Efficient ligation and cloning of DNA fragments with 2-bp overhangs

Various methods of ligation are currently available and routinely used by molecular biologists, such as blunt end ligation, cohesive end (two and four overhangs), and ligation of Taq polymerase-derived products. However, there is no efficient method for the cloning of DNA fragments with 2-bp overhangs. We present a simple method for the efficient ligation of DNA fragments with 2-bp overhanging ends, ranging in size from 0.7 to 2.5 kbp. Our method involves the initial heating and flash freezing of the vector-insert DNA mix, and a subsequent unique ligation reaction. This method provides a new molecular biology tool for researchers.     

Yeast translation elongation factor eEF3 promotes late stages of tRNA translocation

In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA–tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl‐tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slowdown in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo‐EM analysis of native eEF3‐ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non‐rotated ribosomal states, as well as by opening the L1 stalk to release the E‐site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.     

Aggregation-prone Regions in HYPK Help It to Form Sequestration Complex for Toxic Protein Aggregates

Protein aggregates result from altered structural conformations and they can perturb cellular homeostasis. Prevention mechanisms, which function against protein aggregation by modulatory processes, are diverse and redundant. In this study, we have characterized Huntingtin interacting protein K (HYPK) as a global aggregation-regulatory protein. We report the mechanistic details of how HYPK's aggregation-prone regions allow it to sense and prevent other toxic protein's aggregation by forming unique annular-shaped sequestration complexes. Screenings for interacting partners of different aggregation-prone proteins identify HYPK as a global interacting partner/regulator of Huntingtin97Qexon1, α-Synuclein-A53T and Superoxide dismutase1-G93A. C-terminal hydrophobic region in HYPK makes direct contacts with aggregates to initiate the formation of sequestration complexes. HYPK acts as aggregate sensor by existing in a seeded amyloid-like state which also favors its own concentration-dependent self-oligomerization. Oligomerization of HYPK leads to annular and non-fibrillar/amorphous aggregates. Two hydrophobic segments in the C-terminus of HYPK are responsible for its own aggregations. Self-association of HYPK follows seed nucleation, in which oligomeric HYPK seeds nucleate to annular structures. Annular oligomers of HYPK fuse with each other to form amorphous aggregates. HYPK shows differential interactions with aggregation-prone and non-aggregating proteins, as it preferentially binds to aggregation-prone proteins with higher affinity than native/non-aggregating proteins. This favors the formation of HYPK's sequestration complexes both in cytosol and in ribosome. Besides having aggregation-preventive property, HYPK also reduces the cellular level of toxic proteins. In vivo, HYPK sequestration complexes prevent the formation of toxic protein aggregates to physiologically show positive impact on cell survival and restoration of normal cell physiology.     

Chromium and nickel tolerance of Trema orientalis (Blume) L. in tissue culture

Callus of Trema orientalis derived from both contaminated and uncontaminated sources were tested in vitro for their relative tolerance to chromium and nickel. The calluses derived from contaminated source were metal-tolerant and showed better growth than those obtained from uncontaminated plants. The specificity of metal tolerance shown by the parent material was maintained in the calluses. Compared to the uncontaminated explants, the calli derived from contaminated sources exhibited higher catalase and peroxidase activities but a reduced acid phosphatase activity. Biochemical studies, provided evidence that the contaminated sources were physiologically distinct from the uncontaminated ones. Thus, this study indicated that seeds of Trema orientalis collected from contaminated sites were tolerant to chromium and nickel, and may have the advantage of being used in sustainable revegetation programmes on chromiferous minewastes.     

The effect of aqueous plant extracts on growth and aflatoxin production by Aspergillus flavus

The effect of aqueous leaf extracts of four plants, Argemone mexicana, Cyperus rotundus, Euphorbia hirta and Solanum nigrum , on growth and aflatoxin production by Aspergillus flavus was studied in SMKY liquid medium. All the plants inhibited aflatoxin production. No correlation between the growth of the fungus and aflatoxin synthesis was observed. The influence of these plants on the ratio of aflatoxin B₁ to G₁ is discussed.     

Metabolomics of brain and reproductive organs: characterizing the impact of gestational exposure to butylbenzyl phthalate on dams and resultant offspring

Phthalates are plasticizers finding wide spread use in industrial and household products, with measureable levels of phthalate-derived metabolites in the general US population. Phthalates have endocrine disruption potential and have been implicated as obesogens. Our exploratory investigation to reveal the impact of in utero exposure to a phthalate on the biochemical profiles of the brain, testes, and uterus of prepubertal offspring, and of tissues from dams administered butylbenzyl phthalate (BBP). Pregnant rats (three per group) were administered (on gestation day 14?C21) corn oil (control), or 25?mg/kg/day or 750?mg/kg/day BBP in corn oil. Tissues were collected from each of the dams on postnatal day (pnd) 21 (~3?weeks after the end of BBP administration), and from each of the pups on pnd 26 (~4 weeks after birth to dams administered vehicle or BBP during gestation) and processed for metabolomics analysis. Multivariate data analyses revealed metabolites that best distinguished the exposed and control groups. The metabolites most important to distinguishing the study groups were tested for significance using the exact Wilcoxon rank-sum test. Male pups had significant differences (control versus BBP dose groups) in levels of metabolites for both the brain and testes even at the P?<?0.01 level. However, female pups and dams had significant testing for the uterus only at the P?=?0.1 level tested. Female pups also had some significant differences for the brain with P values between 0.5 and 0.1. Amino acid metabolism (male and female pups) and phospholipid metabolism (male pups) were perturbed for the brain. Amino acid metabolism, purine metabolism, and TCA cycle were perturbed for tests and uterus. This study demonstrated the use of metabolomics to reveal metabolic perturbations in tissues of offspring following in utero exposures, and suggests the use of this approach for determining the impact of exposure past the time of the presence of the parent compound and metabolites derived from the parent compound.     

Orthovanadate and fluoroaluminate stimulate inositol phosphate production and in vitro ovulation in goldfish (Carassius auratus) follicles

Both sodium orthovanadate and fluoroaluminate were found to stimulate in vitro ovulation in intact goldfish follicles, suggesting the involvement of G-proteins in ovulation. Although orthovanadate was able to stimulate cAMP production, it probably stimulates ovulation by some other mechanism since cAMP blocks ovulation in this species. These agents also stimulated the accumulation of labeled inositol phosphate in follicle walls. The time course of inositol phosphate production showed a slightly delayed and continuous accumulation for isomers of inositol mono-, bis-, and trisphosphates. No change was observed in inositol tetrakisphosphate levels over time. The accumulation of inositol phosphates in response to orthovanadate was also dose-dependent. Lithium chloride (10 mM) caused varying increases in the levels of most isomers and a decrease in ins-3,4-P2. Inositol phosphate production varied significantly with changes in the maturational stage of follicles. Peak production was observed in follicles 7-8 h after hCG treatment, which corresponds almost exactly with the time of ovulation. This correlation of maximal inositol phosphate production with the time of ovulation, along with the stimulation of ovulation by diacylglycerols, a phorbol ester, and the G-protein-stimulating agents, orthovanadate and fluoroaluminate, suggests a role for polyphosphatidylinositol hydrolysis in ovulation.