The effect of hairpin loop on the structure and gene expression activity of the long-loop G-quadruplex

G-quadruplex (G4), a four-stranded DNA or RNA structure containing stacks of guanine tetrads, plays regulatory roles in many cellular functions. So far, conventional G4s containing loops of 1–7 nucleotides have been widely studied. Increasing experimental evidence suggests that unconventional G4s, such as G4s containing long loops (long-loop G4s), play a regulatory role in the genome by forming a stable structure. Other secondary structures such as hairpins in the loop might thus contribute to the stability of long-loop G4s. Therefore, investigation of the effect of the hairpin-loops on the structure and function of G4s is required. In this study, we performed a systematic biochemical investigation of model G4s containing long loops with various sizes and structures. We found that the long-loop G4s are less stable than conventional G4s, but their stability increased when the loop forms a hairpin (hairpin-G4). We also verified the biological significance of hairpin-G4s by showing that hairpin-G4s present in the genome also form stable G4s and regulate gene expression as confirmed by in cellulo reporter assays. This study contributes to expanding the scope and diversity of G4s, thus facilitating future studies on the role of G4s in the human genome.     

Expression pattern of glucose metabolism genes in relation to development rate of buffalo (Bubalus bubalis) oocytes and in vitro–produced embryos

The expression pattern of glucose metabolism genes (hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase [G6PDH], lactate dehydrogenase [LDH], and pyruvate dehydrogenase [PDH]) were studied in buffalo in vitro–matured oocytes and in vitro–produced embryos cultured under different glucose concentrations (0 mM, 1.5 mM, 5.6 mM, and 10 mM) during in vitro maturation of oocytes and culture of IVF produced embryos. The expression of the genes varied significantly over the cleavage stages under different glucose concentrations. Developmental rate of embryos was highest under a constant glucose level (5.6 mM) throughout during maturation of oocytes and embryo culture. Expression pattern of glucose metabolism genes under optimum glucose level (5.6 mM) indicated that glycolysis is the major pathway of glucose metabolism during oocyte maturation and early embryonic stages (pre-maternal to zygotic transition [MZT]) and shifts to oxidative phosphorylation during post-MZT stages in buffalo embryos. Higher glucose level (10 mM) caused abrupt changes in gene expression and resulted in shifting toward anaerobic metabolism of glucose during post-MZT stages. This resulted in decreased development rate of embryos during post-MZT stages. High expression of LDH and PDH in the control groups (0 mM glucose) indicated that in absence of glucose, embryos try to use available pyruvate and lactate sources, but succumb to handle the post-MZT energy requirement, resulting to poor development rate. Expression pattern of G6PDH during oocyte maturation as well early embryonic development was found predictive of quality and development competence of oocytes/ embryos.     

Medicarpin confers powdery mildew resistance in Medicago truncatula and activates the salicylic acid signalling pathway

Powdery mildew (PM) caused by the obligate biotrophic fungal pathogen Erysiphe pisi is an economically important disease of legumes. Legumes are rich in isoflavonoids, a class of secondary metabolites whose role in PM resistance is ambiguous. Here we show that the pterocarpan medicarpin accumulates at fungal infection sites, as analysed by fluorescein‐tagged medicarpin, and provides penetration and post‐penetration resistance against E. pisi in Medicago truncatula in part through the activation of the salicylic acid (SA) signalling pathway. Comparative gene expression and metabolite analyses revealed an early induction of isoflavonoid biosynthesis and accumulation of the defence phytohormones SA and jasmonic acid (JA) in the highly resistant M. truncatula genotype A17 but not in moderately susceptible R108 in response to PM infection. Pretreatment of R108 leaves with medicarpin increased SA levels, SA‐associated gene expression, and accumulation of hydrogen peroxide at PM infection sites, and reduced fungal penetration and colony formation. Strong parallels in the levels of medicarpin and SA, but not JA, were observed on medicarpin/SA treatment pre‐ or post‐PM infection. Collectively, our results suggest that medicarpin and SA may act in concert to restrict E. pisi growth, providing new insights into the metabolic and signalling pathways required for PM resistance in legumes.     

Sequence to structural analysis of ORF5 protein in Norway rat Hepatitis E Virus

Hepatitis E virus (HEV) is a major causative agent of acute hepatitis in developing countries. The Norway rat HEV genome consists of six open reading frames (ORFs), i.e., ORF1, ORF2, ORF3, ORF4, ORF5 and ORF6. The additional reading frame encoded protein ORF5 is attributed to life cycle of rat HEV. The ORFF5 protein’s function remains undetermined. Therefore, it is of interest to analyze the ORF5 protein for its physiochemical properties, primary structure, secondary structure, tertiary structure and functional characteristics using bioinformatics tools. Analysis of the ORF5 protein revealed it as highly unstable, hydrophilic with basic pI. The ORF5 protein consisted mostly of Arg, Pro, Ser, Leu and Gly. The 3D structural homology model of the ORF5 protein generated showed mixed α/β structural fold with predominance of coils. Structural analysis revealed the presence of clefts, pores and a tunnel. This data will help in the sequence, structure and functional annotation of ORF5.     

