadened towards one end. Roundish depressions are forming two rows on both sides of the narrow axial thickening. The cells are attached to the substratum. Molecular phylogenetic analysis based on the SSU rDNA robustly placed P. ruminata in the family Yogsothothidae. This position is confirmed with the presence of five panacanthocystid increase regions. The morphology of the new genus is in a good accordance with diagnosis of the family. The status of a genus “Heteroraphidiophrys” is discussed. Other potential findings of Pinjata from literature are analyzed. Pinjata represents the third lineage of centrohelids, characterized with the presence of only tangentially oriented plate scales. The halophilic nature of Yogsothothidae is suggested.     

Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors

Mutations in ribosomal RNA (rRNA) have traditionally been detected by the primer extension assay, which is a tedious and multistage procedure. Here, we describe a simple and straightforward fluorescence assay based on binary deoxyribozyme (BiDz) sensors. The assay uses two short DNA oligonucleotides that hybridize specifically to adjacent fragments of rRNA, one of which contains a mutation site. This hybridization results in the formation of a deoxyribozyme catalytic core that produces the fluorescent signal and amplifies it due to multiple rounds of catalytic action. This assay enables us to expedite semi-quantification of mutant rRNA content in cell cultures starting from whole cells, which provides information useful for optimization of culture preparation prior to ribosome isolation. The method requires less than a microliter of a standard Escherichia coli cell culture and decreases analysis time from several days (for primer extension assay) to 1.5 h with hands-on time of ∼10 min. It is sensitive to single-nucleotide mutations. The new assay simplifies the preliminary analysis of RNA samples and cells in molecular biology and cloning experiments and is promising in other applications where fast detection/quantification of specific RNA is required.     

应用RNA干扰沉默猪内源性反转录病毒

目的:尝试应用RNA干扰(RNAi)沉默猪源PK-15细胞中的猪内源性反转录病毒(PERV),并通过反转录酶活性及pol基因相对荧光定量PCR检测沉默效果。方法:依据GenBank公布的PERV pol基因序列,采用Invitro-gen公司的BLOCK-iT RNAi Designer软件设计Stealth小干扰RNA(siRNA)序列;将合成的siRNA转染PK-15细胞,72 h后检测细胞上清PERV反转录酶活性及细胞内pol基因拷贝数并评价沉默效果。结果:反转录酶活性及pol基因拷贝数检测结果表明,设计的3条Stealth siRNA序列中,位于pol基因3272～3296 bp的序列能有效沉默PERV。结论:RNAi方法可有效使猪源PK-15细胞中的PERV沉默,为进一步研究天然抗病毒分子与PERV的相互作用提供了实验基础,同时也为猪源异种移植研究中去除PERV提供了一种可供尝试的方法。     

Molecular dynamics simulation of the substitution of serine for the conserved glycine in the G-loop in the cdc28-srm yeast mutant using the crystal lattice of human CDK2 kinase

Two-nanosecond molecular dynamics simulations of the crystal lattice of an active complex of pT160-CDK2 kinase/cyclin A/ATP-Mg²⁺/substrate were performed. The simulations showed that the structures of the wild-type CDK2 complex and the mutant CDK2 complex involving the substitution G16S-CDK2 corresponding to the yeast substitution G20S-CDC28 differ noticeably and the differences between the structural conformations are most pronounced in the regions that play a key role in the kinase functioning. The results of the computer calculations were used to consider the structural elements that may affect the kinase activity, the regulatory phosphorylation, and the binding of protein kinase with cyclins and substrates.     

Protein trypsin inhibitor from potato tubers

A protein of 22 kDa designated as PKTI-22 was isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and purified to homogeneity using CM-Sepharose CL-6B ion-exchange chromatography. The protein efficiently suppressed the activity of trypsin, affected chymotrypsin less, and did not affect subtilisin Carlsberg. The N-terminal sequence of PKTI-22 (20 amino acid residues) was found to be highly homologous with the amino acid sequences of the potato Kunitz-type proteinase inhibitors of group B (PKPI-B) that were aligned from the corresponding gene sequences and was identical to the sequence (from the 2nd to the 20th residue) of the recombinant protein PKPI-B10. These data together with the observed similarity of the properties of two proteins indicate that the PKTI-22 protein is encoded by the PKPI-B10 gene.