Identification of YfiH (PgeF) as a factor contributing to the maintenance of bacterial peptidoglycan composition

Peptidoglycan (PG) is an essential, envelope‐fortifying macromolecule of eubacterial cell walls. It is a large polymer with multiple glycan strands interconnected by short peptide chains forming a sac‐like structure around cytoplasmic membrane. In most bacteria, the composition of the peptide chain is well‐conserved and distinctive; in E. coli, the peptide chain length varies from two to five amino acids with a tetrapeptide consisting of L‐alanine – D‐glutamic acid – meso‐diaminopimelic acid – D‐alanine. However, it is not known how bacteria conserve the composition and sequence of peptide chains of PG. Here, we find that a conserved open reading frame of unknown function, YfiH (renamed PgeF) contributes to the maintenance of peptide composition in E. coli. Using genetic, biochemical and mass spectrometrical analyses we demonstrate that absence of yfiH results in incorporation of non‐canonical amino acids, L‐serine or glycine in place of L‐alanine in PG sacculi leading to β‐lactam – sensitivity, lethality in mutants defective in PG remodelling or recycling pathways, altered cell morphology and reduced PG synthesis. yfiH orthologs from other Gram‐positive genera were able to compensate the absence of yfiH in E. coli indicating a conserved pathway in bacterial kingdom. Our results suggest editing/quality control mechanisms exist to maintain composition and integrity of bacterial peptidoglycan.     

Transformation and evaluation of different transgenic lines for Glyphosate tolerance and cane borer resistance genes in sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.)

The aim of this study was to ensure the systematic protein expression of two genes (GTG and Cry1Ac) under the influence of two different constitutive promoters i.e. Ubiquitin-1 and CaMV 35S promoters in different sugarcane lines. PCR amplification of GTG and Cry1Ac was achieved from putative transgenic plants through gene specific primers. Qualitative comparisons of GTG and Cry1Ac genes expression under two different promoters were obtained through protein dot blot and dipstick assay. The appearance of comparatively dark color dots in dot blot and dark color bands on dipstick with Ubiquitin as compared to light color bands with CaMV35S promoter, qualitatively confirmed high protein expression of two genes under Ubiquitin promoter. In quantitative gene expression comparisons maximum optical density (OD) at 450 nm of UV-light was obtained for GTG (3.7 OD) and Cry1Ac (3 OD) under Ubiquitin promoter, while for GTG (1.6 OD) and Cry1Ac (2.5 OD) with CaMV 35S promoter. The results indicated higher expression of two genes under Ubiquitin-1 promoter in sugarcane was found as compared to CaMV 35S promoter. This study provides a guide for stable and high expression of transgenes with reference to Ubiquitin-1 promoter which can be utilize in sugarcane as well as in other monocots.     

Purification and characterization of a novel trypsin-like protease from green-seeded chickpea (Cicer arientum)

The present study describes the purification and physicochemical and biochemical characterization of trypsin-like protease from green-seeded chickpea (Cicer arientum). The crude extract of chickpea trypsin (CpT) was obtained by homogenization followed by differential ammonium sulfate precipitation. The CpT was purified by ion-exchange chromatography on diethylaminoethyl (DEAE) column, pre-equilibrated with 20?mM tris-CaCl₂ buffer (pH 8.2) with a flow rate of 0.5?mL min^?1. The molecular weight and purity of ～23?kDa of CpT were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Activity of protease was determined using Nα-benzoyl-DL-arginine-p-nitroanilide as chromogenic substrate and CpT purified showed a specific inhibitor activity of 26978.7697?U?mg^?1, fold purity of 9.8, and the yield of 70.2%. The characterization was performed for thermal stability, pH profile, and effect of various inhibitors on enzymatic activity. The protein isolated showed stability in the neutral to mild alkaline pH range and thermostability up to 50°C. CpT confirmed its serine nature as it was appreciably inhibited by serine protease inhibitors (maximum 6%), whereas metalloprotease inhibitors barely affected the activity of the enzyme (85%). To the best of our knowledge, it is first reported on purification of protease with trypsin-like properties, from this source.     

Primary structure of rabbit lens α-crystallins

The primary structure and posttranslational modifications of rabbit lens α-crystallins were examined using electrospray ionization mass spectrometry to determine the molecular weights of the intact proteins and fast atom bombardment mass spectrometry to analyze proteolytic digests of the αA- and αB-crystallins. The previously determined primary structure of αA-crystallin was confirmed. Posttranslational modifications detected included one phosphorylation site and the presence of a truncated form minus the five C-terminal residues. The previously undetermined amino acid sequence of rabbit αB-crystallin was determined to be the same as the bovine αB-crystallin sequence except at three residues: Thr 40, Thr 132, and Pro 153. Rabbit αB-crystallin showed evidence of phosphorylation at the same three sites as bovine αB-crystallin. The molecular weights of the intact proteins indicated that any one molecule had a maximum of two phosphorylations. Also, there was a truncated form which did not include the five C-terminal residues.     

Comprehensive analysis of the molecular docking of small molecule inhibitors to the Aβ1–40 peptide and its Osaka-mutant: insights into the molecular mechanisms of Aβ-peptide inhibition

Abstract

Alzheimer’s disease (AD) is the most common form of age-related neurodegeneration occurs because of deposition of proteins in the form of extracellular plaques containing aggregated amyloid beta (Aβ) peptide and intracellular neurofibrillary tangles composed of aggregated microtubule-binding protein tau. Amyloid aggregation process can be enhanced by several familial AD-associated mutations in Aβ peptide. In this study, we have unravelled the interactions of 40 small molecule inhibitors with the Osaka-mutant of Aβ_1–40 peptide at atomic level and characterized modes of their binding to mutant Aβ by docking approaches. We have also compared docking energies of these inhibitors with Osaka-mutant with those previously determined for the wild-type and Iowa-mutant peptides and discussed in light of the peptide conformations and non-covalent interactions. We have also discussed inhibition mechanisms of these three peptides. Our analyses revealed that these small molecules can efficiently inhibit Osaka-mutant. The binding modes of drugs with these three peptides are markedly different and so are the mechanisms of inhibition of these three peptides. Overall analysis of the data reveals that binding energy of Iowa-mutant drug complex is lowest and most stable which is followed wild-type peptide-drug complex followed by Osaka-mutant drug complex.

Communicated by Ramaswamy H. Sarma